WO2022192594A2 - Nucleic acid molecules and vaccines comprising same for the prevention and treatment of coronavirus infections and disease - Google Patents

Nucleic acid molecules and vaccines comprising same for the prevention and treatment of coronavirus infections and disease Download PDF

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Publication number
WO2022192594A2
WO2022192594A2 PCT/US2022/019834 US2022019834W WO2022192594A2 WO 2022192594 A2 WO2022192594 A2 WO 2022192594A2 US 2022019834 W US2022019834 W US 2022019834W WO 2022192594 A2 WO2022192594 A2 WO 2022192594A2
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substituted
unsubstituted
bond
independently
nucleic acid
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PCT/US2022/019834
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French (fr)
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WO2022192594A3 (en
Inventor
Runqiang CHEN
Hui Xie
Henry Hongjun Ji
Qidong Hu
Hua Wang
David Francis
Russell F. Ross
Xiaoxuan LYU
Ying Zhao
Peng Wang
Haotian SUN
Ying Zeng
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Sorrento Therapeutics, Inc.
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Publication of WO2022192594A2 publication Critical patent/WO2022192594A2/en
Publication of WO2022192594A3 publication Critical patent/WO2022192594A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates, inter alia , to nucleic acid molecules and compositions, pharmaceutical compositions, vaccines comprising such nucleic acid molecules for preventing or treating coronavirus infections and diseases associated therewith, as well as methods for administering same.
  • Coronaviruses belong to a group of viruses that causes diseases in birds, mammals and humans. Diseases caused by coronavirus infection include respiratory infections and enteric infections, which can be mild or lethal. Coronaviruses are viruses in the subfamily Orthocoronavirinae, in the family Coronaviridae, in the order Nidovirales.
  • the genus Coronavirus includes avian infectious bronchitis virus, bovine coronavirus, canine coronavirus, human coronavirus 299E, human coronavirus OC43, murine hepatitis virus, rat coronavirus, and porcine hemagglutinating encephalomyelitis virus.
  • the genus Torovirus includes Berne virus and Breda virus. Coronaviruses are enveloped viruses having a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry.
  • the genomic size of coronaviruses ranges from approximately 26 to 32 kilobases, which is believed to be the largest for an RNA virus.
  • coronavirus is derived from the Latin corona and the Greek korone (e.g., "garland” or “wreath”), meaning crown or halo.
  • the corona reference relates to the characteristic appearance of virions (the infective form of the virus) by electron microscopy, which have a fringe of large, bulbous surface projections creating an image reminiscent of a royal crown or of the solar corona.
  • This morphology is created by the viral spike (S) peplomers, which are proteins that populate the surface of the virus and determine host tropism. Proteins that contribute to the overall structure of all coronaviruses are the spike protein (also known as “S protein” or “S”, all used interchangeably throughout), envelope (E), membrane (M) and nucleocapsid (N).
  • SARS coronaviruses a defined receptor-binding domain on S mediates the attachment of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • Some coronaviruses also have a shorter spike-like protein called hemagglutinin esterase (HE).
  • HE hemagglutinin esterase
  • SARS-CoV-2 virus has accounted for more than 440 million cases of the coronavirus disease 2019 (COVID-19) and over 6 million fatalities worldwide since its original outbreak in December 2019. This is the 3rd outbreak of a Betacoronavirus since 2002, the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) being its predecessors, and is much more efficiently transmitted person to person.
  • SARS-CoV and Middle East respiratory syndrome coronavirus MERS-CoV
  • SARS-CoV-2 gains entry to human cells by using the angiotensin-converting enzyme 2 (ACE2) protein as a receptor.
  • ACE2 angiotensin-converting enzyme 2
  • S spike protein of SARS-CoV-2, a transmembrane glycoprotein that forms homotrimers, binds ACE2 on host cells leading to internalization of the virus.
  • the SARS-CoV-2 S protein (NCBI Accession QHU79204.1 (SARS-CoV-2 isolate Washington/Wuhan-Hu- 1 ,e.g., SEQ ID NO:20) includes two regions or domains known as SI (the N-terminus to amino acid 685) and S2 (amino acids 686 to 1273) that are cleaved into the SI and S2 subunits by furin, a cellular protease, during the infection process (Peacock et al. (2020) bioRxiv 2020.09.30.318311; doi: https://doi.org/10.1101/2020.09.30.318311).
  • the SI subunit which mediates the interaction between the S protein and ACE2, includes the “N- terminal domain” (NTD) which is followed by the receptor binding domain (RBD) at amino acids 319 to 541 (See, e.g., Huang et al., Acto Pharmacologica Sinica, Vol 41, pages 1 Mi ll 49 (2020)).
  • NTD N- terminal domain
  • RBD receptor binding domain
  • the S2 subunit which includes an extracellular domain, a transmembrane domain, and a cytoplasmic tail, mediates virus-host membrane fusion that results in entry of the virus into the host cell.
  • Vaccines such as mRNA vaccines
  • mRNA vaccines have become a versatile technology for the prevention of infectious diseases and various types of cancer, and thus are attractive candidates for the prevention and/or treatment of coronavirus infection, including infection due to SARS-Cov-2-mediated infections.
  • mRNA-based vaccines are attractive candidates in part because the development process of an mRNA vaccine can be much faster than conventional protein vaccines (DeFrancesco, 2017, Nat. Biotechnol ., 35, 193-197).
  • mRNA vaccine enables the synthesis of antigen proteins in situ, eliminating the need for protein purification and long-term stabilization which are challenging for some antigens.
  • transportation and storage of mRNA may be easier than protein-based vaccines, since RNA, if protected properly against ribonucleases (RNases), is less prone to degradation compared to proteins (Stitz et al., 2017, PLOS Negl. Tropical Dis ., 11, e0006108; Zhang et al., 2019, Front Immuno ., 10, 594). Because of these advantages, mRNA vaccines have great potential to be manufactured and deployed in a timely manner in response to rapid infectious disease outbreaks. Indeed, to date, a total of ten COVID-19 vaccines have been granted for emergency use or fully approved globally, with many of them being mRNA vaccines (https://covidl9irackvaccines.org/agency/who/).
  • VOCs SARS-Cov-2 variants of concern
  • Beta variant exhibits great immune escape and COVID-19 vaccination only showed 75% effectiveness against infection (Abu-Raddad et al., 2021).
  • a new variant B. 1.617.2 (Delta) was identified and subsequentially contributed to a surge in cases in India and worldwide shortly afterwards.
  • Delta variant has been shown to cause more severe disease and result in a poorer prognosis than previously reported VOCs (Farinholt et al., 2021; Mlcochova et al., 2021; Planas et al., 2021).
  • RBD receptor binding domain
  • the high rate of mutations in Omicron RBD may dramatically alter the interaction dynamics between the virus and host cell, which could, at least partially, explain the enhanced transmissibility and breakthrough cases (Liu et al., 2021; Zhao et al., 2021).
  • LNs dermis and draining lymph nodes
  • APCs antigen presentingcells
  • Langerhan cells that reside in the skin (epidermis and dermis) thatare capable of taking up antigen and subsequently trafficking to draining LNs to elicit adaptive immunity.
  • the initial lymphatics are present at high concentrations just below the stratum corneum and provide direct access to draining LNs due to their high permeability and uni-directional flow towards draining LNs.
  • Such devices and methods may advantageously be used with a variety of different drug modalities, including mRNA-based therapeutics for vaccinations.
  • mRNA-based therapeutics for vaccinations.
  • nucleic acid molecules and compositions, pharmaceutical compositions, and vaccines comprising such nucleic acid molecules, for preventing or treating coronavirus infections and diseases associated therewith.
  • the , nucleic acid molecules and compositions, pharmaceutical compositions, and vaccines comprising such nucleic acid molecules are useful for the prevention and treatment of infections caused by SARS-Cov-2 variants, and diseases associated therewith.
  • infection of subjects by a SARS-CoV-2 variant are provided that employ one or more mRNA-comprising compositions, pharmaceutical compositions, and/or vaccines.
  • infection of subjects by a SARS-CoV-2 variant are provided that employ mRNA-comprising compositions, pharmaceutical compositions, and/or vaccines, wherein the one or more mRNA species encode mutant forms of an SARS-CoV-2 variant RBD and/or spike protein, wherein such mutant forms are resistant to furin cleavage and or are stability-enhanced.
  • the mRNAs provided in vaccine compositions herein encode coronavirus receptor binding domain (RBD) from one or more coronavirus variants, and/or a protein comprising such one or more coronavirus RBDs.
  • vaccine compositions herein encode a spike (S) protein of a coronavirus comprising one or more RBDs from one or more coronavirus variant.
  • such vaccine compositions herein encode one or more spike proteins including a QQAQ mutation at the furin cleavage site at the border of the SI and S2 domains.
  • such vaccine compositions herein encode one or more spike proteins including at least one spike-stabilizing mutation, such as a stabilizing PP mutation.
  • such vaccine compositions herein encode one or more spike proteins including a QQAQ mutation at the furin cleavage site at the border of the SI and S2 domains and at least one spike-stabilizing mutation, such as a stabilizing PP mutation.
  • An S protein encoded by a nucleic acid molecule as provided herein can be a variant S protein into which the QQAQ mutation has been introduced.
  • Nonlimiting examples of such variants include the Washington/Wuhan-Hu-1 variant, the alpha variant S protein, the beta variant S protein, the delta variant S protein, the gamma variant S protein, and the kappa variant S protein, the omicron variant SI protein, other variants that include mutations (substitutions, insertions and/or deletions) that correspond to two or more such variants, and combinations of the above.
  • a vaccine composition can include two or more mRNA molecules, e.g., RNA molecules encoding different S protein variants having the QQAQ mutation, as well as, optionally, an mRNA encoding the WA isolate S protein with the QQAQ mutation.
  • the mRNA When delivered to a subject, the mRNA, which may be complexed with or associated with a delivery carrier, is taken up by the subject’s cells, leading to expression of the one or more S protein transcripts by the host cells.
  • host cells transfected with an mRNA composition can display the encoded S proteins on the cell surface, allowing for detection by cells of the host immune system and development of a humoral and/or cellular immune response.
  • the variant S protein can be an S protein of any SARS-CoV-2 variant, including a variant that has arisen naturally in a population or selected for in laboratory experiments. Further the variant S protein can be an engineered variant based on modeling, prediction, and or screens or assays. In some embodiments, the variant S protein includes at least one spike-stabilizing mutation, for example, the PP mutation at the amino acids corresponding to amino acid positions 986 and 987 of the WA1/2020 S protein.
  • nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein, wherein the nucleic acid sequence comprises at least one RBD-encoding sequence of a coronavirus spike protein.
  • nucleic acids comprise at least one RBD-encoding sequence is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an RBD-encoding nucleic acid sequence from a SARS-CoV-2 virus selected the group consisting of: a) a SARS-CoV-2 Wuhan/Washington variant; b) a SARS-CoV-2 Alpha variant; c) a SARS-CoV-2
  • nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein comprising at least one RBD-encoding sequence encoding an RBD amino acid sequence comprising one or more or the following mutations: D614G; ⁇ 69/70- ⁇ 144-N501Y-A570D-D614G-P681H-T716I- S982A-D1118H; D80A-D215G- ⁇ 242/244-K417N-E484K-N501Y-D614G-A701V; D614G, S13I, W152C, L452R; G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, H1101D; T19R, (G142D), ⁇ 156-157, R158G, L452R, T478K, D614G, P681R, D950N;
  • nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a SARS-Cov-2 variant spike protein comprising one or more or the following mutations: D614G; ⁇ 69/70- ⁇ 144-N501Y-A570D- D614G-P681H-T716I-S982A-D1118H; D80A-D215G- ⁇ 242/244-K417N-E484K-N501Y- D614G-A701V; D614G, S13I, W152C, L452R; G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, H1101D; T19R, (G142D), ⁇ 156-157, R158G, L452R, T478K, D614G, P681R, D950N; T19R, (G142D), ⁇ 156-157
  • nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a SARS-Cov-2 variant spike protein viral spike protein wherein at least one RBD-encoding sequence is derived from, or otherwise corresponds to, one or more SARS-CoV-2 virus spike-encoding nucleic acid sequences selected from the group consisting of: a) a SARS-CoV-2 Wuhan/Washington variant; b) a SARS-CoV-2 Alpha variant; c) a SARS-CoV-2 Beta variant; d) a SARS-CoV-2 Gamma variant; e) a SARS-CoV-2 Delta variant; f) a SARS-CoV-2 Delta Plus variant; g) a SARS- CoV-2 Kappa variant; h) a SARS-CoV-2 Lambda variant; i) a SARS-CoV-2 Omicron variant; j) a SARS-CoV-2 Zeta variant;
  • nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein wherein portion of the spike protein comprises at least one RBD-encoding sequence comprises an RBD-encoding sequence present in one or more of SEQ ID Nos: 1-12 and 15-19.
  • nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein wherein portion of the spike protein comprises at least two, at least three, at least four, or at least five RBD-encoding sequences selected from the group consisting of: a SARS-CoV-2 Wuhan/Washington variant; a SARS-CoV-2 Alpha variant; a SARS-CoV-2 Beta variant; a SARS-CoV-2 Gamma variant; a SARS-CoV-2 Delta variant; a SARS-CoV-2 Delta Plus variant; a SARS-CoV-2 Kappa variant; a SARS-CoV-2 Lambda variant; a SARS-CoV-2 Omicron variant; a SARS-CoV-2 Zeta variant; a SARS-CoV-2 Epsilon variant; a SARS-CoV-2 Omicron variant; a SARS- CoV-2 Omicron Plus variant; and n) combinations of a)
  • nucleic acid molecules comprising a nucleic acid sequence encoding a chimeric RBD or a chimeric spike protein.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid comprises: an RBD from a Delta variant and an RBD from an Omicron variant in either order; an RBD from a Beta variant and an RBD from an Omicron variant in either order; or an RBD from a Delta variant, an RBD from a Beta variant, and an RBD from an Omicron variant in any order.
  • nucleic acid molecules comprising an RBD or a spike protein comprising an RBD, which is encoded by a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to a spike protein encoding sequence from a SARS-CoV-2 virus selected the group consisting of: a SARS-CoV-2 Wuhan/Washington variant; a SARS-
  • nucleic acid molecules comprising a nucleic acid sequence encoding a chimeric spike protein as present in SEQ ID NOS: 4, 6, and 7.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a furin site mutation.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a furin site mutation sequence as set forth in SEQ ID NO: 13.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a stabilizing mutation.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a PP spike-stabilizing mutation.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a PP spike-stabilizing mutation as set forth in SEQ ID NO: 14.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule further comprises nucleic acid sequence encoding a furin site mutation and a PP spike-stabilizing mutation.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule further comprises nucleic acid sequence encoding a furin site mutation as set forth in SEQ ID NO: 13 and a PP spike-stabilizing mutation as set forth in SEQ ID NO: 14.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises wherein the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-7.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule consists of a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule consists of a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-7.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule encodes a RBD or a spike protein that comprises an amino acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20- 32.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule encodes a RBD or a spike protein that consists of an amino acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20- 32.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a DNA sequence.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises an RNA sequence.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD and further comprising a promoter operably linked to the nucleic acid sequence.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD and further comprising a promoter operably linked to the nucleic acid sequence, wherein the promoter is an SP6, T3, or T7 promoter.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule includes at least one modified nucleotide.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule includes at least one modified nucleotide, wherein the at least one modified nucleotide is pseudouridine, N1 -methyl-pseudouridine, or 2-thiouridine.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a 5’ cap structure.
  • nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a 3’ polyA sequence.
  • compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules disclosed herein and throughout.
  • compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-19.
  • compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-12.
  • compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-7.
  • compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS:4-12.
  • compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier.
  • compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene;
  • L al and L a2 are each independently each X is independently O or S;
  • R 2 is H or substituted or unsubstituted alkyl
  • R 3 is H or substituted or unsubstituted alkyl
  • R 4 is H or substituted or unsubstituted alkyl
  • such lipids are lipids wherein: R 1 is H, -OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl;
  • B 1 is a bond or an unsubstituted Ci-Cx alkylene
  • B 2 and B 3 are each independently a bond or substituted or unsubstituted Ci-Cx alkylene
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene;
  • L al and L a2 are each independently each X is independently O or S;
  • R 2 is H or substituted or unsubstituted C 1 -C 12 alkyl;
  • R 3 is H or substituted or unsubstituted C 1 -C 12 alkyl;
  • R 4 is H or substituted or unsubstituted C1-C12 alkyl;
  • such lipids are lipids wherein: R 1 is -OH or methoxy; L 1 is a bond; B 1 is an unsubstituted C 1 -C 8 alkylene; B 2 and B 3 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; L 2 is a bond; L 4 is a bond; W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted or unsubstituted C 1 -C 12 alkylene; each X is independently O; L 3 is a bond; L 5 is a bond; L 6 is a bond; L 7 is a bond; R 2 is H or substituted or unsubstituted C1-C12 alkyl; R 3 is H or substituted or unsubstituted C 1 -C 12 alkyl; R 4 is H or substituted or unsubstituted C 1 -C 12 alkyl; and R
  • L a3 is a bond
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl;
  • B 6 and B 7 are each independently a bond or substituted or unsubstituted C2-C4 alkylene
  • L 8 is a bond
  • R 7 is H or methyl
  • R 8 , and R 9 are each independently substituted or unsubstituted C1-C20 alkyl.
  • such lipids are lipids wherein: W 7 and W 8 are each independently a bond or unsubstituted C2-C4 alkylene;
  • L a3 is a bond
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl;
  • B 6 and B 7 are each independently a bond or unsubstituted C2-C4 alkylene
  • L 8 is a bond
  • R 7 is H or methyl
  • R 8 and R 9 are each independently substituted or unsubstituted C1-C20 alkyl.
  • such lipids are lipids wherein: W 7 and W 8 are each independently a bond or unsubstituted C2-C4 alkylene; L a3 is a bond;
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl;
  • B 5 , B 6 , and B 7 are each independently a bond
  • L 8 is a bond
  • L 9 is a bond
  • L 10 is a bond
  • R 7 is H or methyl
  • R 8 and R 9 are each independently substituted or unsubstituted C1-C30 alkyl.
  • such lipids are selected from the group consisting of:
  • compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of formula (III): [0059] [0060] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: [0061] [0062] [0063] Q is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene; V is substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted arylene;
  • such lipids are selected from the group consisting of:
  • compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of formula (IV):
  • B 12 is -W 7 -L a3 -W 8 -;
  • W 7 and W 8 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene;
  • L a3 is a bond
  • W 9 and W 10 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted 2 to 12 membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any combination thereof;
  • compositions disclosed herein and throughout that further comprise lipid nanoparticles.
  • methods of preventing or treating coronavirus infection or disease comprising administering: a nucleic acid molecule; a composition of; and/or or a pharmaceutical composition; as disclosed herein and throughout; to a subject infected with, or at risk of infection, of suspected of having been infected, with a coronavirus.
  • such methods comprise administration via oral, nasal, intrapulmonary, intracavitary, by intra-arterial or intravenous infusion, or by injection.
  • such methods comprise administration via injection.
  • such methods comprise administration via subcutaneous, intramuscular, transdermal, intradermal, subdermal, epidermal, or lymphatic delivery or injection.
  • the nucleic acid molecule, the composition, or the pharmaceutical composition is administered by subdermal injection or delivery.
  • the nucleic acid molecule, the composition, or the pharmaceutical composition administration is administered or delivered into a lymphatic system.
  • the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch.
  • the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch, wherein the patch comprises a polymer.
  • nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch, wherein the patch comprises an absorbable polymer.
  • such methods comprise administering two or more doses of a nucleic acid molecule, a composition of any of and/or or a pharmaceutical composition as disclosed herein and throughout to the subject.
  • such methods comprise administering two or more doses of a nucleic acid molecule, a composition of any of and/or or a pharmaceutical composition as disclosed herein and throughout to the subject, such methods comprising: placing a medical device comprising a plurality of microneedles on the skin of the subject having lymphatic vasculature, wherein the medical device contacts a layer of epidermis with reversible permeability enhancers comprising a chemical, physical or electrical permeability enhancer that induces a-reversible increase in permeability of one or more barrier cells of the epidermis to the nucleic acid molecule, the composition, or the pharmaceutical composition.
  • such methods comprise administering two or more doses of a nucleic acid molecule, a composition of any of and/or or a pharmaceutical composition as disclosed herein and throughout to the subject, such methods comprising: placing a first medical device comprising a plurality of microneedles on the skin of the subject at a first location proximate to a first position under the skin of the subject, wherein the first position is proximate to lymph vessels and/or lymph capillaries that drain into the right lymphatic duct, and wherein the microneedles of the first medical device have a surface comprising nanotopography; placing a second medical device comprising a plurality of microneedles on the skin of the subject at a second location proximate to a second position under the skin of the subject, optionally wherein the first and second medical devices are the same device, wherein the second position is proximate to lymph vessels and/or lymph capillaries that drain into the thoracic duct, and wherein the microneedles of the second medical device have
  • such methods comprises preventing or treating a subject infected, suspected of having been infected, or at risk of being infected, by a SARS-Cov-2 variant.
  • an variant RBD or a variant spike protein has an amino acid sequence that is at least 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.5%, 99.5%, 99.5%, 99.5%, identical to an amino acid sequence selected from the group consisting of those set forth in SEQ ID Nos 20-32, inclusive.
  • a nucleic acid molecule comprises a nucleic acid sequence that encodes a protein having an amino acid sequence that is at least 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.5%, 99.5%, 99.5%, 99.5%, identity to an amino acid sequence selected from the group consisting of those set forth in SEQ ID Nos 20-32, inclusive.
  • a pharmaceutical composition comprising at least one RNA molecule as disclosed herein and a pharmaceutically acceptable carrier.
  • a pharmaceutically-acceptable carrier can be, for example, a buffer or aqueous solution that can include any of the aforementioned ingredients such as salts, buffering agents, chelators, etc.
  • a pharmaceutically-acceptable carrier can also be a delivery carrier that promotes delivery of the mRNA into a cell.
  • delivery carriers include lipids, lipid formulations, lipid nanoparticles (LNPs), polymers, and peptides.
  • a pharmaceutical includes two or more molecules RNA molecules encoding different S proteins, for example, encoding S proteins from different SARS-CoV-2 variants or isolates. In some embodiments, a pharmaceutical includes three or more molecules RNA molecules encoding different S proteins, for example, encoding S proteins from three different SARS-CoV-2 variants or isolates. In some embodiments, the pharmaceutical composition can include mRNAs encoding an isolate such as the Washington/Wuhan-Hu-1 isolate and two different variants, one or both of which may be selected from the alpha, beta, gamma, delta, kappa, and omicron variants.
  • the pharmaceutical composition can include mRNAs encoding an isolate such as the Washington/Wuhan-Hu-1 isolate and three different variants, one or both of which may be selected from the alpha, beta, gamma, delta, kappa, and omicron variants.
  • the pharmaceutical composition can include mRNAs encoding an isolate such as the Washington/Wuhan-Hu-1 isolate and four different variants, one or both of which may be selected from the alpha, beta, gamma, delta, kappa, and omicron variants.
  • a pharmaceutical mRNA composition as provided herein can include at least one delivery carrier, which may be, as nonlimiting examples, a lipid, a lipid formulation, a lipid- based nanoparticle, a polymer, a polymer formulation, or a peptide.
  • a method of preventing SARS-CoV-2 infection comprising administering an mRNA pharmaceutical composition such as any disclosed herein to a subject at risk of infection with SARS-CoV-2.
  • Delivery can be by oral, nasal, intrapulmonary, or intracavitary administration, by intra-arterial or intravenous infusion, or by injection.
  • delivery can be by intramuscular, transdermal, intradermal, or subcutaneous injection.
  • the method comprises administering two or more doses of an mRNA pharmaceutical composition such as any disclosed herein to a subject at risk of infection with SARS-CoV-2.
  • the two or more doses can comprise different RNA molecules.
  • Figure 1 depicts flow cytometry analysis of HEK293 cells one and three days after transfection with an RNA that encodes the “wild type” Washington (Wuhan) SARS-CoV-2 S protein and (bottom panels) HEK293 cells one and three days after transfection with an RNA that encodes the Washington (Wuhan) SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
  • Figure 2 depicts flow cytometry analysis of HEK293 cells one day after transfection with an RNA that encodes the “wild type” alpha variant SARS-CoV-2 S protein and an RNA that encodes the alpha variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
  • Figure 3 depicts flow cytometry analysis of HEK293 cells two and three days after transfection with an RNA that encodes the “wild type” beta variant SARS- CoV-2 S protein and (bottom panels) HEK293 cells two and three days after transfection with an RNA that encodes the beta variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
  • Figure 4 depicts flow cytometry analysis of HEK293 cells two and three days after transfection with an RNA that encodes the “wild type” gamma variant SARS- CoV-2 S protein and (bottom panels) HEK293 cells two and three days after transfection with an RNA that encodes the gamma variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
  • Figure 5 depicts flow cytometry analysis of HEK293 cells one, two, and three days after transfection with an RNA that encodes the “wild type” delta variant SARS-CoV-2 S protein and (bottom panels) HEK293 cells one, two, and three days after transfection with an RNA that encodes the delta variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
  • Figure 6 depicts flow cytometry analysis of HEK293 cells two and three days after transfection with an RNA that encodes the “wild type” kappa variant SARS- CoV-2 S protein and (bottom panels) HEK293 cells two and three days after transfection with an RNA that encodes the kappa variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
  • Figures 7A-7J show results of stability studies comparing lyophilized and liquid SARS-CoA-2 variant mRNA formulations.
  • Figure 7A shows a scatter plot of the control HEK293 cells lacking spike protein expression (no mRNA).
  • Figure 7B shows a scatter plot of HEK293 cells transfected with Washington/Wuhan-Hu-1 variant of mRNA encoding the SARS-CoV-2 spike protein.
  • Figure 7C shows a scatter plot of HEK293 cells, transfected with U.K. (Alpha) variant of mRNA encoding the SARS-CoV-2 spike protein.
  • Figure 7D shows a scatter plot of HEK293 cells, transfected with South Africa (Beta) variant of mRNA encoding the SARS-CoV-2 spike protein.
  • Figure 7E shows a scatter plot of HEK293 cells, transfected with Brazil (Gamma) variant of mRNA encoding the SARS-CoV-2 spike protein.
  • Figure 7F shows a scatter plot of the control HEK293 cells lacking spike protein expression (no mRNA).
  • Figure 7G shows a scatter plot of HEK293 cells transfected with mRNA encoding the SARS-CoV-2 spike protein, stored at -80°C for 11 days.
  • Figure 7H shows a scatter plot of HEK293 cells transfected with mRNA encoding the SARS-CoV-2 spike protein, stored at room temperature for 3 days following lyophilization.
  • Figure 71 shows a scatter plot of HEK293 cells transfected with mRNA encoding the SARS-CoV-2 spike protein, stored at room temperature for 11 days following lyophilization.
  • Figure 7J shows agarose gel electrophoresis of mRNA, encoding the SARS-CoV-2 spike protein, after lyophilization and 11-day storage under various conditions.
  • Figure 8A depicts a schematic of an exemplary STI mRNA vaccine that is optimized for highly efficient translation.
  • Figure 8B depicts flow cytometry results using primary dendritic cells that were transfected with various mRNAs, stained with anti-Spike antibody STI-2020, and evaluated by flow cytometry 24 post-transfection.
  • Figure 9A provides an image of an exemplary MuVaxx device connected to 1 mL syringe.
  • Figure 9B Provides an image of a C57B16 mouse 5 minutes after injection of ICG using the MuVaxx device depicted in FIG. 6A.
  • Figure 9C depicts anti-OVA titers determined by running ELIZA assays on serum collected on days 13 and 34 following either intramuscular injection (IM) or MuZaxx- mediated lymphatic administration (MuVaxx) of C57B16 mice with 10 ⁇ g of OVA (on day zero) and 8 ⁇ g CpG (on day 14).
  • IM intramuscular injection
  • MuZaxx- mediated lymphatic administration MoVaxx
  • Figure 9D provides percentage of CD8 T cells producing the indicated cytokines.
  • Whole blood was collected and stimulated with SIINFEKL peptide followed by ICS to measure IFNy and TNFa in the CD8 T cell compartment.
  • Cytokine statistics represents difference between IFNy+ groups for panel D.
  • Figure 10A illustrates a mouse IM or MuVaxx rRNA vaccination treatment schedule.
  • Figure 11A illustrates a mouse IM or MuVaxx rRNA vaccination treatment schedule.
  • Figure 12A illustrates a mouse IM or MuVaxx rRNA vaccination treatment schedule in which 6 days following booster shot, intracellular cytokine staining was performed in the presence of spike associated peptides (Miltenyi Biotec Peptivator).
  • Figure 12B provides representative flow cytometry plots of IFNy and TNFa production from CD8 T cells.
  • PRNT plaque reduction neutralization test
  • Figure 15A depicts PRNT50 values and anti-Sl IgG produced by long-lived B cells in lungs of mice used to generate the data depicted in Figure 13A.
  • Figures 16A-16B show antibody responses upon immunization with VOC- based vaccines in vivo.
  • Figure 16A shows Day 14 sera post booster was evaluated for antibody binding to recombinant Spike from designated VOCs by ELISA.
  • Figure 16B shows Day 14 sera post booster was evaluated for nAb responses against designated live virus by 50% plaque reduction neutralization test (PRNT).
  • PRNT plaque reduction neutralization test
  • Figures 17A-17C show immunization results with Furin mutant vaccine produces enhanced protection in vivo.
  • Figure 17A provides a schematic illustration of the design of the in vivo study.
  • Figure 17B shows the lung viral titer measured in mice challenged with live SARS-CoV-2 VOCs after full immunization.
  • Figures 18A-18D show immunization with Omicron-specific vaccine produces robust protection against Omicron challenge.
  • Figure 18A shows Day 14 sera post 2 nd vaccine shot were evaluated for nAb responses against Omicron from designated VOC vaccine by pseudovirus assay.
  • Figure 18B shows Day 14 sera post 2 nd Omicron-specific vaccine shot evaluated for antibody binding by ELISA.
  • Figure 18C shows Day 14 sera post 2 nd Omicron- specific vaccine shot evaluated for nAb responses by PRNT.
  • Figure 18D shows the design of the in vivo study for Omicron-specific vaccine booster ⁇ left panel) and the viral titer in the lung from mice challenged with live Omicron virus ⁇ right panel).
  • Figures 19A-19B show Delta RBD-Omicron immunization induces the potent and broadly spectrum neutralization activity against SARS-COV-2 variants.
  • Figure 19A, upper portion provides a schematic drawing of the design of the chimeric Delta RBD- Omicron mRNA.
  • Figure 19A, lower portion shows results with animals that were immunized with the mRNA twice. The Day 14 sera post boost were evaluated for binding antibodies specific to recombinant S proteins from designated VOC vaccines by ELISA.
  • Figures 20A-20F show expression and cleavage of Furin-mutated mRNA.
  • Figure 20A shows the Design of the furin mutant mRNA.
  • Figure 20B shows flow cytometry results, indicating an increase in the surface expression of spike protein from all variants containing a furin cleavage mutation.
  • Figure 20C shows full-length S protein is the dominant species in transfected 293T cells after the removal (mutation) of furin cleavage site.
  • Figure 20D shows the mutation at the furin cleavage site also lowered the level of free SI in the conditioned medium of transfected 293T cells.
  • Figure 20E shows furin-cleavage mutant mRNA elicits higher neutralization antibody titers than wild type mRNA revealed by ELISA on day 14 post boost.
  • Figure 20F shows furin-cleavage mutant mRNA elicits higher neutralization antibody titers than wild type mRNA revealed by and PRNT on day 14 post boost .
  • TM transmembrane domain.
  • FL full-length spike.
  • Figure 21 shows expression of Omicron and Delta RBD- Omicron mRNA.
  • the flow cytometry showed the surface expression of spike protein in transfected 293T cells stained with recombinant human ACE2 receptor.
  • Figure 22 shows results obtained when testing the indicated cholesterol and DSPC molar ratios, either as fresh formulations or as lyophilized and then reconstituted formulations.
  • Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
  • Figure 23 shows results obtained when testing the indicated N/P ratios, PEG molar ratios, and lipid concentrations, either as fresh formulations or as lyophilized and then reconstituted formulations.
  • Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
  • Figure 24 shows results obtained when testing the indicated N/P ratios and PEG molar ratios, and lipid concentrations as lyophilized and then reconstituted formulations.
  • Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
  • Figure 25 shows results obtained in an additional experiment testing the indicated N/P ratios and PEG molar ratios as lyophilized and then reconstituted formulations.
  • Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot, as indicated.
  • Figure 26 shows results obtained in an additional experiment testing the indicated N/P ratios, as well as the presence or absence of HP-b-CD, with samples that were either freeze-thawed or lyophilized and then reconstituted.
  • Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and at day 7, 14, 21, and 28 after booster shot, as indicated.
  • Figure 27 shows results obtained when testing the effect of adding either trehalose or sucrose to samples, as indicated.
  • Mouse SI IgG titers were then measured at day 7, 14, 21, and 28, after first booster shot and at day 14, 21, and 28 after second booster shot, as indicated.
  • Figure 28 shows results obtained when testing the effect of adding Tris buffer or Phosphate buffer to samples, as indicated.
  • Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated.
  • Figure 29 shows results obtained when testing the indicated formulations after various storage conditions/time periods, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated.
  • Figure 30 shows results obtained when testing the indicated formulations at the indicated pHs, as indicated.
  • Mouse SI IgG titers were then measured at day 7 after primary shot, as indicated.
  • Figure 31 shows results obtained when testing the indicated formulations prepared using HPLC pump, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated. Detailed Description
  • B, or C A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example,
  • “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition.
  • Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, all ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as “not including the endpoints”; thus, for example, “ranging from 1 to 10” includes the values 1 and 10 and all integer and (where appropriate) non-integer values greater than 1 and less than 10.
  • coronavirus infection refers to a human or animal that has cells that have been infected by a coronavirus.
  • the infection can be established by performing a detection and/or viral titration from respiratory samples, or by assaying blood-circulating coronavirus-specific antibodies.
  • the detection in the individuals infected with coronavirus is made by conventional diagnostic methods, such as molecular biology (e.g., PCR), which are known to those skilled in the art.
  • subject refers to human and non-human animals, including vertebrates, mammals and non-mammals.
  • the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
  • murine e.g., mice and rats
  • bovine porcine
  • equine canine
  • feline feline
  • caprine caprine
  • lupine ranine or piscine.
  • administering refers to the physical introduction of a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, transdermal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • treatment refers to fighting the coronavirus infection in a human or animal subject.
  • the viral infection rate infectious titer
  • treatment also refers to attenuating symptoms associated with the viral infection (e.g., respiratory syndrome, kidney failure, fever, and other symptoms relating to coronavirus infections).
  • an effective amount may be used interchangeably and refer to an amount of the therapeutic agent that when administered to a subject, is sufficient to affect a measurable improvement or prevention of a disease or disorder associated with coronavirus infection. For example, administering an effective dose sufficient to inhibit the proliferation and/or replication of the coronavirus, and/or the development of the viral infection within the subject.
  • Therapeutically effective amounts of the therapeutic agents provided herein, when used alone or in combination with an antiviral agent, will vary depending upon the relative activity of the therapeutic agent, and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. In one embodiment, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques. In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
  • polypeptide and “protein” and other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids. Polypeptides include recombinant or chemically-synthesized forms. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post- translationally, or otherwise covalently or non-covalently, modified proteins.
  • Polypeptides comprising amino acid sequences of an coronavirus or coronavirus RBD or S protein, corona virus variant RBD or S protein, SAR.S-CoV-2 virus RBD or S protein, or SAR.S-Cov-2 variant RBD or S protein, or a derivative, mutein, or variant thereof , can be prepared using recombinant procedures are described herein.
  • nucleic acid refers to polymers of nucleotides and are not limited to any particular length.
  • Nucleic acids include recombinant and chemically- synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecule can be single-stranded or double-stranded.
  • the nucleic acid molecules comprise a contiguous open reading frame encoding: at least one RBD corresponding to at least one coronavirus variant, such as a SAR.S-Cov-2 variant; one or more spike (S) proteins comprising one or more RBDs corresponding at least one coronavirus variant, such as a SAR.S-Cov-2 variant; or one or more derivatives, muteins, or variants thereof (such as emerging variants of concern and/or combinations of mutations found in two or more currently known SAR.S-Cov-2 variants).
  • nucleic acids comprise one type of polynucleotides or a mixture of two or more different types of polynucleotides.
  • mutation refers to a change in a nucleic acid sequence or amino acid sequence that differs from a reference nucleic acid sequence or a reference amino acid sequence, respectively.
  • mutations includes a point mutation, insertion, deletion, amino acid substitution, inversion, rearrangement, splice, sequence fusion (e.g., gene fusion or RNA fusion), truncation, transversion, translocation, non-sense mutation, sequence repeat, single nucleotide polymorphism (SNP), or other genetic rearrangement.
  • the QQAQ furin site mutation introduced into various coronavirus variants as disclosed herein comprises a series of amino acid substitutions introduced into the wild type Washington/Wuhan-Hu-1 isolate S protein furin site sequence RR.AR. at amino acid positions 682 to 685 and corresponding sites/positions found in other variants.
  • PP spike protein stablilizing mutation as disclosed herein comprises, for example, a pair of amino acid substitutions for the wild type Washington/Wuhan-Hu-1 isolate S protein furin site sequence KV at amino acids 986 and 987 and corresponding sites/positions found in other variants.
  • isolated refers to a protein (e.g., an antibody or an antigen binding portion thereof) or polynucleotide that is substantially free of other cellular material.
  • a protein may be rendered substantially free of naturally associated components (or components associated with a cellular expression system or chemical synthesis methods used to produce the antibody) by isolation, using protein purification techniques well known in the art.
  • isolated also refers in some embodiments to protein or polynucleotides that are substantially free of other molecules of the same species, for example other protein or polynucleotides having different amino acid or nucleotide sequences, respectively.
  • the purity of homogeneity of the desired molecule can be assayed using techniques well known in the art, including low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrophotometry.
  • variant polypeptides and variants of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence.
  • Polypeptide variants include fusion proteins.
  • a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence.
  • Polynucleotide variants include fusion polynucleotides.
  • a “variant” means an isolate of a virus, such as a SARS-Cov-2 Washington/Wuhan-Hu-1 (also referred to as WA1/2020, used interchangeably throughout), as well as currently known, emerging, and/or yet-to-emerge isolates, each independently having one or more mutations with respect to a reference virus, such as a SARS-CoV-2 Washington/Wuhan-Hu-lisolate, from which it is derived.
  • a SARS-CoV-2 variant may be a Washington/Wuhan-Hu-1 isolate, as well as a variant in reference to a Washington/Wuhan-Hu-1 isolate.
  • a variant for example, may typically have multiple mutations with respect to a Washington/Wuhan-Hu-1 isolate which, for the purposes herein, comprises and RBD and/or and S protein comprising such RBD, of a coronavirus isolate, such as a Washington/Wuhan-Hu-1 isolate, and/or variants thereof.
  • a “receptor binding domain” or “RBD” means a portion, region, or domain within a spike protein, such as a spike protein of a coronavirus, such as a SARS-Cov-2 variant, that is involved in the interaction between such spike protein and a cellular receptor of such spike protein, such an angiotensin converting enzyme 2 (“ACE2”) protein.
  • ACE2 angiotensin converting enzyme 2
  • such an RBD corresponds to amino acids 319 through 541, inclusive, of a SARS-Cov-2 spike protein, or to the corresponding amino acids in a variant of such a SARS-Cov-2 virus (see, e.g., Huang etal ., Acto Pharmacologica Sinica, Vol 41, pages 1141-1149 (2020)).
  • such an RBD corresponds to an RBD amino acid sequence encoded by any of SEQ ID Nos: 1-12 and 15-19).
  • a “spike protein,” “S protein’”, or “S” means a spike protein of a coronavirus, such as a SARS-Cov-2 variant, that is involved in the interaction between the coronavirus and a cellular receptor of such spike protein, such an angiotensin converting enzyme 2 (“ACE2”) protein.
  • ACE2 angiotensin converting enzyme 2
  • spike protein corresponds to amino acid sequence of NCBI Accession QHU79204.1 (SARS-CoV-2 isolate Washington/Wuhan-Hu- 1 ,e.g., SEQ ID NO:20), which includes two regions or domains known as SI (the N- terminus to amino acid 685) and S2 (amino acids 686 to 1273) that are cleaved into the SI and S2 subunits by furin, a cellular protease, during the infection process.
  • such a spike protein corresponds to a spike protein amino acid sequence encoded by any of SEQ ID Nos: 4-12 and 15-19.
  • such a spike protein corresponds to a spike protein amino acid sequence according to any of SEQ ID Nos:20-32.
  • Exemplary SARS-Cov-2 variants may also be selected from the group consisting of the following variants: B.1.1.7 (also known as U.K., or Alpha); B.1.351 (also known as South Africa, or Beta); B.1.617.1 (also known as India, or Kappa); B.1.617.2 (also known as India, or Delta); B.1.617.2.1 (also known as AY.l, or Delta Plus); P.l (also known as Brazil/Japan, Brazil, or Gamma ); C.37 (also known as Lambda); P.2 (also known as Brazil, or Zeta); B.1.526 (also known as NY, or Iota); B.1.526.2 (also known as NY, Iota); B.1.1.318 (also known as Mauritius); B.l.1.7 (also known as U.K., or Alpha); B.1.617.1
  • Exemplary SARS-Cov-2 variants may contain mutations selected from the group consisting of the following sets of mutations: D614G; D69/70-D 144-N501 Y-A570D-D614G-P681 H- T716I-S982 A-D 1118H; D80 A-D215G-A242/244-K417N-E484K-N501 Y-D614G- A701 V; D614G, S 131, W152C, L452R; G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, H1101D; T19R, (G142D), D156-157, R158G, L452R, T478K, D614G, P681R, D950N; T19R, (G142D), D156-157, R158G, K417N, L452R, T478K, D614G, P68
  • Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding an RBD found in one or more of SEQ ID Nos: 20-32. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding one or more RBDs found in one or more of SEQ ID Nos. 1-19.
  • Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding spike protein comprising one or more RBDs found in one or more of SEQ ID Nos: 20-32. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding a spike protein comprising one or more RBDs found in one or more of SEQ ID Nos. 1-19. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding spike protein according to one or more of SEQ ID Nos: 20-32. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding a spike protein according to one or more of SEQ ID Nos. 1-19.
  • Exemplary such chimeric spike proteins include, as non limiting examples, variants comprising a spike protein amino acid sequence that includes: an RBD from a Delta variant and an RBD from an Omicron variant (in either order); an RBD from a Beta variant and an RBD from an Omicron variant (in either order); and an RBD from a Delta variant, an RBD from a Beta variant, and an RBD from an Omicron variant (in any order).
  • Exemplary such chimeric spike proteins also include those encoded by any nucleic acid sequence set forth in SEQ ID NOS: 4, 6, and 7.
  • a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to a reference polynucleotide sequence.
  • the term “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • antibody includes, in addition to antibodies comprising two full-length heavy chains and two full- length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.
  • an "antigen binding protein” and related terms used herein refers to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen.
  • antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs.
  • the antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody -derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et ah, 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et ah, 2004, Biotechnol. Prog. 20:639- 654.
  • PAMs peptide antibody mimetics
  • scaffolds based on antibody mimetics utilizing fibronection components as a scaffold.
  • An antigen binding protein can have, for example, the structure of an immunoglobulin.
  • an "immunoglobulin” refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen binding sites.
  • an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens.
  • a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules.
  • variable regions of immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein.
  • An antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently.
  • the CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.
  • the assignment of amino acids to each domain is in accordance with the definitions of Rabat et al. in Sequences of Proteins of Immunological Interest, 5 th Ed., US Dept of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.
  • Other numbering systems for the amino acids in immunoglobulin chains include IMGT.RTM. (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol.
  • an "antibody” and “antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof (or an antigen binding fragment thereof) that binds specifically to an antigen.
  • Antigen binding portions or the antigen binding fragment may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • Antibodies include recombinantly produced antibodies and antigen binding portions.
  • Antibodies include non-human, chimeric, humanized and fully human antibodies.
  • Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities).
  • Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers.
  • Antibodies include F(ab’)2 fragments, Fab’ fragments and Fab fragments.
  • Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide-linked Fvs (sdFv), anti -idiotypic antibodies (anti-id), minibodies.
  • Antibodies include monoclonal and polyclonal populations.
  • a “neutralizing antibody” and related terms refers to an antibody that is capable of specifically binding to the neutralizing epitope of its target antigen (e.g., coronavirus spike protein) and substantially inhibiting or eliminating the biological activity of the target antigen (e.g., coronavirus spike protein).
  • the neutralizing antibody can reduce the biological activity of the target antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher levels of reduced biological activity.
  • an “antigen binding domain,” “antigen binding region,” or “antigen binding site” and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains.
  • telomere binding refers to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens).
  • an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 10 5 M or less, or 10 6 M or less, or 10 7 M or less, or 10 8 M or less, or 10 9 M or less, or 10 10 M or less.
  • a dissociation constant can be measured using a BIACORE surface plasmon resonance (SPR) assay.
  • SPR surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
  • An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof).
  • An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein.
  • An epitope can comprise non-contiguous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen’s primary sequence but that, in the context of the antigen’s tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein).
  • the variable regions, particularly the CDRs, of an antibody interact with the epitope.
  • an "antibody fragment”, “antibody portion”, “antigen-binding fragment of an antibody”, or “antigen-binding portion of an antibody” and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
  • Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.
  • Antigen binding fragments that bind a coronavirus spike protein (S-protein) are described herein.
  • Fab fragment
  • VL variable light chain region
  • CL constant light chain region
  • VH variable heavy chain region
  • CHI first constant region
  • a Fab is capable of binding an antigen.
  • An F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a F(Ab’)2 has antigen binding capability.
  • An Fd fragment comprises VH and CHI regions.
  • An Fv fragment comprises VL and VH regions.
  • An Fv can bind an antigen.
  • a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain (U.S. Patents 6,846,634 and 6,696,245; U.S. published Application Nos. 2002/02512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958; and Ward et ak, Nature 341:544-546, 1989).
  • Fab fragments comprising antigen binding portions from an antibody that binds a coronavirus spike protein (S-protein) are described herein.
  • a single-chain antibody is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain.
  • a linker e.g., a synthetic sequence of amino acid residues
  • the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et ak, 1988, Science 242:423-26 and Huston et ak, 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
  • Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et ak, 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et ak, 1994, Structure 2:1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites.
  • polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites.
  • tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
  • the term “human antibody” refers to antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody).
  • antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes.
  • labeled antibody refers to antibodies and their antigen binding portions thereof that are unlabeled or joined to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
  • detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
  • a variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).
  • the “percent identity” or “percent homology” and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences.
  • the percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences.
  • the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences.
  • a comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm.
  • the "percent identity” or “percent homology” of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters.
  • the amino acid sequence of an RBD e.g., a SARS-CoV-2 RBD or a SARS-CoV-2 variant RBD
  • RBD The similarities between an RBD and the RBDs described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more RBDs found in a SARS-Cov-2 variant.
  • the amino acid sequence of an RBD may be similar but not identical to any of the amino acid sequences of RBD proteins encoded by the nucleic acid molecules described herein.
  • RBD The similarities between an RBD and the RBDs described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more RBDs found one or more of SEQ ID Nos 20-32.
  • amino acid sequence of an S protein may be similar but not identical to any of the amino acid sequences of S proteins encoded by the nucleic acid molecules described herein.
  • the similarities between an S protein and the S proteins described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more S proteins found in a SARS-Cov-2 variant.
  • amino acid sequence of an S protein may be similar but not identical to any of the amino acid sequences of S proteins encoded by the nucleic acid molecules described herein.
  • the similarities between an S protein and the S proteins described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more RBDs found one or more of SEQ ID Nos 1-19.
  • the amino acid substitutions comprise one or more conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
  • R group side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol.
  • Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine.
  • a "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene).
  • Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell).
  • Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector.
  • Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct.
  • Vectors can be single-stranded or double- stranded nucleic acid molecules.
  • Vectors can be linear or circular nucleic acid molecules.
  • vector refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and/or translating the transgene.
  • a viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome.
  • examples of viral vectors include retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • An "expression vector” is a type of vector that can contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers. Expression vectors can include ribosomal binding sites and/or polyadenylation sites. Regulatory sequences direct transcription, or transcription and translation, of a transgene, such as a DNA or RNA transgene encoding an RBD, an S protein, a coronavirus or variant thereof, or a protein encoded by one or more of SEQ ID Nos: 1-19 provided herein, linked to the expression vector which is transduced into a host cell. The regulatory sequence(s) can control the level, timing and/or location of expression of the transgene.
  • the regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). Regulatory sequences can be part of a vector. Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606.
  • a transgene is “operably linked” to a promoter when the linkage between the transgene and the promoter permits functioning or expression of the transgene.
  • a transgene is "operably linked” to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.
  • transfected or “transformed” or “transduced” or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell.
  • a “transfected” or “transformed” or “transduced” host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene).
  • the host cell includes the primary subject cell and its progeny.
  • subject refers to human and non-human animals, including vertebrates, mammals and non-mammals.
  • the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
  • murine e.g., mice and rats
  • bovine porcine
  • equine canine
  • feline feline
  • caprine caprine
  • lupine ranine or piscine.
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • an effective amount refers to an amount of one or more nucleic acid molecules comprising one or more a nucleic acids encoding an RBD, an S protein, a coronavirus or variant thereof, such as one or more RBDs, S proteins, coronaviruses, or variant thereof encoded by a sequence according to one or more of SEQ ID Nos: 1-19, and/or coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein that when administered to a subject, is sufficient to effect a measurable improvement or prevention of a disease associated with coronavirus infection.
  • nucleic acid molecules when used alone or in combination, will vary depending upon the relative activity of the nucleic acid molecules, coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition and symptoms in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques.
  • the nucleic acid molecules, coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein may be administered at about 0.01 g/kg to about 50 mg/kg per day, 0.01 mg/kg to about 30 mg/kg per day, or 0.1 mg/kg to about 20 mg/kg per day.
  • nucleic acid molecules, coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein may be administered daily (e.g., once, twice, three times, or four times daily) or preferably less frequently (e.g., weekly, every two weeks, every three weeks, monthly, or quarterly).
  • adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
  • the vaccine compositions disclosed herein comprise, for example, one or more antigens, neutralizing antibodies, or proteins with imrnuiiostimulatory activity.
  • the vaccine compositions disclosed herein comprise one or more nucleic acid molecules that encode, for example, one or more antigens, neutralizing antibodies, or proteins with immunostimulatory activity.
  • the vaccine compositions disclosed herein comprise one or more nucleic acid molecules comprising messenger ribonucleic acid (mRNA) that encode, for example, one or more antigens, neutralizing antibodies, or proteins with immunostimulatory activity.
  • mRNA messenger ribonucleic acid
  • such vaccine compositions comprise one or more nucleic acid molecules, such as one or more messenger ribonucleic acid (mRNA) molecules, that encode one or more coronavirus antigens, such as one or more RBDs, one or more S proteins, one or more chimeric S proteins, one or more RBDs encoded by a sequence set forth in SEQ ID Nos: 1-19, and or one or more S proteins encoded by a sequence set forth in SEQ ID Nos: 1-19.
  • mRNA messenger ribonucleic acid
  • such vaccine compositions comprise one or more nucleic acid molecules, such as one or more messenger ribonucleic acid (mRNA) molecules, that encode one or more coronavirus antigens, such as one or more RBDs, one or more S proteins, one or more chimeric S proteins, one or more RBDs encoded by a sequence set forth in SEQ ID Nos: 1-19, and or one or more S proteins encoded by a sequence set forth in SEQ ID Nos: 1- 19, which are administered to a subject having, suspected of having, or at risk of having a SARS-CoV-2 variant infection or disease, such that the infection or disease is treated, ameliorated, or prevented.
  • mRNA messenger ribonucleic acid
  • administration which may be by injection or other means, brings the mRNA, typically in association with a delivery carrier, into proximity of cells of the subject so that the mRNA is taken up by cells of the subject.
  • These host cells express display the S protein encoded by the mRNA on their membranes where they are recognized as foreign by the subject’s immune system, promoting humoral and/or cellular immune responses.
  • a pharmaceutical composition e.g., a vaccine composition may include a naked messenger ribonucleic acid (mRNA) encoding an S protein as disclosed herein .
  • mRNA messenger ribonucleic acid
  • a pharmaceutical vaccine composition includes a messenger ribonucleic acid (mRNA), encoding an RED and/or an S protein, where the mRNA is formulated with: a non-lipid nanoparticle, a lipid nanoparticle, a cationic lipid iianoparticle, or a peptide, such as a cationic peptide.
  • mRNA messenger ribonucleic acid
  • lipids and lipid derivatives can be used in lipid formulations and lipid-derived nanoparticles (LNPs), where LNPs can encapsulate the mRNAs.
  • LNPs lipid-derived nanoparticles
  • the lipids and lipid derivatives are positively charged. See, for example, Reichmuth et al. (2016) Ther Deliv 7:319-334.
  • non-lipids can be delivery carriers, including polymers such as poiyamines, dendrimers, and copolymers.
  • the non-lipids can be cationic polymers, such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, and polysaccharide.
  • the non-lipids can be anionic polymers such as PLGA mixed with cationic lipids.
  • amphiphilic block copolymers form non-lipid nanoparticles.
  • the non-lipid nanoparticles are polymeric micelles.
  • the non-lipid nanoparticles are stealth nanoparticles.
  • the nonlipid nanoparticles are polymersomes.
  • the non-lipid nanoparticles are polyrotaxane supramolecular structures.
  • the non-lipid nanoparticles are selfmicro emulsifying systems.
  • amphiphilic block copolymers do not form nonlipid nanoparticles.
  • mRNA is incubated with amphiphilic block copolymers and buffers.
  • amphiphilic block copolymers are branched.
  • the branched amphiphilic block copolymers are poloxamines (Alvarez- Lorenzo et al., 2010, Front. Biosci ., 2, 424-440).
  • amphiphilic block copolymers are linear. In embodiments, the linear amphiphilic block copolymers are poloxamers.
  • the first surfactant is an ethoxylated glyceryl ester. In some aspects, the first surfactant is a copolymer comprising polyethylene glycol units. In some aspects, the first surfactant is a copolymer of polyethylene glycol and polypropylene glycol. In some aspects, the first surfactant is a poloxamer. A poloxamer is a non-ionic surfactant that is a tri-block copolymer with a central polypropylene glycol portion and polyethylene glycol termini. In some aspects, the first surfactant is poloxamer 407. In some aspects, the first surfactant is a combination of surfactants comprising poloxamer 407 and an additional surfactant.
  • the first surfactant is poloxamer 188. In some aspects, the first surfactant is a combination of surfactants comprising poloxamer 188 and an additional surfactant. In some aspects, the first surfactant is poloxamer 407 and poloxamer 188. In some aspects, the first surfactant is poloxamer 407. In some aspects, the first surfactant is a combination of surfactants comprising poloxamer 407 and an additional surfactant.
  • lipids and lipid derivatives formulate lipid and lipid-derived nanoparticles (LNPs).
  • LNPs encapsulate the mRNA vaccine.
  • the lipids and lipid derivatives are positively charged.
  • cationic peptides contain many lysine and arginine residues that provide the positive charge.
  • a cationic peptide is protamine.
  • protamine and mRNA vaccine spontaneously form a complex.
  • cationic peptides are employed, such as cationic cell- penetrating peptides, such as, for example, a RALA peptide or Xentry.
  • An mRNA vaccine as provided herein can include one or more RNA molecules, for example, RNA molecules encoding one or more variant S proteins, optionally in addition to the WA1/2020 S protein.
  • the S protein encoded by an RNA molecule as provided herein can include
  • a vaccine composition includes at least one nucleic acid encoding variant RBD or S protein encoded by any of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, or SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, or may be a different RBD or S protein that is encoded by a SARS-CoV-2 virus that arises in a population or geographical region.
  • a vaccine composition may comprise a nucleic acid encoding an RBD or a spike protein comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.
  • a vaccine composition provided herein includes a nucleotide sequence that encodes one or more variant SARS-CoV-2 RBDs or one or more variant SARS-CoV-2 spike proteins comprising such one or more RBDs, wherein the nucleotide sequence has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to one or more RBDs encoded by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
  • RNA molecules as described herein in a pharmaceutically acceptable carrier.
  • a pharmaceutical composition that includes two or more RNA molecules as described herein in a pharmaceutically acceptable carrier, where the two or more RNA molecules encode different S proteins, e.g., different variants of the SARS-CoV-2 S protein.
  • a pharmaceutical composition can include a first RNA molecule that encodes the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second chimeric adenovirus that encodes the SARS-CoV-2 “UK” isolate (alpha) S protein having a QQAQ furin site mutation.
  • a pharmaceutical composition can include an mRNA encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second chimeric adenovirus that encodes the SARS-CoV-2 “beta” isolate S protein having a QQAQ furin site mutation.
  • a pharmaceutical composition can include a first chimeric adenovirus that includes a gene encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second mRNA that encodes the SARS-CoV-2 “gamma” isolate S protein having a QQAQ furin site mutation.
  • a pharmaceutical composition can include a first chimeric adenovirus that includes a gene encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second mRNA that encodes the SARS-CoV-2 “delta” isolate S protein having a QQAQ furin site mutation.
  • a pharmaceutical composition can include a first chimeric adenovirus that includes a gene encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second mRNA that encodes the SARS-CoV-2 “kappa” isolate S protein having a QQAQ furin site mutation.
  • a first chimeric adenovirus that includes a gene encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation
  • a second mRNA that encodes the SARS-CoV-2 “kappa” isolate S protein having a QQAQ furin site mutation.
  • Lipids, lipid nanoparticles, and mRNA vaccine compositions comprising same
  • Lipids and lipid-containing nanoparticles suitable for use in the compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are disclosed, for example, in U.S. provisional patent application number 63/313,648, filed on February 24, 2022, entitled “Novel Ionizable Cationic Lipids,” the content of which is hereby incorporated by reference in its entirety.
  • lipid or “lipid moiety” are used in accordance with its ordinary meaning in chemistry and refer to a hydrophobic molecule which is typically characterized by an aliphatic hydrocarbon chain.
  • the lipid moiety includes a carbon chain of 3 to 100 carbons.
  • the lipid moiety includes a carbon chain of 5 to 50 carbons.
  • the lipid moiety includes a carbon chain of 5 to 25 carbons.
  • the lipid moiety includes a carbon chain of 8 to 525 carbons.
  • Lipid moieties may include saturated or unsaturated carbon chains, and may be optionally substituted.
  • the lipid moiety is optionally substituted with a charged moiety at the terminal end.
  • the lipid moiety is an alkyl or heteroalkyl optionally substituted with a carboxylic acid moiety at the terminal end.
  • Lipids are also a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. [00213]
  • the terms “cationic lipid” or “ionizable cationic lipid” are used interchangeably herein and refer to lipids that are protonated at low pH, which makes them positively charged, but they remain neutral at physiological pH.
  • lipid nanoparticle includes a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., an mRNA), to a target site of interest (e.g., cell, tissue, organ, and the like).
  • an active agent or therapeutic agent such as a nucleic acid (e.g., an mRNA)
  • a target site of interest e.g., cell, tissue, organ, and the like.
  • the lipid particle described herein is a nucleic acid-lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle.
  • the active agent or therapeutic agent such as a nucleic acid, may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.
  • lipid conjugate refers to a conjugated lipid that inhibits aggregation of lipid particles.
  • lipid conjugates include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dimyristoylglycerols (e.g., PEG-DMG conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides.
  • PEG-lipid conjugates such as, e.g., PEG coupled to dimyristoylglycerols (e.g., PEG-DMG conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides.
  • diacylglycerol or “DAG” includes a compound having 2 fatty acyl chains, R and R 2 , both of which have independently between 2 and 30 carbons bonded to the 1- and 2-position of glycerol by ester linkages.
  • the acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (Ci 6 ), stearoyl (Cis), and icosoyl (C20).
  • R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), R 1 and R 2 are both stearoyl (i.e., distearoyl), etc.
  • Diacylglycerols have the following general formula: [00217]
  • the term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkyl chains, R 1 and R 2 , both of which have independently between 2 and 30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation. Dialkyloxypropyls have the following general formula:
  • “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.
  • substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to - OCH2-.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals.
  • the alkyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons).
  • Alkyl is an uncyclized chain.
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-0-).
  • An alkyl moiety may be an alkenyl moiety.
  • An alkyl moiety may be an alkynyl moiety.
  • An alkyl moiety may be fully saturated.
  • An alkenyl may include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds.
  • An alkynyl may include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.
  • alkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 30 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, B, Se, and S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) e.g., O, N, S, Si, B, Se, or P
  • Heteroalkyl is an uncyclized chain.
  • a heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, B, Se, or P).
  • a heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P).
  • a heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P).
  • a heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P).
  • a heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P).
  • a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P).
  • heteroalkenyl by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond.
  • a heteroalkenyl may optionally include more than one double bond and/or one or more triple bonds in additional to the one or more double bonds.
  • heteroalkynyl by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one triple bond.
  • heteroalkynyl may optionally include more than one triple bond and/or one or more double bonds in additional to the one or more triple bonds.
  • heteroalkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', -NR'R'', -OR', -SR', and/or -SO2R'.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as - NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.
  • cycloalkyl and heterocycloalkyl mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.
  • a “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.
  • cycloalkyl means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system.
  • monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic.
  • cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings.
  • bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CEbjw , where w is 1, 2, or 3).
  • bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane.
  • fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring.
  • cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia.
  • multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
  • multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • a cycloalkyl is a cycloalkenyl.
  • the term “cycloalkenyl” is used in accordance with its plain ordinary meaning.
  • a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system.
  • monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic.
  • monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl.
  • bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings.
  • bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH2)w, where w is 1, 2, or 3).
  • alkylene bridge of between one and three additional carbon atoms
  • bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct 2 enyl.
  • fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring.
  • cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
  • multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • a heterocycloalkyl is a heterocyclyl.
  • heterocyclyl as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle.
  • the heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
  • the 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S.
  • the 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle.
  • heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl
  • the heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl.
  • the heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.
  • bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3- dihydrobenzofuran-3-yl, indolin-l-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-lH-indolyl, and octahydrobenzofuranyl.
  • heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.
  • Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring.
  • multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • multi cyclic heterocyclyl groups include, but are not limited to 1 OH-phenothiazin- 10-yl, 9, 10-dihydroacridin-9-yl, 9, 10-dihydroacridin- 10-yl, lOH-phenoxazin-10-yl, 10,1 l-dihydro-5H-dibenzo[b,f azepin-5-yl, 1, 2,3,4- tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro- lH-carbazol-9-yl.
  • halo or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(Ci-C4)alkyl includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • acyl means, unless otherwise stated, -C(0)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
  • a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring.
  • heteroaryl refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized.
  • heteroaryl includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring).
  • a 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1 -naphthyl, 2- naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3 -pyrazolyl, 2-imidazoly
  • aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.
  • a heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.
  • a fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl.
  • a fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl.
  • a fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.
  • a fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl.
  • Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.
  • Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom.
  • the individual rings within spirocyclic rings may be identical or different.
  • Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings.
  • Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings).
  • Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene).
  • heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring.
  • substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.
  • alkylsulfonyl means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., “C1-C4 alkylsulfonyl”).
  • alkylarylene as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene group has the formula: [00238] [00239] An alkylarylene moiety may be substituted (e.g.
  • the alkylarylene is unsubstituted.
  • Each of the above terms e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,” “heterocycloalkyl,” “aryl,” and “heteroaryl” includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
  • R, R', R'', R'', and R''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.
  • aryl e.g., aryl substituted with 1-3 halogens
  • substituted or unsubstituted heteroaryl substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R'', R''', and R''' group when more than one of these groups is present.
  • R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring.
  • -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4- morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., - C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • haloalkyl e.g., -CF3 and -CH2CF3
  • acyl e.g., - C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like.
  • Substituents for rings may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent).
  • the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings).
  • the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different.
  • a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent)
  • the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency.
  • a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms.
  • the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.
  • Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups.
  • Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure.
  • the ring-forming substituents are attached to adjacent members of the base structure.
  • two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure.
  • the ring-forming substituents are attached to a single member of the base structure.
  • two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure.
  • the ring-forming substituents are attached to non- adjacent members of the base structure.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O) -, -S(O) 2 -, -S(O) 2 NR'-, or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR') s -X'- (C''R''R'') d -, where s and d are independently integers of from 0 to 3, and X' is - O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-.
  • R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • heteroatom or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • a “substituent group,” as used herein, means a group selected from the following moieties: [00248] (A) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO 4 H, -SO 2 NH 2 , ⁇ NHNH 2 , ⁇ ONH 2 , ⁇ NHC(O)NHNH 2 , -NHC(O)NH 2 , -NHSO 2 H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl 3 , -OCF 3 , -OCBr 3 ,
  • a “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C30 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 30 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and each substituted or unsubstituted heteroaryl is
  • a “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-Cs alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3- Ci cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted or un
  • each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.
  • each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C30 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 30 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 - Cio aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C30 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 30 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroaryl ene.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C30 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 30 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C30 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 30 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene.
  • a substituted or unsubstituted moiety e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroaryl ene) is unsubstituted (e.g., is an unsubstituted alkyl, un
  • a substituted or unsubstituted moiety e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroaryl ene) is substituted (e.g., is a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted substituted
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene
  • is substituted with at least one substituent group wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of substituent groups, each substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene
  • is substituted with at least one size-limited substituent group wherein if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group may optionally be different.
  • each size-limited substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene
  • is substituted with at least one lower substituent group wherein if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene
  • the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group, and/or
  • Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure.
  • the compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and/or isolate.
  • the present disclosure is meant to include compounds in racemic and optically pure forms.
  • Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain olefmic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • isomers refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
  • tautomer refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this disclosure.
  • an analog is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
  • salt refers to acid or base salts of the compounds used in the methods of the present invention.
  • acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.
  • salts are meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic,
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge el al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the compounds of the present disclosure may exist as salts, such as with pharmaceutically acceptable acids.
  • the present disclosure includes such salts.
  • Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g.
  • salts may be prepared by methods known to those skilled in the art.
  • the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • the present disclosure provides compounds, which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure.
  • Prodrugs of the compounds described herein may be converted in vivo after administration.
  • prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.
  • Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
  • Y is substituted or unsubstituted C 0 -C 12 alkylene or substituted or unsubstituted 0 to 12 membered heteroalkylene.
  • B 1 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
  • B 2 and B 3 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.
  • L a1 and L a2 are each independently O, S, or CH 2 .
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene.
  • Each R 1A and R 1B is independently H, substituted or unsubstituted C 1 -C 12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • Each R 2A , R 3A , R 4A , and R 5A is independently H, substituted or unsubstituted C1- C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl.
  • Each R 101 , R 102 , R 201 , R 202 , R 301 , R 302 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • a substituent group e.g., C1-C30 alkyl, C1- C 8 alkyl, or C 1 -C 4 alkyl
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heteroalkyl e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl
  • substituted e.g.
  • cycloalkyl e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl
  • substituted e.g.
  • R 1 is substituted with one or more substituent groups. In embodiments, R 1 is substituted with one or more size-limited substituent groups.
  • R 1 is substituted with one or more lower substituent groups.
  • R 1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C 1 -C 30 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl).
  • R 1 is independently unsubstituted alkyl (e.g., C 1 -C 30 alkyl, C1-C8 alkyl, or C1-C4 alkyl).
  • R 1 is independently substituted (e.g.
  • heteroalkyl e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl.
  • R 1 is independently unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 1 is independently substituted (e.g.
  • R 1 is independently unsubstituted cycloalkyl (e.g., C 3 -C8 cycloalkyl, C 3 -C 6 cycloalkyl, or C5-C 6 cycloalkyl).
  • R 1 is independently substituted (e.g.
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl.
  • R 1 is independently unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • R 1 is independently substituted (e.g.
  • R 1 is independently unsubstituted aryl (e.g., C 6 -C1 0 aryl, C10 aryl, or phenyl).
  • R 1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • R 1 is independently unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • R 1 is independently H, -OR 1A or substituted or unsubstituted heteroalkyl.
  • R 1 is independently H, -OR 1A or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 1 is independently H.
  • R 1 is independently -OR 1A .
  • R 1 is independently substituted or unsubstituted heteroalkyl.
  • R 1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 1 is independently H, -OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl. In embodiments, R 1 is independently -OH or methoxy. [00308] In embodiments, R 1 is independently H. In embodiments, R 1 is independently - OH. In embodiments, R 1 is independently methoxy. In embodiments, R 1 is independently ethoxy.
  • R 2 is substituted with one or more substituent groups.
  • R 2 is substituted with one or more size-limited substituent groups.
  • R 2 is substituted with one or more lower substituent groups. [00310] In embodiments, R 2 is substituted (e.g.
  • R 2 is unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl).
  • R 2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 2 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). [00311] In embodiments, R 2 is H or substituted or unsubstituted alkyl. In embodiments, R 2 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 12 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl). In embodiments, R 2 is H.
  • R 2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C 1 -C 4 alkyl).
  • R 2 is unsubstituted alkyl (e.g., C 1 -C 12 alkyl, C 1 -C 8 alkyl, or C1-C4 alkyl).
  • R 2 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 2 is substituted (e.g.
  • R 3 is substituted with one or more substituent groups. In embodiments, R 3 is substituted with one or more size-limited substituent groups.
  • R 3 is substituted with one or more lower substituent groups.
  • R 3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C 1 -C 30 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl).
  • R 3 is unsubstituted alkyl (e.g., C 1 -C 30 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl).
  • R 3 is substituted (e.g.
  • heteroalkyl e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl.
  • R 3 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). [00315] In embodiments, R 3 is H or substituted or unsubstituted alkyl. In embodiments, R 3 is H or substituted (e.g.
  • R 3 is H.
  • R 3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C 1 -C 12 alkyl, C 1 -C 8 alkyl, or C1-C4 alkyl).
  • R 3 is unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C 1 -C 4 alkyl).
  • R 3 is H or substituted or unsubstituted C 1 -C 12 alkyl.
  • R 3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl.
  • R 3 is unsubstituted C1-C12 alkyl.
  • R 4 is substituted with one or more substituent groups.
  • R 4 is substituted with one or more size-limited substituent groups.
  • R 4 is substituted with one or more lower substituent groups. [00318] In embodiments, R 4 is substituted (e.g.
  • R 4 is unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl).
  • R 4 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 4 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). [00319] In embodiments, R 4 is H or substituted or unsubstituted alkyl. In embodiments, R 4 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 12 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl). In embodiments, R 4 is H.
  • R 4 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C 1 -C 4 alkyl).
  • R 4 is unsubstituted alkyl (e.g., C 1 -C 12 alkyl, C 1 -C 8 alkyl, or C1-C4 alkyl).
  • R 4 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 4 is substituted (e.g.
  • R 4 is unsubstituted C 1 -C 12 alkyl.
  • R 5 is substituted with one or more substituent groups. In embodiments, R 5 is substituted with one or more size-limited substituent groups.
  • R 5 is substituted with one or more lower substituent groups.
  • R 5 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl).
  • R 5 is unsubstituted alkyl (e.g., C 1 -C 30 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl).
  • R 5 is substituted (e.g.
  • R 5 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • R 5 is H or substituted or unsubstituted alkyl. In embodiments, R 5 is H or substituted (e.g.
  • R 5 is H.
  • R 5 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C 1 -C 12 alkyl, C 1 -C 8 alkyl, or C1-C4 alkyl).
  • R 5 is unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl).
  • R 5 is H or substituted or unsubstituted C 1 -C 12 alkyl.
  • R 5 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl.
  • R 5 is unsubstituted C1-C12 alkyl.
  • Y is substituted (e.g.
  • Y is substituted with one or more substituent groups. In embodiments, Y is substituted with one or more size-limited substituent groups. In embodiments, Y is substituted with one or more lower substituent groups. [00326] In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C0-C12 alkylene.
  • Y is unsubstituted C0-C12 alkylene. In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 0 to 12 membered heteroalkylene. In embodiments, Y is unsubstituted 0 to 12 membered heteroalkylene. [00327] In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C 1 -C 8 alkylene. In embodiments, Y is unsubstituted C 1 -C 8 alkylene. In embodiments, Y is substituted (e.g.
  • Y is unsubstituted 1 to 8 membered heteroalkylene.
  • Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C4 alkylene.
  • Y is unsubstituted C 1 -C 4 alkylene.
  • Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 1 to 4 membered heteroalkylene.
  • Y is unsubstituted 1 to 4 membered heteroalkylene.
  • Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) methylene, ethylene or propylene.
  • Y is unsubstituted methylene, ethylene or propylene.
  • B 1 is a bond, substituted (e.g.
  • alkylene e.g., C 1 -C 30 alkylene, C1-C8 alkylene, or C1-C4 alkylene
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heteroalkylene e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene
  • substituted e.g.
  • cycloalkylene e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C 5 -C 6 cycloalkylene
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene
  • substituted e.g.
  • B 1 is substituted with one or more substituent groups. In embodiments, B 1 is substituted with one or more size-limited substituent groups.
  • B 1 is substituted with one or more lower substituent groups. In embodiments, B 1 is a bond. [00331] In embodiments, B 1 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C 1 -C 4 alkylene). In embodiments, B 1 is unsubstituted alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene). In embodiments, B 1 is substituted (e.g.
  • heteroalkylene e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene.
  • B 1 is unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 1 is substituted (e.g.
  • cycloalkylene e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene.
  • B 1 is unsubstituted cycloalkylene (e.g., C 3 -C 8 cycloalkylene, C 3 -C 6 cycloalkylene, or C 5 -C 6 cycloalkylene).
  • B 1 is substituted (e.g.
  • heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene.
  • B 1 is unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • B 1 is substituted (e.g.
  • arylene e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene
  • B 1 is unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene).
  • B 1 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • B 1 is unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • B 1 is a bond or a substituted or unsubstituted alkylene.
  • B 1 is a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene).
  • B 1 is a bond or unsubstituted alkylene. In embodiments, B 1 is a bond or unsubstituted C1-C8 alkylene. In embodiments, B 1 is unsubstituted alkylene. In embodiments, B 1 is unsubstituted C 1 -C 8 alkylene. In embodiments, B 1 is a bond. [00334] In embodiments, B 2 and B 3 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. In embodiments, B 2 and B 3 are each independently a bond, substituted (e.g.
  • B 2 is substituted with one or more substituent groups. In embodiments, B 2 is substituted with one or more size-limited substituent groups.
  • B 2 is substituted with one or more lower substituent groups. In embodiments, B 2 is a bond. In embodiments, B 3 is substituted with one or more substituent groups. In embodiments, B 3 is substituted with one or more size-limited substituent groups. In embodiments, B 3 is substituted with one or more lower substituent groups. In embodiments, B 3 is a bond. [00335] In embodiments, B 2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C1-C4 alkylene).
  • alkylene e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C1-C4 alkylene.
  • B 2 is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).
  • B 2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 2 is unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene).
  • B 3 is unsubstituted alkylene (e.g., C 1 -C 30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).
  • B 3 is substituted (e.g.
  • heteroalkylene e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene.
  • B 3 is unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 2 and B 3 are each independently a bond or substituted or unsubstituted alkylene.
  • B 2 and B 3 are each independently a bond or substituted (e.g.
  • B 2 and B 3 are each independently a bond or substituted or unsubstituted Ci-Cs alkylene. In embodiments, B 2 and B 3 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted Ci-Cs alkylene.
  • B 2 is a bond. In embodiments, B 2 is substituted Ci-Cs alkylene.
  • B 2 is unsubstituted Ci-Cs alkylene.
  • B 3 is a bond.
  • B 3 is substituted Ci-Cs alkylene.
  • B 3 is unsubstituted C i-Cx alkylene.
  • B 2 is butylene. In embodiments, B 2 is propylene. In embodiments, B 2 is ethylene. In embodiments, B 2 is methylene. In embodiments, B 3 is butylene. In embodiments, B 3 is propylene. In embodiments, B 3 is ethylene. In embodiments, B 3 is methylene.
  • each R 101 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R 101 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R 101 is substituted with one or more substituent groups. In embodiments, each R 101 is substituted with one or more size-limited substituent groups. In embodiments, each R 101 is substituted with one or more lower substituent groups.
  • each R 101 is independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R 101 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
  • each R 101 is independently H. In embodiments, each R 101 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each R 101 is independently unsubstituted 2 to 12 membered heteroalkyl.
  • L are each independently , where each X is independently O.
  • L al and L a2 are each independently ,
  • L and L are each independently . In embodiments, L and L are each independently . In embodiments, L a and L a are each independently . In embodiments, L al and L a2 are each independently X . In embodiments, L al and L a2 are each independently embodiments, L al and L a2
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkylene.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently substituted with one or more substituent groups.
  • W 3 , W 4 , W 5 , and W 6 are each independently substituted with one or more size-limited substituent groups.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently substituted with one or more lower substituent groups.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently unsubstituted C1-C12 alkylene.
  • each R 1A and R 1B is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
  • each R 1A and R 1B is independently substituted with is independently substituted with one or more substituent groups.
  • each R 1A and R 1B is independently substituted with one or more size-limited substituent groups.
  • each R 1A and R 1B is independently substituted with one or more lower substituent groups.
  • R 1A and R 1B substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered) or substituted or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
  • a substituted or unsubstituted heterocycloalkyl e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered
  • substituted or unsubstituted heteroaryl e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered.
  • a substituted heterocycloalkyl or substituted heteroaryl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted heterocycloalkyl or substituted heteroaryl is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • when a heterocycloalkyl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group.
  • a heterocycloalkyl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom when a heterocycloalkyl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group. In embodiments, when a heteroaryl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group.
  • a heteroaryl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom when a heteroaryl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heteroaryl formed by the joining of R 1A and R 1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group.
  • each R 1A is independently H or substituted or unsubstituted Ci- Ci2 alkyl. In embodiments, each R 1A is independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl. In embodiments, each R 1A is independently H. In embodiments, each R 1A is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each R 1A is independently unsubstituted C1-C12 alkyl.
  • R 1 is H, -OR 1A or substituted or unsubstituted heteroalkyl.
  • B 1 is a bond or a substituted or unsubstituted alkylene.
  • B 2 and B 3 are each independently a bond or substituted or unsubstituted alkylene.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
  • L al and L a2 are each independently each X is independently O or S.
  • R 2 is H or substituted or unsubstituted alkyl.
  • R 3 is H or substituted or unsubstituted alkyl.
  • R 4 is H or substituted or unsubstituted alkyl.
  • R 5 is H or substituted or unsubstituted alkyl.
  • each R 1A is independently H or substituted or unsubstituted C1-C12 alkyl
  • each R 101 is independently H or substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • R 1 is H, -OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl.
  • B 1 is a bond or an unsubstituted Ci-Cs alkylene.
  • B 2 and B 3 are each independently a bond or substituted or unsubstituted Ci-Cx alkylene.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
  • each X is independently
  • R 2 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 3 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 4 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 5 is H or substituted or unsubstituted C1-C12 alkyl, and
  • each R 101 is independently substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • R 1 is -OH or methoxy.
  • L 1 is a bond.
  • B 1 is an unsubstituted Ci-Cs alkylene.
  • B 2 and B 3 are each independently a bond or substituted or unsubstituted Ci-Cs alkylene;
  • L 2 is a bond.
  • L 4 is a bond.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
  • each X is independently
  • L 3 is a bond.
  • L 5 is a bond.
  • L 6 is a bond.
  • L 7 is a bond.
  • R 2 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 3 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 4 is H or substituted or unsubstituted C1-C12 alkyl, and R 5 is H or substituted or unsubstituted C1-C12 alkyl;
  • R 1 is substituted or unsubstituted heteroalkyl.
  • B 1 is a bond.
  • B 2 and B 3 are each independently a bond or substituted or unsubstituted Ci-Cs alkylene.
  • W 1 , W 2 , W 3 , W 4 , W 5 , and W 6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
  • each X is independently
  • O. L 3 is a bond.
  • L 5 is a bond.
  • L 6 is a bond.
  • L 7 is a bond.
  • R 2 is H or substituted or unsubstituted Ci- C12 alkyl.
  • R 3 is H or substituted or unsubstituted C1-C12 alkyl.
  • R 4 is H or substituted or unsubstituted C1-C12 alkyl, and R 5 is H or substituted or unsubstituted C1-C12 alkyl.
  • each R 2A , R 3A , R 4A , and R 5A is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl.
  • each R 2A , R 3A , R 4A , and R 5A is independently H.
  • each R 2A , R 3A , R 4A , and R 5A is independently substituted (e.g.
  • each R 2A , R 3A , R 4A , and R 5A is independently unsubstituted C1-C30 alkyl.
  • each R 2A , R 3A , R 4A , and R 5A is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl.
  • each R 2A , R 3A , R 4A , and R 5A is independently unsubstituted 2 to 30 membered heteroalkyl.
  • each R 102 , R 201 , R 202 , R 301 , R 302 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
  • each R 102 , R 201 , R 202 , R 301 , R 302 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 is independently H.
  • each R 102 , R 201 , R 202 , R 301 , R 302 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl.
  • each R 102 , R 201 , R 202 , R 301 , R 302 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 is independently unsubstituted C1-C12 alkyl.
  • each R 102 , R 201 , R 202 , R 301 , R 302 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 is independently substituted (e.g.
  • each R 102 , R 201 , R 202 , R 301 , R 302 , R 401 , R 402 , R 501 , R 502 , R 601 , R 602 , R 701 , and R 702 is independently unsubstituted 2 to 12 membered heteroalkyl.
  • each s is an integer from 1 to 4. In embodiments, each s is 1. In embodiments, each s is 2. In embodiments, each s is 3. In embodiments, each s is 4.
  • the cationic lipid of formula (I) is: [00426]
  • cationic lipid of formula (II) [00427] (II), [00428] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof.
  • R 10 and R 11 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.
  • B 5 , B 6 , and B 7 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.
  • R 7 , R 8 , and R 9 are each independently H, substituted or unsubstituted C 1 -C 30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl.
  • each R a31 and R a32 is independently H, substituted or unsubstituted C 1 -C 12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • each s is independently an integer from 1 to 4.
  • W 7 and W 8 are each independently a bond or substituted (e.g.
  • W 7 and W 8 are each independently substituted with one or more substituent groups.
  • W 7 and W 8 are each independently substituted with one or more size-limited substituent groups. In embodiments, W 7 and W 8 are each independently substituted with one or more lower substituent groups. [00440] In embodiments, W 7 and W 8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene). In embodiments, W 7 and W 8 are each independently unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).
  • alkylene e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene.
  • W 7 and W 8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • W 7 and W 8 are each independently unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • W 7 and W 8 are each independently a bond.
  • W 7 and W 8 are each independently a bond or substituted or unsubstituted C 1 -C 8 alkylene. In embodiments, W 7 and W 8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C8 alkylene. In embodiments, W 7 and W 8 are each independently unsubstituted C1-C8 alkylene. [00442] In embodiments, W 7 and W 8 are each independently a bond or substituted or unsubstituted C2-C4 alkylene. In embodiments, W 7 and W 8 are each independently substituted (e.g.
  • W 7 and W 8 are each independently unsubstituted C2-C4 alkylene.
  • W 7 and W 8 are each independently a bond or unsubstituted C 2 -C 4 alkylene.
  • W 7 and W 8 are each independently a bond, ethylene, propylene, butylene, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) ethylene, substituted (e.g.
  • L a3 is a bond.
  • R 10 and R 11 are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl), substituted (e.g.
  • R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl) or substituted (e.g.
  • R 10 and R 11 are each independently substituted with one or more substituent groups.
  • R 10 and R 11 are each independently substituted with one or more size-limited substituent groups.
  • R 10 and R 11 are each independently substituted with one or more lower substituent groups. [00446] In embodiments, R 10 and R 11 are each independently substituted (e.g.
  • R 10 and R 11 are each independently unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R 10 and R 11 are each independently substituted (e.g.
  • heteroalkyl e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl.
  • R 10 and R 11 are each independently unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
  • a substituted heterocycloalkyl or substituted heteroaryl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom is substituted with at least one substituent group, size-limited substituent group, or lower substituent group.
  • substituted heterocycloalkyl or substituted heteroaryl is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • a heterocycloalkyl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group.
  • a heterocycloalkyl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group.
  • a heterocycloalkyl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom when a heterocycloalkyl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group. In embodiments, when a heteroaryl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heteroaryl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heteroaryl formed by the joining of R 10 and R 11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group.
  • R 10 and R 11 together with the nitrogen atom to which they are connected form substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl.
  • R 10 and R 11 together with the nitrogen atom to which they are connected form substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • heteroaryl e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl.
  • R 10 and R 11 together with the nitrogen atom to which they are connected form unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
  • R 10 and R 11 are each independently H, substituted or unsubstituted alkyl or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl.
  • R 10 and R 11 are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C 1 -C 30 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl) or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted (e.g.
  • R 10 and R 11 are each independently H [00449] In embodiments, R 10 and R 11 are each independently substituted or unsubstituted alkyl or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl. In embodiments, R 10 and R 11 are each independently substituted (e.g.
  • R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • a substituent group e.g., a size-limited substituent group or a lower substituent group
  • unsubstituted alkyl e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl
  • R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl).
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, butyl, pentyl or hexyl. In embodiments, R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl or propyl. [00451] In embodiments, R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl.
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkyl.
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl.
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted (e.g.
  • R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 5 to 6 membered heterocycloalkyl.
  • R 10 and R 11 together with the nitrogen atom to which they are connected form unsubstituted 5 to 6 membered heterocycloalkyl.
  • B 5 , B 6 , and B 7 are each independently a bond, substituted (e.g.
  • B 5 , B 6 , and B 7 are each independently substituted with one or more substituent groups.
  • B 5 , B 6 , and B 7 are each independently substituted with one or more size-limited substituent groups. In embodiments, B 5 , B 6 , and B 7 are each independently substituted with one or more lower substituent groups. [00457] In embodiments, B 5 , B 6 , and B 7 are each independently a bond. In embodiments, B 5 , B 6 , and B 7 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C 1 -C 4 alkylene).
  • alkylene e.g., C1-C30 alkylene, C1-C8 alkylene, or C 1 -C 4 alkylene.
  • B 5 , B 6 , and B 7 are each independently unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).
  • B 5 , B 6 , and B 7 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 5 , B 6 , and B 7 are each independently unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 5 is a bond
  • B 6 and B 7 are each independently a bond or substituted or unsubstituted alkylene. In embodiments, B 6 and B 7 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, Ci-Cx alkylene, or C1-C4 alkylene).
  • B 6 and B 7 are each independently a bond or substituted or unsubstituted C i-Cx alkylene. In embodiments, B 6 and B 7 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted Ci-Cx alkylene. In embodiments, B 6 and B 7 are each independently a bond. In embodiments, B 6 and B 7 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) Ci-Cx alkylene. In embodiments, B 6 and B 7 are each independently unsubstituted Ci-Cx alkylene.
  • B 6 and B 7 are each independently a bond or substituted or unsubstituted C2-C4 alkylene. In embodiments, B 6 and B 7 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C2-C4 alkylene. In embodiments, B 6 and B 7 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C2-C4 alkylene. In embodiments, B 6 and B 7 are each independently unsubstituted C2-C4 alkylene.
  • B 6 and B 7 are each independently a bond or unsubstituted C2-C4 alkylene. In embodiments, B 6 and B 7 are each independently a bond, ethylene, propylene, or butylene. In embodiments, B 6 and B 7 are each independently a bond. In embodiments, B 6 and B 7 are each independently ethylene. In embodiments, B 6 and B 7 are each independently propylene. In embodiments, B 6 and B 7 are each independently butylene.
  • R 7 , R 8 , and R 9 are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl.
  • R 7 , R 8 , and R 9 are each independently substituted with one or more substituent groups.
  • R 7 , R 8 , and R 9 are each independently substituted with one or more size-limited substituent groups.
  • R 7 , R 8 , and R 9 are each independently substituted with one or more lower substituent groups.
  • R 7 , R 8 , and R 9 are each independently H. In embodiments, R 7 , R 8 , and R 9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C30 alkyl. In embodiments, R 7 , R 8 , and R 9 are each independently unsubstituted C1-C30 alkyl. In embodiments, R 7 , R 8 , and R 9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R 7 , R 8 , and R 9 are each independently unsubstituted 2 to 30 membered heteroalkyl.
  • R 7 , R 8 , and R 9 are each independently H or substituted or unsubstituted C1-C30 alkyl. In embodiments, R 7 , R 8 , and R 9 are each independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl. In embodiments, R 7 , R 8 , and R 9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl.
  • R 7 , R 8 , and R 9 are each independently substituted or unsubstituted C1-C20 alkyl. In embodiments, R 7 , R 8 , and R 9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl. In embodiments, R 7 , R 8 , and R 9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl.
  • R 7 , R 8 , and R 9 are each independently unsubstituted C1-C20 alkyl.
  • each R a31 and R a32 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
  • each R a31 and R a32 is independently H.
  • each R a31 and R a32 is independently substituted (e.g.
  • each R a31 and R a32 is independently unsubstituted C 1 -C 12 alkyl. In embodiments, each R a31 and R a32 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each R a31 and R a32 is independently unsubstituted 2 to 12 membered heteroalkyl.
  • each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C 1 -C 12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
  • each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 is independently H.
  • each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl.
  • each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 is independently unsubstituted C 1 -C 12 alkyl.
  • each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 is independently substituted (e.g.
  • each R 801 , R 802 , R 901 , R 902 , R 110 , and R 111 is independently unsubstituted 2 to 12 membered heteroalkyl.
  • each s is independently an integer from 1 to 4. In embodiments, each s is 1. In embodiments, each s is 2. In embodiments, each s is 3. In embodiments, each s is 4. [00473] In embodiments, W 7 and W 8 are each independently a bond or substituted or unsubstituted alkylene. L a3 is a bond.
  • R 10 and R 11 are each independently H, substituted or unsubstituted alkyl or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl.
  • B 5 is a bond.
  • B 6 and B 7 are each independently a bond or substituted or unsubstituted alkylene.
  • L 8 is a bond.
  • W 7 and W 8 are each independently a bond or substituted or unsubstituted C 1 -C 8 alkylene.
  • L a3 is a bond.
  • R 10 and R 11 are each independently substituted or unsubstituted alkyl or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl.
  • B 5 is a bond.
  • B 6 and B 7 are each independently a bond or substituted or unsubstituted C 1 -C 8 alkylene.
  • L 8 is a bond.
  • W 7 and W 8 are each independently a bond or substituted or unsubstituted C2-C4 alkylene.
  • L a3 is a bond.
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl.
  • B 5 is a bond.
  • B 6 and B 7 are each independently a bond or substituted or unsubstituted C2-C4 alkylene.
  • L 8 is a bond.
  • R 7 is H or methyl
  • R 8 , and R 9 are each independently substituted or unsubstituted C1-C20 alkyl.
  • W 7 and W 8 are each independently a bond or unsubstituted C2-C4 alkylene.
  • L a3 is a bond.
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl.
  • B 5 is a bond.
  • B 6 and B 7 are each independently a bond or unsubstituted C 2 -C 4 alkylene.
  • L 8 is a bond.
  • R 7 is H or methyl, and R 8 and R 9 are each independently substituted or unsubstituted C1-C20 alkyl.
  • W 7 and W 8 are each independently a bond or unsubstituted C 2 -C 4 alkylene.
  • L a3 is a bond.
  • R 10 and R 11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R 10 and R 11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl.
  • B 5 , B 6 , and B 7 are each independently a bond.
  • L 8 is a bond.
  • L 9 is a bond.
  • L 10 is a bond.
  • R 7 is H or methyl
  • R 8 and R 9 are each independently substituted or unsubstituted C 1 -C 30 alkyl.
  • the cationic lipid of formula (II) is: [00487] , , [00508] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof.
  • Q is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene.
  • V is substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted arylene.
  • B 8 , B 9 , B 10 , and B 11 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.
  • R 14 and R 15 are each independently substituted or unsubstituted C2-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl.
  • R 12A , R 12B , R 13A , and R 13B are each independently H, substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted 2 to 20 membered heteroalkyl.
  • each R 210 , R 211 , R 310 , and R 311 is independently H, substituted or unsubstituted C1- C 12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • each n is independently an integer from 0 to 8
  • each s is independently an integer from 1 to 4.
  • L 11 is , where n is an integer from 0 to 8, V is substituted or unsubstituted alkylene, and Q is substituted or unsubstituted alkylene. [00525] In embodiments, L 11 is , where V is substituted or unsubstituted alkylene. In embodiments, L 11 is , where V is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene).
  • L 11 is , where V is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene). In embodiments, L 11 is , where V is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C 1 -C 4 alkylene). [00527] In embodiments, L 11 is where n is an integer from 0 to 8. In embodiments, n is an integer from 0 to 4.
  • L 11 is , where Q is substituted or unsubstituted alkylene. In embodiments, L 11 is , where Q is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). [00533] In embodiments, L 11 where Q is substituted (e.g.
  • alkylene e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene.
  • L 11 is , where Q is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C 1 -C 4 alkylene).
  • Q is substituted (e.g.
  • alkylene e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • heteroalkylene e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene
  • cycloalkylene e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene
  • substituted e.g.
  • arylene e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heteroarylene e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene.
  • Q is substituted with one or more substituent groups.
  • Q is substituted with one or more size-limited substituent groups.
  • Q is substituted with one or more lower substituent groups.
  • Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C 1 -C 4 alkylene).
  • Q is unsubstituted alkylene (e.g., C 1 -C 30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).
  • Q is substituted (e.g.
  • heteroalkylene e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene.
  • Q is unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • Q is substituted (e.g.
  • cycloalkylene e.g., C 3 -C 8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene.
  • Q is unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene).
  • Q is substituted (e.g.
  • heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene.
  • Q is unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • Q is substituted (e.g.
  • arylene e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene
  • Q is unsubstituted arylene (e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene).
  • Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Q is unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).
  • Q is substituted or unsubstituted alkylene.
  • Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).
  • V is substituted (e.g.
  • alkylene e.g., C 1 -C 30 alkylene, C1-C8 alkylene, or C1-C4 alkylene
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted cycloalkylene e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene
  • substituted e.g.
  • V is substituted with one or more substituent groups.
  • V is substituted with one or more size-limited substituent groups.
  • V is substituted with one or more lower substituent groups.
  • V is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C 1 -C 4 alkylene).
  • V is unsubstituted alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene).
  • V is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene).
  • V is unsubstituted cycloalkylene (e.g., C 3 -C 8 cycloalkylene, C 3 -C 6 cycloalkylene, or C 5 -C 6 cycloalkylene).
  • V is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene).
  • V is unsubstituted arylene (e.g., C 6 -C 10 arylene, C 10 arylene, or phenylene).
  • V is substituted or unsubstituted alkylene.
  • V is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene).
  • B 8 , B 9 , B 10 , and B 11 are each independently a bond, substituted (e.g.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted with one or more substituent groups.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted with one or more size-limited substituent groups. In embodiments, B 8 , B 9 , B 10 , and B 11 are each independently substituted with one or more lower substituent groups. [00542] In embodiments, B 8 , B 9 , B 10 , and B 11 are each independently a bond. In embodiments, B 8 , B 9 , B 10 , and B 11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).
  • alkylene e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene.
  • B 8 , B 9 , B 10 , and B 11 are each independently unsubstituted alkylene (e.g., C 1 -C 30 alkylene, C 1 -C 8 alkylene, or C 1 -C 4 alkylene).
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 8 , B 9 , B 10 , and B 11 are each independently unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted alkylene.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted C 1 -C 20 alkylene. In embodiments, B 8 , B 9 , B 10 , and B 11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkylene. In embodiments, B 8 , B 9 , B 10 , and B 11 are each independently substituted (e.g.
  • B 8 , B 9 , B 10 , and B 11 are each independently unsubstituted C1-C20 alkylene.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted C 1 -C 8 alkylene.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C8 alkylene.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C8 alkylene. In embodiments, B 8 , B 9 , B 10 , and B 11 are each independently unsubstituted C1-C8 alkylene.
  • R 12 is H. In embodiments, R 12 is -OR 12A . In embodiments, R 12 is -SR 12A . In embodiments, R 12 is -NR 12A . In embodiments, R 12 is CN. In embodiments, R 12 is -
  • R 12 is -OH, methoxy, or ethoxy.
  • R 13 is -OH, methoxy, or ethoxy.
  • R 12A and R 13A are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl.
  • R 12A and R 13A are each independently substituted with one or more substituent groups.
  • R 12A and R 13A are each independently substituted with one or more size-limited substituent groups.
  • R 12A and R 13A are each independently substituted with one or more lower substituent groups.
  • R 12A and R 13A are each independently H. In embodiments, R 12A and R 13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl. In embodiments, R 12A and R 13A are each independently unsubstituted C1-C20 alkyl. In embodiments, R 12A and R 13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R 12A and R 13A are each independently unsubstituted 2 to 30 membered heteroalkyl.
  • R 12A and R 13A are each independently H, substituted or unsubstituted C1-C20 alkyl. In embodiments, R 12A and R 13A are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl. In embodiments, R 12A and R 13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl. In embodiments, R 12A and R 13A are each independently unsubstituted C1-C20 alkyl.
  • R 12A and R 13A are each independently H, substituted or unsubstituted Ci-Cs alkyl. In embodiments, R 12A and R 13A are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted Ci-Cs alkyl. In embodiments, R 12A and R 13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) Ci-Cs alkyl. In embodiments, R 12A and R 13A are each independently unsubstituted Ci-Cs alkyl.
  • R 12B and R 13B are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl.
  • R 12B and R 13B are each independently H. In embodiments, R 12B and R 13B are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl. In embodiments, R 12B and R 13B are each independently unsubstituted C1-C20 alkyl. In embodiments, R 12B and R 13B are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R 12B and R 13B are each independently unsubstituted 2 to 30 membered heteroalkyl.
  • R 14 and R 15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C2-C30 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl.
  • R 14 and R 15 are each independently substituted with one or more substituent groups.
  • R 14 and R 15 are each independently substituted with one or more size-limited substituent groups.
  • R 14 and R 15 are each independently substituted with one or more lower substituent groups.
  • R 14 and R 15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C2-C30 alkyl. In embodiments, R 14 and R 15 are each independently unsubstituted C2-C30 alkyl. In embodiments, R 14 and R 15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R 14 and R 15 are each independently unsubstituted 2 to 30 membered heteroalkyl.
  • R 14 and R 15 are each independently substituted or unsubstituted C2-C30 alkyl. In embodiments, R 14 and R 15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C2-C30 alkyl.
  • each R 210 , R 211 , R 310 , and R 311 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
  • each R 210 , R 211 , R 310 , and R 311 is independently substituted with one or more substituent groups.
  • each R 210 , R 211 , R 310 , and R 311 is independently substituted with one or more size-limited substituent groups. In embodiments, each R 210 , R 211 , R 310 , and R 311 is independently substituted with one or more lower substituent groups.
  • each R 210 , R 211 , R 310 , and R 311 is independently H. In embodiments, each R 210 , R 211 , R 310 , and R 311 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each R 210 , R 211 , R 310 , and R 311 is independently unsubstituted C1-C12 alkyl.
  • each R 210 , R 211 , R 310 , and R 311 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each R 210 , R 211 , R 310 , and R 311 is independently unsubstituted 2 to 12 membered heteroalkyl. [00567] In embodiments, each n is independently an integer from 0 to 8. In embodiments, each n is independently an integer from 0 to 4. In embodiments, each n is independently 8. In embodiments, each n is independently 7. In embodiments, each n is independently 6. In embodiments, each n is independently 5. In embodiments, each n is independently 4. In embodiments, each n is independently 3. In embodiments, each n is independently 2. In embodiments, each n is independently 1. In embodiments, each n is independently 0.
  • each s is an integer from 1 to 4. In embodiments, each s is 4. In embodiments, each s is 3. In embodiments, each s is 2. In embodiments, each s is 1.
  • n is independently an integer from 0 to 8.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted alkylene.
  • R 12 is H, -OR 12A , or-
  • R 13 is H, -OR 13A , or-NR 13A .
  • R 14 and R 15 are each independently substituted or unsubstituted C2-C30 alkyl.
  • R 12A and R 13A are each independently H, substituted or unsubstituted C1-C20 alkyl.
  • ubstituted or unsubstituted alkylene and each n is independently an integer from 0 to 4.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted C1-C20 alkylene.
  • R 12 is H or -OR 12A .
  • R 13 is H or -OR 13A .
  • R 14 and R 15 are each independently substituted or unsubstituted C2-C20 alkyl.
  • R 12A and R 13A are each independently H, substituted or unsubstituted C 1 -C 8 alkyl.
  • ubstituted or unsubstituted alkylene and each n is independently an integer from 0 to 4.
  • B 8 , B 9 , B 10 , and B 11 are each independently substituted or unsubstituted C 1 -C 8 alkylene.
  • R 12 is -OH, methoxy, or ethoxy.
  • R 13 is -OH, methoxy, or ethoxy.
  • R 14 and R 15 are each independently substituted or unsubstituted C2-C20 alkyl.
  • the cationic lipid of formula (III) is: [00588]
  • [00589] [00590] or a pharmaceutically acceptable salt thereof.
  • cationic lipid of formula (IV) [00592] [00594] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof.
  • B 12 is -W 7 -L a3 -W 8 -.
  • W 7 and W 8 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene.
  • L a3 is a bond
  • W 9 and W 10 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted 2 to 12 membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any combination thereof.
  • R 16 and R 17 are each independently
  • each R 410 , R 411 , R 510 , and R 511 is independently H, substituted or unsubstituted Ci- Ci2 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.
  • each m is independently an integer from 0 to 8
  • each s is independently an integer from 1 to 4.
  • W 7 and W 8 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkylene.
  • W 7 and W 8 are each independently substituted with one or more substituent groups.
  • W 7 and W 8 are each independently substituted with one or more size-limited substituent groups.
  • W 7 and W 8 are each independently substituted with one or more lower substituent groups.
  • W 7 and W 8 are each independently a bond. In embodiments, W 7 and W 8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene. In embodiments, W 7 and W 8 are each independently unsubstituted C1-C12 alkylene. In embodiments, W 7 and W 8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl ene. In embodiments, W 7 and W 8 are each independently unsubstituted 2 to 12 membered heteroalkylene.
  • W 7 and W 8 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
  • W 7 and W 8 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene
  • W 7 and W 8 are each independently a bond or unsubstituted Ci-
  • W 7 and W 8 are each independently a bond or unsubstituted Ci- C8 alkylene. In embodiments, W 7 and W 8 are each independently unsubstituted C i-Cx alkylene. [00613] In embodiments, L a3 is a bond, embodiments,
  • L a3 is a bond. In embodiments, L a3 is -S-S-. In embodiments, L a3 is . In embodiments,
  • W 9 and W 10 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkylene, substituted (e.g.
  • cycloalkylene e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene
  • substituted e.g. with a substituent group, a size-limited substituent group or a lower substituent group
  • unsubstituted heterocycloalkylene e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene
  • W 9 and W 10 are each independently substituted with one or more substituent groups. In embodiments, W 9 and W 10 are each independently substituted with one or more size-limited substituent groups. In embodiments, W 9 and W 10 are each independently substituted with one or more lower substituent groups.
  • W 9 and W 10 are each independently a bond. In embodiments, W 9 and W 10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene. In embodiments, W 9 and W 10 are each independently unsubstituted C1-C12 alkylene. In embodiments, W 9 and W 10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl ene. In embodiments, W 9 and W 10 are each independently unsubstituted 2 to 12 membered heteroalkylene.
  • W 9 and W 10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) cycloalkylene (e.g., C3-C8 cycloalkylene, C 3 - Ce cycloalkylene, or C5-C 6 cycloalkylene).
  • W 9 and W 10 are each independently unsubstituted cycloalkylene (e.g., C 3 -C8 cycloalkylene, C 3 -C 6 cycloalkylene, or C5-C 6 cycloalkylene).
  • W 9 and W 10 are each independently substituted (e.g.
  • heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • W 9 and W 10 are each independently unsubstituted heterocycloalkylene(e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
  • W 9 and W 10 are each independently a bond or substituted or unsubstituted C1-C12 alkylene. In embodiments, W 9 and W 10 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene. In embodiments, W 9 and W 10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene. In embodiments, W 9 and W 10 are each independently unsubstituted C1-C12 alkylene.
  • each R 410 , R 411 , R 510 , and R 511 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C 1 -C 12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
  • each R 410 , R 411 , R 510 , and R 511 is independently substituted with one or more substituent groups. In embodiments, each R 410 , R 411 , R 510 , and R 511 is independently substituted with one or more size-limited substituent groups. In embodiments, each R 410 , R 411 , R 510 , and R 511 is independently substituted with one or more lower substituent groups. [00623] In embodiments, each R 410 , R 411 , R 510 , and R 511 is independently H. In embodiments, each R 410 , R 411 , R 510 , and R 511 is independently substituted (e.g.
  • each R 410 , R 411 , R 510 , and R 511 is independently unsubstituted C 1 -C 12 alkyl.
  • each R 410 , R 411 , R 510 , and R 511 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl.
  • each R 410 , R 411 , R 510 , and R 511 is independently unsubstituted 2 to 12 membered heteroalkyl.
  • each R 410 and R 510 is independently H or substituted or unsubstituted C1-C12 alkyl. In embodiments, each R 410 and R 510 is independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl.
  • each R 410 and R 510 is independently H or unsubstituted C i-Cx alkyl. In embodiments, each R 410 and R 510 is independently unsubstituted Ci-Cx alkyl.
  • each R 410 and R 510 is independently H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl. In embodiments, each R 410 and R 510 is independently H or methyl. In embodiments, each R 410 and R 510 is independently H. In embodiments, each R 410 and R 510 is independently methyl.
  • R 16 and R 17 are each independently
  • cationic lipid of formula (II), , a fragment of cationic lipid of formula (III), a fragment of cationic lipid of formula (III), where B 2 , B 3 , B 4 , B 5 , B 6 , B 7 , B 8 , B 9 , B 10 , B 11 , L 9 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 , L 12 , L 13 , R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are as described herein including embodiments. [00631]
  • R 16 and R 17 are each independently
  • L 9 , L 10 , R 8 , and R 9 are as described herein including embodiments. [00634] In embodiments, R 16 and R 17 are each independently
  • R 16 and R 17 are each independently
  • R 16 and R 17 are each independently
  • each m is independently an integer from 0 to 8. In embodiments, each m is independently 8. In embodiments, each m is independently 7. In embodiments, each m is independently 6. In embodiments, each m is independently 5. In embodiments, each m is independently 4. In embodiments, each m is independently 3. In embodiments, each m is independently 2. In embodiments, each m is independently 1. In embodiments, each m is independently 0.
  • each s is an integer from 1 to 4. In embodiments, each s is 4. In embodiments, each s is 3. In embodiments, each s is 2. In embodiments, each s is 1. [00642] In embodiments, the cationic lipid of formula (IV) is:
  • the lipid is ALC-0315 or JK-0315-C A.
  • lipid nanoparticles comprising one or more of the ionizable cationic lipids or salts thereof described herein.
  • the lipid nanoparticles described herein further include one or more non-cationic lipids.
  • the lipid nanoparticles described herein further include one or more conjugated lipids capable of reducing or inhibiting particle aggregation.
  • the lipid nanoparticles described herein further include one or more therapeutic agents such as nucleic acids (e.g., mRNA).
  • lipid nanoparticles comprising one or more ionizable cationic lipids described herein are used to encapsulate nucleic acids (e.g., mRNA) within the lipid nanoparticles.
  • nucleic acids e.g., mRNA
  • the lipid nanoparticles include a therapeutic agent such as nucleic acid (e.g., mRNA), a cationic lipid (one or more ionizable cationic lipids of formula I-IV or salts thereof, as described herein, or cationic lipids known in the art), a non-cationic lipid (e.g., mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of particles (e.g., one or more PEG-lipid conjugates).
  • a therapeutic agent such as nucleic acid (e.g., mRNA), a cationic lipid (one or more ionizable cationic lipids of formula I-IV or salts thereof, as described herein, or cationic lipids known in the art), a non-cationic lipid (e.g., mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of particles (e.g
  • non-cationic lipids that can be used in the lipid nanoparticles described herein include, without limitation, neutral, zwitterionic or anionic lipids, for example:
  • phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatididyl
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • non-cationic lipids may be sterols such as cholesterol and derivatives thereof.
  • cholesterol derivatives include polar analogues such as 5a-cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl- (4'-hydroxy)-butyl ether.
  • the non-cationic lipids included in the lipid nanoparticles include a mixture of one or more phospholipids and cholesterol or a derivative thereof.
  • non-cationic lipids suitable for use in the lipid nanoparticles include stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • lipid conjugates that can be used in the lipid nanoparticles described herein include, without limitation, PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), and mixtures thereof.
  • the nanoparticles comprise PEG-lipid conjugate.
  • lipid conjugates that can be used in the lipid nanoparticles described herein include, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, mPEG2000-l,2-di-0- alkyl-sn3-carbomoylglyceride (PEG-C-DOMG), l-[8'-(l,2-dimyristoyl-3-propanoxy)- carboxamido-3',6'-dioxaoctanyl]carbamoyl-co-methyl-poly(ethylene glycol) (2 KPEG- DMG), l,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG), PEG conjugated to cholesterol or a derivative
  • PEG-DAA dial
  • lipid nanoparticles described herein are useful for the introduction of therapeutic agents such as nucleic acids (e.g., mRNA) into cells.
  • therapeutic agents such as nucleic acids (e.g., mRNA) into cells.
  • lipid nanoparticle composed of the ionizable cationic lipids of formula I-IV as described herein, to a mammal.
  • the lipid nanoparticles described herein can be administered either alone or in a mixture with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
  • the pharmaceutically acceptable carrier is usually added following lipid nanoparticle formation.
  • the nanoparticle can be diluted into pharmaceutically acceptable carriers such as normal buffered saline.
  • administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation (e.g., intransal or intratracheal), transdermal application, or rectal administration.
  • the pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
  • provided herein is a method for preventing or treating a disease in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a lipid nanoparticle composed of the ionizable cationic lipids of formula I-IV as described herein.
  • a method for preventing a disease in a mammal by administering to the mammal a therapeutically effective amount of a lipid nanoparticle composed of the ionizable cationic lipids of formula I-IV as described herein.
  • a method for treating a disease in a mammal in need thereof, by administering to the mammal a therapeutically effective amount of a lipid nanoparticle composed of the ionizable cationic lipids of formula I-IV as described herein.
  • the mammal is a dog, a cat or a human.
  • the mammal is a dog.
  • the mammal is a cat.
  • the mammal is a human.
  • the present disclosure provides methods for treating a subject who has been infected or is suspected of having been infected with a coronavirus infection, e.g., a MERS- CoV, SARS-CoV, or SARS-CoV-2, or an infection caused by a variant of any of these.
  • a coronavirus infection e.g., a MERS- CoV, SARS-CoV, or SARS-CoV-2, or an infection caused by a variant of any of these.
  • the present disclosure also provides methods for preventing an infection caused by e.g., a MERS- CoV, SARS-CoV, or SARS-CoV-2, or an infection caused by a variant of any of these, in a subject who is at risk of being infected, who has been exposed to such an infection, or who has not yet been infected with a coronavirus infection, e.g., a MERS-CoV, SARS-CoV, or SARS-CoV-2 infection, or an infection caused by a variant of any of these, or who may be in the early stages of infection but is not yet exhibiting symptoms of a coronavirus infection.
  • a coronavirus infection e.g., a MERS-CoV, SARS-CoV, or SARS-CoV-2 infection
  • such treatment can be administered to the asymptomatic subject at about 1-24 hours, about 24-48 hours, or about 48 hours to 3 days following exposure or possible exposure or suspected exposure to the coronavirus.
  • the prophylactic treatment can be administered to the asymptomatic subject at about 3-5 days, or about 5-10 days, or about 10-14 days, or about 14-21 days, or about 21-30 days or longer time ranges following exposure to the coronavirus.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized (i.e. freeze dried).
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized with a stabilizer.
  • the stabilizer is a sugar.
  • the sugar is for example trehalose or sucrose, but other sugars are also contemplated.
  • nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized and stored at room temperature. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized and stored at room temperature for at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days.
  • nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout is stable at room temperature for at least 60, at least 50, at least 40, at least 30, at least 25, at least 20, at least 15, at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4, at least 3, at least 2, or 1 day(s).
  • the administration route for mRNA vaccines plays an important role in determining vaccination efficacy.
  • the most commonly used injection routes include intradermal (ID), subcutaneous (SC), intramuscular (IM), intranodal (IN), and intravenous (IV) administration. Since the immune cells and lymphoid organs are the common vaccination targets, the anatomical and physiological properties of the vaccination sites (skin, muscle, lymphoid organ, and systemic circulation) may affect the safety and efficacy of a vaccine. Such information is useful for the selection of administration route and the delivery format (carrier-mediated, naked, or cell-based) of the mRNA vaccine.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intradermally.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intramuscularly.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intranodally.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered transdermally.
  • nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered subdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered to the epidermal layer. [00686] In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intradermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intramuscularly. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intranodally.
  • nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered transdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered subdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered to the epidermal layer.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered directly to the lymphatic system.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system subdermally.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system intramuscularly.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system intranodally.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system transdermally.
  • Medical devices that comprise an array of microneedles suitable for use herein are known in the art. Exemplary structures and devices comprising a means for controllably delivering one or more agents to a subject are described in International Patent Application Publication Nos. WO 2014/188343, WO 2014/132239, WO 2014/132240, WO 2013/061208, WO 2012/046149, WO 2011/135531, WO 2011/135530, WO 2011/135533, WO
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered by applying one or more medical devices to one or more sites of the skin of a subject.
  • the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered directly to the lymphatic system.
  • a medical device comprising a plurality of microneedles that is suitable for use with of the methods disclosed herein is the SofusaTM drug delivery platform available from Sorrento Therapeutics, Inc. see e.g., US Patent No: 10,737,082; International Patent Application PCT/US2019/034736 (published as WO 2019/232265) which are incorporated by reference herein in their entirety.
  • the medical device is placed in direct contact with the skin of the subject.
  • an intervening layer or structure will be between the skin of the subject and the medical device.
  • surgical tape or gauze may be used to reduce possible skin irritation between the medical device and the skin of the subject.
  • nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout can be to the circulatory system, the lymphatic system, the interstitium, subcutaneous, intramuscular, intradermal or a combination thereof.
  • nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered directly to the lymphatic system of the subject.
  • nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered to the superficial vessels of the lymphatic system.
  • the administration or delivery target is a lymph node, a lymph vessel, an organ that is part of the lymphatic system or a combination thereof.
  • the vaccination target is a lymph node.
  • the administration or delivery target is a specific lymph node as described elsewhere herein.
  • the medical device may comprise a needle array in the form of a patch.
  • the array of needles are able to penetrate a most superficial layer of the stratum corneum and deliver the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout to at least a portion or all of the non-viable epidermis, at least a portion of or all of the viable epidermis, and/or at least a portion of the viable dermis of a subject and subsequently to the lymphatic system of the subject.
  • These needles may further comprise nanotopography on the surface of the needle in a random or organized pattern. In embodiments, the nanotopography pattern may demonstrate fractal geometry.
  • Example 1 Nucleic acid constructs encoding SI proteins.
  • nucleic acid constructs were designed to provide templates for in vitro transcription of mRNAs encoding both “wild type” and mutant forms of the S protein of the Washington strain of SARS-Cov-2 (NCBI Accession QHU79204.1 (SARS-CoV-2 isolate Washington/Wuhan-Hu-1) as well as mutant and wild type forms of the alpha, beta, gamma, delta, and kappa variant forms of the S protein.
  • Each of the mutant genes was designed to encode an S protein having the same mutation of the furin cleavage site, where the amino acid sequence QQAQ was substituted for the RRAR sequence of the wild type S proteins.
  • Genes for each S protein were synthesized by Genewiz to encode the polypeptides of SEQ ID NO:20, SEQ ID NO:22 (WA S/QQAQ), SEQ ID NO:23 (“wild type” alpha S protein), SEQ ID NO:24 (alpha S/QQAQ), SEQ ID NO:25 (“wild type” beta S protein), SEQ ID NO:26 (beta S/QQAQ), SEQ ID NO:27 (“wild type” gamma S protein), SEQ ID NO:28 (gamma S/QQAQ), SEQ ID NO:29 (“wild type” delta S protein), SEQ ID NO:30 (delta S/QQAQ), SEQ ID NO:31(“wild type” kappa S protein), and SEQ ID NO:32 (kappa S/QQAQ).
  • the genes were cloned in the pVAXl vector 3’ of the T7 promoter.
  • Example 2 In vitro transcription of S protein mRNAs.
  • mRNA transcription of mutant S proteins was performed using the Hi ScribeTM T7 High Yield RNA Synthesis Kit kit from NEW ENGLAND BioLabs according to the manufacturer’s protocols.
  • UTP was replaced by Nl-Methyl-Pseudouridine-5'-Triphosphate from TriLink Biotechnologies.
  • CleanCap® Reagent AG from TriLink Biotechnologies was added to the reaction to cap the synthesized RNA at 5 ’-terminus.
  • Full-length mRNAs were purified using the Monarch® RNA Cleanup Kit from NEW ENGLAND BioLabs according to the manufacturer’s protocol.
  • RNA From a standard 20 pL reaction, a typical yield of 100 ug RNA was achieved, with a 260/280 ratio between 1.8-2.0 and a 260/230 ratio between 2.0- 2.2, as determined by NanoDropTM One Spectrophotometer from ThermoFisher Scientific.
  • Example 3 Transfection of cells with mRNA encoding S proteins having QQAQ mutation.
  • HEK293 cells were washed and diluted to 4 x 10 5 cells per ml, with 2 x 10 5 cells used per transfection in a 24-well plate.
  • 1 ⁇ g-2 5 ug of RNA was combined with Lipofectamine MessengerMax (ThermoFisher) and the formulated RNA was added to cell cultures and incubated for 24-72 hours at 37 C.
  • Example 4 Flow cytometry analysis of HEK293 cells transfected with mRNAs encoding mutant S proteins.
  • the cells were washed with FACS buffer and incubated with anti-human Fc antibody conjugated to allophycocyanin (APC). After incubation on ice for 15 minutes, the cells were washed with FACS buffer and analyzed using flow cytometry.
  • APC anti-human Fc antibody conjugated to allophycocyanin
  • Figure 1 shows that approximately 92% of the cells transfected with mRNA encoding either the “wild type” S protein or the furin mutation-containing S protein of the Washington strain expressed the S protein on the cell surface. Expression decreased by day 3 for both mRNAs, although the decrease in expression was somewhat less for the cell population transfected with the furin mutation (to approximately 44% of the cell population expressing the furin mutation S protein versus approximately 36% expressing the wild type S protein).
  • Figure 2 shows that expression of the furin mutation-containing alpha variant S protein in the mutant mRNA-transfected culture was approximately equivalent to the expression of the wild type alpha variant S protein at day 1 post-transfection.
  • Figure 3 shows that the percentage of cells expressing the furin mutation- containing beta variant S protein is similarly approximately equivalent to the percentage of cells expressing the wild type beta variant S protein (in this case analyzed on day 2 post transfection), but interestingly on day 3 the culture transfected with the mRNA encoding the beta S protein with the furin mutation shows only about half as much of a decrease in the percentage of expressing cells as is seen for the culture transfected with mRNA encoding the wild type beta S protein.
  • Figure 5 shows the flow cytometry analysis of cells transfected with mRNA encoding either the wild type delta S protein or mRNA encoding the delta S protein having the furin mutation.
  • culture wide expression dropped approximately 50% in the culture transfected with mRNA encoding the wild type delta S protein between day 1 and day 3, but only about 36% in the culture transfected with mRNA encoding the QQAQ mutant delta S protein over the same time period.
  • the mutant S protein-expressing cultures had a higher percentage of cells expressing the S protein on day 1, by day 3 culture-wide expression of the mutant S protein was approximately 50% higher than expression of the wild type delta protein.
  • Figure 6 shows that when cultures were transfected with mRNAs encoding either the wild type kappa variant S protein or the furin mutation containing kappa variant S protein, the percentage of cells in the cultures expressing the mutant and wild type S proteins was essentially the same.
  • the data shows no detrimental effects of the QQAQ furin mutation in SARS-CoV-2 S proteins, including S protein variants, on expression of the S proteins in cells transfected with nucleic acid constructs that encode the mutant S proteins.
  • inclusion of the QQAQ furin mutation can enhance the expression of SARS-CoV-2 S proteins in transfected cells, as evidenced by both an increased percentage of expressing cells and increased persistence of expression over time.
  • Example 5 Stability study comparing lyophilized mRNA and liquid mRNA.
  • Approximately 10 mg mRNA encoding a SARS-CoV-2 spike protein (one of the four variants: Wuhan/Washington, U.K., South Africa, Brazil) was mixed with 200,000 HEK293 T cells in 100 mL Buffer R included in the Neon Transfection System. The cells were electroporated using the Neon device at 1700V, 20ms, 1 pulse. The 100 mL transfection mix was then added to 400 mL prewarmed complete medium in a 24-well plate. 6 hours post transfection, the cells were collected and washed with FACS buffer (DPBS+0.5%BSA).
  • FACS buffer DPBS+0.5%BSA
  • the spike expression was then assessed by incubation with proprietary anti-spike antibody STI- 5041 (diluted 1 : 120 in FACS buffer) for 30 minutes, followed by incubation with allophycocyanin (APC) anti-human Fc (diluted 1:100 in FACS buffer) for 15 minutes. After washing with FACS buffer, the cells were resuspended in 200 mL FACS buffer and subjected to flow analysis with the Attune NxT Flow Cytometer.
  • proprietary anti-spike antibody STI- 5041 diluted 1 : 120 in FACS buffer
  • APC allophycocyanin
  • Flow cytometry scatter plots confirmed the extremely high expression of each of the variants tested, both in terms of the percentage of cells expressing the transfected mRNAs and the average amount/magnitude of spike protein expressed by cells transfected with each variant (see, e.g., Figures 7A-7E)
  • mRNA that encodes a SARS-CoV-2 spike protein was either in solution or lyophilized.
  • the mRNA solution was stored at -80°C while lyophilized mRNA was stored at room temperature for either 3 or 11 days. Following storage, the mRNA was transfected into HEK293 cells.
  • Control cells did not contain mRNA. Transfection was performed and spike expression was assessed as described above in above. In the control where cells did not generate the spike antigen, only background fluorescence was observed as shown in Figure 7F.
  • Figure 7G shows scatter plot of mRNA solution stored at -80°C for 11 days, prior to transfection into HEK293 cells.
  • Figure 7H and Figure 71 which show scatter plots of lyophilized mRNA stored at room temperature (for 3 days and 11 days, respectively), prior to transfection into HEK293 cells, demonstrate that lyophilized mRNA produces higher fluorescence, indicating better generation of the spike protein on the cells.
  • Example 6 Assessment of safety and efficacy of an exemplary mRNA-LNP vaccine (“Vaccine 1”) administered via lymphatic delivery
  • Vaccine 1 exemplary mRNA-LNP vaccine administered via lymphatic delivery
  • RNA In vitro transcription and purification of RNA.
  • SARS-Cov-2 Washington/Wuhan aka “Wuhan-Hu-1” isolate Spike (S) protein (GenBank: QHD43416.1) was codon optimized and cloned into pVAXl-based backbone which features 5'-UTR, 3'-UTRand Poly-A tail.
  • S SARS-Cov-2 Washington/Wuhan
  • 2P mutations at positions 986-987 were introduced.
  • the plasmid DNA was produced in bacteria, purified and linearized by a single-site restriction enzyme digestion.
  • the template DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase (Cat: M0251, NEB) in the presence of a trinucleotide capl analogue, m7(3OMeG)(5')ppp(5')(2OMeA)pG (Cat: N-7113, TriLink), and of Nl- methylpseudouridine-5’ -triphosphate (Cat: N-1081, TriLink) in place of uridine-5’- triphosphate (UTP).
  • DNase I Cat: M0303, NEB
  • SARS-CoV-2 Virus SARS-CoV-2 viruses were obtained from BEI resources (Washington strain NR-52281; Beta Variant NR-54009) VeroE6 monolayers were infected at anMOI of 0.01 in 5ml virus infection media (DMEM + 2%FCS +1X Pen/Strep). Tissue culture flasks were incubated at 36°C and slowly shaken every 15 minutes for a 90 minute period. Cell growth media (35mL) was added to each flask and infected cultures were incubated at 36°C/5% C02 for 48 hours. Media was then harvested and clarified to remove large cellular debris by room temperature centrifugation at 3000 rpm.
  • OVA vaccine studies 10 ⁇ g of OVA (Cat: VAC- POVA, Invivogen) and 8 ⁇ g of CpG (Cat: TLRL- 1826-1, Invivogen) were administered to mice on days 0 and 14. Peripheral blood was collected from anaesthetized mice once/week via submandibular route.
  • Reference mRNA-LNP vaccine is the same construct as an EUA cleared compound.
  • ELISA assays To asses spike specific antibodies, SI (Cat: 40591-V08H Sino Biological)or RBD (Cat: 40592-V08B, Sino Biological) protein was coated on half-area high bindingplates (Cat: N503 Thermo) at 1 ⁇ g/mL overnight at 4°C. Plates were washed 3 times withELISA wash buffer (Thermo), pre-blocked with casein blocker (Cat: 37528 Thermo) for lhour at room temperature (RT), and washed 1 time with ELISA wash buffer. Mouse serawas diluted in casein blocker and transferred to ELISA plates for 1 hour at RT followed by 3 wash steps.
  • HRP horse radish peroxidase
  • IgG For IgG (Fey), a standard curve was generated using Anti-RBDPAb (Cat: 40592-MP01 Sino Biological) or Anti-Si (Cat: MAB105405 R&D Systems) starting at 3000 ng/mL with 3 fold serial dilutions.
  • IgM m chain
  • IgGl, and IgG2c serial fold dilutions were run and titers were determined using an absorbance cutoff of 0.7OD.
  • Plaque Reduction Neutralization Test PRNT. Simian VeroE6 cells were plated at 18x10 ⁇ cells/well in a flat bottom 96-well plate in a volume of 200 m ⁇ /well. After 24 hours, a serial dilution of seropositive blood serum is prepared in a 100 m ⁇ /well at twice the fmalconcentration desired and live virus was added at 1,000 PFU/IOOmI of SARS-CoV-2 andsubsequently incubated for 1 hour at 37°C in a total volume of 200 m ⁇ /well. Cell culture media was removed from cells and sera/virus premix was added to VeroE6 cells at 100 m ⁇ /well and incubated for 1 hour at 37°C.
  • ICS Peripheral blood T cell intracellular cytokine staining.
  • ICS was performed at the indicated time points following the booster shots for IFNy, TNFa, and IL-4.
  • Whole blood was stimulated for 6 hours with 1 ⁇ g/mL of SIINFEKL (Sigma) or overnight with 1 ⁇ g/peptide per well of spike associated (Cat: 130-127-951, Miltenyi Biotec Peptivator) peptides at 37°C, 5% C02 in the presence of brefeldin A (Biolegend) and monensin (Biolegend).
  • whole blood was incubated with red lysis buffer (Cat: A10492-01 Gibco) at room temperature.
  • Cells were permeabilized using Intracellular Staining Perm Was Buffer (Cat: 421002 Biolegend). Cells were stained with PE anti- mouse IFNy (Cat: 505808 Biolegend), FITC anti-mouse TNFa (Cat: 506304 Biolegend), BV421 anti-mouse IL-4 (Cat: 504127 Biolegend), APC-Cy7 anti-mouse CD3 (Cat: 100222 Biolegend), PE-Cy7 anti-mouse CD4 (Cat: 25-0041-82 Invitrogen), and allophycocyanin anti-mouse CD8a (Cat: 100712 Biolegend). Naive mice (non-vaccinated mice) were usedas negative controls. Cells were then run on a Beckman Coulter CytoFLEX instrument and analyzed via FlowJo VI 0 software.
  • Vaccine 1 comprises a single-stranded mRNA encoding a full-length SARS-CoV-2 S glycoprotein, derived from the strain “Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1”, as an active drug substance encapsulated in lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • mutations were introduced into S protein to substitute residues 986 and 987 to produce prefusion-stabilized SARS-CoV-2 S(2P) protein.
  • the sequence was further codon-optimized and cloned into a pVAXl -based backbone that contains T7 promoter, 5'-UTR, 3'-UTR and optimized Poly-A tail with minimal overhang.
  • an exemplary lymphatic drug delivery device which is connectable to any luer lock syringe ( Figure 9A), and which consists of microneedles that puncture the stratum comeum and release drug at the epidermal/dermal boundary.
  • ICG Indocyanine Green
  • NIRF near-infrared fluorescence
  • mice were injected with OVA and an oligonucleotide adjuvant (CpG) on days 0 and 14 using an IM or MuVaxx administration. All mice treated with MuVaxx generated anti-OVA IgG by day 13 compared to 4 of 8 in the IM cohort.
  • Exemplary mRNA-LNP vaccine “Vaccine 1”, induces anti-spike antibodies with MuVaxx enabling dose sparing.
  • Vaccine 1 favors Thl response over Th2.
  • PRNT Plaque Reduction Neutralization Test
  • an exemplary mRNA-based SARS-CoV-2 vaccine Vaccine 1 that induced similar humoral immunity with elevated cellular immunity compared to a Reference vaccine formulation when administered IM.
  • Immunity generated with the Vaccine 1 formulation was dose dependent as immunity was reduced when going from 10 to 1 ⁇ g IM.
  • dose sparing effects were observed where both humoral and cellular immunity were similar to that of a 10 ⁇ g IM dose highlighting the improved immunogenicity when directing vaccines towards LNs.
  • Previous mRNA-based vaccines have reported dose-dependent side effects with higher doses linked to systemic and local adverse events underscoring an additional advantage of lower dose formulations; lessening side effects while expanding vaccine access to large populations.
  • a vaccine that generates durable immunity is another hallmark of an effective vaccine and is a metric that was investigated.
  • the serum concentrations of both anti-Sl and anti-RBD IgG waned to a lesser degree in mice treated with MuVaxx relative to those treated with the IM same dose, highlighting improved durability.
  • This is of interest as recent reports have shown declines in SARS-CoV-2 neutralizing antibodies 2-3 months after disease onset as short-lived plasma cells stop producing nAbs.
  • a subset of plasma B cells do differentiate into memory B cells following infection and/or vaccination leading to persistent germinal center formation within LNs where somatic hypermutation takes place.
  • efficient delivery of vaccines towards LNs where memory B cells reside may improve memory B cell activation and coverage against emerging variants at lower doses in line with the nAb data shown here.
  • Vaccine 1 delivered via MuVaxx elicited a strong CD8 T cell response towards SARS-CoV-2 peptides which is believed to be advantageous for preventing COVID-19 and providing protection against reinfection.
  • the CD4 Th2 phenotype has previously been associated with vaccine-associated enhanced respiratory disease (VAERD) in those vaccinated against the measles- and respiratory syncytial- virus.
  • VAERD vaccine-associated enhanced respiratory disease
  • IgG2c IgG2c to IgGl antibody response in vaccinated mice.
  • the response of all vaccinated mice favored a CD4 Thl response in line with naive mice.
  • the antibody response also skewed towards a Thl response as measured by the IgG2c to IgGl ratio in vaccinated mice with the Vaccine 1 10 ⁇ g IM and 1 ⁇ g MuVaxx cohorts displaying a stronger IgG2c to IgGl ratio.
  • Mice treated with Vaccine 1 formulations may display enhanced anti-viral activity as mouse IgG2 subclasses have been shown to induce antibody-dependent cellular cytotoxicity.
  • the Thl :Th2 response described above suggested promising activity for avoiding VAERD while promoting anti-viral activity.
  • MuVaxx to deliver an exemplary mRNA vaccine to the lymphatic system, such as by directing the vaccine towards the epidermis and draining lymph nodes, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine producing CD8 T cells, and neutralizing antibodies against the Washington (Wild Type, WT) and South African (beta) variants. Furthermore, a four-fold elevated T cell response was observed in MuVaxx administered vaccination as compared to that of IM administered vaccination.
  • Nanotopography facilitates in vivo transdermal delivery of high molecular weight therapeutics through an integrin-dependent mechanism. Nano Lett.
  • Example 7 Additional mRNA vaccines for the prevention of coronavirus and other viral infections
  • VOCs SARS-CoV-2 variants of concern
  • S spike
  • B.l.1.7 beta
  • Beta B.1.351
  • Gamma P.l
  • Delta B.1.617.2
  • the initial mouse vaccination results showed that the individual VOC mRNAs induced the generation of neutralizing antibody in a VOC-specific manner. Moreover, we discovered that the antibodies produced from mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs cross-reacted with other VOCs. The broad spectrum of generated nAb was further confirmed when vaccinated mice were challenged with the respective live viruses. Interestingly, in a mix-and-match booster experiment, omicron-Furin and WA-Furin mRNA elicited comparable protection against omicron.
  • We also tested the concept of bivalent vaccine by introducing the RBD of Delta strain into the intact S antigen of Omicron. The chimeric mRNA induced potent and broadly acting nAb against Omicron and Delta, and thus serve as vaccine candidates to broadly target current variants of concern as well as emerging variants in the future.
  • RNA sequences of the SARS-Cov-2 Spike protein of VOC were codon optimized and cloned into pVAXl -based backbone which features 5'-UTR, 3'-UTR and Poly- A tail.
  • 2P mutations at positions 986-987 were introduced.
  • the plasmid DNA was produced in bacteria, purified and linearized by a single-site restriction enzyme digestion.
  • the template DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase (Cat: M0251, NEB) in the presence of a trinucleotide capl analogue, m7(30MeG)(5')ppp(5')(20MeA)pG (Cat: N-7113, TriLink), and ofNl- methylpseudouridine-5’ -triphosphate (Cat: N-1081, TriLink) in place of uridine-5’- triphosphate (UTP).
  • DNase I Cat: M0303, NEB
  • LNPs were prepared by microfluidic mixing a buffered solution of mRNA with an ethanol solution of lipids [distearoylphosphatidylcholine (DSPC), cholesterol, 1,2- Dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-2000 (DMG-PEG2000), and ionizable lipid.
  • DSPC disearoylphosphatidylcholine
  • DMG-PEG2000 1,2- Dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-2000
  • ionizable lipid The LNPs were concentrated by dialysis against an aqueous buffer system, following a 0.2 pm sterile filtration. The LNPs were tested for mRNA concentration, encapsulation efficiency, particle size, pH, and osmolality.
  • Monocytes were isolated from PBMCs and differentiated into DCs in presence of GM-CSF (Cat: 300-03, Peprotech) and IL-4 (Cat: 200-04, Peprotech). Between day 6-day 8, cells were transfected with mRNA by the NeonTM electroporation transfection system (Cat: MPK5000, ThermoFisher). 24 hours post-transfection, the cells were collected and subjected to flow cytometry as described below to check the expression of spike.
  • GM-CSF Gibco-CSF
  • IL-4 Cat: 200-04, Peprotech
  • mRNA (2.5ug) of WT vs Mutant from five variants were transfected with LipofectamineTM MessengerMAXTM Transfection Reagent (2ul) and cultured for 72 hours at 37oC using 293T adherent cells with 0.5ml of DMEM media with 10% FBS in each well of a 24-well cell culture-treated plate.
  • Transfected cells from each well were dislodged with 400ul of TrypLE at 72 hours and neutralized with its own media. Cell pellets were collected after spinning down at 550g for 2 minutes by removing supernatant for each well.
  • the cells were spun down and the pellets were washed twice with the same speed and time of centrifugation using 200ul FACS buffer and resuspended in 200ul FACS buffer.
  • the fluorescent intensity of positive cells within the gated population was detected by the Attune NxT Flow Cytometer (Therm oFisher) using lOOul of acquisition volume setting.
  • SARS-COV-2 viruses were obtained from BEI resources (Washington strain NR- 52281; Alpha variant NR-54000; Beta Variant NR-54009; Gamma variant NR-54982; Delta variant NR- 55611 or NR-55672; Lambda variant NR- 55654 and Omicron NR-56461). VeroE6 monolayers were infected at an MOI of 0.01 in 5ml virus infection media (DMEM + 2% FCS + IX Pen/Strep). Tissue culture flasks were incubated at 36°C and slowly shaken every 15 minutes for a 90-minute period. Cell growth media (35mL) was added to each flask and infected cultures were incubated at 36°C/5% C02 for 48 hours. Media was then harvested and clarified to remove large cellular debris by room temperature centrifugation at 3000 rpm.
  • mice 7-week-old BALB/cJ female mice were purchased from the Jackson Laboratory. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC). mRNA formulations were diluted in 50 uL of IX PBS, and mice were inoculated IM into the same hind leg for both prime and boost. There was 3 weeks interval between prime and boost. Two weeks after boost, mice blood was collected from retro-orbital for ELISA and pseudovirus neutralization assay.
  • IACUC Institutional Animal Care and Use Committee
  • Ni-NTA HisSorb plates (Qiagen) were coated with 50ng/well of SI proteins (all from Sino Biological, Cat: 40591-V08H, 40589-V08B6, 40589-V08B7, 40589-V08B8, 40589-V08B16) in IX PBS at 4°C overnight.
  • SI proteins all from Sino Biological, Cat: 40591-V08H, 40589-V08B6, 40589-V08B7, 40589-V08B8, 40589-V08B16
  • IX PBS 4°C overnight.
  • Blocker Casein Cat: 37528 Thermo
  • RT room temperature
  • SARS-CoV-2 Spike pseudotyped AG-VSV-luciferase was generated by nucleofection of BHK cells (maintained in DMEM/F12 with 10%FBS and 5%TPB) with Spike-expressing plasmid followed by transduction with G-pseudotyped AG-luciferase (G*AG-luciferase) rVSV (Kerafast) 18-24 hours later. The supernatant containing pseudovirus was collected following 24 hours and stored at -80°C. Pseudovirus was normalized for luciferase expression using G*AG-luciferase VSV of known titer as the standard.
  • HEK-Blue 293 hACE2-TMPRSS2 cells (Invivogen; maintained in DMEM with 10% FBS) were plated to white-walled 96-well plates at 40,000 cells/well and incubated at 37°C/5% C02. The next day, SARS-CoV-2 Spike pseudotyped ⁇ G-VSV-luciferase was incubated with a dilution series of mouse serum (dilutions as indicated) and anti-VSV-G (Kerafast; 1 ⁇ g/mL) antibody for 30 minutes at room temperature and added to the HEK-Blue 293 hACE2-TMPRSS2 cells.
  • Transduced cells were incubated for 24 hours at 37°C/5% C02 and luminescence measured by addition of 40m1 of ONE-Glo reagent (Promega) with detection using a Tecan Spark plate reader. The percent inhibition was calculated using the formula 1 -([luminescence of serum treated sample]/[average luminescence of untreated samples] x 100. The average of quadruplicate samples were included in the analyses.
  • Simian VeroE6 cells were plated at 18x 103 cells/well in a flat bottom 96-well plate in a volume of 200 m ⁇ /well. After 24 hours, a serial dilution of seropositive blood serum is prepared in a 100 m ⁇ /well at twice the final concentration desired and live virus was added at 1,000 PFU/IOOmI of SARS-CoV-2 and subsequently incubated for 1 hour at 37°C in a total volume of 200 m ⁇ /well. Cell culture media was removed from cells and sera/virus premix was added to VeroE6 cells at 100 m ⁇ /well and incubated for 1 hour at 37°C.
  • K18-hACE2 transgenic mice were purchased from Jackson laboratory and maintained in pathogen-free conditions and handling conforms to the requirements of the National Institutes of Health and the Scripps Research Institute Animal Research Committee. 8-12 weeks old mice were injected with the indicated administration technique under isoflurane anesthesia in the right hind flank area for IM injections. Mice were infected intranasally with 10000 PFU of SARS-CoV-2 in total volume 50 pL.
  • VeroE6 cells were plated at 3xl0e5 cells/well in 24 well plates in volume 400 m ⁇ /well. After 24 h. medium is removed, and serial dilution of homogenized lungs were added to Vero cells and subsequently incubated for 1 h at 37°C. After incubation, an overlay (1:1 of 2% methylcellulose (Sigma) and culture media) is added to each well and incubation commenced for 3 d at 37°C. Plaque staining was performed using Crystal Violet as mentioned above.
  • SARS-CoV-2 spike (S) mRNA vaccines that achieve high expression in mammalian cells (Francis et ak, 2021).
  • One such mRNA vaccine encodes the S protein from the Wuhan/Washington (WA) strain and encodes a polybasic furin cleavage site at the junction of SI and S2 subunits. The feature could affect the stability of spike protein and reduce the pool of antigenic epitopes available to induce cellular and humoral immunity (Peacock et ak, 2021).
  • PRNT Plaque Reduction Neutralization Test
  • VOC mRNA vaccinations led to the production of antibodies capable of binding to a breadth of VOC S proteins, especially Beta-Furin.
  • PRNT was performed where VeroE6 cells were exposed to the live virus of five VOCs in the absence or presence of diluted serum collected from the immunized mice ( Figure 16B).
  • the experiment showed that the individual monovalent mRNA vaccine generally displayed variant-specific protection activity.
  • WA-Furin mRNA induced the highest neutralizing activity against the WA-1 virus.
  • the serum from the Beta-Furin mRNA injected cohort displayed robust and broad protection against all VOCs tested.
  • Beta- Furin mRNA provided a much stronger protection against the highly contagious Delta variant than was elicited by vaccination with the WA-Furin mRNA.
  • VOC-specific strategy can provide strain-specific protection
  • some VOC-based mRNA vaccines have relatively enhanced potential to trigger a broad and potent immune response to the genetically divergent set of existing SARS-CoV-2 variants.
  • the K18-hACE2 transgenic model has been extensively utilized to evaluate the vaccine efficacy and effectiveness in preventing COVID-19 in the preclinical setting (Arce and Costoya, 2021; Dong et al., 2021; Radvak et ak, 2021; Winkler et ah, 2020).
  • Two key metrics to determine the severity of pathogenesis are the virus titer in the lung tissue and body weight loss following virus infection.
  • K18-hACE2 mice were first intramuscularly administrated with 5 ⁇ g of WA-Furin or Beta-Furin mRNA twice with 3-week interval.
  • mice treated with PBS exhibited dramatic weight loss in all challenge settings, regardless of the virus strain.
  • the average body weight in the PBS controls declined to 82%, 78% and 81% on day 5 post infection with WA, Beta and Lambda strains, respectively.
  • none of the mice immunized with Beta-Furin or WA-Furin vaccines showed any sign of weight loss, and some animals in these treatment groups gained weight after infection up to 5 days (Figure 17C).
  • both furin- mutant mRNAs gave effective protection against the Lambda variant although the corresponding spike mRNA was not included among the immunogens, suggesting broad protection capacity of some VOC mRNAs.
  • Serum was collected two weeks post boost and subjected to ELISA assay to measure Omicron-specific binding antibodies.
  • Omicron spike protein as the coating antigen
  • high titers of binding antibodies were observed in the sera of Omicron mRNA-injected mice ( Figure 18B).
  • the protective capability of Omicron-specific mRNA vaccine was further evaluated in PRNT.
  • Figure 18C The protective capability of Omicron-specific mRNA vaccine could induce potent production of Omicron-specific nAbs in vivo.
  • mice showed no detectable viral replication in the lung, suggesting that both WA-1 and Omicron-based booster mRNAs provided substantial and similar protection against Omicron (Figure 18D, right panel).
  • mice were immunized twice with LNP-formulated mRNA as described above.
  • the sera were collected two weeks following the second dose and then analyzed for the titers of binding antibodies and nAbs against various VOCs.
  • the ELISA results showed that the chimeric Delta RBD- Omicron mRNA outperformed the original Omicron mRNA in the generation of binding antibodies against WA, Beta and Gamma variants ( Figure 19A).
  • a moderately higher titer against Delta was seen with the chimeric mRNA vaccination.
  • Prophylactic nucleic acid vaccines can deliver the nucleotide sequence that codes for virus-derived but nonpathogenic proteins into host cells, thus mimicking a native infection to elicit an immune response.
  • mRNA vaccines eradicate the need for nucleic acid to enter the nucleus to achieve expression, and they are less likely to be integrated into the host genome.
  • SARS-CoV-2 vaccines authorized by FDA and widely disseminated. These vaccines encode for the S protein, the major surface protein on the coronavirus virion responsible for anchoring onto target cells, and thus the predominant virus-encoded target for nAb elicited by natural infection.
  • Beta S protein has also been selected by Moderna to test in phase II either as a monovalent antigen or by mixing with mRNA-1273 to tackle the emerging VOCs (Pajon et al., 2022; Waltz, 2021).
  • Omicron S protein has ⁇ 35 mutations, it still exhibits 97% similarity to the ancestral WA strain in the amino acid sequence.
  • most of the epitopes presented to lymphocytes could remain the same, and the consequence of antigenic drift contributed by those ⁇ 35 mutations could be masked by the surge of nAbs generated after the third dose.
  • individuals with prior immunity from vaccination may not necessarily benefit from a change in vaccinating antigens.
  • the homologous boost scheme remains a scientifically proven and economically feasible option at hand in the fight against COVID-19.
  • Another unique feature of our in-house mRNA vaccine is the mutation of furin cleavage site between the SI and S2 domains of S. This cleavage is believed to have emerged during viral transmission from its zoonotic host to humans and is one of the key attributes to explain the high transmissibility of SARS-CoV-2 in humans (Whittaker, 2021). The mutation is mainly to address the concern that circulating SI was detected in the plasma of vaccinated subjects (Ogata et al., 2021).
  • SI can be taken up by many critical organs, such as liver, kidney, spleen, and even cross the blood-brain barrier to gain access to the brain (Rhea et al., 2021).
  • ACE2 binds to ACE2
  • ACE2 downstream signaling events that may lead to inflammation and lung damage
  • furin cleavage mutation is by retaining the full-length S protein within the cell and on the cell surface ( Figure 20B), a larger pool of antigens could become available for presentation to induce the adaptive immunity. Indeed, S protein with furin cleavage mutation even binds with higher affinity to ACE2 (Laczko et al., 2020).
  • our in-house designed mRNA vaccine represents a potentially safer alternative to existing products in use and could induce stronger adaptive immunity against prevalent VOCs, including Omicron and Delta.
  • Our chimeric design will also facilitate the development of next-generation vaccines that achieve the balance between effectiveness and coverage, not only for the variants of SARS-CoV-2 but also for other viruses.
  • Laczko D., Hogan, M.J., Toulmin, S.A., Hicks, P., Lederer, K., Gaudette, B.T., Castano, D., Amanat, F., Muramatsu, H., Oguin, T.H., et al. (2020).
  • a Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity 53, 724-732. e7.
  • Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice. Science 373, 991-998.
  • SARS-CoV-2 Spike Protein Elicits Cell Signaling in Human Host Cells: Implications for Possible Consequences of COVID-19 Vaccines. Vaccines 9, 36.
  • SARS-CoV-2 Omicron RBD shows weaker binding affinity than the currently dominant Delta variant to human ACE2. Signal Transduct. Target. Ther. 7, 1-3.
  • Omicron variant of SARS-CoV-2 imposes a new challenge for the global public health. Biosaf. Health.
  • Example 8 Generation and optimization of mRNA-lipid nanoparticle formulations.
  • lipid containing formulations such as lipid nanoparticle formulations that may be employed to prepare lyophilized formulations comprising the nucleic acid molecules, nucleic acid compositions and mRNA vaccines as disclosed herein and throughout.
  • parameters such as buffer choice, use of cryoprotectants, ration of lipids, N/P ration, and pH, were tested and optimized.
  • JK0315CA/DSPC/Cholesterol/DMG-PEG2000 49.9/10/38.4/0.17)

Abstract

The present disclosure relates, inter alia, to nucleic acid molecules and compositions, pharmaceutical compositions, vaccines comprising such nucleic acid molecules for preventing or treating coronavirus infections and diseases associated therewith, as well as methods for administering same.

Description

NUCLEIC ACID MOLECULES AND VACCINES COMPRISING SAME FOR THE PREVENTION AND TREATMENT OF CORONA VIRUS INFECTIONS AND
DISEASE
Cross Reference to Related Applications
This application claims priority to U.S. provisional patent application number 63/159,972 filed on March 11, 2021, U.S. provisional patent application number 63/228,008, filed on July 30, 2021, U.S. provisional patent application number 63/237,133, filed on August 25, 2021, U.S. provisional patent application number 63/298,755, filed January 12, 2022, and U.S. provisional application number 63/316,329, filed March 3, 2022, the content and disclosure of which are incorporated by reference in their entireties for all purposes.
Technical Field
[0001] The present disclosure relates, inter alia , to nucleic acid molecules and compositions, pharmaceutical compositions, vaccines comprising such nucleic acid molecules for preventing or treating coronavirus infections and diseases associated therewith, as well as methods for administering same.
Background
[0002] Coronaviruses belong to a group of viruses that causes diseases in birds, mammals and humans. Diseases caused by coronavirus infection include respiratory infections and enteric infections, which can be mild or lethal. Coronaviruses are viruses in the subfamily Orthocoronavirinae, in the family Coronaviridae, in the order Nidovirales. The genus Coronavirus includes avian infectious bronchitis virus, bovine coronavirus, canine coronavirus, human coronavirus 299E, human coronavirus OC43, murine hepatitis virus, rat coronavirus, and porcine hemagglutinating encephalomyelitis virus. The genus Torovirus includes Berne virus and Breda virus. Coronaviruses are enveloped viruses having a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry.
The genomic size of coronaviruses ranges from approximately 26 to 32 kilobases, which is believed to be the largest for an RNA virus.
[0003] The name “coronavirus" is derived from the Latin corona and the Greek korone (e.g., "garland” or “wreath"), meaning crown or halo. The corona reference relates to the characteristic appearance of virions (the infective form of the virus) by electron microscopy, which have a fringe of large, bulbous surface projections creating an image reminiscent of a royal crown or of the solar corona. This morphology is created by the viral spike (S) peplomers, which are proteins that populate the surface of the virus and determine host tropism. Proteins that contribute to the overall structure of all coronaviruses are the spike protein ( also known as “S protein” or “S”, all used interchangeably throughout), envelope (E), membrane (M) and nucleocapsid (N).
[0004] In the case of SARS coronaviruses, a defined receptor-binding domain on S mediates the attachment of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). Some coronaviruses (specifically the members of Betacoronavirus subgroup A) also have a shorter spike-like protein called hemagglutinin esterase (HE). The 2019-2020 China pneumonia outbreak in Wuhan was traced to a novel coronavirus, labeled 2019-nCoV by the World Health Organization (WHO) and is also known as SARS-CoV-2.
[0005] The SARS-CoV-2 virus has accounted for more than 440 million cases of the coronavirus disease 2019 (COVID-19) and over 6 million fatalities worldwide since its original outbreak in December 2019. This is the 3rd outbreak of a Betacoronavirus since 2002, the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) being its predecessors, and is much more efficiently transmitted person to person.
[0006] SARS-CoV-2 gains entry to human cells by using the angiotensin-converting enzyme 2 (ACE2) protein as a receptor. The spike (S) protein of SARS-CoV-2, a transmembrane glycoprotein that forms homotrimers, binds ACE2 on host cells leading to internalization of the virus.
[0007] The SARS-CoV-2 S protein (NCBI Accession QHU79204.1 (SARS-CoV-2 isolate Washington/Wuhan-Hu- 1 ,e.g., SEQ ID NO:20) includes two regions or domains known as SI (the N-terminus to amino acid 685) and S2 (amino acids 686 to 1273) that are cleaved into the SI and S2 subunits by furin, a cellular protease, during the infection process (Peacock et al. (2020) bioRxiv 2020.09.30.318311; doi: https://doi.org/10.1101/2020.09.30.318311). The SI subunit, which mediates the interaction between the S protein and ACE2, includes the “N- terminal domain” (NTD) which is followed by the receptor binding domain (RBD) at amino acids 319 to 541 (See, e.g., Huang et al., Acto Pharmacologica Sinica, Vol 41, pages 1 Mi ll 49 (2020)). The S2 subunit, which includes an extracellular domain, a transmembrane domain, and a cytoplasmic tail, mediates virus-host membrane fusion that results in entry of the virus into the host cell. [0008] Vaccines, such as mRNA vaccines, have become a versatile technology for the prevention of infectious diseases and various types of cancer, and thus are attractive candidates for the prevention and/or treatment of coronavirus infection, including infection due to SARS-Cov-2-mediated infections. In particular, mRNA-based vaccines are attractive candidates in part because the development process of an mRNA vaccine can be much faster than conventional protein vaccines (DeFrancesco, 2017, Nat. Biotechnol ., 35, 193-197).
For instance, in response to the pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020, an mRNA vaccine was administrated to the first volunteer in a phase 1 clinical trial within ten weeks after the sequence of the viral genome was revealed (Lurie et al., 2020, New Engl. J. Med., 382, 1969-1973). Second, in vitro transcription reaction is easy to conduct, has a high yield, and can be scaled up (Pardi et al., 2018, Nat. Rev. Drug Discov., 17, 261). Advanced industrial setup can manufacture mRNA up to kilogram scales (Versteeg et al., 2019, Vaccines , 7(4), 122). Third, mRNA vaccine enables the synthesis of antigen proteins in situ, eliminating the need for protein purification and long-term stabilization which are challenging for some antigens. Fourth, transportation and storage of mRNA may be easier than protein-based vaccines, since RNA, if protected properly against ribonucleases (RNases), is less prone to degradation compared to proteins (Stitz et al., 2017, PLOS Negl. Tropical Dis ., 11, e0006108; Zhang et al., 2019, Front Immuno ., 10, 594). Because of these advantages, mRNA vaccines have great potential to be manufactured and deployed in a timely manner in response to rapid infectious disease outbreaks. Indeed, to date, a total of ten COVID-19 vaccines have been granted for emergency use or fully approved globally, with many of them being mRNA vaccines (https://covidl9irackvaccines.org/agency/who/).
[0009] Despite the existence of these vaccines, numerous SARS-Cov-2 variants of concern (VOCs) have caused multiple waves of widespread outbreak with unprecedented speed. Back in 2020, the first VOC Alpha (B.1.1.7) was detected in the United Kingdom, leading to more than 100,000 confirmed cases in the country. Following the emerging of Alpha variant, there were two other outbreaks originating from South Africa and Brazil, triggered by the Beta (B.1.351) and Gamma (P.l) variants, respectively (Adam, 2022; Liu et al., 2021; Otto et al., 2021). With additional mutation in spike protein, both VOCs were estimated to be 40% -80% more transmissible than the wildtype lineage. Furthermore, the Beta variant exhibits great immune escape and COVID-19 vaccination only showed 75% effectiveness against infection (Abu-Raddad et al., 2021). In late 2020, a new variant B. 1.617.2 (Delta) was identified and subsequentially contributed to a surge in cases in India and worldwide shortly afterwards. Besides the increased transmissibility, Delta variant has been shown to cause more severe disease and result in a poorer prognosis than previously reported VOCs (Farinholt et al., 2021; Mlcochova et al., 2021; Planas et al., 2021). To make things even worse, the effectiveness of existing mRNA vaccines against Delta has dropped considerably, from -95% for the original WA strain to 76% (mRNA-1273) and 42% (BNT162b2) (Puranik et al., 2021). By June 2021, global COVID-19-related deaths hit 5 million as Delta variant swept over 161 countries around the world.
[0010] More recently, WHO declared a novel VOC B.1.1.529, designated as Omicron, on 26th Nov 2021, only two days after it was first reported to WHO by South Africa (Xu et al., 2022). Omicron quickly outcompeted the circulating Delta variant within weeks after landing in Europe and US, and has become the dominant VOC around the globe. Scientists found that there was a significant reduction in Omicron virus neutralizing activity in sera obtained from individuals infected by pre-Omicron variants and populations vaccinated with immunogens based on early WA-1 virus (Cao et al., 2021; Lee et al., 2022; Zhao et al., 2021). A huge number of breakthrough infections have been reported even from individuals fully vaccinated with the 3rd booster shot. Although still largely effective in preventing hospitalization and mortality, the two existing mRNA-based vaccines have shown declining capabilities in preventing infection by various VOCs, especially Omicron. A plausible explanation for the increasingly large gap in vaccine coverage is that both EUA-approved mRNA vaccines encode the S protein of the original WA strain as the immunogen.
[0011] Compared to its ancestral strain, Omicron contains more than 30 additional mutations within the S protein coding sequence, 15 of which reside in the receptor binding domain (RBD)(Wu et al., 2022). Capable of binding to the human ACE2 receptor, pioneering structural analysis demonstrated that RBD is the key domain for virus attachment and entry to human cells (Shang et al., 2020a; Shang et al., 2020b; Han et al., 2022; Lupala et al., 2022). Thus, the high rate of mutations in Omicron RBD may dramatically alter the interaction dynamics between the virus and host cell, which could, at least partially, explain the enhanced transmissibility and breakthrough cases (Liu et al., 2021; Zhao et al., 2021).
[0012] The continued exploration and identification of safe and effective vaccines for the in order to protect against current and emerging (VoCs) while generating long lasting immunity therefore continues to hold the world’s attention. To date, most vaccines, including current SARS-Cov-2 vaccines, are administered via intramuscular (IM) injection due to feasibility for healthcare workers, speed of injection, and immunological properties ( i.e . muscle resident lymphocytes and antigen presenting cells). However, directing vaccines toward the dermis and draining lymph nodes (LNs) has been of interest for decades due to the high concentration of antigen presentingcells (APCs) including Langerhan cells that reside in the skin (epidermis and dermis) thatare capable of taking up antigen and subsequently trafficking to draining LNs to elicit adaptive immunity. Moreover, the initial lymphatics are present at high concentrations just below the stratum corneum and provide direct access to draining LNs due to their high permeability and uni-directional flow towards draining LNs. Delivering vaccines directly to LNs provides a promising opportunity for improving vaccine efficacy as 1) lymphocytes reside in LNs at high concentrations, 2) are home to unique and strategically positions APCs that present incoming antigen to T and B cells rapidly to induce immunity, and 3) memory T and B cells reside in LNs during their lifespans. However, although intra-dermal (ID) injections for vaccines offer an approach and have been of interest for decades (Hickling et ah, 2011), prior methods typically require a skilled healthcare worker to successfully administer drug in the dermal layer without injecting into the subcutaneous layer.
[0013] While vaccine efforts have greatly hampered the spread of coronavirus infection and/or disease, questions about their durability and protection against current and emerging VoCs remain. Thus, there is an urgent need to improve vaccine durability and efficacy to emerging VoCs, while balancing costs, stability, and manufacturing speed to scale up for world-wide vaccination efforts.
[0014] Furthermore, despite appealing features associated with mRNA vaccines, in vivo delivery of mRNA remains challenging. In particular, the delivery of mRNA to cells is made difficult by the relative instability and low cell permeability of such species. Thus, there exists a need to develop methods and compositions to facilitate the delivery of therapeutic and/or prophylactics such as mRNAs to cells. Furthermore, a need also exists for devices and methods for effectively administering agents, such as drug substances, vaccines, and formulations comprising the same, into the lymphatic system, such as by directing such compositions toward the dermis and/or to draining LNs, and in a manner that does not require any drug modifications (other than the drug must be solubilized in a liquid). Such devices and methods may advantageously be used with a variety of different drug modalities, including mRNA-based therapeutics for vaccinations. There also exists a need to develop vaccine administration routes, coronavirus vaccine administration routes, that more desirable and/or efficacious than IM or other conventional injections routes.
Summary
[0015] Provided herein are, inter alia , nucleic acid molecules and compositions, pharmaceutical compositions, and vaccines comprising such nucleic acid molecules, for preventing or treating coronavirus infections and diseases associated therewith. In some embodiments, the , nucleic acid molecules and compositions, pharmaceutical compositions, and vaccines comprising such nucleic acid molecules, are useful for the prevention and treatment of infections caused by SARS-Cov-2 variants, and diseases associated therewith. In some embodiments, infection of subjects by a SARS-CoV-2 variant, are provided that employ one or more mRNA-comprising compositions, pharmaceutical compositions, and/or vaccines. In some embodiments, infection of subjects by a SARS-CoV-2 variant, are provided that employ mRNA-comprising compositions, pharmaceutical compositions, and/or vaccines, wherein the one or more mRNA species encode mutant forms of an SARS-CoV-2 variant RBD and/or spike protein, wherein such mutant forms are resistant to furin cleavage and or are stability-enhanced.
[0016] The mRNAs provided in vaccine compositions herein encode coronavirus receptor binding domain (RBD) from one or more coronavirus variants, and/or a protein comprising such one or more coronavirus RBDs. In some embodiments, vaccine compositions herein encode a spike (S) protein of a coronavirus comprising one or more RBDs from one or more coronavirus variant. In some embodiments such vaccine compositions herein encode one or more spike proteins including a QQAQ mutation at the furin cleavage site at the border of the SI and S2 domains. In some embodiments such vaccine compositions herein encode one or more spike proteins including at least one spike-stabilizing mutation, such as a stabilizing PP mutation. In some embodiments such vaccine compositions herein encode one or more spike proteins including a QQAQ mutation at the furin cleavage site at the border of the SI and S2 domains and at least one spike-stabilizing mutation, such as a stabilizing PP mutation. An S protein encoded by a nucleic acid molecule as provided herein can be a variant S protein into which the QQAQ mutation has been introduced. Nonlimiting examples of such variants include the Washington/Wuhan-Hu-1 variant, the alpha variant S protein, the beta variant S protein, the delta variant S protein, the gamma variant S protein, and the kappa variant S protein, the omicron variant SI protein, other variants that include mutations (substitutions, insertions and/or deletions) that correspond to two or more such variants, and combinations of the above. A vaccine composition can include two or more mRNA molecules, e.g., RNA molecules encoding different S protein variants having the QQAQ mutation, as well as, optionally, an mRNA encoding the WA isolate S protein with the QQAQ mutation. When delivered to a subject, the mRNA, which may be complexed with or associated with a delivery carrier, is taken up by the subject’s cells, leading to expression of the one or more S protein transcripts by the host cells. As illustrated by the Examples herein, host cells transfected with an mRNA composition can display the encoded S proteins on the cell surface, allowing for detection by cells of the host immune system and development of a humoral and/or cellular immune response.
[0017] Provided herein in a first aspect is a nucleic acid molecule that encodes a variant S protein having the QQAQ furin site mutation. The variant S protein can be an S protein of any SARS-CoV-2 variant, including a variant that has arisen naturally in a population or selected for in laboratory experiments. Further the variant S protein can be an engineered variant based on modeling, prediction, and or screens or assays. In some embodiments, the variant S protein includes at least one spike-stabilizing mutation, for example, the PP mutation at the amino acids corresponding to amino acid positions 986 and 987 of the WA1/2020 S protein.
[0018] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein, wherein the nucleic acid sequence comprises at least one RBD-encoding sequence of a coronavirus spike protein. In certain embodiments such nucleic acids comprise at least one RBD-encoding sequence is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an RBD-encoding nucleic acid sequence from a SARS-CoV-2 virus selected the group consisting of: a) a SARS-CoV-2 Wuhan/Washington variant; b) a SARS-CoV-2 Alpha variant; c) a SARS-CoV-2 Beta variant; d) a SARS-CoV-2 Gamma variant; e) a SARS-CoV-2 Delta variant; f) a SARS-CoV- 2 Delta Plus variant; g) a SARS-CoV-2 Kappa variant; h) a SARS-CoV-2 Lambda variant; i) a SARS-CoV-2 Omicron variant; j) a SARS-CoV-2 Zeta variant; k) a SARS-CoV-2 Epsilon variant; l) a SARS-CoV-2 Omicron variant; m) a SARS-CoV-2 Omicron Plus variant; and n) combinations of a) – m). [0019] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein comprising at least one RBD-encoding sequence encoding an RBD amino acid sequence comprising one or more or  the following mutations: D614G; Δ69/70-Δ144-N501Y-A570D-D614G-P681H-T716I- S982A-D1118H; D80A-D215G-Δ242/244-K417N-E484K-N501Y-D614G-A701V; D614G, S13I, W152C, L452R; G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, H1101D; T19R, (G142D), Δ156-157, R158G, L452R, T478K, D614G, P681R, D950N; T19R, (G142D), Δ156-157, R158G, K417N, L452R, T478K, D614G, P681R, D950N; L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I; G75V, T76I, Δ246-252, L452Q, F490S, D614G, T859N; E484Q, F565L, D614G, V1176F; L5F, T95I, D253G, E484K, D614G, A701V; L5F, T95I, D253G, S477N, D614G, A701V; T95I, ΔY144, E484K, D614G, P681H, D796H; Δ69/70, D614G, N501Y; D614G, K417N, E484K, N501Y; L452R, E484Q, P681R; D614G, L452R, E484K; D614G, L452R; Δ69/70; D614G-K378Y;  D614G-E406W; D614G-K417E; D614G-N439K; D614G-N440D; D614G-K444Q; D614G- V445A; D614G-G446V; D614G-Y453F; D614G-L455F; D614G-G476S; D614G-S477N; D614G, T478K; D614G-E484K; D614G-E484Q; D614G-F486I; D614G-F486V; D614G- N487R; D614G-N487Y; D614G-Y489H; D614G-F490S; D614G-Q493K; D614G-Q493R; D614G-S494P; D614G-N501Y; D614G-Q677H; and D614G-Q677P.   [0020] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a SARS-Cov-2 variant spike protein comprising one or more or the following mutations: D614G; Δ69/70-Δ144-N501Y-A570D- D614G-P681H-T716I-S982A-D1118H; D80A-D215G-Δ242/244-K417N-E484K-N501Y- D614G-A701V; D614G, S13I, W152C, L452R; G142D, E154K, L452R, E484Q, D614G,  P681R, Q1071H, H1101D; T19R, (G142D), Δ156-157, R158G, L452R, T478K, D614G, P681R, D950N; T19R, (G142D), Δ156-157, R158G, K417N, L452R, T478K, D614G, P681R, D950N; L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G,     H655Y, T 10271; G75V, T76I, D246-252, L452Q, F490S, D614G, T859N; E484Q, F565L, D614G, V1176F; L5F, T95I, D253G, E484K, D614G, A701V; L5F, T95I, D253G, S477N, D614G, A701V; T95I, DU144, E484K, D614G, P681H, D796H; D69/70, D614G, N501Y; D614G, K417N, E484K, N501Y; L452R, E484Q, P681R; D614G, L452R, E484K; D614G, L452R; D69/70; D614G-K378Y; D614G-E406W; D614G-K417E; D614G-N439K; D614G- N440D; D614G-K444Q; D614G-V445A; D614G-G446V; D614G-Y453F; D614G-L455F; D614G-G476S; D614G-S477N; D614G, T478K; D614G-E484K; D614G-E484Q; D614G- F486I; D614G-F486V; D614G-N487R; D614G-N487Y; D614G-Y489H; D614G-F490S; D614G-Q493K; D614G-Q493R; D614G-S494P; D614G-N501Y; D614G-Q677H; and D614G-Q677P.
[0021] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a SARS-Cov-2 variant spike protein viral spike protein wherein at least one RBD-encoding sequence is derived from, or otherwise corresponds to, one or more SARS-CoV-2 virus spike-encoding nucleic acid sequences selected from the group consisting of: a) a SARS-CoV-2 Wuhan/Washington variant; b) a SARS-CoV-2 Alpha variant; c) a SARS-CoV-2 Beta variant; d) a SARS-CoV-2 Gamma variant; e) a SARS-CoV-2 Delta variant; f) a SARS-CoV-2 Delta Plus variant; g) a SARS- CoV-2 Kappa variant; h) a SARS-CoV-2 Lambda variant; i) a SARS-CoV-2 Omicron variant; j) a SARS-CoV-2 Zeta variant; k) a SARS-CoV-2 Epsilon variant; 1) a SARS-CoV-2 Omicron variant; m) a SARS-CoV-2 Omicron Plus variant; and n) combinations of a) - m). [0022] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein wherein portion of the spike protein comprises at least one RBD-encoding sequence comprises an RBD-encoding sequence present in one or more of SEQ ID Nos: 1-12 and 15-19.
[0023] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding at least a portion of a viral spike protein wherein portion of the spike protein comprises at least two, at least three, at least four, or at least five RBD-encoding sequences selected from the group consisting of: a SARS-CoV-2 Wuhan/Washington variant; a SARS-CoV-2 Alpha variant; a SARS-CoV-2 Beta variant; a SARS-CoV-2 Gamma variant; a SARS-CoV-2 Delta variant; a SARS-CoV-2 Delta Plus variant; a SARS-CoV-2 Kappa variant; a SARS-CoV-2 Lambda variant; a SARS-CoV-2 Omicron variant; a SARS-CoV-2 Zeta variant; a SARS-CoV-2 Epsilon variant; a SARS-CoV-2 Omicron variant; a SARS- CoV-2 Omicron Plus variant; and n) combinations of a) - m).
[0024] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding a chimeric RBD or a chimeric spike protein.
[0025] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid comprises: an RBD from a Delta variant and an RBD from an Omicron variant in either order; an RBD from a Beta variant and an RBD from an Omicron variant in either order; or an RBD from a Delta variant, an RBD from a Beta variant, and an RBD from an Omicron variant in any order.
[0026] In certain embodiments, provided are nucleic acid molecules comprising an RBD or a spike protein comprising an RBD, which is encoded by a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to a spike protein encoding sequence from a SARS-CoV-2 virus selected the group consisting of: a SARS-CoV-2 Wuhan/Washington variant; a SARS-CoV-2 Alpha variant; a SARS-CoV-2 Beta variant; a SARS-CoV-2 Gamma variant; a SARS-CoV-2 Delta variant; a SARS-CoV-2 Delta Plus variant; a SARS-CoV-2 Kappa variant; a SARS-CoV-2 Lambda variant; a SARS-CoV-2 Omicron variant; a SARS-CoV-2 Zeta variant; a SARS-CoV-2 Epsilon variant; a SARS- CoV-2 Omicron variant; a SARS-CoV-2 Omicron Plus variant; and combinations of a) - m). [0027] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding a chimeric spike protein as present in SEQ ID NOS: 4, 6, and 7. [0028] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a furin site mutation.
[0029] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a furin site mutation sequence as set forth in SEQ ID NO: 13.
[0030] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a stabilizing mutation.
[0031] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a PP spike-stabilizing mutation. [0032] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a PP spike-stabilizing mutation as set forth in SEQ ID NO: 14.
[0033] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule further comprises nucleic acid sequence encoding a furin site mutation and a PP spike-stabilizing mutation.
[0034] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule further comprises nucleic acid sequence encoding a furin site mutation as set forth in SEQ ID NO: 13 and a PP spike-stabilizing mutation as set forth in SEQ ID NO: 14. [0035] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
[0036] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises wherein the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-7.
[0037] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule consists of a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
[0038] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule consists of a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-7.
[0039] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule encodes a RBD or a spike protein that comprises an amino acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20- 32.
[0040] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule encodes a RBD or a spike protein that consists of an amino acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20- 32.
[0041] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a DNA sequence.
[0042] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises an RNA sequence.
[0043] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD and further comprising a promoter operably linked to the nucleic acid sequence.
[0044] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD and further comprising a promoter operably linked to the nucleic acid sequence, wherein the promoter is an SP6, T3, or T7 promoter.
[0045] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule includes at least one modified nucleotide.
[0046] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule includes at least one modified nucleotide, wherein the at least one modified nucleotide is pseudouridine, N1 -methyl-pseudouridine, or 2-thiouridine.
[0047] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a 5’ cap structure.
[0048] In certain embodiments, provided are nucleic acid molecules comprising a nucleic acid sequence encoding an RBD or a spike protein comprising an RBD, wherein the nucleic acid molecule comprises a 3’ polyA sequence.
[0049] In certain embodiments, provided are compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules disclosed herein and throughout.
[0050] In certain embodiments, provided are compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-19.
[0051] In certain embodiments, provided are compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-12.
[0052] In certain embodiments, provided are compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-7.
[0053] In certain embodiments, provided are compositions comprising at least two, at least three, at least four, or at least five nucleic acid molecules that each encode, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS:4-12.
[0054] In certain embodiments, provided are pharmaceutical compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier.
[0055] In certain embodiments, provided are pharmaceutical compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid. [0056] In certain embodiments, provided are pharmaceutical compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of formula (I):
Figure imgf000017_0001
  or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: R1 is H, -OR1A, -YOR1A, -NR1AR1B, -YNR1AR1B, -SR1A, -YSR1A, -(C=O)R1A, -Y(C=O)R1A, -(C=O)OR1A, -Y(C=O)OR1A, -O(C=O)R1A, -YO(C=O)R1A, -  O(C=O)OR1A, -YO(C=O)OR1A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Y is substituted or unsubstituted C0-C12 alkylene or substituted or unsubstituted 0 to 12 membered heteroalkylene; R2 is H, -OR2A, -SR2A, -(C=O)R2A, -(C=O)OR2A, -O(C=O)R2A, -O(C=O)OR2A, -(C=O)NHR2A, -NH(C=O)R2A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R3 is H, -OR3A, -SR3A, -(C=O)R3A, -(C=O)OR3A, -O(C=O)R3A, -O(C=O)OR3A, -(C=O)NHR3A, -NH(C=O)R3A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;     R4 is H, -OR4A, -SR4A, -(C=O)R4A, -(C=O)OR4A, -O(C=O)R4A, -O(C=O)OR4A, -(C=O)NHR4A, -NH(C=O)R4A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R5 is H, -OR5A, -SR5A, -(C=O)R5A, -(C=O)OR5A, -O(C=O)R5A, -O(C=O)OR5A, -(C=O)NHR5A, -NH(C=O)R5A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; B1 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; B2 and B3 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L1 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR101R102)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR101C(=O)‑, ‑C(=O)NR101‑, ‑NR101C(=S)‑,  ‑C(=S)NR101‑, ‑NR101C(=O)NR102‑, ‑NR101C(=S)NR102‑, ‑OC(=O)NR101‑, ‑NR101C(=O)O‑, ‑SC(=O)NR101‑ or ‑NR101C(=O)S‑; L2 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR201R202)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR201C(=O)‑, ‑C(=O)NR201‑, ‑NR201C(=O)NR202‑, ‑NR201C(=S)‑, ‑C(=S)NR201‑, ‑NR201C(=S)NR202‑, ‑OC(=O)NR201‑, ‑NR201C(=O)O‑, ‑SC(=O)NR201‑ or ‑NR201C(=O)S‑; L3 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR301R302)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR301C(=O)‑, ‑C(=O)NR301‑, ‑NR301C(=O)NR302‑, ‑NR301C(=S)‑, ‑C(=S)NR301‑, ‑NR301C(=S)NR302‑, ‑OC(=O)NR301‑, ‑NR301C(=O)O‑, ‑SC(=O)NR301‑ or ‑NR301C(=O)S‑; L4 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR401R402)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR401C(=O)‑, ‑C(=O)NR401‑, ‑NR401C(=O)NR402‑, ‑NR401C(=S)‑, ‑C(=S)NR401‑, ‑NR401C(=S)NR402‑, ‑OC(=O)NR401‑, ‑NR401C(=O)O‑, ‑SC(=O)NR401‑ or ‑NR401C(=O)S‑; L5 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR501R502)sO-  , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR501C(=O)‑, ‑C(=O)NR501‑, ‑NR501C(=O)NR502‑, ‑NR501C(=S)‑, ‑C(=S)NR501‑, ‑NR501C(=S)NR502‑, ‑OC(=O)NR501‑, ‑NR501C(=O)O‑, ‑SC(=O)NR501‑ or ‑NR501C(=O)S‑;     L6 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR601R602)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR601C(=O)‑, ‑C(=O)NR601‑, ‑NR601C(=O)NR602‑, ‑NR601C(=S)‑, ‑C(=S)NR601‑, ‑NR601C(=S)NR602‑, ‑OC(=O)NR601‑, ‑NR601C(=O)O‑, ‑SC(=O)NR601‑ or ‑NR601C(=O)S‑;   L7 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR701R702)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR701C(=O)‑, ‑C(=O)NR701‑, ‑NR701C(=O)NR702‑, ‑NR701C(=S)‑, ‑C(=S)NR701‑, ‑NR701C(=S)NR702‑, ‑OC(=O)NR701‑, ‑NR701C(=O)O‑, ‑SC(=O)NR701‑ or ‑NR701C(=O)S‑; La1 and La2 are each independently
Figure imgf000019_0001
each X is independently O, S, or CH2; W1, W2, W3, W4, W5, and W6 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene; each R1A and R1B is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; each R2A, R3A, R4A, and R5A is independently H, substituted or unsubstituted C1- C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl; each R101, R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; and each s is independently an integer from 1 to 4. In certain embodiments, such lipids are lipids wherein: R1 is H, -OR1A or substituted or unsubstituted heteroalkyl; L1 is a bond, ‑NR101C(=S)‑, ‑C(=S)NR101‑, -O(C=O)-, -(C=O)O-, or ‑O‑; B1 is a bond or a substituted or unsubstituted alkylene;     B2 and B3 are each independently a bond or substituted or unsubstituted alkylene;
L2 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-;
L4 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-;
W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene;
Lal and La2 are each independently
Figure imgf000020_0001
each X is independently O or S;
L3 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-;
L5 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-;
L6 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-;
L7 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-;
R2 is H or substituted or unsubstituted alkyl;
R3 is H or substituted or unsubstituted alkyl;
R4 is H or substituted or unsubstituted alkyl;
R5 is H or substituted or unsubstituted alkyl; each R1A is independently H or substituted or unsubstituted C1-C12 alkyl; and each R101 is independently H or substituted or unsubstituted 2 to 12 membered heteroalkyl. In certain embodiments, such lipids are lipids wherein: R1 is H, -OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl;
L1 is a bond, -NR101C(=S)-, or -C(=S)NR101-;
B1 is a bond or an unsubstituted Ci-Cx alkylene;
B2 and B3 are each independently a bond or substituted or unsubstituted Ci-Cx alkylene;
L2 is a bond, -0(C=0)-, or -(C=0)0-;
L4 is a bond, -0(C=0)-, or -(C=0)0-;
W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene;
Lal and La2 are each independently
Figure imgf000020_0002
each X is independently O or S; L3 is a bond, -O(C=O)-, or -(C=O)O-; L5 is a bond, -O(C=O)-, or -(C=O)O-; L6 is a bond, -O(C=O)-, or -(C=O)O-; L7 is a bond, -O(C=O)-, or -(C=O)O-; R2 is H or substituted or unsubstituted C1-C12 alkyl; R3 is H or substituted or unsubstituted C1-C12 alkyl; R4 is H or substituted or unsubstituted C1-C12 alkyl; R5 is H or substituted or unsubstituted C1-C12 alkyl; and each R101 is independently substituted or unsubstituted 2 to 12 membered heteroalkyl. In certain embodiments, such lipids are lipids wherein: R1 is -OH or methoxy; L1 is a bond; B1 is an unsubstituted C1-C8 alkylene; B2 and B3 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; L2 is a bond; L4 is a bond; W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene;
Figure imgf000021_0001
each X is independently O; L3 is a bond; L5 is a bond; L6 is a bond; L7 is a bond; R2 is H or substituted or unsubstituted C1-C12 alkyl; R3 is H or substituted or unsubstituted C1-C12 alkyl; R4 is H or substituted or unsubstituted C1-C12 alkyl; and R5 is H or substituted or unsubstituted C1-C12 alkyl. In certain embodiments, such lipids are lipids wherein: R1 is substituted or unsubstituted heteroalkyl;     L1 is ‑C(=S)NR101‑, where the carbon atom is connected to the nitrogen atom in formula (I); B1 is a bond; B2 and B3 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; L2 is a bond, -O(C=O)-, or -(C=O)O-; L4 is a bond, -O(C=O)-, or -(C=O)O-; W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; La1 and La2 are each independently each X is independently O;
Figure imgf000022_0002
L3 is a bond; L5 is a bond; L6 is a bond; L7 is a bond; R2 is H or substituted or unsubstituted C1-C12 alkyl; R3 is H or substituted or unsubstituted C1-C12 alkyl; R4 is H or substituted or unsubstituted C1-C12 alkyl; and R5 is H or substituted or unsubstituted C1-C12 alkyl. In certain embodiments, such lipids are selected from the group consisting of: ,
Figure imgf000022_0001
,    
Figure imgf000023_0001
aceutically acceptable salts thereof.
[0057] In certain embodiments, provided are pharmaceutical compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of formula (II):
Figure imgf000023_0002
or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: B4 is W7-La3-W8; W7 and W8 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; La3 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CRa31Ra32)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NRa31C(=O)‑, ‑C(=O)NRa31‑, ‑NRa31C(=O)NRa32‑, ‑NRa31C(=S)‑, ‑C(=S)NRa31‑, ‑NRa31C(=S)NRa32‑, ‑OC(=O)NRa31‑, ‑NRa31C(=O)O‑, ‑SC(=O)NRa31‑ or ‑NRa31C(=O)S‑; R10 and R11 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; B5, B6, and B7 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L8 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR801R802)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR801C(=O)‑, ‑C(=O)NR801‑, ‑NR801C(=O)NR802‑, ‑NR801C(=S)‑, ‑C(=S)NR801‑, ‑NR801C(=S)NR802‑, ‑OC(=O)NR801‑, ‑NR801C(=O)O‑, ‑SC(=O)NR801‑ or ‑NR801C(=O)S‑; L9 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR901R902)sO- , ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR901C(=O)‑, ‑C(=O)NR901‑, ‑NR901C(=O)NR902‑, ‑NR901C(=S)‑, ‑C(=S)NR901‑, ‑NR901C(=S)NR902‑, ‑OC(=O)NR901‑, ‑NR901C(=O)O‑, ‑SC(=O)NR901‑ or ‑NR901C(=O)S‑; L10 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR110R111)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR110C(=O)‑, ‑C(=O)NR110‑, ‑NR110C(=O)NR111‑, ‑NR110C(=S)‑, ‑C(=S)NR110‑, ‑NR110C(=S)NR111‑, ‑OC(=O)NR110‑, ‑NR110C(=O)O‑, ‑SC(=O)NR110‑ or ‑NR110C(=O)S‑; R7, R8, and R9 are each independently H, substituted or unsubstituted C1-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl;     each Ra31 and Ra32 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; each R801, R802, R901, R902, R110, and R111 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; and each s is independently an integer from 1 to 4. In certain embodiments, such lipids are lipids wherein: W7 and W8 are each independently a bond or substituted or unsubstituted alkylene; La3 is a bond; R10 and R11 are each independently H, substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl; B5 is a bond; B6 and B7 are each independently a bond or substituted or unsubstituted alkylene; L8 is a bond; L9 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑; L10 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑; and R7, R8, and R9 are each independently H or substituted or unsubstituted C1-C30 alkyl. In certain embodiments, such lipids are lipids wherein: W7 and W8 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; La3 is a bond; R10 and R11 are each independently substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl; B5 is a bond; B6 and B7 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; L8 is a bond; L9 is -O(C=O)- or -(C=O)O-; L10 -O(C=O)- or -(C=O)O-; and     R7, R8, and R9 are each independently substituted or unsubstituted C1-C20 alkyl. In certain embodiments, such lipids are lipids wherein: W7 and W8 are each independently a bond or substituted or unsubstituted C2-C4 alkylene;
La3 is a bond;
R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl;
B5 is a bond;
B6 and B7 are each independently a bond or substituted or unsubstituted C2-C4 alkylene;
L8 is a bond;
L9 is -0(C=0)- or -(C=0)0-;
L10 -0(C=0)- or -(C=0)0-;
R7 is H or methyl; and
R8, and R9 are each independently substituted or unsubstituted C1-C20 alkyl. In certain embodiments, such lipids are lipids wherein: W7 and W8 are each independently a bond or unsubstituted C2-C4 alkylene;
La3 is a bond;
R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl;
B5 is a bond;
B6 and B7 are each independently a bond or unsubstituted C2-C4 alkylene;
L8 is a bond;
L9 is -0(C=0)- or -(C=0)0-;
L10 is -0(C=0)- or -(C=0)0-;
R7 is H or methyl; and
R8 and R9 are each independently substituted or unsubstituted C1-C20 alkyl. In certain embodiments, such lipids are lipids wherein: W7 and W8 are each independently a bond or unsubstituted C2-C4 alkylene; La3 is a bond;
R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl;
B5, B6, and B7 are each independently a bond;
L8 is a bond;
L9 is a bond;
L10 is a bond;
R7 is H or methyl; and
R8 and R9 are each independently substituted or unsubstituted C1-C30 alkyl. In certain embodiments, such lipids are selected from the group consisting of:
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
and pharmaceutically acceptable salts thereof.
[0058] In certain embodiments, provided are pharmaceutical compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of formula (III): [0059]
Figure imgf000030_0001
[0060] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: [0061] [0062] [0063]
Figure imgf000030_0002
Figure imgf000030_0003
Q is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene,  substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene; V is substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted arylene; B8, B9, B10, and B11 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L12 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR210R211)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR210C(=O)‑, ‑C(=O)NR210‑, ‑NR210C(=O)NR211‑, ‑NR210C(=S)‑, ‑C(=S)NR210‑, ‑NR210C(=S)NR211‑, ‑OC(=O)NR210‑, ‑NR210C(=O)O‑, ‑SC(=O)NR210‑ or ‑NR210C(=O)S‑; L13 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR310R311)sO-, ‑S‑,  ‑C(=O)S‑, ‑SC(=O)‑, ‑NR310C(=O)‑, ‑C(=O)NR310‑, ‑NR310C(=O)NR311‑, ‑NR310C(=S)‑, ‑C(=S)NR310‑, ‑NR310C(=S)NR311‑, ‑OC(=O)NR310‑, ‑NR310C(=O)O‑, ‑SC(=O)NR310‑ or ‑NR310C(=O)S‑; R12 is H, -OR12A, -SR12A, -NR12A, -CN, -(C=O)R12A, -O(C=O)R12A, -(C=O)OR12A, -NR12A(C=O)-R12B, -(C=O)NR12AR12B;     R13 is H, -OR13A, -SR13A, -NR13A, -CN, -(C=O)R13A, -O(C=O)R13A, -(C=O)OR13A, -NR13A(C=O)-R13B, -(C=O)NR13AR13B; R14 and R15 are each independently substituted or unsubstituted C2-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl;   R12A, R12B, R13A, and R13B are each independently H, substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted 2 to 20 membered heteroalkyl; each R210, R211, R310, and R311 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; each n is independently an integer from 0 to 8; and each s is independently an integer from 1 to 4. In certain embodiments such lipids are lipids wherein:
Figure imgf000031_0001
Q is substituted or unsubstituted alkylene; V is substituted or unsubstituted alkylene; B8, B9, B10, and B11 are each independently substituted or unsubstituted alkylene; L12 is -O(C=O)- or -(C=O)O-; L13 is -O(C=O)- or -(C=O)O-; R12 is H, -OR12A, or-NR12A; R13 is H, -OR13A, or-NR13A; R14 and R15 are each independently substituted or unsubstituted C2-C30 alkyl; R12A and R13A are each independently H, substituted or unsubstituted C1-C20 alkyl; and each n is independently an integer from 0 to 8. In certain embodiments such lipids are lipids wherein:    
Figure imgf000032_0003
V is substituted or unsubstituted alkylene; B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C20  alkylene; L12 is -O(C=O)- or -(C=O)O-; L13 is -O(C=O)- or -(C=O)O-; R12 is H or -OR12A; R13 is H or -OR13A; R14 and R15 are each independently substituted or unsubstituted C2-C20 alkyl; R12A and R13A are each independently H, substituted or unsubstituted C1-C8 alkyl; and each n is independently an integer from 0 to 4. In certain embodiments, such lipids are lipids wherein: is
Figure imgf000032_0001
or  
Figure imgf000032_0002
V is unsubstituted alkylene; B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C8 alkylene; L12 is -O(C=O)- or -(C=O)O-; L13 is -O(C=O)- or -(C=O)O-; R12 is -OH, methoxy, or ethoxy; R13 is -OH, methoxy, or ethoxy; R14 and R15 are each independently substituted or unsubstituted C2-C20 alkyl; and each n is independently an integer from 0 to 4. In certain embodiments, such lipids are selected from the group consisting of:    
Figure imgf000033_0003
and pharmaceutically acceptable salts thereof.
[0064] In certain embodiments, provided are pharmaceutical compositions comprising a nucleic acid molecule or a composition disclosed herein and throughout, and further comprising a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier comprises a lipid, wherein the lipid comprises a cationic lipid of formula (IV):
Figure imgf000033_0001
[0066] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein:
[0067] B12 is -W7-La3-W8-;
[0068] W7 and W8 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene;
[0069] La3 is a bond,
Figure imgf000033_0002
[0070] W9 and W10 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted 2 to 12 membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any combination thereof; [0071] L14 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR410R411)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR410C(=O)‑, ‑C(=O)NR410‑, ‑NR410C(=O)NR411‑, -NR410C(=S)-, -C(=S)NR410‑, ‑NR410C(=S)NR411‑, ‑OC(=O)NR410‑, ‑NR410C(=O)O‑, ‑SC(=O)NR410‑ or ‑NR410C(=O)S‑; [0072] L15 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR510R511)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR510C(=O)‑, ‑C(=O)NR510‑, ‑NR510C(=O)NR511‑, -NR510C(=S)-, -C(=S)NR510‑, ‑NR510C(=S)NR511‑, ‑OC(=O)NR510‑, ‑NR510C(=O)O‑, ‑SC(=O)NR510‑ or ‑NR510C(=O)S‑; [0073] R16 and R17 are each independently [0074]
Figure imgf000034_0001
fragment of cationic lipid of formula (I), [0075]
Figure imgf000034_0002
a     fragment of cationic lipid of formula (III), or a fragment of cationic lipid of formula (III);
Figure imgf000035_0004
[0077] each R410, R411, R510, and R511 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; [0078] each m is independently an integer from 0 to 8; and each s is independently an integer from 1 to 4. In certain embodiments, such lipids are lipids wherein: La3 is a bond,
Figure imgf000035_0001
W7 and W8 are each independently a bond or substituted or unsubstituted C1-C12  alkylene; L14 is -O(C=O)-, -(C=O)O-, ‑C(=O)‑, ‑NR410C(=O)‑, ‑C(=O)NR410‑, - NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑; L15 is -O(C=O)-, -(C=O)O-, ‑C(=O)‑, ‑NR510C(=O)‑, ‑C(=O)NR510‑, -  NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; R16 and R17 are each independently
Figure imgf000035_0002
cationic lipid of formula (II); and each R410 and R510 is independently H or substituted or unsubstituted C1-C12 alkyl. In certain embodiments, such lipids are lipids wherein: La3 is a bond, -S-
Figure imgf000035_0003
W7 and W8 are each independently a bond or unsubstituted C1-C12 alkylene;     L14 is -O(C=O)-, -(C=O)O-, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑; L15 is -O(C=O)-, -(C=O)O-, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; R16 and R17 are each independently
Figure imgf000036_0001
cationic lipid of formula (II); and each R410 and R510 is independently H or substituted or unsubstituted C1-C12  alkyl. In certain embodiments such lipids are lipids wherein: La3 is a bond, -S-S-
Figure imgf000036_0002
W7 and W8 are each independently a bond or unsubstituted C1-C8 alkylene; L14 is -O(C=O)-, -(C=O)O-, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑; L15 is -O(C=O)-, -(C=O)O-, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or unsubstituted C1-C8 alkylene; R16 and R17 are each independently
Figure imgf000036_0003
a fragment of cationic lipid of formula (II); and each R410 and R510 is independently H or unsubstituted C1-C8 alkyl. In certain embodiments such lipids are lipids wherein: La3 is a bond,
Figure imgf000036_0004
W7 and W8 are each independently a bond or unsubstituted C1-C8 alkylene;     L14 is -O(C=O)-, -(C=O)O-, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑; L15 is -O(C=O)-, -(C=O)O-, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or unsubstituted C1-C8 alkylene; R16 and R17 are each independently
Figure imgf000037_0001
each R410 and R510 is independently H or methyl. In certain embodiments, such lipids are selected from the group consisting of:
Figure imgf000037_0002
,    
Figure imgf000038_0001
Figure imgf000039_0001
and pharmaceutically acceptable salts thereof [0079] In certain embodiments, provided are pharmaceutical compositions disclosed herein and throughout that further comprise lipid nanoparticles. [0080] In certain embodiments, provided are methods of preventing or treating coronavirus infection or disease, the methods comprising administering: a nucleic acid molecule; a composition of; and/or or a pharmaceutical composition; as disclosed herein and throughout; to a subject infected with, or at risk of infection, of suspected of having been infected, with a coronavirus. In certain embodiments, such methods comprise administration via oral, nasal, intrapulmonary, intracavitary, by intra-arterial or intravenous infusion, or by injection. In certain embodiments, such methods comprise administration via injection. In certain embodiments, such methods comprise administration via subcutaneous, intramuscular, transdermal, intradermal, subdermal, epidermal, or lymphatic delivery or injection. In certain embodiments such the nucleic acid molecule, the composition, or the pharmaceutical composition is administered by subdermal injection or delivery. In certain embodiments the nucleic acid molecule, the composition, or the pharmaceutical composition administration is administered or delivered into a lymphatic system. In certain embodiments the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch. In certain embodiments the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch, wherein the patch comprises a polymer. In certain embodiments the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch, wherein the patch comprises an absorbable polymer. In certain embodiments, such methods comprise administering two or more doses of a nucleic acid molecule, a composition of any of and/or or a pharmaceutical composition as disclosed herein and throughout to the subject. In certain embodiments, such methods comprise administering two or more doses of a nucleic acid molecule, a composition of any of and/or or a pharmaceutical composition as disclosed herein and throughout to the subject, such methods comprising: placing a medical device comprising a plurality of microneedles on the skin of the subject having lymphatic vasculature, wherein the medical device contacts a layer of epidermis with reversible permeability enhancers comprising a chemical, physical or electrical permeability enhancer that induces a-reversible increase in permeability of one or more barrier cells of the epidermis to the nucleic acid molecule, the composition, or the pharmaceutical composition. In certain embodiments, such methods comprise administering two or more doses of a nucleic acid molecule, a composition of any of and/or or a pharmaceutical composition as disclosed herein and throughout to the subject, such methods comprising: placing a first medical device comprising a plurality of microneedles on the skin of the subject at a first location proximate to a first position under the skin of the subject, wherein the first position is proximate to lymph vessels and/or lymph capillaries that drain into the right lymphatic duct, and wherein the microneedles of the first medical device have a surface comprising nanotopography; placing a second medical device comprising a plurality of microneedles on the skin of the subject at a second location proximate to a second position under the skin of the subject, optionally wherein the first and second medical devices are the same device, wherein the second position is proximate to lymph vessels and/or lymph capillaries that drain into the thoracic duct, and wherein the microneedles of the second medical device have a surface comprising nanotopography; inserting the plurality of microneedles of the first medical device into the subject to a depth whereby at least the epidermis is penetrated and an end of at least one of the microneedles is proximate to the first position; inserting the plurality of microneedles of the second medical device into the subject to a depth whereby at least the epidermis is penetrated and an end of at least one of the microneedles is proximate to the second position; and administering via the microneedles of the first medical device a first dose of the nucleic acid molecule, the composition, or the pharmaceutical composition into the first position; and administering via the microneedles of the second medical device a second dose of the nucleic acid molecule, the composition, or the pharmaceutical composition into a second position. In certain embodiments, such methods comprises preventing or treating a subject infected, suspected of having been infected, or at risk of being infected, bya SARS-Cov-2 variant. [0081] In some embodiments, an variant RBD or a variant spike protein has an amino acid sequence that is at least 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.5%, 99.5%, 99.5%, 99.5%, identical to an amino acid sequence selected from the group consisting of those set forth in SEQ ID Nos 20-32, inclusive. In some embodiments, a nucleic acid molecule comprises a nucleic acid sequence that encodes a protein having an amino acid sequence that is at least 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.5%, 99.5%, 99.5%, 99.5%, identity to an amino acid sequence selected from the group consisting of those set forth in SEQ ID Nos 20-32, inclusive.
[0082] In some embodiments, a pharmaceutical composition comprising at least one RNA molecule as disclosed herein and a pharmaceutically acceptable carrier is provided. A pharmaceutically-acceptable carrier can be, for example, a buffer or aqueous solution that can include any of the aforementioned ingredients such as salts, buffering agents, chelators, etc.
A pharmaceutically-acceptable carrier can also be a delivery carrier that promotes delivery of the mRNA into a cell. Nonlimiting examples of delivery carriers include lipids, lipid formulations, lipid nanoparticles (LNPs), polymers, and peptides.
[0083] In some embodiments, a pharmaceutical includes two or more molecules RNA molecules encoding different S proteins, for example, encoding S proteins from different SARS-CoV-2 variants or isolates. In some embodiments, a pharmaceutical includes three or more molecules RNA molecules encoding different S proteins, for example, encoding S proteins from three different SARS-CoV-2 variants or isolates. In some embodiments, the pharmaceutical composition can include mRNAs encoding an isolate such as the Washington/Wuhan-Hu-1 isolate and two different variants, one or both of which may be selected from the alpha, beta, gamma, delta, kappa, and omicron variants. In some embodiments, the pharmaceutical composition can include mRNAs encoding an isolate such as the Washington/Wuhan-Hu-1 isolate and three different variants, one or both of which may be selected from the alpha, beta, gamma, delta, kappa, and omicron variants. In some embodiments, the pharmaceutical composition can include mRNAs encoding an isolate such as the Washington/Wuhan-Hu-1 isolate and four different variants, one or both of which may be selected from the alpha, beta, gamma, delta, kappa, and omicron variants.
[0084] A pharmaceutical mRNA composition as provided herein can include at least one delivery carrier, which may be, as nonlimiting examples, a lipid, a lipid formulation, a lipid- based nanoparticle, a polymer, a polymer formulation, or a peptide.
[0085] In a further aspect, provide herein is a method of preventing SARS-CoV-2 infection, comprising administering an mRNA pharmaceutical composition such as any disclosed herein to a subject at risk of infection with SARS-CoV-2. Delivery can be by oral, nasal, intrapulmonary, or intracavitary administration, by intra-arterial or intravenous infusion, or by injection.
[0086] For example, delivery can be by intramuscular, transdermal, intradermal, or subcutaneous injection.
[0087] In some embodiments the method comprises administering two or more doses of an mRNA pharmaceutical composition such as any disclosed herein to a subject at risk of infection with SARS-CoV-2. The two or more doses can comprise different RNA molecules.
Brief Description of the Figures
[0088] Figure 1 (top panels) depicts flow cytometry analysis of HEK293 cells one and three days after transfection with an RNA that encodes the “wild type” Washington (Wuhan) SARS-CoV-2 S protein and (bottom panels) HEK293 cells one and three days after transfection with an RNA that encodes the Washington (Wuhan) SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
[0089] Figure 2 depicts flow cytometry analysis of HEK293 cells one day after transfection with an RNA that encodes the “wild type” alpha variant SARS-CoV-2 S protein and an RNA that encodes the alpha variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
[0090] Figure 3 (top panels) depicts flow cytometry analysis of HEK293 cells two and three days after transfection with an RNA that encodes the “wild type” beta variant SARS- CoV-2 S protein and (bottom panels) HEK293 cells two and three days after transfection with an RNA that encodes the beta variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
[0091] Figure 4 (top panels) depicts flow cytometry analysis of HEK293 cells two and three days after transfection with an RNA that encodes the “wild type” gamma variant SARS- CoV-2 S protein and (bottom panels) HEK293 cells two and three days after transfection with an RNA that encodes the gamma variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
[0092] Figure 5 (top panels) depicts flow cytometry analysis of HEK293 cells one, two, and three days after transfection with an RNA that encodes the “wild type” delta variant SARS-CoV-2 S protein and (bottom panels) HEK293 cells one, two, and three days after transfection with an RNA that encodes the delta variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
[0093] Figure 6 (top panels) depicts flow cytometry analysis of HEK293 cells two and three days after transfection with an RNA that encodes the “wild type” kappa variant SARS- CoV-2 S protein and (bottom panels) HEK293 cells two and three days after transfection with an RNA that encodes the kappa variant SARS-CoV-2 S protein with the QQAQ (“Furin”) mutation.
[0094] Figures 7A-7J show results of stability studies comparing lyophilized and liquid SARS-CoA-2 variant mRNA formulations. Figure 7A shows a scatter plot of the control HEK293 cells lacking spike protein expression (no mRNA). Figure 7B shows a scatter plot of HEK293 cells transfected with Washington/Wuhan-Hu-1 variant of mRNA encoding the SARS-CoV-2 spike protein. Figure 7C shows a scatter plot of HEK293 cells, transfected with U.K. (Alpha) variant of mRNA encoding the SARS-CoV-2 spike protein. Figure 7D shows a scatter plot of HEK293 cells, transfected with South Africa (Beta) variant of mRNA encoding the SARS-CoV-2 spike protein. Figure 7E shows a scatter plot of HEK293 cells, transfected with Brazil (Gamma) variant of mRNA encoding the SARS-CoV-2 spike protein. Figure 7F shows a scatter plot of the control HEK293 cells lacking spike protein expression (no mRNA). Figure 7G shows a scatter plot of HEK293 cells transfected with mRNA encoding the SARS-CoV-2 spike protein, stored at -80°C for 11 days. Figure 7H shows a scatter plot of HEK293 cells transfected with mRNA encoding the SARS-CoV-2 spike protein, stored at room temperature for 3 days following lyophilization. Figure 71 shows a scatter plot of HEK293 cells transfected with mRNA encoding the SARS-CoV-2 spike protein, stored at room temperature for 11 days following lyophilization. Figure 7J shows agarose gel electrophoresis of mRNA, encoding the SARS-CoV-2 spike protein, after lyophilization and 11-day storage under various conditions.
[0095] Figure 8A depicts a schematic of an exemplary STI mRNA vaccine that is optimized for highly efficient translation.
[0096] Figure 8B depicts flow cytometry results using primary dendritic cells that were transfected with various mRNAs, stained with anti-Spike antibody STI-2020, and evaluated by flow cytometry 24 post-transfection.
[0097] Figure 9A provides an image of an exemplary MuVaxx device connected to 1 mL syringe.
[0098] Figure 9B Provides an image of a C57B16 mouse 5 minutes after injection of ICG using the MuVaxx device depicted in FIG. 6A.
[0099] Figure 9C depicts anti-OVA titers determined by running ELIZA assays on serum collected on days 13 and 34 following either intramuscular injection (IM) or MuZaxx- mediated lymphatic administration (MuVaxx) of C57B16 mice with 10 μg of OVA (on day zero) and 8 μg CpG (on day 14). Anti-OVA IgG was quantified via serial dilutions run by ELISA assays. The data represents n=8-9 mice per group.
[00100] Figure 9D provides percentage of CD8 T cells producing the indicated cytokines. Whole blood was collected and stimulated with SIINFEKL peptide followed by ICS to measure IFNy and TNFa in the CD8 T cell compartment. The data represents n=8-9 mice per group. Cytokine statistics represents difference between IFNy+ groups for panel D.
[00101] Figure 10A illustrates a mouse IM or MuVaxx rRNA vaccination treatment schedule.
[00102] Figure 10B provides anti-RBD IgM titers determined from serum collected on day 7 from treated mice. Data represents n=5 mice per group. [00103] Figure IOC provides anti-RBD IgG titers determined from serum collected on days 7, 21, and 49 from treated mice. Data represents n=5 mice per group. Statistics represent significance against STI 1 μg IM vs all other groups at that day.
[00104] Figure 10D provides anti-Sl IgG titers determined from serum collected on days 7, 21, and 49 from treated mice. Data represents n=5 mice per group. Statistics represent significance against STI 1 μg IM vs all other groups at that day.
[00105] Figure 10E provides Anti-RBD IgG area under the curve (AUC) plots comparing anti-IgG titers obtained using IM vs. MuVaxx treatment at a 1 μg dose. Data represents n=5 mice per group.
[00106] Figure 10F provides Anti-Sl IgG area under the curve (AUC) plots comparing anti-IgG titers obtained using IM vs. MuVaxx treatment at a 1 μg dose. Data represents n=5 mice per group.
[00107] Figure 11A illustrates a mouse IM or MuVaxx rRNA vaccination treatment schedule.
[00108] Figure 11B depicts the ratio of CD4 Thl (CD4 IFNy+) to Th2 (CD4 IL-4+) phenotypes from whole blood obtained on day 41 of the treatment schedule from treated mice and then incubated with associated peptides (Miltenyi Biotec Peptivator) overnight followed by ICS. Data represents n=5 mice per group.
[00109] Figure 11C depicts end point titers of SI specific IgGl and IgG2c determined by ELISA from whole blood obtained on day 49 from treated mice. Numbers listed above each group represent average IgG2c : IgGl ratio. Data represents n=5 mice per group.
[00110] Figure 12A illustrates a mouse IM or MuVaxx rRNA vaccination treatment schedule in which 6 days following booster shot, intracellular cytokine staining was performed in the presence of spike associated peptides (Miltenyi Biotec Peptivator).
[00111] Figure 12B provides representative flow cytometry plots of IFNy and TNFa production from CD8 T cells.
[00112] Figure 12C provides quantification of results provided in FIG. 9B. Statistical analysis was done using ANOVA with Tukey’s test. **P <0.01 and represents the difference between other IFNy+ groups. Data represents n=5 mice per group.
[00113] Figure 13A depicts plaque reduction neutralization test (PRNT) dilution curves against wild-type virus from mouse serum on day post-prime. Data represents n=5 mice per group. [00114] Figure 13B depicts PRNT dilution curves against the Beta variant from mouse serum on day post-prime. Data represents n=5 mice per group. Statistics represent difference between day 49 STI-7264 1 μg IM group and all other day 49 groups.
[00115] Figure 13C depicts Anti-Si IgG concentrations following ex vivo stimulation of draining lymph nodes for 72 hours after 15 weeks post booster shot. Data represents n=5 mice per group.
[00116] Figure 14 depicts anti-RBD IgG titers from serum collected on day 14 and 21 post prime dose obtained from mice injected with indicated doses and formulations using an IM or MuVaxx administration. Data represents one experiment (n=4-5 mice per group). [00117] Figure 15A depicts PRNT50 values and anti-Sl IgG produced by long-lived B cells in lungs of mice used to generate the data depicted in Figure 13A.
[00118] Figure 15B depicts anti-Sl IgG concentrations following ex vivo stimulation of lungs for 72 hours after 15 weeks post booster shot. Data represents one experiment (n=5 mice per group).
[00119] Figures 16A-16B show antibody responses upon immunization with VOC- based vaccines in vivo. Figure 16A shows Day 14 sera post booster was evaluated for antibody binding to recombinant Spike from designated VOCs by ELISA. Figure 16B shows Day 14 sera post booster was evaluated for nAb responses against designated live virus by 50% plaque reduction neutralization test (PRNT). Plots: mean±SEM; n=6. EPT: endpoint titer.
[00120] Figures 17A-17C show immunization results with Furin mutant vaccine produces enhanced protection in vivo. Figure 17A provides a schematic illustration of the design of the in vivo study. Figure 17B shows the lung viral titer measured in mice challenged with live SARS-CoV-2 VOCs after full immunization. Figure 17C shows the body weight measured in mice challenged with live SARS-CoV-2 VOCs after full immunization. Plots: mean±SEM; n=6. ****p<0.0001 one-way ANOVA.
[00121] Figures 18A-18D show immunization with Omicron-specific vaccine produces robust protection against Omicron challenge. Figure 18A shows Day 14 sera post 2nd vaccine shot were evaluated for nAb responses against Omicron from designated VOC vaccine by pseudovirus assay. Figure 18B shows Day 14 sera post 2nd Omicron-specific vaccine shot evaluated for antibody binding by ELISA. Figure 18C shows Day 14 sera post 2nd Omicron- specific vaccine shot evaluated for nAb responses by PRNT. Figure 18D shows the design of the in vivo study for Omicron-specific vaccine booster {left panel) and the viral titer in the lung from mice challenged with live Omicron virus {right panel). Bars: mean±SEM; n=6; ***p<0.001 by Student’s t-test; plots: mean±SEM; n=6. ****p<0.0001 by one-way ANOVA. [00122] Figures 19A-19B show Delta RBD-Omicron immunization induces the potent and broadly spectrum neutralization activity against SARS-COV-2 variants. Figure 19A, upper portion , provides a schematic drawing of the design of the chimeric Delta RBD- Omicron mRNA. Figure 19A, lower portion shows results with animals that were immunized with the mRNA twice. The Day 14 sera post boost were evaluated for binding antibodies specific to recombinant S proteins from designated VOC vaccines by ELISA. Figure 19B shows the indicated measurement of Day 14 sera post boost when evaluated for nAb responses by pseudovirus assay. Bars: mean±SEM; n=6; **p<0.01 by Student’s t-test plots: mean±SEM; n=6. EPT: endpoint titer.
[00123] Figures 20A-20F show expression and cleavage of Furin-mutated mRNA. Figure 20A shows the Design of the furin mutant mRNA. Figure 20B shows flow cytometry results, indicating an increase in the surface expression of spike protein from all variants containing a furin cleavage mutation. Figure 20C shows full-length S protein is the dominant species in transfected 293T cells after the removal (mutation) of furin cleavage site. Figure 20D shows the mutation at the furin cleavage site also lowered the level of free SI in the conditioned medium of transfected 293T cells. Figure 20E shows furin-cleavage mutant mRNA elicits higher neutralization antibody titers than wild type mRNA revealed by ELISA on day 14 post boost. Figure 20F shows furin-cleavage mutant mRNA elicits higher neutralization antibody titers than wild type mRNA revealed by and PRNT on day 14 post boost . TM: transmembrane domain. FL: full-length spike. Plots: mean±SEM; n=6.
[00124] Figure 21 shows expression of Omicron and Delta RBD- Omicron mRNA. The flow cytometry showed the surface expression of spike protein in transfected 293T cells stained with recombinant human ACE2 receptor.
[00125] Figure 22 shows results obtained when testing the indicated cholesterol and DSPC molar ratios, either as fresh formulations or as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
[00126] Figure 23 shows results obtained when testing the indicated N/P ratios, PEG molar ratios, and lipid concentrations, either as fresh formulations or as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
[00127] Figure 24 shows results obtained when testing the indicated N/P ratios and PEG molar ratios, and lipid concentrations as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
[00128] Figure 25 shows results obtained in an additional experiment testing the indicated N/P ratios and PEG molar ratios as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot, as indicated.
[00129] Figure 26 shows results obtained in an additional experiment testing the indicated N/P ratios, as well as the presence or absence of HP-b-CD, with samples that were either freeze-thawed or lyophilized and then reconstituted. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and at day 7, 14, 21, and 28 after booster shot, as indicated.
[00130] Figure 27 shows results obtained when testing the effect of adding either trehalose or sucrose to samples, as indicated. Mouse SI IgG titers were then measured at day 7, 14, 21, and 28, after first booster shot and at day 14, 21, and 28 after second booster shot, as indicated.
[00131] Figure 28 shows results obtained when testing the effect of adding Tris buffer or Phosphate buffer to samples, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated.
[00132] Figure 29 shows results obtained when testing the indicated formulations after various storage conditions/time periods, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated.
[00133] Figure 30 shows results obtained when testing the indicated formulations at the indicated pHs, as indicated. Mouse SI IgG titers were then measured at day 7 after primary shot, as indicated.
[00134] Figure 31 shows results obtained when testing the indicated formulations prepared using HPLC pump, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated. Detailed Description
[00135] All references cited herein are incorporated by reference in their entireties for all purposes. Headings are purely for the convenience of the reader and should not be construed as limiting the invention described herein or any of its embodiments.
Definitions
[00136] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional procedures well known in the art and as described in various general and more specific references that are cited and discussed herein unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). A number of basic texts describe standard antibody production processes, including, Borrebaeck (ed ) Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995; McCafferty et al. Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England,
1996; and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, N.J., 1995; Paul (ed.), Fundamental Immunology , Raven Press, N.Y, 1993; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Coding Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, N.Y., 1986, and Kohler and Milstein Nature 256: 495-497, 1975. All of the references cited herein are incorporated herein by reference in their entireties. Enzymatic reactions and enrichment/purification techniques are also well known and are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
[00137] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.
[00138] It is understood the use of the alternative (e.g., “or”) herein is taken to mean either one or both or any combination thereof of the alternatives.
[00139] The term “and/or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,”
“A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A,
B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[00140] As used herein, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided.
[00141] As used herein, the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example,
“about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition.
[00142] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, all ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as “not including the endpoints”; thus, for example, “ranging from 1 to 10” includes the values 1 and 10 and all integer and (where appropriate) non-integer values greater than 1 and less than 10.
[00143] The term “coronavirus infection” refers to a human or animal that has cells that have been infected by a coronavirus. The infection can be established by performing a detection and/or viral titration from respiratory samples, or by assaying blood-circulating coronavirus-specific antibodies. The detection in the individuals infected with coronavirus is made by conventional diagnostic methods, such as molecular biology (e.g., PCR), which are known to those skilled in the art.
[00144] The term “subject” as used herein refers to human and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
[00145] The term “administering”, “administered”, and grammatical variants refers to the physical introduction of a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, transdermal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
[00146] The terms “treatment” and “treating” refer to fighting the coronavirus infection in a human or animal subject. By virtue of the administration of at least one embodiment of the compositions described herein, the viral infection rate (infectious titer) in the subject will decrease, and the virus may completely disappear from the subject. The terms “treatment” and “treating” also refers to attenuating symptoms associated with the viral infection (e.g., respiratory syndrome, kidney failure, fever, and other symptoms relating to coronavirus infections).
[00147] The terms "effective amount", “therapeutically effective amount” or “effective dose” or related terms may be used interchangeably and refer to an amount of the therapeutic agent that when administered to a subject, is sufficient to affect a measurable improvement or prevention of a disease or disorder associated with coronavirus infection. For example, administering an effective dose sufficient to inhibit the proliferation and/or replication of the coronavirus, and/or the development of the viral infection within the subject. Therapeutically effective amounts of the therapeutic agents provided herein, when used alone or in combination with an antiviral agent, will vary depending upon the relative activity of the therapeutic agent, and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. In one embodiment, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques. In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
[00148] The terms "peptide", "polypeptide" and "protein" and other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids. Polypeptides include recombinant or chemically-synthesized forms. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post- translationally, or otherwise covalently or non-covalently, modified proteins. Polypeptides comprising amino acid sequences of an coronavirus or coronavirus RBD or S protein, corona virus variant RBD or S protein, SAR.S-CoV-2 virus RBD or S protein, or SAR.S-Cov-2 variant RBD or S protein, or a derivative, mutein, or variant thereof , can be prepared using recombinant procedures are described herein.
[00149] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically- synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecule can be single-stranded or double-stranded.
[00150] In some embodiments, the nucleic acid molecules comprise a contiguous open reading frame encoding: at least one RBD corresponding to at least one coronavirus variant, such as a SAR.S-Cov-2 variant; one or more spike (S) proteins comprising one or more RBDs corresponding at least one coronavirus variant, such as a SAR.S-Cov-2 variant; or one or more derivatives, muteins, or variants thereof (such as emerging variants of concern and/or combinations of mutations found in two or more currently known SAR.S-Cov-2 variants). In one embodiment, nucleic acids comprise one type of polynucleotides or a mixture of two or more different types of polynucleotides.
[00151] The term “mutation”, “modification”, or “variation”, or related terms, refers to a change in a nucleic acid sequence or amino acid sequence that differs from a reference nucleic acid sequence or a reference amino acid sequence, respectively. Examples of mutations includes a point mutation, insertion, deletion, amino acid substitution, inversion, rearrangement, splice, sequence fusion (e.g., gene fusion or RNA fusion), truncation, transversion, translocation, non-sense mutation, sequence repeat, single nucleotide polymorphism (SNP), or other genetic rearrangement. As a nonlimiting example, the QQAQ furin site mutation introduced into various coronavirus variants as disclosed herein comprises a series of amino acid substitutions introduced into the wild type Washington/Wuhan-Hu-1 isolate S protein furin site sequence RR.AR. at amino acid positions 682 to 685 and corresponding sites/positions found in other variants. As another nonlimiting example, PP spike protein stablilizing mutation as disclosed herein comprises, for example, a pair of amino acid substitutions for the wild type Washington/Wuhan-Hu-1 isolate S protein furin site sequence KV at amino acids 986 and 987 and corresponding sites/positions found in other variants.
[00152] The term "isolated" refers to a protein (e.g., an antibody or an antigen binding portion thereof) or polynucleotide that is substantially free of other cellular material. A protein may be rendered substantially free of naturally associated components (or components associated with a cellular expression system or chemical synthesis methods used to produce the antibody) by isolation, using protein purification techniques well known in the art. The term isolated also refers in some embodiments to protein or polynucleotides that are substantially free of other molecules of the same species, for example other protein or polynucleotides having different amino acid or nucleotide sequences, respectively. The purity of homogeneity of the desired molecule can be assayed using techniques well known in the art, including low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrophotometry.
[00153] As used herein, the term “variant” polypeptides and “variants” of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides. [00154] As used herein in referring to coronaviruses, such as SARS-CoV-2, a “variant” means an isolate of a virus, such as a SARS-Cov-2 Washington/Wuhan-Hu-1 (also referred to as WA1/2020, used interchangeably throughout), as well as currently known, emerging, and/or yet-to-emerge isolates, each independently having one or more mutations with respect to a reference virus, such as a SARS-CoV-2 Washington/Wuhan-Hu-lisolate, from which it is derived. Thus, a used herein, a SARS-CoV-2 variant may be a Washington/Wuhan-Hu-1 isolate, as well as a variant in reference to a Washington/Wuhan-Hu-1 isolate. A variant, for example, may typically have multiple mutations with respect to a Washington/Wuhan-Hu-1 isolate which, for the purposes herein, comprises and RBD and/or and S protein comprising such RBD, of a coronavirus isolate, such as a Washington/Wuhan-Hu-1 isolate, and/or variants thereof. [00155] As used herein, a “receptor binding domain” or “RBD” (used interchangeably throughout) means a portion, region, or domain within a spike protein, such as a spike protein of a coronavirus, such as a SARS-Cov-2 variant, that is involved in the interaction between such spike protein and a cellular receptor of such spike protein, such an angiotensin converting enzyme 2 (“ACE2”) protein. In some embodiments, such an RBD corresponds to amino acids 319 through 541, inclusive, of a SARS-Cov-2 spike protein, or to the corresponding amino acids in a variant of such a SARS-Cov-2 virus (see, e.g., Huang etal ., Acto Pharmacologica Sinica, Vol 41, pages 1141-1149 (2020)). In some embodiments, such an RBD corresponds to an RBD amino acid sequence encoded by any of SEQ ID Nos: 1-12 and 15-19).
[00156] As used herein, a “spike protein,” “S protein’”, or “S” means a spike protein of a coronavirus, such as a SARS-Cov-2 variant, that is involved in the interaction between the coronavirus and a cellular receptor of such spike protein, such an angiotensin converting enzyme 2 (“ACE2”) protein. An exemplary such spike protein corresponds to amino acid sequence of NCBI Accession QHU79204.1 (SARS-CoV-2 isolate Washington/Wuhan-Hu- 1 ,e.g., SEQ ID NO:20), which includes two regions or domains known as SI (the N- terminus to amino acid 685) and S2 (amino acids 686 to 1273) that are cleaved into the SI and S2 subunits by furin, a cellular protease, during the infection process. In some embodiments, such a spike protein corresponds to a spike protein amino acid sequence encoded by any of SEQ ID Nos: 4-12 and 15-19. In some embodiments, such a spike protein corresponds to a spike protein amino acid sequence according to any of SEQ ID Nos:20-32. [00157] Exemplary SARS-Cov-2 variants may also be selected from the group consisting of the following variants: B.1.1.7 (also known as U.K., or Alpha); B.1.351 (also known as South Africa, or Beta); B.1.617.1 (also known as India, or Kappa); B.1.617.2 (also known as India, or Delta); B.1.617.2.1 (also known as AY.l, or Delta Plus); P.l (also known as Brazil/Japan, Brazil, or Gamma ); C.37 (also known as Lambda); P.2 (also known as Brazil, or Zeta); B.1.526 (also known as NY, or Iota); B.1.526.2 (also known as NY, Iota); B.1.1.318 (also known as Mauritius); B.l.1.7 (also known as U.K., or Alpha); B.1.617.1/3 (also known as India, or Kappa); B.1.427 (also known as Epsilon); B.1.1.529 (also known as Omicron); BA.2 (also known as Omicron plus); and emerging and/or yet-to-emerge variants thereof. Exemplary SARS-Cov-2 variants may contain mutations selected from the group consisting of the following sets of mutations: D614G; D69/70-D 144-N501 Y-A570D-D614G-P681 H- T716I-S982 A-D 1118H; D80 A-D215G-A242/244-K417N-E484K-N501 Y-D614G- A701 V; D614G, S 131, W152C, L452R; G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, H1101D; T19R, (G142D), D156-157, R158G, L452R, T478K, D614G, P681R, D950N; T19R, (G142D), D156-157, R158G, K417N, L452R, T478K, D614G, P681R, D950N; L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I; G75V,
T76I, D246-252, L452Q, F490S, D614G, T859N; E484Q, F565L, D614G, V1176F; L5F, T95I, D253G, E484K, D614G, A701V; L5F, T95I, D253G, S477N, D614G, A701V; T95I, DU144, E484K, D614G, P681H, D796H; D69/70, D614G, N501Y; D614G, K417N, E484K, N501Y; L452R, E484Q, P681R; D614G, L452R, E484K; D614G, L452R; D69/70; D614G- K378Y; D614G-E406W; D614G-K417E; D614G-N439K; D614G-N440D; D614G-K444Q; D614G-V445A; D614G-G446V; D614G-Y453F; D614G-L455F; D614G-G476S; D614G- S477N; D614G, T478K; D614G-E484K; D614G-E484Q; D614G-F486I; D614G-F486V; D614G-N487R; D614G-N487Y; D614G-Y489H; D614G-F490S; D614G-Q493K; D614G- Q493R; D614G-S494P; D614G-N501Y; D614G-Q677H; D614G-Q677P; and emerging and/or yet-to-emerge variants comprising combinations of such mutation and/or additional mutations. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding an RBD found in one or more of SEQ ID Nos: 20-32. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding one or more RBDs found in one or more of SEQ ID Nos. 1-19.
Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding spike protein comprising one or more RBDs found in one or more of SEQ ID Nos: 20-32. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding a spike protein comprising one or more RBDs found in one or more of SEQ ID Nos. 1-19. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding spike protein according to one or more of SEQ ID Nos: 20-32. Exemplary SARS-Cov-2 variants may also be selected from variants comprising nucleic acid sequence encoding a spike protein according to one or more of SEQ ID Nos. 1-19.
[00158] A “chimeric spike protein,” “chimeric S protein”, “chimeric variant,” and similar terms, used interchangeably throughout, refer to an amino acid sequence corresponding to a spike protein from a coronavirus variant, such as a SARS-Cov-2 variant, and/or a nucleic acid sequence encoding such spike protein, wherein such spike protein comprises at least one RBD from a different variant. Exemplary such chimeric spike proteins include, as non limiting examples, variants comprising a spike protein amino acid sequence that includes: an RBD from a Delta variant and an RBD from an Omicron variant (in either order); an RBD from a Beta variant and an RBD from an Omicron variant (in either order); and an RBD from a Delta variant, an RBD from a Beta variant, and an RBD from an Omicron variant (in any order). Exemplary such chimeric spike proteins also include those encoded by any nucleic acid sequence set forth in SEQ ID NOS: 4, 6, and 7.
[00159] A variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to a reference polynucleotide sequence.
[00160] As used herein, the term “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full- length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.
[00161] An "antigen binding protein" and related terms used herein refers to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody -derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et ah, 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et ah, 2004, Biotechnol. Prog. 20:639- 654. In addition, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimetics utilizing fibronection components as a scaffold. [00162] An antigen binding protein can have, for example, the structure of an immunoglobulin. In one embodiment, an "immunoglobulin" refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen binding sites. In one embodiment, an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens. For example, a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules.
[00163] The variable regions of immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. [00164] One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest. [00165] The assignment of amino acids to each domain is in accordance with the definitions of Rabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for the amino acids in immunoglobulin chains include IMGT.RTM. (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001); Chothia (Al-Lazikani et al., 1997 Journal of Molecular Biology 273:927-948; Contact (Maccallum et al., 1996 Journal of Molecular Biology 262:732-745, and Aho (Honegger and Pluckthun 2001 Journal of Molecular Biology 309:657-670.
[00166] An "antibody" and “antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof (or an antigen binding fragment thereof) that binds specifically to an antigen. Antigen binding portions (or the antigen binding fragment) may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions (or antigen binding fragments) include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
[00167] Antibodies include recombinantly produced antibodies and antigen binding portions. Antibodies include non-human, chimeric, humanized and fully human antibodies. Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers. Antibodies include F(ab’)2 fragments, Fab’ fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide-linked Fvs (sdFv), anti -idiotypic antibodies (anti-id), minibodies. Antibodies include monoclonal and polyclonal populations.
[00168] A “neutralizing antibody” and related terms refers to an antibody that is capable of specifically binding to the neutralizing epitope of its target antigen (e.g., coronavirus spike protein) and substantially inhibiting or eliminating the biological activity of the target antigen (e.g., coronavirus spike protein). The neutralizing antibody can reduce the biological activity of the target antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher levels of reduced biological activity.
[00169] An “antigen binding domain,” “antigen binding region,” or “antigen binding site” and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains.
[00170] The terms "specific binding", "specifically binds" or "specifically binding" and other related terms, as used herein in the context of an antibody or antigen binding protein or antibody fragment, refer to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens). In one embodiment, an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 105 M or less, or 106 M or less, or 107 M or less, or 108 M or less, or 109 M or less, or 10 10 M or less.
[00171] In one embodiment, a dissociation constant (KD) can be measured using a BIACORE surface plasmon resonance (SPR) assay. Surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ). [00172] An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof). An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein. An epitope can comprise non-contiguous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen’s primary sequence but that, in the context of the antigen’s tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein). Generally, the variable regions, particularly the CDRs, of an antibody interact with the epitope.
[00173] An "antibody fragment", "antibody portion", "antigen-binding fragment of an antibody", or "antigen-binding portion of an antibody" and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide. Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment. Antigen binding fragments that bind a coronavirus spike protein (S-protein) are described herein. [00174] The terms “Fab”, “Fab fragment” and other related terms refers to a monovalent fragment comprising a variable light chain region (VL), constant light chain region (CL), variable heavy chain region (VH), and first constant region (CHI). A Fab is capable of binding an antigen. An F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. A F(Ab’)2 has antigen binding capability.
An Fd fragment comprises VH and CHI regions. An Fv fragment comprises VL and VH regions. An Fv can bind an antigen. A dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain (U.S. Patents 6,846,634 and 6,696,245; U.S. published Application Nos. 2002/02512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958; and Ward et ak, Nature 341:544-546, 1989). Fab fragments comprising antigen binding portions from an antibody that binds a coronavirus spike protein (S-protein) are described herein.
[00175] A single-chain antibody (scFv) is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain. Preferably the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et ak, 1988, Science 242:423-26 and Huston et ak, 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
[00176] Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et ak, 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et ak, 1994, Structure 2:1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different. [00177] The term “human antibody” refers to antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody). These antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes.
[00178] The term “labeled antibody” or related terms as used herein refers to antibodies and their antigen binding portions thereof that are unlabeled or joined to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A. A variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).
[00179] The “percent identity” or “percent homology” and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences. A comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. [00180] In some embodiments, the amino acid sequence of an RBD (e.g., a SARS-CoV-2 RBD or a SARS-CoV-2 variant RBD) may be similar but not identical to any of the amino acid sequences of RBD proteins encoded by the nucleic acid molecules described herein. The similarities between an RBD and the RBDs described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more RBDs found in a SARS-Cov-2 variant. In some embodiments, the amino acid sequence of an RBD (e.g., a SARS-CoV-2 RBD or a SARS-CoV-2 variant RBD) may be similar but not identical to any of the amino acid sequences of RBD proteins encoded by the nucleic acid molecules described herein. The similarities between an RBD and the RBDs described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more RBDs found one or more of SEQ ID Nos 20-32.
[00181] In some embodiments, the amino acid sequence of an S protein (e.g., a SAR.S- CoV-2 S protein or a SARS-CoV-2 variant S protein) may be similar but not identical to any of the amino acid sequences of S proteins encoded by the nucleic acid molecules described herein. The similarities between an S protein and the S proteins described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more S proteins found in a SARS-Cov-2 variant. In some embodiments, the amino acid sequence of an S protein (e.g., a SARS-CoV-2 S protein or a SARS-CoV-2 variant S protein) may be similar but not identical to any of the amino acid sequences of S proteins encoded by the nucleic acid molecules described herein. The similarities between an S protein and the S proteins described herein is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to one or more RBDs found one or more of SEQ ID Nos 1-19.
[00182] In some embodiments, the amino acid substitutions comprise one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference in its entirety. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine.
[00183] A "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene). Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell). Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector. Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct. Vectors can be single-stranded or double- stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules.
One type of vector is a "plasmid," which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and/or translating the transgene. A viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome. Examples of viral vectors include retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
[00184] An "expression vector" is a type of vector that can contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers. Expression vectors can include ribosomal binding sites and/or polyadenylation sites. Regulatory sequences direct transcription, or transcription and translation, of a transgene, such as a DNA or RNA transgene encoding an RBD, an S protein, a coronavirus or variant thereof, or a protein encoded by one or more of SEQ ID Nos: 1-19 provided herein, linked to the expression vector which is transduced into a host cell. The regulatory sequence(s) can control the level, timing and/or location of expression of the transgene. The regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). Regulatory sequences can be part of a vector. Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606.
[00185] A transgene is “operably linked” to a promoter when the linkage between the transgene and the promoter permits functioning or expression of the transgene. In one embodiment, a transgene is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.
[00186] The terms "transfected" or "transformed" or "transduced" or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene). The host cell includes the primary subject cell and its progeny.
[00187] The term “subject” as used herein refers to human and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
[00188] The term “administering”, “administered”, and grammatical variants refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. [00189] The terms "effective amount", “therapeutically effective amount” or “effective dose” or related terms may be used interchangeably and refer to an amount of one or more nucleic acid molecules comprising one or more a nucleic acids encoding an RBD, an S protein, a coronavirus or variant thereof, such as one or more RBDs, S proteins, coronaviruses, or variant thereof encoded by a sequence according to one or more of SEQ ID Nos: 1-19, and/or coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein that when administered to a subject, is sufficient to effect a measurable improvement or prevention of a disease associated with coronavirus infection. Therapeutically effective amounts of such nucleic acid molecules, when used alone or in combination, will vary depending upon the relative activity of the nucleic acid molecules, coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition and symptoms in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
[00190] In some embodiments, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques. In general, the nucleic acid molecules, coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein may be administered at about 0.01 g/kg to about 50 mg/kg per day, 0.01 mg/kg to about 30 mg/kg per day, or 0.1 mg/kg to about 20 mg/kg per day. The nucleic acid molecules, coronavirus antigen fusion proteins, nucleic acids, vectors, and/or mRNA vaccines described herein may be administered daily (e.g., once, twice, three times, or four times daily) or preferably less frequently (e.g., weekly, every two weeks, every three weeks, monthly, or quarterly). In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary. mRNA Vaccine Compositions
[00191] In embodiments the vaccine compositions disclosed herein comprise, for example, one or more antigens, neutralizing antibodies, or proteins with imrnuiiostimulatory activity. In embodiments, the vaccine compositions disclosed herein comprise one or more nucleic acid molecules that encode, for example, one or more antigens, neutralizing antibodies, or proteins with immunostimulatory activity. In embodiments, the vaccine compositions disclosed herein comprise one or more nucleic acid molecules comprising messenger ribonucleic acid (mRNA) that encode, for example, one or more antigens, neutralizing antibodies, or proteins with immunostimulatory activity. In embodiments, such vaccine compositions comprise one or more nucleic acid molecules, such as one or more messenger ribonucleic acid (mRNA) molecules, that encode one or more coronavirus antigens, such as one or more RBDs, one or more S proteins, one or more chimeric S proteins, one or more RBDs encoded by a sequence set forth in SEQ ID Nos: 1-19, and or one or more S proteins encoded by a sequence set forth in SEQ ID Nos: 1-19. [00192] In embodiments, such vaccine compositions comprise one or more nucleic acid molecules, such as one or more messenger ribonucleic acid (mRNA) molecules, that encode one or more coronavirus antigens, such as one or more RBDs, one or more S proteins, one or more chimeric S proteins, one or more RBDs encoded by a sequence set forth in SEQ ID Nos: 1-19, and or one or more S proteins encoded by a sequence set forth in SEQ ID Nos: 1-  19, which are administered to a subject having, suspected of having, or at risk of having a SARS-CoV-2 variant infection or disease, such that the infection or disease is treated, ameliorated, or prevented. In certain embodiments, administration, which may be by injection or other means, brings the mRNA, typically in association with a delivery carrier, into proximity of cells of the subject so that the mRNA is taken up by cells of the subject.  These host cells express display the S protein encoded by the mRNA on their membranes where they are recognized as foreign by the subject’s immune system, promoting humoral and/or cellular immune responses. [00193] Without wishing to be bound by any theory, it is believed that expression of the S protein by the cells of a subject can be susceptible to cleavage by the cellular protease furin,  and that such cleavage would result in the S1 subunit being released from the cell membrane, leading to a less effective vaccine and potentially leading to harmful symptoms in the subject, as the S1 subunit would be free to interact with the ACE2 receptor on healthy nontransfected ACE2-expressing cells of the subject. RNAs encoding mutated furin cleavage sites have previously demonstrated poor expression by transfected cells. As disclosed in the Examples  herein, the inventors have found that incorporating a particular mutation (QQAQ) conferring Furin resistance into multiple S proteins resulted in expression that was at least as strong as that of the corresponding non-mutant S proteins. In some cases, expression of the furin-     resistant mutants in RNA-transfected cultures was significantly improved relative to that of the non-mutant S proteins, both in terms of the percentage of S-protein expressing cells and the persistence of expression over three days. Various embodiments of mRNA-based vaccines are contemplated where S proteins of one or more SARS-CoV-2 variants that include the QQAQ mutation are encoded by one or more mRNAs of the vaccine formulation. [00194] In some embodiments, a pharmaceutical composition, e.g., a vaccine composition may include a naked messenger ribonucleic acid (mRNA) encoding an S protein as disclosed herein .
[00195] In some embodiments a pharmaceutical vaccine composition includes a messenger ribonucleic acid (mRNA), encoding an RED and/or an S protein, where the mRNA is formulated with: a non-lipid nanoparticle, a lipid nanoparticle, a cationic lipid iianoparticle, or a peptide, such as a cationic peptide.
[00196] Delivery carriers for mRNA have been disclosed and reviewed extensively, e.g., Zeng et al. (2020) Carr Topics Microbiol Immunol doi.orglO.1007/82 2020 217).
[00i 97] In some embodiments, lipids and lipid derivatives can be used in lipid formulations and lipid-derived nanoparticles (LNPs), where LNPs can encapsulate the mRNAs. In some embodiments the lipids and lipid derivatives are positively charged. See, for example, Reichmuth et al. (2016) Ther Deliv 7:319-334.
[00198] In some embodiments, non-lipids can be delivery carriers, including polymers such as poiyamines, dendrimers, and copolymers. In embodiments, the non-lipids can be cationic polymers, such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, and polysaccharide. In embodiments, the non-lipids can be anionic polymers such as PLGA mixed with cationic lipids.
[00199] In some embodiments, amphiphilic block copolymers form non-lipid nanoparticles. In embodiments, the non-lipid nanoparticles are polymeric micelles. In embodiments, the non-lipid nanoparticles are stealth nanoparticles. In embodiments, the nonlipid nanoparticles are polymersomes. In embodiments, the non-lipid nanoparticles are polyrotaxane supramolecular structures. In embodiments, the non-lipid nanoparticles are selfmicro emulsifying systems. In embodiments, amphiphilic block copolymers do not form nonlipid nanoparticles. In embodiments mRNA is incubated with amphiphilic block copolymers and buffers. [00200] Amphiphilic copolymers that spontaneously aggregate in water lead to supramolecular structures with a hydrophobic core suitable to host poorly soluble drugs, surrounded by a hydrophilic shell that contributes to physically stabilize the amphiphilic aggregate in the aqueous environment (Jones et al., 1999, Eur. J Pharm. Biopharm., 48, 101- 111; Francis et al., 2004, Pure Appl. Chem., 76, 1321-1335).
[00201] In embodiments, the amphiphilic block copolymers are branched. In embodiments, the branched amphiphilic block copolymers are poloxamines (Alvarez- Lorenzo et al., 2010, Front. Biosci ., 2, 424-440).
[00202] In embodiments, the amphiphilic block copolymers are linear. In embodiments, the linear amphiphilic block copolymers are poloxamers.
[00203] In some aspects, the first surfactant is an ethoxylated glyceryl ester. In some aspects, the first surfactant is a copolymer comprising polyethylene glycol units. In some aspects, the first surfactant is a copolymer of polyethylene glycol and polypropylene glycol. In some aspects, the first surfactant is a poloxamer. A poloxamer is a non-ionic surfactant that is a tri-block copolymer with a central polypropylene glycol portion and polyethylene glycol termini. In some aspects, the first surfactant is poloxamer 407. In some aspects, the first surfactant is a combination of surfactants comprising poloxamer 407 and an additional surfactant. In some aspects, the first surfactant is poloxamer 188. In some aspects, the first surfactant is a combination of surfactants comprising poloxamer 188 and an additional surfactant. In some aspects, the first surfactant is poloxamer 407 and poloxamer 188. In some aspects, the first surfactant is poloxamer 407. In some aspects, the first surfactant is a combination of surfactants comprising poloxamer 407 and an additional surfactant.
[00204] In embodiments, lipids and lipid derivatives formulate lipid and lipid-derived nanoparticles (LNPs). In embodiments, LNPs encapsulate the mRNA vaccine. In embodiments the lipids and lipid derivatives are positively charged.
[00205] In some embodiments, cationic peptides contain many lysine and arginine residues that provide the positive charge. In embodiments, a cationic peptide is protamine. In embodiments, protamine and mRNA vaccine spontaneously form a complex.
[00206] In some embodiments, cationic peptides are employed, such as cationic cell- penetrating peptides, such as, for example, a RALA peptide or Xentry. [00207] An mRNA vaccine as provided herein can include one or more RNA molecules, for example, RNA molecules encoding one or more variant S proteins, optionally in addition to the WA1/2020 S protein. In various embodiments the S protein encoded by an RNA molecule as provided herein can include
[00208] In embodiments, a vaccine composition includes at least one nucleic acid encoding variant RBD or S protein encoded by any of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, or SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, or may be a different RBD or S protein that is encoded by a SARS-CoV-2 virus that arises in a population or geographical region. For example, a vaccine composition may comprise a nucleic acid encoding an RBD or a spike protein comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In some a vaccine composition provided herein includes a nucleotide sequence that encodes one or more variant SARS-CoV-2 RBDs or one or more variant SARS-CoV-2 spike proteins comprising such one or more RBDs, wherein the nucleotide sequence has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to one or more RBDs encoded by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
[00209] Also provided herein is a pharmaceutical composition that includes as at least one RNA molecule as described herein in a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition that includes two or more RNA molecules as described herein in a pharmaceutically acceptable carrier, where the two or more RNA molecules encode different S proteins, e.g., different variants of the SARS-CoV-2 S protein. For example, a pharmaceutical composition can include a first RNA molecule that encodes the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second chimeric adenovirus that encodes the SARS-CoV-2 “UK” isolate (alpha) S protein having a QQAQ furin site mutation. Alternatively, a pharmaceutical composition can include an mRNA encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second chimeric adenovirus that encodes the SARS-CoV-2 “beta” isolate S protein having a QQAQ furin site mutation. Further alternatively, a pharmaceutical composition can include a first chimeric adenovirus that includes a gene encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second mRNA that encodes the SARS-CoV-2 “gamma” isolate S protein having a QQAQ furin site mutation. Further alternatively, a pharmaceutical composition can include a first chimeric adenovirus that includes a gene encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second mRNA that encodes the SARS-CoV-2 “delta” isolate S protein having a QQAQ furin site mutation. Further alternatively, a pharmaceutical composition can include a first chimeric adenovirus that includes a gene encoding the SARS-CoV-2 “Wuhan” isolate S protein (Genbank YP 009724390.1) having a QQAQ furin site mutation, and a second mRNA that encodes the SARS-CoV-2 “kappa” isolate S protein having a QQAQ furin site mutation. Various other combinations are within the scope of the invention.
[00210] Lipids, lipid nanoparticles, and mRNA vaccine compositions comprising same
[00211] Lipids and lipid-containing nanoparticles suitable for use in the compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are disclosed, for example, in U.S. provisional patent application number 63/313,648, filed on February 24, 2022, entitled “Novel Ionizable Cationic Lipids,” the content of which is hereby incorporated by reference in its entirety.
[00212] The terms “lipid” or “lipid moiety” are used in accordance with its ordinary meaning in chemistry and refer to a hydrophobic molecule which is typically characterized by an aliphatic hydrocarbon chain. In embodiments, the lipid moiety includes a carbon chain of 3 to 100 carbons. In embodiments, the lipid moiety includes a carbon chain of 5 to 50 carbons. In embodiments, the lipid moiety includes a carbon chain of 5 to 25 carbons. In embodiments, the lipid moiety includes a carbon chain of 8 to 525 carbons. Lipid moieties may include saturated or unsaturated carbon chains, and may be optionally substituted. In embodiments, the lipid moiety is optionally substituted with a charged moiety at the terminal end. In embodiments, the lipid moiety is an alkyl or heteroalkyl optionally substituted with a carboxylic acid moiety at the terminal end. Lipids are also a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. [00213] The terms “cationic lipid” or “ionizable cationic lipid” are used interchangeably herein and refer to lipids that are protonated at low pH, which makes them positively charged, but they remain neutral at physiological pH.
[00214] The term “lipid nanoparticle” includes a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., an mRNA), to a target site of interest (e.g., cell, tissue, organ, and the like). In embodiments, the lipid particle described herein is a nucleic acid-lipid particle, which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle. In other embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.
[00215] The term “lipid conjugate” refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dimyristoylglycerols (e.g., PEG-DMG conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides.
[00216] The term “diacylglycerol” or “DAG” includes a compound having 2 fatty acyl chains, R and R2, both of which have independently between 2 and 30 carbons bonded to the 1- and 2-position of glycerol by ester linkages. The acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (Ci6), stearoyl (Cis), and icosoyl (C20). In preferred embodiments, R1 and R2 are the same, i.e., R1 and R2 are both myristoyl (i.e., dimyristoyl), R1 and R2 are both stearoyl (i.e., distearoyl), etc. Diacylglycerols have the following general formula:
Figure imgf000073_0001
[00217] The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkyl chains, R1 and R2, both of which have independently between 2 and 30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation. Dialkyloxypropyls have the following general formula:
Figure imgf000074_0001
[00218] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.
[00219] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts. Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions.
For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds. [00220] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to - OCH2-.
[00221] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals. The alkyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons). Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-0-). An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully saturated. An alkenyl may include more than one double bond and/or one or more triple bonds in addition to the one or more double bonds. An alkynyl may include more than one triple bond and/or one or more double bonds in addition to the one or more triple bonds.
[00222] The term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 30 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
[00223] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, B, Se, and S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, B, Se, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-  N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH- O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and - CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH- OCH3 and -CH2-O-Si(CH3)3. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, B, Se, or P). The term “heteroalkenyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond. A heteroalkenyl may optionally include more than one double bond and/or one or more triple bonds in additional to the one or more double bonds. The term “heteroalkynyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one triple bond. A   heteroalkynyl may optionally include more than one triple bond and/or one or more double bonds in additional to the one or more triple bonds. [00224] Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For   heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula - C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups, as  used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', -NR'R'', -OR', -SR', and/or -SO2R'. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -     NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.
[00225] The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.
[00226] In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system. In embodiments, monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In embodiments, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CEbjw , where w is 1, 2, or 3). Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclic cycloalkyl groups include, but are not limited to tetradecahydrophenanthrenyl, perhydrophenothiazin-l-yl, and perhydrophenoxazin-l-yl. [00227] In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl” is used in accordance with its plain ordinary meaning. In embodiments, a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system. In embodiments, monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH2)w, where w is 1, 2, or 3). Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct 2 enyl. In embodiments, fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
[00228] In embodiments, a heterocycloalkyl is a heterocyclyl. The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S. The 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
The 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. The heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl. The heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system. Representative examples of bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3- dihydrobenzofuran-3-yl, indolin-l-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-lH-indolyl, and octahydrobenzofuranyl. In embodiments, heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia. Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. The multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring. In embodiments, multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multi cyclic heterocyclyl groups include, but are not limited to 1 OH-phenothiazin- 10-yl, 9, 10-dihydroacridin-9-yl, 9, 10-dihydroacridin- 10-yl, lOH-phenoxazin-10-yl, 10,1 l-dihydro-5H-dibenzo[b,f azepin-5-yl, 1, 2,3,4- tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro- lH-carbazol-9-yl.
[00229] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(Ci-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
[00230] The term “acyl” means, unless otherwise stated, -C(0)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[00231] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1 -naphthyl, 2- naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3 -pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl,
2 -benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.
[00232] A fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.
[00233] Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom. The individual rings within spirocyclic rings may be identical or different. Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring. When referring to a spirocyclic ring system, substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different. [00234] The symbol “ ” denotes the point of attachment of a chemical moiety to the  remainder of a molecule or chemical formula. [00235] he term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom. [00236] The term “alkylsulfonyl,” as used herein, means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R'  may have a specified number of carbons (e.g., “C1-C4 alkylsulfonyl”). [00237] The term “alkylarylene” as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene group has the formula: [00238]
Figure imgf000083_0001
  [00239] An alkylarylene moiety may be substituted (e.g. with a substituent group) on the alkylene moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, -N3, - CF3, -CCl3, -CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2CH3 - SO3H, , -OSO3H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, substituted or unsubstituted C1-C5 alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl). In  embodiments, the alkylarylene is unsubstituted. [00240] Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,” “heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.   [00241] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,     heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, - SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'- C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', - S(O)2R', -S(O)2NR'R'', -NRSO2R', ^NR'NR''R''', ^ONR'R'', ^NR'C(O)NR''NR'''R'''', -CN, - NO2, -NR'SO2R'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such radical. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R''', and R'''' group when more than one of these groups is present. When R' and R'' are attached to the same  nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -  C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like). [00242] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R'', -SR', - halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', - NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', - S(O)2R', -S(O)2NR'R'', -NRSO2R', ^NR'NR''R''', ^ONR'R'', ^NR'C(O)NR''NR'''R'''', -CN, - NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, -NR'SO2R'', - NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R'', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound described herein includes more than one R group, for example,     each of the R groups is independently selected as are each R', R", R", and R"" groups when more than one of these groups is present.
[00243] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent). In such a case, the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings). When a substituent is attached to a ring, but not a specific atom (a floating substituent), and a subscript for the substituent is an integer greater than one, the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different. Where a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent), the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency. Where a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms. Where the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.
[00244] Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non- adjacent members of the base structure. [00245] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may  optionally form a ring of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O) -, -S(O)2-, -S(O)2NR'-, or a single bond, and r is  an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR')s-X'- (C''R''R''')d-, where s and d are independently integers of from 0 to 3, and X' is - O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. [00246] As used herein, the terms “heteroatom” or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). [00247] A “substituent group,” as used herein, means a group selected from the following moieties: [00248] (A) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2,   -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8  membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered     heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and [00249] (B) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl  (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from: [00250] (i) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H,   -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and   [00251] (ii) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from:     [00252] (a) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,   -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and [00253] (b) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, 2 to 6 membered   heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected  from: oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).     [00254] A “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C30 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 30 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.
[00255] A “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-Cs alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3- Ci cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 6 membered heteroaryl.
[00256] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.
[00257] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C30 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 30 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6- Cio aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C30 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 30 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroaryl ene.
[00258] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C30 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 30 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C30 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 30 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth in the Examples section, figures, or tables below. [00259] In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroaryl ene) is unsubstituted (e.g., is an unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and/or unsubstituted heteroaryl ene, respectively). In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroaryl ene) is substituted (e.g., is a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene, respectively).
[00260] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene) is substituted with at least one substituent group, wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of substituent groups, each substituent group is different.
[00261] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene) is substituted with at least one size-limited substituent group, wherein if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group is different.
[00262] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene) is substituted with at least one lower substituent group, wherein if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group is different.
[00263] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroaryl ene) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group is different.
[00264] Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and/or isolate. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefmic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
[00265] As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
[00266] The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
[00267] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure.
[00268] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.
[00269] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure.
[00270] “Analog,” or “analogue” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
[00271] As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.
[00272] The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge el al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
[00273] Thus, the compounds of the present disclosure may exist as salts, such as with pharmaceutically acceptable acids. The present disclosure includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art. [00274] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents. [00275] In addition to salt forms, the present disclosure provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure. Prodrugs of the compounds described herein may be converted in vivo after administration. Additionally, prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.
[00276] Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
[00277]
[00278] Compounds [00279] In an aspect, provided herein is cationic lipid of formula (I):
Figure imgf000095_0001
[00281] (I)
[00282] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof. [00283] R1 is H, -OR1A, -YOR1A, -NR1AR1B, -YNR1AR1B, -SR1A, -YSR1A, -(C=0)R1A, -
Y(C=0)R1A,
-(C=0)OR1a, -Y(C=0)OR1a, -0(C=0)R1a, -YO(C=0)R1a, -0(C=0)0R1a, -YO(C=0)OR1a, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. [00284] Y is substituted or unsubstituted C0-C12 alkylene or substituted or unsubstituted 0 to 12 membered heteroalkylene. [00285] R2 is H, -OR2A, -SR2A, -(C=O)R2A, -(C=O)OR2A, -O(C=O)R2A, -O(C=O)OR2A, - (C=O)NHR2A, -NH(C=O)R2A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. [00286] R3 is H, -OR3A, -SR3A, -(C=O)R3A, -(C=O)OR3A, -O(C=O)R3A, -O(C=O)OR3A, - (C=O)NHR3A,   -NH(C=O)R3A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. [00287] R4 is H, -OR4A, -SR4A, -(C=O)R4A, -(C=O)OR4A, -O(C=O)R4A, -O(C=O)OR4A, - (C=O)NHR4A, -NH(C=O)R4A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalky. R5 is H, -OR5A, -SR5A, -(C=O)R5A, -(C=O)OR5A, -O(C=O)R5A, -O(C=O)OR5A, -  (C=O)NHR5A, -NH(C=O)R5A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. [00288] B1 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted  heteroarylene. [00289] B2 and B3 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. [00290] L1 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR101R102)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR101C(=O)‑, ‑C(=O)NR101‑, ‑NR101C(=S)‑, ‑C(=S)NR101‑,  ‑NR101C(=O)NR102‑, ‑NR101C(=S)NR102‑, ‑OC(=O)NR101‑, ‑NR101C(=O)O‑, ‑SC(=O)NR101‑ or ‑NR101C(=O)S‑. [00291] L2 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR201R202)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR201C(=O)‑, ‑C(=O)NR201‑, ‑NR201C(=O)NR202‑, ‑NR201C(=S)‑, ‑C(=S)NR201‑, ‑NR201C(=S)NR202‑, ‑OC(=O)NR201‑, ‑NR201C(=O)O‑, ‑SC(=O)NR201‑ or ‑NR201C(=O)S‑. [00292] L3 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR301R302)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR301C(=O)‑, ‑C(=O)NR301‑, ‑NR301C(=O)NR302‑, ‑NR301C(=S)‑,     ‑C(=S)NR301‑, ‑NR301C(=S)NR302‑, ‑OC(=O)NR301‑, ‑NR301C(=O)O‑, ‑SC(=O)NR301‑ or ‑NR301C(=O)S‑. [00293] L4 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR401R402)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR401C(=O)‑, ‑C(=O)NR401‑, ‑NR401C(=O)NR402‑, ‑NR401C(=S)‑,  ‑C(=S)NR401‑, ‑NR401C(=S)NR402‑, ‑OC(=O)NR401‑, ‑NR401C(=O)O‑, ‑SC(=O)NR401‑ or ‑NR401C(=O)S‑. [00294] L5 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR501R502)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR501C(=O)‑, ‑C(=O)NR501‑, ‑NR501C(=O)NR502‑, ‑NR501C(=S)‑, ‑C(=S)NR501‑, ‑NR501C(=S)NR502‑, ‑OC(=O)NR501‑, ‑NR501C(=O)O‑, ‑SC(=O)NR501‑ or  ‑NR501C(=O)S‑. [00295] L6 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR601R602)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR601C(=O)‑, ‑C(=O)NR601‑, ‑NR601C(=O)NR602‑, ‑NR601C(=S)‑, ‑C(=S)NR601‑, ‑NR601C(=S)NR602‑, ‑OC(=O)NR601‑, ‑NR601C(=O)O‑, ‑SC(=O)NR601‑ or ‑NR601C(=O)S‑.   [00296] L7 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR701R702)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR701C(=O)‑, ‑C(=O)NR701‑, ‑NR701C(=O)NR702‑, ‑NR701C(=S)‑, ‑C(=S)NR701‑, ‑NR701C(=S)NR702‑, ‑OC(=O)NR701‑, ‑NR701C(=O)O‑, ‑SC(=O)NR701‑ or ‑NR701C(=O)S‑. [00297] La1 and La2 are each independently  
Figure imgf000097_0001
O, S, or CH2. [00299] W1, W2, W3, W4, W5, and W6 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered   heteroalkylene. [00300] Each R1A and R1B is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.     [00301] Each R2A, R3A, R4A, and R5A is independently H, substituted or unsubstituted C1- C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl. [00302] Each R101, R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or   unsubstituted 2 to 12 membered heteroalkyl. [00303] Each s is independently an integer from 1 to 4. [00304] In embodiments, R1 is independently H, -OR1A, -YOR1A, -NR1AR1B, -YNR1AR1B, - SR1A, -YSR1A, -(C=O)R1A, -Y(C=O)R1A, -(C=O)OR1A, -Y(C=O)OR1A, -O(C=O)R1A, -  YO(C=O)R1A, -O(C=O)OR1A, -YO(C=O)OR1A, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C30 alkyl, C1- C8 alkyl, or C1-C4 alkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered  heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl,  3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6   membered heteroaryl). In embodiments, R1 is substituted with one or more substituent groups. In embodiments, R1 is substituted with one or more size-limited substituent groups. In embodiments, R1 is substituted with one or more lower substituent groups. [00305] In embodiments, R1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C30 alkyl, C1-C8  alkyl, or C1-C4 alkyl). In embodiments, R1 is independently unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkyl     (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R1 is independently unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R1 is independently unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl). In embodiments, R1 is independently unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl). In embodiments, R1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl). In embodiments, R1 is independently unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl). In embodiments, R1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R1 is independently unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
[00306] In embodiments, R1 is independently H, -OR1A or substituted or unsubstituted heteroalkyl. In embodiments, R1 is independently H, -OR1A or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R1 is independently H. In embodiments, R1 is independently -OR1A. In embodiments, R1 is independently substituted or unsubstituted heteroalkyl. In embodiments, R1 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl).
[00307] In embodiments, R1 is independently H, -OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl. In embodiments, R1 is independently -OH or methoxy. [00308] In embodiments, R1 is independently H. In embodiments, R1 is independently - OH. In embodiments, R1 is independently methoxy. In embodiments, R1 is independently ethoxy. [00309] In embodiments, R2 is H, -OR2A, -SR2A, -(C=O)R2A, -(C=O)OR2A, -O(C=O)R2A,  -O(C=O)OR2A, -(C=O)NHR2A, -NH(C=O)R2A, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1- C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl), or substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In  embodiments, R2 is substituted with one or more substituent groups. In embodiments, R2 is substituted with one or more size-limited substituent groups. In embodiments, R2 is substituted with one or more lower substituent groups. [00310] In embodiments, R2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4  alkyl). In embodiments, R2 is unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R2 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered  heteroalkyl, or 2 to 4 membered heteroalkyl). [00311] In embodiments, R2 is H or substituted or unsubstituted alkyl. In embodiments, R2 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R2 is H. In embodiments, R2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R2 is unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). [00312] In embodiments, R2 is H or substituted or unsubstituted C1-C12 alkyl. In embodiments, R2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, R2 is unsubstituted C1-C12 alkyl. [00313] In embodiments, R3 is H, -OR3A, -SR3A, -(C=O)R3A, -(C=O)OR3A, -O(C=O)R3A, -O(C=O)OR3A, -(C=O)NHR3A, -NH(C=O)R3A, substituted (e.g. with a substituent group, a     size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1- C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl), or substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R3 is substituted with one or more substituent groups. In embodiments, R3 is substituted with one or more size-limited substituent groups. In embodiments, R3 is substituted with one or more lower substituent groups. [00314] In embodiments, R3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4  alkyl). In embodiments, R3 is unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R3 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). [00315] In embodiments, R3 is H or substituted or unsubstituted alkyl. In embodiments, R3 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R3 is H. In embodiments, R3 is substituted (e.g. with a substituent group, a  size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R3 is unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). [00316] In embodiments, R3 is H or substituted or unsubstituted C1-C12 alkyl. In embodiments, R3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, R3 is unsubstituted C1-C12 alkyl. [00317] In embodiments, R4 is H, -OR4A, -SR4A, -(C=O)R4A, -(C=O)OR4A, -O(C=O)R4A, -O(C=O)OR4A, -(C=O)NHR4A, -NH(C=O)R4A, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1- C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl), or substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R4 is substituted with one or more substituent groups. In embodiments, R4 is     substituted with one or more size-limited substituent groups. In embodiments, R4 is substituted with one or more lower substituent groups. [00318] In embodiments, R4 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R4 is unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R4 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R4 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). [00319] In embodiments, R4 is H or substituted or unsubstituted alkyl. In embodiments, R4 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R4 is H. In embodiments, R4 is substituted (e.g. with a substituent group, a  size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R4 is unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). [00320] In embodiments, R4 is H or substituted or unsubstituted C1-C12 alkyl. In embodiments, R4 is substituted (e.g. with a substituent group, a size-limited substituent group  or a lower substituent group) C1-C12 alkyl. In embodiments, R4 is unsubstituted C1-C12 alkyl. [00321] In embodiments, R5 is H, -OR5A, -SR5A, -(C=O)R5A, -(C=O)OR5A, -O(C=O)R5A, -O(C=O)OR5A, -(C=O)NHR5A, -NH(C=O)R5A, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1- C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl), or substituted (e.g. with a substituent group, a size-  limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R5 is substituted with one or more substituent groups. In embodiments, R5 is substituted with one or more size-limited substituent groups. In embodiments, R5 is substituted with one or more lower substituent groups.   [00322] In embodiments, R5 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R5 is unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4     alkyl). In embodiments, R5 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R5 is unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered   heteroalkyl, or 2 to 4 membered heteroalkyl). [00323] In embodiments, R5 is H or substituted or unsubstituted alkyl. In embodiments, R5 is H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R5 is H. In embodiments, R5 is substituted (e.g. with a substituent group, a  size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R5 is unsubstituted alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). [00324] In embodiments, R5 is H or substituted or unsubstituted C1-C12 alkyl. In embodiments, R5 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, R5 is unsubstituted C1-C12 alkyl. [00325] In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C0-C12 alkylene or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 0 to 12 membered heteroalkylene. In embodiments, Y is substituted with one or more substituent groups. In embodiments, Y is substituted with one or more size-limited substituent groups. In embodiments, Y is substituted with one or more lower substituent groups. [00326] In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C0-C12 alkylene. In embodiments, Y is  unsubstituted C0-C12 alkylene. In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 0 to 12 membered heteroalkylene. In embodiments, Y is unsubstituted 0 to 12 membered heteroalkylene. [00327] In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C8 alkylene. In embodiments, Y is  unsubstituted C1-C8 alkylene. In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 1 to 8 membered heteroalkylene. In embodiments, Y is unsubstituted 1 to 8 membered heteroalkylene.     [00328] In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C4 alkylene. In embodiments, Y is unsubstituted C1-C4 alkylene. In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 1 to 4 membered heteroalkylene. In embodiments, Y is unsubstituted 1 to 4 membered heteroalkylene. [00329] In embodiments, Y is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) methylene, ethylene or propylene. In embodiments, Y is unsubstituted methylene, ethylene or propylene. [00330] In embodiments, B1 is a bond, substituted (e.g. with a substituent group, a size-  limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene), substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene), substituted (e.g. with a substituent group, a size-limited substituent group or  a lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene), substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene), substituted (e.g. with a substituent group, a size-limited  substituent group or a lower substituent group) or unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene). In embodiments, B1 is substituted with one or more substituent groups. In embodiments, B 1 is substituted with one or more size-limited substituent groups. In embodiments, B 1 is substituted with one or more lower substituent groups. In embodiments, B1 is a bond. [00331] In embodiments, B1 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B1 is unsubstituted alkylene (e.g., C1-C30  alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B1 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2     to 4 membered heteroalkylene). In embodiments, B1 is unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, B1 is substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, B1 is unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, B1 is substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered  heterocycloalkylene). In embodiments, B1 is unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene). In embodiments, B1 is substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene). In embodiments, B1 is unsubstituted arylene (e.g., C6-C10 arylene, C10  arylene, or phenylene). In embodiments, B1 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene). In embodiments, B1 is unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene).   [00332] In embodiments, B1 is a bond or a substituted or unsubstituted alkylene. In embodiments, B1 is a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). [00333] In embodiments, B1 is a bond or unsubstituted alkylene. In embodiments, B1 is a bond or unsubstituted C1-C8 alkylene. In embodiments, B1 is unsubstituted alkylene. In embodiments, B1 is unsubstituted C1-C8 alkylene. In embodiments, B1 is a bond. [00334] In embodiments, B2 and B3 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. In embodiments, B2 and B3 are each independently a bond, substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkylene     (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, B2 is substituted with one or more substituent groups. In embodiments, B 2 is substituted with one or more size-limited substituent groups. In embodiments, B 2 is substituted with one or more lower substituent groups. In embodiments,  B2 is a bond. In embodiments, B3 is substituted with one or more substituent groups. In embodiments, B 3 is substituted with one or more size-limited substituent groups. In embodiments, B 3 is substituted with one or more lower substituent groups. In embodiments, B3 is a bond. [00335] In embodiments, B2 is substituted (e.g. with a substituent group, a size-limited  substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B2 is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B2 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2  to 4 membered heteroalkylene). In embodiments, B2 is unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). [00336] In embodiments, B3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8  alkylene, or C1-C4 alkylene). In embodiments, B3 is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B3 is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, B3 is unsubstituted heteroalkylene (e.g., 2  to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). [00337] In embodiments, B2 and B3 are each independently a bond or substituted or unsubstituted alkylene. In embodiments, B2 and B3 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).     [00338] In embodiments, B2 and B3 are each independently a bond or substituted or unsubstituted Ci-Cs alkylene. In embodiments, B2 and B3 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted Ci-Cs alkylene.
[00339] In embodiments, B2 is a bond. In embodiments, B2 is substituted Ci-Cs alkylene.
In embodiments, B2 is unsubstituted Ci-Cs alkylene. In embodiments, B3 is a bond. In embodiments, B3 is substituted Ci-Cs alkylene. In embodiments, B3 is unsubstituted C i-Cx alkylene.
[00340] In embodiments, B2 is butylene. In embodiments, B2 is propylene. In embodiments, B2is ethylene. In embodiments, B2is methylene. In embodiments, B3 is butylene. In embodiments, B3 is propylene. In embodiments, B3 is ethylene. In embodiments, B3 is methylene.
[00341] In embodiments, L2 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-. In embodiments, L2 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-. In embodiments, L2 is a bond,
-0(C=0)- or -(C=0)0-.
[00342] In embodiments, L1 is a bond, -NR101C(=S)-, -C(=S)NR101-, -0(C=0)-, -(C=0)0-, or -0-. In embodiments, L1 is a bond, -NR101C(=S)-, or -C(=S)NR101-.
[00343] In embodiments, L1 is a bond. In embodiments, L1 is -NR101C(=S)-. In embodiments, L1 is -C(=S)NR101. In embodiments, L1 is -0(C=0)-. In embodiments, L1 is - (C=0)0-. In embodiments, L1 is -0-. In embodiments, L1 is -C(=S)NR101, where the carbon atom is connected to the nitrogen atom in formula (I). In embodiments, L1 is -C(=S)NH, where the carbon atom is connected to the nitrogen atom in formula (I).
[00344] In embodiments, each R101 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R101 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R101 is substituted with one or more substituent groups. In embodiments, each R101 is substituted with one or more size-limited substituent groups. In embodiments, each R101 is substituted with one or more lower substituent groups. [00345] In embodiments, each R101 is independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R101 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl.
[00346] In embodiments, each R101 is independently H. In embodiments, each R101 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each R101 is independently unsubstituted 2 to 12 membered heteroalkyl.
[00347] In embodiments, L2 is a bond. In embodiments, L2 is -0(C=0)-. In embodiments, L2 is
-(C=0)0-. In embodiments, L2 is -C(=0)-. In embodiments, L2 is -0(C=0)0-. In embodiments, L2 is -S-. In embodiments, L2 is -0-.
[00348] In embodiments, L3 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-. In embodiments, L3 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-. In embodiments, L3 is a bond,
-0(C=0)- or -(C=0)0-.
[00349] In embodiments, L3 is a bond. In embodiments, L3 is -0(C=0)-. In embodiments, L3 is
-(C=0)0-. In embodiments, L3 is -C(=0)-. In embodiments, L3 is -0(C=0)0-. In embodiments, L3 is -S-. In embodiments, L3 is -0-.
[00350] In embodiments, L4 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-. In embodiments, L4 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-. In embodiments, L4 is a bond,
-0(C=0)- or -(C=0)0-.
[00351] In embodiments, L4 is a bond. In embodiments, L4 is -0(C=0)-. In embodiments, L4 is
-(C=0)0-. In embodiments, L4 is -C(=0)-. In embodiments, L4 is -0(C=0)0-. In embodiments, L4 is -S-. In embodiments, L4 is -0-.
[00352] In embodiments, L5 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-. In embodiments, L5 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-. In embodiments, L5 is a bond, -O(C=O)- or -(C=O)O-. [00353] In embodiments, L5 is a bond. In embodiments, L5 is -O(C=O)-. In embodiments, L5 is 5  -(C=O)O-. In embodiments, L5 is ‑C(=O)‑. In embodiments, L5 is ‑O(C=O)O‑. In embodiments, L5 is ‑S‑. In embodiments, L5 is ‑O‑. [00354] In embodiments, L6 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑. In embodiments, L6 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑. In embodiments, L6 is a bond, 10  -O(C=O)- or -(C=O)O-. [00355] In embodiments, L6 is a bond. In embodiments, L6 is -O(C=O)-. In embodiments, L6 is -(C=O)O-. In embodiments, L6 is ‑C(=O)‑. In embodiments, L6 is ‑O(C=O)O‑. In embodiments, L6 is ‑S‑. In embodiments, L6 is ‑O‑. 15  [00356] In embodiments, L7 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑. In embodiments, L7 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑. In embodiments, L7 is a bond, -O(C=O)- or -(C=O)O-. [00357] In embodiments, L7 is a bond. In embodiments, L7 is -O(C=O)-. In embodiments, 20  L7 is -(C=O)O-. In embodiments, L7 is ‑C(=O)‑. In embodiments, L7 is ‑O(C=O)O‑. In embodiments, L7 is ‑S‑. In embodiments, L7 is ‑O‑. [00358] In embodiments, La1 and La2 are each independently
Figure imgf000109_0001
each X is independently O a1 a2
Figure imgf000109_0002
or S. In embodiments, L and L are each     independently
Figure imgf000110_0009
, where each X is independently O or S. In embodiments, Lal and
L are each independently
Figure imgf000110_0001
, where each X is independently O.
[00359] In embodiments, Lal and La2 are each independently
Figure imgf000110_0002
,
L
Figure imgf000110_0007
and L are each independently . In embodiments, L and L are each independently
Figure imgf000110_0003
. In embodiments, La and La are each independently
Figure imgf000110_0004
Figure imgf000110_0005
. In embodiments, Lal and La2 are each independently X . In embodiments, Lal and La2 are each independently
Figure imgf000110_0006
embodiments, Lal and La2
Figure imgf000110_0008
[00360] In embodiments, W1, W2, W3, W4, W5, and W6 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkylene. In embodiments, W1, W2, W3, W4, W5, and W6 are each independently substituted with one or more substituent groups. In embodiments, W1, W2,
W3, W4, W5, and W6 are each independently substituted with one or more size-limited substituent groups. In embodiments, W1, W2, W3, W4, W5, and W6 are each independently substituted with one or more lower substituent groups.
[00361] In embodiments, W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene. In embodiments, W1, W2, W3, W4, W5, and W6 are each independently a bond. In embodiments, W1, W2, W3, W4, W5, and W6 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene. In embodiments, W1, W2, W3, W4, W5, and W6 are each independently unsubstituted C1-C12 alkylene.
[00362] In embodiments, each R1A and R1B is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R1A and R1B is independently substituted with is independently substituted with one or more substituent groups. In embodiments, each R1A and R1B is independently substituted with one or more size-limited substituent groups. In embodiments, each R1A and R1B is independently substituted with one or more lower substituent groups.
[00363] In embodiments, R1A and R1B substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered) or substituted or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, a substituted heterocycloalkyl or substituted heteroaryl formed by the joining of R1A and R1B substituents bonded to the same nitrogen atom is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted heterocycloalkyl or substituted heteroaryl is substituted with a plurality of groups selected from substituent groups, size- limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when a heterocycloalkyl formed by the joining of R1A and R1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R1A and R1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R1A and R1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group. In embodiments, when a heteroaryl formed by the joining of R1A and R1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heteroaryl formed by the joining of R1A and R1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heteroaryl formed by the joining of R1A and R1B substituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group.
[00364] In embodiments, each R1Ais independently H or substituted or unsubstituted Ci- Ci2 alkyl. In embodiments, each R1Ais independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl. In embodiments, each R1Ais independently H. In embodiments, each R1Ais independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each R1Ais independently unsubstituted C1-C12 alkyl.
[00365] In embodiments, R1 is H, -OR1A or substituted or unsubstituted heteroalkyl. [00366] L1 is a bond, -NR101C(=S)-, -C(=S)NR101-, -0(C=0)-, -(C=0)0-, or -O-.
[00367] B1 is a bond or a substituted or unsubstituted alkylene.
[00368] B2 and B3 are each independently a bond or substituted or unsubstituted alkylene. [00369] L2 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -O-, or -S-.
[00370] L4 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -O-, or -S-.
[00371] W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
[00372] Lal and La2 are each independently
Figure imgf000112_0001
each X is independently O or S. [00373] L3 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-.
[00374] L5 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-.
[00375] L6 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-.
[00376] V is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, -0-, or -S-.
[00377] R2 is H or substituted or unsubstituted alkyl.
[00378] R3 is H or substituted or unsubstituted alkyl.
[00379] R4 is H or substituted or unsubstituted alkyl.
[00380] R5 is H or substituted or unsubstituted alkyl.
[00381] each R1A is independently H or substituted or unsubstituted C1-C12 alkyl, and
[00382] each R101 is independently H or substituted or unsubstituted 2 to 12 membered heteroalkyl.
[00383] In embodiments, R1 is H, -OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl.
[00384] L1 is a bond, -NR101C(=S)-, or -C(=S)NR101-.
[00385] B1 is a bond or an unsubstituted Ci-Cs alkylene.
[00386] B2 and B3 are each independently a bond or substituted or unsubstituted Ci-Cx alkylene.
[00387] L2 is a bond, -0(C=0)-, or -(C=0)0-.
[00388] L4 is a bond, -0(C=0)-, or -(C=0)0-.
[00389] W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
[00390]
Figure imgf000113_0001
each X is independently
O or S.
[00391] L3 is a bond, -0(C=0)-, or -(C=0)0-.
[00392] L5 is a bond, -0(C=0)-, or -(C=0)0-.
[00393] L6 is a bond, -0(C=0)-, or -(C=0)0-.
[00394] L7 is a bond, -0(C=0)-, or -(C=0)0-.
[00395] R2 is H or substituted or unsubstituted C1-C12 alkyl.
[00396] R3 is H or substituted or unsubstituted C1-C12 alkyl.
[00397] R4 is H or substituted or unsubstituted C1-C12 alkyl. [00398] R5 is H or substituted or unsubstituted C1-C12 alkyl, and
[00399] each R101 is independently substituted or unsubstituted 2 to 12 membered heteroalkyl.
[00400] In embodiments, R1 is -OH or methoxy. L1 is a bond. [00401] B1 is an unsubstituted Ci-Cs alkylene.
[00402] B2 and B3 are each independently a bond or substituted or unsubstituted Ci-Cs alkylene;
[00403] L2 is a bond. L4 is a bond.
[00404] W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene.
[00405]
Figure imgf000114_0001
each X is independently
O.
[00406] L3 is a bond. L5 is a bond. L6 is a bond. L7 is a bond. R2 is H or substituted or unsubstituted C1-C12 alkyl. R3 is H or substituted or unsubstituted C1-C12 alkyl. [00407] R4 is H or substituted or unsubstituted C1-C12 alkyl, and R5 is H or substituted or unsubstituted C1-C12 alkyl;
[00408] In embodiments, R1 is substituted or unsubstituted heteroalkyl.
[00409] L1 is -C(=S)NR101-, where the carbon atom is connected to the nitrogen atom in formula (I). [00410] B1 is a bond. B2 and B3 are each independently a bond or substituted or unsubstituted Ci-Cs alkylene. L2 is a bond, -0(C=0)-, or -(C=0)0-. L4 is a bond, -0(C=0)-, or -(C=0)0-.
[00411] W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene. [00412]
Figure imgf000114_0002
each X is independently
O. L3 is a bond.
[00413] L5 is a bond. L6 is a bond. L7 is a bond. R2 is H or substituted or unsubstituted Ci- C12 alkyl. [00414] R3 is H or substituted or unsubstituted C1-C12 alkyl. R4 is H or substituted or unsubstituted C1-C12 alkyl, and R5 is H or substituted or unsubstituted C1-C12 alkyl.
[00415] In embodiments, each R2A, R3A, R4A, and R5Ais independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl. In embodiments, each R2A, R3A, R4A, and R5Ais independently H. In embodiments, each R2A, R3A, R4A, and R5Ais independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C30 alkyl. In embodiments, each R2A, R3A, R4A, and R5Ais independently unsubstituted C1-C30 alkyl. In embodiments, each R2A, R3A, R4A, and R5Ais independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, each R2A, R3A, R4A, and R5Ais independently unsubstituted 2 to 30 membered heteroalkyl.
[00416] In embodiments, each R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently H. In embodiments, each R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently unsubstituted C1-C12 alkyl. In embodiments, each R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently unsubstituted 2 to 12 membered heteroalkyl.
[00417] In embodiments, each s is an integer from 1 to 4. In embodiments, each s is 1. In embodiments, each s is 2. In embodiments, each s is 3. In embodiments, each s is 4.
[00418] In embodiments, the cationic lipid of formula (I) is:
Figure imgf000116_0001
[00426] In an aspect, provided herein is cationic lipid of formula (II): [00427]
Figure imgf000117_0001
(II), [00428] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof. [00429] B4 is W7-La3-W8, where W7 and W8 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene, and La3 is a bond, - O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CRa31Ra32)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NRa31C(=O)‑, ‑C(=O)NRa31‑, ‑NRa31C(=O)NRa32‑, ‑NRa31C(=S)‑, ‑C(=S)NRa31‑, ‑NRa31C(=S)NRa32‑, ‑OC(=O)NRa31‑, ‑NRa31C(=O)O‑, ‑SC(=O)NRa31‑ or ‑NRa31C(=O)S‑. [00430] R10 and R11 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl. [00431] B5, B6, and B7 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. [00432] L8 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR801R802)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR801C(=O)‑, ‑C(=O)NR801‑, ‑NR801C(=O)NR802‑, ‑NR801C(=S)‑, ‑C(=S)NR801‑, ‑NR801C(=S)NR802‑, ‑OC(=O)NR801‑, ‑NR801C(=O)O‑, ‑SC(=O)NR801‑ or ‑NR801C(=O)S‑. [00433] L9 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR901R902)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR901C(=O)‑, ‑C(=O)NR901‑, ‑NR901C(=O)NR902‑, ‑NR901C(=S)‑, ‑C(=S)NR901‑, ‑NR901C(=S)NR902‑, ‑OC(=O)NR901‑, ‑NR901C(=O)O‑, ‑SC(=O)NR901‑ or ‑NR901C(=O)S‑. [00434] L10 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR110R111)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR110C(=O)‑, ‑C(=O)NR110‑, ‑NR110C(=O)NR111‑, ‑NR110C(=S)‑, ‑C(=S)NR110‑, ‑NR110C(=S)NR111‑, ‑OC(=O)NR110‑, ‑NR110C(=O)O‑, ‑SC(=O)NR110‑ or ‑NR110C(=O)S‑. [00435] R7, R8, and R9 are each independently H, substituted or unsubstituted C1-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl. [00436] each Ra31 and Ra32 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.     [00437] each R801, R802, R901, R902, R110, and R111 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl. [00438] each s is independently an integer from 1 to 4. [00439] In embodiments, W7 and W8 are each independently a bond or substituted (e.g.  with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, W7 and  W8 are each independently substituted with one or more substituent groups. In embodiments, W7 and W8 are each independently substituted with one or more size-limited substituent groups. In embodiments, W7 and W8 are each independently substituted with one or more lower substituent groups. [00440] In embodiments, W7 and W8 are each independently substituted (e.g. with a  substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, W7 and W8 are each independently unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, W7 and W8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, W7 and W8 are each independently unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, W7 and W8 are each independently a bond.   [00441] In embodiments, W7 and W8 are each independently a bond or substituted or unsubstituted C1-C8 alkylene. In embodiments, W7 and W8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C8 alkylene. In embodiments, W7 and W8 are each independently unsubstituted C1-C8 alkylene.   [00442] In embodiments, W7 and W8 are each independently a bond or substituted or unsubstituted C2-C4 alkylene. In embodiments, W7 and W8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group)     C2-C4 alkylene. In embodiments, W7 and W8 are each independently unsubstituted C2-C4 alkylene. [00443] In embodiments, W7 and W8 are each independently a bond or unsubstituted C2-C4 alkylene. In embodiments, W7 and W8 are each independently a bond, ethylene, propylene, butylene, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) ethylene, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) propylene, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) butylene. [00444] In embodiments, La3 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, or ‑C(=O)‑. In  embodiments, La3 is a bond. In embodiments, La3 is -O(C=O)-. In embodiments, La3 is - (C=O)O-. In embodiments, La3 is ‑O(C=O)O‑. In embodiments, La3 is ‑C(=O)‑. [00445] In embodiments, R10 and R11 are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl), substituted (e.g. with a  substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl), or R10 and R11 together with the nitrogen atom to which they are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered  heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl) or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R10 and R11 are each independently substituted with one or more substituent groups. In embodiments, R10 and R11 are each independently substituted with one or more size-limited substituent groups. In embodiments, R10 and R11 are each independently substituted with one or more lower substituent groups. [00446] In embodiments, R10 and R11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g.,  C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R10 and R11 are each independently unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl). In embodiments, R10 and R11 are each independently substituted (e.g. with a substituent group, a     size-limited substituent group or a lower substituent group) heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R10 and R11 are each independently unsubstituted heteroalkyl (e.g., 2 to 30 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, a substituted heterocycloalkyl or substituted heteroaryl formed by the joining of R10 and R11 groups bonded to the same nitrogen atom is substituted with at least one substituent group, size-limited substituent group, or lower substituent group. If the substituted heterocycloalkyl or substituted heteroaryl is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when a heterocycloalkyl formed by the joining of R10 and R11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R10 and R11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R10 and R11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group. In embodiments, when a heteroaryl formed by the joining of R10 and R11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heteroaryl formed by the joining of R10 and R11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heteroaryl formed by the joining of R10 and R11 groups bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group.
[00447] In embodiments, R10 and R11 together with the nitrogen atom to which they are connected form substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl). In embodiments, R10 and R11 together with the nitrogen atom to which they are connected form unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl or 5 to 6 membered heterocycloalkyl). In embodiments, R10 and R11 together with the nitrogen atom to which they are connected form substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R10 and R11 together with the nitrogen atom to which they are connected form unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). [00448] In embodiments, R10 and R11 are each independently H, substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl. In embodiments, R10 and R11 are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or  C1-C4 alkyl) or R10 and R11 together with the nitrogen atom to which they are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl. In embodiments, R10 and R11 are each independently H [00449] In embodiments, R10 and R11 are each independently substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl. In embodiments, R10 and R11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C30 alkyl, C1-C8 alkyl, or C1-C4 alkyl) or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl). [00450] In embodiments, R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, butyl, pentyl or hexyl. In embodiments, R10 and R11 are each independently substituted or unsubstituted methyl, ethyl or propyl.   [00451] In embodiments, R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl. [00452] In embodiments, R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they  are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 3 to 8 membered heterocycloalkyl.     [00453] In embodiments, R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl. [00454] In embodiments, R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 5 to 6 membered heterocycloalkyl. [00455] In embodiments, R10 and R11 together with the nitrogen atom to which they are connected form a substituted (e.g. with a substituent group, a size-limited substituent group or  a lower substituent group) 5 to 6 membered heterocycloalkyl. In embodiments, R10 and R11 together with the nitrogen atom to which they are connected form unsubstituted 5 to 6 membered heterocycloalkyl. [00456] In embodiments, B5, B6, and B7 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or  unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, B5, B6, and B7 are each independently substituted with one or more substituent groups. In embodiments, B5, B6, and B7 are each independently substituted with one or more size- limited substituent groups. In embodiments, B5, B6, and B7 are each independently substituted with one or more lower substituent groups. [00457] In embodiments, B5, B6, and B7 are each independently a bond. In embodiments, B5, B6, and B7 are each independently substituted (e.g. with a substituent group, a size-limited  substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B5, B6, and B7 are each independently unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B5, B6, and B7 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to  30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, B5, B6, and B7 are each independently unsubstituted     heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).
[00458] In embodiments, B5 is a bond.
[00459] In embodiments, B6 and B7 are each independently a bond or substituted or unsubstituted alkylene. In embodiments, B6 and B7 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, Ci-Cx alkylene, or C1-C4 alkylene).
[00460] In embodiments, B6 and B7 are each independently a bond or substituted or unsubstituted C i-Cx alkylene. In embodiments, B6 and B7 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted Ci-Cx alkylene. In embodiments, B6 and B7 are each independently a bond. In embodiments, B6 and B7 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) Ci-Cx alkylene. In embodiments, B6 and B7 are each independently unsubstituted Ci-Cx alkylene. [00461] In embodiments, B6 and B7 are each independently a bond or substituted or unsubstituted C2-C4 alkylene. In embodiments, B6 and B7 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C2-C4 alkylene. In embodiments, B6 and B7 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C2-C4 alkylene. In embodiments, B6 and B7 are each independently unsubstituted C2-C4 alkylene.
[00462] In embodiments, B6 and B7 are each independently a bond or unsubstituted C2-C4 alkylene. In embodiments, B6 and B7 are each independently a bond, ethylene, propylene, or butylene. In embodiments, B6 and B7 are each independently a bond. In embodiments, B6 and B7 are each independently ethylene. In embodiments, B6 and B7 are each independently propylene. In embodiments, B6 and B7 are each independently butylene.
[00463] In embodiments, L8 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-. In embodiments, L8 is a bond. In embodiments, L8 is -0(C=0)-. In embodiments, L8 is -(C=0)0-. In embodiments, L8 is -C(=0)-.
[00464] In embodiments, L9 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-. In embodiments, L9 is -0(C=0)- or -(C=0)0-. In embodiments, L9 is a bond. In embodiments, L9 is -0(C=0)-. In embodiments, L9 is -(C=0)0-. In embodiments, L9 is -C(=0)-.
[00465] In embodiments, L10 is a bond, -0(C=0)-, -(C=0)0-, or -C(=0)-. In embodiments, L10 is
-0(C=0)- or -(C=0)0-. In embodiments, L10 is a bond. In embodiments, L10 is -0(C=0)-. In embodiments, L10 is -(C=0)0-. In embodiments, L10 is -C(=0)-.
[00466] In embodiments, R7, R8, and R9 are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl. In embodiments, R7, R8, and R9 are each independently substituted with one or more substituent groups. In embodiments, R7, R8, and R9 are each independently substituted with one or more size-limited substituent groups. In embodiments, R7, R8, and R9 are each independently substituted with one or more lower substituent groups.
[00467] In embodiments, R7, R8, and R9 are each independently H. In embodiments, R7, R8, and R9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C30 alkyl. In embodiments, R7, R8, and R9 are each independently unsubstituted C1-C30 alkyl. In embodiments, R7, R8, and R9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R7, R8, and R9 are each independently unsubstituted 2 to 30 membered heteroalkyl.
[00468] In embodiments, R7, R8, and R9 are each independently H or substituted or unsubstituted C1-C30 alkyl. In embodiments, R7, R8, and R9 are each independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl. In embodiments, R7, R8, and R9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C30 alkyl.
[00469] In embodiments, R7, R8, and R9 are each independently substituted or unsubstituted C1-C20 alkyl. In embodiments, R7, R8, and R9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl. In embodiments, R7, R8, and R9 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl. In embodiments, R7, R8, and R9 are each independently unsubstituted C1-C20 alkyl. [00470] In embodiments, each Ra31 and Ra32 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each Ra31 and Ra32 is independently H. In embodiments, each Ra31 and Ra32 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each Ra31 and  Ra32 is independently unsubstituted C1-C12 alkyl. In embodiments, each Ra31 and Ra32 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each Ra31 and Ra32 is independently unsubstituted 2 to 12 membered heteroalkyl. [00471] In embodiments, each R801, R802, R901, R902, R110, and R111 is independently H,  substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R801, R802, R901, R902, R110, and R111 is independently H. In embodiments, each R801, R802, R901, R902, R110, and R111 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each R801, R802, R901, R902, R110, and R111 is independently unsubstituted C1-C12 alkyl. In embodiments, each R801, R802, R901, R902, R110, and R111 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each  R801, R802, R901, R902, R110, and R111 is independently unsubstituted 2 to 12 membered heteroalkyl. [00472] In embodiments, each s is independently an integer from 1 to 4. In embodiments, each s is 1. In embodiments, each s is 2. In embodiments, each s is 3. In embodiments, each s is 4. [00473] In embodiments, W7 and W8 are each independently a bond or substituted or unsubstituted alkylene. La3 is a bond.     [00474] R10 and R11 are each independently H, substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl. B5 is a bond. B6 and B7 are each independently a bond or substituted or unsubstituted alkylene. L8 is a bond. L9 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑. [00475] L10 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑, and R7, R8, and R9 are each independently H or substituted or unsubstituted C1-C30 alkyl. [00476] In embodiments, W7 and W8 are each independently a bond or substituted or unsubstituted C1-C8 alkylene. La3 is a bond.   [00477] R10 and R11 are each independently substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl. B5 is a bond. B6 and B7 are each independently a bond or substituted or unsubstituted C1-C8 alkylene. L8 is a bond. L9 is -O(C=O)- or -(C=O)O-. L10 - O(C=O)- or -(C=O)O-, and R7, R8, and R9 are each independently substituted or   unsubstituted C1-C20 alkyl. [00478] In embodiments, W7 and W8 are each independently a bond or substituted or unsubstituted C2-C4 alkylene. La3 is a bond. [00479] R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl. B5 is a bond. B6 and B7 are each independently a bond or substituted or unsubstituted C2-C4 alkylene. L8 is a bond. L9 is -O(C=O)- or -(C=O)O-. [00480] L10 -O(C=O)- or -(C=O)O-. R7 is H or methyl, and R8, and R9 are each independently substituted or unsubstituted C1-C20 alkyl.   [00481] In embodiments, W7 and W8 are each independently a bond or unsubstituted C2-C4 alkylene. La3 is a bond. [00482] R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl. B5 is a bond. B6 and B7  are each independently a bond or unsubstituted C2-C4 alkylene. L8 is a bond. L9 is -O(C=O)- or -(C=O)O-. L10 is     -O(C=O)- or -(C=O)O-. R7 is H or methyl, and R8 and R9 are each independently substituted or unsubstituted C1-C20 alkyl. [00483] In embodiments, W7 and W8 are each independently a bond or unsubstituted C2-C4 alkylene. La3 is a bond.   [00484] R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl. B5, B6, and B7 are each independently a bond. [00485] L8 is a bond. L9 is a bond. L10 is a bond. R7 is H or methyl, and R8 and R9 are each  independently substituted or unsubstituted C1-C30 alkyl. [00486] In embodiments, the cationic lipid of formula (II) is: [00487] ,  
Figure imgf000127_0001
,    
Figure imgf000128_0001
Figure imgf000129_0001
[00508] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof.
Figure imgf000130_0001
. [00512] Q is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene. [00513] V is substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted arylene. [00514] B8, B9, B10, and B11 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. [00515] L12 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR210R211)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR210C(=O)‑, ‑C(=O)NR210‑, ‑NR210C(=O)NR211‑, ‑NR210C(=S)‑, ‑C(=S)NR210‑, ‑NR210C(=S)NR211‑, ‑OC(=O)NR210‑, ‑NR210C(=O)O‑, ‑SC(=O)NR210‑ or ‑NR210C(=O)S‑. [00516] L13 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR310R311)sO-,  ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR310C(=O)‑, ‑C(=O)NR310‑, ‑NR310C(=O)NR311‑, ‑NR310C(=S)‑, ‑C(=S)NR310‑, ‑NR310C(=S)NR311‑, ‑OC(=O)NR310‑, ‑NR310C(=O)O‑, ‑SC(=O)NR310‑ or ‑NR310C(=O)S‑. [00517] R12 is H, -OR12A, -SR12A, -NR12A, -CN, -(C=O)R12A, -O(C=O)R12A, -(C=O)OR12A, -NR12A(C=O)-R12B, -(C=O)NR12AR12B. [00518] R13 is H, -OR13A, -SR13A, -NR13A, -CN, -(C=O)R13A, -O(C=O)R13A, -(C=O)OR13A, -NR13A(C=O)-R13B, -(C=O)NR13AR13B.     [00519] R14 and R15 are each independently substituted or unsubstituted C2-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl. [00520] R12A, R12B, R13A, and R13B are each independently H, substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted 2 to 20 membered heteroalkyl. [00521] each R210, R211, R310, and R311 is independently H, substituted or unsubstituted C1- C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl. [00522] each n is independently an integer from 0 to 8, and [00523] each s is independently an integer from 1 to 4. [00524] In embodiments, L11 is
Figure imgf000131_0001
Figure imgf000131_0002
, where n is an integer from 0 to 8, V is substituted or unsubstituted alkylene, and Q is substituted or unsubstituted alkylene. [00525] In embodiments, L11 is
Figure imgf000131_0003
, where V is substituted or unsubstituted alkylene. In embodiments, L11 is
Figure imgf000131_0004
, where V is  substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). [00526] In embodiments, L11 is
Figure imgf000131_0005
, where V is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group)  alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, L11 is
Figure imgf000131_0006
, where V is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).     [00527] In embodiments, L11 is
Figure imgf000132_0009
where n is an integer from 0 to 8. In embodiments,
Figure imgf000132_0001
n is an integer from 0 to 4. [00528] In embodiments,
Figure imgf000132_0002
embodiments,
Figure imgf000132_0003
[00529] In embodiments,
Figure imgf000132_0004
[00530] In embodiments,
Figure imgf000132_0005
[00531] In embodiments,
Figure imgf000132_0006
[00532] In embodiments, L11 is
Figure imgf000132_0007
, where Q is substituted or   unsubstituted alkylene. In embodiments, L11 is
Figure imgf000132_0008
, where Q is substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene).     [00533] In embodiments, L11 where Q is substituted (e.g.
Figure imgf000133_0002
with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, L11 is
Figure imgf000133_0003
, where Q is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8  alkylene, or C1-C4 alkylene).
Figure imgf000133_0001
. [00535] In embodiments, Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30  alkylene, C1-C8 alkylene, or C1-C4 alkylene), substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene), substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted arylene (e.g., C6-C10 arylene,  C10 arylene, or phenylene), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene). In embodiments, Q is substituted with one or more substituent groups. In embodiments, Q is substituted with one or more size-limited substituent groups. In embodiments, Q is   substituted with one or more lower substituent groups.     [00536] In embodiments, Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, Q is unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, Q is unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, Q is substituted (e.g. with a substituent group, a size-  limited substituent group or a lower substituent group) cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, Q is unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkylene (e.g., 3 to 8 membered  heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene). In embodiments, Q is unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene). In embodiments, Q is substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene). In embodiments, Q is unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene). In embodiments, Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to 9 membered heteroarylene, or 5 to 6 membered heteroarylene). In embodiments, Q is unsubstituted heteroarylene (e.g., 5 to 10 membered heteroarylene, 5 to  9 membered heteroarylene, or 5 to 6 membered heteroarylene). [00537] In embodiments, Q is substituted or unsubstituted alkylene. In embodiments, Q is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). [00538] In embodiments, V is substituted (e.g. with a substituent group, a size-limited  substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene), substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted cycloalkylene (e.g.,     C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene). In embodiments, V is substituted with one or more substituent groups. In embodiments, V is substituted with one  or more size-limited substituent groups. In embodiments, V is substituted with one or more lower substituent groups. [00539] In embodiments, V is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, V is unsubstituted alkylene (e.g., C1-C30  alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, V is substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, V is unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, V is substituted (e.g. with a substituent group, a size-limited  substituent group or a lower substituent group) arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene). In embodiments, V is unsubstituted arylene (e.g., C6-C10 arylene, C10 arylene, or phenylene). [00540] In embodiments, V is substituted or unsubstituted alkylene. In embodiments, V is substituted (e.g. with a substituent group, a size-limited substituent group or a lower   substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). [00541] In embodiments, B8, B9, B10, and B11 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower   substituent group) or unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, B8, B9, B10, and B11 are each independently substituted with one or more substituent groups. In embodiments, B8, B9, B10, and B11 are each independently substituted with one or more size- limited substituent groups. In embodiments, B8, B9, B10, and B11 are each independently substituted with one or more lower substituent groups. [00542] In embodiments, B8, B9, B10, and B11 are each independently a bond. In embodiments, B8, B9, B10, and B11 are each independently substituted (e.g. with a substituent     group, a size-limited substituent group or a lower substituent group) alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B8, B9, B10, and B11 are each independently unsubstituted alkylene (e.g., C1-C30 alkylene, C1-C8 alkylene, or C1-C4 alkylene). In embodiments, B8, B9, B10, and B11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene). In embodiments, B8, B9, B10, and B11 are each independently unsubstituted heteroalkylene (e.g., 2 to 30 membered heteroalkylene, 2 to 8 membered heteroalkylene, or 2 to 4 membered heteroalkylene).   [00543] In embodiments, B8, B9, B10, and B11 are each independently substituted or unsubstituted alkylene. In embodiments, B8, B9, B10, and B11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkylene (e.g. with a substituent group, a size-limited substituent group or a lower substituent group).   [00544] In embodiments, B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C20 alkylene. In embodiments, B8, B9, B10, and B11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkylene. In embodiments, B8, B9, B10, and B11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkylene. In embodiments, B8, B9, B10, and B11 are each independently unsubstituted C1-C20 alkylene. [00545] In embodiments, B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C8 alkylene. In embodiments, B8, B9, B10, and B11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C8 alkylene. In embodiments, B8, B9, B10, and B11 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C8 alkylene. In embodiments, B8, B9, B10, and B11 are each independently unsubstituted C1-C8 alkylene. [00546] In embodiments, L12 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, or  ‑O‑. In embodiments, L12 is a bond. In embodiments, L12 is -O(C=O)-. In embodiments, L12 is -(C=O)O-. In embodiments, L12 is ‑O(C=O)O‑. In embodiments, L12 is‑C(=O)‑. In embodiments, L12 is ‑O‑.     [00547] In embodiments, L12 is -0(C=0)- or -(C=0)0-.
[00548] In embodiments, L13 is a bond, -0(C=0)-, -(C=0)0-, -0(C=0)0-, -C(=0)-, or -0-. In embodiments, L13 is a bond. In embodiments, L13 is -0(C=0)-. In embodiments, L13 is -(C=0)0-. In embodiments, L13 is -0(C=0)0-. In embodiments, L13 is-C(=0)-. In embodiments, L13 is -0-.
[00549] In embodiments, L13 is -0(C=0)- or -(C=0)0-.
[00550] In embodiments, R12 is H, -OR12A, -SR12A, -NR12A, -CN, or -(C=0)R12A In embodiments, R12 is H, -OR12A, or -NR12A. In embodiments, R12 is H or -OR12A.
[00551] In embodiments, R12 is H. In embodiments, R12 is -OR12A. In embodiments, R12 is -SR12A. In embodiments, R12 is -NR12A. In embodiments, R12 is CN. In embodiments, R12 is -
(C=0)R12A.
[00552] In embodiments, R12 is -OH, methoxy, or ethoxy.
[00553] In embodiments, R13 is H, -OR13A, -SR13A, -NR13A, -CN, or -(C=0)R13A. In embodiments, R13 is H, -OR13A, or -NR13A. In embodiments, R13 is H or -OR13A. [00554] In embodiments, R13 is H. In embodiments, R13 is -OR13A. In embodiments, R13 is
-SR13A. In embodiments, R13 is -NR13A. In embodiments, R13 is CN. In embodiments, R13 is - (C=0)R13A.
[00555] In embodiments, R13 is -OH, methoxy, or ethoxy.
[00556] In embodiments, R12A and R13A are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl. In embodiments, R12A and R13A are each independently substituted with one or more substituent groups. In embodiments, R12A and R13A are each independently substituted with one or more size-limited substituent groups. In embodiments, R12A and R13A are each independently substituted with one or more lower substituent groups.
[00557] In embodiments, R12A and R13A are each independently H. In embodiments, R12A and R13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl. In embodiments, R12A and R13A are each independently unsubstituted C1-C20 alkyl. In embodiments, R12A and R13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R12A and R13A are each independently unsubstituted 2 to 30 membered heteroalkyl.
[00558] In embodiments, R12A and R13A are each independently H, substituted or unsubstituted C1-C20 alkyl. In embodiments, R12A and R13A are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl. In embodiments, R12A and R13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl. In embodiments, R12A and R13A are each independently unsubstituted C1-C20 alkyl. [00559] In embodiments, R12A and R13A are each independently H, substituted or unsubstituted Ci-Cs alkyl. In embodiments, R12A and R13A are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted Ci-Cs alkyl. In embodiments, R12A and R13A are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) Ci-Cs alkyl. In embodiments, R12A and R13A are each independently unsubstituted Ci-Cs alkyl.
[00560] In embodiments, R12B and R13B are each independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C20 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl.
[00561] In embodiments, R12B and R13B are each independently H. In embodiments, R12B and R13B are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C20 alkyl. In embodiments, R12B and R13B are each independently unsubstituted C1-C20 alkyl. In embodiments, R12B and R13B are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R12B and R13B are each independently unsubstituted 2 to 30 membered heteroalkyl.
[00562] In embodiments, R14 and R15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C2-C30 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 30 membered heteroalkyl. In embodiments, R14 and R15 are each independently substituted with one or more substituent groups. In embodiments, R14 and R15 are each independently substituted with one or more size-limited substituent groups. In embodiments, R14 and R15 are each independently substituted with one or more lower substituent groups.
[00563] In embodiments, R14 and R15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C2-C30 alkyl. In embodiments, R14 and R15 are each independently unsubstituted C2-C30 alkyl. In embodiments, R14 and R15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 30 membered heteroalkyl. In embodiments, R14 and R15 are each independently unsubstituted 2 to 30 membered heteroalkyl.
[00564] In embodiments, R14 and R15 are each independently substituted or unsubstituted C2-C30 alkyl. In embodiments, R14 and R15 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C2-C30 alkyl.
[00565] In embodiments, each R210, R211, R310, and R311 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R210, R211, R310, and R311 is independently substituted with one or more substituent groups. In embodiments, each R210, R211, R310, and R311 is independently substituted with one or more size-limited substituent groups. In embodiments, each R210, R211, R310, and R311 is independently substituted with one or more lower substituent groups.
[00566] In embodiments, each R210, R211, R310, and R311 is independently H. In embodiments, each R210, R211, R310, and R311 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each R210, R211, R310, and R311 is independently unsubstituted C1-C12 alkyl.
In embodiments, each R210, R211, R310, and R311 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl. In embodiments, each R210, R211, R310, and R311 is independently unsubstituted 2 to 12 membered heteroalkyl. [00567] In embodiments, each n is independently an integer from 0 to 8. In embodiments, each n is independently an integer from 0 to 4. In embodiments, each n is independently 8. In embodiments, each n is independently 7. In embodiments, each n is independently 6. In embodiments, each n is independently 5. In embodiments, each n is independently 4. In embodiments, each n is independently 3. In embodiments, each n is independently 2. In embodiments, each n is independently 1. In embodiments, each n is independently 0.
[00568] In embodiments, each s is an integer from 1 to 4. In embodiments, each s is 4. In embodiments, each s is 3. In embodiments, each s is 2. In embodiments, each s is 1.
[00569] In embodiments,
Figure imgf000140_0001
is substituted or unsubstituted alkylene and each n is independently an integer from 0 to 8. [00572] B8, B9, B10, and B11 are each independently substituted or unsubstituted alkylene.
[00573] L12 is -0(C=0)- or -(C=0)0-. L13 is -0(C=0)- or -(C=0)0-. R12 is H, -OR12A, or-
NR12A
[00574] R13 is H, -OR13A, or-NR13A. R14 and R15 are each independently substituted or unsubstituted C2-C30 alkyl. R12A and R13A are each independently H, substituted or unsubstituted C1-C20 alkyl.
[00575] In embodiments,
Figure imgf000140_0002
ubstituted or unsubstituted alkylene and each n is independently an integer from 0 to 4. [00577] B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C20 alkylene. [00578] L12 is -O(C=O)- or -(C=O)O-. L13 is -O(C=O)- or -(C=O)O-. R12 is H or -OR12A. R13 is H or -OR13A. R14 and R15 are each independently substituted or unsubstituted C2-C20 alkyl. [00579] R12A and R13A are each independently H, substituted or unsubstituted C1-C8 alkyl. [00580] [00581] In embodiments,
Figure imgf000141_0002
ubstituted or unsubstituted alkylene and each n is independently an integer from 0 to 4. [00583] B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C8 alkylene. [00584] L12 is -O(C=O)- or -(C=O)O-. L13 is -O(C=O)- or -(C=O)O-. R12 is -OH, methoxy, or ethoxy. [00585] R13 is -OH, methoxy, or ethoxy. R14 and R15 are each independently substituted or unsubstituted C2-C20 alkyl. [00586] In embodiments, the cationic lipid of formula (III) is:
Figure imgf000141_0001
  [00588]    
[00589]
Figure imgf000142_0002
[00590] or a pharmaceutically acceptable salt thereof. [00591] In an aspect, provided herein is cationic lipid of formula (IV): [00592]
Figure imgf000142_0003
[00594] or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof. [00595] B12 is -W7-La3-W8-. [00596] W7 and W8 are each independently a bond, substituted or unsubstituted C1-C12  alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene. [00597] La3 is a bond,
Figure imgf000142_0001
[00598] W9 and W10 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted 2 to 12 membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any   combination thereof. [00599] L14 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR410R411)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR410C(=O)‑, ‑C(=O)NR410‑, ‑NR410C(=O)NR411‑, -NR410C(=S)-, -C(=S)NR410‑, ‑NR410C(=S)NR411‑, ‑OC(=O)NR410‑, ‑NR410C(=O)O‑, ‑SC(=O)NR410‑ or ‑NR410C(=O)S‑. [00600] L15 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR510R511)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR510C(=O)‑, ‑C(=O)NR510‑, ‑NR510C(=O)NR511‑, -NR510C(=S)-, -C(=S)NR510‑, ‑NR510C(=S)NR511‑, ‑OC(=O)NR510‑, ‑NR510C(=O)O‑, ‑SC(=O)NR510‑ or ‑NR510C(=O)S‑.     [00601] R16 and R17 are each independently
[00602]
Figure imgf000143_0001
fragment of cationic lipid of formula (I),
[00603]
Figure imgf000143_0003
, a fragment of cationic lipid of formula (III), or
Figure imgf000143_0002
a fragment of cationic lipid of formula (III).
[00605] each R410, R411, R510, and R511 is independently H, substituted or unsubstituted Ci- Ci2 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl.
[00606] each m is independently an integer from 0 to 8, and [00607] each s is independently an integer from 1 to 4.
[00608] In embodiments, W7 and W8 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene, or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkylene. In embodiments, W7 and W8 are each independently substituted with one or more substituent groups. In embodiments, W7 and W8 are each independently substituted with one or more size-limited substituent groups. In embodiments, W7 and W8 are each independently substituted with one or more lower substituent groups.
[00609] In embodiments, W7 and W8 are each independently a bond. In embodiments, W7 and W8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene. In embodiments, W7 and W8 are each independently unsubstituted C1-C12 alkylene. In embodiments, W7 and W8 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl ene. In embodiments, W7 and W8 are each independently unsubstituted 2 to 12 membered heteroalkylene.
[00610] In embodiments, W7 and W8 are each independently a bond or substituted or unsubstituted C1-C12 alkylene. W7 and W8 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene [00611] In embodiments, W7 and W8 are each independently a bond or unsubstituted Ci-
C12 alkylene.
[00612] In embodiments, W7 and W8 are each independently a bond or unsubstituted Ci- C8 alkylene. In embodiments, W7 and W8 are each independently unsubstituted C i-Cx alkylene. [00613] In embodiments, La3 is a bond,
Figure imgf000144_0001
embodiments,
La3 is a bond. In embodiments, La3 is -S-S-. In embodiments, La3 is
Figure imgf000144_0002
. In embodiments,
Figure imgf000144_0003
[00614] In embodiments, W9 and W10 are each independently a bond, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkylene, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene), substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heterocycloalkylene(e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene), or any combination thereof. In embodiments, W9 and W10 are each independently substituted with one or more substituent groups. In embodiments, W9 and W10 are each independently substituted with one or more size-limited substituent groups. In embodiments, W9 and W10 are each independently substituted with one or more lower substituent groups.
[00615] In embodiments, W9 and W10 are each independently a bond. In embodiments, W9 and W10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene. In embodiments, W9 and W10 are each independently unsubstituted C1-C12 alkylene. In embodiments, W9 and W10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12 membered heteroalkyl ene. In embodiments, W9 and W10 are each independently unsubstituted 2 to 12 membered heteroalkylene. In embodiments, W9 and W10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) cycloalkylene (e.g., C3-C8 cycloalkylene, C3- Ce cycloalkylene, or C5-C6 cycloalkylene). In embodiments, W9 and W10 are each independently unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, W9 and W10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) heterocycloalkylene(e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene). In embodiments, W9 and W10 are each independently unsubstituted heterocycloalkylene(e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene).
[00616] In embodiments, W9 and W10 are each independently a bond or substituted or unsubstituted C1-C12 alkylene. In embodiments, W9 and W10 are each independently a bond or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkylene. In embodiments, W9 and W10 are each independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkylene. In embodiments, W9 and W10 are each independently unsubstituted C1-C12 alkylene.
[00617] In embodiments, W9 and W10 are each independently a bond or unsubstituted Ci- C8 alkylene. In embodiments, W9 and W10 are each independently unsubstituted Ci-Cx alkylene. [00618] In embodiments, L14 is-O(C=O)-, -(C=O)O-, ‑C(=O)‑, ‑NR410C(=O)‑, ‑C(=O)NR410‑, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑. In embodiments, L14 is -O(C=O)-, -(C=O)O-, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑. [00619] In embodiments, L14 is -O(C=O)-. In embodiments, L14 is -(C=O)O-. In embodiments, L14 is‑C(=O)‑. In embodiments, L14 is ‑NR410C(=O)‑. In embodiments, L14 is ‑C(=O)NR410‑. In embodiments, L14 is -NR410C(=S)-. In embodiments, L14 is -C(=S)NR410‑. In embodiments, L14 is ‑OC(=O)NR410‑. In embodiments, L14 is ‑NR410C(=O)O‑. [00620] In embodiments, L15 is-O(C=O)-, -(C=O)O-, ‑C(=O)‑, ‑NR510C(=O)‑, ‑C(=O)NR510‑, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑. In embodiments, L15 is -O(C=O)-, -(C=O)O-, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑. [00621] In embodiments, L15 is -O(C=O)-. In embodiments, L15 is -(C=O)O-. In embodiments, L15 is‑C(=O)‑. In embodiments, L15 is ‑NR510C(=O)‑. In embodiments, L15 is ‑C(=O)NR510‑. In embodiments, L15 is -NR510C(=S)-. In embodiments, L15 is -C(=S)NR510‑. In embodiments, L15 is ‑OC(=O)NR510‑. In embodiments, L5 is ‑NR510C(=O)O‑. [00622] In embodiments, each R410, R411, R510, and R511 is independently H, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl, or substituted (e.g. with a substituent group, a size-limited  substituent group or a lower substituent group) or unsubstituted 2 to 12 membered heteroalkyl. In embodiments, each R410, R411, R510, and R511 is independently substituted with one or more substituent groups. In embodiments, each R410, R411, R510, and R511 is independently substituted with one or more size-limited substituent groups. In embodiments, each R410, R411, R510, and R511 is independently substituted with one or more lower substituent  groups. [00623] In embodiments, each R410, R411, R510, and R511 is independently H. In embodiments, each R410, R411, R510, and R511 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) C1-C12 alkyl. In embodiments, each R410, R411, R510, and R511 is independently unsubstituted C1-C12 alkyl. In embodiments, each R410, R411, R510, and R511 is independently substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) 2 to 12     membered heteroalkyl. In embodiments, each R410, R411, R510, and R511 is independently unsubstituted 2 to 12 membered heteroalkyl.
[00624] In embodiments, each R410 and R510 is independently H or substituted or unsubstituted C1-C12 alkyl. In embodiments, each R410 and R510is independently H or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted C1-C12 alkyl.
[00625] In embodiments, each R410 and R510 is independently H or unsubstituted C i-Cx alkyl. In embodiments, each R410 and R510is independently unsubstituted Ci-Cx alkyl.
[00626] In embodiments, each R410 and R510 is independently H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl. In embodiments, each R410 and R510is independently H or methyl. In embodiments, each R410 and R510is independently H. In embodiments, each R410 and R510is independently methyl.
[00627] In embodiments, R16 and R17 are each independently
[00628]
Figure imgf000147_0001
fragment of cationic lipid of formula (I),
[00629]
[00630]
Figure imgf000147_0002
cationic lipid of formula (II),
Figure imgf000147_0003
, a fragment of cationic lipid of formula (III),
Figure imgf000147_0004
a fragment of cationic lipid of formula (III), where B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, L9, L2, L3, L4, L5, L6, L7, L8, L9, L10, L12, L13, R2, R3, R4, R5, R7, R8, R9, R10, R11, R12, R13, R14, and R15 are as described herein including embodiments. [00631]
[00632] In embodiments, R16 and R17 are each independently
[00633]
Figure imgf000148_0001
fragment of cationic lipid of formula (II), where B6, B7,
L9, L10, R8, and R9 are as described herein including embodiments. [00634] In embodiments, R16 and R17 are each independently
Figure imgf000148_0002
[00636] In embodiments, R16 and R17 are each independently
[00637]
Figure imgf000148_0003
[00638] In embodiments, R16 and R17 are each independently
Figure imgf000148_0004
[00640] In embodiments, each m is independently an integer from 0 to 8. In embodiments, each m is independently 8. In embodiments, each m is independently 7. In embodiments, each m is independently 6. In embodiments, each m is independently 5. In embodiments, each m is independently 4. In embodiments, each m is independently 3. In embodiments, each m is independently 2. In embodiments, each m is independently 1. In embodiments, each m is independently 0.
[00641] In embodiments, each s is an integer from 1 to 4. In embodiments, each s is 4. In embodiments, each s is 3. In embodiments, each s is 2. In embodiments, each s is 1. [00642] In embodiments, the cationic lipid of formula (IV) is:
[00643]
[00644]
[00645]
[00646]
[00647]
[00648]
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
[00658]
[00659] or a pharmaceutically acceptable salt thereof.
[00660] In some embodiments, the lipid is ALC-0315 or JK-0315-C A.
[00661] Lipid Nanoparticles
[00662] In an aspect, provided herein are lipid nanoparticles comprising one or more of the ionizable cationic lipids or salts thereof described herein. In embodiments, the lipid nanoparticles described herein further include one or more non-cationic lipids. In embodiments, the lipid nanoparticles described herein further include one or more conjugated lipids capable of reducing or inhibiting particle aggregation. In other embodiments, the lipid nanoparticles described herein further include one or more therapeutic agents such as nucleic acids (e.g., mRNA).
[00663] In embodiments, lipid nanoparticles comprising one or more ionizable cationic lipids described herein are used to encapsulate nucleic acids (e.g., mRNA) within the lipid nanoparticles.
[00664] In embodiments, the lipid nanoparticles include a therapeutic agent such as nucleic acid (e.g., mRNA), a cationic lipid (one or more ionizable cationic lipids of formula I-IV or salts thereof, as described herein, or cationic lipids known in the art), a non-cationic lipid (e.g., mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of particles (e.g., one or more PEG-lipid conjugates).
[00665] In embodiments, non-cationic lipids that can be used in the lipid nanoparticles described herein include, without limitation, neutral, zwitterionic or anionic lipids, for example:
[00666] phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl- phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
[00667] In embodiments, non-cationic lipids may be sterols such as cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof. In embodiments, the cholesterol derivative is a polar analogue such as cholesteryl- (4'-hydroxy)-butyl ether.
[00668] In embodiments, the non-cationic lipids included in the lipid nanoparticles include a mixture of one or more phospholipids and cholesterol or a derivative thereof.
[00669] In embodiments, non-cationic lipids suitable for use in the lipid nanoparticles include stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
[00670] In embodiments, lipid conjugates that can be used in the lipid nanoparticles described herein include, without limitation, PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. In embodiments, the nanoparticles comprise PEG-lipid conjugate.
[00671] In embodiments, lipid conjugates that can be used in the lipid nanoparticles described herein include, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, mPEG2000-l,2-di-0- alkyl-sn3-carbomoylglyceride (PEG-C-DOMG), l-[8'-(l,2-dimyristoyl-3-propanoxy)- carboxamido-3',6'-dioxaoctanyl]carbamoyl-co-methyl-poly(ethylene glycol) (2 KPEG- DMG), l,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG), PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.
[00672] In embodiments, lipid nanoparticles described herein are useful for the introduction of therapeutic agents such as nucleic acids (e.g., mRNA) into cells.
[00673] In an aspect, provided herein is a method for the in vivo delivery of a therapeutic agent comprising administering the lipid nanoparticle, composed of the ionizable cationic lipids of formula I-IV as described herein, to a mammal.
[00674] In embodiments, the lipid nanoparticles described herein can be administered either alone or in a mixture with a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
[00675] The pharmaceutically acceptable carrier is usually added following lipid nanoparticle formation. Thus, after the lipid nanoparticle is formed, the nanoparticle can be diluted into pharmaceutically acceptable carriers such as normal buffered saline.
[00676] For in vivo administration, administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation (e.g., intransal or intratracheal), transdermal application, or rectal administration.
[00677] In embodiments, the pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
[00678] In an aspect, provided herein is a method for preventing or treating a disease in a mammal in need thereof by administering to the mammal a therapeutically effective amount of a lipid nanoparticle composed of the ionizable cationic lipids of formula I-IV as described herein. In embodiments, provided herein is a method for preventing a disease in a mammal by administering to the mammal a therapeutically effective amount of a lipid nanoparticle composed of the ionizable cationic lipids of formula I-IV as described herein. In embodiments, provided herein is a method for treating a disease in a mammal, in need thereof, by administering to the mammal a therapeutically effective amount of a lipid nanoparticle composed of the ionizable cationic lipids of formula I-IV as described herein. [00679] In embodiments the mammal is a dog, a cat or a human. In embodiments, the mammal is a dog. In embodiments, the mammal is a cat. In embodiments, the mammal is a human.
Methods of Preventing Infection
[00680] The present disclosure provides methods for treating a subject who has been infected or is suspected of having been infected with a coronavirus infection, e.g., a MERS- CoV, SARS-CoV, or SARS-CoV-2, or an infection caused by a variant of any of these. The present disclosure also provides methods for preventing an infection caused by e.g., a MERS- CoV, SARS-CoV, or SARS-CoV-2, or an infection caused by a variant of any of these, in a subject who is at risk of being infected, who has been exposed to such an infection, or who has not yet been infected with a coronavirus infection, e.g., a MERS-CoV, SARS-CoV, or SARS-CoV-2 infection, or an infection caused by a variant of any of these, or who may be in the early stages of infection but is not yet exhibiting symptoms of a coronavirus infection. [00681] In one embodiment, such treatment can be administered to the asymptomatic subject at about 1-24 hours, about 24-48 hours, or about 48 hours to 3 days following exposure or possible exposure or suspected exposure to the coronavirus. In one embodiment, the prophylactic treatment can be administered to the asymptomatic subject at about 3-5 days, or about 5-10 days, or about 10-14 days, or about 14-21 days, or about 21-30 days or longer time ranges following exposure to the coronavirus. mRNA Vaccine Stabilization and Storage
[00682] In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized (i.e. freeze dried). In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized with a stabilizer. In embodiments, the stabilizer is a sugar. In embodiments, the sugar is for example trehalose or sucrose, but other sugars are also contemplated.
[00683] In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized and stored at room temperature. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout may be lyophilized and stored at room temperature for at least 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days. In embodiments, In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout is stable at room temperature for at least 60, at least 50, at least 40, at least 30, at least 25, at least 20, at least 15, at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4, at least 3, at least 2, or 1 day(s).
Methods of mRNA Vaccine Administration [00684] The administration route for mRNA vaccines plays an important role in determining vaccination efficacy. The most commonly used injection routes include intradermal (ID), subcutaneous (SC), intramuscular (IM), intranodal (IN), and intravenous (IV) administration. Since the immune cells and lymphoid organs are the common vaccination targets, the anatomical and physiological properties of the vaccination sites (skin, muscle, lymphoid organ, and systemic circulation) may affect the safety and efficacy of a vaccine. Such information is useful for the selection of administration route and the delivery format (carrier-mediated, naked, or cell-based) of the mRNA vaccine. Vascular and lymphatic vessels In the dermis tissue help transport mRNA vaccines and antigen presenting cells (APCs) to the draining lymph nodes to activate T and B cells, [00685] In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intradermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intramuscularly. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intranodally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered transdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered subdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered to the epidermal layer. [00686] In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intradermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intramuscularly. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered intranodally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered transdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered subdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered to the epidermal layer.
[00687] In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered directly to the lymphatic system. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system subdermally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system intramuscularly. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system intranodally. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered to the lymphatic system transdermally.
[00688] Medical devices that comprise an array of microneedles suitable for use herein are known in the art. Exemplary structures and devices comprising a means for controllably delivering one or more agents to a subject are described in International Patent Application Publication Nos. WO 2014/188343, WO 2014/132239, WO 2014/132240, WO 2013/061208, WO 2012/046149, WO 2011/135531, WO 2011/135530, WO 2011/135533, WO
2014/132240, WO 2015/16821, and International Patent Applications PCT/US2015/028154 (published as WO 2015/168214 Al), PCT/US2015/028150 (published as WO 2015/168210 Al), PCT/US2015/028158 (published as WO 2015/168215 Al), PCT/US2015/028162 (published as WO 2015/168217 Al), PCT/US2015/028164 (published as WO 2015/168219 Al), PCT/US2015/038231 (published as WO 2016/003856 Al), PCT/US2015/038232 (published as WO 2016/003857 Al), PCT/US2016/043623 (published as WO 2017/019526 Al), PCT/US2016/043656 (published as WO 2017/019535 Al), PCT/US2017/027879 (published as WO 2017/189258 Al), PCT/US2017/027891 (published as WO 2017/189259 Al), PCT/US2017/064604 (published as WO 2018/111607 Al), PCT/US2017/064609 (published as WO 2018/111609 Al), PCT/US2017/064614 (published as WO 2018/111611 Al), PCT/US2017/064642 (published as WO 2018/111616 Al), PCT/US2017/064657 (published as WO 2018/111620 Al), and PCT/US2017/064668 (published as WO 2018/111621 Al), all of which are incorporated by reference herein in their entirety.
[00689] In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are administered by applying one or more medical devices to one or more sites of the skin of a subject. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered directly to the lymphatic system. One nonlimiting example of a medical device comprising a plurality of microneedles that is suitable for use with of the methods disclosed herein is the Sofusa™ drug delivery platform available from Sorrento Therapeutics, Inc. see e.g., US Patent No: 10,737,082; International Patent Application PCT/US2019/034736 (published as WO 2019/232265) which are incorporated by reference herein in their entirety.
[00690] In embodiments, the medical device is placed in direct contact with the skin of the subject. In embodiments, an intervening layer or structure will be between the skin of the subject and the medical device. For example, surgical tape or gauze may be used to reduce possible skin irritation between the medical device and the skin of the subject. When the microneedles extend from the apparatus, they will contact and, in some instances, penetrate the epidermis or dermis of the subject in order to deliver the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout to the subject. The delivery of the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout can be to the circulatory system, the lymphatic system, the interstitium, subcutaneous, intramuscular, intradermal or a combination thereof. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered directly to the lymphatic system of the subject. In embodiments, the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout are delivered to the superficial vessels of the lymphatic system.
[00691] In embodiments, the administration or delivery target is a lymph node, a lymph vessel, an organ that is part of the lymphatic system or a combination thereof. In embodiments, the vaccination target is a lymph node. In embodiments, the administration or delivery target is a specific lymph node as described elsewhere herein.
[00692] In embodiments, the medical device may comprise a needle array in the form of a patch. In embodiments, the array of needles are able to penetrate a most superficial layer of the stratum corneum and deliver the nucleic acid molecules, compositions, pharmaceutical compositions, formulations, and vaccines disclosed herein and throughout to at least a portion or all of the non-viable epidermis, at least a portion of or all of the viable epidermis, and/or at least a portion of the viable dermis of a subject and subsequently to the lymphatic system of the subject. These needles may further comprise nanotopography on the surface of the needle in a random or organized pattern. In embodiments, the nanotopography pattern may demonstrate fractal geometry.
EXAMPLES
Example 1. Nucleic acid constructs encoding SI proteins.
[00693] Multiple nucleic acid constructs were designed to provide templates for in vitro transcription of mRNAs encoding both “wild type” and mutant forms of the S protein of the Washington strain of SARS-Cov-2 (NCBI Accession QHU79204.1 (SARS-CoV-2 isolate Washington/Wuhan-Hu-1) as well as mutant and wild type forms of the alpha, beta, gamma, delta, and kappa variant forms of the S protein. Each of the mutant genes was designed to encode an S protein having the same mutation of the furin cleavage site, where the amino acid sequence QQAQ was substituted for the RRAR sequence of the wild type S proteins. Genes for each S protein were synthesized by Genewiz to encode the polypeptides of SEQ ID NO:20, SEQ ID NO:22 (WA S/QQAQ), SEQ ID NO:23 (“wild type” alpha S protein), SEQ ID NO:24 (alpha S/QQAQ), SEQ ID NO:25 (“wild type” beta S protein), SEQ ID NO:26 (beta S/QQAQ), SEQ ID NO:27 (“wild type” gamma S protein), SEQ ID NO:28 (gamma S/QQAQ), SEQ ID NO:29 (“wild type” delta S protein), SEQ ID NO:30 (delta S/QQAQ), SEQ ID NO:31(“wild type” kappa S protein), and SEQ ID NO:32 (kappa S/QQAQ). The genes were cloned in the pVAXl vector 3’ of the T7 promoter.
Example 2. In vitro transcription of S protein mRNAs.
[00694] mRNA transcription of mutant S proteins was performed using the Hi Scribe™ T7 High Yield RNA Synthesis Kit kit from NEW ENGLAND BioLabs according to the manufacturer’s protocols. UTP was replaced by Nl-Methyl-Pseudouridine-5'-Triphosphate from TriLink Biotechnologies. CleanCap® Reagent AG from TriLink Biotechnologies was added to the reaction to cap the synthesized RNA at 5 ’-terminus. Full-length mRNAs were purified using the Monarch® RNA Cleanup Kit from NEW ENGLAND BioLabs according to the manufacturer’s protocol. From a standard 20 pL reaction, a typical yield of 100 ug RNA was achieved, with a 260/280 ratio between 1.8-2.0 and a 260/230 ratio between 2.0- 2.2, as determined by NanoDrop™ One Spectrophotometer from ThermoFisher Scientific. Example 3. Transfection of cells with mRNA encoding S proteins having QQAQ mutation.
[00695] HEK293 cells were washed and diluted to 4 x 105 cells per ml, with 2 x 105 cells used per transfection in a 24-well plate. For each S protein mRNA, 1 μg-2 5 ug of RNA was combined with Lipofectamine MessengerMax (ThermoFisher) and the formulated RNA was added to cell cultures and incubated for 24-72 hours at 37 C.
Example 4. Flow cytometry analysis of HEK293 cells transfected with mRNAs encoding mutant S proteins.
[00696] Twenty-four to seventy-two hours post-transfection, cells were harvested using enzyme free cell dissociation buffer (ThermoFisher), washed once, and resuspended in FACS buffer (DPBS + 0.5% BSA) at 4 x 106 cells/ml. For antibody binding to the cells expressing the S proteins, the cells were dispensed into wells of a 96-well plate and mixed with an equal volume of 2x final concentration of diluted anti-S antibody that recognizes individual variants, STI-2020 for wild-type WA strain S protein, alpha, delta, and kappa variants, STI- 5041 for beta and gamma variants. After incubation on ice for 30 minutes, the cells were washed with FACS buffer and incubated with anti-human Fc antibody conjugated to allophycocyanin (APC). After incubation on ice for 15 minutes, the cells were washed with FACS buffer and analyzed using flow cytometry.
[00697] The FACS results are shown in Figures 1-6. Figure 1 shows that approximately 92% of the cells transfected with mRNA encoding either the “wild type” S protein or the furin mutation-containing S protein of the Washington strain expressed the S protein on the cell surface. Expression decreased by day 3 for both mRNAs, although the decrease in expression was somewhat less for the cell population transfected with the furin mutation (to approximately 44% of the cell population expressing the furin mutation S protein versus approximately 36% expressing the wild type S protein). Regarding expression of the alpha variant S protein from RNA transfection, Figure 2 shows that expression of the furin mutation-containing alpha variant S protein in the mutant mRNA-transfected culture was approximately equivalent to the expression of the wild type alpha variant S protein at day 1 post-transfection.
[00698] Figure 3 shows that the percentage of cells expressing the furin mutation- containing beta variant S protein is similarly approximately equivalent to the percentage of cells expressing the wild type beta variant S protein (in this case analyzed on day 2 post transfection), but interestingly on day 3 the culture transfected with the mRNA encoding the beta S protein with the furin mutation shows only about half as much of a decrease in the percentage of expressing cells as is seen for the culture transfected with mRNA encoding the wild type beta S protein.
[00699] Approximately 50% more cells of the culture transfected with mRNA encoding the gamma S protein having the furin mutation expressed the mutant gamma S protein as compared to cells transfected with mRNA encoding the wild type gamma S protein on day 2 (Figure 4). The difference in culture-wide expression was even more dramatic on day 3 post transfection, when the percentage of cells expressing the wild type gamma S protein dropped precipitously to approximately 9.5%, whereas the percentage of cells expressing the mutant gamma S protein remained high, at approximately 88.5%, a decrease of only about 11%. This pattern of more persistent expression of the S protein having the QQAQ furin site mutation in RNA-transfected cells was also seen for the delta variant S protein. Figure 5 shows the flow cytometry analysis of cells transfected with mRNA encoding either the wild type delta S protein or mRNA encoding the delta S protein having the furin mutation. In this case, culture wide expression dropped approximately 50% in the culture transfected with mRNA encoding the wild type delta S protein between day 1 and day 3, but only about 36% in the culture transfected with mRNA encoding the QQAQ mutant delta S protein over the same time period. Because the mutant S protein-expressing cultures had a higher percentage of cells expressing the S protein on day 1, by day 3 culture-wide expression of the mutant S protein was approximately 50% higher than expression of the wild type delta protein. Finally, Figure 6 shows that when cultures were transfected with mRNAs encoding either the wild type kappa variant S protein or the furin mutation containing kappa variant S protein, the percentage of cells in the cultures expressing the mutant and wild type S proteins was essentially the same. Thus, taken together, the data shows no detrimental effects of the QQAQ furin mutation in SARS-CoV-2 S proteins, including S protein variants, on expression of the S proteins in cells transfected with nucleic acid constructs that encode the mutant S proteins. Further, inclusion of the QQAQ furin mutation can enhance the expression of SARS-CoV-2 S proteins in transfected cells, as evidenced by both an increased percentage of expressing cells and increased persistence of expression over time.
[00700] Example 5. Stability study comparing lyophilized mRNA and liquid mRNA. [00701] Approximately 10 mg mRNA encoding a SARS-CoV-2 spike protein (one of the four variants: Wuhan/Washington, U.K., South Africa, Brazil) was mixed with 200,000 HEK293 T cells in 100 mL Buffer R included in the Neon Transfection System. The cells were electroporated using the Neon device at 1700V, 20ms, 1 pulse. The 100 mL transfection mix was then added to 400 mL prewarmed complete medium in a 24-well plate. 6 hours post transfection, the cells were collected and washed with FACS buffer (DPBS+0.5%BSA). The spike expression was then assessed by incubation with proprietary anti-spike antibody STI- 5041 (diluted 1 : 120 in FACS buffer) for 30 minutes, followed by incubation with allophycocyanin (APC) anti-human Fc (diluted 1:100 in FACS buffer) for 15 minutes. After washing with FACS buffer, the cells were resuspended in 200 mL FACS buffer and subjected to flow analysis with the Attune NxT Flow Cytometer. Flow cytometry scatter plots confirmed the extremely high expression of each of the variants tested, both in terms of the percentage of cells expressing the transfected mRNAs and the average amount/magnitude of spike protein expressed by cells transfected with each variant (see, e.g., Figures 7A-7E) [00702] To assess the effect and stability of mRNA expression when mRNA formulations are lyophilized compared to formulations in solution, mRNA that encodes a SARS-CoV-2 spike protein was either in solution or lyophilized. The mRNA solution was stored at -80°C while lyophilized mRNA was stored at room temperature for either 3 or 11 days. Following storage, the mRNA was transfected into HEK293 cells. Control cells did not contain mRNA. Transfection was performed and spike expression was assessed as described above in above. In the control where cells did not generate the spike antigen, only background fluorescence was observed as shown in Figure 7F. Figure 7G shows scatter plot of mRNA solution stored at -80°C for 11 days, prior to transfection into HEK293 cells. Figure 7H and Figure 71, which show scatter plots of lyophilized mRNA stored at room temperature (for 3 days and 11 days, respectively), prior to transfection into HEK293 cells, demonstrate that lyophilized mRNA produces higher fluorescence, indicating better generation of the spike protein on the cells.
[00703] Stability of lyophilized SARS-Cov-2 spike protein-encoding mRNA compared to liquid mRNA formulations was also evaluated via gel electrophoresis. Control mRNA solution was stored at -80°C for 11 days, while lyophilized mRNA (with trehalose added for stability) was stored at room temperature for 11 days either under ambient conditions, in vacuum, or under nitrogen. As shown in Figure 7J, the mRNAs did not degrade appreciably under any of the conditions tested.
[00704] Example 6. Assessment of safety and efficacy of an exemplary mRNA-LNP vaccine (“Vaccine 1”) administered via lymphatic delivery [00705] Materials and Methods
[00706] In vitro transcription and purification of RNA. To generate the template for RNA synthesis, the sequence of the SARS-Cov-2 Washington/Wuhan (aka “Wuhan-Hu-1”) isolate Spike (S) protein (GenBank: QHD43416.1) was codon optimized and cloned into pVAXl-based backbone which features 5'-UTR, 3'-UTRand Poly-A tail. To increase the protein stability, 2P mutations at positions 986-987 were introduced. The plasmid DNA was produced in bacteria, purified and linearized by a single-site restriction enzyme digestion.
The template DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase (Cat: M0251, NEB) in the presence of a trinucleotide capl analogue, m7(3OMeG)(5')ppp(5')(2OMeA)pG (Cat: N-7113, TriLink), and of Nl- methylpseudouridine-5’ -triphosphate (Cat: N-1081, TriLink) in place of uridine-5’- triphosphate (UTP). After the reaction, DNase I (Cat: M0303, NEB) was added to remove the template DNA and the mRNA was purified by LiCl precipitation (Cat: AM9480, ThermoFisher).
[00707] In vitro mRNA expression. Monocytes are isolated and differentiated into DCs in presence of GM-CSF (Cat: 300-03, Peprotech) and IL-4 (Cat: 200-04, Peprotech). Between day 6-day 8, cells were transfected with mRNA by the Neon^M electroporation transfection system (Cat: MPK5000, ThermoFisher). 24 hours post-transfection, the cells were collected and stained with anti-Spike antibody STI-2020 in FACS buffer (DPBS+0.5%BSA) for 30 minutes on ice. Thereafter, cells were washed twice in FACS buffer and incubated with rat anti-human Fc antibody conjugated to allophycocyanin (Cat: 410712, BioLegend) for 15 minutes on ice. The cells were washed with FACS buffer and analyzed by the Attune NxT Flow Cytometer (ThermoFisher).
[00708] SARS-CoV-2 Virus. SARS-COV-2 viruses were obtained from BEI resources (Washington strain NR-52281; Beta Variant NR-54009) VeroE6 monolayers were infected at anMOI of 0.01 in 5ml virus infection media (DMEM + 2%FCS +1X Pen/Strep). Tissue culture flasks were incubated at 36°C and slowly shaken every 15 minutes for a 90 minute period. Cell growth media (35mL) was added to each flask and infected cultures were incubated at 36°C/5% C02 for 48 hours. Media was then harvested and clarified to remove large cellular debris by room temperature centrifugation at 3000 rpm.
[00709] Animals and in vivo studies. 6- to 12-week old C57B16 mice were purchased from the Jackson Laboratory. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Mice were injected with the indicated administration techniqueunder isoflurane anesthesia in the right hind flank area for IM injections and in the right dorsal area for MuVaxx injections. For imaging studies, ICG (Sigma) was dissolved at 2.5mg/mL in deionized water and injected using MuVaxx with a NIRF camera used to collect images 5 min following injection. For OVA vaccine studies, 10 μg of OVA (Cat: VAC- POVA, Invivogen) and 8 μg of CpG (Cat: TLRL- 1826-1, Invivogen) were administered to mice on days 0 and 14. Peripheral blood was collected from anaesthetized mice once/week via submandibular route. Reference mRNA-LNP vaccine is the same construct as an EUA cleared compound.
[00710] ELISA assays. To asses spike specific antibodies, SI (Cat: 40591-V08H Sino Biological)or RBD (Cat: 40592-V08B, Sino Biological) protein was coated on half-area high bindingplates (Cat: N503 Thermo) at 1 μg/mL overnight at 4°C. Plates were washed 3 times withELISA wash buffer (Thermo), pre-blocked with casein blocker (Cat: 37528 Thermo) for lhour at room temperature (RT), and washed 1 time with ELISA wash buffer. Mouse serawas diluted in casein blocker and transferred to ELISA plates for 1 hour at RT followed by 3 wash steps. Secondary antibody of horse radish peroxidase (HRP)-conjugated rabbitanti-mouse IgM (m chain), HRP-conjugated rabbit anti-mouse IgG (Fey), HRP- conjugatedgoat anti-moues IgGl, or HRP-conjugated goat anti -mouse IgG2c was added to ELISA plates for 1 hour at RT followed by six wash steps. Plates were developed with TMB substrate solution (Cat: 34021 Thermo) for approximately 10 min at RT and stopped with2 normal sulfuric acid. The absorbance was measured at 450 nanometers using a BioTek Cytaktion 5 plate reader. For IgG (Fey), a standard curve was generated using Anti-RBDPAb (Cat: 40592-MP01 Sino Biological) or Anti-Si (Cat: MAB105405 R&D Systems) starting at 3000 ng/mL with 3 fold serial dilutions. For IgM (m chain), IgGl, and IgG2c, serial fold dilutions were run and titers were determined using an absorbance cutoff of 0.7OD.
[00711] Plaque Reduction Neutralization Test (PRNT). Simian VeroE6 cells were plated at 18x10·^ cells/well in a flat bottom 96-well plate in a volume of 200 mΐ/well. After 24 hours, a serial dilution of seropositive blood serum is prepared in a 100 mΐ/well at twice the fmalconcentration desired and live virus was added at 1,000 PFU/IOOmI of SARS-CoV-2 andsubsequently incubated for 1 hour at 37°C in a total volume of 200 mΐ/well. Cell culture media was removed from cells and sera/virus premix was added to VeroE6 cells at 100 mΐ/well and incubated for 1 hour at 37°C. After incubation, 100 mΐ of“overlay” (1:1 of 2% methylcellulose (Sigma) and culture media) is added to each well and incubation commenced for 3 days at 37°C. Plaque staining using Crystal Violet (Sigma) was performed upon 30 min of fixing the cells with 4% paraformaldehyde (Sigma) diluted in PBS. Plaques were assessed using a light microscope (Keyence).
[00712] Peripheral blood T cell intracellular cytokine staining. ICS was performed at the indicated time points following the booster shots for IFNy, TNFa, and IL-4. Whole blood was stimulated for 6 hours with 1 μg/mL of SIINFEKL (Sigma) or overnight with 1 μg/peptide per well of spike associated (Cat: 130-127-951, Miltenyi Biotec Peptivator) peptides at 37°C, 5% C02 in the presence of brefeldin A (Biolegend) and monensin (Biolegend). Following stimulation, whole blood was incubated with red lysis buffer (Cat: A10492-01 Gibco) at room temperature. Cells were permeabilized using Intracellular Staining Perm Was Buffer (Cat: 421002 Biolegend). Cells were stained with PE anti- mouse IFNy (Cat: 505808 Biolegend), FITC anti-mouse TNFa (Cat: 506304 Biolegend), BV421 anti-mouse IL-4 (Cat: 504127 Biolegend), APC-Cy7 anti-mouse CD3 (Cat: 100222 Biolegend), PE-Cy7 anti-mouse CD4 (Cat: 25-0041-82 Invitrogen), and allophycocyanin anti-mouse CD8a (Cat: 100712 Biolegend). Naive mice (non-vaccinated mice) were usedas negative controls. Cells were then run on a Beckman Coulter CytoFLEX instrument and analyzed via FlowJo VI 0 software.
[00713] Statistics. Statistical significance of differences between experimental groups was determined with Prism software (Graphpad). All data are expressed as standard error mean (SEM). ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 by unpaired two- tailed t tests or one- or two- way analysis of variance (ANOVA).
[00714] Results
[00715] Assessment of expression of mRNA component of an exemplary mRNA-LNP vaccine (“Vaccine 1”)
[00716] As described above, Vaccine 1 comprises a single-stranded mRNA encoding a full-length SARS-CoV-2 S glycoprotein, derived from the strain “Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1”, as an active drug substance encapsulated in lipid nanoparticles (LNPs). As described above, mutations were introduced into S protein to substitute residues 986 and 987 to produce prefusion-stabilized SARS-CoV-2 S(2P) protein. To achieve optimal expression in humans, the sequence was further codon-optimized and cloned into a pVAXl -based backbone that contains T7 promoter, 5'-UTR, 3'-UTR and optimized Poly-A tail with minimal overhang. The template was then linearized immediately downstream of the Poly-A tail and used for in vitro transcription (IVT). To facilitate mRNA expression and reduce innate immune response, during the IVT, Cap 1 structure was added to the 5’ terminus of the RNA co-transcriptionally by CleanCap® AG, and UTP completely replaced by Nl-methylpseudo-UTP. This process can be readily scaled up to produce desired amounts of capped mRNA. Figure 8A provides a schematic representation of the elements and residue substitutions described above.
[00717] To confirm the expression of the mRNA component of Vaccine 1, the IVT mRNA was introduced into monocyte-derived dendritic cells (DC) by electroporation. Twenty-four hours post-transfection, the cells were collected and stained with anti-Spike antibody STI- 2020 and detected with allophycocyanin-conjugated anti-human Fc antibody. The flow cytometry showed that the codon-optimized Cap 1 mRNA can be efficiently translated into prefusion-stabilized spike protein in the primary DC (Figure 8B). [00718] MuVaxx effectively delivers antigen to LNs and improves immune response to same.
[00719] To explore the potential of delivering vaccine constituents to draining LNs, MuVaxx, an exemplary lymphatic drug delivery device was utilized, which is connectable to any luer lock syringe (Figure 9A), and which consists of microneedles that puncture the stratum comeum and release drug at the epidermal/dermal boundary.
[00720] To evaluate LN delivery of using MuVaxx, Indocyanine Green (ICG) was used, which can be visualized in vivo with near-infrared fluorescence (NIRF). Following injection, observed ICG accumulation was observed within minutes in the draining brachial LN (Figure 9B). The potential of MuVaxx to augment the immunogenicity of a model antigen, Ovalbumin (OVA), in mice was then explored. Mice were injected with OVA and an oligonucleotide adjuvant (CpG) on days 0 and 14 using an IM or MuVaxx administration. All mice treated with MuVaxx generated anti-OVA IgG by day 13 compared to 4 of 8 in the IM cohort. Additionally, following the booster shot (day 14), anti-OVA IgG was measured on day 34 and mice treated with MuVaxx displayed significantly higher titers compared to mice treated IM resulting in over a 60-fold increase in titers (Figure 9C). The cellular immune response was additionally measured in both cohorts of mice looking at cytokine production in CD8 T cells following the booster shot on days 20 and 28. Interferon-Gamma (IFNy) and Tumor Necrosis Factor-Alpha (TNFa) were assessed following ex vivo stimulation with SIINFEKL, an OVA derived class I peptide. Mice treated with MuVaxx displayed higher proportions of cytokine producing CD8 T cells compared to naive mice on both days 20 and 28 (Figure 9D).
[00721] Taken together, these results demonstrated the capability of MuVaxx to deliver cargo to draining LNs to improve immunity toward immunogenic constituents of vaccines.
[00722] Exemplary mRNA-LNP vaccine, “Vaccine 1”, induces anti-spike antibodies with MuVaxx enabling dose sparing.
[00723] To further evaluate the potential of MuVaxx to enhance immunity, the humoral immunity of Vaccine 1 against a reference vaccine (“Reference”) along with the comparison of intramuscular (IM) vs MuVaxx administrations was compared. C57B16 were immunized with either 10 μg Reference IM, or Vaccine 1 at 10 μg IM, 1 μg IM, or 1 μg MuVaxx on days 0 and day 35 with serum collection every 7 days (Figure 10A). Seven days after the original primer shot, both 10 μg mRNA- LNP formulations (Reference and Vaccine 1) administered IM showed high anti-RBD specific IgM responses with a reduction in IgM observed for the 1 μg dose. When 1 μg of the Vaccine lwas administered using MuVaxx, a similar IgM response was measured Figure 10B). Similar trends were also observed when quantifying the IgG response for both anti-RBD and anti-Sl specific IgG antibodies (Figures IOC and 10D) highlighting the dose sparing potential that is observed when administering 0.01 μg of mRNA via MuVaxx. Additionally, when exploring the IgG response following the initial dose, MuVaxx elicited sustained anti-Spike IgG antibodies in the serum compared to the traditional IM administration which waned at a faster rate (Figures 10E and 10F).
[00724] Overall, these results demonstrated successful generation of a spike encoding mRNA vaccine with delivery toward draining LNs and/or cells within the epidermis via MuVaxx enabling dose sparing activity.
[00725] Vaccine 1 favors Thl response over Th2.
[00726] To assess the Thl/Th2 bias elicited after immunization, whole blood was collected to measure cytokine producing CD4 T cells 6 days after the booster shot via intracellular cytokine staining (ICS) and IgG subclass titers from day 49 serum (Figure 11 A). The ratio of Thl (CD3+CD4+IFNy+) to Th2 (CD3+CD4+IL4+) T cells favored a Thl response and was similar between all cohorts, although the 10 μg Vaccine 1 IM group and 1 μg Vaccine 1 MuVaxx groups had slightly higher ratios favoring an enhanced anti -viral immune response (Figure 11B). Similarly, the ratio of IgG2c to IgGl was skewed towards IgG2c for the 10 μg Vaccine 1 IM group and 1 μg Vaccine 1 MuVaxx cohorts suggesting bias towards a Thl response (Figure 11C) in line with the CD4 T cell cytokine phenotypes.
[00727] Elevated CD8 T cell immunity is elicited following vaccination with Vaccine 1 and MuVaxx.
[00728] In addition to CD4 T cells, the responses in the CD8 T cell compartment were evaluated on day 49 following incubation with spike associated peptides overnight via ICS (Figure 12A). Mice vaccinated with the Reference vaccine displayed a minor increase in cytokine producing CD8 T cells (Figure 12B and 12C), in line with previous literature. However, the Vaccine 1 10 μg formulation when administered IM led to a robust antigen specific CD8 T cell response as measured by IFNy and TNFa. The response was dose dependent as IM administration of 1 μg of Vaccine 1 led to a minimal CD8 T cell response. When 1 μg of Vaccine 1 was administered via MuVaxx toward draining LNs, the CD8 T cell response was restored to similar levels to that of a 10 μg IM dose (Figure 12B and 12C). [00729] Overall, these results highlight the improved CD8 T cell response observed with this Vaccine 1 formulation along with the benefit of directing spike encoding mRNA towards draining LNs to generate CD 8 T cell immunity.
[00730] Neutralizing antibodies and long-lived B cells are generated following Vaccine 1 vaccination.
[00731] To assess neutralizing antibody (nAb) generation, a Plaque Reduction Neutralization Test (PRNT) was performed in vitro , where VeroE6 cells were exposed to the live virus in the absence or presence of diluted mouse serum. PRNT detects plaque formation and is indication of cell infection by the SARS-CoV-2 virus whereas the absence of plaque formation represents nAb presence. Each cohort of treatments led to nAb generation by d49 against the WT strain (Figure 13A). To investigate protection against the Beta variant, VeroE6 cells were incubated with this strain of the virus. The Reference vaccine, Vaccine 1 10 μg IM, and Vaccine 1 1 μg MuVaxx cohorts displayed robust protection against this strain up to a 1 : 120 dilution whereas the lower 1 μg Vaccine 1 IM cohort of mice displayed much lower protection (Figure 13B).
[00732] Taken together, these results show that Vaccine 1 generates nAbs and that lymphatic mediated delivery via MuVaxx can broaden protection at 1/10th the IM dose. [00733] To explore vaccine durability, the generation of long-lived B cells (memory B cells) in the lungs and draining LNs 15 weeks after the booster dose was also explored. Tissues were collected and single cell suspensions were stimulated ex vivo to promote IgG secretion. Elevated concentrations of anti-Sl IgG were observed in the lungs compared to non-vaccinated mice; however anti-Sl concentrations were similar regardless of dose or formulation (Figure 15). Additionally, anti-Sl IgG concentrations in LNs draining the injection site were substantially elevated in MuVaxx treated animals. These data demonstrate enhanced generation and/or survival of long-lived IgG secreting B cells in mice where spike encoding mRNA was delivered more effectively to draining LNs (i.e. administration with MuVaxx).
[00734] As described in this Example, an exemplary mRNA-based SARS-CoV-2 vaccine, Vaccine 1, that induced similar humoral immunity with elevated cellular immunity compared to a Reference vaccine formulation when administered IM. Immunity generated with the Vaccine 1 formulation was dose dependent as immunity was reduced when going from 10 to 1 μg IM. When administering the same 1 μg Vaccine 1 formulation via MuVaxx, dose sparing effects were observed where both humoral and cellular immunity were similar to that of a 10 μg IM dose highlighting the improved immunogenicity when directing vaccines towards LNs. Previous mRNA-based vaccines have reported dose-dependent side effects with higher doses linked to systemic and local adverse events underscoring an additional advantage of lower dose formulations; lessening side effects while expanding vaccine access to large populations.
[00735] A vaccine that generates durable immunity is another hallmark of an effective vaccine and is a metric that was investigated. The serum concentrations of both anti-Sl and anti-RBD IgG waned to a lesser degree in mice treated with MuVaxx relative to those treated with the IM same dose, highlighting improved durability. This is of interest as recent reports have shown declines in SARS-CoV-2 neutralizing antibodies 2-3 months after disease onset as short-lived plasma cells stop producing nAbs. However, a subset of plasma B cells do differentiate into memory B cells following infection and/or vaccination leading to persistent germinal center formation within LNs where somatic hypermutation takes place. Thus, efficient delivery of vaccines towards LNs where memory B cells reside may improve memory B cell activation and coverage against emerging variants at lower doses in line with the nAb data shown here.
[00736] While most emphasis of the available SARS-CoV-2 vaccines has been focused on the humoral response and nAbs, a vaccine that may also generate robust T cell immunity to synergistically protect against infections is desirable. In the context of COVID-19, subsets of patients with preexisting SARS-CoV-2 specific T cells have demonstrated rapid viral clearance and less severe disease highlighting their important role for disease prevention. Additionally, T cell responses against previous betacoronaviruses can persist for decades5,24 and display cross-reactivity against other betacoronaviruses highlighting potential for long term protection and coverage against variants. As demonstrated above, Vaccine 1 delivered via MuVaxx elicited a strong CD8 T cell response towards SARS-CoV-2 peptides which is believed to be advantageous for preventing COVID-19 and providing protection against reinfection. [00737] The CD4 Th2 phenotype has previously been associated with vaccine-associated enhanced respiratory disease (VAERD) in those vaccinated against the measles- and respiratory syncytial- virus. We therefore explored the CD4 Thl vs Th2 response and IgG2c to IgGl antibody response in vaccinated mice. The response of all vaccinated mice favored a CD4 Thl response in line with naive mice. In line with the T cell response, the antibody response also skewed towards a Thl response as measured by the IgG2c to IgGl ratio in vaccinated mice with the Vaccine 1 10 μg IM and 1 μg MuVaxx cohorts displaying a stronger IgG2c to IgGl ratio. Mice treated with Vaccine 1 formulations (high dose IM or low dose MuVaxx) may display enhanced anti-viral activity as mouse IgG2 subclasses have been shown to induce antibody-dependent cellular cytotoxicity. Taken together, the Thl :Th2 response described above suggested promising activity for avoiding VAERD while promoting anti-viral activity.
[00738] Using MuVaxx to deliver an exemplary mRNA vaccine to the lymphatic system, such as by directing the vaccine towards the epidermis and draining lymph nodes, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine producing CD8 T cells, and neutralizing antibodies against the Washington (Wild Type, WT) and South African (beta) variants. Furthermore, a four-fold elevated T cell response was observed in MuVaxx administered vaccination as compared to that of IM administered vaccination. Collectively, the results described above demonstrate improved immunogenicity of an exemplary mRNA-LNP vaccine with dose sparing properties, such as when directed towards the epidermis and draining lymph nodes or otherwise administered via lymphatic administration, using MuVaxx.
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[00759] Ng, O. et al. (2020). Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection.
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Example 7. Additional mRNA vaccines for the prevention of coronavirus and other viral infections
[00776] The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA- approved mRNA vaccines currently in use. Here, we explored various strategies to develop mRNA vaccines that offer potentially safer and wider coverage of VOCs. We first introduced a furin cleavage mutation into the spike (S) protein of predominant VOCs, including Alpha (B.l.1.7), Beta (B.1.351), Gamma (P.l) and Delta (B.1.617.2), and confirmed the expression and cleavage deficiency of furin mutants. We then assessed the capacity of the resultant mRNAs to induce neutralizing antibodies (nAb) in mice following intramuscular administration. The initial mouse vaccination results showed that the individual VOC mRNAs induced the generation of neutralizing antibody in a VOC-specific manner. Moreover, we discovered that the antibodies produced from mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs cross-reacted with other VOCs. The broad spectrum of generated nAb was further confirmed when vaccinated mice were challenged with the respective live viruses. Interestingly, in a mix-and-match booster experiment, omicron-Furin and WA-Furin mRNA elicited comparable protection against omicron. We also tested the concept of bivalent vaccine by introducing the RBD of Delta strain into the intact S antigen of Omicron. The chimeric mRNA induced potent and broadly acting nAb against Omicron and Delta, and thus serve as vaccine candidates to broadly target current variants of concern as well as emerging variants in the future.
[00777] Materials and Methods
[00778] In vitro transcription and purification of RNA [00779] To generate the template for RNA synthesis, the sequences of the SARS-Cov-2 Spike protein of VOC were codon optimized and cloned into pVAXl -based backbone which features 5'-UTR, 3'-UTR and Poly- A tail. To increase the protein stability, 2P mutations at positions 986-987 were introduced. The plasmid DNA was produced in bacteria, purified and linearized by a single-site restriction enzyme digestion. The template DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase (Cat: M0251, NEB) in the presence of a trinucleotide capl analogue, m7(30MeG)(5')ppp(5')(20MeA)pG (Cat: N-7113, TriLink), and ofNl- methylpseudouridine-5’ -triphosphate (Cat: N-1081, TriLink) in place of uridine-5’- triphosphate (UTP). After the reaction, DNase I (Cat: M0303, NEB) was added to remove the template DNA and the mRNA was purified by LiCl precipitation (Cat: AM9480, ThermoFisher).
[00780] mRNA formulation
[00781] LNPs were prepared by microfluidic mixing a buffered solution of mRNA with an ethanol solution of lipids [distearoylphosphatidylcholine (DSPC), cholesterol, 1,2- Dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-2000 (DMG-PEG2000), and ionizable lipid. The LNPs were concentrated by dialysis against an aqueous buffer system, following a 0.2 pm sterile filtration. The LNPs were tested for mRNA concentration, encapsulation efficiency, particle size, pH, and osmolality.
[00782] In vitro mRNA expression
[00783] Monocytes were isolated from PBMCs and differentiated into DCs in presence of GM-CSF (Cat: 300-03, Peprotech) and IL-4 (Cat: 200-04, Peprotech). Between day 6-day 8, cells were transfected with mRNA by the NeonTM electroporation transfection system (Cat: MPK5000, ThermoFisher). 24 hours post-transfection, the cells were collected and subjected to flow cytometry as described below to check the expression of spike. With 293 T adherent cells, mRNA (2.5ug) of WT vs Mutant from five variants (Washington, Alpha, Beta, Gamma and Delta) were transfected with Lipofectamine™ MessengerMAX™ Transfection Reagent (2ul) and cultured for 72 hours at 37oC using 293T adherent cells with 0.5ml of DMEM media with 10% FBS in each well of a 24-well cell culture-treated plate. Transfected cells from each well were dislodged with 400ul of TrypLE at 72 hours and neutralized with its own media. Cell pellets were collected after spinning down at 550g for 2 minutes by removing supernatant for each well. [00784] Flow cytometry
[00785] The collected cell pellets were washed with 250ul of FACS buffer (DPBS + 0.5% BSA) in 96 wells treated plate followed by 30-minute incubation with the in-house STI-2020 (for DCs) or 10A3 (for 293T cells) primary antibody (1 : 1000) to detect SARS Cov-2 spike. 200ul FACS buffer was used to wash the cells twice with the same speed and time after 30- minute incubation, followed by rat anti-human Fc antibody conjugated to APC (Cat: 410712, BioLegend; 1:100 dilution) for 15 min on ice in the dark for secondary detection. The cells were spun down and the pellets were washed twice with the same speed and time of centrifugation using 200ul FACS buffer and resuspended in 200ul FACS buffer. The fluorescent intensity of positive cells within the gated population was detected by the Attune NxT Flow Cytometer (Therm oFisher) using lOOul of acquisition volume setting.
[00786] SARS-CoV-2 Virus
[00787] SARS-COV-2 viruses were obtained from BEI resources (Washington strain NR- 52281; Alpha variant NR-54000; Beta Variant NR-54009; Gamma variant NR-54982; Delta variant NR- 55611 or NR-55672; Lambda variant NR- 55654 and Omicron NR-56461). VeroE6 monolayers were infected at an MOI of 0.01 in 5ml virus infection media (DMEM + 2% FCS + IX Pen/Strep). Tissue culture flasks were incubated at 36°C and slowly shaken every 15 minutes for a 90-minute period. Cell growth media (35mL) was added to each flask and infected cultures were incubated at 36°C/5% C02 for 48 hours. Media was then harvested and clarified to remove large cellular debris by room temperature centrifugation at 3000 rpm.
[00788] Animals and in vivo studies
[00789] 7-week-old BALB/cJ female mice were purchased from the Jackson Laboratory. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC). mRNA formulations were diluted in 50 uL of IX PBS, and mice were inoculated IM into the same hind leg for both prime and boost. There was 3 weeks interval between prime and boost. Two weeks after boost, mice blood was collected from retro-orbital for ELISA and pseudovirus neutralization assay.
[00790] ELISA
[00791] Ni-NTA HisSorb plates (Qiagen) were coated with 50ng/well of SI proteins (all from Sino Biological, Cat: 40591-V08H, 40589-V08B6, 40589-V08B7, 40589-V08B8, 40589-V08B16) in IX PBS at 4°C overnight. To block the plate, Blocker Casein (Cat: 37528 Thermo) was used for 1 hour at room temperature (RT). After standard washes and blocks, plates were incubated with serial dilutions of sera for 1 hour at RT. Following washes, goat anti-mouse IgG (H+L)-HRP conjugate (Cat: #1721011, Bio-Rad) were used as secondary Abs, and Pierce TMB substrate kit (Cat: 34021, Thermo Fisher) was used as the substrate. The absorbance was measured at 450 nanometers using a BioTek Cytaktion 5 plate reader. Endpoint tiers were calculated as the dilution that emitted an optical density exceeding 4X background (secondary Ab alone).
[00792] Pseudovirus neutralization assay
[00793] SARS-CoV-2 Spike pseudotyped AG-VSV-luciferase was generated by nucleofection of BHK cells (maintained in DMEM/F12 with 10%FBS and 5%TPB) with Spike-expressing plasmid followed by transduction with G-pseudotyped AG-luciferase (G*AG-luciferase) rVSV (Kerafast) 18-24 hours later. The supernatant containing pseudovirus was collected following 24 hours and stored at -80°C. Pseudovirus was normalized for luciferase expression using G*AG-luciferase VSV of known titer as the standard. For neutralization testing, HEK-Blue 293 hACE2-TMPRSS2 cells (Invivogen; maintained in DMEM with 10% FBS) were plated to white-walled 96-well plates at 40,000 cells/well and incubated at 37°C/5% C02. The next day, SARS-CoV-2 Spike pseudotyped ΔG-VSV-luciferase was incubated with a dilution series of mouse serum (dilutions as indicated) and anti-VSV-G (Kerafast; 1 μg/mL) antibody for 30 minutes at room temperature and added to the HEK-Blue 293 hACE2-TMPRSS2 cells. Transduced cells were incubated for 24 hours at 37°C/5% C02 and luminescence measured by addition of 40m1 of ONE-Glo reagent (Promega) with detection using a Tecan Spark plate reader. The percent inhibition was calculated using the formula 1 -([luminescence of serum treated sample]/[average luminescence of untreated samples] x 100. The average of quadruplicate samples were included in the analyses.
[00794] Plaque Reduction Neutralization Test (PRNT)
[00795] Simian VeroE6 cells were plated at 18x 103 cells/well in a flat bottom 96-well plate in a volume of 200 mΐ/well. After 24 hours, a serial dilution of seropositive blood serum is prepared in a 100 mΐ/well at twice the final concentration desired and live virus was added at 1,000 PFU/IOOmI of SARS-CoV-2 and subsequently incubated for 1 hour at 37°C in a total volume of 200 mΐ/well. Cell culture media was removed from cells and sera/virus premix was added to VeroE6 cells at 100 mΐ/well and incubated for 1 hour at 37°C. After incubation, 100 mΐ of “overlay” (1:1 of 2% methylcellulose (Sigma) and culture media) is added to each well and incubation commenced for 3 days at 37°C. Plaque staining using Crystal Violet (Sigma) was performed upon 30 min of fixing the cells with 4% paraformaldehyde (Sigma) diluted in PBS. Plaques were assessed using a light microscope (Keyence). [00796] Challenge study
[00797] K18-hACE2 transgenic mice were purchased from Jackson laboratory and maintained in pathogen-free conditions and handling conforms to the requirements of the National Institutes of Health and the Scripps Research Institute Animal Research Committee. 8-12 weeks old mice were injected with the indicated administration technique under isoflurane anesthesia in the right hind flank area for IM injections. Mice were infected intranasally with 10000 PFU of SARS-CoV-2 in total volume 50 pL.
[00798] Plaque assay
[00799] VeroE6 cells were plated at 3xl0e5 cells/well in 24 well plates in volume 400 mΐ/well. After 24 h. medium is removed, and serial dilution of homogenized lungs were added to Vero cells and subsequently incubated for 1 h at 37°C. After incubation, an overlay (1:1 of 2% methylcellulose (Sigma) and culture media) is added to each well and incubation commenced for 3 d at 37°C. Plaque staining was performed using Crystal Violet as mentioned above.
[00800] Statistics [00801] Statistical significance of differences between experimental groups was determined with Prism software (Graphpad). All data are expressed as standard error mean (SEM). ****p < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 by unpaired two-tailed t tests or one- or two- way analysis of variance (ANOVA).
[00802] Results [00803] Immunization with SARS-CoV-2 spike mRNA harboring furin cleavage mutation induces VOC-specific IgG production
[00804] As demonstrated in the Examples above, we have designed several SARS-CoV-2 spike (S) mRNA vaccines that achieve high expression in mammalian cells (Francis et ak, 2021). One such mRNA vaccine encodes the S protein from the Wuhan/Washington (WA) strain and encodes a polybasic furin cleavage site at the junction of SI and S2 subunits. The feature could affect the stability of spike protein and reduce the pool of antigenic epitopes available to induce cellular and humoral immunity (Peacock et ak, 2021). In addition, cleaved SI was detected in the blood of immunized subjects receiving existing mRNA vaccine (Ogata et al., 2022), raising a significant question of whether this free moiety could represent a potential safety issue by mimicking full-length S protein to activate ACE2, and thus trigger some side effects, including myocarditis in healthy subjects. Thus, as discussed and demonstrated in the Examples above, to further optimize the mRNA vaccine and eliminate these potential safety concerns, the furin cleavage site between the SI and S2 domains of the spike was mutated (Figure 20A) in the currently reported build of our mRNA vaccine candidates.
[00805] We then constructed two sets of mRNAs encoding the S protein of all the predominant SARS-CoV-2 VOCs, one with the wild-type (WT) furin cleavage site, the other with mutated site. We examined and compared the expression of these VOC mRNAs, including WA, Alpha, Beta, Gamma and Delta in 293T cells. The flow cytometry results showed that removal of the furin cleavage site increased the surface expression of S proteins derived from several VOCs in transfected 293T cells (Figure 20B). Western blot analysis confirmed that the mutation abolished the furin cleavage and lowered the level of free SI in the conditioned medium (Figure 20C and Figure 20D). We thus hypothesized that the boosted expression of the furin mutant could result in stronger immunogenicity. To test the hypothesis, WA WT or furin mutant mRNAs were formulated with an exemplary lipid nanoparticle (LNP), and six-week-old female B ALB/c mice were intramuscularly injected with two doses of each LNP-mRNA separated by three weeks. ELISA on the sera collected 14 days after boost revealed that mRNA carrying the furin cleavage mutation elicited a higher average endpoint titer (EPT) of total binding antibody than its WT version (Figure 20E). We then performed the Plaque Reduction Neutralization Test (PRNT) using the same antisera to assess neutralizing antibody (nAb)(Nyiro et al., 2019; Schmidt et al., 1976). Consistent with ELISA data, PRNT results confirmed the superior neutralizing activity of sera from WA furin mutant-injected mice (Figure 20F) as compared to that measured in mice vaccinated with the WT version.
[00806] Likewise, to investigate the ability of individual SARS-CoV-2 VOC mRNA vaccines to generate nAb, and to profile their coverage spectrum in vivo , we immunized six- week-old female BALB/c mice with LNP-encapsulated furin-mutant VOC mRNAs and compared the performance of individual mRNAs in vaccinated mice. The EPT of total binding antibodies was first measured by ELISA using the sera day 14 post two-dose injection. As expected, each mRNA induced the strongest antibody response against the corresponding VOC S protein, with the exception of Gamma (Figure 16A). Intriguingly, some VOC mRNA vaccinations led to the production of antibodies capable of binding to a breadth of VOC S proteins, especially Beta-Furin. To further analyse the neutralizing capability of individual sera, PRNT was performed where VeroE6 cells were exposed to the live virus of five VOCs in the absence or presence of diluted serum collected from the immunized mice (Figure 16B). In keeping with the ELISA results, the experiment showed that the individual monovalent mRNA vaccine generally displayed variant-specific protection activity. For example, WA-Furin mRNA induced the highest neutralizing activity against the WA-1 virus. Again, it was observed that the serum from the Beta-Furin mRNA injected cohort displayed robust and broad protection against all VOCs tested. Particularly, the Beta- Furin mRNA provided a much stronger protection against the highly contagious Delta variant than was elicited by vaccination with the WA-Furin mRNA. The data indicates that whereas the VOC-specific strategy can provide strain-specific protection, some VOC-based mRNA vaccines have relatively enhanced potential to trigger a broad and potent immune response to the genetically divergent set of existing SARS-CoV-2 variants.
[00807] Immunization with SARS-CoV-2 spike vaccines carrying furin cleavage mutation produces robust protection in vivo
[00808] The K18-hACE2 transgenic model has been extensively utilized to evaluate the vaccine efficacy and effectiveness in preventing COVID-19 in the preclinical setting (Arce and Costoya, 2021; Dong et al., 2021; Radvak et ak, 2021; Winkler et ah, 2020). Two key metrics to determine the severity of pathogenesis are the virus titer in the lung tissue and body weight loss following virus infection. To investigate the protection capacity of our furin-mutant mRNA vaccines, K18-hACE2 mice were first intramuscularly administrated with 5μg of WA-Furin or Beta-Furin mRNA twice with 3-week interval. Five weeks post full vaccination, the animals were challenged with 1 x 105 half-maximal tissue culture infectious dose (TCID50) of the WA, Beta or Lambda variants, as represented schematically in Figure 17A. Then the virus replication in the lung was quantified to determine the effect of vaccination. The average virus titer was approximated to 1 x 106, 2.1 xlO5, and 4.5 x 106 TCID50/g for WA, Beta and Lambda strains, respectively in the control group injected with PBS. On the other hand, immunization with either WA-Furin or Beta-Furin mRNA almost completely inhibited the replication of virus in the lungs, with virus titers falling below the limit of detection (Figure 17B). As expected, animals treated with PBS exhibited dramatic weight loss in all challenge settings, regardless of the virus strain. The average body weight in the PBS controls declined to 82%, 78% and 81% on day 5 post infection with WA, Beta and Lambda strains, respectively. In contrast, none of the mice immunized with Beta-Furin or WA-Furin vaccines showed any sign of weight loss, and some animals in these treatment groups gained weight after infection up to 5 days (Figure 17C). In addition, both furin- mutant mRNAs gave effective protection against the Lambda variant although the corresponding spike mRNA was not included among the immunogens, suggesting broad protection capacity of some VOC mRNAs.
[00809] Omicron variant undermines the effectiveness of the past VOC based vaccines
[00810] Recently, a new SARS-CoV-2 variant B.1.1.529 (Omicron), was identified in South Africa and declared as VOC by WHO due to its rapid rise in global prevalence. Due in large part to an unusually large number of previously unreported RBD mutations as compared to other VOCs, Omicron exhibited the most significant escape from the serum of both fully vaccinated subjects and convalescents (Planas et al., 2022). To investigate whether our optimized mRNA vaccines could offer effective protection against Omicron, VSV pseudotyped viruses were used to determine the neutralization potency of sera collected from vaccinated animals.
[00811] In agreement with other studies (Muik et al., 2022), we found that the mean neutralization titer (IC50) of Omicron was only 318 for the sera collected at day 14 after boost with WA-Furin mRNA, which represents 7.7-fold decrease of neutralization, as compared to that recorded against Delta variant-based pseudotyped viruses (data not shown). A similar decrease in the Omicron protection capacity of the sera was observed for mRNA vaccine candidates derived from other VOCs (Figure 18A). Surprisingly, even the Beta- Furin mRNA, which has been shown to provide the broadest immune response against all major VOCs (Figure 16), failed to induce sufficient protection against Omicron, prompting us to explore further immunogen designs to overcome this liability.
[00812] To establish the potential of a variant-matched monovalent vaccine to provide protective immunity against Omicron virus, we replaced the coding sequence of the original mRNA vaccines with Omicron spike retaining the furin cleavage mutation. More than 85% of the Omicron-Furin mRNA transfected 293T cells, but not the mock control, could be detected by flow cytometry with Fc-tagged ACE2 recombinant protein (Figure 21). We then performed in vivo studies to assess the immunogenicity and efficacy of the Omicron-specific mRNA vaccine. BALB/c mice were immunized intramuscularly with 5μg Omicron-LNP mRNA or Beta-LNP mRNA two times at a three-week interval. Serum was collected two weeks post boost and subjected to ELISA assay to measure Omicron-specific binding antibodies. Using the recombinant Omicron spike protein as the coating antigen, high titers of binding antibodies were observed in the sera of Omicron mRNA-injected mice (Figure 18B). The protective capability of Omicron-specific mRNA vaccine was further evaluated in PRNT. We confirmed that the sera collected from Omicron-Furin-immunized animals offered superior protection against the infection of Omicron strain (Figure 18C), demonstrating this mRNA vaccine could induce potent production of Omicron-specific nAbs in vivo.
[00813] As the Omicron strain led to increasing number of breakthrough cases even in people already receiving three-dose vaccination, we wanted to investigate whether using Omicron-Furin mRNA solely as a boosting immunogen could provide enhanced protection. Hence, we set up an in vivo challenge study to simulate the real-world scenario, illustrated schematically in Figure 18D, left panel. Two doses of WA-Furin mRNA were administrated into K18-ACE2 transgenic mice as described previously. Five weeks after the second dose, the animals received the booster shot of either 5μg WA-Furin mRNA or Omicron-Furin mRNA. After another five weeks, the animals were challenged with live Omicron strain before lung virus loads were quantified. Surprisingly, while the control group displayed high viral titer, up to 5xl05 PFU per lung, vaccinated mice showed no detectable viral replication in the lung, suggesting that both WA-1 and Omicron-based booster mRNAs provided substantial and similar protection against Omicron (Figure 18D, right panel).
[00814] Chimeric RBD-based mRNA vaccine elicits broad protection against Omicron and Delta
[00815] We next sought to determine whether omicron mRNA vaccine could provide sufficient cross-reactive immunity against other VOCs. The ELISA and pseudovirus assays indicated that the Omicron-Furin mRNA vaccine elicited maderate immunity against VOCs, especially Delta (data not shown). Hence, in an effort to generate a vaccine with broad cross reactivity against other VOCs, we constructed a chimeric VOC immunogen by inserting the RBD domain of the Delta variant directly upstream of the Omicron RBD within the Omicron spike backbone (Figure 19A). We first confirmed that the translation product of the chimeric mRNA could still bind to its natural receptor, ACE2 by flow cytometry (Figure 21, right panel). To evaluate the immunogenicity and efficacy of this chimeric design, mice were immunized twice with LNP-formulated mRNA as described above. The sera were collected two weeks following the second dose and then analyzed for the titers of binding antibodies and nAbs against various VOCs. The ELISA results showed that the chimeric Delta RBD- Omicron mRNA outperformed the original Omicron mRNA in the generation of binding antibodies against WA, Beta and Gamma variants (Figure 19A). A moderately higher titer against Delta was seen with the chimeric mRNA vaccination. As the readout of ELISA assay is not a direct indicator of neutralizing capability, we used the same serum panel to further quantify the nAb titer against Delta and two Omicron pseudoviruses (Figure 19B). The IC50 of neutralization showed that compared to Omicron-Furin mRNA, the Delta RBD-Omicron immunization induced similar neutralization activity towards the original and R346K Omicron sub-variant. The absolute IC50 was 5595 with Omicron-Furin and 4955 with Delta RBD-Omicron against the ancestral omicron strain, while the corresponding readout jumped to 8485 and 7589 against Omicron R346K, respectively. Notably, there was a significant increase in the nAb titer against the Delta variant when mice were immunized with the chimeric Delta RBD-Omicron mRNA as compared to mice immunized with the Omicron- Furin mRNA. Taken together, this chimeric design offers a powerful strategy to develop mRNA vaccines with broad protection capacity against COVID-19 and other infectious diseases.
[00816] Discussion
[00817] Prophylactic nucleic acid vaccines can deliver the nucleotide sequence that codes for virus-derived but nonpathogenic proteins into host cells, thus mimicking a native infection to elicit an immune response. Unlike DNA, mRNA vaccines eradicate the need for nucleic acid to enter the nucleus to achieve expression, and they are less likely to be integrated into the host genome. There are currently two mRNA-based SARS-CoV-2 vaccines authorized by FDA and widely disseminated. These vaccines encode for the S protein, the major surface protein on the coronavirus virion responsible for anchoring onto target cells, and thus the predominant virus-encoded target for nAb elicited by natural infection. Although clinical trials and real-world data have affirmed the safety and effectiveness of these FDA-authorized COVID-19 vaccines, more and more breakthrough infections have been reported for predominant VOCs. For example, the effectiveness of BNT162b2 against Delta-caused infection plummeted to 42%, as compared to 95% against the ancestral WA strain (Puranik et al., 2021), highlighting the need of developing vaccines that can offer wide protection against persistently emerging VOCs.
[00818] As each VOC possesses its unique set of mutations in the S protein, this will invariably result in distinct pools of epitopes being presented to lymphocytes by antigen presenting cells (APCs). Hence, we first evaluated the protection conferred following vaccination with mRNA vaccines encoding the S proteins of VOCs that emerged prior to Omicron. Our in vitro and in vivo data clearly demonstrated the premise that the strongest immunity against individual VOCs can be achieved by vaccination with variant-specific mRNA. For example, immunization with Delta S mRNA provided the best protection against Delta, but not to other VOCs. We also noticed that WA and Beta mRNA, especially the latter, provided the widest breadth of coverage for other VOCs. This could be explained by some mutations within Beta S protein, especially in the N-terminal and RBD. These mutations are also present in other VOCs, for example, L18, K417, E484, N501, D614, some of which are known to cause great immune escape (Grabowski et al., 2021; Winger and Caspari, 2021). Interestingly, Beta S mRNA has also been selected by Moderna to test in phase II either as a monovalent antigen or by mixing with mRNA-1273 to tackle the emerging VOCs (Pajon et al., 2022; Waltz, 2021).
[00819] With the emergence and rampage of Omicron during the course of our candidate vaccine development, we further tested the effectiveness of non-Omicron VOC mRNA vaccines against this new variant. Not surprisingly, none of these candidates induced strong immunity when challenged with Omicron (Figure 18A). This could be explained by the more than 30 mutations in the S protein of Omicron, a mutational burden which far exceeded that of preceding VOCs. This observation is consistent with the complete escape of Omicron from most of EUA-approved neutralizing antibody and convalescent sera therapies (Liu et al., 2022). To recapitulate our previous observation that VOC-matched vaccine induces the strongest nAb response in a VOC-dependent manner, we constructed a new mRNA vaccine encoding the S protein of Omicron and it indeed provided superior protection against this new VOC (Figure 18B and Figure 18C). The notion was reciprocally confirmed by the observation that Omicron mRNA did not induce strong immunity against other VOCs.
[00820] As the world is witnessing an astounding number of Omicron-related breakthrough cases even in people have received a third dose of currently available mRNA vaccines, a serious concern has been raised of whether a booster is adequate to stop its spread. Hence, we compared Omicron-specific booster to WA-specific booster to explore any additive protection. However, our data argued that the heterologous booster scheme is not superior to boosting with the ancestral WA mRNA in terms of establishing immunity against Omicron (Figure 18D). Similar conclusion was drawn by a research group from NIH, when they challenged mRNA- 1273 -vaccinated macaques with live omicron virus (Gagne et al., 2022). A plausible explanation is although Omicron S protein has ~35 mutations, it still exhibits 97% similarity to the ancestral WA strain in the amino acid sequence. As a result, most of the epitopes presented to lymphocytes could remain the same, and the consequence of antigenic drift contributed by those ~35 mutations could be masked by the surge of nAbs generated after the third dose. Hence, individuals with prior immunity from vaccination may not necessarily benefit from a change in vaccinating antigens. Moreover, considering the cost and time needed to put variant specific mRNA vaccine to practical use, the homologous boost scheme remains a scientifically proven and economically feasible option at hand in the fight against COVID-19.
[00821] Nonetheless, the concept of universal vaccine is still very appealing for at least two reasons. One is the virus could keep accumulating more mutations to eventually nullify the effectiveness of existing mRNA vaccines. The other is the idea can be applied to establish immunity against viruses causing different diseases. For example, dimeric RBDs have been ligated in tandem to target MERS, SARS and COVID-19 (Dai et al., 2020). In comparison, our chimeric design included the incorporation of the RBD of Delta variant in the Omicron spike mRNA which offers a larger pool of epitopes. Remarkably, we found that the resultant mRNA restored the strong protection against Delta infection while retaining the effective immunity against Omicron (Figure 19A and Figure 19B). Similar strategies have also been explored to target the Sarbecovirus subgenus with a single-molecule antigen (Martinez et al., 2021), suggesting broad applications of the chimeric vaccine design to deliver effective protection against a wide panel of diseases.
[00822] Another unique feature of our in-house mRNA vaccine is the mutation of furin cleavage site between the SI and S2 domains of S. This cleavage is believed to have emerged during viral transmission from its zoonotic host to humans and is one of the key attributes to explain the high transmissibility of SARS-CoV-2 in humans (Whittaker, 2021). The mutation is mainly to address the concern that circulating SI was detected in the plasma of vaccinated subjects (Ogata et al., 2021). Although the clinical consequence of the free SI moiety has not been full established, studies have suggested that SI can be taken up by many critical organs, such as liver, kidney, spleen, and even cross the blood-brain barrier to gain access to the brain (Rhea et al., 2021). As SI contains the intact RBD, it could still bind to ACE2 and trigger the downstream signaling events that may lead to inflammation and lung damage (Letarov et al., 2021; Suzuki and Gychka, 2021). Hence, we mutated the furin cleavage site and confirmed the abrogation of SI liberation in both the cell lysates and conditioned medium. Although a similar design was adopted in the recombinant protein-based NVX-CoV2373 by Novovax, it is not present in the two mRNA-based Covid-19 vaccines authorized by FDA. Another potential benefit of furin cleavage mutation is by retaining the full-length S protein within the cell and on the cell surface (Figure 20B), a larger pool of antigens could become available for presentation to induce the adaptive immunity. Indeed, S protein with furin cleavage mutation even binds with higher affinity to ACE2 (Laczko et al., 2020).
[00823] Taken together, our in-house designed mRNA vaccine represents a potentially safer alternative to existing products in use and could induce stronger adaptive immunity against prevalent VOCs, including Omicron and Delta. Our chimeric design will also facilitate the development of next-generation vaccines that achieve the balance between effectiveness and coverage, not only for the variants of SARS-CoV-2 but also for other viruses.
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Example 8. Generation and optimization of mRNA-lipid nanoparticle formulations. [00825] It was desirable to generate and optimize lipid containing formulations, such as lipid nanoparticle formulations that may be employed to prepare lyophilized formulations comprising the nucleic acid molecules, nucleic acid compositions and mRNA vaccines as disclosed herein and throughout. Inter alia, it was desirable to generate such formulations that would exhibit stability, nucleic acid molecule integrity, and in vivo efficacy after storage as a lyophilized powder at approximately 2-8 degrees Celsius for over two months. To generate such formulations, parameters such as buffer choice, use of cryoprotectants, ration of lipids, N/P ration, and pH, were tested and optimized.
[00826] Materials and Methods
[00827] Lipid nanoparticle preparation using Precision ignite nanoassemblr
[00828] Preparation of TBT buffer
[00829] To a 6 L beaker equipped with a stir bar (3”) was added Tris base (9.688 g, 0.08 mol), trehalose anhydrous (400.00 g), and water for injection (3.8 L). The mixture was allowed to stir at room temperature for 2 hours or until all materials dissolved in aqueous phase. The pH of the solution was adjusted with HC1 (IN) to pH 7.3 - 7.4. The overall volume of the solution was quantum satis-ed to 4 L, sterile filtered using 0.22 uM filtration, kept at room temperature for further use.
[00830] Preparation of Lipid solution
To a 5 mL Eppendorf tube was added JK0315CA (550.5 uL, 20 mg/mL in EtOH), DSPC (105.4 uL, 20 mg/mL in EtOH), Cholesterol (198.7 uL, 20 mg/mL in EtOH), and DMG- PEG2000 (62.8 uL, 20 mg/mL in EtOH) and EtOH (1796 uL) to make 2.713 mL lipid solution (lipid concentration: 10 mM; molar ratio of lipids:
JK0315CA/DSPC/Cholesterol/DMG-PEG2000 = 49.9/10/38.4/0.17)
[00831] Preparation of mRNA solution
[00832] mRNA (600 uL, 1 mg/mL) was dissolved in NaOAc buffer (pH 5, 25 mM) to make a mRNA NaOAc solution (pH 5, 25 mM, mRNA 73.7 ug/mL)
[00833] LNP formulation
[00834] Draw mRNA solution (8.14 mL, 86.4 ug/mL) in a 10 mL BD syringe, remove air bubble by gently tapping the syringe, Load onto ignite nanoassembr cartridge. Draw Lipid solution (10 mM) into a 3 mL BD syringe, remove air bubble carefully. Load the lipid solution onto cartridge. Set the flow rate as 12 mL/min, ratio of the aqueous solution to EtOH solution as 3/1, 0.1 mL and 0.05 mL waste volume at the beginning and the ending stage. Collect the formulated solution into a dialysis bag (100 KD) for buffer exchange.
[00835] Dialysis
[00836] The dialysis bag with the formulation was dialyzed with TBT buffer (1 L) prepared at step 1. Change buffer every 6 hours for 3 times. The LNP solution after dialysis was collected into an eppendoff tube (15 mL). The size, zeta was measured on Zetasizer Ultra. The mRNA concentration, encapsulation rate was determined using Ribogreen-based mRNA assay using plate reader.
[00837] Addition of Cryoprotectant (HP-b-CD)
After determine the mRNA concentration, the solution was added HP-b-CD (40%). The volume of HP-b-CD is dependent on the mRNA/lipid concentration and the ratio of Lipid/HP-b-CD is 1/8.17
[00838] Lyophilization
[00839] The LNP solution (with HP-b-CD) was aliquoted into 2 mL serum vial (400 uL each vial) with igloo cap on. The solution was frozen in a -80 °C freezer for 4 hours. The frozen sample was then transferred into the shelf of a freeze dryer (the temperature of the shelf was set and precooled to -20 °C.) The frozen sample was lyophilized at -20 °C for 24 hours under vacuum (0.05 mbar), then the shelf temperature was set to 25 °C (1 degree temp increase per min). The sample was then lyophilized at 25 °C for 6 hours, the cap was pushed on under vacuum, allow the freeze dryer chamber pressure to be increased to 1 atm, crimp the serum vial with aluminum cap, then store the capped product at 2- 8 °C for long time storage. [00840] Lipid Nanoparticle preparation using HPLC pump
[00841] Preparation of TBT buffer
[00842] Same as above for Precision ignite nanoassemblr
[00843] Preparation of Lipid solution
[00844] To a 100 mLNelgene bottle was added JK0315CA (11.560 mL, 20 mg/mL in EtOH), DSPC (2.213 mL, 20 mg/mL in EtOH), Cholesterol (4.172 mL, 20 mg/mL in EtOH), and DMG-PEG2000 (1.319 mL, 20 mg/mL in EtOH) and EtOH (37.716 mL) to make 56.979 mL lipid solution (lipid concentration: 10 mM; molar ratio of lipids:
JK0315CA/DSPC/Cholesterol/DMG-PEG2000 = 49.9/10/38.4/0.17)
[00845] Preparation of mRNA solution
To a 200 mL Nelgene bottle was added mRNA (12.6 mL, 1 mg/mL) and NaOAc buffer (158.34 mL, pH 5, 25 mM) to make a mRNANaOAc solution 170.94 mL (pH 5, 25 mM, mRNA 73.7 ug/mL).
[00846] LNP formulation
[00847] The lipid solution (organic) and mRNA solution (aqueous) was loaded onto HPLC pump. The pump was purged with lipid solution and mRNA solution until no bubble shows up. Adjust the percentage of organic to 25% and aqueous to 75% and the flow rate was set up to 12.5 mL/min (organic) and 37.5 mL/min (aqueous). The solution coming out of T-mixer was collected into 500 mL nelgene bottle equipped with a stir bar. After collecting 100 mL of the solution, the pump was stopped, and the bottle was put onto a stir plate. TBT buffer (300 mL) was slowly added into the nelgene bottle while stirring (150 rpm) during 5 min.
[00848] Tangential Flow Filtration (TFF)
[00849] The diluted LNP solution was transferred into Pall minimate EVO system
2 equipped with Repligen hollow fiber filter (mPES, 100KD, 115 cm ). The flow rate of the TFF was set to 60 mL/min, and the permeate flow rate was set to less than 5 mL/min. The solution was concentrated to 20 mL, then diluted to 100 mL with TBT buffer. This process was repeated 5 times and the final mRNA concentration of the LNP was adjusted to 100 ug/mL using TBT buffer.
[00850] Addition of Cryoprotectant (HP-b-CD)
[00851] Same as above for Precision ignite nanoassemblr
[00852] Lyophilization
[00853] Same as above for Precision ignite nanoassemblr [00854] In vivo testing of mRNA lipid nanoparticle formulations
[00855] CD-I or BALB/C mice were obtained and administered formulations at the dosing schedules and regimens indicated throughout Figures 22-31. Each formulation group had 5 mice (to a total of 30 mice).
[00856] Results
[00857] For all results, the following abbreviations have the following meanings:
[00858] Zeta=Zeta potention (mV); N/P= the ratio of positively-chargeable polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups; PEG=polyethylene glycol; EE(%)=encapsulation efficiency (as a percentage); HP-b-CD= 2 Hydroxypropyi- -eyelodextrin (HR-b-CD); PDI^polydi spersity index; DSPO Distearoylphosphatidylcholine.
[00859] Figure 22 shows results obtained when testing the indicated cholesterol and DSPC molar ratios, either as fresh formulations or as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated. [00860] Figure 23 shows results obtained when testing the indicated N/P ratios, PEG molar ratios, and lipid concentrations, either as fresh formulations or as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
[00861] Figure 24 shows results obtained when testing the indicated N/P ratios and PEG molar ratios, and lipid concentrations as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and 7, 14, 21, 28, 35, and 42 days after booster shot, as indicated.
[00862] Figure 25 shows results obtained in an additional experiment testing the indicated N/P ratios and PEG molar ratios as lyophilized and then reconstituted formulations. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot, as indicated.
[00863] Figure 26 shows results obtained in an additional experiment testing the indicated N/P ratios, as well as the presence or absence of HP-b-CD, with samples that were either freeze-thawed or lyophilized and then reconstituted. Mouse SI IgG titers were then measured at day 7, 14, 21, 28, and 35, and 42 after primary shot and at day 7, 14, 21, and 28 after booster shot, as indicated.
[00864] Figure 27 shows results obtained when testing the effect of adding either trehalose or sucrose to samples, as indicated. Mouse SI IgG titers were then measured at day 7, 14, 21, and 28, after first booster shot and at day 14, 21, and 28 after second booster shot, as indicated.
[00865] Figure 28 shows results obtained when testing the effect of adding Tris buffer or Phosphate buffer to samples, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated.
[00866] Figure 29 shows results obtained when testing the indicated formulations after various storage conditions/time periods, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated.
[00867] Figure 30 shows results obtained when testing the indicated formulations at the indicated pH units. Mouse SI IgG titers were then measured at day 7 after primary shot, as indicated. [00868] Figure 31 shows results obtained when testing the indicated formulations prepared using HPLC pump, as indicated. Mouse SI IgG titers were then measured at day 14 after primary shot, as indicated.
[00869] Conclusions [00870] The following conclusions were drawn based on the cumulative results shown in the Figures 22-31 and described above:
[00871] Desirable cholesterol molar ratio ranges for mRNA formulations are within about 38.5% to about 43% (inclusive);
[00872] Desirable DSPC molar ratio ranges for mRNA formulations are within about 10% to about 12% (inclusive);
[00873] Desirable PEG molar ratio ranges for mRNA formulations are within about 1.5%
- to about 2% (inclusive);
[00874] Desirable N/P ratios for mRNA formulations are within about 5 to about 12 (inclusive); [00875] Higher N/P ratios are desired in terms of stability and immune response;
[00876] Freeze dried samples are comparable in performance to freshly prepared samples; [00877] Trehalose showed similar immune response to sucrose;
[00878] Tris buffered trehalose buffer is slightly better than Phosphate buffered trehalose buffer in terms of immune response; [00879] pH approximately or close to 7 or slightly below 7 is beneficial in terms of mRNA integrity.
[00880] For example, freeze dried sample STI-LNP-NP7-PEG1 75-b2.5 (N/P=7;
PEG1.75) showed > 30% mRNA integrity after storage at 25 oC for 45 days; This sample showed acceptable immune response after storage at 25 °C for 30 days. As another example, freeze dried samples STI-LNP-NP6-PEG1 5-b2.5 (N/P = 6; PEG 1.5) and STI-LNP-NP6- PEG1.75 (N/P =6; PEG 1.75) showed > 50% mRNA integrity after storage at 4 oC for 60 days.
[00881] Additionally:
[00882] Optimized cryoprotectants (HP-beta-CD) support the shape of the LNP and prevent the LNP from breaking during freeze dry and reconstitute process;
[00883] One example of a desirable ratio of the lipid components JK-0315- C A/D SPC/Cholesterol/DMG-PEG2000 = 49.9/10/38.4/0.17. [00884] An example of a desirable N/P ration is about 7 or 7. The N/P ratio was optimized to 7.
[00885] DOTAP may be used instead of JK-0315-CA(20%), which may facilitate, for example, intramuscular without compromising the immune response.
[00886] pH value of the final product formulation has been correlated to the mRNA integrity. The pH optimized formulation may provide longer shelf live of the product.
[00887] Tris/Trehalose or similar buffers may provide extra protection to the formulated lipid nanoparticles.
Figure imgf000195_0001
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Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
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Figure imgf000223_0001
Figure imgf000224_0001

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence encoding at least a portion of a viral spike protein, wherein the nucleic acid sequence comprises least one RBD-encoding sequence of a coronavirus spike protein.
2. The nucleic acid molecule of claim 1, wherein the at least one RBD- encoding sequence is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an RBD-encoding nucleic acid sequence from a SAR.S-CoV-2 virus selected the group consisting of: a) a SAR.S-CoV-2 Wuhan/Washington variant; b) a SAR.S-CoV-2 Alpha variant; c) a SAR.S-CoV-2 Beta variant; d) a SAR.S-CoV-2 Gamma variant; e) a SAR.S-CoV-2 Delta variant; f) a SAR.S-CoV-2 Delta Plus variant; g) a SAR.S-CoV-2 Kappa variant; h) a SAR.S-CoV-2 Lambda variant; i) a SAR.S-CoV-2 Omicron variant; j) a SAR.S-CoV-2 Zeta variant; k) a SAR.S-CoV-2 Epsilon variant; l) a SAR.S-CoV-2 Omicron variant; m) a SAR.S-CoV-2 Omicron Plus variant; and n) combinations of a) - m).
3. The nucleic acid molecule of claim 1 or claim 2, wherein the at least one RBD-encoding sequence encodes an RBD amino acid sequence comprising one or more or the following mutations: D614G; D69/70-D144-N501 Y-A570D-D614G- P681H-T716I-S982 A-D 1118H; D80 A-D215G-A242/244-K417N-E484K-N501 Y- D614G-A701V; D614G, S13I, W152C, L452R; G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, H1101D; T19R, (G142D), D156-157, R158G, L452R, T478K, D614G, P681R, D950N; T19R, (G142D), D156-157, R158G, K417N,
L452R, T478K, D614G, P681R, D950N; L18F, T20N, P26S, D138Y, R190S,
K417T, E484K, N501Y, D614G, H655Y, T1027I; G75V, T76I, D246-252, L452Q, F490S, D614G, T859N; E484Q, F565L, D614G, V1176F; L5F, T95I, D253G,
E484K, D614G, A701V; L5F, T95I, D253G, S477N, D614G, A701V; T95I, DU144, E484K, D614G, P681H, D796H; D69/70, D614G, N501Y; D614G, K417N, E484K, N501Y; L452R, E484Q, P681R; D614G, L452R, E484K; D614G, L452R; D69/70; D614G-K378Y; D614G-E406W; D614G-K417E; D614G-N439K; D614G-N440D; D614G-K444Q; D614G-V445A; D614G-G446V; D614G-Y453F; D614G-L455F; D614G-G476S; D614G-S477N; D614G, T478K; D614G-E484K; D614G-E484Q; D614G-F486I; D614G-F486V; D614G-N487R; D614G-N487Y; D614G-Y489H; D614G-F490S; D614G-Q493K; D614G-Q493R; D614G-S494P; D614G-N501Y; D614G-Q677H; and D614G-Q677P.
4. The nucleic acid molecule of any of claims 1-3, wherein the coronavirus spike protein comprises one or more or the following mutations: D614G; A69/70-A144-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H; D80A-D215G- D242/244-K417N-E484K-N 501 Y-D614G- A701 V; D614G, S13I, W152C, L452R; G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, H1101D; T19R,
(G142D), D156-157, R158G, L452R, T478K, D614G, P681R, D950N; T19R, (G142D), D156-157, R158G, K417N, L452R, T478K, D614G, P681R, D950N; L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I; G75V, T76I, D246-252, L452Q, F490S, D614G, T859N; E484Q, F565L, D614G, V1176F; L5F, T95I, D253G, E484K, D614G, A701V; L5F, T95I, D253G, S477N, D614G, A701V; T95I, DU144, E484K, D614G, P681H, D796H; D69/70, D614G, N501Y; D614G, K417N, E484K, N501Y; L452R, E484Q, P681R; D614G, L452R, E484K; D614G, L452R; D69/70; D614G-K378Y; D614G-E406W; D614G-K417E; D614G-N439K; D614G-N440D; D614G-K444Q; D614G-V445A; D614G-G446V; D614G-Y453F; D614G-L455F; D614G-G476S; D614G-S477N; D614G, T478K; D614G-E484K; D614G-E484Q; D614G-F486I; D614G-F486V; D614G-N487R; D614G-N487Y; D614G-Y489H; D614G-F490S; D614G-Q493K; D614G-Q493R; D614G-S494P; D614G-N501Y; D614G-Q677H; and D614G-Q677P.
5. The nucleic acid molecule of any of claims 1-4, wherein the at least one RBD-encoding sequence is derived from, or otherwise corresponds to, one or more SARS-CoV-2 virus spike-encoding nucleic acid sequences selected from the group consisting of: a) a SARS-CoV-2 Wuhan/Washington variant; b) a SARS-CoV-2 Alpha variant; c) a SARS-CoV-2 Beta variant; d) a SARS-CoV-2 Gamma variant; e) a SARS-CoV-2 Delta variant; f) a SARS-CoV-2 Delta Plus variant; g) a SARS-CoV-2 Kappa variant; h) a SARS-CoV-2 Lambda variant; i) a SARS-CoV-2 Omicron variant; j) a SARS-CoV-2 Zeta variant; k) a SARS-CoV-2 Epsilon variant; l) a SARS-CoV-2 Omicron variant; m) a SARS-CoV-2 Omicron Plus variant; and n) combinations of a) - m).
6. The nucleic acid molecule of any of claims 1-5, wherein the at least one RBD-encoding sequence comprises an RBD-encoding sequence present in one or more of SEQ ID Nos: 1-12 and 15-19.
7. The nucleic acid molecule of any of claims 1-6, wherein the nucleic acid molecule comprises at least two, at least three, at least four, or at least five RBD-encoding sequences selected from the group consisting of: a) a SARS-CoV-2 Wuhan/Washington variant; b) a SARS-CoV-2 Alpha variant; c) a SARS-CoV-2 Beta variant; d) a SARS-CoV-2 Gamma variant; e) a SARS-CoV-2 Delta variant; f) a SARS-CoV-2 Delta Plus variant; g) a SARS-CoV-2 Kappa variant; h) a SARS-CoV-2 Lambda variant; i) a SARS-CoV-2 Omicron variant; j) a SARS-CoV-2 Zeta variant; k) a SARS-CoV-2 Epsilon variant; l) a SARS-CoV-2 Omicron variant; m) a SARS-CoV-2 Omicron Plus variant; and n) combinations of a) - m).
8. The nucleic acid molecule of any of claims 1-7, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a chimeric spike protein.
9. The nucleic acid molecule of any of claims 1-8, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a chimeric spike protein comprising: an RBD from a Delta variant and an RBD from an Omicron variant in either order; an RBD from a Beta variant and an RBD from an Omicron variant in either order; or an RBD from a Delta variant, an RBD from a Beta variant, and an RBD from an Omicron variant in any order.
10. The nucleic acid molecule of any of claims 1-9, wherein the viral spike protein is encoded by a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical, at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical , at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to a spike protein encoding sequence from a SARS-CoV-2 virus selected the group consisting of: a) a SARS-CoV-2 Wuhan/Washington variant; b) a SARS-CoV-2 Alpha variant; c) a SARS-CoV-2 Beta variant; d) a SARS-CoV-2 Gamma variant; e) a SARS-CoV-2 Delta variant; f) a SARS-CoV-2 Delta Plus variant; g) a SARS-CoV-2 Kappa variant; h) a SARS-CoV-2 Lambda variant; i) a SARS-CoV-2 Omicron variant; j) a SARS-CoV-2 Zeta variant; k) a SARS-CoV-2 Epsilon variant; l) a SARS-CoV-2 Omicron variant; m) a SARS-CoV-2 Omicron Plus variant; and n) combinations of a) - m).
11. The nucleic acid molecule of any of claims 1-10, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a chimeric spike protein as present in SEQ ID NOS: 4, 6, and 7.
12. The nucleic acid molecule of any of claims 1-11, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a furin site mutation.
13. The nucleic acid molecule of any of claims 1-12, wherein the nucleic acid molecule comprises nucleic acid sequence encoding a furin site mutation sequence as set forth in SEQ ID NO:13.
14. A nucleic acid molecule of any of claims 1-13, wherein he nucleic acid molecule comprises nucleic acid sequence encoding a PP spike-stabilizing mutation.
15. A nucleic acid molecule of any of claims 1-14, the nucleic acid molecule further comprises nucleic acid sequence encoding a PP spike-stabilizing mutation as set forth in SEQ ID NO: 14.
16. The nucleic acid molecule of any of claims 1-15, wherein the nucleic acid molecule further comprises nucleic acid sequence encoding a furin site mutation and a PP spike-stabilizing mutation.
17. The nucleic acid molecule of any of claims 1-16, wherein the nucleic acid molecule further comprises nucleic acid sequence encoding a furin site mutation as set forth in SEQ ID NO: 13 and a PP spike-stabilizing mutation as set forth in SEQ ID NO: 14.
18. The nucleic acid molecule of any one of claims 1-17, wherein the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
19. The nucleic acid molecule of any one of claims 1-18, wherein the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-7.
20. The nucleic acid molecule of any one of claims 1-19, wherein the nucleic acid molecule consists of a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-19.
21. The nucleic acid molecule of any one of claims 1-20, wherein the nucleic acid molecule consists of a nucleic acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-7.
22. The nucleic acid molecule of any one of claims 1-21, wherein the nucleic acid molecule encodes a RBD or a spike protein that comprises an amino acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20- 32.
23. The nucleic acid molecule of any one of claims 1-21, wherein the nucleic acid molecule encodes a RBD or a spike protein that consists of an amino acid sequence that is at least 80% identical, at least 81% identical, least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, least 86% identical, at least 87% identical at least 88% identical, least 89% identical, at least 90% identical, at least 91% identical, least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, least 96% identical, at least 97% identical at least 98% identical, least 99% identical, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20- 32.
24. The nucleic acid molecule of any of claims 1-23, wherein the nucleic acid molecule comprises a DNA sequence.
25. The nucleic acid molecule of any one of claims 1-23, wherein the nucleic acid molecule comprises an RNA sequence.
26. The nucleic acid molecule of any one of claims 1-25, further comprising a promoter operably linked to the nucleic acid sequence.
27. The nucleic acid molecule any one of claims 1-26, further comprising a promoter operably linked to the nucleic acid sequence, wherein the promoter is an SP6, T3, or T7 promoter.
28. The nucleic acid molecule of any one of claims 1-27, wherein the nucleic acid molecule includes at least one modified nucleotide.
29. The nucleic acid molecule of any one of claims 1-28, wherein the nucleic acid molecule includes at least one modified nucleotide, wherein the at least one modified nucleotide is pseudouridine, N1 -methyl-pseudouridine, or 2-thiouridine.
30. The nucleic acid molecule of any one of claims 1-29, wherein the nucleic acid molecule comprises a 5’ cap structure.
31. The nucleic acid molecule of any one of claims 1-30, wherein the nucleic acid molecule comprises a 3’ polyA sequence.
32. A composition comprising at least two, at least three, at least four, or at least five nucleic acid molecules according to any of claims 1-31.
33. The composition of claim 32, wherein each of the nucleic acid molecules in the composition encodes, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-19.
34. The composition of claim 32 or claim 33, wherein each of the nucleic acid molecules in the composition encodes, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-12.
35. The composition of any of claim 32-34, wherein each of the nucleic acid molecules in the composition encodes, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS: 1-7.
36. The composition of any of claim 32-34, wherein each of the nucleic acid molecules in the composition encodes, independently and uniquely, and RBD or a spike protein encoded by a nucleic acid sequence present in one of SEQ ID NOS:4-12.
37. A pharmaceutical composition comprising: the nucleic acid molecule of any of claims 1-31; or the composition of any of claim 32-36; and a pharmaceutically acceptable carrier.
38. The pharmaceutical composition of claim 37, wherein the pharmaceutically acceptable carrier comprises a lipid.
39. The pharmaceutical composition of claim 37 or claim 38, wherein the lipid comprises a cationic lipid of formula (I):
Figure imgf000234_0001
or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: R1 is H, -OR1A, -YOR1A, -NR1AR1B, -YNR1AR1B, -SR1A, -YSR1A, -(C=O)R1A, -Y(C=O)R1A, -(C=O)OR1A, -Y(C=O)OR1A, -O(C=O)R1A, -YO(C=O)R1A, -O(C=O)OR1A, -YO(C=O)OR1A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted   heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Y is substituted or unsubstituted C0-C12 alkylene or substituted or unsubstituted 0 to 12 membered heteroalkylene; R2 is H, -OR2A, -SR2A, -(C=O)R2A, -(C=O)OR2A, -O(C=O)R2A, -O(C=O)OR2A, -(C=O)NHR2A, -NH(C=O)R2A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R3 is H, -OR3A, -SR3A, -(C=O)R3A, -(C=O)OR3A, -O(C=O)R3A, -O(C=O)OR3A, -(C=O)NHR3A, -NH(C=O)R3A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;   R4 is H, -OR4A, -SR4A, -(C=O)R4A, -(C=O)OR4A, -O(C=O)R4A, -O(C=O)OR4A, -(C=O)NHR4A, -NH(C=O)R4A, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R5 is H, -OR5A, -SR5A, -(C=O)R5A, -(C=O)OR5A, -O(C=O)R5A, -O(C=O)OR5A, -(C=O)NHR5A, -NH(C=O)R5A, substituted or unsubstituted alkyl, or substituted or  unsubstituted heteroalkyl; B1 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted     heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; B2 and B3 are each independently a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene; L1 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR101R102)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR101C(=O)‑, ‑C(=O)NR101‑, ‑NR101C(=S)‑, ‑C(=S)NR101‑, ‑NR101C(=O)NR102‑, ‑NR101C(=S)NR102‑, ‑OC(=O)NR101‑, ‑NR101C(=O)O‑, ‑SC(=O)NR101‑ or ‑NR101C(=O)S‑; L2 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR201R202)sO-,  ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR201C(=O)‑, ‑C(=O)NR201‑, ‑NR201C(=O)NR202‑, ‑NR201C(=S)‑, ‑C(=S)NR201‑, ‑NR201C(=S)NR202‑, ‑OC(=O)NR201‑, ‑NR201C(=O)O‑, ‑SC(=O)NR201‑ or ‑NR201C(=O)S‑; L3 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR301R302)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR301C(=O)‑, ‑C(=O)NR301‑, ‑NR301C(=O)NR302‑, ‑NR301C(=S)‑, ‑C(=S)NR301‑, ‑NR301C(=S)NR302‑, ‑OC(=O)NR301‑, ‑NR301C(=O)O‑, ‑SC(=O)NR301‑ or ‑NR301C(=O)S‑; L4 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR401R402)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR401C(=O)‑, ‑C(=O)NR401‑, ‑NR401C(=O)NR402‑, ‑NR401C(=S)‑, ‑C(=S)NR401‑, ‑NR401C(=S)NR402‑, ‑OC(=O)NR401‑, ‑NR401C(=O)O‑,  ‑SC(=O)NR401‑ or ‑NR401C(=O)S‑; L5 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR501R502)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR501C(=O)‑, ‑C(=O)NR501‑, ‑NR501C(=O)NR502‑, ‑NR501C(=S)‑, ‑C(=S)NR501‑, ‑NR501C(=S)NR502‑, ‑OC(=O)NR501‑, ‑NR501C(=O)O‑, ‑SC(=O)NR501‑ or ‑NR501C(=O)S‑; L6 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR601R602)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR601C(=O)‑, ‑C(=O)NR601‑, ‑NR601C(=O)NR602‑, ‑NR601C(=S)‑, ‑C(=S)NR601‑, ‑NR601C(=S)NR602‑, ‑OC(=O)NR601‑, ‑NR601C(=O)O‑, ‑SC(=O)NR601‑ or ‑NR601C(=O)S‑; L7 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR701R702)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR701C(=O)‑, ‑C(=O)NR701‑, ‑NR701C(=O)NR702‑, ‑NR701C(=S)‑, ‑C(=S)NR701‑, ‑NR701C(=S)NR702‑, ‑OC(=O)NR701‑, ‑NR701C(=O)O‑, ‑SC(=O)NR701‑ or ‑NR701C(=O)S‑;     La1 and La2 are each independently
Figure imgf000236_0001
each X is independently O, S, or CH2; W1, W2, W3, W4, W5, and W6 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene; each R1A and R1B is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; each R2A, R3A, R4A, and R5A is independently H, substituted or unsubstituted C1-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl; each R101, R102, R201, R202, R301, R302, R401, R402, R501, R502, R601, R602, R701, and R702 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; and each s is independently an integer from 1 to 4.
40. The pharmaceutical composition of claim 39, wherein the cationic lipid is a lipid wherein: R1 is H, -OR1A or substituted or unsubstituted heteroalkyl; L1 is a bond, ‑NR101C(=S)‑, ‑C(=S)NR101‑, -O(C=O)-, -(C=O)O-, or ‑O‑; B1 is a bond or a substituted or unsubstituted alkylene; B2 and B3 are each independently a bond or substituted or unsubstituted alkylene; L2 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑; L4 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑; W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; La1 and La2 are each independently
Figure imgf000236_0002
    each X is independently O or S; L3 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑; L5 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑; L6 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑;   L7 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, or ‑S‑; R2 is H or substituted or unsubstituted alkyl; R3 is H or substituted or unsubstituted alkyl; R4 is H or substituted or unsubstituted alkyl; R5 is H or substituted or unsubstituted alkyl;   each R1A is independently H or substituted or unsubstituted C1-C12 alkyl; and each R101 is independently H or substituted or unsubstituted 2 to 12 membered heteroalkyl.
41. The pharmaceutical composition of claim 39, wherein the cationic lipid is a  lipid wherein: R1 is H, -OH, methoxy, ethoxy, or substituted or unsubstituted heteroalkyl; L1 is a bond, ‑NR101C(=S)‑, or ‑C(=S)NR101‑; B1 is a bond or an unsubstituted C1-C8 alkylene; B2 and B3 are each independently a bond or substituted or unsubstituted C1-C8 alkylene;   L2 is a bond, -O(C=O)-, or -(C=O)O-; L4 is a bond, -O(C=O)-, or -(C=O)O-; W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; La1 and La2 are each independently
Figure imgf000237_0001
;   each X is independently O or S; L3 is a bond, -O(C=O)-, or -(C=O)O-; L5 is a bond, -O(C=O)-, or -(C=O)O-; L6 is a bond, -O(C=O)-, or -(C=O)O-; L7 is a bond, -O(C=O)-, or -(C=O)O-;   R2 is H or substituted or unsubstituted C1-C12 alkyl;     R3 is H or substituted or unsubstituted C1-C12 alkyl; R4 is H or substituted or unsubstituted C1-C12 alkyl; R5 is H or substituted or unsubstituted C1-C12 alkyl; and each R101 is independently substituted or unsubstituted 2 to 12 membered heteroalkyl.
42. The pharmaceutical composition of claim 39, wherein the cationic lipid is a lipid wherein: R1 is -OH or methoxy; L1 is a bond;   B1 is an unsubstituted C1-C8 alkylene; B2 and B3 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; L2 is a bond; L4 is a bond;   W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; La1 and La2 are each independently
Figure imgf000238_0001
; each X is independently O; L3 is a bond;   L5 is a bond; L6 is a bond; L7 is a bond; R2 is H or substituted or unsubstituted C1-C12 alkyl; R3 is H or substituted or unsubstituted C1-C12 alkyl;   R4 is H or substituted or unsubstituted C1-C12 alkyl; and R5 is H or substituted or unsubstituted C1-C12 alkyl.
43. The pharmaceutical composition of claim 39, wherein the cationic lipid is a lipid wherein: R1 is substituted or unsubstituted heteroalkyl;     L1 is ‑C(=S)NR101‑, where the carbon atom is connected to the nitrogen atom in formula (I); B1 is a bond; B2 and B3 are each independently a bond or substituted or unsubstituted C1-C8  alkylene; L2 is a bond, -O(C=O)-, or -(C=O)O-; L4 is a bond, -O(C=O)-, or -(C=O)O-; W1, W2, W3, W4, W5, and W6 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; La1 and La2 are each independently
Figure imgf000239_0001
; each X is independently O; L3 is a bond; L5 is a bond; L6 is a bond; L7 is a bond; R2 is H or substituted or unsubstituted C1-C12 alkyl; R3 is H or substituted or unsubstituted C1-C12 alkyl; R4 is H or substituted or unsubstituted C1-C12 alkyl; and R5 is H or substituted or unsubstituted C1-C12 alkyl.  
44. The pharmaceutical composition of claim 39, wherein the cationic lipid is:
Figure imgf000239_0002
   
p
Figure imgf000240_0002
45. The pharmaceutical composition of claim 37 or claim 38, wherein the lipid comprises a cationic lipid of formula (II):
Figure imgf000240_0001
or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein: B4 is W7-La3-W8; W7 and W8 are each independently a bond, substituted or unsubstituted  alkylene, or substituted or unsubstituted heteroalkylene; La3 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CRa31Ra32)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NRa31C(=O)‑, ‑C(=O)NRa31‑, ‑NRa31C(=O)NRa32‑, ‑NRa31C(=S)‑, ‑C(=S)NRa31‑, ‑NRa31C(=S)NRa32‑, ‑OC(=O)NRa31‑, ‑NRa31C(=O)O‑, ‑SC(=O)NRa31‑ or ‑NRa31C(=O)S‑;   R10 and R11 are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; B5, B6, and B7 are each independently a bond, substituted or unsubstituted alkylene,  or substituted or unsubstituted heteroalkylene; L8 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR801R802)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR801C(=O)‑, ‑C(=O)NR801‑, ‑NR801C(=O)NR802‑, ‑NR801C(=S)‑, ‑C(=S)NR801‑, ‑NR801C(=S)NR802‑, ‑OC(=O)NR801‑, ‑NR801C(=O)O‑, ‑SC(=O)NR801‑ or ‑NR801C(=O)S‑; L9 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR901R902)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR901C(=O)‑, ‑C(=O)NR901‑, ‑NR901C(=O)NR902‑, ‑NR901C(=S)‑, ‑C(=S)NR901‑, ‑NR901C(=S)NR902‑, ‑OC(=O)NR901‑, ‑NR901C(=O)O‑, ‑SC(=O)NR901‑ or ‑NR901C(=O)S‑; L10 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR110R111)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR110C(=O)‑, ‑C(=O)NR110‑, ‑NR110C(=O)NR111‑, ‑NR110C(=S)‑, ‑C(=S)NR110‑, ‑NR110C(=S)NR111‑, ‑OC(=O)NR110‑, ‑NR110C(=O)O‑, ‑SC(=O)NR110‑ or ‑NR110C(=O)S‑; R7, R8, and R9 are each independently H, substituted or unsubstituted C1-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl;   each Ra31 and Ra32 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl;     each R801, R802, R901, R902, R110, and R111 is independently H, substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; and each s is independently an integer from 1 to 4.
46. The pharmaceutical composition of claim 45, wherein the cationic lipid is a lipid wherein: W7 and W8 are each independently a bond or substituted or unsubstituted alkylene; La3 is a bond;   R10 and R11 are each independently H, substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted heterocycloalkyl; B5 is a bond; B6 and B7 are each independently a bond or substituted or unsubstituted alkylene;  L8 is a bond; L9 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑; L10 is a bond, -O(C=O)-, -(C=O)O-, or ‑C(=O)‑; and R7, R8, and R9 are each independently H or substituted or unsubstituted C1-C30 alkyl.  
47. The cationic lipid of claim 45, wherein W7 and W8 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; La3 is a bond; R10 and R11 are each independently substituted or unsubstituted alkyl or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or   unsubstituted heterocycloalkyl; B5 is a bond; B6 and B7 are each independently a bond or substituted or unsubstituted C1-C8 alkylene; L8 is a bond; L9 is -O(C=O)- or -(C=O)O-; L10 -O(C=O)- or -(C=O)O-; and R7, R8, and R9 are each independently substituted or unsubstituted C1-C20 alkyl.    
48. The pharmaceutical composition of claim 45, wherein the cationic lipid is a lipid wherein: W7 and W8 are each independently a bond or substituted or unsubstituted C2- C4 alkylene; La3 is a bond; R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl; B5 is a bond;   B6 and B7 are each independently a bond or substituted or unsubstituted C2-C4 alkylene; L8 is a bond; L9 is -O(C=O)- or -(C=O)O-; L10 -O(C=O)- or -(C=O)O-; R7 is H or methyl; and R8, and R9 are each independently substituted or unsubstituted C1-C20 alkyl.
49. The pharmaceutical composition of claim 45, wherein the cationic lipid is a lipid wherein: W7 and W8 are each independently a bond or unsubstituted C2-C4 alkylene;  La3 is a bond; R10 and R11 are each independently substituted or unsubstituted methyl, ethyl, propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl; B5 is a bond;   B6 and B7 are each independently a bond or unsubstituted C2-C4 alkylene; L8 is a bond; L9 is -O(C=O)- or -(C=O)O-; L10 is -O(C=O)- or -(C=O)O-; R7 is H or methyl; and R8 and R9 are each independently substituted or unsubstituted C1-C20 alkyl.    
50. The pharmaceutical composition of claim 45, wherein the cationic lipid is a lipid wherein: W7 and W8 are each independently a bond or unsubstituted C2-C4 alkylene; La3 is a bond; R10 and R11 are each independently substituted or unsubstituted methyl, ethyl,  propyl, isopropyl, or R10 and R11 together with the nitrogen atom to which they are connected form a substituted or unsubstituted 5 to 6 membered heterocycloalkyl; B5, B6, and B7 are each independently a bond; L8 is a bond; L9 is a bond;   L10 is a bond; R7 is H or methyl; and R8 and R9 are each independently substituted or unsubstituted C1-C30 alkyl.
51. The pharmaceutical composition of claim 45, wherein the cationic lipid is a lipid wherein the cationic lipid is:
Figure imgf000244_0001
,    
Figure imgf000245_0001
Figure imgf000246_0001
or a pharmaceutically acceptable salt thereof.
52. The pharmaceutical composition of claim 37 or claim 38, wherein the lipid comprises a cationic lipid of formula (III):
Figure imgf000247_0002
or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein:
Figure imgf000247_0001
Q is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted  heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene; V is substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted arylene; B8, B9, B10, and B11 are each independently a bond, substituted or unsubstituted  alkylene, or substituted or unsubstituted heteroalkylene; L12 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR210R211)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR210C(=O)‑, ‑C(=O)NR210‑, ‑NR210C(=O)NR211‑, ‑NR210C(=S)‑, ‑C(=S)NR210‑, ‑NR210C(=S)NR211‑, ‑OC(=O)NR210‑, ‑NR210C(=O)O‑, ‑SC(=O)NR210‑ or ‑NR210C(=O)S‑;   L13 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR310R311)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR310C(=O)‑, ‑C(=O)NR310‑, ‑NR310C(=O)NR311‑, ‑NR310C(=S)‑, ‑C(=S)NR310‑, ‑NR310C(=S)NR311‑, ‑OC(=O)NR310‑, ‑NR310C(=O)O‑, ‑SC(=O)NR310‑ or ‑NR310C(=O)S‑;     R12 is H, -OR12A, -SR12A, -NR12A, -CN, -(C=O)R12A, -O(C=O)R12A, -(C=O)OR12A, -NR12A(C=O)-R12B, -(C=O)NR12AR12B; R13 is H, -OR13A, -SR13A, -NR13A, -CN, -(C=O)R13A, -O(C=O)R13A, -(C=O)OR13A, -NR13A(C=O)-R13B, -(C=O)NR13AR13B; R14 and R15 are each independently substituted or unsubstituted C2-C30 alkyl, or substituted or unsubstituted 2 to 30 membered heteroalkyl; R12A, R12B, R13A, and R13B are each independently H, substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted 2 to 20 membered heteroalkyl; each R210, R211, R310, and R311 is independently H, substituted or unsubstituted C1- C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; each n is independently an integer from 0 to 8; and each s is independently an integer from 1 to 4.
53. The pharmaceutical composition of claim 52, wherein the cationic lipid is a lipid wherein:
Figure imgf000248_0001
Q is substituted or unsubstituted alkylene; V is substituted or unsubstituted alkylene; B8, B9, B10, and B11 are each independently substituted or unsubstituted alkylene; L12 is -O(C=O)- or -(C=O)O-; L13 is -O(C=O)- or -(C=O)O-; R12 is H, -OR12A, or-NR12A; R13 is H, -OR13A, or-NR13A;   R14 and R15 are each independently substituted or unsubstituted C2-C30 alkyl; R12A and R13A are each independently H, substituted or unsubstituted C1-C20 alkyl; and each n is independently an integer from 0 to 8.    
54. The pharmaceutical composition of claim 52, wherein the cationic lipid is a lipid wherein:
Figure imgf000249_0001
; V is substituted or unsubstituted alkylene; B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C20 alkylene; L12 is -O(C=O)- or -(C=O)O-; L13 is -O(C=O)- or -(C=O)O-;   R12 is H or -OR12A; R13 is H or -OR13A; R14 and R15 are each independently substituted or unsubstituted C2-C20 alkyl; R12A and R13A are each independently H, substituted or unsubstituted C1-C8 alkyl;   each n is independently an integer from 0 to 4.
55. The pharmaceutical composition of claim 52, wherein the cationic lipid is a lipid wherein:
Figure imgf000249_0002
  V is unsubstituted alkylene; B8, B9, B10, and B11 are each independently substituted or unsubstituted C1-C8 alkylene; L12 is -O(C=O)- or -(C=O)O-; L13 is -O(C=O)- or -(C=O)O-;   R12 is -OH, methoxy, or ethoxy; R13 is -OH, methoxy, or ethoxy; R14 and R15 are each independently substituted or unsubstituted C2-C20 alkyl; and each n is independently an integer from 0 to 4.    
56. The pharmaceutical composition of claim 52, wherein the cationic lipid is a lipid wherein the cationic lipid is:
Figure imgf000250_0001
or a pharmaceutically acceptable salt thereof.
57. The pharmaceutical composition of claim 37 or claim 38, wherein the lipid comprises a cationic lipid of formula (IV):
Figure imgf000250_0002
or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, or prodrug thereof, wherein:
B12 is -W7-La3-W8-;
W7 and W8 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted 2 to 12 membered heteroalkylene;
La3 is a bond, -S-S-, -0-(CH20)m-,
Figure imgf000250_0003
W9 and W10 are each independently a bond, substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted 2 to 12 membered heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or any combination thereof; L14 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR410R411)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR410C(=O)‑, ‑C(=O)NR410‑, ‑NR410C(=O)NR411‑, - NR410C(=S)-, -C(=S)NR410‑, ‑NR410C(=S)NR411‑, ‑OC(=O)NR410‑, ‑NR410C(=O)O‑, ‑SC(=O)NR410‑ or ‑NR410C(=O)S‑; L15 is a bond, -O(C=O)-, -(C=O)O-, ‑O(C=O)O‑, ‑C(=O)‑, ‑O‑, ‑O(CR510R511)sO-, ‑S‑, ‑C(=O)S‑, ‑SC(=O)‑, ‑NR510C(=O)‑, ‑C(=O)NR510‑, ‑NR510C(=O)NR511‑, - NR510C(=S)-, -C(=S)NR510‑, ‑NR510C(=S)NR511‑, ‑OC(=O)NR510‑, ‑NR510C(=O)O‑, ‑SC(=O)NR510‑ or ‑NR510C(=O)S‑;   R16 and R17 are each independently
Figure imgf000251_0001
a fragment of cationic lipid of formula (I),
Figure imgf000251_0002
a fragment of cationic lipid of formula (II), R
Figure imgf000251_0003
a fragment of cationic lipid of formula (II),
Figure imgf000251_0004
  fragment of cationic lipid of formula (III), or a fragment of cationic lipid of formula (III);
Figure imgf000251_0005
    each R410, R411, R510, and R511 is independently H, substituted or unsubstituted C1- C12 alkyl, or substituted or unsubstituted 2 to 12 membered heteroalkyl; each m is independently an integer from 0 to 8; and each s is independently an integer from 1 to 4.  
58. The pharmaceutical composition of claim 57, wherein the cationic lipid is a lipid wherein: La3 is a bond,
Figure imgf000252_0001
W7 and W8 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; L14 is -O(C=O)-, -(C=O)O-, ‑C(=O)‑, ‑NR410C(=O)‑, ‑C(=O)NR410‑, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑; L15 is -O(C=O)-, -(C=O)O-, ‑C(=O)‑, ‑NR510C(=O)‑, ‑C(=O)NR510‑, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; R16 and R17 are each independently
Figure imgf000252_0002
fragment of cationic lipid of formula (II); and each R410 and R510 is independently H or substituted or unsubstituted C1-C12 alkyl.
59. The pharmaceutical composition of claim 57, wherein the cationic lipid is a lipid wherein: La3 is a bond,
Figure imgf000252_0003
W7 and W8 are each independently a bond or unsubstituted C1-C12 alkylene; L14 is -O(C=O)-, -(C=O)O-, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑;     L15 is -O(C=O)-, -(C=O)O-, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or substituted or unsubstituted C1-C12 alkylene; R16 and R17 are each independently
Figure imgf000253_0001
fragment of cationic lipid of formula (II); and each R410 and R510 is independently H or substituted or unsubstituted C1-C12 alkyl.
60. The pharmaceutical composition of claim 57, wherein the cationic lipid is a  lipid wherein: La3 is a bond,
Figure imgf000253_0002
W7 and W8 are each independently a bond or unsubstituted C1-C8 alkylene; L14 is -O(C=O)-, -(C=O)O-, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑; L15 is -O(C=O)-, -(C=O)O-, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or unsubstituted C1-C8 alkylene; R16 and R17 are each independently
Figure imgf000253_0003
a fragment of cationic lipid of formula (II); and each R410 and R510 is independently H or unsubstituted C1-C8 alkyl.
61. The pharmaceutical composition of claim 57, wherein the cationic lipid is a lipid wherein: La3 is a bond,
Figure imgf000253_0004
    W7 and W8 are each independently a bond or unsubstituted C1-C8 alkylene; L14 is -O(C=O)-, -(C=O)O-, -NR410C(=S)-, -C(=S)NR410‑, ‑OC(=O)NR410‑, or ‑NR410C(=O)O‑; L15 is -O(C=O)-, -(C=O)O-, -NR510C(=S)-, -C(=S)NR510‑, ‑OC(=O)NR510‑, or ‑NR510C(=O)O‑; W9 and W10 are each independently a bond or unsubstituted C1-C8 alkylene; R16 and R17 are each independently
Figure imgf000254_0001
  each R410 and R510 is independently H or methyl.
62. The pharmaceutical composition of claim 57, wherein the cationic lipid is:
Figure imgf000254_0002
,    
Figure imgf000255_0001
Figure imgf000256_0001
or a pharmaceutically acceptable salt thereof.
63. The pharmaceutical composition of any of claims 37-62, wherein the pharmaceutical composition further comprises lipid nanoparticles.
64. A method of preventing or treating coronavirus infection or disease, the method comprising administering: a nucleic acid molecule of any of claims 1-31; a composition of any of claims 32-36; and/or or a pharmaceutical composition of any of claims 37-63; to a subject infected with, or at risk of infection, of suspected of having been infected, with a coronavirus.
65. The method of claim 64, wherein administration is via oral, nasal, intrapulmonary, intracavitary, by intra-arterial or intravenous infusion, or by injection.
66. The method of claim 64 or claim 65, wherein administration is by injection.
67. The method of any of claims 64-66, wherein administration is via subcutaneous, intramuscular, transdermal, intradermal, subdermal, epidermal, or lymphatic delivery or injection.
68. The method of any of claims 64-67, wherein the nucleic acid molecule, the composition, or the pharmaceutical composition is administered by subdermal injection or delivery.
69. The method of any of claims 64-68, wherein the subdermal administration is via injection or delivery into a lymphatic system.
70. The method of any of claims 64-69, wherein the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch.
71. The method of any of claims 64-70, wherein the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch, wherein the patch comprises a polymer.
72. The method of any of claims 64-71, wherein the nucleic acid molecule, the composition, or the pharmaceutical composition is administered or delivered into the lymphatic system via a patch, wherein the patch comprises an absorbable polymer.
73. The method of any of claims 64-72, wherein the method comprises administering two or more doses of a nucleic acid molecule of any of claims 1-31; a composition of any of claims 32-36; and/or or a pharmaceutical composition of any of claims 37-63; to the subject.
74. The method of any of claims 64-73, wherein the nucleic acid molecule of any of claims 1-31; the composition of any of claims 32-36; and/or or a pharmaceutical composition of any of claims 37-63; is administered to the lymphatic system of the subject, the method comprising: placing a medical device comprising a plurality of microneedles on the skin of the subject having lymphatic vasculature, wherein the medical device contacts a layer of epidermis with reversible permeability enhancers comprising a chemical, physical or electrical permeability enhancer that induces a-reversible increase in permeability of one or more barrier cells of the epidermis to the nucleic acid molecule, the composition, or the pharmaceutical composition.
75. The method of any of claims 63-74 wherein the nucleic acid molecule of any of claims 1-31; the composition of any of claims 32-36; and/or or a pharmaceutical composition of any of claims 37-63; is administered to the lymphatic system of the subject, the method comprising: placing a first medical device comprising a plurality of microneedles on the skin of the subject at a first location proximate to a first position under the skin of the subject, wherein the first position is proximate to lymph vessels and/or lymph capillaries that drain into the right lymphatic duct, and wherein the microneedles of the first medical device have a surface comprising nanotopography; placing a second medical device comprising a plurality of microneedles on the skin of the subject at a second location proximate to a second position under the skin of the subject, optionally wherein the first and second medical devices are the same device, wherein the second position is proximate to lymph vessels and/or lymph capillaries that drain into the thoracic duct, and wherein the microneedles of the second medical device have a surface comprising nanotopography; inserting the plurality of microneedles of the first medical device into the subject to a depth whereby at least the epidermis is penetrated and an end of at least one of the microneedles is proximate to the first position; inserting the plurality of microneedles of the second medical device into the subject to a depth whereby at least the epidermis is penetrated and an end of at least one of the microneedles is proximate to the second position; and administering via the microneedles of the first medical device a first dose of the nucleic acid molecule, the composition, or the pharmaceutical composition into the first position; and administering via the microneedles of the second medical device a second dose of the nucleic acid molecule, the composition, or the pharmaceutical composition into a second position.
76. The method of any of claims 63-75, wherein the coronavirus infection comprises an infection mediated by a SARS-Cov-2 variant.
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