Mrna-encoded tat with attenuated cytotoxicity for HIV and siv latency reversal

Abstract

The present invention relates to compositions and methods for targeted delivery of mRNA encoding an attenuated Tat protein and methods of use thereof for treating or preventing HIV and SIV. The compositions and methods are further useful for treatment of latent HIV or SIV.

Description

[0001] mRNA-encoded Tat with Attenuated Cytotoxicity for HIV and SIV Latency Reversal

[0002] CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 511,357, filed June 30, 2023, which is hereby incorporated by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with government support under AH 64570 and AI169633 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE The present application hereby incorporates by reference the entire contents of the XML file named “046483-6270-00WO_SequenceListing.xml” in XML format, which was created on June 28, 2024, and is 51,052 bytes in size.

[0006] BACKGROUND OF THE INVENTION

[0007] Strategies to induce a drug-free HIV cure remain research, clinical, and community priorities (Deeks, et al., 2021, Nature Medicine, 27:2085-2098). Viral latency remains a major obstacle in the development of HIV cure strategies. While currently available antiretroviral therapy (ART) can suppress active viral replication, viral reservoirs persist for years in treated individuals. The latent reservoir is a subset of the reservoir that is infected with HIV or SIV, i.e., harbors an integrated provirus, but does not express viral RNA or proteins. Replication competent virus can be isolated from these latently infected cells through stimulation of the host T cell ex vivo (Ho, Y.-C., et al., 2013, Cell, 155:540-551). As such, persistent HIV-1 reservoirs endure within an infected host despite effective antiretroviral therapy and give rise to virus rebound should therapy be interrupted (Kreider, E. F., and Bar, K. J., 2022, Current HIV / AIDS Reports, 19(3): 194-206; Li, J. Z., et al., 2016, AIDS, 30:343-353). A major contributor to persistent reservoirs are the latently infected resting CD4+ T cells (Cohn, L. B., et al., 2020, Cell Host & Microbe, 27:519-530; Ho, Y.-C., et al., 2013, Cell, 155:540-551). These cells do not express viral proteins and are indistinguishable from their uninfected counterparts (Abdel-Mohsen, M., et al., 2018, Science Translational Medicine, 10(437):eaar6759). By suppressing viral protein expression, latent viruses evade the host immune response, adoptive immunotherapy, and antiretroviral therapy and, thus, remain a major barrier to a drug-free HIV cure.

[0008] Shock and kill eradication strategies target latently infected CD4+ T cells through delivery of latency reversing agents (LRAs) that reactivate quiescent virus and effectors that clear this newly reactivated virus( Margolis, D. M., et al., 2016, Science, 353(6297):aaf6517; Borducchi, E. N., et al., 2018, Nature, 563:360-364). Newer LRAs activate signaling pathways such as NF-kB, which results in transcription of the HIV-1 promoter as well as other host genes. These small molecule agonists suffer from nonspecific pleotropic effects (Zhao, M., et al., 2019, Pharmacological Research, 139:524-534), toxicities (Nixon, C. C., et al., 2020, Nature, 578:160-165; Philip, P. A., et al., 1993, JNCI, 85: 1812-1818; Kulkosky, J., et al., 2002, Journal of Infectious Diseases, 186: 1403-1411), and, importantly, potency limitations (Grau-Exposito, J., et al., 2019, PLoS Pathology, 15:el007991; Singh, V., et al., 2021, Current HI V / AIDS Reports, 18: 117-127). The viral protein Tat antagonizes latency by specifically activating viral transcription through interaction with a viral RNA feature called TAR. 89-100% of latently infected cells express TAR, demonstrating that lack of Tat expression is a major block in latency reversal (Yuki, S. A., et al., 2018, Science Translational Medicine, 10(430):eaap9927). Delivery of Tat to cells harboring latent virus, however, has remained a barrier to its development as a virus-specific LRA.

[0009] Thus, there is a need in the art for improved compositions and methods for treating latent HIV. The present invention addresses this need.

[0010] SUMMARY OF THE INVENTION

[0011] In some embodiments, the invention provides a composition comprising a delivery vehicle conjugated to a targeting domain, wherein the delivery vehicle comprises or encapsulates an mRNA molecule encoding a Tat protein or variant thereof, and further wherein the targeting domain specifically binds to CD4. In some embodiments, the delivery vehicle is a lipid nanoparticle, a liposome, or a micelle. In some embodiments, the delivery vehicle is a lipid nanoparticle. In some embodiments, the mRNA molecule encoding a Tat protein is encapsulated in the lipid nanoparticle.

[0012] In some embodiments, the Tat protein is an attenuated Tat protein about 86 amino acids in length. In some embodiments, the Tat protein comprises at least one mutation relative to a native or wild type Tat protein. Exemplary mutations include, but are not limited to, V36A, Q66A, V67A, S68A, S77A, T23A, I39A, and L69A relative to wildtype Tat-86. In some embodiments, the Tat protein comprises V36A, Q66A, V67A, S68A, and S77A mutations. In some embodiments, the Tat protein comprises the amino acid sequence of SEQ ID NO: 1, 5, 10, and 12. In some embodiments, the mRNA molecule comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the mRNA molecule comprises the nucleotide sequence of SEQ ID NO:2.

[0013] In some embodiments, the targeting domain comprises an antibody or fragment thereof, an aptamer, a nucleic acid, a protein, a peptide, a glycan, a sugar, a hormone, or a small molecule. In some embodiments, the targeting domain comprises an anti-CD4 antibody or CD4-binding fragment thereof. Exemplary anti-CD4 antibodies include, but are not limited to, ibalizumab, A161A1, and CD4-binding fragments thereof.

[0014] In some embodiments, the invention provides a method of treating or preventing a human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in a subject, the method comprising administering to the subject a composition of the invention. In some embodiments, HIV or SIV is latent in the subject. In some embodiments, the method further comprises administering at least one additional therapeutic agent. Exemplary additional therapeutic agents include, but are not limited to, abacavir, emtricitabine, lamuvidine, tenofovir disoproxil fumarate, zidovudine, doravirine, efavirenz, etravirine, nevirapine, rilpivirine, atazanavir, darunavir, fosamprenavir, ritonavir, tipranavir, enfuvitide, maraviroc, cabotegravir, dolutegravir, raltegravir, fostemsavir, idalizumab, lenacapavir, cobicistat, and combinations thereof.

[0015] In some embodiments, the invention provides a method of activating latent HIV or SIV in a subject comprising administering to the subject a composition of the invention. In some embodiments, the invention provides a method of detecting latent viral load in a subject comprising: obtaining a sample from a subject; contacting the sample with a composition of the invention; and detecting the reactivated viral load. In some embodiments, the sample is a lymph sample, a lymphoid tissue sample, a urine sample, a saliva sample, a mucous sample, a whole blood sample, a blood plasma sample, a blood serum sample, a semen sample, or a milk sample obtained from the subject. In some embodiments, the step of obtaining a sample from a subject further includes a step of isolating T-cells from the sample obtained from the subject.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following detailed description of embodiments 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 arrangements and instrumentalities of the embodiments shown in the drawings.

[0018] Figure 1 depicts a schematic representation of the HIV genome and an expanded view of the transcription start site controlled by Tat.

[0019] Figure 2 depicts a schematic representation of HIV transcription stalling without Tat, producing multiple TAR hairpin loops, and with Tat, allowing full-length transcription.

[0020] Figure 3 depicts the results of exemplary experiments showing that latency is maintained through block in elongation with estimated 89-100% of infected CD4+ T cells expressing abortive transcripts (Yuki, S. A., et al., 2018, Science Translational Medicine, 10(430):eaap9927).

[0021] Figure 4 depicts a schematic showing an 86- AA variant of Tat having attenuated activity (Tat-86.R5M4).

[0022] Figure 5 schematic depicting the JC53-bl / TZM-bl cell line used to test Tat-mediated transactivation of the HIV-1 promoter.

[0023] Figure 6 depicts the results of exemplary experiments showing the (Figure 26A) fold luciferase signal (relative light units, RLU) day 2 and day 3 following transfection of TZM-bl cells (luc - positive control; eGFP - negative control). Figure 7 depicts the results of an exemplary experiment showing that Tat- R5M4 potently drives expression from the HIV promoter.

[0024] Figure 8 depicts the results of an exemplary experiment showing Tat- 86. R5M4 (Tat.R5M4) mRNA is as effective as wildtype Tat (Tat.JRCSF) mRNA (Luc - luciferase mRNA positive control; zsGreen - negative control).

[0025] Figure 9 depicts results of an exemplary experiment showing that negative control mutants of Tat.86.R5M4 (Nullbasic and Tat.R5M4.49-51A (partial nullbasic) - impaired membrane permeation; C221 - abolished transactivation) do not drive transcription of the HIV promoter in TZM-bl cells.

[0026] Figure 10 depicts the results of an exemplary experiment showing electroporation of Tat-R5M4 at 1 pg / million cells reactivated I-Latl0.6 with a spectrum of GFP intensity seen among the population.

[0027] Figure 11 depicts the results of an exemplary experiment showing that the GFP signal was correlated to the amount of Tat mRNA electroporated and increased from Day 2 to Day 3.

[0028] Figure 12 depicts the results of an exemplary experiment showing that the percent of cells that are GFP+ is sustained, though decreasing, over 9 days.

[0029] Figure 13 depicts the results of an exemplary experiment showing that the intensity of fluorescence increases in GFP+ cells over 9 days.

[0030] Figure 14 depicts the results of an exemplary experiment showing that Tats derived from different strains of HIV-1 (Tat.l91859.D - subtype D) and SIV-HIV chimera viruses (SHIV - Tat.SHIV.mac766.1919859; Tat. SHIV. mac766. 191859.R5M4). drive HIV-1 promotor expression.

[0031] Figure 15 depicts the results of an exemplary experiment showing that cells expressing Tat mRNA (circles) secrete Tat, which can be taken up by bystander cells (squares) and induce transactivation in those cells.

[0032] Figure 16 depicts the results of an exemplary experiment showing that Tat-86. R5M4, when delivered via nontargeted LNPs, activates the HIV promoter.

[0033] Figure 17 depicts the results of an exemplary experiment showing that treatment with 2 pg / million cells of nontargeted Tat RNA-LNPs results in shift in GFP signal with large spectrum of brightness / proviral expression. Figure 18 depicts the results of an exemplary experiment showing that treatment with nontargeted Tat-R5M4 RNA-LNPs resulted in J-Lat 10.6 reactivation on Day 3.

[0034] Figure 19 depicts the results of an exemplary experiment showing that Tat-86. R5M4 nontargeted LNPs reactivate latent provirus in J-Lat 10.6 cells in a dosedependent manner.

[0035] Figure 20 depicts the results of an exemplary experiment showing that Tat R5M4-LNPs reactivate latent provirus in J-Lat 10.6 cells in a dose-dependent manner.

[0036] Figure 21 is a schematic illustrating CD4 targeting using ibalizumab.

[0037] Figure 22 depicts the results of an exemplary experiment showing that ibalizumab allows targeting of human and rhesus CD4s.

[0038] Figure 23 depicts the results of an exemplary experiment showing that CD4+ T-cells are readily transfected by targeted LNPs.

[0039] Figure 24 depicts the results of an exemplary experiment showing that CD4-targeted ibalizumab can efficiently deliver mRNA to CD4+ T-cells among mixed PBMCs.

[0040] Figure 25 depicts the results of an exemplary experiment showing that CD4+ T-cell transfection is dose dependent.

[0041] Figure 26 depicts the results of an exemplary experiment showing that the transcriptomic profile of cells treated with phorbol-12-myristate-13-acetate and ionomycin (PMA / Iono) is distinct from that of Tat mRNA-LNP -treated and Luc mRNA- LNP-treated cells, which largely overlap.

[0042] Figure 27 depicts the expression of four genes that show statistically significant differences between Tat mRNA-LNP -treated cells and Luc mRNA-LNP - treated cells from Figure 26.

[0043] Figure 28 depicts the results of an exemplary experiment showing that treatment of cells with phorbol-12-myristate-13-acetate and ionomycin (PMA / Iono) induces long-term chromatin accessibility throughout the HIV genome while Tat does not result in such alteration of chromatin accessibility. Figure 29 depicts the results of an exemplary experiment showing that CD4-targeted Tat mRNA-LNPs improve mRNA delivery compared to non-targeted Tat mRNA-LNPs.

[0044] Figure 30 depicts the results of an exemplary experiment showing that CD4-targeted Tat mRNA-LNPs are nontoxic in primary cells. No difference was observed in multiple doses of Tat in two donors (DI and D2, left). General viability (right) in samples from six donors similarly demonstrates no difference between treated and untreated cells.

[0045] Figure 31 depicts the results of an exemplary experiment demonstrating that treatment of primary human CD4+ T cells with targeted Tat mRNA-LNPs does not induce the expression of T cell activation markers CD25, CD38, CD69, and HLA-DR.

[0046] Figure 32 depicts the results of an exemplary experiment demonstrating that primary CD4+ T cells treated with targeted Tat mRNA-LNPs secrete Tat protein which can transactivate TZM-bl cells.

[0047] DETAILED DESCRIPTION

[0048] The invention relates to compositions comprising a delivery vehicle conjugated to a targeting domain, wherein the delivery vehicle comprises at least one agent. In some embodiments, the at least one agent is a therapeutic agent. In some embodiments, the targeting domain specifically binds to a cell surface molecule of a T- cell. For example, in some embodiments, the targeting domain directs the vehicle to CD4+ T-cells.

[0049] In some embodiments, the delivery vehicle serves to deliver at least one therapeutic agent to the T-cells. In some embodiments, at least one therapeutic agent is a nucleic acid molecule encoding a human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) Tat protein, or a variant or fragment thereof.

[0050] The present invention also relates to methods of treating or preventing HIV or SIV using the compositions described herein. In some embodiments, the invention provides a method for treating latent HIV or SIV. In some embodiments, the invention provides a method for reactivating latent HIV or SIV. Definitions

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] As used herein, each of the following terms has the meaning associated with it in this section.

[0053] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0054] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0055] The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen or epitope. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0056] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments. An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0057] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, k and 1 light chains refer to the two major antibody light chain isotypes.

[0058] The term “synthetic antibody” as used herein, means an antibody, which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The term should also be construed to mean an antibody, which has been generated by the synthesis of an RNA molecule encoding the antibody. The RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned) or other technology, which is available and well known in the art.

[0059] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0060] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0061] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0062] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0063] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0064] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. In the context of the present invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0065] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as a human.

[0066] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell. For example, an mRNA where at least one uridine has been replaced with pseudouridine, 1 -methyl pseudouridine, or another modified nucleoside.

[0067] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0068] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0069] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0070] In certain instances, the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0071] In certain embodiments, “pseudouridine” refers, in another embodiment, to m’acp3Y (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the term refers to mxY (1 -methylpseudouridine). In another embodiment, the term refers to Ym (2'-O-methylpseudouridine. In another embodiment, the term refers to m5D (5-methyldihydrouridine). In another embodiment, the term refers to m3Y (3- methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0072] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising at least two amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0073] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. For example, the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.

[0074] By the term “specifically binds,” as used herein with respect to an affinity ligand, in particular, an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from at least one other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.

[0075] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, at least one of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0076] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0077] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0078] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0079] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0080] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains at least one double and / or triple bonds), having from one to twenty-four carbon atoms (C1-C24 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-Ce alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-l-enyl, but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless specifically stated otherwise, an alkyl group is optionally substituted.

[0081] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (i.e., contains at least one double (alkenylene) and / or triple bonds (alkynylene)), and having, for example, from one to twenty-four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (Ci-Cs alkylene), one to six carbon atoms (Ci-Ce alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, / / -butylene, ethenylene, propenylene, / / -butcnylcne, propynylene, 77-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

[0082] “Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, or having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbomyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise, a cycloalkyl group is optionally substituted.

[0083] “Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.

[0084] “Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to 18-membered non-aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms (e.g., nitrogen, oxygen or sulfur). Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise, a heterocyclyl group may be optionally substituted.

[0085] The term “substituted” used herein means any of the above groups (e.g., alkyl, cycloalkyl or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; oxo groups (=0); hydroxyl groups (-0H); alkoxy groups (-0Ra, where Rais C1-C12 alkyl or cycloalkyl); carboxyl groups (-0C(=0)Raor -C(=O)ORa, where Rais H, C1-C12 alkyl or cycloalkyl); amine groups (-NRaRb, where Raand Rbare each independently H, C1-C12 alkyl or cycloalkyl); C1-C12 alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is a oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0086] “Optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0087] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0088] Description

[0089] The invention relates to compositions and methods for T-cell targeted delivery of a therapeutic agent for treating HIV or SIV in a subject. In one aspect, the present invention relates to a composition comprising a delivery vehicle conjugated to a targeting domain. In some embodiments, the delivery vehicle comprises at least one agent, such as a therapeutic agent. In some embodiments, the therapeutic agent is an RNA, including but not limited to mRNA, and nucleoside-modified RNA.

[0090] In some embodiments, the targeting domain binds to a cell surface molecule of an immune cell, such as a T-cell. In some embodiments, the targeting domain binds to CD4. Tn some embodiments, the targeting domain is a CD4-specific antibody, a CD4-binding fragment thereof, or a combination thereof.

[0091] In some embodiments, the delivery vehicle comprises at least one therapeutic agent for delivery to CD4+ cells. In certain embodiments, the at least one therapeutic agent is at least one mRNA molecule encoding at least one HIV Tat protein, SIV Tat protein, or fragment or variant thereof.

[0092] The present invention also relates to methods of treating HIV or SIV in subjects in need thereof, the method comprising the administration of a composition including a delivery vehicle conjugated to a targeting domain.

[0093] In various embodiments, the invention provides a method for treating latent HIV or SIV.

[0094] In some embodiments, the invention provides a method for preventing reactivation of latent HIV or SIV.

[0095] Delivery Vehicle

[0096] In some embodiments, the delivery vehicle is a colloidal dispersion system, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e g., an artificial membrane vesicle).

[0097] The use of lipid formulations is contemplated for the introduction of at least one therapeutic agent into a host cell (in vitro, ex vivo or in vivo). In another aspect, at least one agent may be associated with a lipid. The at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0098] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-agent complexes.

[0099] In some embodiments, delivery of the at least one agent comprises any suitable delivery method, including exemplary delivery methods described elsewhere herein.

[0100] In certain embodiments, the delivery vehicle comprises a complex of the agent and a transfection agent (an agent-transfection agent complex). In some embodiments, the delivery of at least one agent to a subject comprises mixing the agent and a transfection agent to form an agent-transfection agent complex in vitro prior to administration. In other embodiments, the method comprises administering the at least one agent together with a transfection reagent and forming an agent-transfection agent complex in vitro, in vivo, ex vivo, in situ.

[0101] In some embodiments, the transfection reagent is a lipid-based transfection reagent. In some embodiments, the transfection reagent is a cationic lipid. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0102] In another embodiment, the transfection reagent forms a liposome. Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. In some embodiments, the liposomes comprise an internal aqueous space for entrapping water-soluble compounds. In another embodiment, liposomes can deliver the at least one agent to cells in an active form.

[0103] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the LNP encapsulates at least one agent. The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes at least one lipid. In various embodiments, the particle includes a lipid of Formula (I), (II) or (III). In some embodiments, lipid nanoparticles are included in a formulation comprising at least one agent as described herein. In some embodiments, such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or (III)) and at least one excipient. For example, at least one neutral lipid, charged lipid, steroid or polymer conjugated lipid (e.g., a pegylated lipid such as a pegylated lipid of structure (IV), such as compound IVa). In some embodiments, at least one agent is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0104] In various embodiments, the lipid nanoparticles 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, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the lipid nanoparticles have a mean diameter of about 83 nm. In some embodiments, the lipid nanoparticles have a mean diameter of about 102 nm. In some embodiments, the lipid nanoparticles have a mean diameter of about 103 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, the at least one agent, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation by intra- or intercellular enzymes

[0105] The LNP may comprise any lipid capable of forming a particle to which the at least one agent is attached, or in which the at least one agent is encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0106] In some embodiments, the LNP comprises one at least one cationic lipid, and at least one stabilizing lipid. Stabilizing lipids include neutral lipids and pegylated lipids.

[0107] In some embodiments, the LNP comprises a cationic lipid. As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0108] In certain embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)- N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3 -dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3- phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y ); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0109] In some embodiments, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinoley oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-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), 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), and 2,2-dilinoleyl-4-dimethylaminomethyl-[ 1 , 3 ] -di oxolane (DLin-K-DMA).

[0110] Suitable amino lipids include those having the formula: wherein Ri and R2 are either the same or different and independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted Cio-C24acyl;

[0111] R3 and R4 are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2- Ce alkynyl or R3 and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;

[0112] Rs is either absent or present and when present is hydrogen or C1-C6 alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and

[0113] Y and Z are either the same or different and independently O, S, or NH.

[0114] In some embodiments, Ri and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In some embodiments, the amino lipid is a dilinoleyl amino lipid.

[0115] A representative useful dilinoleyl amino lipid has the formula:

[0116] DLIB-K- MA wherein n is 0, 1, 2, 3, or 4.

[0117] In some embodiments, the cationic lipid is a DLin-K-DMA. In some embodiments, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).

[0118] In some embodiments, the cationic lipid component of the LNPs has the structure of Formula (I): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0119] L1and L2are each independently -O(C=O)-, -(C=O)O- or a carbon-carbon double bond;

[0120] Rlaand Rlbare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;

[0121] R2aand R2bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond; R3aand R3bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0122] R4aand R4bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0123] R5and R6are each independently methyl or cycloalkyl;

[0124] R7is, at each occurrence, independently H or C1-C12 alkyl;

[0125] R8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2.

[0126] In certain embodiments of Formula (I), at least one of Rla, R2a, R3aor R4ais C1-C12 alkyl, or at least one of L1or L2is -O(C=O)- or -(C=O)O-. In other embodiments, Rlaand Rlbare not isopropyl when a is 6 or n-butyl when a is 8.

[0127] In still further embodiments of Formula (I), at least one of Rla, R2a, R3aor R4ais C1-C12 alkyl, or at least one of L1or L2is -O(C=O)- or -(OO)O-; and

[0128] Rlaand Rlbare not isopropyl when a is 6 or n-butyl when a is 8.

[0129] In other embodiments of Formula (I), R8and R9are each independently unsubstituted C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;

[0130] In certain embodiments of Formula (I), any one of L1or L2may be -O(C=O)- or a carbon-carbon double bond. L1and L2may each be -O(C=O)- or may each be a carbon-carbon double bond.

[0131] In some embodiments of Formula (I), one of L1or L2is -O(C=O)-. In other embodiments, both L1and L2are -O(C=O)-. In some embodiments of Formula (I), one of L1or L2is -(C=O)O- In other embodiments, both L1and L2are -(C=O)O-

[0132] In some other embodiments of Formula (I), one of L1or L2is a carboncarbon double bond. In other embodiments, both L1and L2are a carbon-carbon double bond.

[0133] In still other embodiments of Formula (I), one of L1or L2is -O(C=O)- and the other of L1or L2is -(C=O)O-. In more embodiments, one of L1or L2is -O(C=O)- and the other of L1or L2is a carbon-carbon double bond. In yet more embodiments, one of L1or L2is -(C=O)O- and the other of L1or L2is a carbon-carbon double bond.

[0134] It is understood that “carbon-carbon” double bond, as used throughout the specification, refers to one of the following structures: wherein Raand Rbare, at each occurrence, independently H or a substituent. For example, in some embodiments Raand Rbare, at each occurrence, independently H, Ci- C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.

[0135] In other embodiments, the lipid compounds of Formula (I) have the following structure (

[0136] In other embodiments, the lipid compounds of Formula (I) have the following structure (lb):

[0137]

[0138] In yet other embodiments, the lipid compounds of Formula (I) have the following structure (Ic):

[0139] In certain embodiments of the lipid compound of Formula (I), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0140] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16. In some more embodiments of Formula (I), c is 1 . In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0141] In some certain other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0142] In some other various embodiments of Formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0143] The sum of a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties. In some embodiments, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.

[0144] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0145] The substituents at Rla, R2a, R3aand R4aof Formula (I) are not particularly limited. In certain embodiments Rla, R2a, R3aand R4aare H at each occurrence. In certain other embodiments at least one of Rla, R2a, R3aand R4ais C1-C12 alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Cs alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0146] In certain embodiments of Formula (I), Rla, Rlb, R4aand R4bare C1-C12 alkyl at each occurrence.

[0147] In further embodiments of Formula (I), at least one of Rlb, R2b, R3band R4bis H or Rlb, R2b, R3band R4bare H at each occurrence.

[0148] In certain embodiments of Formula (I), Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0149] The substituents at R5and R6of Formula (I) are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R5or R6is methyl. In certain other embodiments one or both of R3or R6is cycloalkyl for example cyclohexyl. In these embodiments the cycloalkyl may be substituted or not substituted. In certain other embodiments the cycloalkyl is substituted with C1-C12 alkyl, for example tert-butyl.

[0150] The substituents at R7are not particularly limited in the foregoing embodiments of Formula (I). In certain embodiments at least one R7is H. In some other embodiments, R7is H at each occurrence. In certain other embodiments R7is C1-C12 alkyl.

[0151] In certain other of the foregoing embodiments of Formula (I), one of R8or R9is methyl. In other embodiments, both R8and R9are methyl.

[0152] In some different embodiments of Formula (I), R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.

[0153] In various different embodiments, exemplary lipid of Formula (I) can include:

[0154]

[0155] In some embodiments, the LNPs comprise a lipid of Formula (I), at least one agent, and at least one excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (I) is compound 1-5. In some embodiments the lipid of Formula (I) is compound 1-6. In some other embodiments, the cationic lipid component of the I.NPs has the structure of Formula ( or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0156] L1and L2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa, -OC(=O)NRa-, -NRaC(=O)O-, or a direct bond;

[0157] G1is C1-C2 alkylene, -(C=O)- , -O(C=O)-, -SC(=O)-, -NRaC(=O)- or a direct bond;

[0158] G2is -C(=O)- , -(C=O)O-, -C(=O)S-, -C(=O)NRaor a direct bond;

[0159] G3is Ci-Ce alkylene;

[0160] Rais H or C1-C12 alkyl;

[0161] Rlaand Rlbare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;

[0162] R2aand R2bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0163] R3aand R3bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond; R4aand R4bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0164] R5and R6are each independently H or methyl;

[0165] R7is C4-C20 alkyl;

[0166] R8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

[0167] In some embodiments of Formula (II), L1and L2are each independently -O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G1and G2are each independently -(C=O)- or a direct bond. In some different embodiments, L1and L2are each independently -O(C=O)-, -(C=O)O- or a direct bond; and G1and G2are each independently -(C=O)- or a direct bond.

[0168] In some different embodiments of Formula (II), L1and L2are each independently -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRa-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa, -OC(=O)NRa-, -NRaC(=O)O-, -NRaS(O)xNRa-, -NRaS(O)x- or -S(O)xNRa-.

[0169] In other of the foregoing embodiments of Formula (II), the lipid compound has one of the following structures (IIA) or (IIB):

[0170] (IIA) (IIB) In some embodiments of Formula (II), the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).

[0171] In any of the foregoing embodiments of Formula (II), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-.

[0172] In some different embodiments of Formula (II), one of L1or L2is -(C=O)O-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0173] In different embodiments of Formula (II), one of L1or L2is a direct bond. As used herein, a “direct bond” means the group (e.g., L1or L2) is absent. For example, in some embodiments each of L1and L2is a direct bond.

[0174] In other different embodiments of Formula (II), for at least one occurrence of Rlaand Rlb, Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond.

[0175] In still other different embodiments of Formula (II), for at least one occurrence of R4aand R4b, R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0176] In more embodiments of Formula (II), for at least one occurrence of R2aand R2b, R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0177] In other different embodiments of Formula (II), for at least one occurrence of R3aand R3b, R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0178] In various other embodiments of Formula (II), the lipid compound has one of the following structures (IIC) or (IID) :

[0179] wherein e, f, g and h are each independently an integer from 1 to 12.

[0180] In some embodiments of Formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).

[0181] In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[0182] In certain embodiments of Formula (II), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0183] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0184] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0185] In some certain embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0186] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.

[0187] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

[0188] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

[0189] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.

[0190] In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.

[0191] The sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties. In some embodiments, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[0192] The substituents at Rla, R2a, R3aand R4aof Formula (II) are not particularly limited. In some embodiments, at least one of Rla, R2a, R3aand R4ais H. In certain embodiments Rla, R2a, R3aand R4aare H at each occurrence. In certain other embodiments at least one of Rla, R2a, R3aand R4ais C1-C12 alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Cs alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0193] In certain embodiments of Formula (II), Rla, Rlb, R4aand R4bare C1-C12 alkyl at each occurrence.

[0194] In further embodiments of Formula (II), at least one of Rlb, R2b, R3band R4bis H or Rlb, R2b, R3band R4bare H at each occurrence. In certain embodiments of Formula (II), Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0195] The substituents at R3and R6of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments one of R5or R6is methyl. In other embodiments each of R or R6is methyl.

[0196] The substituents at R7of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments R7is Ce-Ci6 alkyl. In some other embodiments, R7is C6-C9 alkyl. In some of these embodiments, R7is substituted with -(C=O)ORb, -O(C=O)Rb, -C(=O)Rb, -ORb, -S(O)xRb, -S-SRb, -C(=O)SRb, -SC(=O)Rb, -NRaRb, -NRaC(=O)Rb, -C(=O)NRaRb, -NRaC(=O)NRaRb, -OC(=O)NRaRb, -NRaC(=O)ORb, -NRaS(O)xNRaRb, -NRaS(O)xRbor -S(O)xNRaRb, wherein: Rais H or C1-C12 alkyl; Rbis C1-C15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R7is substituted with -(C=O)ORbor -O(C=O)Rb.

[0197] In various of the foregoing embodiments of Formula (II), Rbis branched C1-C15 alkyl. For example, in some embodiments Rbhas one of the following structures:

[0198] In certain other of the foregoing embodiments of Formula (II), one of R8or R9is methyl. In other embodiments, both R8and R9are methyl.

[0199] In some different embodiments of Formula (II), R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring. In still other embodiments of the foregoing lipids of Formula (II), G3is

[0200] C2-C4 alkylene, for example C3 alkylene.

[0201] In various different embodiments, the lipid compound has one of the following structures:

[0202]

[0203]

[0204] In some embodiments, the LNPs comprise a lipid of Formula (II), at least one agent, and at least one excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (II) is compound II-9. In some embodiments, the lipid of Formula (II) is compound II- 10. In some embodiments, the lipid of Formula (II) is compound II- 11. In some embodiments, the lipid of Formula (II) is compound 11-12. In some embodiments, the lipid of Formula (II) is compound 11-32. In some other embodiments, the cationic lipid component of the I.NPs has the structure of Formula (III): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, ,NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;

[0205] G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0206] G3is C1-C24 alkylene, C1-C24 alkenylene, Ca-Cs cycloalkylene, Ca-Cs cycloalkenylene;

[0207] Rais H or C1-C12 alkyl;

[0208] R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0209] R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;

[0210] R4is C1-C12 alkyl;

[0211] R5is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0212] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

[0213] (IIIA) (IIIB) wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

[0214] R6is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0215] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0216] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):

[0217] (IIIC) (IIID) wherein y and z are each independently integers ranging from 1 to 12.

[0218] In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0219] In some different embodiments of Formula (III), the lipid has one of the following structures (IHE) or (IIIF):

[0220] (IIIE) (IIIF)

[0221] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ) :

[0222]

[0223] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0224] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0225] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0226] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0227] In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: wherein: R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0228] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis Ci-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0229] In different embodiments of Formula (III), R1or R2, or both, has one of

[0230] In some of the foregoing embodiments of Formula (III), R3is OH, CN, -C(=O)OR4, -OC(=O)R4or -NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0231] In various different embodiments, the cationic lipid of Formula (III) has one of the following structures:

[0232]

[0233] In some embodiments, the LNPs comprise a lipid of Formula (III), at least one agent, and at least one excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound III-7. In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In some embodiments, the LNP comprises only cationic lipids.

[0234] In certain embodiments, the LNP comprises at least one additional lipid which stabilizes the formation of particles during their formation.

[0235] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0236] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0237] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl -phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl ethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearioyl-2-oleoyl- phosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In some embodiments, the neutral lipid is l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).

[0238] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to the neutral lipid ranges from about 2: 1 to about 8: 1. In various embodiments, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[0239] In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to cholesterol ranges from about 2: 1 to 1 : 1.

[0240] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0241] In certain embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GMi). In certain embodiments, the LNP comprises a sterol, such as cholesterol.

[0242] In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.

[0243] In certain embodiments, the LNP comprises an additional, stabilizing - lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycollipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In some embodiments, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2ooo)carbamyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-l-O-((0- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG- cer), or a PEG dialkoxypropylcarbamate such as comethoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co- methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25: 1.

[0244] In some embodiments, the LNPs comprise a pegylated lipid having the following structure (IV): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0245] R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by at least one ester bond; and z has mean value ranging from 30 to 60.

[0246] In some of the foregoing embodiments of the pegylated lipid (IV), R10and R11are not both n-octadecyl when z is 42. In some other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R10is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R11is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0247] In various embodiments, z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0248] In other embodiments, the pegylated lipid has one of the following structures: wherein n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g / mol.

[0249] In certain embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In some embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In some embodiments, the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent. In some embodiments, the LNPs comprise a lipid of Formula (I), a nucleoside-modified RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments the lipid of Formula (I) is compound 1-6. In different embodiments, the neutral lipid is DSPC. In other embodiments, the steroid is cholesterol. In still different embodiments, the pegylated lipid is compound IVa.

[0250] In certain embodiments, the LNP comprises at least one targeting moiety that targets the LNP to a cell or cell population. For example, in some embodiments, the targeting domain is a ligand which directs the LNP to a receptor found on a cell surface.

[0251] In certain embodiments, the LNP comprises at least one internalization domain. For example, in some embodiments, the LNP comprises at least one domain which binds to a cell to induce the internalization of the LNP. For example, in some embodiments, the at least one internalization domain bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP -bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in some embodiments, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.

[0252] Other exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e!39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

[0253] The following Reaction Schemes illustrate methods to make lipids of Formula (I), (II) or (III). GENERAL REACTION SCHEME 1

[0254] Embodiments of the lipid of Formula (I) (e.g., compound A-5) can be prepared according to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, compounds of structure A-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of A-l, A-2 and DMAP is treated with DCC to give the bromide A-3. A mixture of the bromide A-3, a base (e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessarily workup and or purification step.

[0255] GENERAL REACTION SCHEME 2

[0256] Other embodiments of the compound of Formula (I) (e.g., compound B-5) can be prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of B-l (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinum chlorochromate) and intermediate product B-3 is recovered. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and / or purification.

[0257] It should be noted that although starting materials A-l and B-l are depicted above as including only saturated methylene carbons, starting materials which include carbon-carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds.

[0258] GENERAL REACTION SCHEME 3

[0259] Different embodiments of the lipid of Formula (I) (e.g., compound C-7 or C9) can be prepared according to General Reaction Scheme 3 (“Method C”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C- l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. GENERAL REACTION SCHEME 4

[0260] D-7

[0261] Embodiments of the compound of Formula (II) (e.g., compounds D-5 and D-7) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein Rla, Rlb, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R8, R9, L1, L2, G1, G2, G3, a, b, c and d are as defined herein, and R7represents R7or a C3-C19 alkyl. Referring to General Reaction Scheme 1, compounds of structure D-l and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of D-l and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up. A solution of D-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride D-4 (or carboxylic acid and DCC) to obtain D-5 after any necessary work up and / or purification. D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary work up and / or purification. GENERAL REACTION SCHEME 5

[0262] Embodiments of the lipid of Formula (II) (e.g., compound E-5) can be prepared according to General Reaction Scheme 5 (“Method E”), wherein Rla, Rlb, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8, R9, L1, L2, G3, a, b, c and d are as defined herein. Referring to General Reaction Scheme 2, compounds of structure E-1 and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of E-1 (in excess), E-2 and a base (e.g., potassium carbonate) is heated to obtain E-3 after any necessary work up. A solution of E-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride E-4 (or carboxylic acid and DCC) to obtain E-5 after any necessary work up and / or purification.

[0263] GENERAL REACTION SCHEME 6

[0264] General Reaction Scheme 6 provides an exemplary method (Method F) for preparation of Lipids of Formula (III). G1, G3, R1and R3in General Reaction Scheme 6 are as defined herein for Formula (III), and GL refers to a one-carbon shorter homologue of GL Compounds of structure F-l are purchased or prepared according to methods known in the art. Reaction of F-l with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester / alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).

[0265] It should be noted that various alternative strategies for preparation of lipids of Formula (III) are available to those of ordinary skill in the art. For example, other lipids of Formula (III) wherein L1and L2are other than ester can be prepared according to analogous methods using the appropriate starting material. Further, General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G1and G2are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G1and G2are different.

[0266] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, Z-butyldimethylsilyl, Z-butyldiphenylsilyl or trimethyl silyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include / -butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.

[0267] Agents

[0268] In some embodiments, the delivery vehicle comprises at least one agent. In some embodiments, the agent is a therapeutic agent. In some embodiments, the therapeutic agent is a nucleic acid, a peptide, or a small molecule. In some embodiments, the therapeutic agent is a nucleic acid molecule encoding an HIV Tat protein, an SIV Tat protein, an HIV-SIV hybrid (SHIV) Tat protein, or a fragment or variant thereof.

[0269] In some embodiments, the therapeutic agent is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA or miRNA molecule. In some embodiments, the isolated nucleic acid molecule encodes a therapeutic peptide. In some embodiments, the isolated nucleic acid encodes an HIV Tat protein, an SIV Tat protein, an SHIV Tat protein, or a fragment or variant thereof.

[0270] In some embodiments, at least one therapeutic agent encodes an HIV Tat protein, SIV Tat protein, SHIV Tat protein, or a fragment or variant thereof. In some embodiments, the HIV Tat protein, SIV Tat protein, SHIV Tat protein, or a fragment or variant thereof has reduced cytotoxicity relative to a wildtype HIV Tat protein or SIV Tat protein. In some embodiments, the HIV Tat protein, SIV Tat protein, or fragment or variant thereof comprises at least one mutation relative to a wildtype HIV Tat protein or SIV Tat protein. In some embodiments, the HIV Tat protein is a Tat protein from HIV-1 or HIV-2. In some embodiments, the Tat protein from HIV-1 is a Tat protein from a Group M, Group N, Group O, or Group P HIV-1. In some embodiments, the Group M HIV-1 Tat protein is a Tat protein from a subtype A, subtype B, subtype C, subtype D, subtype F, subtype G, subtype H, subtype J, or subtype K Group M HIV-1 , or circulating recombinant forms thereof. In some embodiments, the HIV-2 Tat protein is from a Type A or a Type B HIV-2.

[0271] In some embodiments, the Tat protein comprises at least one mutation relative to a native or wild type Tat protein sequence. In some embodiments, the isolated nucleic acid encodes an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to an amino acid sequence as set forth in any of SEQ ID NO: 1, 3-6 or 10-53. Exemplary mutations that can be incorporated into a variant Tat protein include, but are not limited to, mutations corresponding to T23A, V36A, I39A, Q66A, V67A, S68A, L69A, and S77A relative to the native or wild type Tat protein sequence. In some embodiments, the HIV Tat protein or SIV Tat protein comprises a combination of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or more than 8 mutations relative to a native or wild type Tat protein sequence. For example, in one embodiment, the variant Tat protein comprises a combination of V36A, Q66A, V67A, S68A, and S77A mutations relative to the native or wild type Tat protein sequence. In some embodiments, the HIV Tat protein comprises an amino acid sequence as set forth in any of SEQ ID NOs: 1, 5, 10, and 12. In some embodiments, the HIV Tat protein comprises an amino acid sequence of SEQ ID NO:1. In some embodiments, the isolated nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to SEQ ID NO:2. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO:2.

[0272] Tat-86. R5M4 AA MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQACFITKALGISYGRKKRRQ RRRPPQGSQTHAAALSKQPTSQARGDPTGPKE (SEQ ID NO: 1)

[0273] Tat-86. R5M4 mRNA ATGGAGCCCGTGGACCCCCGCCTGGAGCCCTGGAAGCACCCCGGCTCCCAGC CCAAGACCGCCTGCACCAACTGCTACTGCAAGAAGTGCTGCTTCCACTGCCA GGCCTGCTTCATCACCAAGGCCCTGGGCATCTCCTACGGCCGCAAGAAGCGC CGCCAGCGCCGCCGCCCCCCCCAGGGCTCCCAGACCCACGCCGCCGCCCTGT CCAAGCAGCCCACCTCCCAGGCCCGCGGCGACCCCACCGGCCCCAAGGAGTG

[0274] A (SEQ ID N0:2)

[0275] TAT.HXB2 AA

[0276] MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQ

[0277] RRRAHQNSQTHQASLSKQPTSQPRGDPTGPKE (SEQ ID N0:3)

[0278] TAT.HRCSF

[0279] MEPVDPSLEPWKHPGSQPKTACTNCYCKKCCLHCQVCFTTKGLGISYGRKKRRQ

[0280] RRRPPQDSQTHQVSLPKQPSSQQRGDPTGPKESKKKVERETETDPDN (SEQ ID N0:4)

[0281] TAT-86.R5M4 (GENG) AA

[0282] MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQACFITKALGISYGRKKRRQ RRRPPQGSQTHAAALSKQPTSQARGDPTGPKE (SEQ ID NO: 1)

[0283] TAT-86.R5M4-A101

[0284] MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQACFITKALGISYGRKKRRQ

[0285] RRRPPQGSQTHAAALSKQPTSQARGDPTGPKE (SEQ ID NO:5)

[0286] TAT-86.R5M4.NULLBASIC

[0287] MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQACFITKALGISYGGGGGGA

[0288] GGGPPQGSQTHAAALSKQPTSQARGDPTGPKE (SEQ ID NO:6)

[0289] TAT-86.R5M4.49-51

[0290] MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQACFITKALGISYGAAARRQ

[0291] RRRPPQGSQTHAAALSKQPTSQARGDPTGPKE (SEQ ID NO:7)

[0292] TAT-86.R5M4.C22A

[0293] MEPVDPRLEPWKHPGSQPKTAATNCYCKKCCFHCQACFITKALGISYGRKKRRQ

[0294] RRRPPQGSQTHAAALSKQPTSQARGDPTGPKE (SEQ ID NO:8)

[0295] TAT.191859.D

[0296] MEPVDPSLEPWNHPGSQPGTPCNSCYCKQCCYHCQLCFITKGLGISYGRKKRRQ

[0297] RRRTPQGGQAHQDHISKQPSSQPHGDPPGPKE (SEQ ID NO:9)

[0298] TAT.191859.D.R5M4

[0299] MEPVDPSLEPWNHPGSQPGTPCNSCYCKQCCYHCQACFITKGLGISYGRKKRRQ RRRTPQGGQAHAAAISKQPSSQAHGDPPGPKE (SEQ ID NO: 10)

[0300] TAT.SHIV.MAC766.191859D

[0301] METPLREQENSLESSNEHSSC1SEADASTPESANLGEEILSQLYRPLEACYNSCYC

[0302] KQCCYHCQLCFITKGLGISYGRKKRRQRRRTPQGGQAHQDHISKQPSSQPHGDPP GPKE (SEQ ID NO: 11) TAT.SHIV.MAC766.191859D.R5M4

[0303] METPLREQENSLESSNEHSSCISEADASTPESANLGEEILSQLYRPLEACYNSCYC

[0304] KQCCYHCQACFITKGLGISYGRKKRRQRRRTPQGGQAHAAAISKQPSSQAHGDP PGPKE (SEQ ID NO: 12)

[0305] TAT.MAC766

[0306] METPLREQENSLESSNEHSSCISEADASTPESANLGEEILSQLYRPLEACYNTCYC

[0307] KKCCYHCQFCFLKKGLGICYEQSRKRRRTPKKAKANTPSASNKPISNRTRHCQPE

[0308] KAKKETVEKAVATAPGLGR (SEQ ID NO: 13)

[0309] TAT.02B.FR.09.URF3.JN882651

[0310] MEPVDPRLEPWKHPGSQPRTACTNCYCKNCCFHCQVCFIRKGLGISYGRKKRRQ

[0311] RRRTPQDSQTHKVSPSKQPTSQPGGDPTGPKESKKKVERERETPPSD (SEQ ID NO: 14)

[0312] TAT.02A1.PK.14.PK012.KX232605

[0313] MELVDPNLEPWNHPGSQPTTACNTCYCKKCCWHCQLCFLKKGLGISYGRKKRS

[0314] KRRGTPQSNKDHQNPIPKQPIPRISGISTGPKESKKKVEGETETDRFD (SEQ ID NO: 15)

[0315] TAT.02F2.CM.11.NYU6541.6.BULK.MK086126

[0316] MELVDPSLEPWNHPGSQPTTACNNCYCKVCCWHCQLCFLNKGLGISYGRKKRR

[0317] HRRGPPQSRQDHQNPVPKQPLPIIRGNHQTGPEESKKEMESKTKTDQ (SEQ ID NO: 16)

[0318] TAT.B.FR.83.HXB2_LAI_IIffi_BRU.K03455

[0319] MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQ

[0320] RRRAHQNSQTHQASLSKQPTSQPRGDPTGPKE (SEQ ID NO: 17)

[0321] TAT.A1.RW.17.KIN_18F_CON.MZ642264

[0322] MDPVDPNLEPWNHPGSQPSTPCSTCYCKVCCYHCQSCFLRKGLGISYGRKKRQQ

[0323] RRGTPSSSKSHQDPVPKQPLPQTQGISTGSKESKKTVESETETDRFD (SEQ ID

[0324] NO: 18)

[0325] TAT. Al RW.19.GWE_68F_CON.MZ642269

[0326] MEPVDPKLEPWNHPGSQPATACTKCYCKKCCYHCPVCFLTKGLGISYGRKKRR

[0327] QRRGTPYSNKDHQNPIPEQSIPQTQGISTGPEESKKKVESQAETDRYA (SEQ ID NO: 19)

[0328] TAT.A6.BY.13.PV85.KT983615

[0329] MDPVDPSLDPWNHPGSQPKTACSSCYCKRCCLHCQICFLKKGLGISYGRKKRRH

[0330] RRGTSHNSEDHQNHISKQPLPHTQRDQTGPEESTKKVESKAEAAQLD (SEQ ID NO:20)

[0331] TAT.A6.CY.05.CY021 FJ388892 MDPVDPNLEPWNHPGSQPKTACSNCYCKKCCWHCQICFLKKGLGISYGRKKRR HRRGTPHSSKDHQIPIPKQPLPPTQRDQTGPEESKKKVESKAEPDRLD (SEQ ID N0:21)

[0332] TAT.A6.IT.02.60000.EU861977

[0333] MDPVDPNLEPWNHPGSQPKTACSKCYCKKCCWHCQVCFLNKGLG1SYGRKKRR RRRGTPQNNKDHQNPIPNQPLPRTQRVQTGPEESKKKVESKAETDRFD (SEQ ID NO: 22)

[0334] TAT.A6.RU.19.RU_ERS_2647_2019.MZ427749

[0335] MDPVDPNLEPWKHPGSQPKTACSNCYCKNCCWHCQVCFLKKGLGISYGRKKRK HRRGTPQSSKDHQDLISKQPLPHTQRDQTGPEKSKKKVESKTETDQCA (SEQ ID NO:23)

[0336] TAT. B.BE.20.P5_2020_CD4_4.MW881676

[0337] MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFTKKALGISYGRKKRR QRRRAPQGNQNNQVPLSEQPASQPRGDSTGPKESKEKVERETENDQLD (SEQ ID NO: 24)

[0338] TAT.B.RU.20.RU_ERS_3596_2020.MZ427708

[0339] MEPVDPRLEPWKHPGSQPKTACNNCYCKRCCFHCQLCFTKKGLGISYGRKKRRQ RRRPPPDSQTNQVSLSKQPASQPRGDPTGPKESKKKVERETETDPDH (SEQ ID NO:25)

[0340] TAT. B.SE.03.003SE.MF373125

[0341] MEPVDPRLEPWKHPGSQPSTACTNCYCKKCCFHCQVCFTTKGLGISYGRKKRRQ RRRAPQSSQNHQDSLPKQSASQSRGDPTGPKESKKKVEKETETDPID (SEQ ID NO:26)

[0342] TAT. B.US.19.P3_CMV_FBXO22.MW309889

[0343] MEPVDPRLEPWKHPGSQPRTPCTTCFCKKCCYHCQVCFITKGLGISYGRKKRRQ RRRTPQNSQTHQVSLSKQPASQPGGDPTGPEESKKKVERETETDPDH (SEQ ID NO:27)

[0344] TAT. B.US.19.P5 CMV DELEC1.MW309891

[0345] MEPVDPSLEPWKHPGSQPRTACNNCYCKQCCFHCQVCFIKKGLGISYGRKKRRQ RRRAPQDSQTHQEYLPKQPASQPRGDPTGQKKSKEKVERETETDTVD (SEQ ID NO:28)

[0346] TAT. B.X.X.HYPERVIRULENTDUTCHHIV l 1 MW689460

[0347] MEPVDPRLEPWKHPGSRPKTECTNCYCKICCFHCQVCFMKKGLGISYGRKKRRQ RRRAPQYSQNNQASISKQPASQPRGDPTGPKESKKKVETETEVDPVH (SEQ ID NO: 29)

[0348] TAT. C.IN.15.SC008.KY713229 MEPVDPNLEPWNHPGSQPETACNNCYCKHCSYHCLVCFQKKGLGISYGRKKRR QRRSAPQSSEDHQNPISKQPLPRTQGDSTGSEESKKKVESKTKTDPFD (SEQ ID NO:30)

[0349] TAT. C.IN.15.SC013.KY713230

[0350] MEPVDPNLEPWNHPGSQPRTACNNCFCKRCSYHCLVCFQRKGLGISYGRKKRRQ RRSAPQSSEDHQNLISKQPLSRPRGDQTGSEESKKEVESKTETDPFD (SEQ ID N0:31)

[0351] TAT. C.MW.09.DEMC09MW008.KP109525

[0352] MEPIDPNLKPWEHPGSQPKTACNTCYCKRCCYHCLVCFQTKGLGISYGRKKRRQ RRRTPQSSEDHQNLISKQPLPRTQGDQTGSEESKKKVESKTETDQFDL (SEQ ID NO:32)

[0353] TAT. D.SE.12.077UG.MF373180

[0354] MDPVDPNLEPWNHPGSQPRTPCNKCYCKKCCYHCQVCFITKGLGISYGRKKRRQ RRRTPQGGQTHQDPIPKQPSSQLRGDQTGPKE (SEQ ID NO 33)

[0355] TAT. D.SN.90.SE365A2.L22945

[0356] MDPVDPNLEPWNHPGSQPKTPCNNCYCKKCCYHCQNCFITKGLGISYGRKKRR QRRRTPQGNQTHQVSVPKQPSSQHRGDPTGPKE (SEQ ID NO:34)

[0357] TAT. D.UG.05.P190049.JX236668

[0358] MEPVDPNLEPWNHPGSQPSSPCNKCYCKRCCYHCQVCFITKGLGISYGRKKRRQ RRRTPQGGQAHQDSIPKQPSSQSRGDQTGPKEQKTKVESKAKANPFDW (SEQ ID NO 35)

[0359] TAT. F2.CM.95.95CM_MP257.AJ249237

[0360] MEVVDPNLDPWKHPGSQPETPCNKCYCKKCCFHCQLCFTRKGLGISYGRKKRR QRRRTPQSGEVHQDPVSKQPLSQTRGDPKGPEESKKKVESKTKTDPSD (SEQ ID NO:36)

[0361] TAT. F2.CM.97.CM53657.AF377956

[0362] MEMVDPKLEPWNHPGSQPETPCNKCYCKKCCFHCQLCFTRKGLGISYGRKKRR QRRRASQSSEIHQNPVPKQPLPQAGGNPNGPEESKKKVESKTKPDPSD (SEQ ID NO:37)

[0363] TAT. G.CM.11 DEMG11 CM046.KY658701

[0364] MDPVDPKLEPWNHPGSQPRTPCNKCYCKVCCWHCQVCFLNKGLGISYGRKKRR PRRGTSSGNKEHQNPVPKQPLPTTSGNPTGSKEQKKEVASKTETDLCA (SEQ ID NO:38)

[0365] TAT. H.CD.01.CG_0538_02_NGSID15.KY392778

[0366] MDPVDPNLEPWNHPGSQPKTACNNCYCKKCSYHCELCFLKKGLGISYGRKKRS QRRGTPAGVQDHQDNIPKQPLSRTHGDPTGPKEQKKKVESQTEPDPRD (SEQ ID NO:39) TAT. 02_AG.CM.02.NYU129_5.MK086121 MELVDPSLEPWKHPGSQPTTACNNCYCKICCWHCQICFLKKGLVFSYGRKKRRQ RRGTPQSHQDHQNPVPKQPLPTTKGDPTGPKKPKKEVASKTETDQCD (SEQ ID NO: 40)

[0367] TAT. 07_BC.CN.09.09LNA446.JX960600

[0368] MEPVDPSLEPWKHPGSQPKTACTNCYCKQCCFHCQVCFIKKGLGIFYGRKKRRP RRRAPQSSEDHQNPISKQPLSRTQGDPTGSEESKKKVESKAETDPYD (SEQ ID NO:41)

[0369] TAT. 15_01B.TH.05.05TH522586.JN248351

[0370] MELVDPNLEPWKHPGSQPTTPCGKCYCKKCCWHCQLCFLKKGLGLSYGRKKRK HRRGPPQSSKDHQNPLPKQSLPISRGNPTDPKESKEKVASKAETDPCD (SEQ ID NO: 42)

[0371] TAT. 16_A2D.KE.05.05KE493170V5.KT022403

[0372] MDPVDPNLEPWNHPGSQPRTACNKCYCKKCCYHCQVCFLNKGLGISYGRKKRR PRRGTPPSNTDRQNPIPEQSLPHTQRVSTGPEESKKEVESKAETDRFD (SEQ ID NO:43)

[0373] TAT. A1D.UG.92.UG035.AY352656

[0374] MDPVDPSLEPWNHPGSQPTTACSKCYCKRCCFHCQKCFLNKGLGISYGRKKRKQ RRRTPYSNKDHQNPIPKQPSSQPRGDPTGPEESKKRVESKAETDRLD (SEQ ID NO:44)

[0375] TAT. BC.CN.09.09YNYJ217016SG.KC899014

[0376] XEPIDPNLEPWNHPGSQPETACNNCYCKRCSYHCQVCFLKKGLGISYGRKKRRQ RRSTPQSSEDHQNLISKQPLSRTQGDPTGSEESKKKVESKTKTGPFA (SEQ ID NO:45)

[0377] TAT. BC.CN.09.09YNYJ479SG.KC899015

[0378] MEPVDPNLEPWNHPGSQPETACNNCYCKRCSYHCLVCFQKKGLGISHGRKKRRQ RRSAPQNSEDHQTLISKQPIPRTQGDPTGSEESKKKVESKTEADPFA (SEQ ID NO:46)

[0379] TAT. BF1.PY.O3.O3PY_PSPO114.JN251905

[0380] MDPVDPRVDPWNHPGSQPTTPCTSCYCKRCCFHCYLCFAKKGLGISYGRKKRRQ RRPAPQDSQTHQGSLSKQPISQARGNPTGQKEPKKEVESKTKTDPCD (SEQ ID NO:47)

[0381] TAT. BF1.SE.15.103MK.MF373205

[0382] MEPIDPNLDPWNHPGSKPTTPCNKCYCKHCCFHCYHCFATKGLGISYGRKKRRQ RHRKHPHSSPTHQIPVPEQPLSQSRGDPTGPEEQKKKVESKTETDPFD (SEQ ID NO:48) TAT. U.NL.01 ,U_NL_01_H10986_Cl 1 .EF029069 MEPIDPKIEPWNQPGSQPKTACNQCYCKKCCYHCQLCFLKKGLGISNGRKKRRP RRTTPYGSKNHQDPIPEQPLSQARGDPTGPKEPKKEVESKTEADPFD (SEQ ID NO:49)

[0383] TAT. O.BE.87.ANT70.L20587 MDPVDPEVPPWHHPGSQPQIPCNNCYCKRCCYHCYVCFVRKGLGISYGRKKRG RPAAASHPDHKDPVPKQSPTITKRKQERQEEQEEEVEKKAGPGGYPR (SEQ ID NO:50)

[0384] TAT. O.DE.X.DEOXXDE004.KF859742 MDPVDPEVPPWQHPGSRPQTPCNNCYCKSCSYHCHVCFTRKGLGISYGRKKRRR PAAARHPDHKDPVPKQPPPIIERKQKRQEEQEKEVEKKAGPG (SEQ ID NO: 51)

[0385] TAT. O.FR.03.LA31BCF108.KU168283 MDPVEPEMPPWHHPGSQPQTPCNKCYCKRCCYHCYVCFTRKGLGISYGRKKRR HPAAAASRPDNKDLVRQQPPSSTSREQKRQEEQEKEVEKKAGPD (SEQ ID NO:52)

[0386] In some embodiments, the nucleic acid comprises a promoter / regulatory sequence such that the nucleic acid is capable of directing expression of the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous nucleic acid into cells with concomitant expression of the exogenous nucleic acid in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

[0387] In order to assess the expression of the mRNA, an expression vector to be introduced into a cell can also contain either a selectable marker gene, a reporter gene, or both to facilitate identification of expressing cells from the population of cells sought to be transfected or infected using a delivery vehicle of the invention. In other embodiments, the selectable marker may be carried on a separate piece of DNA and also be contained within the delivery vehicle. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibioticresistance genes, such as neomycin resistance and the like. Therefore, in one aspect, the delivery vehicle may contain at least one vector, comprising the at least one nucleotide sequence or construct to be delivered. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In a particular embodiment, the vector of the invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. Exemplary expression vectors include viral vectors, bacterial vectors, and mammalian cell vectors. Prokaryote- and / or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0388] By way of illustration, a vector in which the nucleic acid sequence is introduced can be a plasmid, which is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.

[0389] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

[0390] In some embodiments, the recombinant expression vectors may also contain nucleic acid molecules, which encode a peptide or peptidomimetic.

[0391] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0392] Naturally, it will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0393] The recombinant expression vectors may also contain a selectable marker gene, which facilitates the selection of host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin such as IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0394] In vitro transcribed RNA

[0395] In some embodiments, the therapeutic agent comprises in vitro transcribed (IVT) RNA. In some embodiments, the composition of the invention comprises in vitro transcribed (IVT) RNA encoding a therapeutic protein. In some embodiments, the composition of the invention comprises IVT RNA encoding a plurality of therapeutic proteins. In some embodiments, an IVT RNA can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. In some embodiments, the desired template for in vitro transcription is a therapeutic protein, as described elsewhere herein.

[0396] In some embodiments, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In some embodiments, the DNA is a full-length gene of interest of a portion of a gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In some embodiments, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including the 5' and 3' UTRs. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0397] Genes that can be used as sources of DNA for PCR include genes that encode polypeptides that induce or enhance an adaptive immune response in an organism. In some embodiments, genes which are useful are for a short-term treatment, or where there are safety concerns regarding dosage or the expressed gene.

[0398] In various embodiments, a plasmid is used to generate a template for in vitro transcription of RNA which is used for transfection.

[0399] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. In some embodiments, the RNA has 5' and 3' UTRs. In some embodiments, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0400] The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0401] In some embodiments, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA.

[0402] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0403] In a one embodiment, the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized RNA which is effective in eukaryotic transfection when it is polyadenylated after transcription.

[0404] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).

[0405] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.

[0406] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E- PAP) or yeast polyA polymerase. In some embodiments, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase RNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0407] 5' caps on also provide stability to RNA molecules. In a one embodiment, RNAs produced by the methods to include a 5' capl structure. Such capl structure can be generated using Vaccinia capping enzyme and 2 ’-O-methyl transferase enzymes (CellScript, Madison, WI). Alternatively, 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958-966 (2005)).

[0408] Nucleoside-modified RNA

[0409] In some embodiments, the composition of the present invention comprises a nucleoside-modified nucleic acid. In some embodiments, the composition of the invention comprises a nucleoside-modified RNA encoding a therapeutic protein. In some embodiments, the nucleoside-modified RNA encodes an HIV Tat protein, an SIV Tat protein, a fragment thereof, a variant thereof, or a combination thereof.

[0410] For example, in some embodiments, the composition comprises a nucleoside-modified RNA. In some embodiments, the composition comprises a nucleoside-modified mRNA. Nucleoside-modified mRNAs have particular advantages over non-modified mRNAs, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent No. 8,278,036, which is incorporated by reference herein in its entirety.

[0411] In certain embodiments, nucleoside-modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days (Kariko et al., 2008, Mol Ther 16: 1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small and that makes it applicable for human therapy.

[0412] In certain instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viralbased vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA. In certain embodiments, using mRNA rather than the protein also has many advantages. Half-lives of proteins in the circulation are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days. Purification of proteins is problematic and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0413] In certain embodiments, the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In certain embodiments, inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16: 1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23: 165-175).

[0414] It has been demonstrated that the presence of modified nucleosides, including pseudouridines in RNA suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23: 165-175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16: 1833-1840). Subsequently, it is shown that the presence of pseudouridine improves the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). A preparative HPLC purification procedure has been established that was critical to obtain pseudouridine-containing RNA that has superior translational potential and no innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:el42). Administering HPLC-purified, pseudouridine-containing RNA coding for erythropoietin into mice and macaques resulted in a significant increase of serum EPO levels (Kariko et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0415] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises an isolated nucleic acid molecule, wherein the nucleic acid molecule comprises at least one pseudouridine or modified nucleoside.

[0416] In some embodiments, the nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein. For example, in certain embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In another embodiment, the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0417] In some embodiments, the modified nucleoside is nfacp3'!' (l-methyl-3- (3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the modified nucleoside is m1'P (1 -methylpseudouridine). In another embodiment, the modified nucleoside is Tm (2'-O-methylpseudouridine). In another embodiment, the modified nucleoside is m5D (5-methyldihydrouridine). In another embodiment, the modified nucleoside is m3T (3 -methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0418] In some embodiments, the nucleoside that is modified in the nucleoside- modified RNA of the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

[0419] In one embodiment, the modified nucleoside of the present invention is m5C (5-methylcytidine). In another embodiment, the modified nucleoside is m5U (5- methyluridine). In another embodiment, the modified nucleoside is m6A (N6- methyladenosine). In another embodiment, the modified nucleoside is s2U (2- thiouridine). In another embodiment, the modified nucleoside is T (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0420] In other embodiments, the modified nucleoside is m’A (1- methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2- methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyl adenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m1! (1 -methylinosine); nflm (l,2'-O-dimethylinosine); m3C (3- methylcytidine); Cm (2'-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4- acetylcytidine); fC (5-formylcytidine); m5Cm (5,2'-O-dimethylcytidine); ac4Cm (N4- acetyl-2'-O-methylcytidine); k2C (lysidine); mxG (1 -methylguanosine); m2G (N2- methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2- dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O- trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); O2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G+(archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5- methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3- carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo’U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5- (carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm’Um (5- methoxycarbonylmethyl-2'-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2- thiouridine); nm3s2U (5-aminomethyl-2-thiouridine); mnm5U (5- methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2 -thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmmrfU (5- carboxymethylaminomethyluridine); cmnm5Um (5-carboxymethylaminomethyl-2'-O- methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6- dimethyladenosine); Im (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2'-O- dimethylcytidine); htrPC (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,2'-O-dimethyladenosine); m Am (N6,N6,O-2'- trimethyladenosine); m2 7G (N2,7-dimethylguanosine); m2’2,7G (N2,N2,7- trimethylguanosine); m3Um (3,2'-O-dirnethyluridine); m’D (5-methyldihydrouridine); f^Cm (5-formyl-2'-O-methylcytidine); nfGm (l,2'-O-dimethylguanosine); m'Am (1,2'- O-dimethyladenosine); rm5U (5-taurinomethyluridine); rm5s2U (5-taurinomethyl-2- thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac6A (N6- acetyladenosine).

[0421] In one embodiment, a nucleoside-modified RNA of the present invention comprises a combination of at least 2 of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of at least 3 of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

[0422] In some embodiments, between 0.1% and 100% of the residues in the nucleoside-modified of the present invention are modified (e.g. either by the presence of pseudouridine or another modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0423] In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0424] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of the given nucleotide that is modified is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0425] In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0426] In some embodiments, a nucleoside-modified RNA molecule of the present invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence. For example, in one embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In another embodiment, translation is enhanced by a 3-fold factor. In another embodiment, translation is enhanced by a 5-fold factor. In another embodiment, translation is enhanced by a 7-fold factor. In another embodiment, translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100- fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10- 1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000- fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0427] Polypeptide therapeutic agents

[0428] In some embodiments, the invention encompasses peptidic therapeutic agents (e.g., Tat peptides), or variants thereof. The variants of the polypeptide therapeutic agents may be (i) one in which at least one of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there is at least one modified amino acid residue, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and / or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein. In some embodiments, the polypeptide therapeutic agent comprises an HIV Tat protein, an SIV Tat protein, fragments thereof, variants thereof, or a combination thereof.

[0429] Combinations

[0430] In some embodiments, the composition of the present invention comprises a combination of agents, or a combination of delivery vehicles for delivery of a combination of agents. In some embodiments, the invention relates to compositions and methods for delivery of a combination of at least two mRNA molecules or nucleoside modified mRNA molecules encoding at least two HIV Tat proteins, SIV Tat proteins, and / or fragments and variants thereof. In some embodiments, the invention relates to compositions and methods for delivery of a combination of an HIV Tat protein, an SIV Tat protein, and / or fragments and variants thereof and an mRNA molecule or nucleoside modified mRNA molecule encoding the same.

[0431] In certain embodiments, a composition comprising a combination of agents described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual agent. In other embodiments, a composition comprising a combination of agents described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual agent.

[0432] A composition comprising a combination of agents comprises individual agents in any suitable ratio. For example, in some embodiments, the composition comprises a 1 : 1 ratio of two individual agents. However, the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.

[0433] Conjugation

[0434] In various embodiments of the invention, the delivery vehicle is conjugated to a targeting domain. Exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, hydrophobic interactions and “van der Waals” interactions. In some embodiments, the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain upon exposure to certain conditions or chemical agents. In another embodiment, the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate from the targeting domain.

[0435] In some embodiments, the conjugation comprises a covalent bond between an activated polymer conjugated lipid and the targeting domain. The term “activated polymer conjugated lipid” refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group. In some embodiments, the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In some embodiments, the activated polymer conjugated lipid is an activated pegylated lipid. In some embodiments, the first coupling group is bound to the lipid portion of the pegylated lipid. In another embodiment, the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid. In some embodiments, the second functional group is covalently attached to the targeting domain.

[0436] The first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. Exemplary first coupling groups or second coupling groups include maleimides, N-hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. Exemplary first coupling groups or second coupling groups include free amines (-NH2), free sulfhydryl groups (-SH), free hydroxide groups (-OH), carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl. In some embodiments, the first coupling group is a maleimide.

[0437] In some embodiments, the second coupling group is a sulfhydryl group. The sulfhydryl group can be installed on the targeting domain using any method known to those of skill in the art. In some embodiments, the sulfhydryl group is present on a free cysteine residue. In some embodiments, the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2-mercaptoethylamine. In some embodiments, the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S- acetylthioacetate (SATA).

[0438] In some embodiments, the polymer conjugated lipid and targeting domain are functionalized with groups used in “click” chemistry. Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles. Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cy cl cooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone

[0439] Targeting Domain

[0440] In some embodiments, the composition comprises a targeting domain that directs the delivery vehicle to a specific target cell or tissue. In some embodiments, the target cell or tissue is a target cell or tissue in need of the agent within the delivery vehicle. The targeting domain may comprise a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, and the like that targets the particle to a site in particular need of the therapeutic agent. In certain embodiments, the particle comprises multivalent targeting, wherein the particle comprises multiple targeting mechanisms described herein. In certain embodiments, the targeting domain of the delivery vehicle specifically binds to a target associated with a site in need of an agent comprised within the delivery vehicle. For example, the targeting domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Such a target can be a protein, protein fragment, antigen, or other biomolecule that is associated with the targeted site. In some embodiments, the targeting domain is an affinity ligand which specifically binds to a target. In certain embodiments, the target (e g. antigen) is associated with a site in need of a treatment with an agent. In some embodiments, the targeting domain may be copolymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to the composition comprising the delivery vehicle, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle. Targeting domains of the instant invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0441] In various embodiments, the targeting domain binds to a cell surface molecule of a T-cell. In some embodiments, the targeting domain binds to CD4.

[0442] Peptides In some embodiments, the targeting domain of the invention comprises a peptide. In certain embodiments, the peptide targeting domain specifically binds to a target of interest.

[0443] The peptide of the present invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0444] The peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

[0445] The variants of the peptides according to the present invention may be (i) one in which at least one of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there is at least one modified amino acid residue, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and / or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post- translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0446] As known in the art the “similarity” between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide to a sequence of a second peptide. Variants are defined to include peptide sequences different from the original sequence, such as different from the original sequence in less than 40% of residues per segment of interest, or different from the original sequence in less than 25% of residues per segment of interest, or different by less than 10% of residues per segment of interest, or different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences can be determined by using the BLASTP algorithm [BLAST Manual, Altschul, S„ et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0447] The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.

[0448] The peptides of the invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

[0449] Nucleic acids

[0450] In some embodiments, the targeting domain of the invention comprises an isolated nucleic acid, including for example a DNA oligonucleotide and / or an RNA oligonucleotide. In certain embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, in some embodiments, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0451] The nucleotide sequences of a nucleic acid targeting domain can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and / or deletions of one at least one nucleotide, with the condition that the resulting nucleic acid functions as the original and specifically binds to the target of interest.

[0452] In the sense used in this description, a nucleotide sequence is “substantially homologous” to any of the nucleotide sequences describe herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, of at least 70%, of at least 85%, and of at least 95%. Other examples of possible modifications include the insertion of at least one nucleotide in the sequence, the addition of at least one nucleotide in any of the ends of the sequence, or the deletion of at least one nucleotide in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences can be determined by using the BLASTN algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0453] Antibodies

[0454] In some embodiments, the targeting domain of the invention comprises an antibody, or antibody fragment. In certain embodiments, the targeting domain specifically binds to a target of interest. Such antibodies, or antibody fragments, include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

[0455] In some embodiments, the antibodies are intact monoclonal or polyclonal antibodies, immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0456] Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species. Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity.

[0457] In some embodiments, the targeting domain of the instant invention is an antibody that specifically binds to T-cells.

[0458] In some embodiments, the targeting domain is an antibody which specifically binds to CD4.

[0459] Exemplary antibodies or antibody fragments that bind to a T-cell marker described herein and thus may be used as a targeting domain are well known in the art. Exemplary antibodies that bind to CD4 include keliximab and ibalizumab.

[0460] Therapeutic Methods

[0461] The invention provides methods of delivering at least one Tat peptide, or RNA molecule encoding the same, to T-cells. In certain embodiments, the disclosure provides a method of treating or preventing HIV or SIV in a subject. In some embodiments, the HIV or SIV is latent in the subject. Therefore, in certain embodiments, the disclosure provides a method of preventing reactivation of latent HIV or SIV in a subject.

[0462] It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited to treatment of a disease or disorder that is already established. Particularly, the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of a disease or disorder do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing a disease or disorder, in that a composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of a disease or disorder, thereby preventing the disease or disorder.

[0463] One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of a disease or disorder, encompasses administering to a subject a composition as a preventative measure against the development of, or progression of, a disease or disorder. As more fully discussed elsewhere herein, methods of modulating the level or activity of a gene, or gene product, encompass a wide plethora of techniques for modulating not only the level and activity of polypeptide gene products, but also for modulating expression of a nucleic acid, including either transcription, translation, or both.

[0464] The invention encompasses delivery of a delivery vehicle, comprising at least one agent, conjugated to a targeting domain. To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate composition to a subject. The present invention is not limited to any particular method of administration or treatment regimen.

[0465] One of skill in the art will appreciate that the compositions of the invention can be administered singly or in any combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and / or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that the compositions of the invention can be used to prevent or to treat a disease or disorder, and that a composition can be used alone or in any combination with another composition to affect a therapeutic result. In various embodiments, any of the compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with a disease or disorder.

[0466] In some embodiments, the invention is a method of treating HIV or SIV in a subject, comprising reactivating latent HIV or SIV in the subject and administering to the subject at least one anti -HIV therapy or drug. In some embodiments, the step of reactivating latent HIV or SIV in the subject comprises administering to the subject a composition of the invention. In some embodiments, the composition of the present invention is administered to the subject to reactivate latent HIV or SIV prior to administration of the at least one anti-HIV drug. In some embodiments, the at least one anti-HIV drug is administered to the subject while the composition of the present invention is administered to the subject to reactivate latent HIV or SIV. In some embodiments, an anti-HIV drug is regularly being administered to the subject prior to, during, and after administration of the composition of the present invention. Exemplary anti-HIV drugs that can be administered in combination with a T cell targeted LNP comprising an RNA molecule encoding a Tat peptide include, but are not limited to, abacavir, emtricitabine, lamuvidine, tenofovir disoproxil fumarate, zidovudine, doravirine, efavirenz, etravirine, nevirapine, rilpivirine, atazanavir, darunavir, fosamprenavir, ritonavir, tipranavir, enfuvitide, maraviroc, cabotegravir, dolutegravir, raltegravir, fostemsavir, idalizumab, lenacapavir, and cobicistat.

[0467] In some embodiments, the invention includes a method comprising administering a combination of compositions described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of the administering a combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.

[0468] The method comprises administering a combination of compositions in any suitable ratio. For example, in some embodiments, the method comprises administering two individual compositions at a 1 :1 ratio. However, the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.

[0469] Diagnostic Methods

[0470] The invention also provides methods of determining latent viral reservoir loading in a subject. In some embodiments, the method is used to determine the latent HIV or SIV viral reservoir loading in a subject. It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited any method of detecting or quantifying viral mRNA, proteins, or particles.

[0471] In some embodiments, the method comprises obtaining a sample from a subject suspected of having latent HIV or SIV, contacting the sample with a composition of the present invention to reactivate the latent virus, and detecting the reactivated viral load. Exemplary samples include, but are not limited to, a lymph sample, a lymphoid tissue sample, a urine sample, a saliva sample, a mucous sample, a whole blood sample, a blood plasma sample, a blood serum sample, a semen sample, and a milk sample obtained from the subject. In some embodiments, the method includes a step of isolating T-cells from the sample obtained from the subject and utilizing the obtained T-cells as the sample for determining latent viral reservoir loading.

[0472] Methods of detecting and quantifying viral loading include, but are not limited to, sequencing, immunoassays, mass spectrometry (MS), and culturing. In some embodiments, the viral load is detected by sequencing. Examples of sequencing useful in the present invention include, but are not limited to, fragment analysis, first generation next generation sequencing (NGS), Sanger sequencing, second generation NGS, shotgun sequencing, pyrosequencing, bridge amplification sequencing, reversible terminator sequencing, sequencing-by-ligation, ion semiconductor sequencing, sequencing by synthesis, combinatorial probe anchor synthesis, emulsion PCR, complementary metal oxide semiconductor sequencing, third generation NGS, nanopore sequencing, singlemolecule sequencing, single-molecule real-time sequencing, massively parallel signature sequencing, polony sequencing, microfluidic sequencing, and metagenomic NGS.

[0473] Immunoassays that may be useful in the present invention include, but are not limited to, monoclonal antibody detection, enzyme-linked immunosorbent assay (ELISA), direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, colorimetric ELISA, chemiluminescent ELISA, Radioimmunoassay (RIA), Western blot, and Northern blot.

[0474] Mass spectrometric methods include, but are not limited to, protein sequencing, nucleotide sequencing, and metabolomic analysis. Culturing methods that may be useful in the present invention include, but are not limited to, transfection with reporter genes, flow cytometry, fluorescence- activated cell sorting (FACS), magnetic-activated cell sorting (MACS), buoyancy- activated cell sorting (BACS), and bead-based affinity capture.

[0475] In some embodiments, the viral load is detected by a combination of methods. In some embodiments, these methods are additionally be combined with other preparative techniques including, but not limited to, capillary electrophoresis, gel electrophoresis, liquid chromatography, gas chromatography, affinity chromatography, size-exclusion chromatography, and ion exchange chromatography. For example, viral load can be detected using liquid chromatography-mass spectrometry (LC-MS) to quantify multiple viral molecules. Examples of virus-derived molecules that can be detected include, but are not limited to, RNA molecules, proteins, and fragments thereof, as well as small molecule metabolites.

[0476] Pharmaceutical Compositions

[0477] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or at least one other accessory ingredient, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0478] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0479] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0480] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0481] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0482] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise at least one additional pharmaceutically active agent.

[0483] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0484] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[0485] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, suspending, stabilizing, or dispersing agents. In some embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0486] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0487] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In some embodiments, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In some embodiments, dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0488] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (having a particle size of the same order as particles comprising the active ingredient).

[0489] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, suspending, stabilizing, or dispersing agents. In some embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0490] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0491] As used herein, “additional ingredients” include, but are not limited to, at least one of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0492] EXPERIMENTAL EXAMPLES

[0493] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0494] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0495] Example 1 : mRNA Encoding an Attenuated Tat Protein Reactivates Latent HIV

[0496] Current data suggest that a major block to reversing latency in HIV is the lack of expression of a virally encoded protein called Tat. In the absence of Tat, the viral promoter initiates transcription, but RNA polymerization halts after ~60 nucleotides. In contrast, if Tat is expressed, it binds to these 60-nucleotide RNA hairpins, called TAR elements, and induces changes to the transcriptional machinery and epigenetic landscape resulting in upregulation of transcription of viral RNA (Figure 1 and Figure 2). Ex vivo studies have suggested that 89-100% of HIV-infected cells from people living with HIV express abortive transcripts (Yuki, S. A., et al., 2018, Science Translational Medicine, 10(430):eaap9927, Figure 3). Further, studies that apply modeling with experimental validation have found that the control of viral latency is autonomously regulated by Tat expression and is irrespective of cellular activation state (Razooky, B. S., et al., 2015, Cell, 160(5):990-l 001 ). Based on these and many other lines of evidence, it has been proposed that delivery of Tat to latently infected cells could drive elongation of abortive transcripts, reversing latency.

[0497] Hurdles exist to delivering Tat to the latent reservoir. First, Tat is itself cytotoxic to host cells. This includes direct toxicity to CD4+ T cells and even more pronounced neurotoxicity. Second, delivery of the protein to cells, including resting CD4+ T cells known to harbor the latent reservoir, requires a platform that can penetrate lymphoid tissues and impact the quiescent immune cells known to harbor the latent reservoir.

[0498] Selection of Tat: An 86-amino acid variant that does not adversely impact T cell viability was selected for investigation The variant is based on a lab-adapted virus that has been studied extensively and incorporates 5 alanine substitutions at positions 36, 66-68, and 77 (Figure 4). Further, because it is an 86-amino acid-long variant, it lacks certain dispensable protein domains that may induce off-target effects, including an NF- kb signaling motif ESKKKV (positions 86-91). It does retain essential domains for membrane permeation and viral transactivation, such as the basic domain (positions SO- 57) and cysteine-rich domain (positions 22-37). In contrast to other shorter protein truncations, TatR5M4 retains an RGD motif that permits binding to integrins (positions 78-80). Thus, compared to other unpublished mRNA-Tat approaches, this construct includes engineered changes that reduce adverse effects while maintaining essential Tat functions. The amino acid sequence of this protein, called Tat86.R5M4, was codon optimized using a codon frequency usage to enhance expression from mRNA.

[0499] Additional versions of Tat proteins, that incorporate mutations known to impact transactivation or membrane permeation (as negative controls), have been prepared based on primary HIV-1 sequences (Subtype D sequence 191859), or using SIV or SIV / HIV chimeric sequences (SIVmac766 or SHIV.C.CH505-V2, sequence identical to all SHIVs described in Li, J. Z., et al., 2016, PNAS, AIDS, 30:343-353). Each of these Tat variants have been successfully expressed as mRNA using in vitro transcription.

[0500] Expression of Tat-86, R5M4 To test the ability of Tat-86. R5M4 to reactivate HIV, mRNA encoding Tat-86. R5M4 was transfected via Lipofectamine® into TZM-bls, a reporter cell line that expresses luciferase and beta-galactosidase under the control of the HIV promoter (Figure 5). As controls, mRNA encoding GFP (negative control) and mRNA encoding luciferase (positive control) were also transfected into TZM-bls. Two days after transfection, cells transfected with Tat-86.R5M4 produced robust luciferase (Figure 6) and beta-galactosidase activity (Figure 7). The Tat-86.R5M4 variant exhibited high potency, with signal intensity comparable to the positive luciferase mRNA control being observed at a Tat-86.R5M4 mRNA dose 0.2% of the luciferase mRNA dose.

[0501] Tat-86.R5M4 was further compared to wildtype Tat in order to verify that no activity was lost in selection. TZM-bl cells were transactivated with either Tat- 86.R5M4 or wildtype Tat-JRCSF mRNA, with luciferase mRNA as a positive control, and zsGreen mRNA and RNA-free as negative controls. Transactivation activity of Tat- 86. R5M4 was observed to be the same as wildtype Tat (Figure 8). Three mutants of Tat- 86.R5M4 were developed as additional negative control Tats, including a Nullbasic mutant with impaired permeation, a Nullbasic partial mutant (49-51 A) with impaired permeation, and a C22A mutant, which abolished transactivation (Figure 9).

[0502] The ability of Tat-86. R5M4 to successfully reactivate latent HIV was further examined in J-Lat 10.6 cells, a common model for latent infection as it harbors a single copy of a replication-defective, GFP-encoding virus (disruption of the nef and env reading frames). Tat-86.R5M4 mRNA and GFP mRNA were transfected into J-Lat 10.6 cells via electroporation and examined for GFP fluorescence two and three days after transfection. Cells were examined and gated for GFP fluorescence. Tat-86.R5M4 mRNA was found to produce a spectrum of GFP intensities, with some cells even yielding more intense fluorescence than the positive controls (Figure 10). Next, the dose-response was examined, with serial 2-fold dilutions of Tat-86.R5M4 mRNA demonstrating a clear dose-dependency (Figure 11). The sustainability of the reactivation was subsequently evaluated, with 20-30% of cells retaining GFP expression up to 9 days after transfection, whereas cells transfected with GFP mRNA displayed extinction of fluorescence after seven days (Figure 12). Interestingly, while the population of cells transfected with Tat- 86. R5M4 that displayed fluorescence decreased over time, the cells that did express GFP displayed an increase in the intensity of the fluorescence over time (Figure 13). Additional Tats derived from other HIV strains (subtype D) and SIV-HIV chimera viruses (SHIV) similarly drove transactivation of HIV-1 (Figure 14).

[0503] The ability of Tat to transactivate non-transfected cells was also investigated. Cells transfected with mRNA encoding Tat or luciferase were plated above a semipermeable membrane (transwell) with un-transfected “bystander” cells plated in the bottom of the transwell, below the semipermeable membrane. Examination of Tat transfected and un-transfected bystander cells after 24 hours revealed luciferase in transfected cells, as well as un-transfected bystander cells that were cultured in proximity to the transfected cells, demonstrating that Tat is secreted and induces transactivation in neighboring cells (Figure 15).

[0504] Nanoparticle Delivery of Tat-86. R5M4 mRNA: With verification that Tat- 86. R5M4 mRNA can reactivate latent HIV, the ability of nanoparticles to successfully introduce Tat-86.R5M4 mRNA was next investigated. As an initial experiment, TZM- bl / JC53-bl cells were treated with various amounts of nontargeted lipid nanoparticles (LNPs) loaded with Tat-86.R5M4 mRNA. Two days after transfection, luminescence was observed in the cells, indicating that the LNPs successfully delivered into the cells (Figure 16).

[0505] Next, J-Lat 10.6 cells were transfected with non-targeted LNPs containing Tat-86. R5M4 mRNA or luciferase mRNA. The cells were cultured for three days, at which point the cells were examined for GFP fluorescence, with cells transfected with Tat-86. R5M4 mRNA displaying a GFP fluorescence while those transfected with the negative control luciferase mRNA displayed the same profile as untreated cells (Figure 17). Subsequently, J-Lat 10.6 cells were treated with varying amounts of nontargeted LNPs encapsulating Tat-86. R5M4 mRNA to determine whether the dose-response relationship previously observed remained. LNPs encapsulating luciferase mRNA were again utilized as a negative control, with the addition of phytohemagglutinin (PHA), known to activate latent HIV in human lymphocytes, as a positive control. While PHA induced only a small fraction of cells, only about 20% activation at 20 pg / million cells (Figure 18 and Figure 19), the intensity of the induced fluoresced was considerable (Figure 20). The LNPs encapsulating Tat-86.R5M4, on the other hand, induced reactivation in over 80% of cells with a similar intensity at a treatment of only 4 pg / million cells (Figure 18 through Figure 20).

[0506] Targeted LNPs for Delivery to HIV Latency Reservoirs: As lymphoid tissue, including CD4+ T-cells, is believed to be the reservoir for latent HIV, it is of great importance to target delivery of LNPs encapsulating Tat-86.R5M4 to these CD4+ cells. As such, LNPs were decorated with anti-CD4 antibodies (Figure 21). Among the anti- CD4 antibodies available, ibalizumab and A161A1 were selected for investigation. Important features of ibalizumab include its previous approval as an HIV anti-retroviral drug, its rapid internalization, and its minimal immunostimulation.

[0507] To test the targeting capability of the specificity of anti-CD4-LNPs, nontargeted LNPs encapsulating GFP mRNA were compared to LNPs conjugated to ibalizumab encapsulating GFP mRNA or luciferase mRNA. These LNPs were then introduced to T-cells expressing human or rhesus CD4. Examination of the cells revealed that the ibalizumab-conjugated LNPs induced GFP fluorescence in both cells expressing human CD4 and cells expressing rhesus CD4, while the non-targeted LNPs and targeted LNPs encapsulating luciferase mRNA did not (Figure 22). Interestingly, no discernable difference was observed in the fluorescence of cells expressing human CD4 treated with 1 pg / million cells versus 2 pg / million cells, but a significant shift was observed in cells expressing rhesus CD4.

[0508] It was examined whether the targeted LNPs could transfect a variety of cells. Ibalizumab-conjugated LNPs encapsulating luciferase mRNA were administered to fresh non-activated CD4+ T-cells, activated CD4+ T-cells, post-activation CD4+ T-cells, and peripheral blood mononuclear cells (PBMCs), and luminescence measured for four days. Luminescence was observed in all cell four cell populations at similar levels, with activated T-cells initially having an increased luminescence that later dropped to the levels of the other three populations (Figure 23).

[0509] PBMCs were then further investigated to determine the ability of CD4- targeted ibalizumab to target CD4+ T-cells within a mixed population. PBMCs were treated with varying concentrations of ibalizumab-conjugated LNPs encapsulating GFP mRNA or luciferase mRNA. PBMCs were treated with ibalizumab-LNPs then sorted with gating for single cells. The single cells obtained were then sorted with gating for live cells. The obtained live cells were then sorted with gating for CD3+ / CD8- cells. The resulting cells were then sorted with gating for GFP fluorescence. While all doses of LNPs encapsulating GFP yielded a shift in GFP fluorescence, the previously observed dose-dependent response was observed again (Figure 24).

[0510] With successful delivery of ibalizumab-conjugated LNPS to CD4+ cells, the efficacy of ibalizumab targeting was then compared to that of Al 61 Al. Here, resting CD4+ cells were treated with varying concentrations of A161Al-LNPs encapsulating GFP mRNA, ibalizumab-LNPs encapsulating GFP mRNA, or ibalizumab-LNPs encapsulating luciferase mRNA. As before with ibalizumab-LNPs encapsulating GFP mRNA, there was no discernable difference in GFP fluorescence of cells treated with 1 pg / million cells versus 2 pg / million cells (Figure 22 and Figure 25). A161Al-LNPs, at doses of 1 pg / million cells or less, displayed a dose-dependent response similar to that of ibalizumab-LNPs when at concentrations less than 1 pg / million cells, with both antibody- LNPs having similar fluorescence profdes at 1 pg / million cells (Figure 24 and Figure

[0511] 25).

[0512] Alterations in the transcriptomic profile of cells treated with LNPs delivering Tat-86. R5M4 mRNA were also examined. J-Lat 10.6 cells were treated with Tat-86. R5M4 mRNA-LNPs at a dose that results in 60% reactivation (2 pg), luciferase mRNA-LNPs (control), or phorbol-12-myri state- 13 -acetate (PMA) and ionomycin and analyzed by RNAseq and ATACseq. UMAP analysis of RNAseq data demonstrated that, while PMA / ionomycin-treated cells have a distinct transcriptomic profile, cells treated with Tat-86.R5M4 mRNA-LNPs and luciferase mRNA-LNPs largely co-localize (Figure

[0513] 26). From this transcriptomic analysis, four genes showed statistically significant difference in expression between Tat-86. R5M4 mRNA-LNPs and luciferase mRNA- LNPs. In Tat-treated cells, a slight decline was observed in the expression of MTRNR2L12, RPS10, and SNHG2S compared to luciferase-treated cells, while an increase in the expression of the HIV genome was observed (Figure 27). This change in expression indicates that Tat treatment is relatively specific for driving expression of the HIV genome, with fe off-target genes showing a change in mRNA levels. ATACseq of these cells demonstrated that Tat-treated cells do not exhibit increased chromatin accessibility two and three days after treatment, as opposed to PMA / ionomycin, which shows significantly increased chromatin accessibility throughout the entirety of the HIV genome (Figure 28). While a small signal was observed at the 5’ end of the genome, this was similarly found in the luciferase-treated cells, and is consistent with the previously described finding that the 5’ LTR of the HIV genome remains accessible. This indicates that Tat treatment transiently, rather than permanently, alters the epigenetic landscape of the HIV genome.

[0514] Targeted Delivery of LNPs Carrying Tat-86,R5M4 mRNA: With Tat- 86. R5M4 mRNA successfully reactivating HIV and LNPs conjugated to an anti-CD4 antibody, targeted LNPs are prepared for delivery of Tat-86.R5M4 mRNA.

[0515] Ibalizumab-conjugated LNPs and nontargeted LNPs were prepared with Tat-86. R5M4 mRNA. Cells expressing CD4 and carrying a Tat-inducible reporter gene were treated with the prepared LNPs at varying concentrations and cultured. Luciferase fluorescence of the cells was measured, with targeted LNPs resulting in significantly improved transactivation compared to non-targeted LNPs (Figure 28).

[0516] Cytotoxicity of CD4-targeted Tat was examined by treating CD4+ T cells or PBMCs from healthy donors were treated with one or multiple doses of targeted CD4- targeted Tat mRNA-LNPs, and cell viability was measured by Annexin-V staining. As positive controls, cells were also activated and lysed. While no difference in Tat-treated and untreated cells was observed, significant cell death was observed in activated cells (Figure 29).

[0517] CD4+ T cells treated with targeted Tat mRNA-LNPs were then examined for activation markers via flow cytometry in comparison to untreated and control activated cells. Expression of CD69, CD25, CD38, and HLA-DR in Tat-treated cells were all consistent with untreated cells, while activated cells showed significant upregulation of all four markers (Figure 30).

[0518] Finaly, the ability of CD4+ T cells treated with Tat mRNA-LNP to transactivate cells harboring dormant HIV was examined. CD4+ T cells were treated with targeted Tat mRNA-LNPs, direct Tat mRNA transfection, or left untreated, and incubated for one or two days, after which the supernatant was harvested. The supernatants were then used to treat TZM-bl cells, where it was observed that the supernatant from Tat mRNA-LNP -treated T cells resulted in high transactivation of TMZ-bl cells (Figure 31).

[0519] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a delivery vehicle conjugated to a targeting domain, wherein the delivery vehicle comprises or encapsulates an mRNA molecule encoding a Tat protein or variant thereof, and further wherein the targeting domain specifically binds to CD4.

2. The composition of claim 1, wherein the delivery vehicle is selected from the group consisting of a lipid nanoparticle, a liposome, and a micelle.

3. The composition of claim 1, wherein the delivery vehicle is a lipid nanoparticle.

4. The composition of claim 1, wherein the mRNA molecule encoding a Tat protein is encapsulated in the lipid nanoparticle.

5. The composition of claim 1, wherein the Tat protein is an attenuated Tat protein about 86 amino acids in length.

6. The composition of claim 1, wherein the Tat protein comprises at least one mutation selected from the group consisting of V36A, Q66A, V67A, S68A, S77A, T23A, I39A, and L69A relative to wildtype Tat-86.

7. The composition of claim 1, wherein the Tat protein comprises V36A, Q66A, V67A, S68A, and S77A mutations.

8. The composition of claim 1, wherein the Tat protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: l, 5, 6, 11, and 13.

9. The composition of claim 1, wherein the Tat protein comprises the amino acid sequence of SEQ ID NO: 1.

10. The composition of claim 1, wherein the mRNA molecule comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO:2.

11. The composition of claim 1, wherein the mRNA molecule comprises a nucleotide sequence of SEQ ID NO:2.

12. The composition of claim 1, wherein the targeting domain comprises an antibody or fragment thereof, an aptamer, a nucleic acid, a protein, a peptide, a glycan, a sugar, a hormone, or a small molecule.

13. The composition of claim 12, wherein the targeting domain comprises an anti-CD4 antibody or CD4-binding fragment thereof.

14. The composition of claim 13, wherein the anti-CD4 antibody or fragment thereof is selected from the group consisting of ibalizumab, A161A1, and CD4-binding fragments thereof.

15. A method of treating or preventing a human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in a subject, the method comprising administering to the subject the composition of any one of claims 1-14.

16. The method of claim 1 , wherein the HIV or SIV is latent in the subject.

17. The method of claim 15 or 16, wherein the method further comprises administering at least one additional therapeutic agent.

18. The method of claim 17, wherein the at least one additional therapeutic agent is selected from the group consisting of abacavir, emtricitabine, lamuvidine, tenofovir disoproxil fumarate, zidovudine, doravirine, efavirenz, etravirine, nevirapine, rilpivirine, atazanavir, darunavir, fosamprenavir, ritonavir, tipranavir, enfuvirtide, maraviroc, cabotegravir, dolutegravir, raltegravir, fostemsavir, idalizumab, lenacapavir, cobicistat, and combinations thereof.

19. A method of activating latent HIV or SIV in a subject comprising administering to the subject the composition of any one of claims 1-14.

20. A method of treating a subject with HIV or SIV comprising reactivating latent HIV or SIV in the subject and administering to the subject at least one anti-HIV therapy or drug.

21. The method of claim 20, wherein the reactivating latent HIV or SIV in the subject comprises administering to the subject the composition of any one of claims 1-14.

22. The method of claim 20, wherein the at least one anti-HIV drug comprises at least one selected from the group consisting of abacavir, emtricitabine, lamuvidine, tenofovir disoproxil fumarate, zidovudine, doravirine, efavirenz, etravirine, nevirapine, rilpivirine, atazanavir, darunavir, fosamprenavir, ritonavir, tipranavir, enfuvirtide, maraviroc, cabotegravir, dolutegravir, raltegravir, fostemsavir, idalizumab, lenacapavir, and cobicistat.

23. A method of detecting latent viral load in a subject comprising: a) obtaining a sample from a subject; b) contacting the sample with a composition of any one of claims 1- 14; and c) detecting the reactivated viral load.

24. The method of claim 23, wherein the sample is at least one selected from the group consisting of a lymph sample, a lymphoid tissue sample, a urine sample, asaliva sample, a mucous sample, a whole blood sample, a blood plasma sample, a blood serum sample, a semen sample, and a milk sample obtained from the subject.

25. The method of claim 23, wherein the step a) further includes a step of isolating T-cells from the sample obtained from the subject.

Images