EP4680595A2 - Lipidnanopartikel mit codierenden rna-molekülen zur verwendung bei der geneditierung und als impfstoffe und therapeutika - Google Patents

Lipidnanopartikel mit codierenden rna-molekülen zur verwendung bei der geneditierung und als impfstoffe und therapeutika

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Publication number
EP4680595A2
EP4680595A2 EP24719912.8A EP24719912A EP4680595A2 EP 4680595 A2 EP4680595 A2 EP 4680595A2 EP 24719912 A EP24719912 A EP 24719912A EP 4680595 A2 EP4680595 A2 EP 4680595A2
Authority
EP
European Patent Office
Prior art keywords
optionally substituted
certain embodiments
formula
compound
glycero
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24719912.8A
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English (en)
French (fr)
Inventor
Muthusamy Jayaraman
Ganapathy Subramanian SANKARAN
Karolina Anna KOSAKOWSKA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Renagade Therapeutics Management Inc
Original Assignee
Renagade Therapeutics Management Inc
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Filing date
Publication date
Application filed by Renagade Therapeutics Management Inc filed Critical Renagade Therapeutics Management Inc
Publication of EP4680595A2 publication Critical patent/EP4680595A2/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/34—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/32—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D207/325—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms with substituted hydrocarbon radicals directly attached to the ring nitrogen atom
    • C07D207/327—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present disclosure generally relates to the field of nucleic acid lipid nanoparticle (LNP) compositions and delivery thereof for use as vaccines and/or therapeutics for the treatment of disease.
  • the disclosure further relates to compositions comprising LNPs formulated with coding RNAs, including linear and/or circular mRNAs, for the delivery of encoded vaccine antigens and/or therapeutic proteins for the vaccination against infectious agents and/or treatment of disease, including infectious disease and cancer.
  • Nucleic acid-based therapeutics and vaccines are generally composed of DNA or RNA.
  • DNA is known to be relatively stable and easy to handle, however, the use of DNA bears the risk of undesired insertion into a cell’s genome which potentially may produce mutagenic events.
  • the delivery of DNA is associated with unwanted immunogenicity and the production of anti-DNA antibodies.
  • Yet another concern in the use of DNA is the limited expression level of the encoded peptide or protein that is achievable due to the requirement that the administered DNA must first enter the nucleus to undergo transcription prior to translation into a desired protein product (e.g., antigen or therapeutic protein).
  • RNA-based agents do not require extraneous promoter sequences for effective expression of an encoded protein and are also less immunogenic than DNA-based agents, in part because RNA has a relatively short half-life unlike DNA.
  • DNA must enter the nuclease to perform its function, RNA performs its function outside of the nucleus and is therefore more efficient.
  • RNA e.g., mRNA
  • DNA DNA
  • RNA-degrading enzymes e.g., RNA-degrading enzymes
  • LNPs lipid nanoparticles
  • LNPs have emerged as the most promising nonviral delivery vehicle for exogenous mRNA (see e.g., Guan et al., “Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems,” Gene Ther, 24 (2017), pp.133-143).
  • the LNP is a complex nanostructured body that provides protection to payload RNA molecules encapsulated within from the harshly degrading nuclease environment in vivo while facilitating intracellular delivery.
  • LNPs are formed through self-assembly by combining the RNA payload with several lipid components, including an ionizable lipid that plays a central role in delivery efficacy (e.g., Miao et al., “Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation,” Nat. Biotechnol., 27 (2019), pp.1174-1185).
  • an ionizable lipid that plays a central role in delivery efficacy
  • RNA Entrapment of RNA is achieved by mixing RNA with lipids at an acidic pH at which the ionizable lipid is positively charged, thus ensuring a charge-driven interaction with the negatively charged RNA molecules (e.g., Mindy et al., “Mechanism of macromolecular structure evolution in self-assembled lipid nanoparticles for siRNA delivery,” Langmuir, 20 (2014), pp.4613-4622).
  • the pH is then adjusted to above the pKa of the ionizable lipid, which results in a near-neutral surface charge desirable for clinical administration (see Id.).
  • RNA payloads to cells in vivo in a targeted manner that also allows for sufficient levels of protein production (e.g., production of vaccine antigens or therapeutic proteins) remains an important and significant challenge.
  • Genome editing tools encompass a diverse set of technologies that can make many types of genomic alterations in various contexts.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • CRISPR-Cas9 CRISPR-associated proteins
  • CRISPR-Cas9 CRISPR-associated proteins
  • CRISPR-Cas9 has been derivatized in numerous ways to expand upon its guide RNA-based programmable double-strand cutting activity to form systems ranging from finding alternative CRISPR Cas nuclease enzymes having different PAM requirements and cutting properties (e.g., engineered Cas9 proteins and other naturally-occurring Cas9 homologs, including, but not limited to, Cas12a, Cas12f, Cas13a, and Cas13b, and their engineered variants) to base editing ( Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, May 19, 2016, 533 (7603); pp.420- 424 [cytosine base editors or CBEs] and Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature, Vol.551, pp.464-471 [adenine base editors or ABEs]) to prime editing (
  • LNPs lipid nanoparticles
  • improved LNPs including better performing ionizable lipids, that will enhance the targeted delivery of LNP-based gene editing tools.
  • such improved LNPs would protect payloads from degradation and clearance while achieving targeted delivery, be suitable for systemic or local delivery, and provide delivery of RNA cargo, including those relating to a wide variety of gene editing tools, such as those mentioned above.
  • improved LNP-based therapeutics should exhibit low toxicity and provide an adequate therapeutic index, such that patient treatment at an effective dose of the LNP minimizes risk to the patient while maximizing therapeutic benefit.
  • improved LNPs that enhance the delivery of LNP-based RNA vaccines and therapeutics to cells, tissues, and bodily sites and which are more protective of RNA payloads would advance the art.
  • such improved LNPs would protect RNA payloads from degradation and clearance while achieving delivery, be suitable for ex vivo or in vivo delivery , and provide delivery of any target, including RNA in linear and/or circular and/or modified form.
  • such improved LNP-based RNA vaccines and therapeutics should exhibit low toxicity and provide an adequate therapeutic index, such that patient treatment at an effective dose of the LNP minimizes risk to the patient while maximizes therapeutic benefit.
  • the present disclosure provides these and related advantages.
  • compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA medicines e.g., vaccines and gene-editing therapeutics.
  • LNP-based RNA medicines e.g., vaccines and gene-editing therapeutics.
  • LNP-based RNA medicines e.g., vaccines and gene editing therapeutics
  • the non- coding RNAs may comprise one or more guide RNAs relating to a gene editing system, such as one based on CRISPR-Cas9 or CRISPR-Casl2a, each of which require complexing with a guide RNA that facilitates the localizing the protein-RNA complex to a target sequence having an enzyme- specific PAM site (protospaccr adjacent motif - recognized by the CRISPR enzyme) and a target nucleotide sequence (i.e., the protospacer) that is complementary to a portion of the guide RNA (i.e., to the spacer region).
  • a gene editing system such as one based on CRISPR-Cas9 or CRISPR-Casl2a, each of which require complexing with a guide RNA that facilitates the localizing the protein-RNA complex to a target sequence having an enzyme-specific PAM site (protospaccr adjacent motif - recognized by the CRISPR enzyme) and a target nucleotide sequence (i.e
  • the coding RNA may encode any protein component of LNP-based RNA medicine, such as, but limited to a virus antigen (e.g., a viral envelope spike protein), a therapeutic protein (e.g., a functional version of a defective protein), or one or more gene editing components (e.g., a programmable nuclease or other effector protein, such as a deaminase or reverse transcriptase).
  • a virus antigen e.g., a viral envelope spike protein
  • a therapeutic protein e.g., a functional version of a defective protein
  • gene editing components e.g., a programmable nuclease or other effector protein, such as a deaminase or reverse transcriptase.
  • compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA medicines for the delivery of one or more RNA molecules, e.g., a coding RNA that codes for one or more therapeutic proteins for the prophylactic and/or therapeutic treatment of one or more diseases or a symptom thereof, or a non-coding RNA, such as, but not limited to a guide RNA for a gene editing system.
  • the RNA molecule delivered by the herein disclosed LNPs can be a linear mRNA.
  • the RNA molecule delivered by the herein disclosed LNPs can be a circular mRNA.
  • the RNA molecule delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA.
  • the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and/or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures).
  • chemical modifications e.g., ribonucleotide analogs, alternative phosphate chain linkers
  • sequence modification e.g., relative to a wild type sequence
  • structural modification e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary
  • the disclosure provides novel lipid components of the herein disclosed LNPs, including, but not limited to, novel ionizable lipids.
  • improved LNP-based RNA medicines e.g., vaccines and therapeutics
  • improved LNPs including better performing ionizable lipids, that enhance the targeted delivery of LNP-based RNA vaccines and therapeutics based on linear and/or circular mRNAs.
  • the improved LNPs protect linear and/or circular mRNA cargos (i.e., the circular and/or linear mRNA molecules encapsulated by the LNPs) from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced vaccines and/or therapeutic agents.
  • a pharmaceutical composition comprising: a) at least one lipid nanoparticle comprising at least one compound having a structure of any of Formulae (S- A’), (S-A), (S-B), (S-C), (S-D), (S-E), (S-F), (S-G), (S-H), (S-I), (S-Ia), (S-Ib), (S-J), (S-K), (S-L), (AT), (AT-A), (AT-A1), (AT-A2), (AT-B), (AT-B’), (AT-C), (AT-D), (AT-D’), (AT-D’a), (AT-D’b), (AT-E), (AT-E’’), (AT-F), (AT-F’), (AT-F’’), (AT-F’’’), (AT-F’’’’), (AT-F’’’’), (AT-F’’’’’), (AT-F’’’’’), (AT-F’’
  • lipid nanoparticle comprising a compound having a structure of any of Formulae (S-A’), (S-A), (S-B), (S-C), (S-D), (S- E), (S-F), (S-G), (S-H), (S-I), (S-Ia), (S-Ib), (S-J), (S-K), (S-L), (AT), (AT-A), (AT-A1), (AT-A2), (AT-B), (AT-B’), (AT-C), (AT-D), (AT-D’), (AT-D’a), (AT-D’b), (AT-E), (AT-E’’), (AT-F), (AT- F’), (AT- F’), (AT- F’), (AT-A-
  • provided herein is a method for delivering a nucleic acid to a cell comprising contacting the cell with a LNP disclosed herein or a pharmaceutical composition disclosed herein.
  • a method for treating a disease characterized by a deficiency of a functional protein comprising administering to a subject having the disease, a LNP formulation comprising a LNP disclosed herein, wherein the mRNA encodes the functional protein or a protein having the same biological activity as the functional protein.
  • FIG.1 is a diagram illustrating the LNP-based RNA vaccines and therapeutics disclosed herein which are encapsulated with RNA payloads (e.g., linear and/or circular mRNAs).
  • FIG.2 is a diagram illustrating an originator polynucleotide construct of the present disclosure which may be linear or circular.
  • the instant specification describes compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA medicines (e.g., vaccines, gene therapies, or gene-editing therapeutics).
  • the LNP-based RNA medicines comprise an LNP delivery system (as described in detail herein) and an encapsulated cargo/payload (e.g., RNA in the case of RNA medicines).
  • the LNP delivery vehicle is a complex nanostructured body that provides protection to an encapsulated RNA payload (i.e., one or more RNA molecules) environmental damage (e.g., an intracellular environment).
  • an encapsulated RNA payload i.e., one or more RNA molecules
  • environmental damage e.g., an intracellular environment
  • LNPs are formed through self-assembly of multiple lipid components, including (i) an ionizable lipid (e.g., ALC-0315 as in COMIRNATY® (Pfizer-BioNTech), SM-102 as in SPIKEVAX® (Moderna), or MC3 as in ONPATTRO® (Alnylam), or those ionizable lipids described herein), (ii) a helper lipid (such as, but not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), (iii) a sterol (e.g., cholesterol), and (iv) a PEG-lipid (e.g., PEG-DSPE).
  • an ionizable lipid e.g., ALC-0315 as in COMIRNATY® (Pfizer-BioNTech
  • SM-102 as in SPIKEVAX® (Moderna)
  • the RNA payload in the herein described LNP-based medicines may comprise coding and/or non-coding RNA, and/or mixtures thereof.
  • the particular RNA payload constituents will generally reflect the medicine.
  • an LNP-based vaccine or therapeutic may comprise only coding RNA for expressing a vaccine antigen or a therapeutic protein, respectively.
  • an LNP-based gene editing medicine may comprise a combination of coding RNA (e.g., encoding a CRISPR nuclease) and non-coding RNAs (e.g., guide RNAs).
  • the RNA molecule delivered by the herein disclosed LNPs can be a linear mRNA.
  • the RNA molecule delivered by the herein disclosed LNPs can be a circular mRNA.
  • the RNA molecule delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA.
  • the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and/or structural modification (e.g., secondary- folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures).
  • chemical modifications e.g., ribonucleotide analogs, alternative phosphate chain linkers
  • sequence modification e.g., relative to a wild type sequence
  • structural modification e.g., secondary- folded structures, such as, but not limited to, stem-loop
  • improved LNP-based RNA vaccines for use in immunization against disease.
  • the disclosure describes improved LNPs, including better performing ionizable lipids, that enhance the targeted delivery of LNP-based RNA vaccines and therapeutics based on linear and/or circular mRNAs.
  • the improved LNPs protect linear and/or circular mRNA cargos (i.e., the circular and/or linear mRNA molecules encapsulated by the LNPs) from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced vaccines.
  • compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA vaccines are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA vaccines for the delivery of an RNA molecule that codes for one or more immunogenic viral antigens for use as vaccine and/or immunogenic compositions.
  • the RNA molecules delivered by the herein disclosed LNPs can be linear mRNA. In other embodiments, the RNA molecules delivered by the herein disclosed LNPs can be circular mRNA.
  • the RNA molecule delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA.
  • the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and/or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures).
  • the disclosure provides novel lipid components of the herein disclosed LNPs, including, but not limited to, novel ionizable lipids.
  • improved LNP-based RNA therapeutics for use in treating disease or a symptom thereof.
  • the disclosure describes improved LNPs, including better performing ionizable lipids, that enhance the targeted delivery of LNP-based RNA therapeutics based on linear and/or circular mRNAs.
  • the improved LNPs protect linear and/or circular mRNA cargos (i.c., the circular and/or linear mRNA molecules encapsulated by the LNPs) from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced therapeutic agents.
  • compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA therapeutics are described herein.
  • described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA therapeutics for the delivery of an RNA molecule that codes for one or more therapeutic proteins for use treating a disease or a symptom thereof.
  • the RNA molecules delivered by the herein disclosed LNPs can be linear mRNA.
  • the RNA molecules delivered by the herein disclosed LNPs can be circular mRNA.
  • the RNA molecules delivered by the herein disclosed LNPs can include both linear and circular forms of mRNA.
  • the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and/or structural modification (e.g., secondary- folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures).
  • the disclosure provides novel lipid components of the herein disclosed LNPs, including, but not limited to, novel ionizable lipids.
  • LNP compositions comprising gene editing systems for use in treating disease and/or otherwise modifying the sequence and/or expression of target nucleotide sequences.
  • the disclosure provides LNPs capable of delivering a gene editing system to a target organ, tissue, and/or cell.
  • the gene editing systems may be delivered to cells under in vitro or ex vivo conditions and to organs, tissues, or cells under in vivo conditions (e.g., administered to a subject in an effective amount).
  • the disclosure also provides in various aspects therapeutic or pharmaceutical compositions comprising LNPs comprising gene editing systems or one or more components thereof.
  • the gene editing systems may comprise DNA components, RNA components, protein components, nucleoprotein components, polysaccharide components, or combinations thereof.
  • the disclosure provides nucleic acid molecules (e.g., RNA or DNA) that encode and/or constitute various componentry of the deliverable gene editing systems contemplated herein.
  • nucleic acid molecules as components of the herein contemplated gene editing systems, such as, but not limited to plasmids or vectors encoding one or more components of a gene editing system, RNAs encoding one or more components of a gene editing system (e.g., mRNAs coding for a nuclease domain of a gene editing system), and non-coding RNAs (e.g., guide RNAs capable of complexing with and targeting a nucleic acid-programmable DNA binding domain to a specific target nucleotide sequence or a retron ncRNAs).
  • plasmids or vectors encoding one or more components of a gene editing system
  • RNAs encoding one or more components of a gene editing system e.g., mRNAs coding for a nuclease domain of a gene editing system
  • non-coding RNAs e.g., guide RNAs capable of complexing with and targeting a nucleic acid-programmable DNA binding domain
  • the nucleic acid components may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and/or structural modification (e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple- stranded structures).
  • chemical modifications e.g., ribonucleotide analogs, alternative phosphate chain linkers
  • sequence modification e.g., relative to a wild type sequence
  • structural modification e.g., secondary-folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes
  • tertiary structural elements such as, but not limited to, helical duplexes and triple- stranded
  • the disclosure in other aspects, describes various protein components (which may be encoded by the nucleic acid components described herein) of the various gene editing systems contemplated herein, including, but not limited to, user-programmable DNA binding proteins and various effector proteins, such as nucleases, polymerases, reverse transcriptases, recombinases, integrases, endonucleases, exonucleases, transposases, and deaminases.
  • various protein components which may be encoded by the nucleic acid components described herein of the various gene editing systems contemplated herein, including, but not limited to, user-programmable DNA binding proteins and various effector proteins, such as nucleases, polymerases, reverse transcriptases, recombinases, integrases, endonucleases, exonucleases, transposases, and deaminases.
  • the disclosure also describes nucleoprotein components of the gene editing systems contemplated herein, such as, but not limited to nuclease-guide RNA complexes.
  • the disclosure also provides methods of modifying the sequence and/or expression level of a target nucleic acid molecule through the delivery and/or administration of an LNP described herein that comprises a gene editing system or components thereof.
  • the disclosure provides methods of treating a disease by administering a therapeutically effective amount of an LNP-based gene editing system that results in the modification in the sequence and/or expression level of a target nucleic acid molecule (e.g., a disease-associated gene or regulatory sequence, such as a promoter, transcription factor binding site, or gene enhancer site).
  • a target nucleic acid molecule e.g., a disease-associated gene or regulatory sequence, such as a promoter, transcription factor binding site, or gene enhancer site.
  • the gene editing systems deliverable by the herein disclosed LNPs can be any type of gene editing system.
  • the gene editing systems contemplated herein can include (A) nucleobase gene editing systems which result in one or more the changes to the sequence of a target nucleic acid molecule (e.g., a gene or gene regulatory sequence) (sequence modifications may include, but are not limited to, an insertion of one or more base pairs, a deletion of one of more base pairs, a substitution or one or more base pairs, a conversion of a base pair to another base pair (e.g., a G:C pair converted to an A:T pair), an inversion, or a translocation), (B) an epigenetic editing system which results in one or more modifications to the epigenome to bring about an effect on gene expression without altering the sequence of a nucleic acid molecule, and (C) gene editing systems that combine the features of nucleobase editing systems and epigenetic editing systems (e.g., combining components from both types of systems
  • Nucleobase editing systems include a wide array of configurations with various combinations of protein functionalities and/or nucleic acid molecule components, all of which are contemplated herein.
  • nucleobase editing systems comprise at least a (i) DNA binding domain that is user-programmable to target a specific sequence in a nucleic acid molecule and optionally (ii) one or more effector domains that facilitate the modification of the sequence of the nucleic acid molecule.
  • epigenetic editing systems comprise at least a (i) DNA binding domain that targets a specific sequence in a nucleic acid molecule and (ii) one or more effector domains that facilitates the modification of one or more epigenomic features of the nucleic acid molecule.
  • Gene editing systems may comprise one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and/or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.
  • deaminases e.g., cytidine deaminases or adenosine deaminases
  • polymerases e.g., reverse transcriptases
  • integrases e.g., recombinases, etc.
  • fusion proteins comprising one or more functional domains linked together.
  • gene editing systems that utilize a nucleic acid sequence-programmable DNA binding domain or protein (naspDBP) may also comprise one or more non-coding nucleic acids, such as, one or more guide RNAs which complex with the nucleic acid programmable DNA binding protein (naspDBP) and target the complex to a specific nucleotide sequence.
  • the guide RNA may be a prime editing guide RNA (“pegRNA”) which comprises a specialized RNA template molecule that provides a template or coding sequence for a reverse transcriptase of the prime editing system.
  • the RNA template molecule may be coupled to a guide RNA as an extension arm at the 5’ or 3’ end of the guide RNA.
  • the RNA template molecule may be provided in trans as a separate molecule in a manner such that the RNA template molecule may itself become localized and associated with the target sequence and/or the gene editing system at the site of editing.
  • co- localization of an in trans RNA template molecule may be achieved with an aptamer or other RNA structure which binds to a binding partner that is coupled to, integrated with, or otherwise associated with the editing complex.
  • naspDBP DNA binding protein
  • appropriate guides may be designed and synthesized using methods, software, and commercial sources which are well known to those having ordinary skill in the art such that guide RNAs for any given naspDBP may be obtained without undue experimentation.
  • PMCID PMC4602062; (11) Creutzburg SCA, Wu WY, Mohanraju P, Swartjes T, Alkan F, Gorodkin J, Staals RHJ, van der Oost J. Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Casl2a. Nucleic Acids Res. 2020 Apr 6;48(6):3228-3243. doi: 10.1093/nar/gkzl240. PMID: 31989168; PMCID: PMC7102956; (12) Heigwer F, Boutros M. Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments. Methods Mol Biol. 2021;2162:3-22.
  • CRISPR-Cas9 gRN A efficiency prediction an overview of predictive tools and the role of deep learning. Nucleic Acids Res. 2022 Apr 22:50(7):3616-3637. doi: 10.1093/nar/gkacl92. PMID: 35349718; PMCID: PMC9023298; (19) Wang J, Zhang X, Cheng 1.., Luo Y. An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools. RNA Biol. 2020 Jan;17(l):13-22. doi: 10.1080/15476286.2019.1669406. Epub 2019 Sep 27.
  • guide RNAs for CRISPR editing applications (including base editing and prime editing) and provide various tools and instruction for the ordering, design, synthesis, modification, and structural configuration of guide RNAs: GENSCRIPT, SYNTHEGO, TAKARA BIO, INTEGRATED DNA TECHNOLOGIES, LC SCIENCES, HORIZON DISCOVERY; SIGMA-ALDRICH; ORIGENE, and TWIST BIOSCIENCES, among others.
  • guide RNA may be modified with chemical modifications and/or structural modifications for enhancing various properties thereof, including specificity, stability, and limiting off-target activity.
  • One of ordinary skill in the art will be able to modify a guide RNA with any known modification without undue experimentation. Guide modifications are discussed in the following references: (1) Ke Y, Ghalandari B, Huang S, Li S, Huang C, Zhi X, Cui D, Ding X. 2'- O- Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CR1SPR-Casl2a systems. Chem Sei. 2022 Feb 1; 13(7):2050-2061.
  • SNP single nucleotide polymorphism
  • pegRNAs may be modified with chemical modifications and/or structural modifications for enhancing various properties thereof, including specificity, stability, and limiting off-target activity.
  • One of ordinary skill in the art will be able to modify a pegRNA for prime editing with any known modification without undue experimentation.
  • pegRNA modifications are discussed in the following references: (1) Nelson JW, Randolph PB, Siren SP, Everette KA, Chen PJ, Anzalone AV, An M, Newby GA, Chen JC, Hsu A, Liu DR. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. 2022 Mar;40(3):402-410. doi: 10.1038/S41587-021-01039-7.
  • RNAs may be included depending upon the requirements and/or nature of the gene editing system and the cognate nucleic acid programmable proteins.
  • TnpB enzymes require a specialized guide RNA referred to as reRNA.
  • guide RNAs have different characteristics (e.g., PAM preferences, the spacer length, and the scaffold portion that binds to the nuclease protein) depending upon the programmable nuclease requirements.
  • the gene editing systems contemplated here may introduce a wide variety of changes, including (A) a change in the sequence of the target nucleic acid molecule, such as, but not limited to, (i) a nucleobase substitution (e.g., a purine to a pyrimidine), (ii) a deletion of one or more nucleobases, (iii) an insertion of one or more nucleobases, (iv) a combination of a deletion and insertion of one or more nucleobases, (v) an inversion of a nucleobase sequence, a (vi) translocation of a nucleobase sequence, and (vii) a combination or two or more such modifications, and (B) one or more modifications to the epigenome to bring about an effect on gene expression without altering the sequence of a nucleic acid molecule wherein said epigenetic change results in altered gene expression through altered chromatin structure or accessibility.
  • a nucleobase substitution e.g., a purine to
  • the LNP compositions and/or gene editing systems described herein may include a variety of coding RNA molecules that code for the various components of gene editors.
  • the coding RNA may be linear mRNA.
  • the coding RNA may be circular mRNA.
  • the improved LNPs protect linear and/or circular mRNA cargos from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced gene editing platforms and/or therapeutic agents.
  • the LNP compositions and/or gene editing systems described herein may also include a repair template, e.g., an homology -directed repair (HDR) -dependent repair template (or HDR template).
  • HDR templates are well-known in the art and can include single- strand or double-stranded DNA (e.g., oligos) or RNA. Further information regarding HDR and HDR templates for use in editing systems for various applications, such as gene knock-in, may be found in Fu YW. Dai XY, Wang WT, Yang ZX, Zhao JJ, Zhang JP, Wen W, Zhang F, Oberg KC, Zhang I... Cheng T, Zhang XB.
  • compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based gene editing systems as therapeutic compositions. Further described herein are compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based gene editing therapeutics for the prophylactic and/or therapeutic treatment of one or more diseases or a symptom thereof.
  • LNP payloads may include all of the biological materials described above, including DNA molecules, RNA molecules (coding and/or non-coding), proteins, and nucleoproteins (e.g., Cas/guide RNA complexes).
  • RNA payloads e.g., linear and circular mRNAs
  • LNPs lipid nanoparticles
  • LNPs that may be used as the RNA payload delivery vehicles contemplated herein, as well as the various ionizable lipids, structural lipids, PEGylated lipids, and phospholipids that may be used to make the herein LNPs for delivery RNA payloads to cells.
  • LNP components that are contemplated, such as targeting moieties and other lipid components.
  • the present disclosure further provides delivery systems for delivery of a therapeutic payload (e.g., the RNA payloads described herein which may encode a polypeptide of interest, e.g., an antigen or a therapeutic protein) disclosed herein.
  • a delivery system suitable for delivery of the therapeutic payload disclosed herein comprises a lipid nanoparticle (LNP) formulation.
  • LNP lipid nanoparticle
  • an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid.
  • an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid.
  • an LNP further comprises a 5th lipid, besides any of the aforementioned lipid components.
  • the LNP encapsulates one or more elements of the active agent of the present disclosure.
  • an LNP further comprises a targeting moiety covalently or non-covalently bound to the outer surface of the LNP.
  • the targeting moiety is a targeting moiety that binds to, or otherwise facilitates uptake by, cells of a particular organ system.
  • an LNP has a diameter of at least about 20nm, 30 nm, 40nm, 50nm, 60nm, 70nm, 80nm, or 90nm. In some embodiments, an LNP has a diameter of less than about lOOnm, HOnm, 120nm, 130nm, 140nm, 150nm, or 160nm.
  • an LNP has a diameter of less than about 120 nm. In some embodiments, an LNP has a diameter of less than about lOOnm. In some embodiments, an LNP has a diameter of less than about 90nm. In some embodiments, an LNP has a diameter of less than about 80nm. In some embodiments, an LNP has a diameter of about 60-100nm. In some embodiments, an LNP has a diameter of about 50-120nm. In some embodiments, an LNP has a diameter of about 75-80nm.
  • the lipid nanoparticle compositions of the present disclosure are described according to the respective molar ratios of the component lipids in the formulation.
  • the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol- %.
  • the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%.
  • the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%.
  • the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%.
  • the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol-%.
  • the mol-% of the ionizable lipid may be from about 30 mol-% to about 40 mol-%.
  • the mol-% of the ionizable lipid may be from about 25 mol-% to about 35 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 10 mol-%.
  • the mol-% of the ionizable lipid is about 15 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 20 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 25 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 30 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 33 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 35 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 40 mol-%.
  • the mol-% of the ionizable lipid is about 45 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 55 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 60 mol-%.
  • the mol-% of the phospholipid may be from about 1 mol-% to about 50 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 3 mol- % to about 40 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 30 mol-%.
  • the mol-% of the phospholipid may be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid is from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the phospholipid is from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the phospholipid is from about 35 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid is about 10 mol-%.
  • the mol-% of the phospholipid is about 15 mol-%. In some embodiments, the mol-% of the phospholipid is about 20 mol-%. In some embodiments, the mol-% of the phospholipid is about 25 mol-%. In some embodiments, the mol-% of the phospholipid is about 30 mol-%. In some embodiments, the mol-% of the phospholipid is about 35 mol-%. In some embodiments, the mol-% of the phospholipid is about 40 mol-%. In some embodiments, the mol-% of the phospholipid is about 45 mol-%. In some embodiments, the mol-% of the phospholipid is about 55 mol-%. In some embodiments, the mol-% of the phospholipid is about 60 mol-%.
  • the mol-% of the phospholipid as described above comprises two or more phospholipids at an individual mol-% that totals to an aforementioned amount.
  • the mol-% of the phospholipid is about 20 mol-% each of two phospholipids.
  • the mol-% of the phospholipid is about 15 mol-% each of two phospholipids.
  • the mol-% of the phospholipid is about 25 mol-% each of two phospholipids.
  • the mol-% of the phospholipid is about 30 mol-% each of two phospholipids.
  • the mol-% of the phospholipid is about 15 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 30 mol-% of a first phospholipid and about 10 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 25 mol-% of a first phospholipid and about 10 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 25 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 15 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid.
  • the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%.
  • the mol-% of the PEG lipid may be from about 0.1 mol-% to about 10 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 1 .5 mol-%.
  • the mol-% of the PEG lipid may be about 2.5 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 3.5 mol-%. [0062] Where reference is made above to “mol-%” or “mol %”, the amount of the noted LNP component is intended to be the mol% of the specific component as compared to the total lipid component content of the lipid nanoparticle. i. Ionizable lipids [0063] In some embodiments, an LNP disclosed herein comprises an ionizable lipid. In some embodiments, an LNP comprises two or more ionizable lipids.
  • Lipids of the Disclosure have a structure of Formula (S-A), or (S-A’), wherein the Lipids of the Disclosure have a structure of Formula (S-B): (S-B), or a pharmaceutically acceptable salt thereof.
  • Formula (S-C) [0068] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), or (S-A’), wherein the Lipids of the Disclosure have a structure of Formula (S-C): (S-C), or a pharmaceutically acceptable salt thereof.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 2 and/or X 2a are/is optionally substituted C 2 -C 14 alkylenyl (e.g., C 4 - C10 alkylenyl, C5-C7 alkylenyl, C5, C6, or C7 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 2 is C4-C10 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 2a is C4-C10 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 2 is C 5 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 2 is C 6 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 2a is C5 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 2a is C6 alkylenyl. [0073] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 and/or Y 1a are/is .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 is .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1a is .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 and/or Y 1a are/is .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 is .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1a is .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 and/or Y 1a are/is , wherein Z 2 is hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 is , wherein Z 2 is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1a is , wherein Z 2 is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 and/or Y 1a are/is , wherein Z 2 is hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 is , wherein Z 2 is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1a is , wherein Z 2 is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 and Y 1a are independently or .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein Y 1 is independently or .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S- A’), (S-B), or (S-C), wherein Y 1a is independently or .
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 3 is optionally substituted C2-C14 alkylenyl (e.g., C4-C10 alkylenyl, C5-C7 alkylenyl, C5, C6, or C7 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 3 is C 5 -C 7 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 3 is C 5 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2 , R 3 , R 2' , and/or R 3' are hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2 is hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S- C), wherein R 3 , is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2' is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 3' is hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2 , R 3 , R 2' , and/or R 3' are optionally substituted C1-C14 alkyl (e.g., C5- C 14 , C 5 -C 10 , C 6 -C 9 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2 is C 5 -C 10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 3 is C 5 -C 10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2’ is C5-C10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 3’ is C5-C10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2 is C 8 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 3 is C 8 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 2’ is C 8 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 3’ is C8 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-A) or (S-C), wherein R 4 is optionally substituted C 4 -C 14 alkyl (e.g., C 6 -C 12 , C 8 -C 12 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-A) or (S-C), wherein R 4 is C 6 -C 12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 4 is C11 alkyl. [0077] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein R 1 is OH.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 1 is C 2-4 alkylenyl (e.g., C 2, C 3 , or C 4 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 1 is C2 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-A), (S-A’), (S-B), or (S-C), wherein X 1 is C4 alkylenyl.
  • Z 1 is optionally substituted C1-C6 alkyl;
  • R 10 is C1-C6 alkylenyl;
  • R 7b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1-C6 alkyl;
  • R 7c is hydrogen or C 1 -C 6 alkyl;
  • R 8b is C1-C6 alkyl, (hydroxy)C1-C6 alkyl, or (amino)C1
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein A is -C(R ' )(-L 1 N(R")R 6 )-. [0081] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein A is -C(R')(-OR 7a )-. [0082] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein A is -C(R')(-N(R")R 8a ).
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein X 2 and/or X 2a are/is optionally substituted C 2 -C 14 alkylenyl (e.g., C 2 -C 10 alkylenyl, C 2 -C 8 alkylenyl, C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , or C 8 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein X 2 is C 2 -C 14 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein X 2a is C 2 -C 14 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1 and/or Y 1a are/is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S- D), wherein Y 1 is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1a is .
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1 and/or Y 1a are/is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1 is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1a is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1 and/or Y 1a are/is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1 is .
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1a is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1 and/or Y 1a are/is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1 is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein Y 1a is .
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein X 3 is optionally substituted C 1 -C 14 alkylenyl (e.g., C 1 -C 6 , C 1 -C 4 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein X 3 is C 1 -C 14 alkylenyl. [0090] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 2 , R 3 , R 2' , and/or R 3' are hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein R 2 is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 3 is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 2’ is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 3’ is hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein R 2 , R 3 , R 2' , and/or R 3' are optionally substituted C1-C14 alkyl (e.g., C4-C10 alkyl, C5, C6. C7. C8, C 9 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein R 2 is C 4 -C 10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein R 3 is C 4 -C 10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein R 2’ is C 4 -C 10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 3’ is C4-C10 alkyl. [0092] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 4 is optionally substituted C 4 -C 14 alkyl (e.g., C 8 -C 14 alkyl, linear C 8 -C 14 alkyl, C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , or C 14 alkyl).
  • R 4 is optionally substituted C 4 -C 14 alkyl (e.g., C 8 -C 14 alkyl, linear C 8 -C 14 alkyl, C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , or C 14 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-D), wherein R 4 is linear C 8 -C 14 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 4 is linear C 11 alkyl. [0093] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein L 1 is C1-C3 alkylenyl. [0094] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-D), wherein R 6 is (hydroxy)C 1 -C 6 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein R 1 is , wherein Z 1 is methyl and Z 1a is hydrogen or methyl. [00106] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein R 1 is , wherein Z 1 is methyl. [00107] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein R 1 is -NR"C(O)OR 20 . [00108] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein R 1 is -NR"R 21 .
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein R 20 is t-butyl or benzyl. [00110] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein X 2 and/or X 2a are/is optionally substituted C 2 -C 14 alkylenyl (e.g., C 4 -C 8 alkylenyl, C 4 , C 5 , C 6 , C 7 , C 8 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein X 2 is C4-C8alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein X 2a is C4-C8alkylenyl. [00111] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 and/or Y 1a are/is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1a is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 and/or Y 1a are/is .
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1a is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 and/or Y 1a are/is , wherein Z 3 is C 2 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 is , wherein Z 3 is C 2 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1a is , wherein Z 3 is C 2 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 and/or Y 1a are/is , wherein Z 3 is C 2 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1 is , wherein Z 3 is C2 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein Y 1a is , wherein Z 3 is C 2 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein R 2 , R 3 , R 2' , and R 3' are independently hydrogen, optionally substituted linear C 1 -C 14 alkyl (e.g., C 4 -C 10 alkyl, C 6 -C 8 alkyl, C 5 , C 6 , C 7 , C 8 , C 9 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein R 2 is hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein R 3 is hydrogen.
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein R 2’ is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein R 3’ is hydrogen. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein R 2 is linear C 4 -C 10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein R 3 is linear C 4 -C 10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-E), wherein R 2’ is linear C4-C10alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-E), wherein R 3’ is linear C4- C10alkyl.
  • Formula (S-F) [00112]
  • Lipids of the Disclosure have a structure of Formula (S-F): (S-F), or a pharmaceutically acceptable salt thereof, wherein R 1 is , or ; Z 1 is optionally substituted C1-C6 alkyl; X 1 is optionally substituted C2-C6 alkylenyl; X 2 and X 2a are independently optionally substituted C 2 -C 14 alkylenyl; Y 1 and Y 1a are independently or , Z 3 is independently optionally substituted C 2 -C 6 alkylenyl; R 2 and R 3 are independently optionally substituted C 4 -C 14 alkyl; R 2' and R 3' are independently optionally substituted C 4 -C 14 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-F), wherein R 1 is , wherein Z 1 is methyl.
  • Lipids of the Disclosure have a structure of Formula (S-F), wherein X 1 is C2-C4 alkylenyl (e.g., C3 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-F), wherein X 1 is C3 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-F), wherein X 2 is C4-C10 alkylenyl (e.g., C6 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-F), wherein X 2 is C6 alkyl. [00116] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-F), wherein R 2 and R 3 are independently optionally substituted C 4 -C 10 alkyl (e.g., C 8 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-F), wherein R 2 and R 3 are independently C 8 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-G): (S-G), or a pharmaceutically acceptable salt thereof, wherein R 20 is C 1 -C 6 alkylenyl-NR 20' C(O)OR 20'' ; R 20' is hydrogen or optionally substituted C 1 -C 6 alkyl; R 20'' is optionally substituted C1-C6 alkyl, phenyl, or benzyl; Z 1 is optionally substituted C1-C6 alkyl; X 2 and X 2a are independently optionally substituted C2-C14 alkylenyl; Y 1 and Y 1a are independently or ; wherein the bond marked with an "*" is attached to X 2 or X 2a ; Z 3 is independently optionally substituted C2-C6 alkylenyl; R 2 and R 3 are independently optionally substituted C 4 -C 14 alkyl; and R 2' and R 3' are independently optionally optional
  • Lipids of the Disclosure have a structure of Formula (S-G), wherein R 20 is -CH2CH2CH2NHC(O)O-t-butyl or -CH2CH2CH2NHC(O)O-benzyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein R 20 is - CH2CH2CH2NHC(O)O-t-butyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein R 20 is -CH 2 CH 2 CH 2 NHC(O)O-benzyl.
  • Lipids of the Disclosure have a structure of Formula (S-G), wherein X 2 and X 2a are independently C4-C8 alkylenyl (e.g., C5, C6, C7 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein X 2 is C6 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-G), wherein X 2a is C 6 alkyl [00120] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein Y 1 and Y 1a are , wherein Z 3 is C2-C4alkylenyl (e.g., C2 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein Y 1 is , wherein Z 3 is C 2 - C4alkylenyl (e.g., C2 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-G), wherein Y 1a is , wherein Z 3 is C 2 -C 4 alkylenyl (e.g., C 2 alkylenyl). [00121] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein R 2 , R 3 , R 2' and R 3' are independently optionally substituted C4-C10 alkyl (e.g., C6-C9alkyl, C6, C7, C8, C9 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein R 2 is C6-C9alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-G), wherein R 3 is C 6 -C 9 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein R 2’ is C 6 -C 9 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-G), wherein R 3’ is C 6 -C 9 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-H): (S-H), or a pharmaceutically acceptable salt thereof, wherein R 1 is -OH or ; X 1 is optionally substituted C 4 alkylenyl; X 2 and X 2a are independently optionally substituted C 2 -C 14 alkylenyl; Y 1 and Y 1a are independently or ; Z 3 is independently optionally substituted C 2 -C 6 alkylenyl; R 2 and R 3 are independently optionally substituted C 4 -C 14 alkyl or C 1 -C 2 alkyl substituted with optionally substituted cyclopropyl; or R 2' and R 3' are independently optionally substituted C4-C14 alkyl or C1-C2 alkyl substituted with optionally substituted cyclopropyl.
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein X 1 is C 4 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein X 2 and X 2a are independently optionally substituted C4-C10 alkylenyl (e.g., C5, C6, C7, C8, C9, or C10 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein X 2 is C4-C10 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein X 2a is C 4 -C 10 alkylenyl. [00125] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein Y 1 and Y 1a are independently , wherein Z 3 is independently C 2 -C 4 alkylenyl (e.g., C 2 , C 4 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2 , R 3 , R 2' and R 3' are independently C6-C14 alkyl (e.g., C6, C7, C8, C9, C10, C11, C12, C13, or C14 alkyl) or C1-C2 alkyl substituted with optionally substituted cyclopropyl.
  • R 2 , R 3 , R 2' and R 3' are independently C6-C14 alkyl (e.g., C6, C7, C8, C9, C10, C11, C12, C13, or C14 alkyl) or C1-C2 alkyl substituted with optionally substituted cyclopropyl.
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2 , R 3 , R 2' and R 3' are independently C 6 -C 14 alkyl (e.g., C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , or C 14 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2 is C 6 -C 14 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 3 is C 6 -C 14 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2’ is C6- C14 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein R 3’ is C6-C14 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2 is C1-C2 alkyl substituted with substituted cyclopropyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein R 3 is C 1 -C 2 alkyl substituted with substituted cyclopropyl.
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2' is C 1 -C 2 alkyl substituted with substituted cyclopropyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein R 3' is C1-C2 alkyl substituted with substituted cyclopropyl [00127] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2 , R 3 , R 2' and R 3' are independently C 1 -C 2 alkyl substituted with cyclopropylene-(C 1 - C 6 alkylenyl optionally substituted with cyclopropylene substituted with C 1 -C 6 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2 is C 1 -C 2 alkyl substituted with cyclopropylene-(C1-C6alkylenyl optionally substituted with cyclopropylene substituted with C 1 -C 6 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein R 3 is C 1 -C 2 alkyl substituted with cyclopropylene-(C 1 -C 6 alkylenyl optionally substituted with cyclopropylene substituted with C1-C6alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-H), wherein R 2' is C1-C2 alkyl substituted with cyclopropylene-(C1-C 6 alkylenyl optionally substituted with cyclopropylene substituted with C1- C 6 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-H), wherein R 3' is C 1 -C 2 alkyl substituted with cyclopropylene-(C 1 -C 6 alkylenyl optionally substituted with cyclopropylene substituted with C 1 -C 6 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-J): (S-J), or a pharmaceutically acceptable salt thereof, wherein R 1 is -OH or ; X 1 is branched C 2 -C 8 alkylenyl X 2 and X 2a are independently optionally substituted C 2 -C 14 alkylenyl; Y 1 and Y 1a are independently or ; Z 3 is independently optionally substituted C 2 -C 6 alkylenyl; R 2 and R 3 are independently optionally substituted C 4 -C 14 alkyl; R 2' and R 3' are independently optionally substituted C 4 -C 14 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-J), wherein X 1 is branched C6 alkylenyl. [00130] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein X 2 and X 2a are independently C 4 -C 10 alkylenyl (e.g., C 6 , C 7 , C 8 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein X 2 is C 4 -C 10 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-J), wherein X 2a is C4-C10 alkylenyl [00131] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein Y 1 and Y 1a are , wherein Z 3 is independently optionally substituted C 2 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein Y 1 is , wherein Z 3 is independently optionally substituted C 2 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-J), wherein Y 1a is , wherein Z 3 is independently optionally substituted C2 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-J), wherein R 2 , R 3 , R 2' and R 3' are independently C 6 -C 12 alkyl (e.g., C 9 alkyl) or C 4 -C 10 alkyl (e.g., C 4, C 6 alkyl) optionally substituted with C2-C8alkenylene (e.g., C4, C6 alkenylene).
  • Lipids of the Disclosure have a structure of Formula (S-J), wherein R 2 is C6-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein R 3 is C6-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein R 2’ is C6- C 12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein R 3’ is C 6 -C 12 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-J), wherein R 2 is C 4 -C 10 alkyl optionally substituted with C 2 -C 8 alkenylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-J), wherein R 3 is C4-C10 alkyl optionally substituted with C2-C8alkenylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (S- J), wherein R 2’ is C4-C10 alkyl optionally substituted with C2-C8alkenylene.
  • Lipids of the Disclosure have a structure of Formula (S-J), wherein R 3’ is C 4 -C 10 alkyl optionally substituted with C 2 -C 8 alkenylene.
  • Formula (S-K) [00133]
  • Lipids of the Disclosure have a structure of Formula (S-K): (S-K), or a pharmaceutically acceptable salt thereof, wherein R 1 is -OH; X 1 is optionally substituted C2-C6 alkylenyl; X 2 and X 2a are independently optionally substituted C2-C14 alkylenyl; each of Y 1 and Y 1a is a bond; R 2 and R 3 are independently optionally substituted C 4 -C 14 alkyl; and R 2' and R 3' are independently optionally substituted C 4 -C 14 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-K), wherein X 1 is C4 alkylenyl. [00135] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-K), wherein X 2 and X 2a are independently C 4 -C 10 alkylenyl (e.g., C 6 -C 8 alkylenyl, C 6 , C 7 , C 8 alkylenyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-K), wherein X 2 is C 4 -C 10 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-K), wherein X 2a is C4-C10 alkylenyl. [00136] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-K), wherein R 2 , R 3 , R 2' and R 3' are independently C6-C10 alkyl (e.g., C7. C8 alkyl). In some embodiments, Lipids of the Disclosure have a structure of Formula (S-K), wherein R 2 is C 6 -C 10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-K), wherein R 3 is C 6 -C 10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-K), wherein R 2’ is C 6 - C10 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-K), wherein R 3’ is C6-C10 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-L): (S-L), or a pharmaceutically acceptable salt thereof, wherein R 1 is -OH, -R 1a , X 1 is optionally substituted C2-C6 alkylenyl; (i) Y 1 is ; Z 3 is optionally substituted C2-C6 alkylenyl; and R 2 and R 3 are independently optionally substituted C4-C14 alkyl; X 2 and X 3 are C5 alkylenyl; or (ii) Y 1 is a bond R 2 and R 3 are independently C 4 -C 7 alkyl; X 2 is optionally substituted C 2 -C 14 alkylenyl; X 3 is optionally substituted C5 alkylenyl; R 4 is optionally substituted C4-C14 alkyl; R 1a is: , , , or ; R 2a , R 2b , and
  • Lipids of the Disclosure have a structure of Formula (S-L), wherein R 1 is OH. [00139] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein X 1 is C 2 alkylenyl. [00140] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein Y 1 is , wherein Z 3 is C 2 alkylenyl. [00141] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein R 2 and R 3 are independently C6-C12 alkyl (C7, C8, C9, C10, C11 alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-L), wherein R 2 is C6-C12 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein R 3 is C 6 -C 12 alkyl. [00142] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein Y 1 is a bond. [00143] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein R 2 and R 3 are C4-C7alkyl (e.g., C7alkyl).
  • Lipids of the Disclosure have a structure of Formula (S-L), wherein R 2 is C 4 -C 7 alkyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein R 3 is C 4 -C 7 alkyl. [00144] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-L), wherein X 2 is C6-C12 alkylenyl (e.g., C7, C8, C9, C10 alkylenyl).
  • Lipids of the Disclosure have a structure of Formula (S-I’): (S-I’), or a pharmaceutically acceptable salt thereof, wherein: X is N or CH; Y is a bond, , , or , wherein bond marked with an “**” is attached to X; each Z is independently selected from the group consisting of: , , , , , , and wherein the bond marked with an "*” is attached to L; each L is independently C 2 -C 10 alkylenyl; R 1 is OH, -N(R 3 ) 2, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), or (S-I), wherein X is N. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), or (S-I), wherein X is CH. Y [00148] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), or (S-I), wherein Y is a bond.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein Z is , wherein bond marked with an “*” is attached to X.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein Z is , wherein bond marked with an “*” is attached to X.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein Z is , wherein bond marked with an “*” is attached to X.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein Z is , wherein bond marked with an “*” is attached to X. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein Z is , wherein bond marked with an “*” is attached to X. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein Z is , wherein bond marked with an “*” is attached to X.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein Z is , wherein bond marked with an “*” is attached to X.
  • L [00150]
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein L is C 2 -C 10 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein L is C 5 -C 8 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein L is C 5 alkylenyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein L is C6 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein L is C7 alkylenyl. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein L is C 8 alkylenyl. R 1 [00151] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein R 1 is OH.
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein R 1 is N(R 3 )2. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein R 1 is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein R 1 is selected from the group consisting of , , , , , , , and , wherein each R is independently -H or C 1 -C 6 aliphatic. In certain embodiments, R 1 is . [00152] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or
  • Lipids of the Disclosure have a structure of Formula (S-I), or
  • Lipids of the Disclosure have a structure of Formula (S-I), or (S-I'), wherein R 1 is
  • Lipids of the Disclosure have a structure of Formula (S-Ia): or a pharmaceutically acceptable salt thereof, wherein: each R 2 is independently selected from optionally substituted C 2-14 alkyl and C 2-14 alkenyl, wherein any –(CH 2 ) 2 - of the C 2 -C 14 alkyl can be optionally replaced with C 3 -C 6 cycloalkylenyl; n is selected from 1 to 4; each m is independently selected from 2 to 10; and each p is independently selected from 2 to 6.
  • Lipids of the Disclosure have a structure of Formula (S-Ib): (S-Ib), or a pharmaceutically acceptable salt thereof, wherein: each R 2 is independently selected from optionally substituted C2-14alkyl and C2-14alkenyl, wherein any –(CH2)2- of the C2-C14 alkyl can be optionally replaced with C3-C6 cycloalkylenyl; each R 3 independently selected from is H and C 1-6 alkylene; n is selected from 0 to 4; each m is independently selected from 2 to 10; and each p is independently selected from 2 to 6.
  • Lipids of the Disclosure have a structure of Formula (S-Ic): (S-Ic), or a pharmaceutically acceptable salt thereof, wherein: each R 2 is independently selected from optionally substituted C 2-14 alkyl and C 2-14 alkenyl, wherein any –(CH2)2- of the C2-C14 alkyl can be optionally replaced with C3-C6 cycloalkylenyl; n is selected from 1 to 4; each m is independently selected from 2 to 10; and each p is independently selected from 2 to 6.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is optionally substituted C2-14alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is optionally substituted C 7-12 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S- Ia), or Formula (S-Ib), wherein R 2 is independently selected from the group consisting of: , , , , and .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S- Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is optionally substituted C 2-14 alkenyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S- Ia), or Formula (S-Ib), wherein R 2 is independently selected from: and .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is optionally substituted C8-9alkenyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 2 is .
  • Lipids of the Disclosure have a structure of Formula (S-I) or Formula (S-Ib), wherein R 3 is hydrogen. [00161] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein R 3 is C 1-6 alkylene. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I’), wherein each R 3 is C 1 alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, or C 6 alkyl.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein n is 3.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein n is 4.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein n is 1, 2, 5, or 6.
  • Lipids of the Disclosure have a structure of Formula (S-Ia), or Formula (S-Ib), wherein m is selected from 5 to 8. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-Ia), or Formula (S-Ib), wherein m is 5. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-Ia) or Formula (S-Ib), wherein m is 6. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-Ia) or Formula (S-Ib), wherein m is 7.
  • Lipids of the Disclosure have a structure of Formula (S-Ia) or Formula (S-Ib), wherein m is 8. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-Ia), or Formula (S-Ib), wherein m is 2, 3, 4, 9, or 10.
  • p [00164] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein p is independently selected from 2 to 4. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein p is 2.
  • Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein p is 3. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein p is 4. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), Formula (S-I’), Formula (S-Ia), or Formula (S-Ib), wherein p is 5 or 6. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-I), or (S-I'), wherein p is 1.
  • Lipids of the Disclosure have a structure of Formula (S-M’): or a pharmaceutically acceptable salt thereof, wherein:
  • X is N or CH
  • Y is a bond, O wherein bond marked with an
  • each Z is independently selected from the group consisting of: wherein the bond marked with an is attached to L; each L is independently C2-C10 alkylenyl;
  • each R is independently -H or C1-C6 aliphatic;
  • R Z is NR2 or OH;
  • X Z is optionally substituted C2-C14 alkylenyl or optionally substituted C2-C14 alkenylenyl;each R 3 independently selected from is H and C1-6alkyl;
  • R 4 is -CH(SR 6 )(SR 7 );
  • R 5 is -CH(OR 8 )(OR 9 ); -CH(SR 8 )(SR 9 ); -CH(R 8 )(R 9 ) or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl, phenyl, -O-,
  • Lipids of the Disclosure have a structure of Formula (S-M): (S-M), or a pharmaceutically acceptable salt thereof, wherein: X is N or CH; Y is a bond, , , or , wherein bond marked with an “**” is attached to X; each Z is independently selected from the group consisting of: , , , , , , and wherein the bond marked with an "*” is attached to L; each L is independently C2-C10 alkylenyl; R 1 is OH, N(R 3 )2, , , , , , , , , , , and ; each R is independently -H or C1-C6 aliphatic; each R 3 independently selected from is H and C 1-6 alkyl; R 4 is -CH(SR 6 )(SR 7 ); R 5 is -CH(OR 8 )(OR 9 ); -CH(SR 8 )(SR 9 );
  • Lipids of the Disclosure have a structure of Formula (S-Ma) (S-Ma) or a pharmaceutically acceptable salt thereof, wherein: n is selected from 1 to 4; each R 4 and R 5 is as described in Formula S-M or S-M’; each m is independently selected from 2 to 10; and each p is independently selected from 2 to 6.
  • Lipids of the Disclosure have a structure of Formula (S-Mb) (S-Mb), or a pharmaceutically acceptable salt thereof, wherein: each R 3 independently selected from is H and C 1-6 alkyl; n is selected from 1 to 4; each R 4 and R 5 is as described in Formula S-M or S-M’; each m is independently selected from 2 to 10; and each p is independently selected from 2 to 6. R 1 [00169] In some embodiments, Lipids of the Disclosure have a structure of Formula (S-M), or (S-M’), wherein R 1 is OH.
  • Lipids of the Disclosure have a structure of Formula (S-M), or (S-M’), wherein R 1 is N(R 3 ) 2 . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-M), or (S-M’), wherein R 1 is . In some embodiments, Lipids of the Disclosure have a structure of Formula (S-M), or (S-M’),wherein R 1 is , , , , , , , and , wherein each R is independently -H or C 1 -C 6 aliphatic. In certain embodiments, R 1 is .
  • Lipids of the Disclosure have a structure of Formula (S-M), or (S-M’), wherein R 1 is selected from the group consisting of OH, -N(R 3 )2, , , , , , , , , , , , , , , , , ,
  • Lipids of the Disclosure have a structure of Formula (S-M’), wherein R 1 is selected from the group consisting of OH, N(R 3 )2, and .
  • Lipids of the Disclosure have a structure of Formula (S-M’), wherein R 1 is . n
  • Lipids of the Disclosure have a structure of Formula (S-M), Formula (S-M’), Formula (S-Ma), or Formula (S-Mb), wherein n is 3.
  • Lipids of the Disclosure have a structure of Formula (S-M), Formula (S-M’), Formula (S-Ma), or Formula (S-Mb), wherein n is 4.
  • Lipids of the Disclosure have a structure of Formula (S-M), Formula (S-M’), Formula (S-Ma), or Formula (S-Mb), wherein n is 1, 2, 5, or 6.
  • p is independently selected from 2 to 4.
  • Lipids of the Disclosure have a structure of Formula (S-M), Formula (S- M’), Formula (S-Ma), or Formula (S-Mb), wherein p is 2. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-M), Formula (S-M’), Formula (S-Ma), or Formula (S-Mb), wherein p is 3. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-M), Formula (S-M’), Formula (S-Ma), or Formula (S-Mb), wherein p is 4.
  • Lipids of the Disclosure have a structure of Formula (S-M), Formula (S-M’), Formula (S-Ma), or Formula (S-Mb), wherein p is 5 or 6. In some embodiments, Lipids of the Disclosure have a structure of Formula (S-M), or (S-M’), wherein p is 1.
  • R 4 [00174] As disclosed in Formula (S-M), in certain embodiments, R 4 is -CH(SR 6 )(SR 7 ). In certain embodiments, R 4 is selected from , , , , and .
  • R 5 is -CH(OR 8 )(OR 9 ); - CH(SR 8 )(SR 9 ); -CH(R 8 )(R 9 ) or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 5 is optionally substituted C 1 -C 14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S- , -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 5 is optionally substituted C1-C14 aliphatic.
  • R 5 is -CH(OR 8 )(OR 9 ) . In certain embodiments, R 5 is -CH(R 8 )(R 9 ). In certain embodiments, R 5 is -CH(SR 8 )(SR 9 ). [00176] In certain embodiments, R 4 and R 5 are the same. In certain embodiments, R 4 and R 5 are different. [00177] In certain embodiments, R 5 is selected from , , , , , , , , , , , , , and .
  • R 6 and R 7 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, - SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 6 and R 7 are the same. In certain embodiments, R 6 and R 7 are different. [00180] In certain embodiments, R 6 is optionally substituted C1-C14 aliphatic. In certain embodiments, R 6 is optionally substituted C 1 -C 14 alkylene. In certain embodiments, R 6 is optionally substituted C 1 -C 14 branched alkylene. In certain embodiments, R 6 is optionally substituted C 1 -C 14 straight chain alkylene. In certain embodiments, R 6 is optionally substituted C 1 -C 14 alkenylene. In certain embodiments, R 6 is optionally substituted C 1 -C 14 branched alkenylene.
  • R 6 is optionally substituted C1-C14 straight chain alkenylene. In certain embodiments, R 6 is optionally substituted C6-C10 alkylene. In certain embodiments, R 6 is optionally substituted – (CH2)5CH3. In certain embodiments, R 6 is optionally substituted –(CH2)6CH3. In certain embodiments, R 6 is optionally substituted –(CH 2 ) 7 CH 3 . In certain embodiments, R 6 is optionally substituted – (CH 2 ) 8 CH 3 . In certain embodiments, R 6 is optionally substituted –(CH 2 ) 9 CH 3 .
  • one of the methylene linkages of R 6 is replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , , , , , and .
  • R 7 is optionally substituted C 1 -C 14 aliphatic.
  • R 7 is optionally substituted C 1 -C 14 alkylene.
  • R 7 is optionally substituted C 1 -C 14 branched alkylene. In certain embodiments, R 7 is optionally substituted C 1 -C 14 straight chain alkylene. In certain embodiments, R 7 is optionally substituted C1-C14 alkenylene. In certain embodiments, R 7 is optionally substituted C1-C14 branched alkenylene. In certain embodiments, R 7 is optionally substituted C1-C14 straight chain alkenylene. In certain embodiments, R 7 is optionally substituted C6-C10 alkylene. In certain embodiments, R 7 is optionally substituted – (CH 2 ) 5 CH 3 . In certain embodiments, R 7 is optionally substituted –(CH 2 ) 6 CH 3 .
  • R 7 is optionally substituted –(CH 2 ) 7 CH 3 . In certain embodiments, R 7 is optionally substituted – (CH 2 ) 8 CH 3 . In certain embodiments, R 6 is optionally substituted –(CH 2 ) 9 CH 3 . [00183] In certain embodiments, one of the methylene linkages of R 7 is replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl.
  • R 7 is an optionally substituted bridged bicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from adamantyl, bicyclo[2.2.2]octyl, cubanyl, bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.1]heptyl, and bicyclo[3.2.1]octyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from: , , , and .
  • R 8 and R 9 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, - SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 8 and R 9 are the same. In certain embodiments, R 8 and R 9 are different. [00189] In certain embodiments, R 8 is optionally substituted C 1 -C 14 aliphatic. In certain embodiments, R 8 is optionally substituted C1-C14 alkylene. In certain embodiments, R 8 is optionally substituted C1-C14 branched alkylene. In certain embodiments, R 8 is optionally substituted C1-C14 straight chain alkylene. In certain embodiments, R 8 is optionally substituted C1-C14 alkenylene. In certain embodiments, R 8 is optionally substituted C 1 -C 14 branched alkenylene.
  • R 9 is optionally substituted C1-C14 branched alkylene. In certain embodiments, R 9 is optionally substituted C1-C14 straight chain alkylene. In certain embodiments, R 9 is optionally substituted C1-C14 alkenylene. In certain embodiments, R 9 is optionally substituted C 1 -C 14 branched alkenylene. In certain embodiments, R 9 is optionally substituted C 1 -C 14 straight chain alkenylene. In certain embodiments, R 9 is optionally substituted C 6 -C 10 alkylene. In certain embodiments, R 9 is optionally substituted – (CH 2 ) 5 CH 3 . In certain embodiments, R 9 is optionally substituted –(CH 2 ) 6 CH 3 .
  • R 9 is optionally substituted –(CH2)7CH3. In certain embodiments, R 9 is optionally substituted – (CH2)8CH3. In certain embodiments, R 9 is optionally substituted –(CH2)9CH3. [00192] In certain embodiments, one of the methylene linkages of R 9 is replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl. In certain embodiments, the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , , , , , , and .
  • R 8 and R 9 are selected from , , , , , and .
  • R 8 and R 9 taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.
  • each R 8 and R 9 are each independently selected from an optionally substituted bridged bicyclic C 5 -C 12 cycloalkylenyl.
  • R 8 is an optionally substituted bridged multicyclic C 5 -C 12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from: , , , , , , , and .
  • the substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is a structure selected from , , , , , , , , and , wherein one or more C-H bonds are substituted.
  • R 8 and R 9 taken together form an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , and .
  • Lipids of the Disclosure comprise an acyclic core.
  • Lipids of the Disclosure are selected from any lipid in Table (I-A) below or a pharmaceutically acceptable salt thereof: Table (I-A).
  • Non-Limiting Examples of Ionizable Lipids Cmpd Structure N S [ ] escr e eow are a num er o exempary onza e p s o e presen scosure.
  • Lipids of the Disclosure have a structure of Formula (AT) (AT), or a pharmaceutically acceptable salt thereof, wherein: i) A is N; Z is a bond; X 1 is optionally substituted C1-C6 aliphatic, wherein the optional substituent is not oxo when X 1 is C1 aliphatic; and R 1 is selected from the group consisting of: , , and ; or ii) A is CH; Z is , , , , , , , , , or ; wherein the bond marked with an "*" is attached to X 1 ; X 1 is a bond or optionally substituted C 1 -C 6 aliphatic; R 1 is selected from the group consisting of: , , , , , , , and ; X 4 is a bond or optionally substituted C 1 -C 6 aliphatic; R Z is NR 2 or OH;
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-A): (AT-A), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-A1): (AT-A1), or a pharmaceutically acceptable salt thereof, wherein Z is or , wherein the bond marked with an "*" is attached to X 1 ; Y 1 and Y 2 are independently selected from the group consisting of , , , , , and ; wherein the bond marked with an "*" is attached to R 2 for Y 1 or R 3 for Y 2 ; and R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-A2): (AT-A2), or a pharmaceutically acceptable salt thereof, wherein Z is or , wherein the bond marked with an "*" is attached to X 1 ; Y 1 and Y 2 are each ,wherein the bond marked with an "*” is attached to R 2 for Y 1 or R 3 for Y 2 ; and R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-B): (AT-B), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-B’): (AT-B’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-C): (AT-C), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-D): (AT-D), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-D’): (AT-D’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-D’a): (AT-D’a), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-D’b): (AT-D’b), Formula (AT-E) [00210]
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-E): (AT-E), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-E’): (AT-E’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-E’’): (AT-E’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-F): (AT-F), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-F’): (AT-F’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-F’’): (AT-F’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-F’’’): (AT-F’’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Formula (AT-F’’’’) [00217]
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-F’’’’):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-F’’’’): (AT-F’’’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-G): (AT-G), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , Y 1 , Y 2 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-G’): (AT-G’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , Y 1 , Y 2 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-H): (AT-H), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-H’): (AT-H’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-H’’): (AT-H’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-H’’’): (AT-H’’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-I): (AT-I), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-J): (AT-J), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-J’): (AT-J’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-K):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-K’):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-L):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-L’):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-L’’):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-L’”): (AT-L’’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-M): (AT-M), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-N): (AT-N), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-N’): (AT-N’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-O): (AT-O), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , Y 1 , Y 2 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-O’): (AT-O’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , Y 1 , Y 2 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-P): (AT-P), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-P’): (AT-P’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-P’’): (AT-P’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-P’’’): (AT-P’’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , X 4 , R Z , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-Q): (AT-Q), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-Q1): (AT-Q1), or a pharmaceutically acceptable salt thereof, wherein Y 1 and Y 2 are independently selected from the group consisting of , , , , , and ; wherein the bond marked with an "*" is attached to R 2 for Y 1 or R 3 for Y 2 ; and R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-Q2): (AT-Q2), or a pharmaceutically acceptable salt thereof, wherein R 1 is ; Y 1 and Y 2 are ; wherein the bond marked with an "*" is attached to R 2 for Y 1 or R 3 for Y 2 ; and R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-R): O R 2 X 2 R 4 R 1 1 N 3 O X X O R 3 R 5 O (AT-R), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-R’): (AT-R’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-S): (AT-S), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-S’): (AT-S’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-S’’): (AT-S’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-T): (AT-T), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-T’): (AT-T’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Formula (AT-T’’) [00253]
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-T’’):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-T’’’): (AT-T’’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • Formula (AT-T’’’’) [00255]
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-T’’’’):
  • Lipids of the Disclosure have a structure of Formula (AT), wherein the Lipids of the Disclosure have a structure of Formula (AT-T’’’’): (AT-T’’’’’), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , R Z , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AT) or as otherwise described in any embodiments below.
  • A As disclosed in Formula (AT), in certain embodiments, A is CH or N. In certain embodiments, A is CH.
  • A is N.
  • Z As disclosed in Formula (AT), in certain embodiments wherein A is CH, Z is , , , , , , , , or ; wherein the bond marked with an "*" is attached to X 1 . In certain embodiments wherein A is CH, Z is , , , , , , , or . In certain embodiments wherein A is CH, Z is or . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is .
  • Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . In certain embodiments, Z is . As disclosed in Formula (AT), in certain embodiments wherein A is N, Z is a bond.
  • X 1 [00259] As disclosed in Formula (AT), in certain embodiments wherein A is N, X 1 is optionally substituted C1-C6 aliphatic. In certain embodiments wherein A is N, X 1 is unsubstituted C1- C 6 aliphatic. In certain embodiments, X 1 is optionally substituted C 1 -C 6 alkylene. In certain embodiments, X 1 is unsubstituted C 1 -C 6 alkylene.
  • X 1 is unsubstituted C 2 -C 6 alkylene. In certain embodiments, X 1 is optionally substituted methylene. In certain embodiments, R 2 is optionally substituted C2 alkylene. In certain embodiments, X 1 is optionally substituted C3 alkylene. In certain embodiments, X 1 is optionally substituted C4 alkylene. In certain embodiments, X 1 is optionally substituted C5 alkylene. In certain embodiments, X 1 is optionally substituted C6 alkylene. In certain embodiments, X 1 is –(CH 2 )-. In certain embodiments, X 1 is –(CH 2 ) 2 -.
  • X 1 is –(CH 2 ) 3 -. In certain embodiments, X 1 is –(CH 2 ) 4 -. In certain embodiments, X 1 is – (CH 2 ) 5 -. In certain embodiments, X 1 is –(CH 2 ) 6 -. [00260] As disclosed in Formula (AT), in certain embodiments wherein A is CH, X 1 is a bond or optionally substituted C1-C6 aliphatic. In certain embodiments, X 1 is a bond. In certain embodiments, X 1 is optionally substituted C1-C6 alkylene. In certain embodiments, X 1 is unsubstituted C1-C6 alkylene.
  • X 1 is unsubstituted C2-C6 alkylene. In certain embodiments, X 1 is optionally substituted methylene. In certain embodiments, R 2 is optionally substituted C 2 alkylene. In certain embodiments, X 1 is optionally substituted C 3 alkylene. In certain embodiments, X 1 is optionally substituted C 4 alkylene. In certain embodiments, X 1 is optionally substituted C 5 alkylene. In certain embodiments, X 1 is optionally substituted C 6 alkylene. In certain embodiments, X 1 is –(CH2)-. In certain embodiments, X 1 is –(CH2)2-. In certain embodiments, X 1 is –(CH2)3-.
  • X 1 is –(CH2)4-. In certain embodiments, X 1 is –(CH2)5-. In certain embodiments, X 1 is –(CH2)6-.
  • R 1 [00261] As disclosed in Formula (AT), in certain embodiments wherein A is N, R 1 is selected from the group consisting of , , and . As disclosed in Formula (AT), in certain embodiments wherein A is CH, R 1 is selected from the group consisting of , , , , , , , and . [00262] In certain embodiments, R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is .
  • R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is . [00263] In certain embodiments, R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is . In certain embodiments, R 1 is . X 2 and X 3 [00264] As disclosed in Formula (AT), in certain embodiments, X 2 and X 3 are each independently optionally substituted C 1 -C 12 aliphatic. In certain embodiments, X 2 and X 3 are the same. In certain embodiments, X 2 and X 3 are different.
  • X 2 is an optionally substituted C1-C12 alkylene. In certain embodiments, X 2 is an optionally substituted C 1 -C 12 alkenylene. In certain embodiments, X 2 is an optionally substituted C 1 -C 10 aliphatic. In certain embodiments, X 2 is an optionally substituted C 1 -C 10 alkylene. In certain embodiments, X 2 is an optionally substituted C 1 -C 10 alkenylene. In certain embodiments, X 2 is an optionally substituted C 1 -C 8 aliphatic. In certain embodiments, X 2 is an optionally substituted C1-C8 alkylene.
  • X 2 is an optionally substituted C1-C8 alkenylene. In certain embodiments, X 2 is an optionally substituted C1-C6 aliphatic. In certain embodiments, X 2 is an optionally substituted C1-C6 alkylene. In certain embodiments, X 2 is an optionally substituted C 1 -C 6 alkenylene. In certain embodiments, X 2 is an optionally substituted C 2 - C 12 aliphatic. In certain embodiments, X 2 is an optionally substituted C 2 -C 12 alkylene. In certain embodiments, X 2 is an optionally substituted C 2 -C 12 alkenylene.
  • X 2 is an optionally substituted C4-C12 aliphatic. In certain embodiments, X 2 is an optionally substituted C4-C12 alkylene. In certain embodiments, X 2 is an optionally substituted C 4 -C 12 alkenylene. In certain embodiments, X 2 is an optionally substituted C 4 -C 10 aliphatic. In certain embodiments, X 2 is an optionally substituted C4-C10 alkylene. In certain embodiments, X 2 is an optionally substituted C4-C10 alkenylene. In certain embodiments, X 2 is an optionally substituted C6-C8 aliphatic. In certain embodiments, X 2 is an optionally substituted C6-C8 alkylene.
  • X 2 is an optionally substituted C 6 -C 8 alkenylene. In certain embodiments, X 2 is –(CH 2 )-. In certain embodiments, X 2 is –(CH 2 ) 2 -. In certain embodiments, X 2 is –(CH 2 ) 3 -. In certain embodiments, X 2 is – (CH 2 ) 4 -. In certain embodiments, X 2 is –(CH 2 ) 5 -. In certain embodiments, X 2 is –(CH 2 ) 6 -. In certain embodiments, X 2 is –(CH2)7-. In certain embodiments, X 2 is –(CH2)8-.
  • X 2 is – (CH2)9-. In certain embodiments, X 2 is –(CH2)10-.
  • X 3 is an optionally substituted C1-C12 alkylene. In certain embodiments, X 3 is an optionally substituted C 1 -C 12 alkenylene. In certain embodiments, X 3 is an optionally substituted C 1 -C 10 aliphatic. In certain embodiments, X 3 is an optionally substituted C 1 -C 10 alkylene. In certain embodiments, X 3 is an optionally substituted C 1 -C 10 alkenylene. In certain embodiments, X 3 is an optionally substituted C1-C8 aliphatic.
  • X 3 is an optionally substituted C1-C8 alkylene. In certain embodiments, X 3 is an optionally substituted C1-C8 alkenylene. In certain embodiments, X 3 is an optionally substituted C1-C6 aliphatic. In certain embodiments, X 3 is an optionally substituted C1-C6 alkylene. In certain embodiments, X 3 is an optionally substituted C 1 -C 6 alkenylene. In certain embodiments, X 3 is an optionally substituted C 2 - C 12 aliphatic. In certain embodiments, X 3 is an optionally substituted C 2 -C 12 alkylene.
  • X 3 is an optionally substituted C 2 -C 12 alkenylene. In certain embodiments, X 3 is an optionally substituted C4-C12 aliphatic. In certain embodiments, X 3 is an optionally substituted C4-C12 alkylene. In certain embodiments, X 3 is an optionally substituted C4-C12 alkenylene. In certain embodiments, X 3 is an optionally substituted C4-C10 aliphatic. In certain embodiments, X 3 is an optionally substituted C 4 -C 10 alkylene. In certain embodiments, X 3 is an optionally substituted C 4 -C 10 alkenylene.
  • X 3 is an optionally substituted C 6 -C 8 aliphatic. In certain embodiments, X 3 is an optionally substituted C 6 -C 8 alkylene. In certain embodiments, X 3 is an optionally substituted C 6 -C 8 alkenylene. In certain embodiments, X 3 is –(CH 2 )-. In certain embodiments, X 3 is –(CH2)2-. In certain embodiments, X 3 is –(CH2)3-. In certain embodiments, X 3 is – (CH2)4-. In certain embodiments, X 3 is –(CH2)5-. In certain embodiments, X 3 is –(CH2)6-.
  • X 3 is –(CH2)7-. In certain embodiments, X 3 is –(CH2)8-. In certain embodiments, X 3 is – (CH 2 ) 9 -. In certain embodiments, X 3 is –(CH 2 ) 10 -. [00267] In certain embodiments, X 2 and X 3 are both –(CH 2 ) 8 -. In certain embodiments, X 2 and X 3 are both –(CH 2 ) 6 -.
  • X 4 [00268] As disclosed in Formula (AT), in certain embodiments, X 4 is a bond or C 2 -C 6 aliphatic. In certain embodiments, X 4 is a bond.
  • X 4 is C2-C6 aliphatic. In certain embodiments, X 4 is C2 aliphatic. In certain embodiments, X 4 is C3 aliphatic. In certain embodiments, X 4 is C4 aliphatic. In certain embodiments, X 4 is C5 aliphatic. In certain embodiments, X 4 is C 6 aliphatic.
  • Y 1 and Y 2 are each independently , , , , , , , , or , wherein the bond marked with an "*" is attached to X 2 for Y 1 or X 3 for Y 2 .
  • Y 1 and Y 2 are the same. In certain embodiments, Y 1 and Y 2 are different. [00270] In certain embodiments, Y 1 and Y 2 are each independently , , , , , or . In certain embodiments, Y 1 and Y 2 are each independently or . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is .
  • Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 1 and Y 2 are both . In certain embodiments, Y 1 and Y 2 are both . R 2 [00271] As disclosed in Formula (AT), in certain embodiments, R 2 is optionally substituted C1-C6 aliphatic. In certain embodiments, R 2 is optionally substituted C1-C6 alkylene. In certain embodiments, R 2 is optionally substituted methylene. In certain embodiments, R 2 is optionally substituted C 2 alkylene.
  • R 2 is optionally substituted C 3 alkylene. In certain embodiments, R 2 is optionally substituted C 4 alkylene. In certain embodiments, R 2 is optionally substituted C 5 alkylene. In certain embodiments, R 2 is optionally substituted C 6 alkylene. In certain embodiments, R 2 is –(CH2)-. In certain embodiments, R 2 is –(CH2)2-. In certain embodiments, R 2 is – (CH2)3-. In certain embodiments, R 2 is –(CH2)4-. In certain embodiments, R 2 is –(CH2)5-. In certain embodiments, R 2 is –(CH2)6-.
  • R 3 is optionally substituted C 1 -C 6 aliphatic. In certain embodiments, R 3 is optionally substituted C 1 -C 6 alkylene. In certain embodiments, R 3 is optionally substituted methylene. In certain embodiments, R 3 is optionally substituted C2 alkylene. In certain embodiments, R 3 is optionally substituted C3 alkylene. In certain embodiments, R 3 is optionally substituted C4 alkylene. In certain embodiments, R 3 is optionally substituted C5 alkylene. In certain embodiments, R 3 is optionally substituted C6 alkylene. In certain embodiments, R 3 is –(CH 2 )-.
  • R 3 is –(CH 2 ) 2 -. In certain embodiments, R 3 is – (CH 2 ) 3 -. In certain embodiments, R 3 is –(CH 2 ) 4 -. In certain embodiments, R 3 is –(CH 2 ) 5 -. In certain embodiments, R 3 is –(CH2)6-. [00273] In certain embodiments, R 2 and R 3 are the same. In certain embodiments, R 2 and R 3 are different.
  • R 4 is -CH(OR 6 )(OR 7 ), - CH(SR 6 )(SR 7 ), -CH(R 6 )(R 7 ), or optionally substituted C 1 -C 14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 4 is optionally substituted C 1 -C 14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl, phenyl, -O-, -NH-, -S- , -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 4 is optionally substituted C 1 -C 14 aliphatic.
  • R 4 is -CH(OR 6 )(OR 7 ) . In certain embodiments, R 4 is -CH(R 6 )(R 7 ). In certain embodiments, R 4 is -CH(SR 6 )(SR 7 ). [00275] In certain embodiments, one of the methylene linkages of R 4 is replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from: , , , , , , , and .
  • R 4 is selected from is selected from , , , , , , , , , , and .
  • R 4 is selected from is selected from and .
  • R 5 is -CH(OR 8 )(OR 9 ), - CH(SR 8 )(SR 9 ), -CH(R 8 )(R 9 ), or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 5 is optionally substituted C 1 -C 14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S- , -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 5 is optionally substituted C1-C14 aliphatic.
  • R 5 is -CH(OR 8 )(OR 9 ) . In certain embodiments, R 5 is -CH(R 8 )(R 9 ). In certain embodiments, R 5 is -CH(SR 8 )(SR 9 ). [00279] In certain embodiments, one of the methylene linkages of R 5 is replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , , , , , and .
  • R 4 and R 5 are the same. In certain embodiments, R 4 and R 5 are different.
  • R 5 is selected from , , , , , , , , , and .
  • R 5 is selected from is selected from and .
  • R 6 and R 7 are each independently optionally substituted C 1 -C 14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, - SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 6 and R 7 are the same. In certain embodiments, R 6 and R 7 are different. [00285] In certain embodiments, R 6 is optionally substituted C 1 -C 14 aliphatic. In certain embodiments, R 6 is optionally substituted C 1 -C 14 alkyl. In certain embodiments, R 6 is optionally substituted C 1 -C 14 branched alkyl. In certain embodiments, R 6 is optionally substituted C 1 -C 14 straight chain alkyl. In certain embodiments, R 6 is optionally substituted C1-C14 alkenylene. In certain embodiments, R 6 is optionally substituted C1-C14 branched alkenyl.
  • R 6 is optionally substituted C1-C14 straight chain alkenyl. In certain embodiments, R 6 is optionally substituted C 6 -C 10 alkyl. In certain embodiments, R 6 is optionally substituted –(CH 2 ) 5 CH 3 . In certain embodiments, R 6 is optionally substituted –(CH 2 ) 6 CH 3 . In certain embodiments, R 6 is optionally substituted –(CH 2 ) 7 CH 3 . In certain embodiments, R 6 is optionally substituted –(CH 2 ) 8 CH 3 . In certain embodiments, R 6 is optionally substituted –(CH 2 ) 9 CH 3 .
  • one of the methylene linkages of R 6 is replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from: , , , , , , , and .
  • R 7 is optionally substituted C1-C14 aliphatic. In certain embodiments, R 7 is optionally substituted C1-C14 alkyl.
  • R 7 is optionally substituted C 1 -C 14 branched alkyl. In certain embodiments, R 7 is optionally substituted C 1 -C 14 straight chain alkyl. In certain embodiments, R 7 is optionally substituted C 1 -C 14 alkenylene. In certain embodiments, R 7 is optionally substituted C 1 -C 14 branched alkenyl. In certain embodiments, R 7 is optionally substituted C 1 -C 14 straight chain alkenyl. In certain embodiments, R 7 is optionally substituted C6-C10 alkyl. In certain embodiments, R 7 is optionally substituted –(CH2)5CH3. In certain embodiments, R 7 is optionally substituted –(CH2)6CH3.
  • R 7 is optionally substituted –(CH2)7CH3. In certain embodiments, R 7 is optionally substituted –(CH2)8CH3. In certain embodiments, R 6 is optionally substituted –(CH 2 ) 9 CH 3 . [00288] In certain embodiments, one of the methylene linkages of R 7 is replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , , , , , and .
  • each R 6 and R 7 are selected from , , , , , , and .
  • each R 6 and R 7 are each independently selected from an optionally substituted bridged bicyclic C5-C12 cycloalkylenyl.
  • R 6 is an optionally substituted bridged multicyclic C5-C12 cycloalkylenyl.
  • R 7 is an optionally substituted bridged bicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from adamantyl, bicyclo[2.2.2]octyl, cubanyl, bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.1]heptyl, and bicyclo[3.2.1]octyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , , , , , and .
  • the substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is a structure selected from , , , , , , , , and , wherein one or more C-H bonds are substituted.
  • R 6 and R 7 taken together form an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , and .
  • R 8 and R 9 are each independently optionally substituted C 1 -C 14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, - SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 8 and R 9 are the same. In certain embodiments, R 8 and R 9 are different. [00295] In certain embodiments, R 8 is optionally substituted C 1 -C 14 aliphatic. In certain embodiments, R 8 is optionally substituted C1-C14 alkyl. In certain embodiments, R 8 is optionally substituted C1-C14 branched alkyl. In certain embodiments, R 8 is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R 8 is optionally substituted C1-C14 alkenyl. In certain embodiments, R 8 is optionally substituted C 1 -C 14 branched alkenyl.
  • R 8 is optionally substituted C 1 -C 14 straight chain alkenyl. In certain embodiments, R 8 is optionally substituted C 6 -C 10 alkyl. In certain embodiments, R 8 is optionally substituted –(CH 2 ) 5 CH 3 . In certain embodiments, R 8 is optionally substituted –(CH 2 ) 6 CH 3 . In certain embodiments, R 8 is optionally substituted –(CH2)7CH3. In certain embodiments, R 8 is optionally substituted –(CH2)8CH3. In certain embodiments, R 8 is optionally substituted –(CH2)9CH3.
  • one of the methylene linkages of R 8 is replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from: , , , , , , , and .
  • R 9 is optionally substituted C 1 -C 14 aliphatic. In certain embodiments, R 9 is optionally substituted C 1 -C 14 alkyl.
  • R 9 is optionally substituted C 1 -C 14 branched alkyl. In certain embodiments, R 9 is optionally substituted C 1 -C 14 straight chain alkyl. In certain embodiments, R 9 is optionally substituted C 1 -C 14 alkenyl. In certain embodiments, R 9 is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R 9 is optionally substituted C1-C14 straight chain alkenyl. In certain embodiments, R 9 is optionally substituted C6-C10 alkyl. In certain embodiments, R 9 is optionally substituted –(CH2)5CH3. In certain embodiments, R 9 is optionally substituted –(CH 2 ) 6 CH 3 .
  • R 9 is optionally substituted –(CH 2 ) 7 CH 3 . In certain embodiments, R 9 is optionally substituted –(CH 2 ) 8 CH 3 . In certain embodiments, R 9 is optionally substituted –(CH 2 ) 9 CH 3 . [00298] In certain embodiments, one of the methylene linkages of R 9 is replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl. In certain embodiments, the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from:
  • each R 8 and R 9 are each independently selected from an optionally substituted bridged bicyclic C5-C12 cycloalkylenyl.
  • R 8 is an optionally substituted bridged multicyclic C5-C12 cycloalkylenyl.
  • R 9 is an optionally substituted bridged bicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from adamantyl, bicyclo[2.2.2]octyl, cubanyl, bicyclo[l.l.l]pcntyl, bicyclo[2.2.1]hcptyl, bicyclo[3.1.1]hcptyl, and bicyclo[3.2.1]octyL In certain embodiments, the optionally substituted bridged bicyclic or multicyclic
  • the substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is a structure selected from , and , wherein one or more C-H bonds are substituted.
  • Lipids of the Present Disclosure are selected from any lipid in
  • Lipids of the Disclosure have a structure of Formula (AC’) (AC’), or a pharmaceutically acceptable salt thereof, wherein: R 1 is selected from the group consisting of -NR2, , , , , , , , , , , , , , , , , , , , , , , , and ; each R is independently -H or C1-C6 aliphatic; R Z is NR2 or OH; X Z is optionally substituted C 2 -C 14 alkylenyl or optionally substituted C 2 -C 14 alkenylenyl;each R 3 independently selected from is H and C 1-6 alkyl; X 1 is a bond or optionally substituted C 2 -C 6 aliphatic; Z is , , , ,
  • Lipids of the Disclosure have a structure of Formula (AC) (AC), or a pharmaceutically acceptable salt thereof, wherein: R 1 is selected from the group consisting of -NR2, , , , , , , , , and ; each R is independently -H or C1-C6 aliphatic; X 1 is a bond or optionally substituted C2-C6 aliphatic; Z is , , , , , , , or ; wherein the bond marked with an "*" is attached to X 1 ; X 2 and X 3 are each independently optionally substituted C1-C12 aliphatic; X 4 is a bond or C 2 -C 6 aliphatic; Y 1 and Y 2 are independently selected from the group consisting of , , , , , , and ; wherein the bond marked with an "*" is attached to X 2 for Y 1 or
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-A): (AC-A), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AC) or (AC’) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-B): (AC-B), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AC) or (AC’) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-C): Y 1 R 4 O X 2 R 2 2 5 1 Y R X O X 3 R 3 R 1 (AC-C), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AC) or (AC’) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-D): (AC-D), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AC) or (AC’) or as otherwise described in any embodiments below.
  • Formula (AC-D1) Formula (AC-D1)
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-D1): o
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-D2): o
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-E):
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-F):
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-H): (AC-H), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AC) or (AC’) or as otherwise described in any embodiments below.
  • Lipids of the Disclosure have a structure of Formula (AC), wherein the Lipids of the Disclosure have a structure of Formula (AC-I): O 2 2 R O X R 6 R 1 Z O O X 1 X 4 X 3 R 7 O R 3 O R 8 O O R 9 (AC-I), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , Z, X 2 , X 3 , X 4 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (AC) or (AC’) or as otherwise described in any embodiments below.
  • R 1 is selected from the group consisting of -NR2,
  • R 1 is selected from the group consisting of -NR2.
  • R 1 is -NR 2 . In certain embodiments, R 1 is
  • R . In certain embodiments, R 1 . In certain embodiments, R 1 is . In certain embodiments,
  • R 1 is R . In certain embodiments, R 1 is R In certain embodiments, R 1 is
  • R 1 is In certain embodiments, R 1 is
  • R 1 is R . In certain embodiments, R 1 is R . In certain embodiments, R 1 is selected from the group consisting of -N(Et) 2 , -N(Me)(Et), I , and ⁇ . In certain embodiments, R 1 is -N(Et) 2 .
  • R 1 is -N(Me) 2 . In certain embodiments, R 1 is -N(Me)(Et In certain embodiments, R 1 is -NHz. In certain embodiments, R 1 is -N(nPr) 2 . In certain embodiments, R 1 is -
  • R 1 is -N(Me)(Et). In certain embodiments, R 1 is I . In certain N ⁇
  • Lipids of the Disclosure have a structure of Formula (AC) or (AC-I), wherein X 4 is a bond or C2-C6 aliphatic. In certain embodiments, Lipids of the Disclosure have a structure of Formula (AC) or (AC-I), wherein X 4 is a bond. In certain embodiments, Lipids of the Disclosure have a structure of Formula (AC) or (AC-I), wherein X 4 is C 2 -C 6 aliphatic.
  • Lipids of the Disclosure have a structure of Formula (AC) or (AC-I), wherein X 4 is C 2 aliphatic. In certain embodiments, Lipids of the Disclosure have a structure of Formula (AC) or (AC- I), wherein X 4 is C3 aliphatic. In certain embodiments, Lipids of the Disclosure have a structure of Formula (AC) or (AC-I), wherein X 4 is C4 aliphatic. In certain embodiments, Lipids of the Disclosure have a structure of Formula (AC) or (AC-I), wherein X 4 is C 5 aliphatic.
  • Lipids of the Disclosure have a structure of Formula (AC) or (AC-I), wherein X 4 is C 6 aliphatic.
  • Y 1 and Y 2 are each independently , , , , , , , or , wherein the bond marked with an "*" is attached to X 2 for Y 1 or X 3 for Y 2 ..
  • Y 1 and Y 2 are the same.
  • Y 1 and Y 2 are different.
  • Y 1 is .
  • Y 1 is .
  • Y 1 is .
  • Y 1 is .
  • R 2 is –(CH2)3-. In certain embodiments, R 2 is –(CH2)4-. In certain embodiments, R 2 is –(CH2)5-. In certain embodiments, R 2 is –(CH2)6-.
  • R 3 is optionally substituted C 1 -C 6 aliphatic. In certain embodiments, R 3 is optionally substituted C 1 -C 6 alkylene. In certain embodiments, R 3 is optionally substituted methylene. In certain embodiments, R 3 is optionally substituted C2 alkylene. In certain embodiments, R 3 is optionally substituted C3 alkylene. In certain embodiments, R 3 is optionally substituted C4 alkylene.
  • R 6 is optionally substituted C6-C10 alkylene. In certain embodiments, R 6 is optionally substituted – (CH2)5CH3. In certain embodiments, R 6 is optionally substituted –(CH2)6CH3. In certain embodiments, R 6 is optionally substituted –(CH 2 ) 7 CH 3 . In certain embodiments, R 6 is optionally substituted – (CH 2 ) 8 CH 3 . In certain embodiments, R 6 is optionally substituted –(CH 2 ) 9 CH 3 .
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from: , , , , , , , and .
  • R 6 and R 7 are selected from , , , , , and .
  • R 8 is optionally substituted C 6 -C 10 alkylene. In certain embodiments, R 8 is optionally substituted – (CH 2 ) 5 CH 3 . In certain embodiments, R 8 is optionally substituted –(CH 2 ) 6 CH 3 . In certain embodiments, R 8 is optionally substituted –(CH2)7CH3. In certain embodiments, R 8 is optionally substituted – (CH2)8CH3. In certain embodiments, R 8 is optionally substituted –(CH2)9CH3.
  • R 9 is optionally substituted C1-C14 branched alkylene. In certain embodiments, R 9 is optionally substituted C1-C14 straight chain alkylene. In certain embodiments, R 9 is optionally substituted C1-C14 alkenylene. In certain embodiments, R 9 is optionally substituted C1-C14 branched alkenylene. In certain embodiments, R 9 is optionally substituted C 1 -C 14 straight chain alkenylene. In certain embodiments, R 9 is optionally substituted C 6 -C 10 alkylene. In certain embodiments, R 9 is optionally substituted – (CH 2 ) 5 CH 3 . In certain embodiments, R 9 is optionally substituted –(CH 2 ) 6 CH 3 .
  • R 1 is selected from the group consisting of -OH, -N(R) 2 , , , , , , , , , , , , , , , , , , , and ; each R is independently -H or C 1 -C 6 aliphatic;
  • R Z is NR2 or OH;
  • X Z is optionally substituted C2-C14 alkylenyl or optionally substituted C2-C14 alkenylenyl;each R 3 independently selected from is H and C1-6alkyl; each R is independently -H or C 1 -C 6 aliphatic;
  • X 1 is optionally substituted C 2 -C 6 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O
  • R 1 is selected from the group consisting of -NR 2 , , , , , , , , , , and ; each R is independently -H or C 1 -C 6 aliphatic; X 1 is optionally substituted C 2 -C 6 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; X 2 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X 3 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X 4 and X 5 are each independently optionally substitute
  • the compound of Formula (CO) is a compound of Formula (CO-A): (CO-A), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 2 , X 3 , X 4 , X 5 , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-B): (CO-B), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 4 , X 5 , Y 1 , Y 2 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-C): (CO-C), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 4 , X 5 , Y 1 . Y 2 , R 2 , R 3 , R 4 , R 3 , R°, R', R s , and R-' are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-D):
  • the compound of Formula (CO) is a compound of Formula (CO-E):
  • the compound of Formula (CO) is a compound of Formula (CO-F):
  • the compound of Formula (CO) is a compound of Formula
  • the compound of Formula (CO) is a compound of Formula
  • the compound of Formula (CO) is a compound of Formula
  • the compound of Formula (CO) is a compound of Formula (CO-H):
  • the compound of Formula (CO) is a compound of Formula
  • the compound of Formula (CO) is a compound of Formula (CO-I): (CO-I), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , X 4 , X 5 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-I’):
  • the compound of Formula (CO) is a compound of Formula (CO-J): (CO-J), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-J’):
  • the compound of Formula (CO) is a compound of Formula (CO-K): (CO-K), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-K): (CO-K), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-L): (CO-L), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-L’):
  • the compound of Formula (CO) is a compound of Formula (CO-M): (CO-M), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-M): (CO-M), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-M’):
  • the compound of Formula (CO) is a compound of Formula (CO-N): (CO-N), or a pharmaceutically acceptable salt thereof, wherein R, X 1 , X 4 , X 5 , Y 1 , Y 2 , R 2 , R 3 , R 6 , R 7 , R 8 , and R 9 are as described in Formula (CO) or (CO’) or as otherwise described in any embodiments below.
  • the compound of Formula (CO) is a compound of Formula (CO-N’):
  • the compound of Formula (CO) is a compound of Formula
  • the compound of Formula (CO) is a compound of Formula (CO-O’):
  • R 1 is selected from the group
  • R 1 is selected from the group consisting of
  • R 1 is -NR2. In certain embodiments, R 1 is . In certain embodiments,
  • R 'N ⁇ N ⁇ embodiments R 1 is R . In certain embodiments, R is R . In certain embodiments, R 1 is In certain embodiments, R
  • X 5 is an optionally substituted C1-C10 alkenylene. In certain embodiments, X 5 is an optionally substituted C1-C6 alkylene. In certain embodiments, X 5 is an optionally substituted C1-C6 alkenylene. In certain embodiments, X 5 is –(CH2)-. In certain embodiments, X 5 is –(CH2)2-. In certain embodiments, X 5 is –(CH 2 ) 3 -. In certain embodiments, X 5 is –(CH 2 ) 4 -. In certain embodiments, X 5 is – (CH 2 ) 5 -. In certain embodiments, X 5 is –(CH 2 ) 6 -.
  • R 8 is optionally substituted C 1 -C 14 straight chain alkenyl. In certain embodiments, R 8 is optionally substituted C6-C10 alkyl. In certain embodiments, R 8 is optionally substituted –(CH2)5CH3. In certain embodiments, R 8 is optionally substituted –(CH2)6CH3. In certain embodiments, R 8 is optionally substituted –(CH2)7CH3. In certain embodiments, R 8 is optionally substituted –(CH2)8CH3. In certain embodiments, R 8 is optionally substituted –(CH 2 ) 9 CH 3 .
  • one of the methylene linkages of R 8 is replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from: , , , , , , , and .
  • R 9 is optionally substituted C1-C14 aliphatic. In certain embodiments, R 9 is optionally substituted C1-C14 alkyl.
  • each R 8 and R 9 are each independently selected from an optionally substituted bridged bicyclic C 5 -C 12 cycloalkylenyl.
  • R 8 is an optionally substituted bridged multicyclic C 5 -C 12 cycloalkylenyl.
  • R 9 is an optionally substituted bridged bicyclic C5-C12 cycloalkylenyl.
  • Lipids of the Present Disclosure are selected from any lipid in
  • the compound of Formula (CC) is a compound of Formula (CC-A): (CC-A), or a pharmaceutically acceptable salt thereof, wherein R‘, R, X‘, X 4 , X 5 , Y‘, Y 2 , R 2 , R 4 , R 4 , R 4 , R 6 , R', R 8 , R 9 ,R 10 , R 1 1 , are as described in Formula (CC) or (CC*) or as otherwise described in any embodiments below.
  • the compound of Formula (CC) is a compound of Formula (CC-G):
  • the compound of Formula (CC) is a compound of Formula (CC-I): (CC-I), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 4 , X 5 , Y 1 , Y 2 , R 2 , R 3 , R 8 , R 9 , R 11 , are as described in Formula (CC) or (CC’) or as otherwise described in any embodiments below.
  • the compound of Formula (CC) is a compound of Formula (CC-J): (CC-J), or a pharmaceutically acceptable salt thereof, wherein R 1 , R, X 1 , X 4 , X 5 , R 2 , R 3 , R 8 , R 9 , R 11 , are as described in Formula (CC) or (CC’) or as otherwise described in any embodiments below.
  • the compound of Formula (CC) is a compound of Formula (CC-K):
  • the compound of Formula (CC) is a compound of Formula (CC-L): (CC-L), or a pharmaceutically acceptable salt thereof, wherein X 1 , X 4 , X 5 , R 2 , R 3 , R 8 , R 9 , R 11 , are as described in Formula (CC) or (CC’) or as otherwise described in any embodiments below.
  • the compound of Formula (CC) is a compound of Formula (CC-L): (CC-L), or a pharmaceutically acceptable salt thereof, wherein X 1 , X 4 , X 5 , R 2 , R 3 , R 8 , R 9 , R 11 , are as described in Formula (CC) or (CC’) or as otherwise described in any embodiments below.
  • the compound of Formula (CC) is a compound of Formula (CC-M):
  • Lipids of the Disclosure have a structure of Formula (CC’),
  • R 1 is selected from the group
  • R 1 is -OH. In certain embodiments, R 1 is -OAc. In certain embodiments, R 1 is -NRi. In certain embodiments, R 1 is In certain embodiments,
  • R 1 is R . In certain embodiments, R 1 is certain embodiments, R 1 is In certain embodiments, R 1 is
  • R 1 is R . In certain embodiments, R 1 is R
  • R 1 is -N(Et)2. In certain embodiments, R 1 is -N(Me)2. In certain embodiments, R 1 is -NH2. In certain embodiments, R 1 is -N(nPr)2. In certain embodiments, R 1 is -N(iPr) 2 . In certain embodiments, R 1 is -N(Me)(Et). In certain embodiments, R 1 is OH. In certain embodiments, R 1 is .
  • X 1 is optionally substituted C2-C6 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -NHC(O)- or -C(O)O-.
  • X 1 is optionally substituted C2-C6 aliphatic.
  • X 1 is optionally substituted C 2 -C 6 alkylene.
  • X 1 is optionally substituted C 2 alkylene.
  • X 1 is optionally substituted C 3 alkylene. In certain embodiments, X 1 is optionally substituted C 4 alkylene. In certain embodiments, X 1 is optionally substituted C 5 alkylene. In certain embodiments, X 1 is optionally substituted C 6 alkylene. In certain embodiments, X 1 is – (CH2)2-. In certain embodiments, X 1 is –(CH2)3-. In certain embodiments, X 1 is –(CH2)4-. In certain embodiments, X 1 is –(CH2)5-. In certain embodiments, X 1 is –(CH2)6-.
  • X 2 is selected from the group consisting of a bond, -CH 2 - and -CH 2 CH 2 -. In certain embodiments, X 2 is a bond. In certain embodiments, X 2 is -CH2-. In certain embodiments, X 2 is -CH2CH2-. X 2’ [00451] As disclosed in Formula (CC), in certain embodiments, X 2’ is selected from the group consisting of a bond, -CH 2 - and -CH 2 CH 2 -. In certain embodiments, X 2’ is a bond. In certain embodiments, X 2’ is -CH 2 -.
  • X 2’ is -CH 2 CH 2 -.
  • X 3 [00452] As disclosed in Formula (CC), in certain embodiments, X 3 is selected from the group consisting of a bond, -CH2- and -CH2CH2-. In certain embodiments, X 3 is a bond. In certain embodiments, X 3 is -CH2-. In certain embodiments, X 3 is -CH2CH2-. X 3’ [00453] As disclosed in Formula (CC), in certain embodiments, X 3’ is selected from the group consisting of a bond, -CH2- and -CH2CH2-. In certain embodiments, X 3’ is a bond. In certain embodiments, X 3’ is -CH2-.
  • X 4 is an optionally substituted C 1 -C 10 alkylene. In certain embodiments, X 4 is an optionally substituted C1-C10 alkenylene. In certain embodiments, X 4 is an optionally substituted C1-C6 alkylene. In certain embodiments, X 4 is an optionally substituted C1-C6 alkenylene. In certain embodiments, X 4 is –(CH2)-. In certain embodiments, X 4 is –(CH2)2-. In certain embodiments, X 4 is –(CH 2 ) 3 -. In certain embodiments, X 4 is –(CH 2 ) 4 -. In certain embodiments, X 4 is – (CH 2 ) 5 -.
  • X 4 is –(CH 2 ) 6 -.
  • X 5 is an optionally substituted C 1 -C 10 alkylene. In certain embodiments, X 5 is an optionally substituted C1-C10 alkenylene. In certain embodiments, X 5 is an optionally substituted C1-C6 alkylene. In certain embodiments, X 5 is an optionally substituted C1-C6 alkenylene. In certain embodiments, X 5 is –(CH2)-. In certain embodiments, X 5 is –(CH2)2-. In certain embodiments, X 5 is –(CH 2 ) 3 -. In certain embodiments, X 5 is –(CH 2 ) 4 -.
  • X 5 is – (CH 2 ) 5 -. In certain embodiments, X 5 is –(CH 2 ) 6 -. [00458] In certain embodiments, X 4 and X 5 are both –(CH 2 )-. In certain embodiments, X 4 and X 5 are both –(CH2)2-.
  • Y 1 and Y 2 [00459] As disclosed in Formula (CC), in certain embodiments, Y 1 and Y 2 are each independently , , , , , , or , wherein the bond marked with an "*" is attached to X 4 or X 5 . In certain embodiments, Y 1 and Y 2 are the same.
  • Y 1 and Y 2 are different. [00460] In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 1 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is . In certain embodiments, Y 2 is
  • Y 2 is . In certain embodiments, Y 1 and Y 2 are both . In certain embodiments, Y 1 and Y 2 are both . R 2 [00461] As disclosed in Formula (CC), in certain embodiments, R 2 is optionally substituted C 1 -C 6 aliphatic. In certain embodiments, R 2 is optionally substituted C 1 -C 6 alkylene. In certain embodiments, R 2 is optionally substituted methylene. In certain embodiments, R 2 is optionally substituted C 2 alkylene. In certain embodiments, R 2 is optionally substituted C 3 alkylene. In certain embodiments, R 2 is optionally substituted C4 alkylene. In certain embodiments, R 2 is optionally substituted C5 alkylene.
  • R 2 is optionally substituted C6 alkylene. In certain embodiments, R 2 is –(CH2)-. In certain embodiments, R 2 is –(CH2)2-. In certain embodiments, R 2 is – (CH 2 ) 3 -. In certain embodiments, R 2 is –(CH 2 ) 4 -. In certain embodiments, R 2 is –(CH 2 ) 5 -. In certain embodiments, R 2 is –(CH 2 ) 6 -.
  • R 3 [00462] As disclosed in Formula (CC), in certain embodiments, R 3 is optionally substituted C1-C6 aliphatic. In certain embodiments, R 3 is optionally substituted C1-C6 alkylene.
  • R 3 is optionally substituted methylene. In certain embodiments, R 3 is optionally substituted C 2 alkylene. In certain embodiments, R 3 is optionally substituted C 3 alkylene. In certain embodiments, R 3 is optionally substituted C 4 alkylene. In certain embodiments, R 3 is optionally substituted C 5 alkylene. In certain embodiments, R 3 is optionally substituted C 6 alkylene. In certain embodiments, R 3 is –(CH 2 )-. In certain embodiments, R 3 is –(CH 2 ) 2 -. In certain embodiments, R 3 is – (CH2)3-. In certain embodiments, R 3 is –(CH2)4-. In certain embodiments, R 3 is –(CH2)5-.
  • R 3 is –(CH2)6-. [00463] In certain embodiments, R 2 and R 3 are the same. In certain embodiments, R 2 and R 3 are different. In certain embodiments, R 2 and R 3 are both –(CH 2 ) 2 -.
  • R 4 is -CH(OR 6 )(OR 7 ); - CH(SR 6 )(SR 7 ); -CH(SR 8 )(SR 9 ); -CH(R 6 )(R 7 ); -R 10 ; or optionally substituted C1-C14 aliphatic-R 10 wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C 3 -C 8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)- , -NHC(O)- or -C(O)O-.
  • R 4 is optionally substituted C1-C14 aliphatic-R 10 , wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic
  • R 4 is optionally substituted C1-C14 aliphatic-R 10 .
  • R 4 is -CH(OR 6 )(OR 7 ).
  • R 4 is -CH(R 6 )(R 7 ).
  • R 4 is -CH(SR 6 )(SR 7 ).
  • R 4 is -CH(SR 8 )(SR 9 ).
  • R 4 is R 10 .
  • R 5 is -CH(OR 8 )(OR 9 ); - CH(SR 8 )(SR 9 ); -CH(R 8 )(R 9 ); optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted Cs-Cg cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -CIO)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; - R 11 ; or optionally substituted C1-C14 aliphatic-R 11 , wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-
  • R 5 is optionally substituted C1-C14 aliphatic. In certain embodiments, R 5 is -CII(OR 8 )(OR 9 ) . In certain embodiments, R 5 is -CH(R 8 )(R 9 ). In certain embodiments, R 5 is -CH(SR 8 )(SR 9 ). In certain embodiments, R 5 is R 11 .
  • R 4 and R 5 are the same. In certain embodiments, R 4 and R 5 are different.
  • R 5 is selected from
  • R 6 and R 7 are each independently -R 1U ; optionally substituted -Ci-Cu aliphatic-R 10 ; wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted Cs-Cg cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -CIO)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-.
  • R 6 and R 7 are the same. In certain embodiments, R 6 and R 7 are different. [00473] In certain embodiments, R 6 is R 10 . In certain embodiments, R 6 is optionally substituted C1-C14 aliphatic-R 10 . In certain embodiments, R 6 is optionally substituted C1-C14 alkyl-R 10 . In certain embodiments, R 6 is optionally substituted C1-C14 branched alkyl-R 10 . In certain embodiments, R 6 is optionally substituted C1-C14 straight chain alkyl-R 10 . In certain embodiments, R 6 is optionally substituted C1-C14 alkenyl-R 10 .
  • R 6 is optionally substituted Ci- C14 branched alkenyl-R 10 . In certain embodiments, R 6 is optionally substituted C1-C14 straight chain alkenyl-R 10 . In certain embodiments, R 6 is optionally substituted C1-C5 alkyl-R 10 . In certain embodiments, R 6 is optionally substituted -(CI bi-R 10 . In certain embodiments, R 6 is optionally substituted -(CFhh-R 10 .
  • R 6 is optionally substituted -(CH2)3-R 10 - In certain embodiments, R 6 is optionally substituted -(CH2)4-R 10 - In certain embodiments, R 6 is optionally substituted -(CF ⁇ s-R 10 -
  • R 7 is R 10 . In certain embodiments, R 7 is optionally substituted CI-CH aliphatic-R 10 . In certain embodiments, R 7 is optionally substituted CI-CH alkyl-R 10 . In certain embodiments, R 7 is optionally substituted C -Ci ; branched alkyl-R 10 . In certain embodiments, R 7 is optionally substituted C1-C14 straight chain alkyl-R 10 . In certain embodiments, R 7 is optionally substituted C1-C14 alkenyl-R 10 . In certain embodiments, R 7 is optionally substituted CI- CH branched alkenyl-R 10 .
  • R 7 is optionally substituted C1-C14 straight chain alkenyl-R 10 . In certain embodiments, R 7 is optionally substituted C1-C5 alkyl-R 10 . In certain embodiments, R 7 is optionally substituted -(CH2)-R 10 . In certain embodiments, R' is optionally substituted - (CH2)2-R 10 - In certain embodiments, R 7 is optionally substituted -(CH2)3-R 10 . In certain embodiments, R 7 is optionally substituted -(CH2)4-R 10 . In certain embodiments, R 7 is optionally substituted -(CH2)5-R 10 .
  • R 6 and R 7 are selected from and
  • R 8 and R 9 are each independently R 11 ; optionally substituted -C1-C14 aliphatic wherein one or more methylene linkages arc each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylcnyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; or optionally substituted -C1-C14 aliphatic-R 11 wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, - SS-, -C(O)-, -OC(O)O-, -OC
  • R 8 and R 9 are the same. In certain embodiments, R 8 and R 9 are different. [00478] In certain embodiments, R 8 is R 11 . In certain embodiments, R 8 is optionally substituted C1-C14 aliphatic. In certain embodiments, R 8 is optionally substituted C1-C14 alkyl. In certain embodiments, R 8 is optionally substituted C1-C14 branched alkyl. In certain embodiments, R 8 is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R 8 is optionally substituted C 1 -C 14 alkenyl. In certain embodiments, R 8 is optionally substituted C 1 -C 14 branched alkenyl.
  • R 8 is optionally substituted C 1 -C 14 straight chain alkenyl. In certain embodiments, R 8 is optionally substituted C 6 -C 10 alkyl. In certain embodiments, R 8 is optionally substituted –(CH 2 ) 5 CH 3 . In certain embodiments, R 8 is optionally substituted –(CH 2 ) 6 CH 3 . In certain embodiments, R 8 is optionally substituted –(CH2)7CH3. In certain embodiments, R 8 is optionally substituted –(CH2)8CH3. In certain embodiments, R 8 is optionally substituted –(CH2)9CH3. [00479] In certain embodiments, R 8 is optionally substituted C 1 -C 14 aliphatic-R 11 .
  • R 8 is optionally substituted C 1 -C 14 alkylene-R 11 . In certain embodiments, R 8 is optionally substituted C 1 -C 14 branched alkylene-R 11 . In certain embodiments, R 8 is optionally substituted C 1 -C 14 straight chain alkylene-R 11 . In certain embodiments, R 8 is optionally substituted C 1 - C14 alkenylene-R 11 . In certain embodiments, R 8 is optionally substituted C1-C14 branched alkenylene- R 11 . In certain embodiments, R 8 is optionally substituted C1-C14 straight chain alkenylene-R 11 . In certain embodiments, R 8 is optionally substituted C1-C5 alkylene-R 11 .
  • R 8 is optionally substituted –(CH 2 )-R 11 . In certain embodiments, R 8 is optionally substituted –(CH 2 ) 2 -R 11 . In certain embodiments, R 8 is optionally substituted –(CH 2 ) 3 -R 11 . In certain embodiments, R 8 is optionally substituted –(CH 2 ) 4 -R 11 . In certain embodiments, R 8 is optionally substituted –(CH 2 ) 5 -R 11 . [00480] In certain embodiments, R 9 is R 11 . In certain embodiments, R 9 is optionally substituted C1-C14 aliphatic. In certain embodiments, R 9 is optionally substituted C1-C14 alkyl.
  • R 9 is optionally substituted C1-C14 branched alkyl. In certain embodiments, R 9 is optionally substituted C 1 -C 14 straight chain alkyl. In certain embodiments, R 9 is optionally substituted C 1 -C 14 alkenyl. In certain embodiments, R 9 is optionally substituted C 1 -C 14 branched alkenyl. In certain embodiments, R 9 is optionally substituted C 1 -C 14 straight chain alkenyl. In certain embodiments, R 9 is optionally substituted C6-C10 alkyl. In certain embodiments, R 9 is optionally substituted –(CH2)5CH3. In certain embodiments, R 9 is optionally substituted –(CH2)6CH3.
  • R 9 is optionally substituted –(CH2)7CH3. In certain embodiments, R 9 is optionally substituted –(CH2)8CH3. In certain embodiments, R 9 is optionally substituted –(CH2)9CH3. [00481] In certain embodiments, R 9 is optionally substituted C 1 -C 14 aliphatic-R 11 . In certain embodiments, R 9 is optionally substituted C 1 -C 14 alkylene-R 11 . In certain embodiments, R 9 is optionally substituted C1-C14 branched alkylene-R 11 . In certain embodiments, R 9 is optionally substituted C1-C14 straight chain alkylene-R 11 .
  • R 9 is optionally substituted C1- C14 alkenylene-R 11 . In certain embodiments, R 9 is optionally substituted C1-C14 branched alkenylene- R 11 . In certain embodiments, R 9 is optionally substituted C 1 -C 14 straight chain alkenylene-R 11 . In certain embodiments, R 9 is optionally substituted C 1 -C 5 alkylene-R 11 . In certain embodiments, R 9 is optionally substituted –(CH 2 )-R 11 . In certain embodiments, R 9 is optionally substituted –(CH 2 ) 2 -R 11 . In certain embodiments, R 9 is optionally substituted –(CH2)3-R 11 .
  • R 9 is optionally substituted –(CH2)4-R 11 . In certain embodiments, R 9 is optionally substituted –(CH2)5-R 11 . [00482] In certain embodiments, R 8 and R 9 are selected from , , , , , and . In certain embodiments, R 8 and R 9 are selected from and . R 10 and R 11 [00483] As disclosed in Formula (CC), in certain embodiments, each R 10 and R 11 are an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, or two R 10 or two R 11 taken together form an optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl.
  • each R 10 and R 11 are the same. In certain embodiments, each R 10 and R 11 are different. [00485] In some embodiments, each R 10 and R 11 is independently an optionally substituted cyclic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cyclic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R 10 or two R 11 taken together form an optionally substituted bridged bicyclic or multicyclic C 4 -C 14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl.
  • each R 10 is an optionally substituted bridged bicyclic C 5 -C 12 cycloalkylenyl. In certain embodiments, each R 10 is an optionally substituted bridged multicyclic C 5 - C 12 cycloalkylenyl. In certain embodiments, each R 11 is an optionally substituted bridged bridged bicyclic C 5 - C12 cycloalkylenyl. In certain embodiments, each R 11 is an optionally substituted bridged multicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is selected from adamantyl, bicyclo[2.2.2]octyl, cubanyl, bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.1]heptyl, and bicyclo[3.2.1]octyl.
  • the optionally substituted bridged bicyclic or multicyclic C 5 -C 12 cycloalkylenyl is selected from:
  • the substituted bridged bicyclic or multicyclic are substituted.
  • two R 10 taken together form an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl.
  • two R 11 taken together form an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl.
  • the optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl is
  • Lipids of the Present Disclosure are selected from any lipid in
  • an LNP comprises a structural lipid.
  • an LNP comprises two or more structural lipids.
  • Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, olcanolic acid, bcta-sitostcrol-acctatc and mixtures thereof.
  • the structural lipid is cholesterol.
  • the structural lipid is a cholesterol analogue disclosed by Patel, et aL, Nat Common., 11, 983 (2020), which is incorporated herein by reference in its entirety.
  • the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combinations thereof.
  • a structural lipid is described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.
  • the structural lipid is cholesteryl hemisuccinate (CHEMS). In some embodiments, the structural lipid is 3-(4-((2-(4-morpholinyl)ethyl)amino)-4-oxobutanoate) (Mochol).
  • the targeting moiety targets a receptor selected from CD2, CD3, CD5 and CD7. In some embodiments, the targeting moiety targets a receptor selected from CD2, CD3, CD5, CD7, CD8, CD4, beta 7 integrin, beta 2 integrin, and Clq. In some embodiments, the targeting moiety targets CD117. In some embodiments, the targeting moiety targets CD90. In some embodiments, the targeting moiety targets a receptor selected from a mannose receptor, CD206 and Clq.
  • the targeting moiety is any one described or contemplated in US20230312713A1, US20230203538A1, US20230320995A1, US20160145348, and US20110038941, each of which is incorporated by reference herein in its entirety.
  • the PEG-lipid is selected from the group consisting of PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.
  • the non-ionizable lipid is a phospholipid selected from the group consisting of Egg Sphingomyelin (Egg SM I ESM I (2S,3R,E)-3-hydroxy-2- palmitamidooctadec-4-en-l-yl (2-(trimethylammonio)ethyl) phosphate). Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3-hydroxy-2-stearamidooctadec-4-en-l -yl (2- (trimethylammonio)ethyl) phosphate).
  • Egg SM I ESM I (2S,3R,E)-3-hydroxy-2- palmitamidooctadec-4-en-l-yl (2-(trimethylammonio)ethyl) phosphate Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3-hydroxy-2-stearamidooct
  • Milk or Bovine Sphingomyelin (Milk SM / (2S,3R,E)-3- hydroxy-2-tricosanamidooctadec-4-en-l-yl (2-(trimethylammonio)ethyl) phosphate), 28:0 SM (N- octacosanoyl-D-erythro-sphingosylphosphorylcholine), 14:0 SM (N-myristoyl-D-erythro- sphingosylphosphorylcholine), 16:1 SM (N-palmitoleoyl-D-erythro-sphingosylphosphorylcholine), 12:0 Dihydro SM (N-lauroyl-D-erythro-sphinganylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lys
  • the PEG lipid is PEG2k-DMG or PEG2k-DSPE or a mixture thereof;
  • the structural lipid is cholesterol; and
  • the non-ionizable lipid or zwitterionic lipid is a sphingolipid or DSPC or a mixture thereof.
  • the lipid component of the nanoparticle comprises: (a) about 0 mol% to about 10 mol% of PEG lipid; (b) about 0 mol% to about 30 mol% structural lipid; (c) about 20 mol% to about 45 mol% non-ionizablc lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of a Lipid of the Disclosure.
  • the lipid component of the nanoparticle composition comprises about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component of the nanoparticle composition comprises about 25 mol % to about 45 mol % ionizable lipid, about 35 mol % to about 50 mol % phospholipid, about 10 mol % to about 25 mol % structural lipid, and about 1 mol% to about 5 mol% of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component comprises about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid.
  • the lipid component comprises about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid.
  • the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG- DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPE. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPC.
  • the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 30 mol% of the total lipid component of the LNP.
  • the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 25 mol% of the total lipid component of the LNP.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % sphingomyelin, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPE, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DLPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPS, about 25 mol % cholesterol, and about 2 mol % of PEG lipid.
  • LNP comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % cholesterol. and about 2 mol % of PEG lipid.
  • LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE.
  • the LNP comprises about 43mol% ionizable lipid, about 15mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid.
  • the LNP comprises about 33mol% ionizable lipid, about 15mol% of a sphingolipid, about 25mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid.
  • the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin.
  • the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K- DSPE. In some embodiments, the LNP further comprises a targeting moiety. In some embodiments, the targeting moiety is an antibody or a fragment thereof.
  • the PEG lipid is PEG2K-DPPE
  • the structural lipid is cholesterol
  • the phospholipid is a mixture of DSPC and sphingomyelin.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DPPE.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DPPE.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K- DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DPPE.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DMG.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DMG.
  • a phospholipid such as DSPC
  • the PEG lipid is PEG2K-DSPE
  • the structural lipid is cholesterol
  • the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin.
  • the PEG lipid is PEG2K- DSPE
  • the structural lipid is cholesterol
  • the phospholipid is a mixture of DSPC and sphingomyelin.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K- DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE.
  • a phospholipid such as DSPC
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE.
  • the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DSPE.
  • the LNP further comprises an active agent.
  • the active agent is a nucleic acid.
  • the nucleic acid is a ribonucleic acid.
  • the ribonucleic acid is at least one ribonucleic acid selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (IncRNA).
  • siRNA small interfering RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • mRNA messenger RNA
  • IncRNA long non-coding RNA
  • the nucleic acid is a messenger RNA (mRNA) or a circular RNA.
  • the mRNA includes an open reading frame encoding a cancer antigen.
  • the mRNA includes an open reading frame encoding an immune checkpoint modulator.
  • the mRNA includes at least one motif selected from the group consisting of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadcnylation signal, and a 5' cap structure.
  • the nucleic acid is suitable for a genome editing technique.
  • the genome editing technique is clustered regularly interspaced short palindromic repeats (CRISPR) or transcription activator-like effector nuclease (TALEN).
  • the nucleic acid is at least one nucleic acid suitable for a genome editing technique selected from the group consisting of a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a single guide RNA (sgRNA), and a DNA repair template.
  • the mRNA is at least 30 nucleotides in length.
  • the mRNA is at least 300 nucleotides in length.
  • the nucleic acid encodes a therapeutic protein.
  • the therapeutic protein is a CAR or TCR complex protein.
  • the CAR or TCR complex protein comprises an antigen binding domain specific for an antigen selected from the group: CD 19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule- 1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disaloganglioside GD3, TNF receptor family member, B cell maturation antigen (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)), prostate- specific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (RORl), Fms-Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (EPCAM), B7
  • the instant specification describes compositions, methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and/or use of LNP-based RNA medicines (e.g., vaccines, gene therapies, or gene-editing therapeutics).
  • the LNP-based RNA medicines comprise an LNP delivery system (as described in detail herein) and an encapsulated cargo/payload (e.g., RNA in the case of RNA medicines).
  • the payload can be one or more RNA molecules, including coding RNA (e.g., linear or circular mRNA) or non-coding RNA (e.g., guide RNA, pegRNA, or retron ncRNA).
  • coding RNA e.g., linear or circular mRNA
  • non-coding RNA e.g., guide RNA, pegRNA, or retron ncRNA
  • the payloads can include any type of nucleic acid molecule, including coding RNA molecules (e.g., mRNA), guide RNAs for editing systems (e.g., Cas9 guides, Casl2a guides, base editor guides, and prime editor guides), other non-coding RNAs relating to editing systems (e.g., retron ncRNAs), small RNAs (sRNAs) — which refer to a wide variety of polymeric RNA molecules that are generally less than 200 nucleotides in length with various functionalities, such as RNA interference, and include small-interfering RNA (siRNA), microRNAs (miRNA), piwi-interacting RNA (piRNA), repeat associated small interfering RNA (rasiRNA), small nuclear RNA (snRNA or U-RNA), small nucleolar RNA (snoRNA), small rDNA- derived RNA (srRNA), rRNA fragment (tRF), and Y RNA-derived small RNA,
  • coding RNA molecules e.
  • the cargo nucleic acid molecules may be single-stranded or double-stranded.
  • Such nucleic acid cargo may comprise exactly one molecule.
  • Such nucleic acid cargo may comprise exactly two molecules.
  • Such nucleic acid cargo may comprise exactly three molecules.
  • Such nucleic acid cargo may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 distinct molecules.
  • Such nucleic acid cargo may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 distinct molecules.
  • Such nucleic acid cargo may comprise between 1-25, or 5-30, or 10-35, or 20-40, or up to 100, or more distinct molecules.
  • the LNPs described herein may be used to deliver any payload of interest to a biological target, e.g., to a cell or a bodily tissue.
  • payload refers to an active substance (i.e., not limited to RNA or DNA), such as a small molecule, polypeptide, peptide, carbohydrate, or nucleic acid molecule, and includes, without limitation, mRNA molecules (including linear and circular mRNA) or non-coding RNA molecules (e.g., guide RNAs, pegRNAs, retron ncRNAs) which are encapsulated within the LNPs described herein.
  • the LNP cargo may comprise an RNP or ribonucleoprotein, such as a gene editing nuclease protein complexed with a cognate guide RNA.
  • the payload is an RNA molecule, which may be linear or circular and may comprise one or more functional nucleotide sequences of interest, which may include, but are not limited to coding and non-coding nucleotide sequences.
  • the non-coding nucleotide sequences may comprise regulatory elements that influence RNA post- transcriptional processing, nuclear translation control sequences, and sequences which encode one or more biological products of interest, e.g., a therapeutic protein or antigen, among other sequence elements that may impact the functioning of the RNA or its encoded products.
  • the term “coding region of interest” or “product coding region” or the like may be used to refer to the encoded one or more biological products of interest. Equivalently, a product coding region may be referred to as a “product expression sequence.”
  • oiler constructs or “originator polynucleotide constructs” and “benchmark constructs” (or “benchmark polynucleotide constructs”), which are embodiments of payloads comprising nucleic acid molecules, i.e., embodiments of linear and/or circular mRNA payloads, and which may comprise a product coding region that encodes a polypeptide, such as, but not limited to an antigen or a therapeutic protein or to components of a gene editing system (e.g., a programmable nuclease).
  • a polypeptide such as, but not limited to an antigen or a therapeutic protein or to components of a gene editing system (e.g., a programmable nuclease).
  • FIG. 2 shows an example of an originator construct 100, which may be a linear or circular mRNA molecule.
  • the originator construct 100 may include at least one product coding region 10 which is or encodes a polypeptide of interest, such as, but not limited to a vaccine antigen or a therapeutic protein.
  • the originator construct 100 may contain 1 or 2 flanking regions 20.
  • the flanking regions 20 may be located 5' to the product coding region 10 and/or 3' to the product coding region 10. In some instances the originator construct 100 does not contain a flanking region 20.
  • the flanking region 20 of the originator construct 100 may include at least one regulatory region 30.
  • At least one flanking region 20 of the originator polynucleotide construct 100 may include at least one identifier region 40.
  • the identifier region 40 may be, but is not limited to, a barcode, label, signal and/or tag. Additionally, the identifier region 40 may be located within the product coding region 10 or may be located in the product coding region 10 and at least one flanking region 20.
  • the originator construct comprises from about 5 to about 10,000 nucleotides in length.
  • the length of the originator construct may be from 5 to 30, from 5 to 50, from 5 to 100, from 5 to 250, from 5 to 500, from 5 to 1,000, from 5 to 1,500, from 5 to 3,000 from 5 to 5,000, from 5 to 7,000, from 5 to 10,000 from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 1,000 to
  • the flanking region may range independently from 0 to 10,000 nucleotides in length such as, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 nucleotides in length.
  • the regulatory region may range independently from 0 to 3,000 nucleotides in length such as, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the identifier region may range independently from 1 to 3,000 nucleotides in length such as, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000.
  • the identifier region overlaps with the product coding region by 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 2- 7 nucleotides, 3-7 nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides, 7-10 nucleotides, 1-15 nucleotides, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10-15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12-20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nucleotides, 3-25 nucleotides, 5-25 nucleotides,
  • the benchmark polynucleotide construct comprises a product coding region and two identifier regions.
  • Each identifier region may independently be located 5' to the product coding region, 3’ to the product coding region, or the identifier region may overlap with the 5' end or the 3'end of the product coding region.
  • the first and second identifier regions are located 5' to the product coding region and the first identifier region is inverted.
  • the first and second identifier regions are located 5' to the product coding region and the second identifier region is inverted.
  • the first and second identifier region are both inverted and located 3' to the product coding region.
  • the first and second identifier regions are located 3' to the product coding region and the first identifier region is inverted.
  • the first and second identifier regions arc located 3' to the product coding region and the second identifier region is inverted.
  • the first identifier region is located 5' to the product coding region and overlaps with the product coding region and the second identifier region is located 3' to the product coding region.
  • the first identifier region is located 5' to the product coding region and the second identifier region is located 3' to the product coding region and overlaps with the product coding region.
  • the first and second identifier regions are located 5' to the product coding region and the second identifier region overlaps with the product coding region.
  • the first and second identifier regions are located 3' to the product coding region and the first identifier region overlaps with the product coding region.
  • the first identifier region is inverted, is located 5' to the product coding region and overlaps with the product coding region, and the second identifier region is located 3' to the product coding region.
  • the first identifier region is inverted and is located 5' to the product coding region and the second identifier region is located 3' to the product coding region and overlaps with the product coding region.
  • the first identifier region is inverted, is located 5' to the product coding region, the second identifier region is located 3' to the product coding region, and both of the first and second identifier regions overlap with the product coding region.
  • the first identifier region is inverted, is located 5' to the product coding region and overlaps with the product coding region, and the second identifier region is inverted and is located 3' to the product coding region.
  • the first identifier region is inverted and is located 5' to the product coding region and the second identifier region is inverted, is located 3' to the product coding region and overlaps with the product coding region.
  • the first identifier region is located 5' to the product coding region and overlaps with the product coding region, and the second identifier region is inverted and is located 3' to the product coding region.
  • the first identifier region is located 5' to the product coding region and the second identifier region is inverted, is located 3’ to the product coding region and overlaps with the product coding region.
  • the first identifier region is located 5' to the product coding region and the second identifier region is inverted and is located 3' to the product coding region, and both of the first and second identifier regions overlap with the product coding region.
  • the first and second identifier regions are both inverted and are located 5' to the product coding region, and the second identifier region overlaps with the product coding region.
  • the first and second identifier regions are located 5' to the product coding region and the first identifier region is inverted, and the second identifier region overlaps with the product coding region.
  • the first and second identifier regions are located 5' to the product coding region and the second identifier region is inverted and overlaps with the product coding region.
  • the first and second identifier region are both inverted and located 3' to the product coding region, and the first identifier region overlap with the product coding region.
  • the first and second identifier regions are located 3' to the product coding region and the first identifier region is inverted and overlaps with the product coding region.
  • the first and second identifier regions are located 3' to the product coding region and the second identifier region is inverted, and the first product coding region overlap with the product coding region.
  • At least one identifier moiety may be associated with the benchmark polynucleotide construct.
  • the benchmark polynucleotide construct may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more identifier moieties associated with the benchmark polynucleotide construct which may be the same moiety or different moieties associated with the benchmark polynucleotide construct.
  • Each identifier moiety may independently be located on the flanking region 5’ to the product coding region, on the flanking region 3' to the product coding region, or the location of the identifier moiety may span the 5' end or the 3'cnd of the product coding region and a flanking region.
  • the location of the identifier moiety may include one or more nucleotides of the product coding region such as, but not limited to, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleo
  • the location of the identifier moiety may include one or more nucleotides of the product coding region such as, but not limited to, 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 2-7 nucleotides, 3-7 nucleotides, 1-10 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 5-10 nucleotides, 7-10 nucleotides, 1-15 nucleotides, 2-15 nucleotides, 3-15 nucleotides, 5-15 nucleotides, 7-15 nucleotides, 10-15 nucleotides, 12-15 nucleotides, 1-20 nucleotides, 2-20 nucleotides, 3-20 nucleotides, 5-20 nucleotides, 7-20 nucleotides, 10-20 nucleotides, 12-20 nucleotides, 15-20 nucleotides, 17-20 nucleotides, 1-25 nucleotides, 2-25 nucleo
  • one identifier moiety may be associated with the benchmark polynucleotide construct.
  • the identifier moiety may be associated with the benchmark polynucleotide construct on the 5' end of the benchmark polynucleotide construct.
  • the identifier moiety may be associated with the benchmark polynucleotide construct on the 5' flanking region.
  • the identifier moiety may be associated with the benchmark polynucleotide construct on the 3' flanking region.
  • the identifier moiety may be associated with the benchmark polynucleotide construct on the 3' end of the benchmark polynucleotide construct.
  • the identifier moiety may be associated with the benchmark polynucleotide construct on the product coding region.
  • the benchmark polynucleotide construct comprises an identifier moiety and the location of the identifier moiety spans the 5' end of the product coding region and the 5' flanking region.
  • the benchmark polynucleotide construct comprises an identifier moiety and the location of the identifier moiety spans the 3' end of the product coding region and the 3' flanking region.
  • two identifier moieties are associated with the benchmark polynucleotide construct.
  • the first identifier moiety and the second identifier moiety are located on the 5' flanking region.
  • the first identifier moiety and the second identifier moiety are located on the product coding region.
  • the first identifier moiety and the second identifier moiety are located on the 3' flanking region.
  • the first identifier moiety and the second identifier moiety are located on the 5' end of the benchmark polynucleotide construct.
  • the first identifier moiety and the second identifier moiety arc located on the 3' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 5' flanking region.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the product coding region.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 3' flanking region.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 5' flanking region and the product coding region.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 3' flanking region and the product coding region.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the product coding region.
  • the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the 3' flanking region.
  • the first identifier moiety is located on the 5' flanking region and the location of the second identifier moiety spans the 5' flanking region and the product coding region.
  • the first identifier moiety is located on the 5' flanking region and the location of the second identifier moiety spans the 3' flanking region and the product coding region.
  • the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the 5' flanking region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 5' flanking region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the product coding region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the location of the second identifier moiety spans the 3' flanking region and the product coding region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' flanking region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the product coding region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the product coding region and the second identifier moiety is located on the 5' flanking region.
  • the first identifier moiety is located on the product coding region and the location of the second identifier moiety spans the 5' flanking region and the product coding region.
  • the first identifier moiety is located on the product coding region and the location of the second identifier moiety spans the 3' flanking region and the product coding region.
  • the first identifier moiety is located on the product coding region and the second identifier moiety is located on the 3' flanking region.
  • the first identifier moiety is located on the product coding region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.
  • the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct.
  • the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 5' flanking region.
  • the location of the first identifier moiety spans the 3' flanking region and the product coding region and the location of the second identifier moiety spans the 5' flanking region and the product coding region.
  • the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the product coding region.
  • the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 3' flanking region.
  • the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 3'end of the benchmark polynucleotide construct.
  • the location of the first identifier moiety spans the 3' flanking region and the product coding region and the second identifier moiety is located on the 5' flanking region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the product coding region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the location of the second identifier moiety spans the 3' flanking region and the product coding region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' flanking region.
  • the location of the first identifier moiety spans the 5' flanking region and the product coding region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the 5' flanking region.
  • the first identifier moiety is located on the 3' flanking region and the location of the second identifier moiety spans the 5' flanking region and the product coding region.
  • the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the product coding region.
  • the first identifier moiety is located on the 3' flanking region and the location of the second identifier moiety spans the 3' flanking region and the product coding region.
  • the first identifier moiety is located on the 3' flanking region and the second identifier moiety is located on the 3' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 5' end of the benchmark polynucleotide construct.
  • the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 5' flanking region.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 5' flanking region and the product coding region.
  • the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the product coding region.
  • the first identifier moiety is located on the 5' end of the benchmark polynucleotide construct and the location of the second identifier moiety spans the 3' flanking region and the product coding region.
  • the first identifier moiety is located on the 3' end of the benchmark polynucleotide construct and the second identifier moiety is located on the 3' flanking region.
  • three identifier moieties are associated with the benchmark polynucleotide construct.
  • four identifier moieties are associated with the benchmark polynucleotide construct.
  • five identifier moieties are associated with the benchmark polynucleotide construct.
  • six identifier moieties are associated with the benchmark polynucleotide construct.
  • seven identifier moictics arc associated with the benchmark polynucleotide construct.
  • eight identifier moieties are associated with the benchmark polynucleotide construct.
  • nine identifier moieties are associated with the benchmark polynucleotide construct.
  • ten identifier moieties are associated with the benchmark polynucleotide construct.
  • the product coding region encodes a biologically active molecule such as, but not limited to a therapeutic protein or an antigen.
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • the CROI encodes one or more prophylactically- or therapeutically-active proteins, polypeptides, or other factors.
  • the CROI may encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer.
  • the CROI may encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., CROI encodes Dystrophin), cardiovascular disease (e.g., CROI encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., CROI encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., CROI encodes GlyRal), an enkephalin, or a glutamate decarboxylase (e.g., CROI encodes GAD65, GAD67, or another isoform), lung disease (e.g., CROI encodes CFTR), hemophilia (e.g., CROI encodes Factor VIII or Factor IX), neoplasia (e.g., CROI encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB
  • Neuregulin (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplxl), Tphl Tryptophan hydroxylase, Tph2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, DTNBPI, Dao (Daol)), trinucleotide repeat disorders (e.g., HTT (Huntington’s Dx), SBMA/SMAXI/AR (Kennedy's Dx), FXN/X25 (Friedrich’s Ataxia), ATX3 (Machado-Joseph's Dx), ATXNI and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and AtnKDRPLA Dx), CBP (Creb-BP
  • the product coding region of the RNA payloads described herein encodes a factor that can affect the differentiation of a cell.
  • a factor that can affect the differentiation of a cell.
  • the expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, mir-302/367, or other miRNAs can cause the cell to become an induced pluripotent stem (iPS) cell.
  • iPS induced pluripotent stem
  • the product coding region of the RNA payloads described herein encodes a factor for transdifferentiating cells.
  • factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurrl, LmxlA, Bm2, Mytll, NeuroDl, FoxA2 for neural cells; and Hnf4a, Foxal, Foxa2 or Foxa3 for hepatic cells.
  • the LNP compositions described herein can be used to deliver a nucleic acid or polynucleotide payload, e.g., a DNA HDR donor, a linear or circular mRNA, or a chimeric DNA/RNA guide.
  • a nucleic acid or polynucleotide payload e.g., a DNA HDR donor, a linear or circular mRNA, or a chimeric DNA/RNA guide.
  • a LNP is capable of delivering a polynucleotide to a target cell, tissue, or organ.
  • a polynucleotide in its broadest sense of the term, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger mRNA
  • RNAi-inducing agents RNAi agents
  • siRNAs siRNAs
  • shRNAs shRNAs
  • miRNAs miRNAs
  • antisense RNAs antisense RNAs
  • ribozymes catalytic DNA
  • RNAs that induce triple helix formation aptamers, vectors, etc.
  • RNAs useful in the compositions and methods described herein can be selected from the group consisting of but are not limited to, shortimers, antagomirs, antisense, ribozymes, short interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.
  • a polynucleotide is mRNA.
  • a polynucleotide is circular RNA.
  • a polynucleotide encodes a protein, e.g., a vaccine antigen, a therapeutic protein, or a nucleobase editing enzyme.
  • a polynucleotide may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide.
  • a polypeptide may be of any size and may have any secondary structure or activity.
  • a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.
  • a polynucleotide may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the first region (e.g., a 5 -UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3'-stabilizing region.
  • a polypeptide of interest e.g., a coding region
  • a first flanking region located at the 5'-terminus of the first region
  • a second flanking region located at the 3'-terminus of the first region
  • at least one 5'-cap region e.g., a 3'-UTR
  • the nucleic acid payloads may contain one or more modifications.
  • modifications include various chemical and/or structural modifications.
  • the RNA may comprise one or more modifications, including chemical modifications (e.g., ribonucleotide analogs, alternative phosphate chain linkers), sequence modification (e.g., relative to a wild type sequence), and/or structural modification (e.g., secondary- folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes, and tertiary structural elements, such as, but not limited to, helical duplexes and triple-stranded structures).
  • chemical modifications e.g., ribonucleotide analogs, alternative phosphate chain linkers
  • sequence modification e.g., relative to a wild type sequence
  • structural modification e.g., secondary- folded structures, such as, but not limited to, stem-loops, hairpins, and G-quadruplexes
  • tertiary structural elements such
  • RNA modifications including N 5 -methyladenosine (m e 'A), N 6 ,2'-O-dimethyiadenosine (m°Am), 8-oxo-7,8- dihydroguanosine (8-oxoG), pseudouridine (T), 5-methylcytidine (m 3 C), and N 4 -acetylcytidine (ac 4 C), have been shown to regulate mRNA stability, consequently affecting diverse cellular and biological processes. Any known modification to RNA or DNA is contemplated herein.
  • a polynucleotide contains only naturally occurring nucleosides. Nucleic acid modifications are well known in the art and are further discussed in the following references: (1) Crookc ST, Witztum JL, Bennett CF, Baker BF. RNA-Targeted Therapeutics. Cell Metab. 2018 Apr 3;27(4):714-739. doi: 10.1016/j.cmet.2018.03.004. Erratum in: Cell Metab. 2019 Feb 5;29(2):501. PMID: 29617640; (2) JP, Wen W, Zhang F, Oberg KC, Zhang L, Cheng T, Zhang XB.
  • a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the poly nucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides.
  • the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides.
  • the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides.
  • the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.
  • a polynucleotide molecule, formula, composition or method associated therewith comprises one or more polynucleotides comprising features as described in W02002/098443, W02003/051401, W02008/052770, W02009/127230, WO2006/122828, W02008/083949, W02010/088927, W02010/037539, W02004/004743, W02005/016376, W02006/024518, W02007/095,976, W02008/014979, W02008/077592, W02009/030481, W02009/095226, WO2011/069586, WO2011/026641, WO2011/144358, W02012/019780, WO2012/013326, WO2012/089338, WO2012/113513, WO2012/116811, WO2012/116810, WO2013/113502, WO2013/113501, WO2013
  • a polynucleotide comprises one or more microRNA binding sites.
  • a microRNA binding site is recognized by a microRNA in a non-target organ.
  • a microRNA binding site is recognized by a microRNA in the liver.
  • a microRNA binding site is recognized by a microRNA in hepatic cells.
  • the LNP-based RNA vaccines, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver an RNA payload that is a linear mRNA molecule.
  • the LNP-based pharmaceutical compositions described herein may include one or more linear mRNA molecules or linear mRNA payloads.
  • the mRNA payloads may encode one or more components of the herein described gene editing systems.
  • an mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., TALENS and zinc finger- binding domains) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Casl2a, CRISPR Casl2f, CRISPR Casl3a, CRISPR Casl3b, or TnpB).
  • mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and/or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.
  • deaminases e.g., cytidine deaminases or adenosine deaminases
  • polymerases e.g., reverse transcriptases
  • integrases e.g., recombinases, etc.
  • fusion proteins comprising one or more functional domains linked together.
  • RNA Ribonucleic acid
  • the nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C).
  • A adenine
  • G guanine
  • U uracil
  • C cytosine
  • RNA mostly exists in the single- stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA.
  • RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., IncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., IncRNA) sequence.
  • the LNP-based RNA vaccines, RNA therapeutics, gene editing systems and pharmaceutical compositions thereof described herein can be used to deliver a mRNA payload that is a linear mRNA molecule.
  • the mRNA payload may comprise one or more nucleotide sequences that encode a product of interest, such as, but not limited to a vaccine antigen, a component of a gene editing system (e.g., an endonuclease, a prime editor, etc.) and/or a therapeutic protein.
  • the RNA payload may be a linear mRNA.
  • mRNA messenger RNA
  • mRNA refers to any polynucleotide which encodes a protein of interest and which is capable of being translated to produce the encoded protein of interest in vitro, in vivo, in situ or ex vivo.
  • a mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail.
  • UTR 5' untranslated region
  • 3' UTR 3' UTR
  • 5' cap 5' cap
  • poly-A tail one or more structural and/or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced.
  • a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a nucleic acid without significant chemical modification to the nucleotides themselves.
  • a coding region of interest in an mRNA used herein may encode a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.
  • the mRNA may encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids.
  • the length of the region of the mRNA encoding a product of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).
  • the mRNA has a total length that spans from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 500 to 3,000, from 500 to 5,000
  • the region or regions flanking the region encoding the product of interest may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
  • the mRNA comprises a tailing sequence which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides).
  • the tailing region is a polyA tail
  • the length may be determined in units of or as a function of polyA Binding Protein binding.
  • the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein.
  • PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.
  • the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap.
  • the capping sequence may be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length.
  • the caping sequence is absent.
  • the mRNA comprises a region comprising a start codon.
  • the region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA comprises a region comprising a stop codon.
  • the region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA comprises a region comprising a restriction sequence.
  • the region comprising the restriction sequence may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.
  • the mRNA payloads of the LNP-bascd RNA vaccines, RNA therapeutics, nucleobase editing systems and pharmaceutical compositions thereof described herein may comprise at least one untranslated region (UTR) which flanks the region encoding the product of interest and/or is incorporated within the mRNA molecule. UTRs are transcribed by not translated.
  • the mRNA payloads can include 5’ UTR sequences and 3’ UTR sequences, as well as internal UTRs.
  • the RNA payloads of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region.
  • the nucleic acid may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation.
  • RNA payload molecules e.g., linear and circular mRNA molecules
  • the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • 5'UTR and 3'UTR sequences are known and available in the art.
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein may comprise at least one UTR that may be selected from any UTR sequence listed in Tables 19 or 20 of U.S. Patent No. 10,709,779, which is incorporated herein by reference.
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein may comprise at least one 5' UTR.
  • the 5’ UTR comprises a sequence provided in Table (II) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a 5’ UTR sequence provided in Table (II), or a variant or a fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, or six nucleotides of the 5’ UTR sequence provided in Table (II)).
  • the 5’ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, 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, or SEQ ID NO: 28.
  • 5'UTR also have been known to form secondary structures which are involved in elongation factor binding. 5' UTR sequences are also known to be important for ribosome recruitment to the mRNA and have been reported to play a role in translation (Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6). In addition, 5’ UTR sequences may confer increased half-life, increased expression and/or increased activity of a polypeptide encoded by the RNA payload described herein.
  • the RNA payload constructs contemplated herein may include 5'UTRs that are found in nature and those that are not.
  • the 5’UTRs can be synthetic and/or can be altered in sequence with respect to a naturally occurring 5'UTR.
  • Such altered 5'UTRs can include one or more modifications relative to a naturally occurring 5'UTR, such as, for example, an insertion, deletion, or an altered sequence, or the substitution of one or more nucleotide analogs in place of a naturally occurring nucleotide.
  • the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3 'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, the UTRs may have a regulatory role in terms of translation and stability of the nucleic acid.
  • a 5' UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different mRNA.
  • a 5' UTR is a synthetic UTR, i.e., does not occur in nature.
  • Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic.
  • Exemplary 5' UTRs include Xenopus or human derived alpha-globin or beta-globin (e.g., US8,278,063 and US9,012,219), human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus.
  • CMV immediate-early 1 (IE1) gene (see US20140206753 and WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 29) (WO2014144196) may also be used.
  • 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g.,
  • an internal ribosome entry site IRS is used as a substitute for a 5' UTR.
  • a 5' UTR of the present disclosure comprises SEQ ID NO: 30 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC).
  • AU rich elements can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class 11 AREs possess two or more overlapping UUAU(JUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
  • 3' UTRs are known to have stretches of adenosines and uridines embedded in them.
  • AU rich elements can be separated into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM- CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
  • AREs 3' UTR AU rich elements
  • one or more copies of an ARE can be introduced to make mRNA less stable and thereby curtail translation and decrease production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
  • the introduction of features often expressed in genes of target organs the stability and protein production of the mRNA can be enhanced in a specific organ and/or tissue.
  • the feature can be a UTR.
  • the feature can be introns or portions of introns sequences.
  • 5' UTRs that are heterologous or synthetic may be used with any desired 3' UTR sequence.
  • a heterologous 5' UTR may be used with a synthetic 3' UTR with a heterologous 3' UTR.
  • Non-UTR sequences may also be used as regions or subregions within an RNA payload construct.
  • introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.
  • any UTR from any gene may be incorporated into the regions of an RNA payload molecule (e.g., a linear mRNA).
  • multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3' UTR or 5' UTR may be altered relative to a wild- type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.
  • patterned UTRs are those UTRs which reflect a repeating or alternating pattern, such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.
  • flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property.
  • polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
  • the untranslated region may also include translation enhancer elements (TEE).
  • TEE translation enhancer elements
  • the TEE may include those described in US Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art.
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein may comprise a 5’ cap structure.
  • Additional modified guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and/or 5'-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'- hydroxyl group of the sugar ring.
  • Multiple distinct 5 '-cap structures can be used to generate the 5 '- cap of a nucleic acid molecule, such as an mRNA molecule.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3’-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5 '-guanosine (m 7 G-3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G).
  • the 3’-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA).
  • the N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA).
  • cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
  • mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures.
  • more authentic refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
  • Non-limiting examples of more authentic 5 'cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5’ endonucleases and/or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0- methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-mcthyL
  • Capl structure Such a structure is termed the Capl structure.
  • Cap structures include, but are not limited to, 7mG(5*)ppp(5*)N,pN2p (cap 0), 7mG(5*)ppp(5*)NlmpNp (cap 1), and 7mG(5*)-ppp(5')NlmpN2mp (cap 2).
  • a 5' terminal cap may comprise a guanine analog.
  • Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7- deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. IRES Sequences
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein may comprise one or more IRES sequences.
  • the mRNA may contain an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • An IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure.
  • An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
  • An mRNA that contains more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes.
  • IRES sequences that can be used include without limitation, those from picornaviruses (e.g.
  • FMDV pest viruses
  • CFFV pest viruses
  • PV polio viruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot-and-mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV murine leukemia virus
  • SIV simian immune deficiency viruses
  • CrPV cricket paralysis viruses
  • Human enterovirus 71 Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human cIF4G, Mouse NDST4L, Human LEF1, Mouse IIIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein may comprise a poly-A tail.
  • a long chain of adenine nucleotides may be added to a polynucleotide such as an mRNA molecules in order to increase stability.
  • a polynucleotide such as an mRNA molecules
  • the 3' end of the transcript may be cleaved to free a 3' hydroxyl.
  • poly-A polymerase adds a chain of adenine nucleotides to the free 3' hydroxyl end.
  • the process called polyadenylation, adds a poly-A tail of a certain length.
  • the length of a poly-A tail is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides) and no more than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 nucleotides in length.
  • the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300,
  • the mRNA includes a poly-A tail from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1 ,000, from 30 to 1 ,500, from 30 to 2,000, from 30 to
  • the poly-A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the region coding for a target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the ultimate product expressed from the mRNA.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the mRNA or feature thereof.
  • the poly-A tail may also be designed as a fraction of mRNA to which it belongs.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail.
  • engineered binding sites and conjugation of mRNA for poly-A binding protein may enhance expression.
  • multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3'-end using modified nucleotides at the 3 '-terminus of the poly-A tail.
  • Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.
  • the mRNA are designed to include a polyA-G quartet.
  • the G- quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail.
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein may comprise one or more translation stop codons.
  • Translational stop codons UAA, UAG, and UGA, are an important component of the genetic code and signal the termination of translation of an mRNA.
  • stop codons interact with protein release factors and this interaction can modulate ribosomal activity thus having an impact translation (Tate WP, et al., (2016) Biochem Soc Trans, 46(6):1615-162).
  • a stop element as used herein refers to a nucleic acid sequence comprising a stop codon.
  • the stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA.
  • a stop element comprises two consecutive stop codons.
  • a stop clement comprises three consecutive stop codons.
  • a stop element comprises four consecutive stop codons.
  • a stop element comprises five consecutive stop codons.
  • the mRNA may include one stop codon.
  • the mRNA may include two stop codons.
  • the mRNA may include three stop codons.
  • the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As non-limiting examples, the stop codon may be selected from TGA, TAA and TAG.
  • the stop codon may be selected from one or more of the following stop elements of Table (III):
  • the mRNA includes the stop codon TGA and one additional stop codon.
  • the addition stop codon may be TAA.
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein may comprise one or more regulatory elements, including, but not limited to microRNA (miRNA) binding sites, structured mRNA sequences and/or motifs, artificial binding sites to bind to endogenous nucleic acid binding molecules, and combinations thereof.
  • miRNA microRNA
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein are not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine.
  • nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U).
  • nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).
  • the mRNA payloads of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions thereof described herein comprise, in some embodiments, comprises at least one chemical modification.
  • the terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5'- terminal mRNA cap moieties.
  • modification refers to a modification relative to the canonical set 20 amino acids. Polypeptides, as provided herein, are also considered “modified” of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.
  • Polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides comprise various (more than one) different modifications.
  • a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications.
  • a modified RNA polynucleotide e.g., a modified mRNA polynucleotide
  • introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide.
  • a modified RNA polynucleotide e.g., a modified mRNA polynucleotide
  • introduced into a cell or organism may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).
  • Polynucleotides include, without limitation, those described herein.
  • Polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides may comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications.
  • Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).
  • Polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties.
  • the modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.
  • Modified nucleotide base pairing encompasses not only the standard adenosine- thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into polynucleotides of the present disclosure.
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modified nucleobases in polynucleotides are selected from the group consisting of pseudouridine ( ⁇
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (c.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise pseudouridine (vp) and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 1-methyl- pseudouridine (m'y).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 2-thiouridine (s 2 U).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • 2-thiouridine and 5-methyl-cytidine m 3 C.
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • methoxy-uridine mimethoxy-uridine
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 5-methoxy-uridine (mo 5 U) and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 2'-O-mcthyl uridine.
  • polynucleotides c.g., RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides comprise N6-methyl-adenosine (m 6 A).
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • N6-methyl-adenosine m 6 A
  • 5-methyl-cytidine mC
  • polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with 5-methyl-cytidine (m'C). meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m’C).
  • m'C 5-methyl-cytidine
  • m 5-methyl-cytidine
  • m 5-methyl-cytidine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • nucleobases and nucleosides having a modified cytosine include N4- acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5- hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5- methyl-cytidine.
  • a modified nucleobase is a modified uridine.
  • Exemplary nucleobases and In some embodiments, a modified nucleobase is a modified cytosine
  • nucleosides having a modified uridine include 5-cyano uridine, and 4'-thio uridine.
  • the polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • one or more or all or a given type of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotides X in a polynucleotide of the present disclosure are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • the polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 20% to 95%, from 20% to 100%, from
  • the polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
  • the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine.
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil).
  • the modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine).
  • the modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (c.g., 2, 3, 4 or more unique structures).
  • the LNP-based RNA vaccines, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver an RNA payload that is a circular mRNA molecule or “oRNA.”
  • the circular mRNA molecule may encode a CROI, such as a vaccine antigen, cancer antigen, or therapeutic protein as described in this specification.
  • the LNP-based pharmaceutical compositions described herein may include one or more circular mRNA molecules or “oRNAs.”
  • the circular mRNA payloads may encode one or more components of the herein described gene editing systems or other therapeutic protein of interest.
  • a circular mRNA payload may encode an amino acid sequence-programmable DNA binding domain (e.g., TALENS and zinc finger-binding domains) or a nucleic acid sequence-programmable DNA binding domain (e.g., CRISPR Cas9, CRISPR Casl2a, CRISPR Casl2f, CRISPR Casl3a, CRISPR Casl3b, or TnpB).
  • amino acid sequence-programmable DNA binding domain e.g., TALENS and zinc finger-binding domains
  • a nucleic acid sequence-programmable DNA binding domain e.g., CRISPR Cas9, CRISPR Casl2a, CRISPR Casl2f, CRISPR Casl3a, CRISPR Casl3b, or TnpB.
  • the circular mRNA payloads may also encode, depending upon the nature of the gene editing system, one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and/or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together.
  • deaminases e.g., cytidine deaminases or adenosine deaminases
  • polymerases e.g., reverse transcriptases
  • integrases e.g., recombinases, etc.
  • fusion proteins comprising one or more functional domains linked together.
  • the RNA payload is a circular RNA (oRNA).
  • oRNA circular RNA
  • the terms “oRNA” or “circular RNA” are used interchangeably and can refer to a RNA that forms a circular structure through covalent or non-covalent bonds.
  • Circular RNA described herein are polyribonucleotides that form a continuous structure through covalent or non-covalent bonds. Due to the circular structure, oRNAs have improved stability, increased half-life, reduced immunogenicity, and/or improved functionality (e.g., of a function described herein) compared to a corresponding linear RNA.
  • an oRNA binds a target. In some embodiments, an oRNA binds a substrate. In some embodiments, an oRNA binds a target and binds a substrate of the target. In some embodiments, an oRNA binds a target and mediates modulation of a substrate of the target. In some embodiments, an oRNA brings together a target and its substrate to mediate modification of the substrate, e.g., post-translational modification. In some embodiments, an oRNA brings together a target and its substrate to mediate a cellular process (c.g., alters protein degradation or signal transduction) involving the substrate.
  • a cellular process c.g., alters protein degradation or signal transduction
  • a target is a target protein and a substrate is a substrate protein.
  • an oRNA comprises a conjugation moiety for binding to chemical compound.
  • the conjugation moiety can be a modified polyribonucleotide.
  • the chemical compound can be conjugated to the oRNA by the conjugation moiety.
  • the chemical compound binds to a target and mediates modulation of a substrate of the target.
  • an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring together the target and its substrate to mediate modification of the substrate, e.g., post-translational modification.
  • an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring together the target and its substrate to mediate modification of the substrate to mediate a cellular process (e.g., alters protein degradation or signal transduction) involving the substrate.
  • a target is a target protein and a substrate is a substrate protein.
  • the oRNA may be non-immunogenic in a mammal (e.g., a human, non-human primate, rabbit, rat, and mouse).
  • a mammal e.g., a human, non-human primate, rabbit, rat, and mouse.
  • the oRNA may be capable of replicating or replicates in a cell from an aquaculture animal (e.g., fish, crabs, shrimp, oysters etc.), a mammalian cell, a cell from a pet or zoo animal (e.g., cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (e.g., horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof.
  • an aquaculture animal e.g., fish, crabs, shrimp, oysters etc.
  • a mammalian cell e.g., a cell from a
  • a pharmaceutical composition comprising: a circular RNA comprising, in the following order, a 3’ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (e.g., a vaccine antigen, therapeutic protein, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein or a nucleobase editing system or component thereof), and a 5’ group I intron fragment, and a transfer vehicle comprising at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular RNA polynucleotide to a cell (e.g., a human cell, such as an immune cell present in a human subject), such that the polypeptide is translated in the cell.
  • a transfer vehicle comprising at least one of (i) an ionizable lipid, (ii)
  • the pharmaceutical composition is formulated for intravenous administration to the human subject in need thereof.
  • the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments.
  • the 3’ intron fragment and 5’ intron fragment arc defined by the L9a-5 permutation site in the intact intron. In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron.
  • the IRES is from Taura syndrome virus, Tiiatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus , Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black
  • the IRES comprises a CVB3 IRES or a fragment or variant thereof.
  • the pharmaceutical composition comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment.
  • the first and second internal spacers each have a length of about 10 to about 60 nucleotides.
  • the circular mRNA comprises a nucleotide sequence encoding a polypeptide of interest, such as a vaccine antigen, nucleobase editing system, or therapeutic protein (e.g., a CAR or TCR complex protein).
  • a polypeptide of interest such as a vaccine antigen, nucleobase editing system, or therapeutic protein (e.g., a CAR or TCR complex protein).
  • the CAR or TCR complex protein comprises an antigen binding domain specific for an antigen selected from the group: CD 19, CD 123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule- 1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disaloganglioside GD3, TNF receptor family member, B cell maturation antigen (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)), prostate- specific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms- Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonic
  • the CAR or TCR complex protein comprises a CAR comprising an antigen binding domain specific for CD 19.
  • the CAR or TCR complex protein comprises a CAR comprising a costimulatory domain selected from the group CD28, 4-1BB, 0X40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations thereof.
  • the CAR or TCR complex protein comprises a CAR comprising a CD3zeta signaling domain.
  • the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and/or CD8 spacer domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain. [00701] In some embodiments, the CAR or TCR complex protein comprises a CAR comprising: an antigen binding domain, a spacer domain, a transmembrane domain, a costimulatory domain, and an intracellular T cell signaling domain.
  • the CAR or TCR complex protein comprises a multispecific CAR comprising antigen binding domains for at least two different antigens.
  • the CAR or TCR complex protein comprises a TCR complex protein selected from the group TCRalpha, TCRbeta, TCRgamma, and TCRdelta.
  • the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing system, and pharmaceutical compositions described herein further comprise a targeting moiety.
  • the targeting moiety mediates receptor-mediated endocytosis or direct fusion of the delivery vehicle (LNPs) into selected cells of a selected cell population or tissue in the absence of cell isolation or purification.
  • the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, CDS, CD7, PD-1, 4-1BB, CD28, Clq, and CD2.
  • the targeting moiety comprises an antibody specific for a macrophage, dendritic cell, NK cell, NKT, or T cell antigen.
  • the targeting moiety comprises a scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof.
  • the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing system, and pharmaceutical compositions described herein are administered in an amount effective to treat a disease in the human subject (e.g., wherein the disease can be cancer, muscle disorder, or CNS disorder, etc.).
  • the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing system, and pharmaceutical compositions have an enhanced safety profile when compared to a pharmaceutical composition comprising T cells or vectors comprising exogenous DNA encoding the same polypeptide, e.g., a CAR complex protein.
  • the LNP-based RNA vaccines and pharmaceutical compositions thereof are administered in an amount effective to mount an immunogenic response in a human subject for the vaccination against an infectious agent and/or cancer.
  • the LNP-based RNA vaccines and pharmaceutical compositions have an enhanced safety profile when compared to state of the art vaccine compositions.
  • the LNP-based nucleobase editing systems and pharmaceutical compositions thereof are administered in an amount effective to induce a desire precise edit in a genome.
  • the LNP-based nucleobase editing systems and pharmaceutical compositions have an enhanced safety profile when compared to state of the art gene editing delivery compositions.
  • the present disclosure provides a circular RNA comprising, in the following order, a 3' group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (c.g., a vaccine antigen, nuclcobasc editing system or component thereof, therapeutic protein, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein), and a 5’ group I intron fragment.
  • a polypeptide c.g., a vaccine antigen, nuclcobasc editing system or component thereof, therapeutic protein, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments.
  • the 3' intron fragment and 5’ intron fragment are defined by the L9a-5 permutation site in the intact intron.
  • the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron.
  • the IRES comprises a CVB3 IRES or a fragment or variant thereof.
  • the circular RNA comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment.
  • the first and second internal spacers each have a length of about 10 to about 60 nucleotides.
  • the circular RNA payload of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions described herein consists of natural nucleotides.
  • the circular RNA further comprises a second expression sequence encoding a therapeutic protein.
  • the therapeutic protein comprises a checkpoint inhibitor.
  • the therapeutic protein comprises a cytokine.
  • the circular RNA payload of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing system, and pharmaceutical compositions described herein consists of natural nucleotides.
  • the circular RNA payload LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions described herein comprises a nucleotide sequence that is codon optimized, either partially or fully.
  • the circular RNA is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide.
  • the circular RNA is optimized to lack at least one endonuclease susceptible site present in an equivalent pre-optimized polynucleotide.
  • the circular RNA is optimized to lack at least one RNA-editing susceptible site present in an equivalent pre-optimized polynucleotide.
  • the circular RNA payload of the LNP-based RNA vaccines, RNA therapeutics, nuclcobasc editing system, and pharmaceutical compositions described herein has an in vivo functional half- life in humans greater than that of an equivalent linear RNA having the same expression sequence.
  • the circular RNA has a length of about 100 nucleotides to about 10 kilobases.
  • the circular RNA has a functional half-life of at least about 20 hours.
  • the circular RNA has a duration of therapeutic effect in a human cell of at least about 20 hours.
  • the circular RNA has a duration of therapeutic effect in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence. In some embodiments, the circular RNA has a functional half-life in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence.
  • the oRNA has a half-life or persistence in a cell for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.
  • the oRNA has a half-life or persistence in a cell for no more than about 10 mins to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.
  • the circular RNA payload of the LNP-based RNA vaccines, nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein has a half-life or persistence in a cell while the cell is dividing.
  • the oRNA has a half-life or persistence in a cell post division.
  • the circular RNA payload of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions described herein has a half-life or persistence in a dividing cell for greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.
  • the circular RNA payload of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions described herein modulates a cellular function, e.g., transiently or long term.
  • the cellular function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer.
  • the cellular function is transiently altered, e.g., such as a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours(3 days), 4 days, 5 days, 6 days, or 7 days.
  • a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours
  • the circular RNA payload of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions described herein is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides,
  • the maximum size of the circular RNA payload of the LNP- based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions described herein may be limited by the ability of packaging and delivering the RNA to a target.
  • the size of the oRNA is a length sufficient to encode polypeptides, and thus, lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be useful.
  • the circular RNA payload of the LNP-based RNA vaccines, RNA therapeutics, nucleobase editing systems, and pharmaceutical compositions described herein comprises one or more elements described elsewhere herein.
  • the elements may be separated from one another by a spacer sequence or linker.
  • the elements may be separated from one another by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides.
  • one or more elements are contiguous with one another, e.g., lacking a spacer element.
  • one or more elements is conformationally flexible.
  • the conformational flexibility is due to the sequence being substantially free of a secondary structure.

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