WO2023218420A1 - Mrna compositions for inducing latent hiv-1 reversal - Google Patents

Mrna compositions for inducing latent hiv-1 reversal Download PDF

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Publication number
WO2023218420A1
WO2023218420A1 PCT/IB2023/054932 IB2023054932W WO2023218420A1 WO 2023218420 A1 WO2023218420 A1 WO 2023218420A1 IB 2023054932 W IB2023054932 W IB 2023054932W WO 2023218420 A1 WO2023218420 A1 WO 2023218420A1
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seq
hiv
polynucleotide sequence
utr
lipid
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PCT/IB2023/054932
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French (fr)
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Daniel BODEN
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Janssen Pharmaceuticals, Inc.
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Publication of WO2023218420A1 publication Critical patent/WO2023218420A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present disclosure relates generally to the field of molecular biology and genetic engineering, including nucleic acid molecules useful for regulating gene expression, and the use of the nucleic acid molecules for, for example, production of desired products in suitable host cells in cell culture or in a subject, and for conferring beneficial characteristics to the host cells or subjects.
  • HAART Highly active antiretroviral therapy
  • HAART can suppress HIV-1 levels in plasma to below the limit of detection of clinical assays ( ⁇ 50 copies/ml) and reduce the morbidity and mortality of HIV - 1 infection.
  • HAART alone fails to cure HIV infection.
  • HAART leaves latent integrated proviruses unaffected.
  • Latent viral genomes reside in a small pool of infected resting memory CD4+ T-cells that constitute a stable viral reservoir. In these cells, the provirus remains transcriptionally silent as long as the host cells are in a quiescent state. This allows the virus to evade host immune surveillance and rebound quickly following discontinuation of HAART.
  • the remarkable stability of the latent viral reservoir necessitates lifelong HAART. Elimination of the latent reservoir leading to the cure of HIV-1 infected individuals of the latent reservoir has been proposed as a goal worthy of a major scientific effort.
  • HIV transcription is driven by its native 5’-LTR (long terminal repeat) promoter whereby transcriptional activity is auto-induced by the HIV-1 tat protein (transactivator of transcription) leading to a powerful positive feedback loop (Kam 1999).
  • LTR long terminal repeat
  • Tat levels of Tat expression can regulate the fate of HIV-infected cells by favoring latency establishment or latency reversal (Jordan 2001).
  • Tat is a small basic protein with length varying between 86 to 101 amino acids.
  • the invention satisfies this need by providing reliable, safe and convenient compounds able to lead to reactivation of latent HIV that avoid overall T-cell activation.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a ribonucleic acid (RNA) molecule encapsulated in a pharmaceutically acceptable encapsulating carrier, wherein the ribonucleic acid molecule comprises a non-naturally occurring polynucleotide sequence comprising a polynucleotide sequence encoding a wildtype HIV-derived accessory protein tat for the reactivation of latent HIV.
  • the pharmaceutically acceptable encapsulating carrier is a lipid nanoparticle.
  • the lipid nanoparticle comprises a cationic lipid, such as one having the Formula I, II, III, IV, V, VI, VII or VIII described herein.
  • the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat encodes at least the first 57 N-terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV.
  • the nucleotide sequence encoding a wild-type HIV -derived accessory protein tat encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3.
  • the encoded wildtype HIV-derived accessory protein tat is a protein consisting of the amino acid sequence SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3, or a variant thereof.
  • the variant contains one or more amino acid substitutions as shown in Table 1.
  • the ribonucleic acid molecule comprises, a 5 ’-UTR, a nucleotide sequence encoding a wildtype HIV-derived accessory protein tat for the reactivation of latent HIV, a 3’-UTR and a poly A tail.
  • the ribonucleic acid molecule consists essentially of, a 5’- UTR, a nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, a 3’-UTR and a poly A tail, more particularly arranged in the order from the 5’- end to the 3 ’-end of the ribonucleic acid molecule.
  • the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises at least one sequence from table 2.
  • the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises a 5’-UTR, the T66 cds, and a 3’-UTR from table 2.
  • the 5’-UTR has a sequence of SEQ ID No. 4.
  • the 3’-UTR has a sequence of SEQ ID No. 5.
  • the poly A tail comprises 100 to 140 adenosine monophosphates. In some embodiments, the poly A tail comprises 120 adenosine monophosphates.
  • the 5’-UTR has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID No: 4 or SEQ ID NO: 6. In some embodiments, the 3’-UTR has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID No: 5 or SEQ ID NO: 7.
  • the encoded wild-type HIV-derived accessory protein is a deletion mutant selected from the group consisting of tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl- 64, tatl-60, tatl-57, and tat2-72.
  • the RNA molecule is an mRNA molecule.
  • the present disclosure relates to an RNA molecule comprising a 5’-UTR, a nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for reactivation of latent HIV, a 3’-UTR, and a poly A tail as described herein.
  • the RNA molecule is an mRNA molecule.
  • the application is related to an isolated ribonucleic acid molecule comprising: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
  • the application relates to a DNA molecule encoding a ribonucleic acid molecule of the application.
  • the DNA molecule comprises a promoter sequence, such as a T7 promoter sequence, more particularly, a T7 promoter comprises the sequence of TAATACGACTCACTATAG (SEQ ID NO: 14) or TAATACGACTCACTATAAG (SEQ ID NO: 15).
  • Additional embodiments include a method of reactivating latent HIV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to an embodiment of the application.
  • the method further comprises administering to the subject an effective amount of one or more anti-viral agent.
  • the method further comprises administering to the subject an effective amount of one or more latency reversing agents.
  • the latency reversing agent is selected from Phytohemagglutinin (PHA), Phorbol 12-Myristate 13-Acetate (PMA), and anti-CD3 + anti-CD28 antibodies.
  • the subject is a human.
  • the subject is a human infected with the HIV and/or is under antiretroviral therapy treatment for at least 6 months, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months prior to the administration of a pharmaceutical composition according to an embodiment of the application.
  • a small amount of HIV or only latent HIV is detected in the plasma of the human prior to the administration of the pharmaceutical composition.
  • the human has an HIV viral load of about 100 copies/ml or less (e.g., about 90, 80, 70, 60, 50, 40, 30, 20, 10 dml or less)or only latent HIV detected in plasma prior to the initial administration of the pharmaceutical composition.
  • Additional embodiments include a method of increasing the induction of latent HIV-1 reversal in a cell, comprising contacting the cell with a pharmaceutical composition according to an embodiment of the application, wherein the increase is an increase in induction of latent HIV-1 reversal over contacting the cell with an equivalent wild-type HIV- derived accessory protein tat for the reactivation of latent HIV.
  • the application relates to an isolated host cell comprising a ribonucleic acid molecule of the application.
  • the application further relates to an isolated host cell comprising a nucleic acid encoding the ribonucleic acid molecule of the application.
  • the application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a ribonucleic acid molecule of the application encapsulated in a pharmaceutically acceptable carrier, such as a lipid nanoparticle.
  • a pharmaceutically acceptable carrier such as a lipid nanoparticle.
  • the ribonucleic acid molecule is an mRNA molecule.
  • the application relates to a pharmaceutical composition of the application for use in reactivation of latent HIV in a subject in need thereof, preferably the subject has HIV-1 infection, optionally in combination with another therapeutic agent, preferably an anti-HIV agent, a latency reversing agent and/or another antiretroviral therapy.
  • another therapeutic agent preferably an anti-HIV agent, a latency reversing agent and/or another antiretroviral therapy.
  • FIG. 1 shows an embodiment of latency reversal activity of tat deletion mutants.
  • FIG. 2A shows an embodiment of a dose-dependent latency reversal activity of T66 protein in three different cell lines.
  • FIG. 2B shows latency reversal activity of T66 protein in CD4+ T-cells obtained from 10 subjects.
  • FIG. 3A and 3B show an embodiment of increased cell-associated HIV-1 RNA copies from resting CD4+ T-cells treated with T66 protein. The cells were isolated from ART-treated individuals and the RNA copied were measured by RT-PCR.
  • FIG. 3C and 3D show an embodiment of the level of p24 secretion on CD4+ T cells isolated from ART- treated individuals due to reactivation of HIV-1.
  • FIG. 3E shows an embodiment of T66 protein-induced latency reversal activity on CD4+ T cells isolated from ART-treated indivisibles measured by the quantitative viral outgrowth assay.
  • FIG. 4A shows no up-regulation of CD69 and CD25 in CD4+ T-cells.
  • FIG. 4A shows no up-regulation of CD69 and CD25 in CD4+ T-cells.
  • FIG. 4B shows no significant impact of T66 treatment on the host transcriptome in the HIV -infected CD4+ T-cells isolated from ART-treated individuals.
  • FIG. 4C shows HIV reactivation of Tat- 66 protein in FIG. 4A and 4B.
  • Fig. 4D shows a Volcano plots which allows to identify 8 genes which are differently expressed in T66-treated CD4+ T-cells in comparison to PBS- treated cells.
  • FIG. 5 shows an embodiment of an in vitro transcription template comprising a Tat-66 coding sequence.
  • FIG. 6 shows an embodiment of agarose gel analysis, wherein lane 1 : 1 kb DNA ladder; lane 2: T66 in vitro transcription; lane 3: purified T66 mRNA.
  • FIG. 7 shows evaluation of an embodiment of T66 mRNA HIV-1 reactivation (reversal) on a latent HIV reporter T cell line using a variety of transfection reagents.
  • FIG. 8 A shows an embodiment of latency reversal activity comparison in MT4 reporter cells between T66 mRNA and its protein counterpart as determined by the increase of GFP (+) cells through HIV LTR activation.
  • FIG. 8B shows an embodiment of the same comparison between T66 mRNA and its protein counterpart as determined by HIV LTR driven increased luciferase expression.
  • FIG. 9 shows an embodiment of T66 mRNA LTR activation in Jurkat HIV-GFP reporter cells along with activators such as Phorbol 12-Myristate 13-Acetate (PMA), CD3/CD28 and T66 protein.
  • PMA Phorbol 12-Myristate 13-Acetate
  • CD3/CD28 CD3/CD28
  • FIG. 10 shows an embodiment of a T66 mRNA dose response HIV activation in latent T-cell clone Jurkat cl50.
  • any numerical value such as a % sequence identity or a % sequence identity range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a dosage of 10 mg includes 9 mg to 11 mg.
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
  • sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acid or nucleic acid. Suitable programs for aligning protein sequences are known to the skilled person.
  • the percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g., using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).
  • the terms and phrases “in combination,” “in combination with,” “co-delivery,” and “administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration of two or more therapies or components, such as two nucleic acid molecules, e.g., ribonucleic acid molecules and an adjuvant. “Simultaneous administration” can be administration of the two components at least within the same day.
  • a first therapy or component e.g., a first therapy or component
  • first ribonucleic acid molecule can be administered prior to (e.g., 5 minutes to one hour before), concomitantly with or simultaneously with, or subsequent to (e.g., 5 minutes to one hour after) the administration of a second therapy or component (e.g., second ribonucleic acid molecule).
  • a first therapy or component e.g. first ribonucleic acid molecule
  • a second therapy or component e.g., e.g., second ribonucleic acid molecule
  • a first therapy or component e.g. first ribonucleic acid molecule
  • a second therapy or component e.g., second ribonucleic acid molecule
  • a first therapy or component e.g. first ribonucleic acid molecule
  • a second therapy or component e.g., second ribonucleic acid molecule
  • a “non-naturally occurring” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature.
  • a “non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting.
  • a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment.
  • a “non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source.
  • a “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
  • operably linked refers to a linkage or a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing the transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest over a membrane.
  • subject means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the application.
  • mammal encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human.
  • a human subject can include a patient.
  • ribonucleic acid molecules of the application described herein may contain particular components, including, but not limited to, 5’ untranslated region (5’ UTR), 3’ UTR, coding sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, polyadenylation signal sequences, etc. arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in ribonucleic acid molecules of the application.
  • the application contemplates use of any of the applicable components in any combination having any sequence that can be used in ribonucleic acid molecules of the application, whether or not a particular combination is expressly described.
  • HIV-1 means the human immunodeficiency virus type-1. HIV-1 includes but is not limited to extracellular virus particles and the forms of HIV- 1 found in HIV- 1 infected cells.
  • HIV-1 infection means the introduction of HIV- 1 genetic information into a target cell, such as by fusion of the target cell membrane with HIV- 1 or an HIV-1 envelope glycoprotein + cell.
  • the target cell may be a bodily cell of a subject.
  • the target cell is a bodily cell from a human subject.
  • the application provides a ribonucleic acid molecule (e.g., a messenger RNA (mRNA) molecule or circular RNA (circRNA) molecule) comprising a non- naturally occurring polynucleotide sequence encoding a mutant of the HIV-derived accessory protein tat (trans-activator of transcription) for the reactivation and subsequent eradication of latent HIV.
  • the ribonucleic acid molecule can, for example, be a non-self replicating ribonucleic acid molecule (i.e., a non-self replicating RNA), more particularly a non-self replicating mRNA or circRNA.
  • a ribonucleic acid molecule can comprise any non-naturally occurring polynucleotide sequence encoding the mutant of the HIV-derived accessory protein tat of the application, which can be made using methods known in the art in view of the present disclosure.
  • the ribonucleic acid molecule comprises a non-naturally occurring polynucleotide sequence comprising a polynucleotide sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV but more preferably said polynucleotide sequence encoding a protein comprising at least the first 57 N- terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV.
  • the polynucleotide sequence encoding a protein comprising a protein having at least the first 60 N-terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV.
  • tatl-57 which contains the 57 N-terminal amino acids, is represented by the following SEQ ID No. 1: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQR RR.
  • tatl-60 which contains the first 60 N- terminal amino acids, is represented by the following SEQ ID No; 2: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQR RRAHQ.
  • the polynucleotide sequence encoding a protein comprising the 66 amino acid deletion mutant [T66 or tatl-66] according to an embodiment of the invention with a reactivation capacity close to full length exon 1 Tat72 has the amino acid sequence of SEQ ID No. 3: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQR RRAHQNSQTHQ
  • the polynucleotide sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity to one or more of the following sequences within the RNA molecule: Table 2
  • the ribonucleic acid molecule further comprises a 5 ’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR), and/or a polyadenylation sequence.
  • a “5’ untranslated region” or “5’ UTR” refers to a region of an mRNA or circRNA that is directly upstream (i.e., 5’) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
  • 3’ untranslated region refers to a region of an mRNA or circRNA that is directly downstream (i.e., 3’) from the stop codon (i.e., the codon of an mRNA or circRNA transcript that signals a termination of translation) that does not encode a polypeptide.
  • an open reading frame is a continuous stretch of codons beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG, or TGA) that encodes a polypeptide.
  • a start codon e.g., methionine (ATG)
  • a stop codon e.g., TAA, TAG, or TGA
  • polyA tail or “polyadenylation sequence” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, conservative adenosine monophosphates.
  • a poly A tail can contain 10 to 300 adenosine monophosphates, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates.
  • the polyA tail can, e.g., function to protect mRNA from enzymatic degradation, e.g., in the cytoplasm of a cell, and can, e.g., aid in transcription termination, export of the mRNA from the nucleus, and translation.
  • the application also relates to a DNA molecule encoding an RNA molecule of the application, preferably the DNA molecule contains a promoter operationally linked to a nucleic acid sequence encoding the RNA molecule.
  • promoters include, but are not limited to, a T3 promoter, a T7 promoter, and an SP6 promoter.
  • a promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein.
  • a promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic.
  • a promoter is a T7 promoter.
  • a nucleotide sequence of an exemplary T7 promoter comprises the sequence TAATACGACTCACTATAG or TAATACGACTCACTATAAG.
  • an enhancer sequence can comprise an untranslated region.
  • the polynucleotides encoding the wild-type HIV-derived accessory protein tat for the reactivation of latent HIV of the application can be made by any method known in the art in view of the present disclosure.
  • a polynucleotide encoding a wild-type HIV- derived accessory protein tat for the reactivation of latent HIV can be introduced or “cloned” into an expression vector using standard molecular biology techniques, e.g., polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.
  • PCR polymerase chain reaction
  • the nucleic acid molecule is transcribed in vivo.
  • the nucleic acid molecule is transcribed in vitro.
  • the mRNA or circRNA is formed by transcribing from a DNA template, wherein the following elements are introduced in a pDNA vector: a T7 promoter, followed by a nucleotide sequence corresponding to a 5’-UTR and a 3’-UTR from either the human a-globin [HAG] or the frog (Xenopus) a-globin [XBG], terminated by a 120 nt polyA tail. [Fig5] .
  • a capping reagent such as AG CleanCap (Trilink) is applied and the wild type T7 promoter sequence is changed in the DNA template, e g., from TAATACGACTCACTATAGG to TAATACGACTCACTATAAG.
  • the sequences are selected from those in Table 3. In other embodiments, the sequences have 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 point mutations from the sequences in Table 2.
  • a polynucleotide sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV (also referred to in some embodiments as HIV-1 T66 codon-optimized gene sequence) is introduced via restriction enzymes-based cloning into the DNA template plasmid between the coding sequence of the 5 -UTR and 3’-UTR, as shown in FIG.5.
  • the application relates to an isolated ribonucleic acid molecule comprising: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
  • the wild-type HIV-derived accessory protein tat comprises at least the first 57 N-terminal amino acids of a wild-type tat (86-101 aa) for the reactivation of latent HIV.
  • the wild-type HIV- derived accessory protein tat is tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl-64, tatl-60, tatl-57, or tat2-72.
  • the wild-type HIV-derived accessory protein tat comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3.
  • the wild-type HIV-derived accessory protein tat comprises one or more substitutions in Table 1 in the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3.
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail containing 120 adenosine monophosphates.
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail containing 120 adenosine monophosphates.
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail containing 120 adenosine monophosphates.
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail
  • the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail containing 120 adenosine monophosphates.
  • the application relates to a DNA molecule encoding the isolated ribonucleic acid molecule.
  • an RNA molecule of the application is transcribed from a DNA molecule comprising a DNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 11, which is transcribed into a 5’UTR; a DNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 13, which is transcribed into a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV; and a DNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ
  • the ribonucleic acid molecule contains one or more modified nucleobase.
  • Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5 -methylcytosine (5- me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6- methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and
  • compositions and pharmaceutical compositions comprising one or more polynucleotide encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, polynucleotides, and/or vectors encoding one more polynucleotide encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV according to the application.
  • polynucleotide encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV
  • polynucleotides including RNA, e.g., mRNA or circRNA
  • RNA e.g., mRNA or circRNA
  • compositions comprising any nucleic acid molecule and/or vector described herein, together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient.
  • Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings.
  • suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like.
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • the aqueous solution/ suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
  • compositions of the application can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections.
  • the parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection.
  • Pharmaceutical compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
  • compositions of the application are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
  • pharmaceutical compositions for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS).
  • a pharmaceutically acceptable carrier e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the compositions can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents.
  • a pharmaceutical composition of the application comprising a ribonucleic acid molecule can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier.
  • PBS phosphate buffered saline
  • the ribonucleic acid molecule can be administered at 1-1000 pg/dose, e.g., 1 pg/dose, 10 pg/dose, 20 pg/dose, 30 pg/dose, 40 pg/dose, 50 pg/dose, 60 pg/dose, 70 pg/dose, 80 pg/dose, 90 pg/dose, 100 pg/dose, 200 pg/dose, 300 pg/dose, 400 pg/dose, 500 pg/dose, 600 pg/dose, 700 pg/dose, 800 pg/dose, 900 pg/dose, 1000 pg/dose, or any number in between.
  • an adjuvant is included in a pharmaceutical composition of the application or co-administered with a pharmaceutical composition of the application.
  • Use of an adjuvant is optional and can further enhance immune responses when the composition is used for vaccination purposes.
  • Adjuvants suitable for co-administration or inclusion in compositions in accordance with the application should preferably be ones that are potentially safe, well tolerated and effective in humans.
  • An adjuvant can be an anti-viral agent such as a small molecule and/or antibody directed towards HIV, but not limited to Nucleoside/Nucleotide Reverse Transcriptase Inhibitors, such as Abacavir, or Didanosine, Emtricitabine, Lamivudine, Stavudine, Tenofovir alafenamide, Tenofovir disoproxil fumarate, Zidovudine; Non-nucleoside Reverse Transcriptase Inhibitors, such as, Cabotegravir/rilpivirine, Delavirdine, Doravirine, Efavirenz, Etravirine, Nevirapine, Rilpivirine; Protease Inhibitors, such as, Atazanavir, Darunavir, Fosamprenavir, Indinavir, Lopinavir + ritonavir, Nelfmavir, Ritonavir, Saquinavir, Tipranavir; Integrase Inhibitors, such
  • an active compound of the application is administered with an epigenetic drug, such as a histone deacetylase (HDAC) inhibitor (e.g. Vorinostat, Romidepsin, Panobinostat, Belinostat) or a BET inhibitor (e.g. JQ1).
  • HDAC histone deacetylase
  • BET BET inhibitor
  • the application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an active compound of the application (e.g., a polynucleotide encoding a wildtype HIV-derived accessory protein tat for the reactivation of latent HIV, e.g., mRNA or circRNA), and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is an encapsulating carrier, such as a nanoparticle encapsulating an active compound of the application.
  • an active compound of the application e.g., a polynucleotide encoding a wildtype HIV-derived accessory protein tat for the reactivation of latent HIV, e.g., mRNA or circRNA
  • the pharmaceutically acceptable carrier is an encapsulating carrier, such as a nanoparticle encapsulating an active compound of the application.
  • encapsulating nanoparticles include lipid nanoparticles (LNPs), liposomes, lipoplexes, etc.
  • the active compound of the application is formulated using one or more liposomes, lipoplexes, polymer-based encapsulating formulation, and/or lipid nanoparticles.
  • liposome or lipid nanoparticle formulations described herein can comprise a polycationic composition.
  • the formulations comprising a polycationic composition can be used for the delivery of the active compound of the application described herein in vivo and/or ex vitro.
  • the term “lipid” refers to any fatty acid derivative or other amphiphilic compound which is capable of forming a lyotropic lipid phase, or more preferentially, a lamellar lyotropic phase.
  • lipid refers to any fatty acid derivative which is capable of forming a bilayer such that a hydrophobic part of the lipid molecule orients toward the bilayer while a hydrophilic part orients toward the aqueous phase.
  • the term “lipid” comprises neutral, anionic or cationic lipids and combinations thereof. Lipids preferably comprise a hydrophobic domain with at least one, preferably two, or more, alkyl chains or a cholesterol moiety and a polar head group.
  • the alkyl chains of the fatty acids in the hydrophobic domain of the lipid are not limited to a specific length or number of double bonds. Nevertheless, it is preferred that the fatty acid has a length of 10 to 30, preferably 14 to 25 carbon atoms.
  • the lipid may also comprise two different fatty acids.
  • a lipid-based delivery vehicle typically serves to transport a desired RNA to a target cell or tissue.
  • the lipid- based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and an RNA of the present disclosure.
  • the lipid bilayer preferably further comprises a neutral lipid or a polymer.
  • neutral lipid means a lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.
  • the lipid formulation preferably comprises a liquid medium.
  • the formulation preferably further encapsulates a nucleic acid.
  • the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or a polymer.
  • the lipid formulation preferably encapsulates the nucleic acid.
  • lipid formulations comprising one or more RNA encapsulated within the lipid formulation.
  • the lipid formulation comprises liposomes.
  • the lipid formulation comprises cationic liposomes.
  • the lipid formulation comprises lipid nanoparticles.
  • the RNA or combination of nucleic acid molecules is fully encapsulated within the lipid portion of the lipid formulation such that the RNA or combination of nucleic acid molecules in the lipid formulation is resistant in aqueous solution to nuclease degradation.
  • the term “fully encapsulated” means that the nucleic acid (e.g., mRNA or circRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free RNA.
  • nucleic acid in the particle When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10%, and most preferably less than 5% of the nucleic acid in the particle is degraded.
  • “Fully encapsulated” as used herein also means that the nucleic acid-lipid particles do not rapidly decompose into their component parts upon in vivo administration.
  • the lipid formulations described herein are substantially non-toxic to mammals such as humans.
  • the combination of nucleic acids is encapsulated within the same lipid nanoparticle.
  • each nucleic acid molecule in the combination of nucleic acid molecules is independently encapsulated in individual lipid nanoparticles.
  • the lipid formulations of the disclosure also typically have a total lipid: RNA ratio (mass/mass ratio) of from about 1 : 1 to about 100: 1, from about 1 : 1 to about 50: 1, from about 2: 1 to about 45: 1, from about 3: 1 to about 40: 1, from about 5: 1 to about 38: 1, or from about 6: 1 to about 40: 1, or from about 7: 1 to about 35: 1, or from about 8: 1 to about 30: 1; or from about 10: 1 to about 25: 1; or from about 8: 1 to about 12: 1; or from about 13: 1 to about 17: 1; or from about 18 : 1 to about 24 : 1 ; or from about 20 : 1 to about 30 : 1.
  • RNA ratio mass/mass ratio
  • the total lipid:RNA ratio is from about 10: 1 to about 25: 1.
  • the ratio may be any value or sub value within the recited ranges, including endpoints.
  • the lipid formulations of the present disclosure typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 n
  • the diameter may be any value or subvalue within the recited ranges, including endpoints.
  • nucleic acids when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.
  • the lipid formulations comprise an RNA or combination of nucleic acid molecules, a cationic lipid (e.g. , one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates).
  • the lipid formulations can also include cholesterol.
  • lipid conjugate means a conjugated lipid that inhibits aggregation of lipid particles.
  • Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, and mixtures thereof.
  • PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties.
  • non-ester-containing linker moieties such as amides or carbamates, are used.
  • the PEG-lipid conjugate is 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (i.e., ALC-0159).
  • cationic lipid refers to amphiphilic lipids and salts thereof having a positive, hydrophilic head group; one, two, three, or more hydrophobic (e.g., having apolar groups) fatty acid or fatty alkyl chains; and a connector between these two domains.
  • An ionizable or protonatable cationic lipid is typically protonated (i.e., positively charged) at a pH below its pKa and is substantially neutral at a pH above the pKa.
  • Preferred ionizable cationic lipids are those having a pKa that is less than physiological pH, which is typically about 7.4.
  • the cationic lipids of the disclosure may also be termed titratable cationic lipids.
  • the cationic lipids can be an “amino lipid” having a protonatable tertiary amine (e.g., pH-titratable) head group.
  • Some amino exemplary amino lipid can include Cis alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains.
  • Such cationic lipids include, but are not limited to, (4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2 -hexyldecanoate (also known as ALC-0315), LipofectinTM also known as DOTMA (N-D-(2,3-dioleyloxy) propyls N,N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB (dimethyldioctadecyl-ammonium bromide), DOGS (dioctadecylamidologlycyl spermine), DSDMA, DODMA, DLinDMA, DLenDMA, y- DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C
  • the cationic lipid is ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate), i.e., ALC-0315.
  • anionic lipid refers to a lipid that is negatively charged at physiological pH.
  • these lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • phosphatidylglycerols include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succin
  • the RNA or combination of nucleic acid molecules may be fully encapsulated within the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation.
  • a lipid formulation comprising an RNA or combination of nucleic acid molecules is fully encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation.
  • the RNA or combination of nucleic acid molecules in the lipid formulation is not substantially degraded after exposure of the particle to a nuclease at 37 °C for at least 20, 30, 45, or 60 minutes.
  • the RNA or combination of nucleic acid molecules in the lipid formulation is not substantially degraded after incubation of the formulation in serum at 37 °C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.
  • the RNA or combination of nucleic acid molecules is complexed with the lipid portion of the formulation.
  • full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with a nucleic acid. Encapsulation is determined by adding the dye to a lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent.
  • the present disclosure provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles.
  • the nucleic acid-lipid composition comprises a plurality of RNA liposomes.
  • the nucleic acid-lipid composition comprises a plurality of RNA cationic liposomes.
  • the nucleic acid-lipid composition comprises a plurality of RNA lipid nanoparticles.
  • the lipid formulations comprise an RNA or combination of nucleic acid molecules that is fully encapsulated within the lipid portion of the formulation, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%
  • the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.
  • the expressible polynucleotides and RNA described herein are lipid formulated.
  • the lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes, and lipid nanoparticles.
  • a lipid formulation is a cationic liposome or a lipid nanoparticle (LNP) comprising:
  • an aggregation reducing agent such as polyethylene glycol (PEG) lipid or PEG- modified lipid
  • lipid optionally a non-cationic lipid (such as a neutral lipid), and
  • the lipid nanoparticle encapsulating the RNA or combination of nucleic acid molecules comprises a cationic lipid and at least one other lipid selected from the group consisting of anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer conjugated lipids, phospholipids, glycolipids, and combinations thereof.
  • the cationic lipid is an ionizable cationic lipid.
  • the lipid nanoparticle formulation consists of (i) at least one cationic lipid; (ii) a helper lipid; (iii) a sterol (e.g. , cholesterol); and (iv) a PEG-lipid, in a molar ratio of about 30% to about 60% ionizable cationic lipid: about 5% to about 20% helper lipid: about 35% to about 50% sterol: about 0.5-5% PEG-lipid.
  • Example cationic lipids including ionizable cationic lipids, helper lipids (e.g., neutral lipids), sterols, and ligand-containing lipids (e.g., PEG-lipids) are described herein below.
  • lipids and their relative % compositions depends on several factors including the desired therapeutic effect, the intended in vivo delivery target, and the planned dosing regimen and frequency. Generally, lipids that correspond to both high potency (i.e., therapeutic effect such as knockdown activity or translation efficiency) and biodegradability resulting in rapid tissue clearance are most preferred. However, biodegradability may be less important for formulations that are intended for only one or two administrations within the subject. In addition, the lipid composition may require careful engineering so that the lipid formulation preserves its morphology during in vivo administration and its journey to the intended target, but will then be able to release the active agent upon uptake into target cells. Thus, several formulations typically need to be evaluated in order to find the best possible combination of lipids in the best possible molar ratio of lipids as well as the ratio of total lipid to active ingredient.
  • Suitable lipid components and methods of manufacturing lipid nanoparticles are well known in the art and are described for example in PCT/US2020/023442, U.S. 8,058,069, U.S. 8,822,668, U.S. 9,738,593, U.S. 9,139,554, PCT/US2014/066242, PCT/US2015/030218, PCT/2017/015886, and PCT/US2017/067756, the contents of which are incorporated by reference.
  • Additional lipid components and compositions embodied in the present disclosure include those in WO/2015/199952, WO/2017/004143, WO/2017/075531 , WO/2020/081938, WO/2017/201091, WO/2019/246203, WO/2020/051223, and WO/2022/040641, the contents of which are incorporated by reference.
  • the lipid formulation preferably includes a cationic lipid suitable for forming a cationic liposome or lipid nanoparticle.
  • Cationic lipids are widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake.
  • cationic lipids are amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails, or a steroid portion and a connector between these two domains.
  • the cationic lipid carries a net positive charge at about physiological pH.
  • Cationic liposomes have been traditionally the most commonly used non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA/small hairpin RNA- shRNA.
  • Cationic lipids such as DOTAP, (l,2-dioleoyl-3- trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids by electrostatic interaction, providing high in vitro transfection efficiency.
  • the cationic lipid may be, for example, ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate) (also known as ALC-0315), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl- N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and l,2-Dioleyloxy-3-trimethylaminopropane chloride salt), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),
  • DODAC N,N-dioleyl
  • cationic lipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P- (N-(N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Choi), N-(l-(2,3- dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dileoyl-sn- 3 -phosphoethanolamine (DOPE), l,2-dioleoyl-3 -dimethylammonium propane (DODAP), N- (l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE),
  • cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN including DOTMA and DOPE, available from GIBCO/BRL
  • Lipofectamine comprising DOSPA and DOPE, available from GIBCO/BRL
  • Suitable cationic lipids are disclosed in International Publication Nos. WO 09/086558, WO 09/127060, WO 10/048536, WO 10/054406, WO 10/088537, WO 10/129709, and WO 2011/153493; U.S. Patent Publication Nos. 2011/0256175, 2012/0128760, and 2012/0027803; U.S. Patent No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are herein incorporated by reference.
  • Suitable cationic lipids include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N-ethyl- N-methylamino-, and N-propyl-N -ethylamino-). These lipids are part of a subcategory of cationic lipids referred to as amino lipids.
  • the cationic lipid is an amino lipid.
  • amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of fdter sterilization.
  • Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of Ci4 to C22 may be used.
  • Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.
  • the lipid formulation comprises the cationic lipid with Formula I according to the patent application PCT/EP2017/064066.
  • PCT/EP2017/064066 the disclosure of PCT/EP2017/064066 is also incorporated herein by reference.
  • amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH.
  • physiological pH e.g., pH 7.4
  • second pH preferably at or above physiological pH.
  • Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the disclosure.
  • the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11.
  • the ionizable cationic lipid has a pKa of about 5 to about 7.
  • the pKa of an ionizable cationic lipid is about 6 to about 7.
  • the lipid formulation comprises an ionizable cationic lipid of Formula I: or a pharmaceutically acceptable salt or solvate thereof, wherein R 5 and R 6 are each independently selected from the group consisting of a linear or branched C1-C31 alkyl, C2-C31 alkenyl or C2-C31 alkynyl and cholesteryl; L 5 and L 6 are each independently selected from the group consisting of a linear C1-C20 alkyl and C2-C20 alkenyl; X 5 is -C(O)O-, whereby -C(O)O- R 6 is formed or -OC(O)- whereby -OC(O)-R 6 is formed; X 6 is -C(O)O- whereby -C(O)O-R 5 is formed or
  • -OC(O)- whereby -OC(O)-R 5 is formed;
  • X 7 is S or O;
  • L 7 is absent or lower alkyl;
  • R 4 is a linear or branched Ci-Ce alkyl; and
  • R 7 and R 8 are each independently selected from the group consisting of a hydrogen and a linear or branched Ci-Ce alkyl.
  • X 7 is S.
  • X 5 is -C(O)O-, whereby -C(O)O-R 6 is formed and X 6 is - C(O)O- whereby -C(O)O-R 5 is formed.
  • R 7 and R 8 are each independently selected from the group consisting of methyl, ethyl and isopropyl.
  • L 5 and L 6 are each independently a C1-C10 alkyl. In some embodiments, L 5 is C1-C3 alkyl, and L 6 is C1-C5 alkyl. In some embodiments, L 6 is C1-C2 alkyl. In some embodiments, L 5 and L 6 are each a linear C7 alkyl. In some embodiments, L 5 and L 6 are each a linear C9 alkyl.
  • R 5 and R 6 are each independently an alkenyl. In some embodiments, R 6 is alkenyl. In some embodiments, R 6 is C2-C9 alkenyl. In some embodiments, the alkenyl comprises a single double bond. In some embodiments, R 5 and R 6 are each alkyl. In some embodiments, R 5 is a branched alkyl. In some embodiments, R 5 and R 6 are each independently selected from the group consisting of a C9 alkyl, C9 alkenyl and C9 alkynyl. In some embodiments, R 5 and R 6 are each independently selected from the group consisting of a C11 alkyl, C11 alkenyl and C11 alkynyl.
  • R 5 and R 6 are each independently selected from the group consisting of a C7 alkyl, C7 alkenyl and C7 alkynyl.
  • R 5 is -CH((CH2)pCH3)2 or -CH((CH2)pCH3)((CH2)p-iCH3), wherein p is 4-8.
  • p is 5 and L 5 is a C1-C3 alkyl.
  • p is 6 and L 5 is a C3 alkyl.
  • p is 7.
  • p is 8 and L 5 is a C1-C3 alkyl.
  • R 5 consists of -CH((CH2)pCH3)((CH2)p-iCH3), wherein p is 7 or 8.
  • R 4 is ethylene or propylene. In some embodiments, R 4 is n- propylene or isobutylene.
  • L 7 is absent, R 4 is ethylene, X 7 is S and R 7 and R 8 are each methyl. In some embodiments, L 7 is absent, R 4 is n-propylene, X 7 is S and R 7 and R 8 are each methyl. In some embodiments, L 7 is absent, R 4 is ethylene, X 7 is S and R 7 and R 8 are each ethyl.
  • X 7 is S
  • X 5 is -C(O)O-, whereby -C(O)O-R 6 is formed
  • X 6 is -C(O)O- whereby -C(O)O-R 5 is formed
  • L 5 and L 6 are each independently a linear C3-C7 alkyl
  • L 7 is absent
  • R 5 is -CH((CH2)pCH3)2
  • R 6 is C7-C12 alkenyl.
  • p is 6 and R 6 is C9 alkenyl.
  • the lipid formulation comprises an ionizable cationic lipid selected from the group consisting of
  • cationic lipids have been described in the literature, many of which are commercially available.
  • suitable cationic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP), 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane (DLinDMA), and 1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA).
  • DOTAP 1,2-dioleoyl-3 -trimethylammonium -propane
  • DLinDMA 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane
  • DLenDMA 1,2-Dilinolenyloxy-N,N- dimethylaminopropane
  • the pKa of formulated cationic lipids is correlated with the effectiveness of lipid particles for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)).
  • the preferred range of pKa is ⁇ 5 to ⁇ 7.
  • the cationic lipid is a compound of Formula (I’):
  • Ri is a substituted alkyl consisting of 10 to 31 carbons
  • R2 is a linear alkyl, alkenyl or alkynyl consisting of 2 to 20 carbons
  • Rs is a linear or branched alkane consisting of 1 to 6 carbons
  • R4 and Rs are the same or different, each a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons
  • Li and L2 are the same or different, each a linear alkane of 1 to 20 carbons or a linear alkene of 2 to 20 carbons
  • Xi is S or O; or a salt or solvate thereof.
  • Exemplary compounds of formula (I), their synthesis and uses thereof are described in US2018/0169268, all of which are herein incorporated by reference.
  • the cationic lipid is a compound of formula (II’):
  • Ri is a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons
  • Li is linear alkane of 1 to 15 carbons
  • R2 is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons or a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons
  • L2 is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons
  • X is O or S
  • Rs is a linear alkane of 1, 2, 3, 4, 5, or 6 carbons
  • R4 and Rs are the same or different, each a linear or branched, noncyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons; or a pharmaceutically acceptable salt or solvate thereof.
  • the cationic lipid is a compound of formula (III’), (IV’) or (V’):
  • R comprises a biologically active molecule
  • Li, L2 and Ls independently for each occurrence comprise a ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile; a pharmaceutically acceptable salt thereof; or a pharmaceutical composition thereof.
  • Exemplary compounds of formula (III’), (IV’) and (V’), their synthesis and uses thereof are described in US2017/0028074, all of which are herein incorporated by reference.
  • the cationic lipid is a compound of formula (VI’):
  • the cationic lipid is a compound of formula (VII’): Formula (VIE) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G 1 or
  • the cationic lipid is a compound of formula (VIII’):
  • X is CR a ;
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or
  • Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
  • R a is, at each occurrence, independently H, C1-C12 alkyl, Ci-C 12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C
  • R' is, at each occurrence, independently H or C1-C12 alkyl; a 1 and a 2 are, at each occurrence, independently an integer from 3 to 12; b 1 and b 2 are, at each occurrence, independently 0 or 1; c 1 and c 2 are, at each occurrence, independently an integer from 2 to 12; d 1 and d 2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a 1 , a 2 , c 1 , c 2 , d 1 and d 2 are selected such that the sum of a ⁇ +d 1 is an integer from 18 to 30, and the sum of a 2 +c 2 +d 2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alky
  • Additional cationic lipids that can be used in compositions of the application include, but are not limited to, those described in W02019/036030, W02019/036028, W02019/036008, WO2019/036000, US2016/0376224, US2017/0119904, W02018/200943 and WO2018/191657, the relevant contents on the lipids, their synthesis and uses are herein incorporated by reference in their entireties.
  • any one or more lipids recited herein may be expressly excluded.
  • the RNA lipid formulations of the present disclosure can comprise a helper lipid, which can be referred to as a neutral lipid, a neutral helper lipid, non-cationic lipid, noncationic helper lipid, anionic lipid, anionic helper lipid, or a zwitterionic lipid. It has been found that lipid formulations, particularly cationic liposomes and lipid nanoparticles have increased cellular uptake if helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9):882-92).
  • lipids such as l,2-dioleoyl-sn-glycero-3 -phosphatidylcholine (DOPC), Di-Oleoyl- Phosphatidyl-Ethanoalamine (DOPE) and l,2-DiStearoyl-sn-glycero-3-PhosphoCholine (DSPC), being more fusogenic (i.e., facilitating fusion) than cationic lipids, can affect the polymorphic features of lipid-nucleic acid complexes, promoting the transition from a lamellar to a hexagonal phase, and thus inducing fusion and a disruption of the cellular membrane.
  • DOPC Di-Oleoyl- Phosphatidyl-Ethanoalamine
  • DSPC l,2-DiStearoyl-sn-glycero-3-PhosphoCholine
  • helper lipids can help to reduce any potential detrimental effects from using many prevalent cationic
  • Non-limiting examples of non-cationic lipids suitable for lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatid
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • non-cationic lipids include sterols such as cholesterol and derivatives thereof.
  • sterols such as cholesterol and derivatives thereof.
  • cholesterol increases the spacing of the charges of the lipid layer interfacing with the nucleic acid making the charge distribution match that of the nucleic acid more closely.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy) -butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.
  • the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid formulation. In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation.
  • helper lipids include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine -lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.
  • nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate
  • the helper lipid comprises from about 30 mol% to about 60 mol%, from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, about 35 mol% to about 54 mol%, from about 36 mol% to about 52 mol%, from about 37 mol% to about 51 mol%, from about 38 mol% to about 50 mol%, or about 39 mol%, about 50 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, or about 49 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.
  • the total of helper lipid in the formulation comprises two or more helper lipids and the total amount of helper lipid comprises from about 30 mol% to about 60 mol%, from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, about 35 mol% to about 54 mol%, from about 36 mol% to about 52 mol%, from about 37 mol% to about 51 mol%, from about 38 mol% to about 50 mol%, or about 39 mol%, about 50 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, or about 49 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.
  • the helper lipids are a combination of DSPC and DOTAP.
  • the helper lipids are
  • the cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 50 mol%, about 35 mol%, about 40 mol%, about 45 mol%, or about 50 mol% of the total lipid present in the lipid formulation.
  • the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 45 mol%, about 30 mol% to about 45 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, or about 45 mol% of the total lipid present in the lipid formulation.
  • the percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ⁇ 5 mol%.
  • a lipid formulation containing a cationic lipid compound or ionizable cationic lipid compound may be on a molar basis about 30-60% cationic lipid compound, about 35-50 % cholesterol, about 5-20% helper lipid, and about 0.5-5% of a polyethylene glycol (PEG) lipid, wherein the percent is of the total lipid present in the formulation.
  • the composition is about 40-50% cationic lipid compound, about 35-45% cholesterol, about 5-15% helper lipid, and about 0.5-3% of a PEG-lipid, wherein the percent is of the total lipid present in the formulation.
  • the lipid formulations described herein may further comprise a lipid conjugate.
  • the conjugated lipid is useful for preventing the aggregation of particles.
  • Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic-polymer-lipid conjugates, and mixtures thereof.
  • lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front. Pharmacol. 2015 Dec 1; 6:286).
  • the lipid conjugate is a PEG-lipid.
  • PEG polyethylene glycol
  • PEGylation has been widely used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms.
  • Detergent-like PEG lipids e.g., PEG-DSPE
  • PEG-DSPE can enter the lipid formulation to form a hydrated layer and steric barrier on the surface.
  • the surface layer can be generally divided into two types, brush-like and mushroom-like layers.
  • PEG-DSPE-stabilized formulations PEG will take on the mushroom conformation at a low degree of PEGylation (usually less than 5 mol%) and will shift to brush conformation as the content of PEG-DSPE is increased past a certain level (J. Nanomaterials. 2011 ;2011 : 12). It has been shown that increased PEGylation leads to a significant increase in the circulation half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15; 13:507-30; J. Control Release. 2010 Aug 3; 145(3): 178-81).
  • PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.
  • PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups.
  • PEGs are classified by their molecular weights and include the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol- succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NEE), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as well as such compounds containing a terminal hydroxyl group instead of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NEE).
  • MePEG-OH monomethoxypolyethylene glycol
  • MePEG-S monomethoxypolyethylene glycol- succinate
  • the PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight may be any value or subvalue within the recited ranges, including endpoints.
  • the PEG monomers can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group.
  • the PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties.
  • the linker moiety is a non-ester-containing linker moiety.
  • Suitable non-ester-containing linker moieties include, but are not limited to, amido (- C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CEEC(O)-), succinamidyl (- NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).
  • a carbamate linker is used to couple the PEG to the lipid.
  • an ester-containing linker moiety is used to couple the PEG to the lipid.
  • Suitable ester-containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.
  • Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those of skill in the art.
  • Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of Cio to C20 are preferred. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).
  • DMPE dimyristoylphosphatidylethanolamine
  • DPPE dipalmitoyl-phosphatidylethanolamine
  • DOPE dioleoyl-phosphatidylethanolamine
  • DSPE distearoyl-phosphatidylethanolamine
  • the PEG-DAA conjugate is a PEG-didecyloxypropyl (Cio) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (Cie) conjugate, or a PEG-distearyloxypropyl (Cis) conjugate.
  • the PEG preferably has an average molecular weight of about 750 to about 2,000 daltons.
  • the terminal hydroxyl group of the PEG is substituted with a methyl group.
  • hydrophilic polymers can be used in place of PEG.
  • suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%, from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 0.9 mol% to about 1.6 mol%, from about 0.9 mol% to about 1.8 mol%, from about 1 mol% to about 1.8 mol%, from about 1 mol% to about 1.7 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, or from about 1.4 mol% to about 1.6 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation.
  • the lipid conjugate (e.g., PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5%, (or any fraction thereof or range therein) of the total lipid present in the lipid formulation.
  • the amount may be any value or subvalue within the recited ranges, including endpoints.
  • the PEG-lipid is PEG550-PE. In some embodiments, the PEG-lipid is PEG750-PE. In some embodiments, the PEG-lipid is PEG2000-DMG. In some preferred embodiments, the PEG-lipid is 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (also known as ALC-0159).
  • the percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid formulations of the disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ⁇ 0.5 mol%.
  • concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid formulation is to become fusogenic.
  • Lipid formulations for the intracellular delivery of nucleic acids are designed for cellular uptake by penetrating target cells through exploitation of the target cells’ endocytic mechanisms where the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell.
  • nucleic Acid Therapeutics 28(3): 146-157, 2018.
  • the mRNA or circRNA-lipid formulation enters hepatocytes through receptor mediated endocytosis.
  • lipid delivery vehicle Prior to endocytosis, functionalized ligands such as PEG-lipid at the surface of the lipid delivery vehicle are shed from the surface, which triggers internalization into the target cell.
  • functionalized ligands such as PEG-lipid at the surface of the lipid delivery vehicle are shed from the surface, which triggers internalization into the target cell.
  • some part of the plasma membrane of the cell surrounds the vector and engulfs it into a vesicle that then pinches off from the cell membrane, enters the cytosol and ultimately undergoes the endolysosomal pathway.
  • the increased acidity as the endosome ages results in a vehicle with a strong positive charge on the surface. Interactions between the delivery vehicle and the endosomal membrane then result in a membrane fusion event that leads to cytosolic delivery of the payload.
  • the cell’s own internal translation processes will then translate the RNA or combination of nucleic acid molecules into the encoded protein (e.g., a wild-type HIV- derived accessory protein tat for the reactivation of latent HIV).
  • the encoded protein can further undergo post-translational processing, including transportation to a targeted organelle or location within the cell.
  • composition and concentration of the lipid conjugate By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate exchanges out of the lipid formulation and, in turn, the rate at which the lipid formulation becomes fusogenic.
  • other variables including, e.g., pH, temperature, or ionic strength, can be used to vary and/or control the rate at which the lipid formulation becomes fusogenic.
  • Other methods which can be used to control the rate at which the lipid formulation becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.
  • composition and concentration of the lipid conjugate one can control the liposomal or lipid particle size.
  • lipid formulations comprising a nucleic acid, e.g. RNA or combination of nucleic acid molecules.
  • RNA nucleic acid
  • the techniques of thin fdm hydration, double emulsion, reverse phase evaporation, microfluidic preparation, dual asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.
  • MLVs Multilamellar Vesicles
  • Lipid formulations can also be prepared through the Double Emulsion technique, which involves lipids dissolution in a water/organic solvent mixture.
  • the organic solution, containing water droplets is mixed with an excess of aqueous medium, leading to a water-in- oil-in-water (W/O/W) double emulsion formation. After mechanical vigorous shaking, part of the water droplets collapse, giving Large Unilamellar Vesicles (LUVs).
  • Double Emulsion technique involves lipids dissolution in a water/organic solvent mixture.
  • the organic solution containing water droplets, is mixed with an excess of aqueous medium, leading to a water-in- oil-in-water (W/O/W) double emulsion formation. After mechanical vigorous shaking, part of the water droplets collapse, giving Large Unilamellar Vesicles (LUVs).
  • LUVs Large Unilamellar Vesicles
  • Reverse Phase Evaporation also allows one to achieve LUVs loaded with nucleic acid.
  • REV Reverse Phase Evaporation
  • a two-phase system is formed by phospholipids dissolution in organic solvents and aqueous buffer.
  • the resulting suspension is then sonicated briefly until the mixture becomes a clear one-phase dispersion.
  • the lipid formulation is achieved after the organic solvent evaporation under reduced pressure. This technique has been used to encapsulate different large and small hydrophilic molecules including nucleic acids.
  • the Microfluidic method unlike other bulk techniques, gives the possibility of controlling the lipid hydration process.
  • the method can be classified in continuous-flow microfluidic and droplet-based microfluidic, according to the way in which the flow is manipulated.
  • MHF microfluidic hydrodynamic focusing
  • lipids are dissolved in isopropyl alcohol which is hydrodynamically focused in a microchannel cross junction between two aqueous buffer streams.
  • Vesicles size can be controlled by modulating the flow rates, thus controlling the lipids solution/buffer dilution process.
  • the method can be used for producing oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three-inlet and one-outlet ports.
  • Dual Asymmetric Centrifugation differs from more common centrifugation as it uses an additional rotation around its own vertical axis.
  • An efficient homogenization is achieved due to the two overlaying movements generated: the sample is pushed outwards, as in a normal centrifuge, and then it is pushed towards the center of the vial due to the additional rotation.
  • VPC viscous vesicular phospholipid gel
  • the lipid formulation size can be regulated by optimizing DAC speed, lipid concentration and homogenization time.
  • the Ethanol Injection (El) method can be used for nucleic acid encapsulation. This method provides the rapid injection of an ethanolic solution, in which lipids are dissolved, into an aqueous medium containing nucleic acids to be encapsulated, through the use of a needle. Vesicles are spontaneously formed when the phospholipids are dispersed throughout the medium. Detergent Dialysis
  • the Detergent dialysis method can be used to encapsulate nucleic acids. Briefly lipid and plasmid are solubilized in a detergent solution of appropriate ionic strength, after removing the detergent by dialysis, a stabilized lipid formulation is formed. Unencapsulated nucleic acid is then removed by ion-exchange chromatography and empty vesicles by sucrose density gradient centrifugation. The technique is highly sensitive to the cationic lipid content and to the salt concentration of the dialysis buffer, and the method is also difficult to scale.
  • Stable lipid formulations can also be produced through the Spontaneous Vesicle Formation by Ethanol Dilution method in which a stepwise or dropwise ethanol dilution provides the instantaneous formation of vesicles loaded with nucleic acid by the controlled addition of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing the nucleic acid.
  • nucleic acids can also be obtained starting with preformed liposomes through two different methods: (1) a simple mixing of cationic liposomes with nucleic acids which gives electrostatic complexes called “lipoplexes”, where they can be successfully used to transfect cell cultures, but are characterized by their low encapsulation efficiency and poor performance in vivo,' and (2) a liposomal destabilization, slowly adding absolute ethanol to a suspension of cationic vesicles up to a concentration of 40% v/v followed by the dropwise addition of nucleic acids achieving loaded vesicles; however, the two main steps characterizing the encapsulation process are too sensitive, and the particles have to be downsized.
  • examples of lipids and lipid nanoparticles, pharmaceutical compositions comprising the lipids, methods of making the lipids or formulating pharmaceutical compositions comprising the lipids and nucleic acid molecules, and methods of using the pharmaceutical compositions for treating or preventing diseases are described in U.S.
  • the application also provides methods of inducing reactivation of latent HIV in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of the application.
  • a pharmaceutical composition of the application described herein can be used in the methods of the application.
  • a subject to be treated according to the methods of the application is an HIV-infected subject. More preferably, the HIV-infected subjects are under ART-treatment for at least 6 months, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months.
  • a small amount of HIV or only latent HIV can be detected in plasma isolated from the human prior to the administration of the pharmaceutical composition.
  • Methods according to embodiments of the application further comprises administering to the subject in need thereof another an antiviral agent such as a small molecule and/or antibody directed towards HIV or another anti-HIV agent in combination with a pharmaceutical composition of the application. Administration may be concurrent or sequential.
  • another an antiviral agent such as a small molecule and/or antibody directed towards HIV or another anti-HIV agent in combination with a pharmaceutical composition of the application. Administration may be concurrent or sequential.
  • compositions of the application can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration.
  • parenteral administration e.g., intramuscular, subcutaneous, intravenous, or intradermal injection
  • oral administration e.g., oral administration
  • transdermal administration e.g., transdermal administration
  • nasal administration e.g., by intramuscular injection or intradermal injection
  • parenterally e.g., by intramuscular injection or intradermal injection
  • administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection.
  • Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the ribonucleic acid molecules to cells.
  • electroporation refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane.
  • in vivo electroporation electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules from one side of a cellular membrane to the other.
  • In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site. As a result, transfection efficiency and immune response are significantly improved (e.g., up to 1,000 fold and 100 fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection.
  • Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used.
  • Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipoplexes, nanoparticles, etc.
  • a ribonucleic acid molecule of the application can be formulated in a composition that comprises one or more lipid molecules, preferably positively charged lipid molecules.
  • a ribonucleic acid molecule of the disclosure can be formulated using one or more liposomes, lipoplexes, and/or lipid nanoparticles.
  • liposome or lipid nanoparticle formulations described herein can comprise a polycationic composition.
  • the formulations comprising a polycationic composition can be used for the delivery of the ribonucleic acid molecule described herein in vivo and/or ex vitro.
  • a deletion exercise is performed to delineate the essential Tat amino acid sequence that would result in the same activation capacity as the 72 aa Tat exon 1 protein.
  • the rationale to reduce the overall size of HIV Tat was based on the following assumptions: (i) to remove potential anti-HIV Tat antibody epitopes, (ii) to diminish off target effects and improve cellular uptake, (iii) the absence of potential tat contamination by TLR agonists, and (iv) to facilitate chemical synthesis of the protein.
  • the HIV derived accessory protein Tat 86 to 101 amino acids (aa) long, is translated from two different exons where exon 1 (1-72 aa) contains all the domains essential for trans-activation (Jeang 1997).
  • a DNA sequence encoding a Tat deletion mutant, with a length ranging from 57 aa up to 86 aa e.g., tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl-64, tatl-60, tatl-57, or tat2-72
  • apDNA expression vector e.g., tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl-64, tatl-60, tatl-57, or tat2-72
  • Tatl-86 and Tatl- 72 showed similar transactivation activity confirming the reported finding that exon-1 [72 aa] contains all elements essential for transactivation (Arya 1985, Muesing 1987).
  • the Tat variant comprising only 66 amino acids was identified to have a comparable latency reversal activity as the full-length exon Tat-72 construct (Fig. 1). The latency reversal potential of the T66 variant was further studied in various in vitro and ex vivo latency reactivation assays.
  • Example 2 Strong and consistent T66 protein mediated LTR activation of latent HIV in a variety of latent T-cell lines
  • the MT4 T-cells expressing LTR-GFP and LTR-Luc (Firefly luciferase) upon T66 protein-induced LTR promoter activation were used to compare T66 protein-induced LTR activation.
  • Other reporter cell lines, Jurkat E6.1 clone 32 and Jurkat E6.1 clone 50, that include latent HIV with a GFP reporter were also used to measure latency reversal activity.
  • T66 protein was synthesized as trifluoroacetate salt by Bachem. The lyophilized T66 protein was resuspended in sterile water containing 1 mM DTT for use.
  • the latency reversal activity with different doses (ranging from 5.1 to 9 pM) of the T66 protein was evaluated in three different cell lines indicated above (FIG. 2A).
  • the percentage of GFP positive cells reached a plateau at a concentration of 6.2pM in the three cell lines, with 87%, 63% and 51% of positive cells in the MT-4-LTR-GFP, Jurkat clone 50 and Jurkat clone 32, respectively.
  • CD4+ T cells were purified from PBMCs by negative selection (Easy SepTM Human CD4+ T Cell Isolation Kit). The assay is based on the co-infection of isolated CD4+ T cells from HIV-negative donors with an HIV reporter construct and a virus expressing two antiapoptotic proteins (MCL7 and Bcl2).
  • FIG. 3A and 3B show cell-associated HIV-1 RNA copies measured by qRT-PCR using gag-specific primers (1: GGACCAAAGGAACCCTTTAGAGA (SEQ ID NO: 16); 2: GGACCAACAAGGTTTCTGTCATC (SEQ ID NO: 17)). HIV-1 intracellular RNA was evaluated 24h after the addition of T66 protein.
  • T66 does not induce global T cell activation nor modifications in the CD4+ T cells transcriptome
  • T66-treated cells cluster closely positioned to PBS-treated cells for all participants, suggesting insignificant impact of T66 treatment on the host transcriptome (Fig. 4B). This is in sharp contract with PHA-stimulated cells which formed a second cluster, confirming the profound impact of the mitogen PHA on the human transcriptome. It is confirmed that T66 (4 pM) was present and active in the HIV-infected CD4+ T cells, observing latency reversal determined by HIV RNA qPCR using the gagspecific primers in T66-treated cells (Fig. 4C).
  • T66 protein showed robust latency reversal in multiple assays while no global T- cell activation nor any significant transcriptome perturbation was observed.
  • a potential pitfail of the T66 protein is the relatively high concentration required for HIV reactivation, which might be challenging for large scale production and in vivo administration.
  • inefficient cellular protein transduction and substantial endosomal trapping of the T66 protein are likely the cause for the large amount of protein needed to achieve maximum HIV activation.
  • One potential strategy to address the poor cytosolic delivery is to use T66 mRNA.
  • a DNA template for the in vitro transcription of mRNA was generated by introducing the following elements in a standard pDNA vector: a T7 promoter, followed by the coding sequences of the 5 ’-and 3’-UTRs from either the human a-globin [HAG] or the frog (Xenopus) a-globin [XBG] (Table 2), terminated by a 120 nt polyA tail.
  • a T7 promoter followed by the coding sequences of the 5 ’-and 3’-UTRs from either the human a-globin [HAG] or the frog (Xenopus) a-globin [XBG] (Table 2), terminated by a 120 nt polyA tail.
  • HOG human a-globin
  • XBG a-globin
  • HIV-1 T66 codon-optimized gene sequence as shown in Fig.5, was introduced via restriction enzymes-based cloning into the DNA template plasmid between the 5’-UTR and 3’-UTR coding sequences. Sequence-verified DNA constructs were upscaled, linearized and column-purified with the Pure Link Gel Extraction Kit (Thermofisher).
  • the in vitro transcript was purified by column purification using the RNA RNA miniprep/maxiprep kit (Qiagen) according to the provided protocol.
  • the RNA was quantified with the Nanodrop spectrophotometer and its integrity verified by agarose gel analysis, as shown in Fig.6.
  • the following transfection reagents were tested in a cellular reporter assay to identify the optimal delivery system of T66 mRNA into the cytosol: JetPEI (Polyplus), JetMessenger (Polyplus), Viromer (Lipocalyx), TransIT mRNA (Minis), RNAiMax (Thermofisher), and Messenger Max (Thermofisher).
  • JetPEI Polyplus
  • JetMessenger Polyplus
  • Viromer Lipocalyx
  • TransIT mRNA Minis
  • RNAiMax Thermofisher
  • Messenger Max Thermofisher
  • JetMessenger® (Polyplus) turned out to be the most efficient transfection reagent for T66 mRNA showing up to 97 % of GFP (+) cells down to a concentration of 2.5 nM T66 mRNA, as shown in Fig.7.
  • Example6 Evaluation of T66 mRNA HIV-1 reactivation (reversal) potential on a variety of latent T-cell lines
  • T66 mRNA mediated activation was used to compare T66 mRNA mediated activation with T66 protein induced LTR activation.
  • T66 mRNA turned out to be more than 10,000-fold m5ore active when compared to its protein counterpart, as shown in Fig.8A and 8B. Maximal reporter expression is achieved with 1 nM T66 mRNA while it takes 10 pM of protein to have the same effect.
  • T66 protein induces luciferase expression by only 28-fold whereas T66 mRNA enhances luciferase expression by 68-fold (Fig. 8B).
  • T66 mRNA were profiled in an engineered Jurkat T-cell line harboring a latent HIV-1 GFP reporter pro virus side by side with the strongest ex vivo HIV-1 latency reversal agents known to date such as PMA and anti-CD3/-CD28 costimulation (anti- CD3/CD28 magnetic Dynabeads, Invitrogen).
  • PMA and anti-CD3/-CD28 costimulation anti- CD3/CD28 magnetic Dynabeads, Invitrogen.
  • T66 mRNA LTR activation determined by the increase of GFP (+) cells exceeded the reactivation achieved by T66 protein or CD3/CD28, as shown in Fig.9.
  • PMA nor CD3/CD28 costimulation may be used in vivo due to their induction of global T-cell activation.
  • no primary CD4 T-cell activation determined by upregulation of CD25 and CD69 activation markers was observed with T66 mRNA delivered by JetMessenger (data not shown).
  • NSG mice or NOD/shi-scid/ccnull (NOG) mice will be transplanted with CD34+ cells by iv injection.
  • the mice will be bled at 12 and 24 weeks post-transplantation to determine human immune cell reconstitution by flow cytometry.
  • Animals with > 20% of circulating human CD45+ cells at 24 weeks post-transplantation will be infected by intraperitoneal injections of a HIV-1 laboratory adapted strain. Infection will be monitored every 2 weeks on blood samples for CD4+ T cell counting using flow cytometry analysis and for viral load measurement.
  • cART treatment will be initiated 1 week (early treated) or 8 weeks (late treated) post infection and to be continued for a total of 8 weeks in both groups.
  • T66 mRNA or another latency reversal agent will be added and viral rebound will be evaluated by measuring viral loads in blood samples of the mice.
  • Example 8 Evaluation of T66 mRNA HIV-1 reactivation (reversal) potential in rhesus macaques
  • Rhesus macaques will be infected with SIVmac239 and treated with a potent ART regimen.
  • viral load in blood is below 60 copies/ml
  • the animal are treated with T66 mRNA or another or another latency reversal agent.
  • the viral rebound will be evaluated by measuring viral load in blood samples of the animal.
  • Example 9 Identification of nanoparticle formulations for the efficient mRNA delivery
  • a panel of commercial transfection reagents were tested in a cellular HIV-GFP reporter assay to identify the optimal delivery system of T66 mRNA into Jurkat T lymphocyte cells: JetPEI (Polyplus), JetMessenger (Polyplus), Viromer (Lipocalyx), TransIT mRNA (Minis), RNAiMax (Thermofisher), and Messenger Max (Thermofisher).
  • the transfection was performed on IxlO 5 Jurkat cells plated in 100 pl medium in 96-well plates using lOOng T66 mRNA formulated with the different transfection reagents according to the supplier’s instructions.

Abstract

The present disclosure includes mRNA compositions and pharmaceutical formulations thereof, combinations therewith and methods of reactivation of latent HIV that avoid overall T-cell activation.

Description

MRNA COMPOSITIONS FOR INDUCING LATENT HIV-1 REVERSAL
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 63/369,133 filed July 22, 2022, and U .S. Provisional Application No. 63/341,668 filed May 13, 2022, the disclosures of which are incorporated herein by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] This application contains a sequence listing, which is submitted electronically. The content of the electronic sequence listing (sequence listing.xml; Size: 20,480 bytes; and Date of Creation: July 22, 2022) is herein incorporated by reference in its entirety.
FIELD OF INVENTION
[0003] The present disclosure relates generally to the field of molecular biology and genetic engineering, including nucleic acid molecules useful for regulating gene expression, and the use of the nucleic acid molecules for, for example, production of desired products in suitable host cells in cell culture or in a subject, and for conferring beneficial characteristics to the host cells or subjects.
BACKGROUND
[0001] Highly active antiretroviral therapy (HAART) can suppress HIV-1 levels in plasma to below the limit of detection of clinical assays (<50 copies/ml) and reduce the morbidity and mortality of HIV - 1 infection. However, HAART alone fails to cure HIV infection. In particular, HAART leaves latent integrated proviruses unaffected. Latent viral genomes reside in a small pool of infected resting memory CD4+ T-cells that constitute a stable viral reservoir. In these cells, the provirus remains transcriptionally silent as long as the host cells are in a quiescent state. This allows the virus to evade host immune surveillance and rebound quickly following discontinuation of HAART. The remarkable stability of the latent viral reservoir necessitates lifelong HAART. Elimination of the latent reservoir leading to the cure of HIV-1 infected individuals of the latent reservoir has been proposed as a goal worthy of a major scientific effort.
[0002] Therapies targeting the latent reservoir generally involve reactivation of latent virus. Expression of viral genes renders infected cells susceptible to viral cytopathic effects and immune clearance. Along with HAART, this reactivation strategy could ultimately purge latent virus from infected individuals. While latent viruses respond to T-cell activation signals initial attempts to deplete the latent reservoir through T-cell reception (TCR) stimulation using anti-CD3 antibodies proved toxic. The toxicity likely resulted from global T-cell activation with subsequent release of pro-inflammatory cytokines. Therefore, an ideal treatment should reactivate latent HIV-1, but avoid overall T-cell activation.
[0003] However, despite global extensive research efforts, there still exists a high unmet medical need for reliable, safe and convenient compounds able to lead to reactivation of latent HIV from above mentioned reservoir in HIV-infected patients. HIV transcription is driven by its native 5’-LTR (long terminal repeat) promoter whereby transcriptional activity is auto-induced by the HIV-1 tat protein (transactivator of transcription) leading to a powerful positive feedback loop (Kam 1999). Several studies have shown that levels of Tat expression can regulate the fate of HIV-infected cells by favoring latency establishment or latency reversal (Jordan 2001). Tat is a small basic protein with length varying between 86 to 101 amino acids.
SUMMARY OF THE INVENTION
[0004] Accordingly, there is an unmet medical need in the treatment of HIV-1. The invention satisfies this need by providing reliable, safe and convenient compounds able to lead to reactivation of latent HIV that avoid overall T-cell activation.
[0005] In a general aspect, the present disclosure relates to a pharmaceutical composition comprising a ribonucleic acid (RNA) molecule encapsulated in a pharmaceutically acceptable encapsulating carrier, wherein the ribonucleic acid molecule comprises a non-naturally occurring polynucleotide sequence comprising a polynucleotide sequence encoding a wildtype HIV-derived accessory protein tat for the reactivation of latent HIV. In some embodiments, the pharmaceutically acceptable encapsulating carrier is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, such as one having the Formula I, II, III, IV, V, VI, VII or VIII described herein. In some embodiments, the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat encodes at least the first 57 N-terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV. In some embodiments, the nucleotide sequence encoding a wild-type HIV -derived accessory protein tat encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3. In some embodiments, the encoded wildtype HIV-derived accessory protein tat is a protein consisting of the amino acid sequence SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3, or a variant thereof. Preferably, the variant contains one or more amino acid substitutions as shown in Table 1. In some embodiments, the ribonucleic acid molecule comprises, a 5 ’-UTR, a nucleotide sequence encoding a wildtype HIV-derived accessory protein tat for the reactivation of latent HIV, a 3’-UTR and a poly A tail. In some embodiments, the ribonucleic acid molecule consists essentially of, a 5’- UTR, a nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, a 3’-UTR and a poly A tail, more particularly arranged in the order from the 5’- end to the 3 ’-end of the ribonucleic acid molecule. In some embodiments, the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises at least one sequence from table 2. In some embodiments, the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises a 5’-UTR, the T66 cds, and a 3’-UTR from table 2. In some embodiments, the 5’-UTR has a sequence of SEQ ID No. 4. In some embodiments, the 3’-UTR has a sequence of SEQ ID No. 5. In some embodiments, the poly A tail comprises 100 to 140 adenosine monophosphates. In some embodiments, the poly A tail comprises 120 adenosine monophosphates. In some embodiments, the 5’-UTR has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID No: 4 or SEQ ID NO: 6. In some embodiments, the 3’-UTR has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID No: 5 or SEQ ID NO: 7. In some embodiments, the encoded wild-type HIV-derived accessory protein is a deletion mutant selected from the group consisting of tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl- 64, tatl-60, tatl-57, and tat2-72. In some embodiments, the RNA molecule is an mRNA molecule.
[0006] In another general aspect, the present disclosure relates to an RNA molecule comprising a 5’-UTR, a nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for reactivation of latent HIV, a 3’-UTR, and a poly A tail as described herein. In some embodiments, the RNA molecule is an mRNA molecule. In some embodiments, the application is related to an isolated ribonucleic acid molecule comprising: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
[0007] In another general aspect, the application relates to a DNA molecule encoding a ribonucleic acid molecule of the application. In some embodiments, the DNA molecule comprises a promoter sequence, such as a T7 promoter sequence, more particularly, a T7 promoter comprises the sequence of TAATACGACTCACTATAG (SEQ ID NO: 14) or TAATACGACTCACTATAAG (SEQ ID NO: 15).
[0008] Additional embodiments include a method of reactivating latent HIV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to an embodiment of the application. In some embodiments, the method further comprises administering to the subject an effective amount of one or more anti-viral agent. In some embodiments, the method further comprises administering to the subject an effective amount of one or more latency reversing agents. In some embodiments, the latency reversing agent is selected from Phytohemagglutinin (PHA), Phorbol 12-Myristate 13-Acetate (PMA), and anti-CD3 + anti-CD28 antibodies. In some embodiments, the subject is a human. In some embodiments, the subject is a human infected with the HIV and/or is under antiretroviral therapy treatment for at least 6 months, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months prior to the administration of a pharmaceutical composition according to an embodiment of the application. In some embodiments, a small amount of HIV or only latent HIV is detected in the plasma of the human prior to the administration of the pharmaceutical composition. In some embodiment, the human has an HIV viral load of about 100 copies/ml or less (e.g., about 90, 80, 70, 60, 50, 40, 30, 20, 10 dml or less)or only latent HIV detected in plasma prior to the initial administration of the pharmaceutical composition.
[0009] Additional embodiments include a method of increasing the induction of latent HIV-1 reversal in a cell, comprising contacting the cell with a pharmaceutical composition according to an embodiment of the application, wherein the increase is an increase in induction of latent HIV-1 reversal over contacting the cell with an equivalent wild-type HIV- derived accessory protein tat for the reactivation of latent HIV.
[0010] In another general aspect, the application relates to an isolated host cell comprising a ribonucleic acid molecule of the application. The application further relates to an isolated host cell comprising a nucleic acid encoding the ribonucleic acid molecule of the application.
[0011] In another general aspect, the application relates to a pharmaceutical composition comprising a ribonucleic acid molecule of the application encapsulated in a pharmaceutically acceptable carrier, such as a lipid nanoparticle. In some embodiments, the ribonucleic acid molecule is an mRNA molecule.
[0012] In another general aspect, the application relates to a pharmaceutical composition of the application for use in reactivation of latent HIV in a subject in need thereof, preferably the subject has HIV-1 infection, optionally in combination with another therapeutic agent, preferably an anti-HIV agent, a latency reversing agent and/or another antiretroviral therapy.
[0013] Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.
[0015] FIG. 1 shows an embodiment of latency reversal activity of tat deletion mutants.
[0016] FIG. 2A shows an embodiment of a dose-dependent latency reversal activity of T66 protein in three different cell lines. FIG. 2B shows latency reversal activity of T66 protein in CD4+ T-cells obtained from 10 subjects.
[0017] FIG. 3A and 3B show an embodiment of increased cell-associated HIV-1 RNA copies from resting CD4+ T-cells treated with T66 protein. The cells were isolated from ART-treated individuals and the RNA copied were measured by RT-PCR. FIG. 3C and 3D show an embodiment of the level of p24 secretion on CD4+ T cells isolated from ART- treated individuals due to reactivation of HIV-1. FIG. 3E shows an embodiment of T66 protein-induced latency reversal activity on CD4+ T cells isolated from ART-treated indivisibles measured by the quantitative viral outgrowth assay. [0018] FIG. 4A shows no up-regulation of CD69 and CD25 in CD4+ T-cells. FIG. 4B shows no significant impact of T66 treatment on the host transcriptome in the HIV -infected CD4+ T-cells isolated from ART-treated individuals. FIG. 4C shows HIV reactivation of Tat- 66 protein in FIG. 4A and 4B. Fig. 4D shows a Volcano plots which allows to identify 8 genes which are differently expressed in T66-treated CD4+ T-cells in comparison to PBS- treated cells.
[0019] FIG. 5 shows an embodiment of an in vitro transcription template comprising a Tat-66 coding sequence.
[0020] FIG. 6 shows an embodiment of agarose gel analysis, wherein lane 1 : 1 kb DNA ladder; lane 2: T66 in vitro transcription; lane 3: purified T66 mRNA.
[0021] FIG. 7 shows evaluation of an embodiment of T66 mRNA HIV-1 reactivation (reversal) on a latent HIV reporter T cell line using a variety of transfection reagents.
[0022] FIG. 8 A shows an embodiment of latency reversal activity comparison in MT4 reporter cells between T66 mRNA and its protein counterpart as determined by the increase of GFP (+) cells through HIV LTR activation. FIG. 8B shows an embodiment of the same comparison between T66 mRNA and its protein counterpart as determined by HIV LTR driven increased luciferase expression.
[0023] FIG. 9 shows an embodiment of T66 mRNA LTR activation in Jurkat HIV-GFP reporter cells along with activators such as Phorbol 12-Myristate 13-Acetate (PMA), CD3/CD28 and T66 protein. The increase of GFP (+) cells incubated with T66 mRNA exceeded the reactivation achieved by T66 protein or CD3/CD28.
[0024] FIG. 10 shows an embodiment of a T66 mRNA dose response HIV activation in latent T-cell clone Jurkat cl50.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed. [0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.
[0027] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
[0028] Unless otherwise stated, any numerical value, such as a % sequence identity or a % sequence identity range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a dosage of 10 mg includes 9 mg to 11 mg. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
[0029] As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
[0030] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.
[0031] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.”
[0032] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the invention can be replaced with the term “consisting of’ or “consisting essentially of’ to vary scopes of the disclosure.
[0033] The phrases “percent (%) sequence identity” or “% identity” or “% identical to” when used with reference to an amino acid or nucleic acid sequence describe the number of matches (“hits”) of identical amino acids or nucleic acids of two or more aligned am amino acid or nucleic acid sequences as compared to the number of amino acid or nucleic acid residues making up the overall length of the amino acid or nucleic acid sequences. In other terms, using an alignment, for two or more sequences the percentage of amino acid or nucleic acid residues that are the same (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full-length of the amino acid or nucleic acid sequences) may be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The same determination may be made for nucleotide sequences. The sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acid or nucleic acid. Suitable programs for aligning protein sequences are known to the skilled person. The percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g., using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).
[0034] As used herein, the terms and phrases “in combination,” “in combination with,” “co-delivery,” and “administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration of two or more therapies or components, such as two nucleic acid molecules, e.g., ribonucleic acid molecules and an adjuvant. “Simultaneous administration” can be administration of the two components at least within the same day. When two components are “administered together with” or “administered in combination with,” they can be administered in separate compositions sequentially within a short time period, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour, or they can be administered in a single composition at the same time. The use of the term “in combination with” does not restrict the order in which therapies or components are administered to a subject. For example, a first therapy or component (e.g. first ribonucleic acid molecule) can be administered prior to (e.g., 5 minutes to one hour before), concomitantly with or simultaneously with, or subsequent to (e.g., 5 minutes to one hour after) the administration of a second therapy or component (e.g., second ribonucleic acid molecule). In some embodiments, a first therapy or component (e.g. first ribonucleic acid molecule) and a second therapy or component (e.g., e.g., second ribonucleic acid molecule) are administered in the same composition. In other embodiments, a first therapy or component (e.g. first ribonucleic acid molecule) and a second therapy or component (e.g., second ribonucleic acid molecule) are administered in separate compositions.
[0035] As used herein, a “non-naturally occurring” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature. A “non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting. In some cases, a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment. As used herein, a “non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source. A “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
[0036] As used herein, the term “operably linked” refer to a linkage or a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing the transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest over a membrane.
[0037] As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the application. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human. A human subject can include a patient.
[0004] In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. For example, while embodiments of ribonucleic acid molecules of the application described herein may contain particular components, including, but not limited to, 5’ untranslated region (5’ UTR), 3’ UTR, coding sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, polyadenylation signal sequences, etc. arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in ribonucleic acid molecules of the application. The application contemplates use of any of the applicable components in any combination having any sequence that can be used in ribonucleic acid molecules of the application, whether or not a particular combination is expressly described.
[0038] As used herein, “HIV-1” means the human immunodeficiency virus type-1. HIV-1 includes but is not limited to extracellular virus particles and the forms of HIV- 1 found in HIV- 1 infected cells.
[0039] As used herein, “HIV-1 infection” means the introduction of HIV- 1 genetic information into a target cell, such as by fusion of the target cell membrane with HIV- 1 or an HIV-1 envelope glycoprotein+ cell. The target cell may be a bodily cell of a subject. In the preferred embodiment, the target cell is a bodily cell from a human subject.
Polynucleotides
[0040] In a general aspect, the application provides a ribonucleic acid molecule (e.g., a messenger RNA (mRNA) molecule or circular RNA (circRNA) molecule) comprising a non- naturally occurring polynucleotide sequence encoding a mutant of the HIV-derived accessory protein tat (trans-activator of transcription) for the reactivation and subsequent eradication of latent HIV. The ribonucleic acid molecule can, for example, be a non-self replicating ribonucleic acid molecule (i.e., a non-self replicating RNA), more particularly a non-self replicating mRNA or circRNA. A ribonucleic acid molecule can comprise any non-naturally occurring polynucleotide sequence encoding the mutant of the HIV-derived accessory protein tat of the application, which can be made using methods known in the art in view of the present disclosure.
[0041] In certain embodiments, the ribonucleic acid molecule comprises a non-naturally occurring polynucleotide sequence comprising a polynucleotide sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV but more preferably said polynucleotide sequence encoding a protein comprising at least the first 57 N- terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV. In some embodiments, the polynucleotide sequence encoding a protein comprising a protein having at least the first 60 N-terminal amino acids of wild-type tat (86-101 aa) for the reactivation of latent HIV.
[0042] In an embodiment of the application, tatl-57, which contains the 57 N-terminal amino acids, is represented by the following SEQ ID No. 1: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQR RR.
[0043] In an embodiment of the application, tatl-60, which contains the first 60 N- terminal amino acids, is represented by the following SEQ ID No; 2: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQR RRAHQ.
[0044] In another embodiment, the polynucleotide sequence encoding a protein comprising the 66 amino acid deletion mutant [T66 or tatl-66] according to an embodiment of the invention with a reactivation capacity close to full length exon 1 Tat72 has the amino acid sequence of SEQ ID No. 3: MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQR RRAHQNSQTHQ
[0045] In Table 1 below substitutions are provided for each position in these SEQ ID Nos. 1, 2 and 3 respectively for which a polynucleotide sequence capable of encoding said protein is within the scope of the present invention. Table 1
Figure imgf000013_0001
Figure imgf000014_0001
[0046] In some embodiments, the polynucleotide sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity to one or more of the following sequences within the RNA molecule: Table 2
Figure imgf000015_0001
Components of ribonucleic acid
[0047] In certain embodiments, the ribonucleic acid molecule further comprises a 5 ’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR), and/or a polyadenylation sequence.
[0048] As used herein a “5’ untranslated region” or “5’ UTR” refers to a region of an mRNA or circRNA that is directly upstream (i.e., 5’) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
[0049] As used herein a “3’ untranslated region” or “3’ UTR” refers to a region of an mRNA or circRNA that is directly downstream (i.e., 3’) from the stop codon (i.e., the codon of an mRNA or circRNA transcript that signals a termination of translation) that does not encode a polypeptide.
[0050] As used herein “an open reading frame” is a continuous stretch of codons beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG, or TGA) that encodes a polypeptide.
[0051] As used herein a “polyA tail” or “polyadenylation sequence” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, conservative adenosine monophosphates. A poly A tail can contain 10 to 300 adenosine monophosphates, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. The polyA tail can, e.g., function to protect mRNA from enzymatic degradation, e.g., in the cytoplasm of a cell, and can, e.g., aid in transcription termination, export of the mRNA from the nucleus, and translation.
[0052] In certain embodiments, the application also relates to a DNA molecule encoding an RNA molecule of the application, preferably the DNA molecule contains a promoter operationally linked to a nucleic acid sequence encoding the RNA molecule. Examples of promoters that can be used include, but are not limited to, a T3 promoter, a T7 promoter, and an SP6 promoter. A promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. A promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Preferably, a promoter is a T7 promoter. In some embodiments, a nucleotide sequence of an exemplary T7 promoter comprises the sequence TAATACGACTCACTATAG or TAATACGACTCACTATAAG.
[0053] Any enhancer sequence known to those skilled in the art in view of the present disclosure can be used. For example, an enhancer sequence can comprise an untranslated region.
[0054] The polynucleotides encoding the wild-type HIV-derived accessory protein tat for the reactivation of latent HIV of the application can be made by any method known in the art in view of the present disclosure. For example, a polynucleotide encoding a wild-type HIV- derived accessory protein tat for the reactivation of latent HIV can be introduced or “cloned” into an expression vector using standard molecular biology techniques, e.g., polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.
[0055] Also provided are methods of producing a ribonucleic acid molecule of the application, comprising transcribing a nucleic acid molecule comprising a DNA sequence encoding a ribonucleic acid molecule disclosed herein. In some embodiments, the nucleic acid molecule is transcribed in vivo. In some embodiments, the nucleic acid molecule is transcribed in vitro.
[0056] In certain embodiments, the mRNA or circRNA is formed by transcribing from a DNA template, wherein the following elements are introduced in a pDNA vector: a T7 promoter, followed by a nucleotide sequence corresponding to a 5’-UTR and a 3’-UTR from either the human a-globin [HAG] or the frog (Xenopus) a-globin [XBG], terminated by a 120 nt polyA tail. [Fig5] . In some embodiments, a capping reagent, such as AG CleanCap (Trilink), is applied and the wild type T7 promoter sequence is changed in the DNA template, e g., from TAATACGACTCACTATAGG to TAATACGACTCACTATAAG. In some embodiments, the sequences are selected from those in Table 3. In other embodiments, the sequences have 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 point mutations from the sequences in Table 2.
Table 3
Figure imgf000017_0001
[0057] In some embodiments, a polynucleotide sequence encoding at least a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV (also referred to in some embodiments as HIV-1 T66 codon-optimized gene sequence) is introduced via restriction enzymes-based cloning into the DNA template plasmid between the coding sequence of the 5 -UTR and 3’-UTR, as shown in FIG.5.
[0058] In another general aspect, the application relates to an isolated ribonucleic acid molecule comprising: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; and (4) a poly A tail containing 100 to 140 adenosine monophosphates. In certain embodiment, the wild-type HIV-derived accessory protein tat comprises at least the first 57 N-terminal amino acids of a wild-type tat (86-101 aa) for the reactivation of latent HIV. In certain embodiment, the wild-type HIV- derived accessory protein tat is tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl-64, tatl-60, tatl-57, or tat2-72. In certain embodiment, the wild-type HIV-derived accessory protein tat comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3. In certain embodiment, the wild-type HIV-derived accessory protein tat comprises one or more substitutions in Table 1 in the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3.
[0059] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
[0060] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail containing 120 adenosine monophosphates.
[0061] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
[0062] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail containing 120 adenosine monophosphates.
[0063] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
[0064] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 4; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail containing 120 adenosine monophosphates.
[0065] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
[0066] In some embodiment, the isolated ribonucleic acid molecule comprises: (1) a 5’ UTR having the polynucleotide sequence of SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) a 3’ UTR having the polynucleotide sequence of SEQ ID NO: 7; and (4) a poly A tail containing 120 adenosine monophosphates.
[0067] In another general aspect, the application relates to a DNA molecule encoding the isolated ribonucleic acid molecule. In some embodiment, an RNA molecule of the application is transcribed from a DNA molecule comprising a DNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 11, which is transcribed into a 5’UTR; a DNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 13, which is transcribed into a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV; and a DNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 12, which is transcribed into a 3’UTR. A poly A tail containing 100 to 140 adenosine monophosphates is then added to the 3 ’-end of the transcribed RNA.
Modified Nucleotides
[0068] In some embodiments, the ribonucleic acid molecule contains one or more modified nucleobase. Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5 -methylcytosine (5- me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6- methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), Nl- Methypseudouridine, 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5 -trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3- deazaadenine. In preferred embodiments, the ribonucleic acid molecule contains one or more Nl-Methypseudouridine nucleobases.
Compositions
[0069] The application also relates to compositions and pharmaceutical compositions comprising one or more polynucleotide encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, polynucleotides, and/or vectors encoding one more polynucleotide encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV according to the application. Any of the polynucleotide encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, polynucleotides (including RNA, e.g., mRNA or circRNA) of the application described herein can be used in the compositions and pharmaceutical compositions of the application.
[0070] The application provides, for example, a pharmaceutical composition comprising any nucleic acid molecule and/or vector described herein, together with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/ suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
[0071] Pharmaceutical compositions of the application can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Pharmaceutical compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
[0072] In a preferred embodiment of the application, pharmaceutical compositions of the application are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection. [0073] According to embodiments of the application, pharmaceutical compositions for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS). The compositions can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents. For example, a pharmaceutical composition of the application comprising a ribonucleic acid molecule can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier. The ribonucleic acid molecule can be administered at 1-1000 pg/dose, e.g., 1 pg/dose, 10 pg/dose, 20 pg/dose, 30 pg/dose, 40 pg/dose, 50 pg/dose, 60 pg/dose, 70 pg/dose, 80 pg/dose, 90 pg/dose, 100 pg/dose, 200 pg/dose, 300 pg/dose, 400 pg/dose, 500 pg/dose, 600 pg/dose, 700 pg/dose, 800 pg/dose, 900 pg/dose, 1000 pg/dose, or any number in between.
[0074] In certain embodiments, an adjuvant is included in a pharmaceutical composition of the application or co-administered with a pharmaceutical composition of the application. Use of an adjuvant is optional and can further enhance immune responses when the composition is used for vaccination purposes. Adjuvants suitable for co-administration or inclusion in compositions in accordance with the application should preferably be ones that are potentially safe, well tolerated and effective in humans. An adjuvant can be an anti-viral agent such as a small molecule and/or antibody directed towards HIV, but not limited to Nucleoside/Nucleotide Reverse Transcriptase Inhibitors, such as Abacavir, or Didanosine, Emtricitabine, Lamivudine, Stavudine, Tenofovir alafenamide, Tenofovir disoproxil fumarate, Zidovudine; Non-nucleoside Reverse Transcriptase Inhibitors, such as, Cabotegravir/rilpivirine, Delavirdine, Doravirine, Efavirenz, Etravirine, Nevirapine, Rilpivirine; Protease Inhibitors, such as, Atazanavir, Darunavir, Fosamprenavir, Indinavir, Lopinavir + ritonavir, Nelfmavir, Ritonavir, Saquinavir, Tipranavir; Integrase Inhibitors, such as, Bictegravir, Cabotegravir and rilpivirine, Cabotegravir, Dolutegravir, Elvitegravir, Raltegravir; Fusion Inhibitors, such as, Enfuvirtide; gpl20 Attachment Inhibitors, CCR5 Antagonist; Post-Attachment Inhibitors or Monoclonal Antibodies, such as, Ibalizumab-uiyk; Pharmacologic Enhancers, and the like. In some embodiments, an active compound of the application is administered with an epigenetic drug, such as a histone deacetylase (HDAC) inhibitor (e.g. Vorinostat, Romidepsin, Panobinostat, Belinostat) or a BET inhibitor (e.g. JQ1). Pharmaceutically Acceptable Encapsulating Carrier
[0075] In another general aspect, the application relates to a pharmaceutical composition comprising an active compound of the application (e.g., a polynucleotide encoding a wildtype HIV-derived accessory protein tat for the reactivation of latent HIV, e.g., mRNA or circRNA), and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is an encapsulating carrier, such as a nanoparticle encapsulating an active compound of the application. Nonlimiting examples of encapsulating nanoparticles include lipid nanoparticles (LNPs), liposomes, lipoplexes, etc.
[0076] In some embodiments, the active compound of the application is formulated using one or more liposomes, lipoplexes, polymer-based encapsulating formulation, and/or lipid nanoparticles. In some embodiments, liposome or lipid nanoparticle formulations described herein can comprise a polycationic composition. In some embodiments, the formulations comprising a polycationic composition can be used for the delivery of the active compound of the application described herein in vivo and/or ex vitro.
[0077] According to the present invention, the term "lipid" refers to any fatty acid derivative or other amphiphilic compound which is capable of forming a lyotropic lipid phase, or more preferentially, a lamellar lyotropic phase. In particular, the term "lipid" refers to any fatty acid derivative which is capable of forming a bilayer such that a hydrophobic part of the lipid molecule orients toward the bilayer while a hydrophilic part orients toward the aqueous phase. The term "lipid" comprises neutral, anionic or cationic lipids and combinations thereof. Lipids preferably comprise a hydrophobic domain with at least one, preferably two, or more, alkyl chains or a cholesterol moiety and a polar head group. The alkyl chains of the fatty acids in the hydrophobic domain of the lipid are not limited to a specific length or number of double bonds. Nevertheless, it is preferred that the fatty acid has a length of 10 to 30, preferably 14 to 25 carbon atoms. The lipid may also comprise two different fatty acids.
[0078] In the context of the present disclosure, a lipid-based delivery vehicle typically serves to transport a desired RNA to a target cell or tissue. In some embodiments, the lipid- based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and an RNA of the present disclosure. In some embodiments, the lipid bilayer preferably further comprises a neutral lipid or a polymer. The term “neutral lipid” means a lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols. In some embodiments, the lipid formulation preferably comprises a liquid medium. In some embodiments, the formulation preferably further encapsulates a nucleic acid. In some embodiments, the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or a polymer. In some embodiments, the lipid formulation preferably encapsulates the nucleic acid.
[0079] The description provides lipid formulations comprising one or more RNA encapsulated within the lipid formulation. In some embodiments, the lipid formulation comprises liposomes. In some embodiments, the lipid formulation comprises cationic liposomes. In some embodiments, the lipid formulation comprises lipid nanoparticles.
[0080] In some embodiments, the RNA or combination of nucleic acid molecules is fully encapsulated within the lipid portion of the lipid formulation such that the RNA or combination of nucleic acid molecules in the lipid formulation is resistant in aqueous solution to nuclease degradation. The term “fully encapsulated” means that the nucleic acid (e.g., mRNA or circRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free RNA. When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10%, and most preferably less than 5% of the nucleic acid in the particle is degraded. “Fully encapsulated” as used herein also means that the nucleic acid-lipid particles do not rapidly decompose into their component parts upon in vivo administration. In other embodiments, the lipid formulations described herein are substantially non-toxic to mammals such as humans. In some embodiments, the combination of nucleic acids is encapsulated within the same lipid nanoparticle. In some embodiments, each nucleic acid molecule in the combination of nucleic acid molecules is independently encapsulated in individual lipid nanoparticles.
[0081] The lipid formulations of the disclosure also typically have a total lipid: RNA ratio (mass/mass ratio) of from about 1 : 1 to about 100: 1, from about 1 : 1 to about 50: 1, from about 2: 1 to about 45: 1, from about 3: 1 to about 40: 1, from about 5: 1 to about 38: 1, or from about 6: 1 to about 40: 1, or from about 7: 1 to about 35: 1, or from about 8: 1 to about 30: 1; or from about 10: 1 to about 25: 1; or from about 8: 1 to about 12: 1; or from about 13: 1 to about 17: 1; or from about 18 : 1 to about 24 : 1 ; or from about 20 : 1 to about 30 : 1. In some preferred embodiments, the total lipid:RNA ratio (mass/mass ratio) is from about 10: 1 to about 25: 1. The ratio may be any value or sub value within the recited ranges, including endpoints. [0082] The lipid formulations of the present disclosure typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or subvalue within the recited ranges, including endpoints. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.
[0083] In preferred embodiments, the lipid formulations comprise an RNA or combination of nucleic acid molecules, a cationic lipid (e.g. , one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The lipid formulations can also include cholesterol. The term “lipid conjugate” means a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, and mixtures thereof. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties, such as amides or carbamates, are used. In certain preferred embodiments, the PEG-lipid conjugate is 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (i.e., ALC-0159).
[0084] The term “cationic lipid” as used herein refers to amphiphilic lipids and salts thereof having a positive, hydrophilic head group; one, two, three, or more hydrophobic (e.g., having apolar groups) fatty acid or fatty alkyl chains; and a connector between these two domains. An ionizable or protonatable cationic lipid is typically protonated (i.e., positively charged) at a pH below its pKa and is substantially neutral at a pH above the pKa. Preferred ionizable cationic lipids are those having a pKa that is less than physiological pH, which is typically about 7.4. The cationic lipids of the disclosure may also be termed titratable cationic lipids. The cationic lipids can be an “amino lipid” having a protonatable tertiary amine (e.g., pH-titratable) head group. Some amino exemplary amino lipid can include Cis alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains. Such cationic lipids include, but are not limited to, (4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2 -hexyldecanoate (also known as ALC-0315), Lipofectin™ also known as DOTMA (N-D-(2,3-dioleyloxy) propyls N,N, N-trimethylammonium chloride), DOTAP (1,2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB (dimethyldioctadecyl-ammonium bromide), DOGS (dioctadecylamidologlycyl spermine), DSDMA, DODMA, DLinDMA, DLenDMA, y- DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin- M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), (DLin-MP- DMA)(also known as 1-B1 1), and cholesterol derivatives such as DCChol (3 beta-(N — (N',N'-dimethyl aminomethane)-carbamoyl) cholesterol). In certain preferred embodiments, the cationic lipid is ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate), i.e., ALC-0315.
[0085] The term “anionic lipid” as used herein refers to a lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
[0086] In the nucleic acid-lipid formulations, the RNA or combination of nucleic acid molecules may be fully encapsulated within the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation. In preferred embodiments, a lipid formulation comprising an RNA or combination of nucleic acid molecules is fully encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the RNA or combination of nucleic acid molecules in the lipid formulation is not substantially degraded after exposure of the particle to a nuclease at 37 °C for at least 20, 30, 45, or 60 minutes. In certain other instances, the RNA or combination of nucleic acid molecules in the lipid formulation is not substantially degraded after incubation of the formulation in serum at 37 °C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the RNA or combination of nucleic acid molecules is complexed with the lipid portion of the formulation.
[0087] In the context of nucleic acids, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with a nucleic acid. Encapsulation is determined by adding the dye to a lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent.
Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E = (Io - I)/Io, where I and Io refer to the fluorescence intensities before and after the addition of detergent.
[0088] In other embodiments, the present disclosure provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA lipid nanoparticles.
[0089] In some embodiments, the lipid formulations comprise an RNA or combination of nucleic acid molecules that is fully encapsulated within the lipid portion of the formulation, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction thereof or range therein) of the particles have the RNA or combination of nucleic acid molecules encapsulated therein. The amount may be any value or sub value within the recited ranges, including endpoints.
[0090] Depending on the intended use of the lipid formulation, the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.
[0091] According to some embodiments, the expressible polynucleotides and RNA described herein are lipid formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes, and lipid nanoparticles. In one preferred embodiment, a lipid formulation is a cationic liposome or a lipid nanoparticle (LNP) comprising:
(a) an RNA or combination of nucleic acid molecules of the present disclosure,
(b) a cationic lipid,
(c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG- modified lipid),
(d) optionally a non-cationic lipid (such as a neutral lipid), and
(e) optionally, a sterol.
[0092] Preferably, the lipid nanoparticle encapsulating the RNA or combination of nucleic acid molecules comprises a cationic lipid and at least one other lipid selected from the group consisting of anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer conjugated lipids, phospholipids, glycolipids, and combinations thereof.
[0093] In some embodiments, the cationic lipid is an ionizable cationic lipid. In one embodiment, the lipid nanoparticle formulation consists of (i) at least one cationic lipid; (ii) a helper lipid; (iii) a sterol (e.g. , cholesterol); and (iv) a PEG-lipid, in a molar ratio of about 30% to about 60% ionizable cationic lipid: about 5% to about 20% helper lipid: about 35% to about 50% sterol: about 0.5-5% PEG-lipid. Example cationic lipids (including ionizable cationic lipids), helper lipids (e.g., neutral lipids), sterols, and ligand-containing lipids (e.g., PEG-lipids) are described herein below.
[0094] The selection of specific lipids and their relative % compositions depends on several factors including the desired therapeutic effect, the intended in vivo delivery target, and the planned dosing regimen and frequency. Generally, lipids that correspond to both high potency (i.e., therapeutic effect such as knockdown activity or translation efficiency) and biodegradability resulting in rapid tissue clearance are most preferred. However, biodegradability may be less important for formulations that are intended for only one or two administrations within the subject. In addition, the lipid composition may require careful engineering so that the lipid formulation preserves its morphology during in vivo administration and its journey to the intended target, but will then be able to release the active agent upon uptake into target cells. Thus, several formulations typically need to be evaluated in order to find the best possible combination of lipids in the best possible molar ratio of lipids as well as the ratio of total lipid to active ingredient.
[0095] Suitable lipid components and methods of manufacturing lipid nanoparticles are well known in the art and are described for example in PCT/US2020/023442, U.S. 8,058,069, U.S. 8,822,668, U.S. 9,738,593, U.S. 9,139,554, PCT/US2014/066242, PCT/US2015/030218, PCT/2017/015886, and PCT/US2017/067756, the contents of which are incorporated by reference. Additional lipid components and compositions embodied in the present disclosure include those in WO/2015/199952, WO/2017/004143, WO/2017/075531 , WO/2020/081938, WO/2017/201091, WO/2019/246203, WO/2020/051223, and WO/2022/040641, the contents of which are incorporated by reference.
Cationic Uipids
[0096] The lipid formulation preferably includes a cationic lipid suitable for forming a cationic liposome or lipid nanoparticle. Cationic lipids are widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails, or a steroid portion and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at about physiological pH. Cationic liposomes have been traditionally the most commonly used non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA/small hairpin RNA- shRNA. Cationic lipids, such as DOTAP, (l,2-dioleoyl-3- trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids by electrostatic interaction, providing high in vitro transfection efficiency.
[0097] In the presently disclosed lipid formulations, the cationic lipid may be, for example, ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate) (also known as ALC-0315), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl- N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and l,2-Dioleyloxy-3-trimethylaminopropane chloride salt), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), l,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y- linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA), l,2-Dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1, 2-Dilinoley oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), l,2-Dilinoleoyl-3- dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S- DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2- Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-Dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl- [l,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2- di((9Z, 12Z)-octadeca-9, 12-dienyl)tetrahydro-3aH-cyclopenta[d] [l,3]dioxol-5-amine, (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2- hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethylazanediyl)didodecan-2-ol (Cl 2-200), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28 3 l-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3- ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N,N-dimethylpropan-l -amine (MC3 Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N- dimethylbutan-l-amine (MC4 Ether), or any combination thereof. Other cationic lipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P- (N-(N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Choi), N-(l-(2,3- dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dileoyl-sn- 3 -phosphoethanolamine (DOPE), l,2-dioleoyl-3 -dimethylammonium propane (DODAP), N- (l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO/BRL).
[0098] Other suitable cationic lipids are disclosed in International Publication Nos. WO 09/086558, WO 09/127060, WO 10/048536, WO 10/054406, WO 10/088537, WO 10/129709, and WO 2011/153493; U.S. Patent Publication Nos. 2011/0256175, 2012/0128760, and 2012/0027803; U.S. Patent No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are herein incorporated by reference.
[0099] Other suitable cationic lipids include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N-ethyl- N-methylamino-, and N-propyl-N -ethylamino-). These lipids are part of a subcategory of cationic lipids referred to as amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of fdter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of Ci4 to C22 may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.
[0100] In some embodiments, the lipid formulation comprises the cationic lipid with Formula I according to the patent application PCT/EP2017/064066. In this context, the disclosure of PCT/EP2017/064066 is also incorporated herein by reference.
[0101] In some embodiments, amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. Of course, it will be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the disclosure. In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of an ionizable cationic lipid is about 6 to about 7.
[0102] In some embodiments, the lipid formulation comprises an ionizable cationic lipid of Formula I:
Figure imgf000032_0001
or a pharmaceutically acceptable salt or solvate thereof, wherein R5 and R6 are each independently selected from the group consisting of a linear or branched C1-C31 alkyl, C2-C31 alkenyl or C2-C31 alkynyl and cholesteryl; L5 and L6 are each independently selected from the group consisting of a linear C1-C20 alkyl and C2-C20 alkenyl; X5 is -C(O)O-, whereby -C(O)O- R6 is formed or -OC(O)- whereby -OC(O)-R6 is formed; X6 is -C(O)O- whereby -C(O)O-R5 is formed or
-OC(O)- whereby -OC(O)-R5 is formed; X7 is S or O; L7 is absent or lower alkyl; R4 is a linear or branched Ci-Ce alkyl; and R7 and R8 are each independently selected from the group consisting of a hydrogen and a linear or branched Ci-Ce alkyl.
[0103] In some embodiments, X7 is S.
[0104] In some embodiments, X5 is -C(O)O-, whereby -C(O)O-R6 is formed and X6 is - C(O)O- whereby -C(O)O-R5 is formed.
[0105] In some embodiments, R7 and R8 are each independently selected from the group consisting of methyl, ethyl and isopropyl.
[0106] In some embodiments, L5 and L6 are each independently a C1-C10 alkyl. In some embodiments, L5 is C1-C3 alkyl, and L6 is C1-C5 alkyl. In some embodiments, L6 is C1-C2 alkyl. In some embodiments, L5 and L6 are each a linear C7 alkyl. In some embodiments, L5 and L6 are each a linear C9 alkyl.
[0107] In some embodiments, R5 and R6 are each independently an alkenyl. In some embodiments, R6 is alkenyl. In some embodiments, R6 is C2-C9 alkenyl. In some embodiments, the alkenyl comprises a single double bond. In some embodiments, R5 and R6 are each alkyl. In some embodiments, R5 is a branched alkyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a C9 alkyl, C9 alkenyl and C9 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a C11 alkyl, C11 alkenyl and C11 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a C7 alkyl, C7 alkenyl and C7 alkynyl. In some embodiments, R5 is -CH((CH2)pCH3)2 or -CH((CH2)pCH3)((CH2)p-iCH3), wherein p is 4-8. In some embodiments, p is 5 and L5 is a C1-C3 alkyl. In some embodiments, p is 6 and L5 is a C3 alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L5 is a C1-C3 alkyl. In some embodiments, R5 consists of -CH((CH2)pCH3)((CH2)p-iCH3), wherein p is 7 or 8.
[0108] In some embodiments, R4 is ethylene or propylene. In some embodiments, R4 is n- propylene or isobutylene.
[0109] In some embodiments, L7 is absent, R4 is ethylene, X7 is S and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is n-propylene, X7 is S and R7 and R8 are each methyl. In some embodiments, L7is absent, R4 is ethylene, X7 is S and R7and R8 are each ethyl.
[0110] In some embodiments, X7 is S, X5 is -C(O)O-, whereby -C(O)O-R6 is formed, X6 is -C(O)O- whereby -C(O)O-R5 is formed, L5 and L6 are each independently a linear C3-C7 alkyl, L7 is absent, R5 is -CH((CH2)pCH3)2, and R6 is C7-C12 alkenyl. In some further embodiments, p is 6 and R6 is C9 alkenyl.
[OlH] In some embodiments, the lipid formulation comprises an ionizable cationic lipid selected from the group consisting of
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
[0112] Several cationic lipids have been described in the literature, many of which are commercially available. For example, suitable cationic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP), 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane (DLinDMA), and 1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA). The pKa of formulated cationic lipids is correlated with the effectiveness of lipid particles for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). The preferred range of pKa is ~5 to ~7.
[0113] In one embodiment, the cationic lipid is a compound of Formula (I’):
Figure imgf000041_0002
Formula (I’) wherein Ri is a substituted alkyl consisting of 10 to 31 carbons, R2 is a linear alkyl, alkenyl or alkynyl consisting of 2 to 20 carbons, Rs is a linear or branched alkane consisting of 1 to 6 carbons, R4 and Rs are the same or different, each a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; Li and L2 are the same or different, each a linear alkane of 1 to 20 carbons or a linear alkene of 2 to 20 carbons, and Xi is S or O; or a salt or solvate thereof. Exemplary compounds of formula (I), their synthesis and uses thereof are described in US2018/0169268, all of which are herein incorporated by reference.
[0114] In another embodiment, the cationic lipid is a compound of formula (II’):
Figure imgf000042_0001
Formula (IF) wherein Ri is a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; Li is linear alkane of 1 to 15 carbons; R2 is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons or a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; L2 is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons; X is O or S; Rs is a linear alkane of 1, 2, 3, 4, 5, or 6 carbons; and R4 and Rs are the same or different, each a linear or branched, noncyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons; or a pharmaceutically acceptable salt or solvate thereof.
Exemplary compounds of formula (II), their synthesis and uses thereof are described in US2018/0170866, all of which are herein incorporated by reference.
[0115] In another embodiment, the cationic lipid is a compound of formula (III’), (IV’) or (V’):
Figure imgf000042_0002
(III’) (IV’) (V’) wherein R comprises a biologically active molecule, and Li, L2, and Ls independently for each occurrence comprise a ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile; a pharmaceutically acceptable salt thereof; or a pharmaceutical composition thereof. Exemplary compounds of formula (III’), (IV’) and (V’), their synthesis and uses thereof are described in US2017/0028074, all of which are herein incorporated by reference.
[0116] In another embodiment, the cationic lipid is a compound of formula (VI’):
Figure imgf000043_0001
Formula (VI’) wherein X is a linear or branched alkylene or alkenylene, monocyclic, bicyclic, or tricyclic arene or heteroarene; Y is a bond, an ethene, or an unsubstituted or substituted aromatic or heteroaromatic ring; Z is S or O; L is a linear or branched alkylene of 1 to 6 carbons; R and R4 are independently a linear or branched alkyl of 1 to 6 carbons; Ri and R2 are independently a linear or branched alkyl or alkenyl of 1 to 20 carbons; r is 0 to 6; and m, n, p, and q are independently 1 to 18; wherein when n=q, m=p, and Ri=Ra, then X and Y differ; wherein when X=Y, n=q, m=p, then Ri and R2 differ; wherein when X=Y, n=q, and Ri=Ra, then m and p differ; and wherein when X=Y, m=p, and Ri=Ra, then n and q differ; or a pharmaceutically acceptable salt thereof. Exemplary compounds of formula (VI’), their synthesis and uses thereof are described in US2017/0190661, all of which are herein incorporated by reference.
[0117] In another embodiment, the cationic lipid is a compound of formula (VII’):
Figure imgf000043_0002
Formula (VIE) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or
G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — ,
— S(O)y-, — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— , and the other of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)y-, — S — S‘, — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Rais, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000044_0001
a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 5 to 10; d1 and d2are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
[0118] In another embodiment, the cationic lipid is a compound of formula (VIII’):
Figure imgf000045_0001
Formula (VIII’) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — ,
— S(O)y-, — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— , and the other of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)y-, — S— S— , — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— ,
— N(Ra)C(=O)N(Ra) — , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Rais, at each occurrence, independently H, C1-C12 alkyl, Ci-C 12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, Ci-C 12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000045_0002
R' is, at each occurrence, independently H or C1-C12 alkyl; a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 2 to 12; d1 and d2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a1, a2, c1, c2, d1 and d2 are selected such that the sum of a^^+d1 is an integer from 18 to 30, and the sum of a2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
[0119] Exemplary compounds of formula (VII’) and (VIII’), their synthesis and uses thereof are described in US20190022247, all of which are herein incorporated by reference.
[0120] Additional cationic lipids that can be used in compositions of the application include, but are not limited to, those described in W02019/036030, W02019/036028, W02019/036008, WO2019/036000, US2016/0376224, US2017/0119904, W02018/200943 and WO2018/191657, the relevant contents on the lipids, their synthesis and uses are herein incorporated by reference in their entireties.
[0121] In some embodiments, any one or more lipids recited herein may be expressly excluded.
Helper Lipids and Sterols
[0122] The RNA lipid formulations of the present disclosure can comprise a helper lipid, which can be referred to as a neutral lipid, a neutral helper lipid, non-cationic lipid, noncationic helper lipid, anionic lipid, anionic helper lipid, or a zwitterionic lipid. It has been found that lipid formulations, particularly cationic liposomes and lipid nanoparticles have increased cellular uptake if helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9):882-92). For example, some studies have indicated that neutral and zwitterionic lipids such as l,2-dioleoyl-sn-glycero-3 -phosphatidylcholine (DOPC), Di-Oleoyl- Phosphatidyl-Ethanoalamine (DOPE) and l,2-DiStearoyl-sn-glycero-3-PhosphoCholine (DSPC), being more fusogenic (i.e., facilitating fusion) than cationic lipids, can affect the polymorphic features of lipid-nucleic acid complexes, promoting the transition from a lamellar to a hexagonal phase, and thus inducing fusion and a disruption of the cellular membrane. (Nanomedicine (Lond). 2014 Jan; 9(1): 105-20). In addition, the use of helper lipids can help to reduce any potential detrimental effects from using many prevalent cationic lipids such as toxicity and immunogenicity.
[0123] Non-limiting examples of non-cationic lipids suitable for lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
[0124] Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof. One study concluded that as a helper lipid, cholesterol increases the spacing of the charges of the lipid layer interfacing with the nucleic acid making the charge distribution match that of the nucleic acid more closely. (J. R. Soc. Interface. 2012 Mar 7; 9(68): 548-561). Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy) -butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.
[0125] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid formulation. In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation.
[0126] Other examples of helper lipids include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine -lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.
[0127] In some embodiments, the helper lipid comprises from about 30 mol% to about 60 mol%, from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, about 35 mol% to about 54 mol%, from about 36 mol% to about 52 mol%, from about 37 mol% to about 51 mol%, from about 38 mol% to about 50 mol%, or about 39 mol%, about 50 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, or about 49 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.
[0128] In some embodiments, the total of helper lipid in the formulation comprises two or more helper lipids and the total amount of helper lipid comprises from about 30 mol% to about 60 mol%, from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, about 35 mol% to about 54 mol%, from about 36 mol% to about 52 mol%, from about 37 mol% to about 51 mol%, from about 38 mol% to about 50 mol%, or about 39 mol%, about 50 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, or about 49 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation. In some embodiments, the helper lipids are a combination of DSPC and DOTAP. In some embodiments, the helper lipids are a combination of DSPC and DOTMA.
[0129] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 50 mol%, about 35 mol%, about 40 mol%, about 45 mol%, or about 50 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 45 mol%, about 30 mol% to about 45 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, or about 45 mol% of the total lipid present in the lipid formulation.
[0130] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ± 5 mol%.
[0131] A lipid formulation containing a cationic lipid compound or ionizable cationic lipid compound may be on a molar basis about 30-60% cationic lipid compound, about 35-50 % cholesterol, about 5-20% helper lipid, and about 0.5-5% of a polyethylene glycol (PEG) lipid, wherein the percent is of the total lipid present in the formulation. In some embodiments, the composition is about 40-50% cationic lipid compound, about 35-45% cholesterol, about 5-15% helper lipid, and about 0.5-3% of a PEG-lipid, wherein the percent is of the total lipid present in the formulation.
Lipid Conjugates
[0132] The lipid formulations described herein may further comprise a lipid conjugate. The conjugated lipid is useful for preventing the aggregation of particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic-polymer-lipid conjugates, and mixtures thereof. Furthermore, lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front. Pharmacol. 2015 Dec 1; 6:286).
[0133] In a preferred embodiment, the lipid conjugate is a PEG-lipid. The inclusion of polyethylene glycol (PEG) in a lipid formulation as a coating or surface ligand, a technique referred to as PEGylation, helps protect nanoparticles from the immune system and their escape from RES uptake (Nanomedicine (Lond). 2011 Jun; 6(4): 715-28) . PEGylation has been widely used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms. Detergent-like PEG lipids (e.g., PEG-DSPE) can enter the lipid formulation to form a hydrated layer and steric barrier on the surface. Based on the degree of PEGylation, the surface layer can be generally divided into two types, brush-like and mushroom-like layers. For PEG-DSPE-stabilized formulations, PEG will take on the mushroom conformation at a low degree of PEGylation (usually less than 5 mol%) and will shift to brush conformation as the content of PEG-DSPE is increased past a certain level (J. Nanomaterials. 2011 ;2011 : 12). It has been shown that increased PEGylation leads to a significant increase in the circulation half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15; 13:507-30; J. Control Release. 2010 Aug 3; 145(3): 178-81).
[0134] Suitable examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof. [0135] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights and include the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol- succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NEE), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as well as such compounds containing a terminal hydroxyl group instead of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NEE).
[0136] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight may be any value or subvalue within the recited ranges, including endpoints.
[0137] In certain instances, the PEG monomers can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester-containing linker moieties include, but are not limited to, amido (- C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CEEC(O)-), succinamidyl (- NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In a preferred embodiment, a carbamate linker is used to couple the PEG to the lipid.
[0138] In other embodiments, an ester-containing linker moiety is used to couple the PEG to the lipid. Suitable ester-containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof. [0139] Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those of skill in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of Cio to C20 are preferred. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).
[0140] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (Cio) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (Cie) conjugate, or a PEG-distearyloxypropyl (Cis) conjugate. In these embodiments, the PEG preferably has an average molecular weight of about 750 to about 2,000 daltons. In particular embodiments, the terminal hydroxyl group of the PEG is substituted with a methyl group.
[0141] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
[0142] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%, from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 0.9 mol% to about 1.6 mol%, from about 0.9 mol% to about 1.8 mol%, from about 1 mol% to about 1.8 mol%, from about 1 mol% to about 1.7 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, or from about 1.4 mol% to about 1.6 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5%, (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. The amount may be any value or subvalue within the recited ranges, including endpoints.
[0143] In some embodiments, the PEG-lipid is PEG550-PE. In some embodiments, the PEG-lipid is PEG750-PE. In some embodiments, the PEG-lipid is PEG2000-DMG. In some preferred embodiments, the PEG-lipid is 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (also known as ALC-0159).
[0144] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid formulations of the disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ± 0.5 mol%. One of ordinary skill in the art will appreciate that the concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid formulation is to become fusogenic.
Mechanism of Action for Cellular Uptake of Lipid Formulations
[0145] Lipid formulations for the intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes, and lipid nanoparticles, are designed for cellular uptake by penetrating target cells through exploitation of the target cells’ endocytic mechanisms where the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3): 146-157, 2018). Specifically, in the case of an RNA lipid formulation targeting hepatocytes described herein, the mRNA or circRNA-lipid formulation enters hepatocytes through receptor mediated endocytosis. Prior to endocytosis, functionalized ligands such as PEG-lipid at the surface of the lipid delivery vehicle are shed from the surface, which triggers internalization into the target cell. During endocytosis, some part of the plasma membrane of the cell surrounds the vector and engulfs it into a vesicle that then pinches off from the cell membrane, enters the cytosol and ultimately undergoes the endolysosomal pathway. For ionizable cationic lipid-containing delivery vehicles, the increased acidity as the endosome ages results in a vehicle with a strong positive charge on the surface. Interactions between the delivery vehicle and the endosomal membrane then result in a membrane fusion event that leads to cytosolic delivery of the payload. For RNA payloads, the cell’s own internal translation processes will then translate the RNA or combination of nucleic acid molecules into the encoded protein (e.g., a wild-type HIV- derived accessory protein tat for the reactivation of latent HIV). The encoded protein can further undergo post-translational processing, including transportation to a targeted organelle or location within the cell.
[0146] By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate exchanges out of the lipid formulation and, in turn, the rate at which the lipid formulation becomes fusogenic. In addition, other variables including, e.g., pH, temperature, or ionic strength, can be used to vary and/or control the rate at which the lipid formulation becomes fusogenic. Other methods which can be used to control the rate at which the lipid formulation becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure. Also, by controlling the composition and concentration of the lipid conjugate, one can control the liposomal or lipid particle size.
Lipid Formulation Manufacture
[0147] There are many different methods for the preparation of lipid formulations comprising a nucleic acid, e.g. RNA or combination of nucleic acid molecules. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). The techniques of thin fdm hydration, double emulsion, reverse phase evaporation, microfluidic preparation, dual asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.
Thin Film Hydration
[0148] In Thin Film Hydration (TFH) or the Bangham method, the lipids are dissolved in an organic solvent, then evaporated through the use of a rotary evaporator leading to a thin lipid layer formation. After the layer hydration by an aqueous buffer solution containing the compound to be loaded, Multilamellar Vesicles (MLVs) are formed, which can be reduced in size to produce Small or Large Unilamellar vesicles (LUV and SUV) by extrusion through membranes or by the sonication of the starting MLV.
Double Emulsion
[0149] Lipid formulations can also be prepared through the Double Emulsion technique, which involves lipids dissolution in a water/organic solvent mixture. The organic solution, containing water droplets, is mixed with an excess of aqueous medium, leading to a water-in- oil-in-water (W/O/W) double emulsion formation. After mechanical vigorous shaking, part of the water droplets collapse, giving Large Unilamellar Vesicles (LUVs).
Reverse Phase Evaporation [0150] The Reverse Phase Evaporation (REV) method also allows one to achieve LUVs loaded with nucleic acid. In this technique a two-phase system is formed by phospholipids dissolution in organic solvents and aqueous buffer. The resulting suspension is then sonicated briefly until the mixture becomes a clear one-phase dispersion. The lipid formulation is achieved after the organic solvent evaporation under reduced pressure. This technique has been used to encapsulate different large and small hydrophilic molecules including nucleic acids.
Microfluidic Preparation
[0151] The Microfluidic method, unlike other bulk techniques, gives the possibility of controlling the lipid hydration process. The method can be classified in continuous-flow microfluidic and droplet-based microfluidic, according to the way in which the flow is manipulated. In the microfluidic hydrodynamic focusing (MHF) method, which operates in a continuous flow mode, lipids are dissolved in isopropyl alcohol which is hydrodynamically focused in a microchannel cross junction between two aqueous buffer streams. Vesicles size can be controlled by modulating the flow rates, thus controlling the lipids solution/buffer dilution process. The method can be used for producing oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three-inlet and one-outlet ports.
Dual Asymmetric Centrifugation
[0152] Dual Asymmetric Centrifugation (DAC) differs from more common centrifugation as it uses an additional rotation around its own vertical axis. An efficient homogenization is achieved due to the two overlaying movements generated: the sample is pushed outwards, as in a normal centrifuge, and then it is pushed towards the center of the vial due to the additional rotation. By mixing lipids and an NaCl-solution a viscous vesicular phospholipid gel (VPC) is achieved, which is then diluted to obtain a lipid formulation dispersion. The lipid formulation size can be regulated by optimizing DAC speed, lipid concentration and homogenization time.
Ethanol Injection
[0153] The Ethanol Injection (El) method can be used for nucleic acid encapsulation. This method provides the rapid injection of an ethanolic solution, in which lipids are dissolved, into an aqueous medium containing nucleic acids to be encapsulated, through the use of a needle. Vesicles are spontaneously formed when the phospholipids are dispersed throughout the medium. Detergent Dialysis
[0154] The Detergent dialysis method can be used to encapsulate nucleic acids. Briefly lipid and plasmid are solubilized in a detergent solution of appropriate ionic strength, after removing the detergent by dialysis, a stabilized lipid formulation is formed. Unencapsulated nucleic acid is then removed by ion-exchange chromatography and empty vesicles by sucrose density gradient centrifugation. The technique is highly sensitive to the cationic lipid content and to the salt concentration of the dialysis buffer, and the method is also difficult to scale.
Spontaneous Vesicle Formation by Ethanol Dilution
[0155] Stable lipid formulations can also be produced through the Spontaneous Vesicle Formation by Ethanol Dilution method in which a stepwise or dropwise ethanol dilution provides the instantaneous formation of vesicles loaded with nucleic acid by the controlled addition of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing the nucleic acid.
Encapsulation in Preformed Liposomes
[0156] The entrapment of nucleic acids can also be obtained starting with preformed liposomes through two different methods: (1) a simple mixing of cationic liposomes with nucleic acids which gives electrostatic complexes called “lipoplexes”, where they can be successfully used to transfect cell cultures, but are characterized by their low encapsulation efficiency and poor performance in vivo,' and (2) a liposomal destabilization, slowly adding absolute ethanol to a suspension of cationic vesicles up to a concentration of 40% v/v followed by the dropwise addition of nucleic acids achieving loaded vesicles; however, the two main steps characterizing the encapsulation process are too sensitive, and the particles have to be downsized.
[0157] In certain embodiments, examples of lipids and lipid nanoparticles, pharmaceutical compositions comprising the lipids, methods of making the lipids or formulating pharmaceutical compositions comprising the lipids and nucleic acid molecules, and methods of using the pharmaceutical compositions for treating or preventing diseases are described in U.S. or International Patent Application Publications, such as US2017/0190661, US2006/0008910, US2015/0064242, US2005/0064595, W02019/036030, US2019/0022247, W02019/036028, W02019/036008, W02019/036000, US2016/0376224, US2017/0119904, WO2018/200943, WO2018/191657, WO2018/118102, US2018/0169268 , W02018/118102 , WO2018/119163, US2014/0255472, and US2013/0195968, the relevant content of each of which is hereby incorporated by reference in its entirety. Methods of Inducing Reactivation of Latent HIV
[0158] The application also provides methods of inducing reactivation of latent HIV in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of the application. Any of the pharmaceutical compositions of the application described herein can be used in the methods of the application. Preferably, a subject to be treated according to the methods of the application is an HIV-infected subject. More preferably, the HIV-infected subjects are under ART-treatment for at least 6 months, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months. In some embodiments, a small amount of HIV or only latent HIV can be detected in plasma isolated from the human prior to the administration of the pharmaceutical composition.
[0159] Methods according to embodiments of the application further comprises administering to the subject in need thereof another an antiviral agent such as a small molecule and/or antibody directed towards HIV or another anti-HIV agent in combination with a pharmaceutical composition of the application. Administration may be concurrent or sequential.
Methods of Delivery
[0160] Pharmaceutical compositions of the application can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. Preferably, pharmaceutical compositions are administered parenterally (e.g., by intramuscular injection or intradermal injection) or transdermally.
[0161] In some embodiments of the application in which a pharmaceutical composition comprises one or more ribonucleic acid molecules, administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the ribonucleic acid molecules to cells. As used herein, the term “electroporation” refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane. During in vivo electroporation, electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules from one side of a cellular membrane to the other. In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site. As a result, transfection efficiency and immune response are significantly improved (e.g., up to 1,000 fold and 100 fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection.
[0162] Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used. Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipoplexes, nanoparticles, etc. For example, a ribonucleic acid molecule of the application can be formulated in a composition that comprises one or more lipid molecules, preferably positively charged lipid molecules. In some embodiments, a ribonucleic acid molecule of the disclosure can be formulated using one or more liposomes, lipoplexes, and/or lipid nanoparticles. In some embodiments, liposome or lipid nanoparticle formulations described herein can comprise a polycationic composition. In some embodiments, the formulations comprising a polycationic composition can be used for the delivery of the ribonucleic acid molecule described herein in vivo and/or ex vitro.
EXAMPLES
The following examples are offered to illustrate but not to limit the invention(s) of the present disclosure. One of skill in the art will recognize that the following procedures may be modified using methods known to one of ordinary skill in the art.
Example 1: Identifying minimal HIV Tat activation domain
[0163] A deletion exercise is performed to delineate the essential Tat amino acid sequence that would result in the same activation capacity as the 72 aa Tat exon 1 protein. The rationale to reduce the overall size of HIV Tat was based on the following assumptions: (i) to remove potential anti-HIV Tat antibody epitopes, (ii) to diminish off target effects and improve cellular uptake, (iii) the absence of potential tat contamination by TLR agonists, and (iv) to facilitate chemical synthesis of the protein.
[0164] The HIV derived accessory protein Tat, 86 to 101 amino acids (aa) long, is translated from two different exons where exon 1 (1-72 aa) contains all the domains essential for trans-activation (Jeang 1997). A DNA sequence encoding a Tat deletion mutant, with a length ranging from 57 aa up to 86 aa (e.g., tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl-64, tatl-60, tatl-57, or tat2-72), was introduced into a pDNA expression vector. These expression constructs were used to co-transfect HEK293 cells along with pLTR-FLuc (Firefly luciferase) and pEF-RLuc (Renilla luciferase) reporter plasmids. Tatl-86 and Tatl- 72 showed similar transactivation activity confirming the reported finding that exon-1 [72 aa] contains all elements essential for transactivation (Arya 1985, Muesing 1987). The Tat variant comprising only 66 amino acids was identified to have a comparable latency reversal activity as the full-length exon Tat-72 construct (Fig. 1). The latency reversal potential of the T66 variant was further studied in various in vitro and ex vivo latency reactivation assays.
Example 2: Strong and consistent T66 protein mediated LTR activation of latent HIV in a variety of latent T-cell lines
[0165] The MT4 T-cells expressing LTR-GFP and LTR-Luc (Firefly luciferase) upon T66 protein-induced LTR promoter activation were used to compare T66 protein-induced LTR activation. Other reporter cell lines, Jurkat E6.1 clone 32 and Jurkat E6.1 clone 50, that include latent HIV with a GFP reporter were also used to measure latency reversal activity. T66 protein was synthesized as trifluoroacetate salt by Bachem. The lyophilized T66 protein was resuspended in sterile water containing 1 mM DTT for use. The latency reversal activity with different doses (ranging from 5.1 to 9 pM) of the T66 protein was evaluated in three different cell lines indicated above (FIG. 2A). The percentage of GFP positive cells reached a plateau at a concentration of 6.2pM in the three cell lines, with 87%, 63% and 51% of positive cells in the MT-4-LTR-GFP, Jurkat clone 50 and Jurkat clone 32, respectively.
[0166] To confirm these results in a more physiologically relevant model, a transient HIV latency assay was established in primary CD4+ T cells. PBMCs were isolated from blood of HIV-infected individuals (n=10) who were all on ART for at least 1.5 years and had a small HIV level inn plasma (a viral load <50 copies/ml). CD4+ T cells were purified from PBMCs by negative selection (Easy Sep™ Human CD4+ T Cell Isolation Kit). The assay is based on the co-infection of isolated CD4+ T cells from HIV-negative donors with an HIV reporter construct and a virus expressing two antiapoptotic proteins (MCL7 and Bcl2). Following overnight incubation of these CD4+ T cells with PBS, T66 protein (4 pM) or PMA (Sigma Aldrich, 5 ng/mL), substantial levels of reactivation were obtained with T66 and PMA in all donors tested, shown as % increase in GFP compared to PBS stimulated control (Fig. 2B). In 5/10 participants, the T66-treated cells reactivated even more compared to PMA stimulation. Example 3: Strong and consistent latency reversal activity in CD4 T-cells from ART- treated individuals
[0167] Next the latency reversal capacity of the T66 protein was investigated on resting CD4+ T-cells from the subject described above. Fig. 3A and 3B show cell-associated HIV-1 RNA copies measured by qRT-PCR using gag-specific primers (1: GGACCAAAGGAACCCTTTAGAGA (SEQ ID NO: 16); 2: GGACCAACAAGGTTTCTGTCATC (SEQ ID NO: 17)). HIV-1 intracellular RNA was evaluated 24h after the addition of T66 protein. T66 treatment (4 pM) resulted in a significant elevation (p=0.0002 negative control versus T66) of cell-associated RNA compared to nonstimulated controls, surpassing HIV RNA levels achieved by PMA stimulation (5 ng/mL) in all ten individuals (Fig. 3A). (n=10; medians: 242, 2180, 422 gag RNA copies in the nonstimulated, T66 and PMA-treated cells, respectively). Reactivation by T66 protein (4 pM) was also evaluated in resting CD4 T-cells (Fig. 3B). T66 increased the levels of cell associated RNA compared to the non-stimulated condition in 3 out of 4 patients. Those levels were comparable to the ones observed following Phytohemagglutinin (PHA, Sigma Aldrich) stimulation (10 pg/mL). To evaluate viral particles, p24 secretion in the supernatant due to HIV-1 reactivation was tested by the ultrasensitive SIMOA® (HIV P24 assay, Quanterix). CD4+ T-cells obtained from the subject described above were incubated for 10 days either with T66 protein (4 pM) or anti-CD3/-CD28 antibodies. Both treatments led to similar p24 induction in all five donors (Fig. 3C). An additional study using a similar methodology confirmed T66-mediated p24 induction in resting CD4+ T-cells collected from four HIV- infected individuals. (Fig. 3D). p24 was detected in all samples 24h after the addition of T66 protein when compared to PBS treated cells. Finally, the quantitative viral outgrowth assay (qVOA, Crooks 2015, Archin 2017) was applied to investigate T66 induced latency reversal on CD4+ T cells from six ART-treated individuals (Fig. 3E).
Example 4: T66 does not induce global T cell activation nor modifications in the CD4+ T cells transcriptome
[0168] Clinically acceptable LRAs shall not induce global T-cell activation. In contrast to the well-known mitogen PHA, T66 protein (4pM) did not induce global T cell activation, as shown by the absence of CD69 (early activation marker) and CD25 (late activation marker) up-regulation (Fig. 4A). CD25 and CD69 were labeled with anti-CD25 PerCp-cy5.5 and anti- CD69 APC and the cells were analyzed by flow cytometry (BD biosciences). Moreover, micro-array experiments were conducted on CD4+ T-cells from 4 subject described above to study the impact of the T66 protein on the transcriptome of the cells (Fig. 4B). PBS and PHA (10 pg/mL)-treated samples were taken along as negative and positive controls, respectively. Micro-array experiments revealed T66-treated cells cluster closely positioned to PBS-treated cells for all participants, suggesting insignificant impact of T66 treatment on the host transcriptome (Fig. 4B). This is in sharp contract with PHA-stimulated cells which formed a second cluster, confirming the profound impact of the mitogen PHA on the human transcriptome. It is confirmed that T66 (4 pM) was present and active in the HIV-infected CD4+ T cells, observing latency reversal determined by HIV RNA qPCR using the gagspecific primers in T66-treated cells (Fig. 4C). Based on Volcano plots, we identified 8 genes as differentially expressed between PBS-treated samples and T66-treated samples (p<0.05, Fig. 4D). However, for these 8 differentially expressed genes, the log2 fold change was lower than 2, indicating that treatment with the T66 protein does not perturbate the transcriptome of CD4+ T-cells.
Example5: Synthesis of mRNA
[0169] T66 protein showed robust latency reversal in multiple assays while no global T- cell activation nor any significant transcriptome perturbation was observed. However, a potential pitfail of the T66 protein is the relatively high concentration required for HIV reactivation, which might be challenging for large scale production and in vivo administration. While not wishing to be bound by theories, inefficient cellular protein transduction and substantial endosomal trapping of the T66 protein are likely the cause for the large amount of protein needed to achieve maximum HIV activation. One potential strategy to address the poor cytosolic delivery is to use T66 mRNA.
[0170] A DNA template for the in vitro transcription of mRNA was generated by introducing the following elements in a standard pDNA vector: a T7 promoter, followed by the coding sequences of the 5 ’-and 3’-UTRs from either the human a-globin [HAG] or the frog (Xenopus) a-globin [XBG] (Table 2), terminated by a 120 nt polyA tail. [Fig5] . When the capping reagent AG CleanCap (Trilink) was applied, the wild type T7 promoter sequence was changed in the DNA template from TAATACGACTCACTATAGG to TAATACGACTCACTATAAG. The coding sequence for the PolyA tail contains a final restriction enzyme site for linearization.
[0171] The HIV-1 T66 codon-optimized gene sequence, as shown in Fig.5, was introduced via restriction enzymes-based cloning into the DNA template plasmid between the 5’-UTR and 3’-UTR coding sequences. Sequence-verified DNA constructs were upscaled, linearized and column-purified with the Pure Link Gel Extraction Kit (Thermofisher).
[0172] In vitro transcription was performed with either HiScribe T7 ARCA mRNA kit or the HiScribe In Vitro Transcription Kit (NEB) according to the manufacturer’s recommendation. 500 ng linearized plasmid DNA was added per 20 microliter transcription reaction. The ribonucleotide uridine was in some cases replaced with Nl- Methypseudouridine. The capping reagent AG Cleancap (Trilink) was added at 5 mM concentration to the transcription reaction along with 0.1 U of inorganic pyrophophatase YIPP (NEB). In vitro transcription was typically carried out for 3 hours at 37C. The in vitro transcript was purified by column purification using the RNA RNA miniprep/maxiprep kit (Qiagen) according to the provided protocol. The RNA was quantified with the Nanodrop spectrophotometer and its integrity verified by agarose gel analysis, as shown in Fig.6.
[0173] The following transfection reagents were tested in a cellular reporter assay to identify the optimal delivery system of T66 mRNA into the cytosol: JetPEI (Polyplus), JetMessenger (Polyplus), Viromer (Lipocalyx), TransIT mRNA (Minis), RNAiMax (Thermofisher), and Messenger Max (Thermofisher). In this assay the translated T66 protein MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFMTKALGISYGRKKRRQR RRAHQNSQTHQ would activate transcription from an HIV-1 LTR promoter driving GFP protein expression quantified by flow cytometry. JetMessenger® (Polyplus) turned out to be the most efficient transfection reagent for T66 mRNA showing up to 97 % of GFP (+) cells down to a concentration of 2.5 nM T66 mRNA, as shown in Fig.7.
Example6: Evaluation of T66 mRNA HIV-1 reactivation (reversal) potential on a variety of latent T-cell lines
[0174] The MT4 T-cell lines LTR-GFP and LTR-Luc expressing either GFP or Firefly luciferase upon T66 induced LTR promoter activation were used to compare T66 mRNA mediated activation with T66 protein induced LTR activation. To our big surprise T66 mRNA turned out to be more than 10,000-fold m5ore active when compared to its protein counterpart, as shown in Fig.8A and 8B. Maximal reporter expression is achieved with 1 nM T66 mRNA while it takes 10 pM of protein to have the same effect. Furthermore T66 protein induces luciferase expression by only 28-fold whereas T66 mRNA enhances luciferase expression by 68-fold (Fig. 8B). [0175] Next, T66 mRNA were profiled in an engineered Jurkat T-cell line harboring a latent HIV-1 GFP reporter pro virus side by side with the strongest ex vivo HIV-1 latency reversal agents known to date such as PMA and anti-CD3/-CD28 costimulation (anti- CD3/CD28 magnetic Dynabeads, Invitrogen). T66 mRNA LTR activation determined by the increase of GFP (+) cells exceeded the reactivation achieved by T66 protein or CD3/CD28, as shown in Fig.9. Neither PMA nor CD3/CD28 costimulation may be used in vivo due to their induction of global T-cell activation. Of note no primary CD4 T-cell activation determined by upregulation of CD25 and CD69 activation markers was observed with T66 mRNA delivered by JetMessenger (data not shown).
[0176] Lastly the ability of T66 mRNA to induce latent HIV-1 reversal in a difficult to reactivate Jurkat cl50 T-cell clone was assessed. This clone has been generated and employed for a large latency reversal agent screening campaign. It was engineered with a full length HIV-GFP infection followed by four consecutive FACS rounds aiming to achieve minimum basal activity but significant PMA-induced HIV-1 reactivation. In the Jurkat cl50 assay we have never identified any latency reversal agent that would achieve an activation profile that would approach the one of PMA. However when T66 mRNA was tested in Jurkat C50 it reached equipotency to PMA at an mRNA concentration of only 6 nM, as shown in Fig. 10.
Example 7: Evaluation of T66 mRNA HIV-1 reactivation (reversal) potential in mice
[0177] NSG mice or NOD/shi-scid/ccnull (NOG) mice will be transplanted with CD34+ cells by iv injection. The mice will be bled at 12 and 24 weeks post-transplantation to determine human immune cell reconstitution by flow cytometry. Animals with > 20% of circulating human CD45+ cells at 24 weeks post-transplantation will be infected by intraperitoneal injections of a HIV-1 laboratory adapted strain. Infection will be monitored every 2 weeks on blood samples for CD4+ T cell counting using flow cytometry analysis and for viral load measurement. cART treatment will be initiated 1 week (early treated) or 8 weeks (late treated) post infection and to be continued for a total of 8 weeks in both groups. T66 mRNA or another latency reversal agent will be added and viral rebound will be evaluated by measuring viral loads in blood samples of the mice.
Example 8: Evaluation of T66 mRNA HIV-1 reactivation (reversal) potential in rhesus macaques
[0178] Rhesus macaques will be infected with SIVmac239 and treated with a potent ART regimen. When viral load in blood is below 60 copies/ml, the animal are treated with T66 mRNA or another or another latency reversal agent. The viral rebound will be evaluated by measuring viral load in blood samples of the animal.
Example 9: Identification of nanoparticle formulations for the efficient mRNA delivery
[0179] A panel of commercial transfection reagents were tested in a cellular HIV-GFP reporter assay to identify the optimal delivery system of T66 mRNA into Jurkat T lymphocyte cells: JetPEI (Polyplus), JetMessenger (Polyplus), Viromer (Lipocalyx), TransIT mRNA (Minis), RNAiMax (Thermofisher), and Messenger Max (Thermofisher). The transfection was performed on IxlO5 Jurkat cells plated in 100 pl medium in 96-well plates using lOOng T66 mRNA formulated with the different transfection reagents according to the supplier’s instructions.
[0180] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.
[0181] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
[0182] Other embodiments are set forth in the following claims.

Claims

Claims We claim:
1. A pharmaceutical composition comprising a ribonucleic acid molecule encapsulated in a pharmaceutically acceptable encapsulating carrier, wherein ribonucleic acid molecule comprises a non-naturally occurring polynucleotide sequence comprising a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable encapsulating carrier is a lipid nanoparticle.
3. The pharmaceutical composition of claim 1 or 2, wherein the wild-type HIV-derived accessory protein tat comprises at least the first 57 N-terminal amino acids of a wild-type tat (86-101 aa) for the reactivation of latent HIV.
4. The pharmaceutical composition of claim 1 or 2, wherein the wild-type HIV-derived accessory protein tat is tatl-86, tatl-72, tatl-70, tatl-68, tatl-66, tatl-65, tatl-64, tatl-60, tatl-57, or tat2-72.
5. The pharmaceutical composition of claim 1 or 2, wherein the wild-type HIV-derived accessory protein tat comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
6. The pharmaceutical composition of claim 1 or 2, wherein the wild-type HIV-derived accessory protein tat comprises one or more substitutions in Table 1 in the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3.
7. The pharmaceutical composition of any of the foregoing claims, wherein the ribonucleic acid molecule comprises a 5’-UTR, a nucleotide sequence encoding the wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, 3’-UTR and a poly A tail.
8. The pharmaceutical composition of claim 7, wherein the ribonucleic acid molecule consists essentially of the 5’-UTR, the nucleotide sequence encoding the wild-type HIV- derived accessory protein tat for the reactivation of latent HIV, the 3’-UTR and the poly A tail.
9. The pharmaceutical composition of claim 7, wherein the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises at least one sequence from table 2.
10. The pharmaceutical composition of claim 6, wherein the nucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV comprises a 5 ’-UTR sequence, the T66 coding sequence and a 3 ’-UTR sequence from table 2.
11. The pharmaceutical composition of any one of claims 7 to 10, wherein the 5’-UTR has the sequence of SEQ ID No. 4.
12. The pharmaceutical composition of any one of claims 7 to 11, wherein the 3’-UTR has the sequence of SEQ ID No. 5.
13. The pharmaceutical composition of any one of claims 7 to 12, wherein the poly A tail comprises 100 to 140 adenosine monophosphates.
14. The pharmaceutical composition of any one of claims 7 to 12, wherein the poly A tail comprises 120 adenosine monophosphates.
15. The pharmaceutical composition of any of the foregoing claims, wherein the ribonucleic acid molecule is an mRNA molecule.
16. An isolated ribonucleic acid molecule comprising: (1) a 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6; (2) a polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV; (3) a 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
17. The isolated ribonucleic acid molecule of claim 16, comprising: (1) the 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly the polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%,
97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5; and (4) the poly A tail containing 100 to 140 adenosine monophosphates.
18. The isolated ribonucleic acid molecule of claim 16, comprising: (1) the 5’ UTR having the polynucleotide sequence of SEQ ID NO: 4; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having the polynucleotide sequence of SEQ ID NO: 5; and (4) the poly A tail containing 120 adenosine monophosphates.
19. The isolated ribonucleic acid molecule of claim 16, comprising: (1) the 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly the polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5; and (4) a poly A tail containing 100 to 140 adenosine monophosphates.
20. The isolated ribonucleic acid molecule of claim 19, comprising: (1) the 5’ UTR having the polynucleotide sequence of SEQ ID NO: 6; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having the polynucleotide sequence of SEQ ID NO: 5; and (4) the poly A tail containing 120 adenosine monophosphates.
21. The isolated ribonucleic acid molecule of claim 16, comprising: (1) the 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly the polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7; and (4) the poly A tail containing 100 to 140 adenosine monophosphates.
22. The isolated ribonucleic acid molecule of claim 21, comprising: (1) the 5’ UTR having the polynucleotide sequence of SEQ ID NO: 4; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having the polynucleotide sequence of SEQ ID NO: 7; and (4) the poly A tail containing 120 adenosine monophosphates.
23. The isolated ribonucleic acid molecule of claim 16, comprising: (1) the 5’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV, particularly the polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having a polynucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7; and (4) the poly A tail containing 100 to 140 adenosine monophosphates.
24. The isolated ribonucleic acid molecule of claim 23, comprising: (1) the 5’ UTR having the polynucleotide sequence of SEQ ID NO: 6; (2) the polynucleotide sequence encoding a wild-type HIV-derived accessory protein tat for the reactivation of latent HIV and comprising the nucleotide sequence of SEQ ID NO: 8; (3) the 3’ UTR having the polynucleotide sequence of SEQ ID NO: 7; and (4) the poly A tail containing 120 adenosine monophosphates.
25. A nucleic acid molecule comprising a polynucleotide sequence encoding the isolated ribonucleic acid molecule of any one of claims 16-24.
26. The nucleic acid molecule of claim 25 is a DNA molecule, further comprising a promoter operably linked the polynucleotide sequence, such as a T7 promoter comprising a sequence of TAATACGACTCACTATAG or TAATACGACTCACTATAAG.
27. A method of producing a ribonucleic acid molecule, comprising transcribing the nucleic acid molecule of claims 25 or 26, preferably the transcription is conducted in vitro.
28. A pharmaceutical composition comprising the ribonucleic acid molecule of any one of claims 16-24 encapsulated in a pharmaceutically acceptable encapsulating carrier
29. The pharmaceutical composition of claim 27, wherein the pharmaceutically acceptable encapsulating carrier is a lipid nanoparticle.
30. The pharmaceutical composition of any one of claims 1-15 and 28-29, wherein the pharmaceutically acceptable encapsulating carrier comprises a lipid nanoparticle comprising one or more of cationic lipid selected from: a. l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), b. l,2-DiLinoleyloxy-,N,N-dimethylaminopropane (DLinDMA), c. l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), d . a compound of F ormula (I) :
Figure imgf000068_0001
Formula (I) wherein Ri is a substituted alkyl consisting of 10 to 31 carbons, R2 is a linear alkyl, alkenyl or alkynyl consisting of 2 to 20 carbons, Rs is a linear or branched alkane consisting of 1 to 6 carbons, R4 and Rs are the same or different, each a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; Li and L2 are the same or different, each a linear alkane of 1 to 20 carbons or a linear alkene of 2 to 20 carbons, and Xi is S or O; or a salt or solvate thereof; e. a compound of formula (II):
Figure imgf000068_0002
Formula (II) wherein Ri is a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; Li is linear alkane of 1 to 15 carbons; R2 is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons or a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; L2 is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons; X is O or S; Rs is a linear alkane of 1, 2, 3, 4, 5, or 6 carbons; and R4and Rs are the same or different, each a linear or branched, noncyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons; or a pharmaceutically acceptable salt or solvate thereof; f. a compound of formula (III), (IV) or (V):
Figure imgf000069_0001
wherein R comprises a biologically active molecule, and Li, L2, and L independently for each occurrence comprise a ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile; a pharmaceutically acceptable salt thereof; or a pharmaceutical composition thereof; g- a compound of formula (VI):
Figure imgf000069_0002
Formula (VI) wherein X is a linear or branched alkylene or alkenylene, monocyclic, bicyclic, or tricyclic arene or heteroarene; Y is a bond, an ethene, or an unsubstituted or substituted aromatic or heteroaromatic ring; Z is S or O; L is a linear or branched alkylene of 1 to 6 carbons; Rs and R4 are independently a linear or branched alkyl of 1 to 6 carbons; Ri and R2 are independently a linear or branched alkyl or alkenyl of 1 to 20 carbons; r is 0 to 6; and m, n, p, and q are independently 1 to 18; wherein when n=q, m=p, and Ri=Rs, then X and Y differ; wherein when X=Y, n=q, m=p, then Ri and R2 differ; wherein when X=Y, n=q, and RI=R2, then m and p differ; and wherein when X=Y, m=p, and RI=R2, then n and q differ; or a pharmaceutically acceptable salt thereof; h. a compound of formula (VII):
Figure imgf000070_0001
Formula (VII) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , _ C(=O)— , — O— , — S(O)y-, — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — , and the other of G1 or G2 is, at each occurrence, — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)y-, — S— S‘, — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Rais, at each occurrence, independently H, C1-C12 alkyl, Ci- C 12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, Ci-C 12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000070_0002
a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 5 to 10; d1 and d2are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituents; and a compound of formula (VIII) :
Figure imgf000071_0001
Formula (VIII) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , _ C(=O)— , — O— , — S(O)y-, — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — , and the other of G1 or G2 is, at each occurrence, — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)y-, — S— S— , — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Rais, at each occurrence, independently H, C1-C12 alkyl, Ci- C 12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, Ci-C 12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000072_0001
R' is, at each occurrence, independently H or C1-C12 alkyl; a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 2 to 12; d1 and d2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a1, a2, c1, c2, d1 and d2 are selected such that the sum of a^^+d1 is an integer from 18 to 30, and the sum of a2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
31. A combination comprising the pharmaceutical composition of any one of claims 1-15 and 28-30 and at least one of a Latency Reversing Agent and an antiretroviral agent, in particular the Latency Reversing Agent is Phytohemagglutinin (PHA), Phorbol 12-Myristate 13-Acetate (PMA), or anti-CD3 + anti-CD28 antibodies.
32. A method of reactivating latent HIV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of claims 1 to 15 and 28-30.
33. The method of claim 32, comprising further administering to the subject an effective amount of one or more anti-viral agent.
34. The method of claim 32, comprising further administering to the subject an effective amount of one or more Latency Reversing Agent, such as Phytohemagglutinin (PHA), Phorbol 12-Myristate 13-Acetate (PMA), or anti-CD3 + anti-CD28 antibodies.
35. The method of any one of claims 32-34, wherein the subject is a human.
36. The method of claim 35, wherein the human is infected with HIV and/or is under antiretroviral therapy treatment for at least 6 months, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 months, prior to the administration of the pharmaceutical composition.
37. The method of any one of claims 32-36, wherein the human has an HIV viral load of 100 copies/ml or less or only latent HIV detected in plasma prior to the initial administration of the pharmaceutical composition.
38. A method of increasing the induction of latent HIV-1 reversal in a cell, comprising contacting the cell with a pharmaceutical composition of any one of claims 1 to 15 and 28-30, wherein the increase is an increase in induction of latent HIV-1 reversal over contacting the cell with an equivalent wild-type HIV-derived accessory protein tat for the reactivation of latent HIV.
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