Transitory modulation of gene transcription through targeted MRNA-LNP delivery of modified transcription activator-like effectors (TALE)

Abstract

Compositions are provided for transient transcriptional modulation of genes through delivery of RNA molecules encoding site-specific DNA binding molecules linked to transcriptional modulation domains, as well as methods for treating or preventing a disease or disorder, including a cancer, infectious disease, or immunological disease.

Description

TRANSITORY MODULATION OF GENE TRANSCRIPTION THROUGH TARGETEDMRNA-LNP DELIVERY OF MODIFIED TRANSCRIPTION ACTIVATOR-LIKEEFFECTORS (TALE)CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 662,296, filed June 20, 2024, which is hereby incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under All 76597 and All 72629 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “046483- 6288-OOWO Sequence Listing.xml,” having a creation date of June 20, 2025, and having a size of 739,862 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0004] The ability to selectively modulate gene transcription at the DNA level is of vital importance in the treatment of various diseases. The relatively recent advances in gene editing with technology such as zinc nuclear finger (ZNF) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) paired with nucleases (i.e., Cas9) have allowed for researchers to selectively target a locus in the cellular genome. These nuclease-based methods have shown promise in clinical trials for permanent modulation of gene transcription including one for sickle cell disease by knocking out transcription of the BCL11 A gene (Frangoul et al., 2020, NEJM 384:252-260) and one for improving the capability of cancer-targeting CAR-Tcells (Zhang et al., 2022, Nature 609:369-374). While permanent gene modulation has its benefits, the potential long-term side effects remain understudied and there are several disease contexts where permanent editing may come with risks for other diseases. One of these examples is in HIV infection where knockout of the CCR5 gene protects CD4+ T-cells from HIV-1 infection. However, CCR5 is an important chemokine receptor for immune responses and may be relevant in limiting the pathogenesis of West Nile Virus and other related flaviviruses (Lim et al., 2006, Trends in Immunology 7:308-12; Ellwanger et al., 2020, Virus Research 286: 198040).

[0005] The latent HIV reservoir is a pool of HIV- 1 infected cells with viral DNA integrated into the host cellular genome that remains even during long-term antiretroviral therapy (ART) where there is undetectable viral load. Upon cessation of ART, viral rebound and resumption of disease progression occurs in most individuals. This latent reservoir is the most pressing obstacle against an accessible HIV cure. One of the most promising theories for a HIV cure is known as “shock and kill” where HIV-1 infected cells are induced (“shock”) to produce viral protein to make them identifiable for downstream elimination by immunological or pharmacological means (“kill”). To date, there have been no “shock” strategies that can completely reawaken the latent reservoir despite the use of non-specific gene modulation (i.e. HD AC inhibitor, SMAC mimetics to induce non-canonical NFKB signaling, cytokines) and targeted gene modulation (i.e. CRISPR-dCas9 with activation domains). Non-specific gene modulation raises the risks of off-target side effects, and the failure of targeted gene modulation highlights the insufficiency of CRISPR-based technologies for targeted gene modulation in the HIV context.

[0006] As such, alternative therapeutic routes with nonpermanent gene modulation can be more palatable as therapies with reduced risk of long-term side effects. Additionally, transitory gene modulation has benefits outside of the HIV context and can be used with other transitory expression systems (i.e. recent mRNA vaccines that temporarily introduce antigens for priming the immune system).

[0007] Thus, there is need in the art for improved compositions and methods for transitory gene modifications. The present invention satisfies this unmet need.SUMMARY OF THE INVENTION

[0008] The invention is based, in part, on the development of compositions for transient modulation of gene transcription comprising delivery of a mRNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional modulator for locus-specific gene transcription modulation, and methods of use thereof for the treatment of a disease or disorder.

[0009] In one embodiment, the invention relates to a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator.

[0010] In one embodiment, the site-specific DNA binding molecule comprises a transcription activator-like effector (TALE).

[0011] In one embodiment, the transcriptional modulator comprises a transcriptional activation domain or a transcriptional repression domain.

[0012] In one embodiment, the transcriptional activation domain is selected from the group consisting of: VP64, VPR, p300, and p65-HSFl.

[0013] In one embodiment, the nucleotide sequence comprises a sequence encoding SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47 retains the function of transcriptional activation.

[0014] In one embodiment, the nucleotide sequence comprises a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48 retains the function of transcriptional activation.

[0015] In one embodiment, the composition comprises a fusion of the transcriptional modulator and Tat. In one embodiment, the Tat comprises an amino acid sequence of SEQ ID NO:51 or a fragment or variant thereof. In one embodiment, the Tat comprises a nucleotide sequence of SEQ ID NO: 52 or a fragment or variant thereof.

[0016] In one embodiment, the transcriptional repression domain comprises a Kriippel- associated box (KRAB) domain comprising a sequence as set forth in SEQ ID NO:49, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:49 retains the function of transcriptional repression.

[0017] In one embodiment, the nucleotide sequence comprises the sequence as set forth in SEQ ID NO: 50, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:50 retains the function of transcriptional repression.

[0018] In one embodiment, the site-specific DNA binding molecule specifically binds to a viral gene, an oncogene, or a human gene, or a regulatory region thereof.

[0019] In one embodiment, the site-specific DNA binding molecule specifically binds to a viral latency gene, or a regulator region thereof.

[0020] In one embodiment, the viral latency gene is an HIV gene, an EBV lytic gene, a human CMV gene, a Kaposi sarcoma-associated herpesvirus gene or hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), or a regulatory region thereof.

[0021] In one embodiment, the TALE is an HIV-specific TALE and further wherein the activation domain is selected from the group consisting of VP64, VPR, p300 and p65- HSF1.

[0022] In one embodiment, the RNA molecule comprises a nucleic acid sequence encoding SEQ ID NO:5, SEQ ID N0:7, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID N0: 15, SEQ ID NO:17, SEQ ID NO 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO 41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47.

[0023] In one embodiment, the RNA molecule comprises a nucleotide sequence comprising SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 or a fragment or variant thereof linked to a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48.

[0024] In one embodiment, the RNA molecule further comprises a nucleotide sequence encoding Tat, or a fragment or variant thereof.

[0025] In one embodiment, the Tat comprises SEQ ID NO:51, or a fragment or variant thereof.

[0026] In one embodiment, the RNA molecule comprises a nucleic acid sequence comprising SEQ ID NO:52, or a fragment or variant thereof.

[0027] In one embodiment, the RNA comprises a nucleic acid sequence encoding SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 or SEQ ID NO:91 or a fragment or variant thereof.

[0028] In one embodiment, the RNA comprises a nucleic acid sequence of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90 or SEQ ID NO:92, or a fragment or variant thereof.

[0029] In one embodiment, the TALE is an HIV-specific TALE and further wherein the transcriptional repression domain comprises a Kriippel-associated box (KRAB) domain.

[0030] In one embodiment, the RNA molecule comprises a nucleic acid sequence encoding SEQ ID NO 5, SEQ ID N0:7, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:49.

[0031] In one embodiment, the RNA molecule comprises a nucleotide sequence comprising SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO 34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 or a fragment or variant thereof linked to a sequence of SEQ ID NO:50.

[0032] In one embodiment, the RNA comprises a nucleic acid sequence encoding SEQ ID NO:77, SEQ ID NO:79 or SEQ ID NO:81 or a fragment or variant thereof.

[0033] In one embodiment, the RNA comprises a nucleic acid sequence of SEQ ID NO:78, SEQ ID NO:80 or SEQ ID NO:82, or a fragment or variant thereof.

[0034] In one embodiment, the site-specific DNA binding molecule specifically binds to CCR5, FOXP3, RORC, IL4, TOX, TOX2, MS4A1, FOXR2, LMO1 or LYL1, or a regulatory region of CCR5, FOXP3, RORC, IL4, TOX, TOX2, MS4A1, FOXR2, LMO1 or LYL1.

[0035] In one embodiment, the TALE is a MS4A1 -specific TALE and further wherein the transcriptional activation domain is VPR, VP64, p300 or p65-HSFl.

[0036] In one embodiment, the RNA comprises a nucleic acid sequence encoding SEQ ID NO: 1 or SEQ ID NO:3 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO 41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47.

[0037] In one embodiment, the RNA comprises a nucleotide sequence comprising SEQ ID NO:2 or SEQ ID NO:4 or a fragment or variant thereof linked to a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48.

[0038] In one embodiment, the RNA comprises a nucleic acid sequence encoding SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:57 or a fragment or variant thereof.

[0039] In one embodiment, the RNA comprises a nucleic acid sequence of SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO:58, or a fragment or variant thereof.

[0040] In one embodiment, the TALE is an MS4A1 -specific TALE and the transcriptional repression domain comprises a Kriippel-associated box (KRAB) domain.

[0041] In one embodiment, the RNA molecule comprises a nucleic acid sequence encoding SEQ ID NO: 1 or SEQ ID NO:3, or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:49.

[0042] In one embodiment, the RNA molecule comprises a nucleotide sequence comprising SEQ ID NO:2, or SEQ ID NO:4 or a fragment or variant thereof linked to a sequence of SEQ ID NO:50.

[0043] In one embodiment, the composition comprises a lipid nanoparticle (LNP) encapsulating the RNA molecule.

[0044] In one embodiment, the RNA molecule comprises an mRNA molecule.

[0045] In one embodiment the invention relates to a method of transiently modulating the transcription of at least one gene in a subject in need thereof, the method comprising administering a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator to the subject.

[0046] In one embodiment, the gene comprises a viral gene, an oncogene, or a human gene.

[0047] In one embodiment the invention relates to a method of inducing expression of latent viral genes in a subject, the method comprising administering to the subject a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator, wherein the composition comprises a site-specific DNA binding molecule that specifically binds to a viral latency gene, or a regulatory region thereof, operably linked to a transcriptional activation domain.

[0048] In one embodiment, the viral latency gene is an HIV gene, an EBV lytic gene, a human CMV gene, a Kaposi sarcoma-associated herpesvirus gene or hepatitis B virus (HBV) covalently closed circular DNA (cccDNA).

[0049] In one embodiment the invention relates to a method of reducing or preventing viral entry into cells in a subject, the method comprising administering to the subject a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator, wherein the composition comprises a site-specific DNA binding molecule that specifically binds to CCR5, or a regulatory region thereof, operably linked to a transcriptional activation domain.

[0050] In one embodiment the invention relates to a method of transiently modulating expression of CD20 in a subject, the method comprising administering to the subject a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator, wherein the composition comprises a site-specific DNA binding molecule specific that specifically binds to MS4A1, or a regulatory region thereof, operably linked to a transcriptional modulation domain.

[0051] In one embodiment the invention relates to a method of improving T cell or NK cell functionality, the method comprising administering a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising adelivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator to the subject.

[0052] In one embodiment, the composition comprises an RNA encoding a site-specific TALE that specifically binds to TOX or TOX2, or a regulatory region thereof, linked to a transcriptional repressor domain.

[0053] In one embodiment the invention relates to a method of increasing the efficacy of a vaccine or cancer therapy, the method comprising administering a composition for sitespecific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator prior to, concurrent with, or following administration of the vaccine or cancer therapy.

[0054] In one embodiment, the composition comprises an RNA encoding a site-specific TALE that specifically binds to IL4 or RORC, or a regulatory region thereof, linked to a transcriptional activation domain.

[0055] In one embodiment the invention relates to a method of inducing the formation of regulatory T cells in a subject in need thereof, the method comprising administering a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator to the subject.

[0056] In one embodiment, the composition comprises an RNA encoding a site-specific TALE that specifically binds to FOXP3, or a regulatory region thereof, linked to a transcriptional activation domain.

[0057] In one embodiment the invention relates to a method of suppressing tumor growth in a subject in need thereof, the method comprising administering a composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator to the subject.

[0058] In one embodiment, the composition comprises an RNA encoding a site-specific TALE specific for binding to an oncogene, or a regulatory region thereof, linked to a transcriptional repression domain

[0059] In one embodiment, the oncogene is selected from the group consisting of FOXR2, LM01 and LYLE

[0060] In one embodiment the invention relates to a plasmid for expression of an RNA molecule encoding a site-specific transient transcriptional modulation of a gene of interest, encoding a site-specific DNA binding molecule linked to a transcriptional modulator. In one embodiment, the plasmid comprises a nucleotide sequence of any one of SEQ ID NO:97 to SEQ ID NO: 146.BRIEF DESCRIPTION OF THE DRAWINGS

[0061] 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.

[0062] Figure 1, comprising Figure 1A through Figure IF, depicts general schematics of TALE constructs. Gene specific TALEs contain 18.5 repeat modules that each confer specificity towards a DNA nucleotide. Combined, a TALE with these repeat modules will confer specificity to a 20bp DNA sequence, thus enabling targeting towards a particular locus. The effector domain can be any domain used to modulate RNA transcription - such as activation (VP64, VPR, p300, p65-HSFl, Tat, or a fusion molecule (e.g., p65-HSFl Tat fusion)), repressive (KRAB), or knockout (TevI, FokI) used to modulate or eliminate RNA transcription. (Figure 1A and Figure IB) Testing versions of TALEs will contain fluorescent protein reporters in either N-terminal end or the C-terminal end. These fluorescent proteins will be separated by a T2A or P2A peptide sequence. (Figure 1C) General schematic of pre-clinical version of TALE without T2A / P2A and reporter domains. (Figure ID) General schematic of next-generation TALE with modified N / C terminal regions. The modifications only occur in the constant regions while the 18.5 repeat modules are unchanged. (Figure IE) General schematic of next-generation TALEs with modified N / C terminal regions and effector domain that is connected to Tat via a linker (including GGGGGS6). For sub-figures (Figure 1A - Figure IE), the domain and construct widths are not to scale. (Figure IF) Example mRNA sequence following the construct shown in (Figure ID) to be used in mRNA LNPs. Axis scale and legend are specific to Figure IF only.

[0063] Figure 2, comprising Figure 2A and Figure 2B, depicts schematic diagrams of specific targeting of TALEs. Figure 2A depicts specific targeting of various TALEs in the 5’ LTR region of integrated HIV-1 DNA in JLat 10.6 cells. Figure 2B depicts specific targeting of varioues TALEs in the intergenic region of integrated HIV-1 DNA in JLat 10.6 cells. All basepair (bp) coordinates are relative to the proviral JLat 10.6 sequence.

[0064] Figure 3 depicts representative flow plots demonstrating HIV reactivation potential from different regions that are shown in Figure 2. Samples shown are JLat 10.6 cells which contain integrated HIV DNA with an GFP reporter. X-axis indicates GFP signal as a marker of HIV reactivation while Y-axis represents cell viability. TALE2 and TALE3 have the strongest reactivation potential.

[0065] Figure 4 depicts representative experimental results demonstrating that, as introduced in Figure 1, different generations of TALE mRNA constructs were electroporated into JLat 10.6 cells. For this figure, the base TALE was the HIV-specific TALE2 and the effector domain was VP64. These data indicate that modifications / truncations to the N and C terminal constant regions do not impact the HIV reactivation potential as shown on the X-axis.

[0066] Figure 5, comprising Figure 5A and Figure 5B, depicts various N / C terminal truncated TALE mRNAs were electroporated into different latent HIV-infected cell lines (each contains a GFP reporter as a measure of HIV reactivation). As positive controls, viral Tat mRNA and Tnfa (100 ng / ml) were used. Neg represents untreated cells. Figure 5 A depicts the efficiency of HIV reactivation as a percentage of GFP positive cells from gated live cells. Figure 5B depicts the strength of HIV reactivation, shown as the brightness of GFP signal. Error bars for Figure 5A and Figure 5B represent mean + / - standard error.

[0067] Figure 6 depicts representative experimental results demonstrating that nextgeneration TALE2 with p65-HSFl fused with Tat is able to reactivate HIV at higher levels than each individual component (TALE2-p65-HSFl and Tat). mRNA encoding various components or fusion TALE-Tat were electroporated into JLat 6.3 and 8.4 cells. HIV reactivation was most potent in the fusion TALE-Tat condition.

[0068] Figure 7 depicts representative experimental results demonstrating that TALEs can also target other loci, including in the human genome. Representative flow plots are shown for a mRNA encoding a CD20-specific TALE (N and C terminal truncated) fused with thep65-HSFl effector domain that was transfected in various HIV-infected cell lines. Strong CD20 signal is detected in all three cell lines.

[0069] Figure 8 depicts representative experimental results demonstrating that JLat 10.6 cells were treated with 2 pg mRNA-LNP encoding either the HIV-specific TALE3 (unmodified) or viral protein Tat. TALE3 mRNA-LNP can potently reactivate proviral transcription above the baseline (untreated) control and at higher levels compared to viral Tat. A positive control with non-specific TNFα (100ng / ml) treatment is included for comparison.

[0070] Figure 9 depicts data demonstrating that CD4-targeted mRNA LNP delivery of HIV-specific TALE reactivates provirus within 24 hours in ex vivo samples. Memory CD4+ T cells were isolated from three donors with HIV that were taking antiretroviral therapy. Cells were treated with 1 pg ibalizumab (anti-CD4) conjugated mRNA-LNP per million cells for 24 hours. mRNALNPs either encoded HIV specific TALE3 (unmodified) with VP64 activation domain or a negative control sequence (tdTomato). A positive control (16 pM PMA + 1 pM ionomycin) is included for comparison. In two of the donors, mRNA-LNPs encoding TALE3 increases HIV transcription, while mRNA-LNPs encoding tdTomato are not different from untreated conditions. The donor that did not have increased HIV transcription has a consensus sequence with mismatches to the TALE3 recognition site.DETAILED DESCRIPTION

[0071] In various embodiments, the invention relates to compositions for transient modulation of gene transcription comprising delivery of a mRNA molecule encoding a sitespecific DNA binding molecule linked to a transcriptional modulator for locus-specific gene transcription modulation.

[0072] In some embodiments, the site-specific DNA binding molecule comprises a transcription activator-like effector (TALE). In some embodiments, the TALE is fused to a transcriptional activation domain or a transcriptional repression domain. In some embodiments, the activation domain is VP64 comprising 4 tandem repeats of the trans activator VP16 protein from herpes simplex virus. In some embodiments, the activation domain is VPR comprising VP64, p65 from human NFKB, and Rta from Epstein Barr Virus. In some embodiments the TALE is HIV specific and the activation domain is VPR. In some embodiments the TALE is HIV specific and the activation domain is VP64. In some embodiments the TALE is HIVspecific and the activation domain is p65-HSFl . In some embodiments, the TALE is CD20 specific and the activation domain is VPR. In some embodiments the TALE is CD20 specific and the activation domain is p65-HSFl.

[0073] In some embodiments, the site-specific DNA binding molecule comprises a TALE fused to a transcriptional activation domain and Tat. In some embodiments, the Tat is HIV Tat or a variant thereof. In some embodiments the TALE is HIV specific. In some embodiments, the activation domain is VPR, VP64, p65-HSFl or p300.

[0074] In some embodiments, the invention also relates methods of treating a disease or disorder in a subject in need thereof, the method comprising administration of a delivery vehicle comprising an mRNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional modulator. In some embodiments, the disease or disorder includes, but is not limited to, cancer, viral infection, HIV, or HIV latency.Definitions

[0075] 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.

[0076] As used herein, each of the following terms has the meaning associated with it in this section.

[0077] 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.

[0078] “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.

[0079] 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 of the 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).

[0080] 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.

[0081] 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.

[0082] 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.

[0083] By the term “synthetic antibody” as used herein, is meant 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.

[0084] 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 itwould 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.

[0085] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0086] “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 noncoding 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.

[0087] “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.

[0088] “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.

[0089] “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.

[0090] 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.

[0091] 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. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0092] 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.

[0093] 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 translation by translational machinery in a cell. For example, in some aspects, the nucleotide sequence comprises an mRNA where some or all of the uridines have been replaced with pseudouridine, 1 -methyl pseudouridine, or another modified nucleoside.

[0094] 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 secondnucleic 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.

[0095] 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.

[0096] 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.

[0097] 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).

[0098] In some embodiments, “pseudouridine” refers to (l-methyl-3-(3-amino-3 -carboxypropyl) pseudouridine). In another embodiment, the term refers to (1-methylpseudouridine). In another embodiment, the term refers to (2’-O-methylpseudouridine. In another embodiment, the term refers to m5D (5- methyldihydrouridine). In another embodiment, the term refers to(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, theterm refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.

[0099] 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 two or more 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.

[0100] As used herein, “fragment” is defined as at least a portion of a sequence. For example, in one embodiment, the term “fragment” refers to a portion of the variable region of the immunoglobulin molecule which binds to its target, i.e. the antigen binding region. Some of the constant region of the immunoglobulin may be included.

[0101] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by at least one substitution, addition, or deletion, or any combination thereof. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or bydirect synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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, whenadministered, 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] “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 bond), having from one to twenty-four carbon atoms (C1-C24alkyl), one to twelve carbon atoms (C1-C12alkyl), one to eight carbon atoms (C1-C8alkyl) or one to six carbon atoms (C1-C6alkyl) 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 1 enyl, but-l-enyl, pent-1-enyl, penta- 1 ,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless specifically stated otherwise, an alkyl group is optionally substituted.

[0111] “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 bond (alkynylene)), and having, for example, from one to twenty- four carbon atoms (C1-C24alkylene), one to fifteen carbon atoms (C1-C15alkylene), one to twelve carbon atoms (C1-C12alkylene), one to eight carbon atoms (C1-C8alkylene), one to six carbon atoms (C1-C6alkylene), two to four carbon atoms (C2-C4alkylene), one to two carbon atoms (C1-C2alkylene), e.g., methylene, ethylene, propylene, n butylene, ethenylene, propenylene, n butenylene, propynylene, n 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.

[0112] “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, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise, a cycloalkyl group is optionally substituted.

[0113] “Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.

[0114] “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 selected from the group consisting of nitrogen, oxygen and 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, thienylf 1 ,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 di oxo thiomorpholinyl. Unless specifically stated otherwise, a heterocyclyl group may be optionally substituted.

[0115] 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 nonhydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; oxo groups (=0); hydroxyl groups (-OH); alkoxy groups ( ORa, where Rais C1-C12alkyl or cycloalkyl); carboxyl groups ( 0C(=0) Raor -C(=0)0Ra, where Rais H, C1-C12alkyl or cycloalkyl); amine groups ( NRaRb, where Raand Rbare each independently H, C1-C12alkyl or cycloalkyl); C1-C12alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12alkyl 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.

[0116] “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.

[0117] As used herein, the term “genome editing vector” refers to a nucleic acid molecule which encodes the components of a genome editing system, such as, but not limited to, a CRISPR / Cas9 protein, a base editor, or a prime editor, and any associated requiredcomponents, such as an appropriate guide RNA (gRNA). See Kantor et al., “CRISPR-Cas9 DNA Base-Editing and Prime Editing,” Int J Mol Sci, 2020; 21; p. 6240, the contents of which are incorporated by reference.

[0118] 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.Description

[0119] The invention relates to the development of compositions for site-specific transient transcription modulation. In some embodiments, the composition comprises a delivery vehicle comprising or encapsulating an RNA molecule encoding a site-specific DNA binding molecule fused with a transcriptional activation or repression domains for locusspecific gene transcription modulation.

[0120] In some embodiments, the site-specific DNA binding molecule comprises a zinc finger domain, an antisense oligonucleotide or a transcription activator-like effector (TALE).

[0121] In some embodiments, the composition of the invention comprises an RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional repressor which transiently decreases expression of a specific gene or genomic region. In some embodiments, the composition of the invention comprises a TALE linked to a transcriptional repressor which transiently increases expression of a specific gene or genomic region.

[0122] In some embodiments, the composition of the invention comprises an RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional activator which transiently increases expression of a specific gene or genomic region. In some embodiments, the composition of the invention comprises a TALE linked to a transcriptional activator which transiently increases expression of a specific gene or genomic region.

[0123] In one embodiment, the composition comprises a TALE specific for binding to a latent viral gene. Exemplary genes that can be regulated using the compositions and methods of the invention include, but are not limited to, latent HIV genes, EBV lytic genes (e.g., BZLF1 and BRLF1), human CMV (e.g., major IE promoter and major IE enhancer), Kaposi sarcoma- associated herpesvirus (e.g., RTA gene and LANA latency gene) and HBV (targeting HBV cccDNA). In one embodiment, the composition comprises a TALE that specially binds to a transcriptional activator to induce expression of latent viral genes. In one embodiment, the composition comprises a TALE that specifically binds to a transcriptional activator to induce expression of latent viral genes. In one embodiment, the composition comprises a TALE that specifically binds to a transcriptional repressor to repress expression of genes required for viral latency.

[0124] In one embodiment, the composition comprises a TALE that specifically binds to a human gene target or human genomic locus (e.g., a promoter or enhancer region of a human gene).

[0125] In one embodiment, the composition comprises a TALE that specifically binds to CCR5 linked to a transcriptional repressor to transiently inhibit expression of CCR5, thereby protecting cells from HIV-1 infection. In such an embodiment, it is envisioned that the delivery vehicle comprises a targeting molecule for targeted delivery to CD4+ T cells. In some embodiments, the targeting molecule comprises an anti-CD4 antibody.

[0126] In one embodiment, the composition comprises a TALE that specifically binds to a human gene target. In some embodiment, the composition comprises a TALE that specifically binds to FOXP3 operably linked to a transcriptional activator for increasing expression of FOXP3. In some embodiments, the composition is used for increasing regulatory T cell formation. In some embodiments, a TALE that specifically binds to FOXP3 operably linked to a transcriptional activator can be administered to increase regulatory T cells for the treatment of an autoimmune disease or disorder.

[0127] In some embodiment, the composition comprises a TALE that specifically binds to RORC operably linked to a transcriptional activator for increasing expression of RORC. In some embodiments, the composition is used for increasing Thl7 induction. In some embodiments, a TALE that specifically binds to RORC operably linked to a transcriptional activator can be administered to increase Th 17 induction as an adjuvant therapy forvaccination. For example, in some embodiments, the composition can be administered as an additional element to prime and boost vaccine efficacy.

[0128] In some embodiments, the composition comprises a TALE that specifically binds to IL4 operably linked to a transcriptional activator for increasing expression of IL4. In some embodiments, the composition is used for Th2 cell induction. In some embodiments, a TALE that specifically binds to IL4 operably linked to a transcriptional activator can be administered to increase Th2 induction as an adjuvant therapy for vaccination. For example, in some embodiments, the composition can be administered as an additional element to prime and boost vaccine efficacy.

[0129] In some embodiments, the composition comprises a TALE that specifically binds to TOX and / or TOX2 operably linked to a transcriptional repressor for decreasing expression of TOX and / or TOX2. In some embodiments, the composition is used to reduce T cell exhaustion. In some embodiments, a TALE that specifically binds to TOX and / or TOX2 operably linked to a transcriptional repressor can be administered to improve T cell and / or NK cell functionality.

[0130] In one embodiment, the composition comprises a TALE that specifically binds to an oncogene or oncogene regulatory region (e.g., a promoter or enhancer region of an oncogene).

[0131] In some embodiments, the composition comprises a TALE that specifically binds to MS4A1 operably linked to a transcriptional activator for increasing expression of MS4A1. In some embodiments, the composition is used to increase CD20 expression. In some embodiments, a TALE that specifically binds to MS4A1 operably linked to a transcriptional activator can be administered to improve CD20 CAR-T therapy for the treatment of B cell lymphoma.

[0132] In some embodiments, the composition comprises a TALE that specifically binds to FOXR2 operably linked to a transcriptional repressor for decreasing expression of FOXR2. In some embodiments, the composition is used to decrease aberrant expression of FOXR2 in tumor cells, thus suppressing tumor growth.

[0133] In some embodiments, the composition comprises a TALE that specifically binds to LM01 or LYL1 operably linked to a transcriptional repressor for decreasing expression of LM01 or LYL1. In some embodiments, the composition is used to decreaseaberrant expression of LM01 or LYL1 in tumor cells, thus suppressing tumor growth. In some embodiments, the tumor is acute lymphoblastic leukemia and / or acute myeloid leukemia.Transcriptional Modulator

[0134] In some embodiments, the composition comprises an RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional activator which transiently increases expression of a specific gene or genomic region. Exemplary transcriptional activators include, but are not limited to, VP64, VPR, p300 or p65-HSFl, or a fragment or variant thereof. In some embodiments, the VPR activation domain comprises VP64, p65 from human NFKB, and Rta from Epstein Barr Virus.

[0135] In one embodiment, the nucleotide sequence comprises a sequence encoding SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47 retains the function of transcriptional activation.

[0136] In one embodiment, the nucleotide sequence comprises a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48 retains the function of transcriptional activation.

[0137] In some embodiments, the composition comprises an RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional repressor which transiently decreases expression of a specific gene or genomic region. In some embodiments, the transcriptional repressor comprises a Kriippel-associated box (KRAB) domain or a fragment or variant thereof.

[0138] In one embodiment, the transcriptional repression domain comprises a Kriippel- associated box (KRAB) domain comprising a sequence as set forth in SEQ ID NO:49, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:49 retains the function of transcriptional repression.

[0139] In one embodiment, the nucleotide sequence comprises the sequence as set forth in SEQ ID NO: 50, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:50 retains the function of transcriptional repression.

[0140] In some embodiments, the RNA encoding the HIV-specific TALE encodes a protein that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQIDNO:13, SEQIDNO:15, SEQIDNO:17, SEQIDNO:19, SEQ IDNO:21, SEQIDNO:23, SEQIDNO25, SEQ ID NO: 27, SEQIDNO:29, SEQIDNO:31, SEQ ID NO:33, SEQ ID NO 35, SEQ ID NO: 37, or SEQ ID NO: 39, or a fragment thereof. In some embodiments, the fragment or variant of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQIDNO:11, SEQIDNO:13, SEQIDNO:15, SEQIDNO:17, SEQIDNO:19, SEQ ID NO:21, SEQIDNO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 retains site-specific binding to HIV.

[0141] In some embodiments, the RNA encoding the HIV-specific TALE comprises a nucleotide sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQIDNO:12, SEQIDNO:14, SEQIDNO:16, SEQIDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO 24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or a fragment thereof. In some embodiments, the fragment or variant of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO 24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 encodes an HIV-specific TALE which retains site-specific binding to HIV.

[0142] In some embodiments, the RNA encoding an HIV-specific TALE linked to an activation domain encodes SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQIDNO:21, SEQIDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, or a fragment or variant thereof. In some embodiments, the RNA encoding the HIV-specific TALE linked to an activation domain comprises a nucleotide sequence of SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 or a fragment or variant thereof linked to a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, or a fragment or variant thereof.

[0143] In one embodiment, the RNA comprises a nucleic acid sequence encoding SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, or SEQ ID NO:75, or a fragment or variant thereof.

[0144] In one embodiment, the RNA comprises a nucleic acid sequence of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO: 72, SEQ ID NO: 74, or SEQ ID NO: 76, or a fragment or variant thereof.

[0145] In some embodiments, the RNA encoding an HIV-specific TALE linked to a transcriptional repression domain encodes SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:49, or a fragment or variant thereof. In some embodiments, the RNA encoding the HIV-specific TALE linked to a transcriptional repression domain comprises a nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID N0: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 or a fragment or variant thereof linked to a sequence of SEQ ID NO:50, or a fragment or variant thereof.

[0146] In one embodiment, the RNA comprises a nucleic acid sequence encoding SEQ ID NO:77, SEQ ID NO:79 or SEQ ID NO:81 or a fragment or variant thereof.

[0147] In one embodiment, the RNA comprises a nucleic acid sequence of SEQ ID NO:78, SEQ ID NO:80 or SEQ ID NO:82, or a fragment or variant thereof.

[0148] In some embodiments, the RNA encoding the MS4A1 -specific TALE encodes a protein that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity SEQ ID NO: 1 or SEQ ID NO:3, or a fragment thereof. In some embodiments, the fragment or variant of SEQ ID NO: 1 or SEQ ID NO:3 retains site-specific binding to MS4A1.

[0149] In some embodiments, the RNA encoding the MS4A1 -specific TALE comprises a nucleotide sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2 or SEQ ID NO:4, or a fragment thereof. In some embodiments, the fragment or variant of SEQ ID NO:2 or SEQ ID NO:4 encodes a MS4Al-specific TALE which retains sitespecific binding to MS4A1.

[0150] In some embodiments, the RNA encoding a MS4A1 -specific TALE linked to an activation domain encodes SEQ ID NO: 1 or SEQ ID NO:3 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, or a fragment or variant thereof.

[0151] In some embodiments, the RNA encoding the MS4Al-specific TALE linked to an activation domain comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or a fragment or variant thereof, linked to a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, or a fragment or variant thereof.

[0152] In one embodiment, the RNA comprises a nucleic acid sequence encoding SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:57 or a fragment or variant thereof.

[0153] In one embodiment, the RNA comprises a nucleic acid sequence of SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO:58, or a fragment or variant thereof.

[0154] In some embodiments, the RNA encoding a MS4A1 -specific TALE linked to a transcriptional repression domain encodes SEQ ID NO: 1 or SEQ ID NO:3 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:49, or a fragment or variant thereof. In some embodiments, the RNA encoding the MS4A1 -specific TALE linked to a transcriptional repression domain comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or a fragment or variant thereof linked to a sequence of SEQ ID NO:50, or a fragment or variant thereof.

[0155] In some embodiments, the composition comprises a lipid nanoparticle (LNP) encapsulating an RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional modulator. In some embodiments, the RNA molecule is encapsulated in the LNP.Tat Fusion

[0156] In some embodiments, the invention comprises a comprises an RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional activator which transiently increases expression of a specific gene or genomic region fused to an HIV Tat protein, SIV Tat protein, SHIV Tat protein, or a fragment or variant thereof. Exemplary transcriptional activators include, but are not limited to, VP64, VPR, p300 or p65-HSFl, or a fragment or variant thereof.

[0157] 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.

[0158] 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:51. 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. Exemplary Tat sequences that can be included in a fusion molecule include, but are not limitedto, those described in WO 2025 / 007009, which is incorporated herein by reference in its entirety. In some embodiments, the HIV Tat protein comprises an amino acid sequence as set forth in SEQ ID NO:51, or a fragment or variant thereof. 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:52. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO:52.

[0159] In some embodiments, the RNA molecule encodes an HIV-specific TALE operably linked to an activation domain fused to Tat.

[0160] In some embodiments, the RNA encoding an HIV-specific TALE-activation domain-Tat fusion encodes SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID N0:13, SEQ ID NO:15, SEQ ID NO 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 linked to SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47 linked to SEQ ID NO:51, or a fragment or variant thereof. In some embodiments, the RNA encoding the HIV-specific TALE-activation domain-Tat fusion comprises a nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 linked to SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48 linked to SEQ ID NO:52, or a fragment or variant thereof. In some embodiments, the variant of SEQ ID NO:51 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:51. In some embodiments, the variant of SEQ ID NO:52 comprises a nucleotide sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:52.

[0161] In some embodiments, the RNA molecule encodes an HIV-specific TALE operably linked to a transcriptional activation domain linked to Tat. In some embodiments, the RNA encoding an HIV-specific TALE operably linked to a transcriptional activation domainlinked to Tat encodes SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 or SEQ ID NO:91 or a fragment or variant thereof. In some embodiments, the RNA encoding the HIV- specific TALE operably linked to a transcriptional activation domain linked to Tat comprises a nucleotide sequence of SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90 or SEQ ID NO: 92, or a fragment or variant thereof. In some embodiments, the RNA encoding the HIV-specific TALE operably linked to a transcriptional activation domain linked to Tat encodes a protein that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 or SEQ ID NO:91, or a fragment thereof. In some embodiments, the RNA encoding the HIV-specific TALE operably linked to a transcriptional activation domain linked to Tat comprises a nucleotide sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90 or SEQ ID NO: 92 or a fragment thereof.Delivery Vehicle

[0162] The invention relates in part to compositions for delivery of a gene editing molecule. In some embodiments, the composition comprises a delivery vehicle comprising a targeting molecule for targeted delivery of a gene editing molecule to a specific cell of interest. In some embodiments, the composition comprises at least one RNA molecule encoding a gene editing agent comprising a site-specific DNA binding domain linked to a transcriptional modulatory domain.

[0163] In various embodiments, the composition comprises lipids or a derivative thereof. In various embodiments, the composition comprises a nanoparticle (LNP).

[0164] 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, aldehydes, and polymers (e g., PEGylated lipids).

[0165] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetylphosphate (“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.

[0166] In one embodiment, the LNP comprises at least one cationic lipid, and at least one stabilizing lipid. Stabilizing lipids include neutral lipids and pegylated lipids.

[0167] In one embodiment, 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.

[0168] 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 carboxy spermine (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 -di oleyloxy )propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipidscomprising di octadecyl amidoglycyl 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), N,N-dimethyl-2,3-bis(((9Z, 12Z, 15Z)-octadeca-9, 12, 15- trien- 1 -yl)oxy)propan- 1 -amine (DLenDMA).

[0169] In one embodiment, 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- dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1, 2-dilinoley oxy-3 - morpholinopropane (DLin-MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), l,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-[l,3]-dioxolane (DLin-K-DMA).

[0170] Suitable amino lipids include, but are not limited to, those having the formula:wherein Ri and R2 are either the same or different and independently optionally substituted C10-C24alkyl, optionally substituted C10-C24alkenyl, optionally substituted C10-C24alkynyl, or optionally substituted C10-C24acyl;R3and R4are either the same or different and independently optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, or optionally substituted C2-C6alkynyl or R3and 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;R5is either absent or present and when present is hydrogen or C1-C6alkyl; 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 Y and Z are either the same or different and independently O, S, or NH.

[0171] In one embodiment, Ri and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0172] A representative useful dilinoleyl amino lipid has the formula:wherein n is 0, 1, 2, 3, or 4.

[0173] In one embodiment, the cationic lipid is a DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).

[0174] In one embodiment, the cationic lipid component of the LNPs has the structure of Formula (I):or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:! / and L2are each independently -O(C=O)-, -(C=O)O- or a carbon-carbon double bond;Rlaand Rlbare, at each occurrence, independently either (a) H or C1-C12alkyl, or (b) Rlais H or C1-C12alkyl, 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 carboncarbon double bond;R2aand R2bare, at each occurrence, independently either (a) H or C1-C12alkyl, or (b) R2ais H or C1-C12alkyl, 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 carboncarbon double bond;R3aand R3bare, at each occurrence, independently either (a) H or C1-C12alkyl, or (b) R3ais H or C1-C12alkyl, 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 carboncarbon double bond;R4aand R4bare, at each occurrence, independently either (a) H or C1-C12alkyl, or (b) R4ais H or C1-C12alkyl, 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 carboncarbon double bond;R5and R6are each independently methyl or cycloalkyl;R7is, at each occurrence, independently H or C1-C12alkyl;R8and R9are each independently C1-C12alkyl; 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.

[0175] In certain embodiments of Formula (I), at least one of Rla, R2a, R3aor R4ais Ci- C12alkyl, 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.

[0176] In still further embodiments of Formula (I), at least one of Rla, R2a, R3aor R4ais C1-C12alkyl, or at least one of L1or L2is -O(C=O)- or -(C=O)O-; and Rlaand Rlbare not isopropyl when a is 6 or n-butyl when a is 8.

[0177] In other embodiments of Formula (I), R8and R9are each independently unsubstituted C1-C12alkyl; 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;

[0178] 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 carboncarbon double bond.

[0179] In some embodiments of Formula (I), one of L1or L2is -O(C=O)-. In other embodiments, both L1and L2are -O(C=O)-.

[0180] In some embodiments of Formula (I), one of L1or L2is -(C=O)O- In other embodiments, both L1and L2are -(C=O)O-

[0181] In some other embodiments of Formula (I), one of L1or L2is a carbon-carbon double bond. In other embodiments, both L1and L2are a carbon-carbon double bond.

[0182] 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.

[0183] 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, C1-C12alkyl or cycloalkyl, for example H or C1-C12alkyl.

[0184] In other embodiments, the lipid compounds of Formula (I) have the following structure (la):

[0185] In other embodiments, the lipid compounds of Formula (I) have the following structure (lb):

[0186] In yet other embodiments, the lipid compounds of Formula (I) have the following structure (Ic):

[0187] 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.

[0188] 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.

[0189] 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. Insome 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.

[0190] 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.

[0191] 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.

[0192] 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 one embodiment, 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.

[0193] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0194] 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-C12alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais C1-C8alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais C1-C6alkyl. In some of the foregoing embodiments, the C1-C8alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0195] In certain embodiments of Formula (I), Rla, Rlb, R4aand R4bare C1-C12alkyl at each occurrence.

[0196] In further embodiments of Formula (I), at least one of Rlb, R2b, R3band R4bis H or Rlb, R2b, R3band R4bare H at each occurrence.

[0197] 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.

[0198] The substituents at R5and R6of Formula (I) are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R3or R6is methyl. In certain other embodiments one or both of R5or 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-C12alkyl, for example tert-butyl.

[0199] 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-C12alkyl.

[0200] In certain other of the foregoing embodiments of Formula (I), one of R8or R9is methyl. In other embodiments, both R8and R9are methyl.

[0201] 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.

[0202] In various different embodiments, exemplary lipid of Formula (I) can include

[0203] In some embodiments, the LNP comprises 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 (1) is compound 1-6.

[0204] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (II):or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:LI and L2 are 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)NR, -OC(=O)NRa-, -NRaC(=O)O-, or a direct bond;G1is C1-C2 alkylene, -(C=O)- , -O(C=O)-, -SC(=O)-, -NRaC(=O)- or a direct bond;G2is -C(=O)- , -(C=O)O-, -C(=O)S-, -C(=O)NRaor a direct bond;G3is C1-C6alkylene;Rais H or C1-C12alkyl;Rlaand Rlbare, at each occurrence, independently either: (a) H or C1-C12alkyl; or (b) Rlais H or C1-C12alkyl, 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 carboncarbon double bond;R2aand R2bare, at each occurrence, independently either: (a) H or C1-C12alkyl; or (b) R2ais H or C1-C12alkyl, 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 carboncarbon double bond;R3aand R3bare, at each occurrence, independently either: (a) H or C1-C12alkyl; or (b) R3ais H or C1-C12alkyl, 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 carboncarbon double bond;R4aand R4bare, at each occurrence, independently either: (a) H or C1-C12alkyl; or (b) R4ais H or C1-C12alkyl, 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 carboncarbon double bond;R5and R6are each independently H or methyl;R7is C4-C20 alkyl;R8and R9are each independently C1-C12alkyl; 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.

[0205] 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.

[0206] 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-

[0207] In other of the foregoing embodiments of Formula (II), the lipid compound has one of the following structures (IIA) or (IIB):

[0208] In some embodiments of Formula (II), the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).

[0209] 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)-.

[0210] 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-.

[0211] 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.

[0212] In other different embodiments of Formula (II), for at least one occurrence of Rlaand Rlb, Rlais H or C1-C12alkyl, 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.

[0213] In still other different embodiments of Formula (II), for at least one occurrence of R4aand R4b, R4ais H or C1-C12alkyl, 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.

[0214] In more embodiments of Formula (II), for at least one occurrence of R2aand R2b, R2ais H or C1-C12alkyl, 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 carboncarbon double bond.

[0215] In other different embodiments of Formula (II), for at least one occurrence of R3aand R3b, R3ais H or C1-C12alkyl, 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.

[0216] In various other embodiments of Formula (II), the lipid compound has one of the following structures (IIC) or (IID):wherein e, f, g and h are each independently an integer from 1 to 12.

[0217] In some embodiments of Formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).

[0218] In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[0219] 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 otherembodiments, a is 10. In more embodiments, a is 1 1 . 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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. Insome embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 1 1 . In yet other embodiments, f is 12.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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 one embodiment, 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.

[0229] 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-C12alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais C1-C8alkyl. In certain other embodiments at least one of Rla, R2a, R3aand R4ais C1-C6alkyl. In some of the foregoing embodiments, the C1-C8alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0230] In certain embodiments of Formula (II), Rla, Rlb, R4aand R4bare C1-C12alkyl at each occurrence.

[0231] In further embodiments of Formula (II), at least one of Rlb, R2b, R3band R4bis H or Rlb, R2b, R3band R4bare H at each occurrence.

[0232] 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.

[0233] The substituents at R5and R6of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments one of R5or R6is methyl. In other embodiments each of R5or R6is methyl.

[0234] The substituents at R7of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments R7is C6-C16alkyl. In some other embodiments, R7is C6-C9alkyl. 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-C12alkyl; Rbis C1-C15alkyl; and x is 0, 1 or 2. For example, in some embodiments R7is substituted with -(C=O)ORbor -O(C=O)Rb.

[0235] In various of the foregoing embodiments of Formula (II), Rbis branched C1-C15alkyl. For example, in some embodiments Rbhas one of the following structures:

[0236] In certain other of the foregoing embodiments of Formula (II), one of R8or R9is methyl. In other embodiments, both R8and R9are methyl.

[0237] 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.

[0238] In still other embodiments of the foregoing lipids of Formula (II), G3is C2-C4alkylene, for example C3alkylene.

[0239] In various different embodiments, the lipid compound has one of the following structures:

[0240] In some embodiments, the LNP comprises a lipid of Formula (II), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. 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-l 1. In some embodiments, the lipid of Formula (II) is compound 11-12. In some embodiments, the lipid of Formula (II) is compound 11-32.

[0241] In some other embodiments, the cationic lipid component of the LNP 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)-, -(OO)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;G1and G2are each independently unsubstituted C1-C12alkylene or C1-C12alkenylene;G3is C1-C24alkylene, C1-C24alkenylene, C3-C8cycloalkylene, C3-C8cycloalkenylene;Rais H or C1-C12alkyl;R1and R2are each independently C6-C24alkyl or C6-C24alkenyl;R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;R4is C1-C12alkyl;R5is H or C1-C6alkyl; and x is 0, 1 or 2.

[0242] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):wherein:A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;R6is, at each occurrence, independently H, OH or C1-C24alkyl; n is an integer ranging from 1 to 15.

[0243] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0244] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):wherein y and z are each independently integers ranging from 1 to 12.

[0245] 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 eachindependently -(C=0)0- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0246] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

[0247] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

[0248] 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 is3, 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.

[0249] 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.

[0250] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24alkyl. In other embodiments, R6is OH.

[0251] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24alkylene or linear C1-C24alkenylene.

[0252] 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-C12alkyl; 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.

[0253] 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 C1-C8alkyl. For example, in some embodiments, C1-C8alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

[0254] In different embodiments of Formula (III), R1or R2, or both, has one of theCN, -C(=O)OR4, -OC(=O)R4or -NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0256] In various different embodiments, the cationic lipid of Formula (III) has one of the following structures:

[0257] In some embodiments, the LNP comprises a lipid of Formula (III), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound III-7.

[0258] In some other embodiments, the cationic lipid component of the LNP has the structure of Formula (IV):or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,o is an integer from 1 to 4, p is an integer from 1 to 4, wherein when p = 1 , each R is independently C6to C16straight-chain alkyl; C6to C16branched alkyl; C6to C16straight-chain alkenyl; C6to C16branched alkenyl; C9to C16cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C8to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each R is independently C6to C14straight-chain alkyl; C6to C14straight-chain alkenyl; C6to C14branched alkyl; C6to C14branched alkenyl; C9to C14cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at the either end orwithin the alkyl chain; or C8to C16aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 3, each R is independently C6to C12straight-chain alkyl; C6to C12straight-chain alkenyl; C8to C12branched alkyl; C6to C12branched alkenyl; C9to C12cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C8to C14aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each R is independently C6to C10straight-chain alkyl; C6to C10straight-chain alkenyl; C6to C10branched alkyl; C6to C10branched alkenyl; C9to C10cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl; or Cs to C12 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0259] Some embodiments specifically include one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and / or carbon chain length, structure, or saturation. In some embodiments, when p is 1, each R is independently C6to C12, C13, or C14straight-chain alkyl. In some embodiments, each R from a nearest common branch point is the same. In some embodiments, each R is the same.

[0260] In some embodiments, the ionizable cationic lipid has a structure of Formula IVawherein each R is independently C6to C16straight-chain alkyl; C6to C16branched alkyl; C6to C16straight-chain alkenyl; C6to C16branched alkenyl; C9to C16cycloalkyl -alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C8to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chainY is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,o is an integer from 1 to 4.

[0261] In some other embodiments, the cationic lipid component of the LNP has the structure of Formula (V):n is an integer from 0 to 4,m is an integer from 1 to 3, and o is an integer from 1 to 4, p is an integer from 1 to 4, wherein when p = 1 , each R is independently C6to C16straight-chain alkyl; C6to C16branched alkyl; C6to C16straight-chain alkenyl; C6to C16branched alkenyl; C9to C16cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C8to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each R is independently C6to C14straight-chain alkyl; C6to C14straight-chain alkenyl; C6to C14branched alkyl; C6to C14branched alkenyl; C9to C14cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at the either end or within the alkyl chain; or C8to C16aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 3, each R is independently C6to C12straight-chain alkyl; C6to C12straight-chain alkenyl; C6to C12branched alkyl; C6to C12branched alkenyl; C9to C12cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C8to C14aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each R is independently C6to C10straight-chain alkyl; C6to C10straight-chain alkenyl; C6to C10branched alkyl; C6to C10branched alkenyl; C9to C10cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl; or Cs to C12 aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0262] Some embodiments include one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of R, X, Y, m, n, 0, p, and / or carbon chain length, structure, or saturation. In some embodiments, each R from a nearest common branch point is the same. In some embodiments, each R is the same.

[0263] In some embodiments, the ionizable cationic lipid has a structure of Formula Vawherein R is C6to C16straight-chain alkyl; C6to C16straight-chain alkenyl; C6to C16branched alkyl; branched C6to C16alkenyl; C9to C16cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C8to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain,Y is O, NH, N-CH3, or CH2, n is an integer from 0 to 4,m is an integer from 1 to 3, and o is an integer from 1 to 4.57 ; 9b In some embodiments, the ionizable cationic lipid has a structure of FormulaVI: %wherein W is C=0 or CH2, n is an integer from 0 to 4,m is an integer from 1 to 3, o is an integer from 1 to 4, p is an integer from 1 to 4, wherein when p = 1 , each Rcis independently C8to C18straight-chain alkyl; C8to C18straight-chain alkenyl; C8to C18branched alkyl; C8to C18branched alkenyl; C11to C18cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C10to C20 aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 2, each Rcis independently C8to C16straight-chain alkyl; C8to C16straight-chain alkenyl; C8to C16branched alkyl; C8to C16branched alkenyl; C11to C16cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at the either end or within the alkyl chain; or C10to C18aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein when p = 3, each Rcis independently C8to C14straight-chain alkyl; C8to C14straight-chain alkenyl; C8to C14branched alkyl; C8to C14branched alkenyl; C11to C14cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end orwithin the alkyl chain; or C10to C16aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain, and wherein when p = 4, each Rcis independently C8to C12straight-chain alkyl; C8to C12straight-chain alkenyl; C8to C12branched alkyl; C8to C12branched alkenyl; C11to C12cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl; or C10to C14aryl-alky in which the aryl is phenyl or naphthalenyl and is positioned at the either end or within the alkyl chain.

[0265] Some embodiments include one or more species or subgenera based on specific choices of Rc, W, X, m, n, 0, p, and / or carbon chain length, structure, or saturation. Other embodiments specifically exclude one or more species or subgenera based on specific choices of Rc, W, X, m, n, 0, p, and / or carbon chain length, structure, or saturation. In some embodiments, each Rc from a nearest common branch point is the same. In some embodiments, each Rc is the same.

[0266] In some embodiments, the ionizable cationic lipid has a structure of Formula Via:wherein Rcis C8to C18straight-chain alkyl; C8to C18straight-chain alkenyl; C8to C18branched alkyl; C8to C18branched alkenyl; C11to C18cycloalkyl-alkyl in which the cycloalkyl is C3to C8cycloalkyl positioned at either end or within the alkyl chain; or C10to C20aryl-alkyl in which the aryl is phenyl or naphthalenyl and is positioned at either end or within the alkyl chain, wherein W is C=O or CH2,n is an integer from 0 to 4,m is an integer from 1 to 3, and o is an integer from 1 to 4.%

[0267] With respect to each of the forgoing aspects, in some embodiments, all four R groups are identical. In other embodiments, the two Rc groups stemming from a first branchpoint are identical to each other and the two Rc groups from a second branchpoint are identical to each other, but the Rc groups stemming from the first branchpoint are different than the R groups stemming from the second branchpoint.

[0268] With respect to each of the forgoing aspects, some embodiments are limited to one, or a subset, of the alternatives for Rc, W, X, Y, m, n, 0, and / or p, as applicable. Other embodiments specifically exclude one, or a subset, of the alternatives for Rc, W, X, Y, m, n, o, p, and / or carbon chain length, structure, or saturation, as applicable. Each range of carbon chain length is meant to convey embodiments of all individual lengths and subranges therein.

[0269] With respect to each of the foregoing aspects and embodiments, in some instances Rc is straight-chain alkyl and in further instances the chain is unsubstituted. In still further instances, Rc is C8or C9or C10to C12.

[0270] With respect to each of the foregoing aspects and embodiments, in some / N instances With respect to each of the foregoing aspects and embodiments, in someinstances Y is O and in other instances Y is NH or N-CH3.

[0271] Ionizable cationic lipids of this disclosure have a branched structure to give the lipid a conical rather than cylindrical shape and such structure helps promote endosomolytic activity. The greater the endosomolytic activity, the more efficient release of the nucleotide cargo.

[0272] To promote biodegradability and minimize the accumulation of ionizable cationic lipids of this disclosure, the fatty acid tails are designed to comprise esters in a position that minimizes steric hinderance of ester cleavage. For example, while a single fattyacid tail will tend to extend away from the ester carbonyl, the presence of two tails leads to the tails extending in opposite directions as this is an energetically favorable conformation. This means one of the tails may extend toward the carbonyl and sterically hinder cleavage of the ester. Accordingly, large branches immediately adjacent to the ester carbonyl were avoided. In positioning the ester(s) within the lipid, consideration was also given to potential degradation products to avoid the generation of toxic compounds, such as formaldehyde.

[0273] In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only cationic lipids.

[0274] In certain embodiments, the LNP comprises at least one additional lipid which stabilizes the formation of particles during their formation.

[0275] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0276] 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.

[0277] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanol amine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyLsn- glycero-3 -phosphocholine (DSPC).

[0278] In some embodiments, the LNP comprises 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 VI)) to the neutral lipid ranges from about 2: 1 to about 8: 1.

[0279] In various embodiments, the LNP further comprises a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[0280] 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 VI)) to cholesterol ranges from about 2:1 to 1 :1.

[0281] 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.

[0282] In certain embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GMi). In certain embodiments, the LNP comprises a sterol, such as cholesterol.

[0283] In some embodiments, the LNP comprises 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.

[0284] In certain embodiments, the LNP comprises a polyethylene glycol-lipid (pegylated lipid). The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. In some embodiments, a PEG is of 500-5000 or IODO- 5000 Da molecular weight (MW). In some embodiments, the PEG unit has a MW of 2000 Da. In some instances, the MW2000 PEG-lipid comprises DMG-PEG2000 (1 ,2- dimyristoyl-glycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1 ,2- dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1 ,2- di stearoyl -glycero-3- methoxypolyethylene glycol-2000), DOG-PEG2000 (1 ,2-dioleoyl- glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1 ,2-dimyristoyl- glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE- PEG2000 (1 ,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE- PEG2000 (1 ,2-distearoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol- 2000), DGPE-PEG2000 (1 ,2-dioleoyl-glycero-3- phosphoethanolamine-3 - methoxypolyethylene glycol-2000), or combinations thereof. In some embodiments, the PEG unit has a MW of 2000 Da. In some instances, the MW2000 PEG-lipid comprises DMrG- PEG2000 (1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DPrG- PEG2000 (1 ,2-dipalmitoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DSrG- PEG2000 (1 ,2-distearoyl-rac-glycero-3- methoxypolyethylene glycol-2000), DGrG-PEG2000 (1 ,2-dioleoyl-glycero-3- methoxypolyethylene-rac-glycol-2000), DMPEr-PEG200 (1 ,2- dimyristoyl-rac-glycero- 3 -phosphoethanolamine-3 -methoxypolyethylene glycol-2000), DPPEr-PEG2000 (1 ,2- dipalmitoyl-rac-glycero-3 -phosphoethanolamine-3 - methoxypolyethylene glycol-2000), DSPEr-PEG2000 (1 ,2-distearoyl-rac-glycero-3- phosphoethanolamine-3- methoxypolyethylene glycol-2000), DOPEr-PEG2000 (1 ,2-dioleoyl- rac-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), or combinations thereof. Suitable polyethylene glycol-lipids 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 one embodiment, the polyethylene glycol-lipid is N-[(m ethoxy poly(ethylene glycol)2ooo)carbamyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as1 -(m onom ethoxy -poly ethyleneglycol)-2, 3 -dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate di acylglycerol (PEG-S-DAG) such as 4-O-(2’,3 ’-di(tetradecanoyloxy)propyl- 1 -O-(« -methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as <B-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-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.

[0285] A PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity. Additionally, a PEG moiety may impede binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the reticuloendothelial system. The PEG-moiety can also be functionalized to serve as an attachment point for a targeting moiety. Conjugating a cell- or tissue-specific binding moiety to the PEG-moiety enables a tLNP to avoid the liver and bind to its target tissue or cell type, greatly increasing the proportion of LNP that reaches the targeted tissue or cell type. PEG-lipid can thus serve as means for inhibiting LNP binding, and PEG- lipid conjugated to a binding moiety can serve as means for LNP-targeting.

[0286] In some embodiments, the LNP comprises a pegylated lipid having the following structure (VII):or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: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.

[0287] In some of the foregoing embodiments of the pegylated lipid (VII), 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 orbranched, 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.

[0288] In various embodiments, z spans a range that is selected such that the PEG portion has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0289] 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.

[0290] In certain embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.

[0291] In some embodiments, the LNP comprises a lipid of Formula (I to VI), a nucleoside-modified RNA, a neutral lipid, a steroid and a pegylated lipid. 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.

[0292] 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, el39; 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.

[0293] The following Reaction Schemes illustrate methods to make lipids of Formula (I), (II) or (III).

[0294] 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-C24alkyl 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-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of A-1, 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.

[0295] 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-C24alkyl 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.

[0296] 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 carboncarbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds.

[0297] 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-C24alkyl 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.

[0298] 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, andR7represents R7or a C3-C19alkyl. 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.

[0299] 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-l 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-l (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.

[0300] 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 GT 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).

[0301] 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.

[0302] 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, Lbutyldimethylsilyl, / -butyldiphenylsilyl or trimethyl silyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t- butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), -m ethoxy benzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protectinggroups 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.

[0303] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., l-1000nm) which includes at least one lipid. In some embodiments, the LNP comprises at least one agent that is either organized within inverse lipid micelles and encased within a lipid monolayer envelope or intercalated between adjacent lipid bilayers (e.g. lipid bilayer-agent-lipid bilayer). In some embodiments, the morphology of the LNP is distinct from that of a traditional liposome, characterized by a lipid bilayer surrounding an aqueous core, as the LNP possesses an electron-dense core, where the cationic / ionizable lipids are organized into inverted micelles around the encapsulated agent (e.g. mRNA molecules)(Cullis and Hope, 2017; Guevara et al., 2019b). In various embodiments, the LNP includes a lipid of Formula (I to VI). In some embodiments, the LNP is included in a formulation comprising at least one agent as described herein. In some embodiments, the LNP comprises a cationic lipid (e.g., a lipid of Formula (I to VI)) and at least one excipient selected from a neutral lipid, charged lipid, steroid and lipid-anchored polyethylene glycol (e.g., a pegylated lipid such as a pegylated lipid of structure (VII). In some embodiments, the at least one agent is encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or all of the lipid portion of the at least one LNP, 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.

[0304] In various embodiments, the LNP has a mean diameter 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 one embodiment, the LNP has a mean diameter of about 83 nm. In oneembodiment, the LNP has a mean diameter of about 102 nm. In one embodiment, the LNP has a mean diameter of about 103 nm. In some embodiments, the LNP is substantially non-toxic.In certain embodiments, the at least one agent, when present in the at least one LNP, is resistant in aqueous solution to degradation by intra- or intercellular enzymes

[0305] 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 or complexed. 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. Exemplary lipids are shown elsewhere herein.

[0306] In one embodiment, the LNP comprises at least one cationic lipid, and at least one stabilizing lipid. Stabilizing lipids include neutral lipids, anionic lipids and pegylated lipids.

[0307] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term “cationic or ionizable lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pKa, 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.

[0308] In various embodiments, the LNP comprises a cationic or ionizable lipids, stabilizing lipids, sterol, and a lipid-anchored polyethylene glycol (i.e PEGylated lipids).

[0309] In some embodiments, the LNP comprises an ionic lipid of Formula (I), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments the lipid of Formula (I) is compound 1-5. In some embodiments the lipid of Formula (I) is compound 1-6.

[0310] In some embodiments, the LNP comprises an ionic lipid of Formula (II), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (II) is compound II-9. In some embodiments, the lipid of Formula (II) is compound 11-10. In some embodiments, the lipid of Formula (II) is compound 11-11. In some embodiments, the lipid of Formula (II) is compound 11-12. In some embodiments, the lipid of Formula (II) is compound 11-32.

[0311] In some embodiments, the LNP comprises an ionic lipid of Formula (III), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound III-7.

[0312] In some embodiments, the LNP comprises an ionic lipid of Formula (IV), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (IV) comprises a lipid of Formula IVa.

[0313] In some embodiments, the LNP comprises an ionic lipid of Formula (V), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (IV) comprises a lipid of Formula Va.

[0314] In some embodiments, the LNP comprises an ionic lipid of Formula (VI), at least one agent, and at least one excipient selected from a neutral lipid, steroid and pegylated lipid. In some embodiments, the lipid of Formula (IV) comprises a lipid of Formula Via.

[0315] In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only cationic lipids.

[0316] In certain embodiments, the LNP comprises at least one stabilizing lipid (e.g. a neutral or anionic lipid) which helps to encapsulate the agent and stabilize the formation of particles during their formation.

[0317] Any suitable format of the at least one delivery vehicle is contemplated. In some embodiments, the at least one 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, liposomes, and lipid nanoparticles. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo include liposomes (e.g., an artificial membrane vesicle) and lipid nanoparticles.

[0318] The use of lipid formulations, as described above, is contemplated for the introduction of the at least one agent into the host cell (in vitro, ex vivo, or in vivo). In another aspect, the at least one agent may be associated with a lipid. The at least one agent associatedwith 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, complexed with 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.

[0319] In one embodiment, delivery of the at least one agent comprises any suitable delivery method, including exemplary delivery methods described elsewhere herein. In certain embodiments, delivery of the at least one agent to a subject comprises mixing the at least one agent with a transfection reagent prior to the step of contacting. In another embodiment, a method of the present invention further comprises administering the at least one agent together with the transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent.

[0320] In another embodiment, the transfection reagent is a lipid-based transfection reagent. 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.

[0321] In some embodiments, delivery of the at least one agent comprises liposomes. “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 etal., 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.

[0322] In one embodiment, 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.

[0323] 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.Cell Targeting

[0324] In one embodiment, the invention relates to compositions having at least one delivery vehicle conjugated to at least one cell targeting molecule, or a variant or fragment thereof, wherein the at least one delivery vehicle comprises at least one RNA molecule encoding a site-specific DNA binding molecule operably linked to a transcriptional modulator, and wherein the at least one RNA molecule encoding a site-specific DNA binding molecule operably linked to a transcriptional modulator is delivered to the at least one target cell. In some embodiments, the at least one delivery vehicle comprises an LNP.

[0325] In certain embodiments, the targeting molecule is specific for at least one cell surface receptor expressed on the target cell, i.e., are expressed and / or are contained only onthe target cell, or at least, are minimally present in cells that are not the target cells. For example, in some embodiments, the receptor on the at least one target cell comprises a receptor expressed on the surface of a specific cell subset. Suitable targeting ligands can be selected so that the targeted LNPs are specifically targeted to specific subset(s) of cells (e.g., CD4+ immune cells or oncogene expressing cells). This aspect may be referred to as “selective delivery” of a delivery vehicle to a target cell of interest. The term “selective delivery” means that delivery vehicles are localized by binding covalently or non-covalently to a target cell (e g., a particular T-cell subpopulation) through the binding interaction between the at least one ligand of the at least one delivery vehicle and the at least one receptor on the surface of the at least one target cell, but wherein the at least one delivery vehicle does not bind, or binds minimally, to cells that do not express the at least one receptor (i.e., such cells may be referred to as “non-target cells”). By “bind minimally,” it is meant that binding of the at least one delivery vehicle to non-target cells ranges between undetected to less than 1%, or less than 2%, or less than 3%, or less than 4%, or less than 5%, or less than 6%, or less than 7%, or less than 8%, or less than 9%, or less than 10% increased binding relative to a negative control (which can be a cell type known not to bind to the delivery vehicle).

[0326] Thus, the at least one delivery vehicle of the present disclosure may be localized or targeted to a particular type of cell (e.g., a natural killer (NK) cell, a macrophage, a B cell, a dendritic cell (DC), a particular T-cell subpopulation, a particular type of immune cell, a specific tumor cell etc.) by utilizing at least one targeting molecule which is conjugated to the at least one delivery vehicle. In some embodiments, the at least one targeting molecule is conjugated such that the targeting molecule is presented or otherwise exposed on the outer surface of the at least one delivery vehicle such that the targeting molecule may bind to at least one receptor on the surface of at least one target cell, thereby promoting or facilitating the binding of the at least one delivery vehicle to the at least one target cell (such as, an immune cell, a CD3+ T cell, a CD4+ T cell, a CD5+ T cell, a CD8+ T cell, a CD16+ NK cell, a Treg cell, effector T cells, oncogene expressing cells etc.), where it would then become internalized (e.g., through active internalization, such as endocytosis, etc.) with the concomitant release of the at least one agent (e.g., mRNA, etc.) carried by the delivery vehicle once inside the target cell.Conjugation

[0327] In some embodiments, the delivery vehicle is conjugated to a targeting molecule. In one embodiment, the invention relates to compositions having at least one delivery vehicle conjugated to at least one targeting molecule comprising a domain that specifically binds a target molecule (e.g., an antibody or antibody fragment), or a variant or fragment thereof, wherein the at least one delivery comprises at least one RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional modulator, and wherein the at least one RNA molecule encoding a site-specific DNA binding molecule linked to a transcriptional modulator is delivered to the at least one target cell. In some embodiments, the at least one delivery vehicle comprises an LNP.

[0328] Exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, hydrophobic interactions, and “van der Waals” interactions. In one embodiment, 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.

[0329] 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 one embodiment, the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer conjugated lipid is an activated, pegylated lipid. In one embodiment, 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 one embodiment, the second functional group is covalently attached to the targeting domain.

[0330] 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. In some embodiments, the first coupling group or second coupling group are selected from the group consisting of 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. In some embodiments, the first coupling group or second coupling group is selected from the group consisting of 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 one embodiment, the first coupling group is a maleimide.

[0331] In one embodiment, 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 one embodiment, the sulfhydryl group is present on a free cysteine residue. In one embodiment, the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2-mercaptoethylamine. In one embodiment, 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).

[0332] 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 cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone.

[0333] In some embodiments, the at least one delivery vehicle or compositions comprising the at least one delivery vehicle may further include at least one additional targeting molecule that enhances the localization of the delivery vehicles to a target cell. Such additional targeting molecule may include other peptides, aptamers, oligonucleotides, vitamins or other molecules that facilitate the localization of a delivery vehicle to a target cell, but which are not necessarily directly coupled to the delivery vehicle.Target Cells

[0334] In some embodiments, the targeted delivery vehicles of the invention are specifically targeted for binding to a surface receptor expressed on at least one target cell. In some embodiments, the at least one target cell comprises a lymphocyte. In some embodiments, the at least one target cell comprises at least one natural killer (NK) cell, at least one T cell, at least one B cell, at least one monocyte, at least one macrophage, at least one dendritic cell, or at least one neutrophil, or a combination thereof. In some embodiments, the at least one target cell comprises a tumor cell.

[0335] In one embodiment, the T cells that can be targeted using the compositions of the invention comprise T helper cells (CD4+), cytotoxic T cells (also referred to as cytotoxic T lymphocytes, CTL; CD8+ T cells), and memory T cells, including effector memory T cells, for example, TEM cells and TEMRA (CD45RA+) cells, Thl7 cells, Th2 cells, Tfh (follicular helper) cells, regulatory T cells (Tregs), and early differentiated T cells.

[0336] In one embodiment, the tumor cells that can be targeted using the compositions of the invention comprise oncogene expressing tumor cells. In some embodiments, the oncogene comprises FOXR2, LM01 or LYL1.

[0337] In some embodiments, the delivery vehicle is operably linked to a targeting molecule that specifically binds to a protein or receptor on the surface of a target cell. In some embodiments, the target cell is an immune cell or a cancer cell. Exemplary targeting molecules for targeting a B cell include, but are not limited to, antibodies or antigen binding molecules that specifically bind to at least one of CD40, BAFFR, TACI, BCMA, CD 19 or CD79a.

[0338] One of ordinary skill in the art will be able to identify at least one appropriate cell surface receptor on any cell type of interest such that the at least one delivery vehicle conjugated to at least one targeting molecule becomes localized to any cell type of interest due to specific and selective interaction between the targeting molecule and the at least one appropriate cell surface receptor on any cell type of interest that will allow the targeting of the at least one delivery vehicle of the present invention to any cell of interest.Delivery vehicle agents

[0339] In some embodiments, the delivery vehicle comprises or encapsulates at least one agent for delivery to a target cell subpopulation. In some embodiments, the agent comprises at least one nucleoside-modified mRNA molecule. In some embodiments, the agentcomprises at least one in vitro transcribed (IVT) mRNA molecule. In some embodiments, the mRNA molecule encodes a DNA targeting module and a gene modulatory agent.Nucleoside-modified RNA

[0340] In one aspect, the at least one agent comprises at least one nucleoside-modified nucleic acid, wherein the at least one nucleoside-modified nucleic acid comprises at least one nucleoside-modified RNA, wherein the at least one nucleoside-modified RNA comprises as least one nucleoside-modified mRNA molecule. In one embodiment, the nucleoside-modified mRNA encodes a DNA targeting module and a gene modulatory agent.

[0341] For example, in one embodiment, the composition comprises at least one nucleoside-modified RNA. Nucleoside-modified mRNA have particular advantages over nonmodified mRNA, 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 Nos. 8,278,036, 8,691,966, and 8,835,108, each of which is incorporated by reference herein in its entirety.

[0342] 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.

[0343] In certain instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. For example, when expressing a protein by delivering the encoding mRNA, 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 viral -based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery.

[0344] In certain embodiments, the at least one nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In certain embodiments, inclusion of pseudouridine makes the at least one mRNA more stable, non-immunogenic, and highlytranslatable (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).

[0345] 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). Similar effects as described for pseudouridine have also been observed for RNA containing 1-methyl-pseudouridine.

[0346] In some embodiments, the at least one nucleoside-modified nucleic acid molecule is a purified nucleoside-modified nucleic acid molecule. For example, in some embodiments, the composition is purified to remove double-stranded contaminants. In some instances, a preparative HPLC purification procedure is used 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.

[0347] In some embodiments, the at least one nucleoside-modified nucleic acid molecule is purified using non-HPLC methods. In some instances, the nucleoside-modified nucleic acid molecule is purified using chromatography methods, including but not limited to HPLC and fast protein liquid chromatography (FPLC). An exemplary FPLC -based purification procedure is described in Weissman et al., 2013, Methods Mol Biol, 969: 43-54. Exemplary purification procedures are also described in U.S. Patent Application Publication No. US2016 / 0032316, which is hereby incorporated by reference in its entirety.

[0348] In one embodiment, the at least one nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein. For example, in certain embodiments, the at leastone nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. Tn another embodiment, the at least one nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the at least one nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0349] In one embodiment, the at least one nucleoside-modified RNA of the invention comprises at least one modified nucleoside. In one embodiment, the at least one modified nucleoside is m1acp3'P (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the at least one modified nucleoside is m1'P (1-methylpseudouridine). In another embodiment, the at least one modified nucleoside is m (2'-O-methylpseudouridine. In another embodiment, the at least one modified nucleoside is m5D (5-methyldihydrouridine). In another embodiment, the at least one modified nucleoside is m3T (3-methylpseudouridine). In another embodiment, the at least one modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the at least one modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the at least one modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0350] In another embodiment, the at least one nucleoside that is modified in the at least one nucleoside-modified RNA the present invention is uridine (U). In another embodiment, the at least one modified nucleoside is cytidine (C). In another embodiment, the at least one modified nucleoside is adenosine (A). In another embodiment, the at least one modified nucleoside is guanosine (G).

[0351] In another embodiment, the at least one modified nucleoside of the present invention is m5C (5-methylcytidine). In another embodiment, the at least one modified nucleoside is m5U (5-methyluridine). In another embodiment, the at least one modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the at least one modified nucleoside is s2U (2-thiouridine). In another embodiment, the at least one modified nucleoside is ψ (pseudouridine). In another embodiment, the at least one modified nucleoside is Um (2'-O- methyluridine).

[0352] In other embodiments, the at least one modified nucleoside is m1A (1- methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2- methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); 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- hydroxynorvalyl carbamoyladenosine); 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); PC (5-formylcytidine); m5Cm (5,2'-O-dimethylcytidine); ac4Cm (N4-acetyl-2'-O-methylcytidine); k2C (lysidine); m1G (l- 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); 02yW (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); mo’U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5- methoxycarbonylmethyluridine); mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (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); cmnrn5U (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); hm5C (5-hydroxymethylcytidine); m3U (3 -methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,2'-O-dimethyladenosine); m62Am (N6,N6,O-2'- trimethyladenosine); m2,7G (N2,7-dimethylguanosine); m2,2’7G (N2,N2,7-trimethylguanosine);m3Um (3,2'-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O- methylcytidine); m1Gm (l,2'-O-dimethylguanosine); irdAm (l,2'-O-dimethyladenosine); τm5U (5-taurinomethyluridine); τmVU (5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2 (isowyosine); or ac6A (N6-acetyladenosine).

[0353] In another embodiment, the at least one nucleoside-modified RNA of the present invention comprises a combination of 2 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

[0354] In another embodiment, between 0.1% and 100% of the residues in the at least one nucleoside-modified mRNA of the present invention are modified (e.g. either by the presence of pseudouridine or a 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%.

[0355] 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 anotherembodiment, 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%.

[0356] In some embodiments, the at least one agent comprises a purified preparation of at least one single-stranded nucleoside modified RNA. For example, in some embodiments, the purified preparation of at least one single-stranded nucleoside modified RNA is substantially free of double stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, or at least 91%, or at least 92%, or at least 93 % or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0357] 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 anotherembodiment, the fraction is 50%. Tn 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%.

[0358] 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%.

[0359] In another embodiment, the at least one nucleoside-modified RNA of the present invention is translated in the at least one target cell more efficiently than an unmodified RNA molecule with the same sequence. In another 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.

[0360] In another embodiment, the at least one nucleoside-modified RNA of the present invention exhibits significantly less innate immunogenicity than an unmodified in vitro-synthesized RNA molecule of the same sequence. In another embodiment, the at least one modified RNA molecule exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In another embodiment, innate immunogenicity is reduced by a 3-fold factor. In another embodiment, innate immunogenicity is reduced by a 4-fold factor. In another embodiment, innate immunogenicity is reduced by a 5-fold factor. In another embodiment, innate immunogenicity is reduced by a 6-fold factor. In another embodiment, innate immunogenicity is reduced by a 7-fold factor. In another embodiment, innate immunogenicity is reduced by a 8-fold factor. In another embodiment, innate immunogenicity is reduced by a 9- fold factor. In another embodiment, innate immunogenicity is reduced by a 10-fold factor. In another embodiment, innate immunogenicity is reduced by a 15-fold factor. In another embodiment, innate immunogenicity is reduced by a 20-fold factor. In another embodiment, innate immunogenicity is reduced by a 50-fold factor. In another embodiment, innate immunogenicity is reduced by a 100-fold factor. In another embodiment, innate immunogenicity is reduced by a 200-fold factor. In another embodiment, innate immunogenicity is reduced by a 500-fold factor. In another embodiment, innate immunogenicity is reduced by a 1000-fold factor. In another embodiment, innate immunogenicity is reduced by a 2000-fold factor. In another embodiment, innate immunogenicity is reduced by another fold difference.

[0361] In another embodiment, “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity. In another embodiment, the term refers to a fold decrease in innate immunogenicity (e.g., 1 of the fold decreases enumerated above). In another embodiment, the term refers to a decrease such that an effective amount of the nucleoside-modified RNA can be administered without triggering a detectable innate immune response. In another embodiment, the term refers to a decrease such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the modified RNA. In another embodiment, the decrease is such that the nucleoside-modified RNA can berepeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the modified RNA.In vitro transcribed mRNA

[0362] In one embodiment, the nucleic acid molecule encoding the DNA binding domain linked to the transcriptional modulator comprises an in vitro transcribed (IVT) RNA molecule, wherein the IVT RNA is messenger RNA (mRNA). In one embodiment, the at least one in vitro transcribed (IVT) RNA encodes a fusion protein comprising a DNA binding domain linked to a transcriptional modulator.

[0363] In one embodiment, at least one mRNA is produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA can be from any source, and 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.

[0364] In one embodiment, 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 one embodiment, 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 one embodiment, 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.

[0365] 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. Exemplarygenes are genes which are useful for a short-term treatment, or where there are safety concerns regarding dosage or the expressed gene.

[0366] In various embodiments, a plasmid is used to generate a template for in vitro transcription of mRNA.

[0367] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. In some embodiments, the mRNA has 5' and 3' UTRs. In one embodiment, 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 of the transcribed mRNA.

[0368] 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 mRNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed mRNA based on properties of UTRs that are well known in the art.

[0369] In one embodiment, 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 mRNA transcripts, but does not appear to be required for all mRNAs 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 mRNA virus whose mRNA 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 mRNA.

[0370] To enable synthesis of mRNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of thesequence to be transcribed. When a sequence that functions as a promoter for an mRNA 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 exemplary 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.

[0371] In an exemplary embodiment, the mRNA 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.

[0372] 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).

[0373] 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.

[0374] Poly(A) tails of mRNAs 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 one embodiment, 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 mRNA. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA 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 mRNA.

[0375] 5' caps also provide stability to mRNA molecules. In an exemplary embodiment, mRNAs produced by the methods include a 5' cap-1 structure. Such cap-1structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase 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)).Methods

[0376] The invention relates, in part, to compositions of the invention are used to transiently modulating transcription. Therefore in one embodiment, the present disclosure provides methods for transiently modulating transcription of at least one gene in vivo. In some embodiments, the method comprises administering a composition comprising an RNA molecule encoding a site-specific DNA binding molecule operably linked to a transcriptional modulator. In some embodiments, the method comprises transiently activating transcription of at least one gene in vivo. In some embodiments, the method comprises transiently repressing transcription of at least one gene in vivo. In some embodiments, the gene is a disease- associated gene. Therefore, in some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder (e.g., cancer, infection, immune disease etc.) in a subject by administering a composition that transiently modulates transcription of at least one disease-associated gene in vivo. In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder comprising administering composition comprising a site-specific DNA binding molecule that specifically binds to a gene, or a regulatory region thereof (e.g., a region upstream of the promoter), operably linked to a transcriptional modulator to transiently modulate expression of the gene. In some embodiments, the composition comprises an LNP encapsulating an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to a gene, or a regulatory region thereof (e.g., a region upstream of the promoter), operably linked to a transcriptional modulator to transiently modulate expression of the gene. In some embodiments, the disease or disorder is cancer, an autoimmune disease or a viral disease.

[0377] Exemplary disease-associated genes that can be modulated using the methods of the invention include, but are not limited to, viral genes, human genes required for viral entry into cells, human genes involved in the immune response and oncogenes. Exemplary geneswhose expression can be modulated include, but are not limited to, CCR5, FOXP3, RORC, IL4, TOX, TOX2, MS4A1, FOXR2, LM01 and LYL1. In some embodiments, the invention provides methods of transiently increasing expression of FOXP3 in a subject comprising administering a TALE specific for binding to FOXP3, or a region upstream of the FOXP3 promoter, linked to a transcriptional activator. In some embodiments, the invention provides methods of transiently increasing expression of IL4 in a subject comprising administering a TALE specific for binding to IL4, or a region upstream of the IL4 promoter, linked to a transcriptional activator. In some embodiments, the invention provides methods of transiently increasing expression of RORC in a subject comprising administering a TALE specific for binding to RORC, or a region upstream of the RORC promoter, linked to a transcriptional activator. In some embodiments, the invention provides methods of transiently increasing expression of TOX in a subject comprising administering a TALE specific for binding to TOX, or a region upstream of the TOX promoter, linked to a transcriptional activator. In some embodiments, the invention provides methods of transiently increasing expression of TOX2 in a subject comprising administering a TALE specific for binding to TOX2, or a region upstream of the TOX2 promoter, linked to a transcriptional activator. In some embodiments, the invention provides methods of transiently increasing expression of CD20 in a subject comprising administering a TALE specific for binding to MS4A1, or a region upstream of the MS4A1 promoter, linked to a transcriptional activator. In some embodiments, the invention provides methods of transiently decreasing expression of CCR5 in a subject comprising administering a TALE specific for binding to CCR5, or a region upstream of the CCR5 promoter, linked to a transcriptional repressor. In some embodiments, the invention provides methods of transiently decreasing expression of FOXR2 in a subject comprising administering a TALE specific for binding to FOXR2, or a region upstream of the FOXR2 promoter, linked to a transcriptional repressor. In some embodiments, the invention provides methods of transiently decreasing expression of LM01 in a subject comprising administering a TALE specific for binding to LM01, or a region upstream of the LM01 promoter, linked to a transcriptional repressor. In some embodiments, the invention provides methods of transiently decreasing expression of LYL1 in a subject comprising administering a TALE specific for binding to LYL1, or a region upstream of the LYL1 promoter, linked to a transcriptional repressor.

[0378] In some embodiments, the disclosure provides a method for inducing expression of latent viral genes to promote viral clearance. In some embodiments, the method includes inducing viral activation in a subject by administering a site-specific DNA binding molecule that specifically binds to a viral latency gene operably linked to a transcriptional activation domain. For example, in one embodiment, the method comprises administration of a composition comprising an RNA molecule encoding a latent viral gene-specific TALE linked to a transcriptional activator to induce expression of the latent virus. In some embodiments, induction of the latent virus induces viral activation in the subject and promotes clearance of the latent virus. In some embodiments, the latent virus is HIV, EBV, human CMV, or Kaposi sarcoma-associated herpesvirus (KSHV). In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to an integrated HIV gene, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is linked to a transcriptional activator for activation of the lytic phase and reduction of the latent viral reservoir. In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to an integrated EBV lytic gene (e.g., BZLF1 and BRLF1), or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is linked to a transcriptional activator for activation of the lytic phase and reduction of the latent viral reservoir. In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to an integrated human CMV gene (e.g., major IE promoter (MIEP) and / or MIE enhancer), or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is linked to a transcriptional activator for activation of the lytic phase and reduction of the latent viral reservoir. In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to an integrated KSHV RTA gene, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is linked to a transcriptional activator for activation of the lytic phase and reduction of the latent viral reservoir.

[0379] In some embodiments, the disclosure provides a method for reducing expression of viral genes required for viral latency, thereby promoting viral clearance. Forexample, in one embodiment, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to an integrated KSHV LANA latency gene, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is linked to a transcriptional repressor. In some embodiments, repression of a gene required for viral latency promotes activation of the lytic phase and reduction of the latent viral reservoir.

[0380] In some embodiments, the disclosure provides a method for reducing or preventing viral entry into cells. For example, in one embodiment, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to a gene, or regulatory region, encoding a receptor or protein expressed on human cells that is used for viral entry, wherein the site-specific DNA binding molecule is operably linked to a transcriptional repressor to transiently reduce expression of the receptor or protein. In one embodiment, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to CCR5, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is linked to a transcriptional repressor. In some embodiments, repression of CCR5 prevents HIV entry into immune cells. In one embodiment, the composition further comprises a targeting molecule to target the composition to CD4+ immune cells. In some embodiments, targeted delivery of the CCR5-TALE-repressor fusion allows for transient repression of CCR5 in CD4+ immune cells to prevent HIV entry, thus reducing or preventing viral infection. In some embodiments, the composition is administered in combination with at least one additional HIV treatment. For example, in some embodiments, the composition is administered in combination with at least one RNA molecule encoding a site-specific DNA binding molecule that specifically binds to a latent HIV gene, or regulatory region thereof, wherein the site-specific DNA binding molecule is linked to a transcriptional activator. In some embodiments, the combination activates the latent viral reservoir while at the same time preventing viral entry into CD4+ immune cells.

[0381] In some embodiments, the disclosure provides a method for transiently modulating expression of genes that mediate the immune response. In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to at least one gene, or regulatory region thereof, that promotesformation or induction of a specific immune cell subset (e.g., regulatory T cells, T helper 17 cells (Thl7), or T helper 2 cells (Th2)).

[0382] In some embodiments, the disclosure provides a method for inducing formation of regulatory T cells. In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to FOXP3, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is operably linked to a transcriptional activator to transiently activate expression of FOXP3 therefore inducing formation of regulatory T cells. Induction of regulatory T cells is beneficial for the treatment of autoimmune disease, therefore, in some embodiments, provided is a method of treating or preventing an autoimmune disease or disorder by administering a composition comprising an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to FOXP3, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is operably linked to a transcriptional activator to transiently activate expression of FOXP3 thereby inducing formation of regulatory T cells.

[0383] In some embodiments, the disclosure provides a method of improving T cell or NK cell functionality. In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to TOX or TOX2, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is operably linked to a transcriptional repressor to transiently repress expression of TOX or TOX2 therefore reducing expression of factors for T cell exhaustion. Reduction of TOX or TOX2 increases T cell or NK cell longevity and therefore increases T cell or NK cell functionality. Increased T cell or NK cell functionality is beneficial for the treatment of infectious disease, therefore, in some embodiments, provided is a method of treating an infectious disease or disorder by administering a composition comprising an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to TOX or TOX2, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is operably linked to a transcriptional repressor to transiently repress expression of TOX or TOX2 thereby delaying or reducing T cell or NK cell exhaustion. In some embodiments, the composition is administered in combination with at least one additional treatment. For example, in some embodiments, thecomposition is administered in combination with at least one vaccine. In some embodiments, the combination increases the vaccine efficacy by reducing T cell or NK cell exhaustion.

[0384] In some embodiments, the disclosure provides a method for increasing the efficacy of a vaccine. In some embodiments, the method comprises administering an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to RORC or IL4, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the sitespecific DNA binding molecule is operably linked to a transcriptional activator to transiently activate induction of helper T cells (e.g., Thl7 or Th2). Induction of helper T cells may boost vaccine efficacy, therefore, in some embodiments, provided is a method of increasing vaccine efficacy by administering a composition comprising an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to RORC or IL4, or a regulatory region thereof (e.g., a region upstream of the promoter), wherein the site-specific DNA binding molecule is operably linked to a transcriptional activator to transiently activate expression of RORC or IL4 thereby inducing helper T cells. In some embodiments, the composition is administered in combination with at least one additional vaccine treatment. In some embodiments, the method comprises administering the composition as a vaccine priming agent. In some embodiments, the method comprises administering the composition as a vaccine boosting agent. In some embodiments, the method comprises administering the composition as an additional agent in a vaccine prime-boost regimen.

[0385] In some embodiments, the method comprises modulating expression of a gene for the treatment of cancer comprising administering a composition comprising an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to an oncogene gene, or a regulatory region thereof (e.g., a region upstream of the promoter), operably linked to a transcriptional activator to transiently modulate expression of the gene. In some embodiments, the composition comprises a targeting domain to target a specific cell for delivery of the site-specific transcriptional modulator. In some embodiments, delivery of a sitespecific transcriptional activator can transiently increase expression of a gene in cells allowing targeting of the cells by additional therapeutic methods. In some embodiments, delivery of a site-specific transcriptional repressor can transiently decrease expression of an oncogene in cells preventing tumor growth.

[0386] In some embodiments, the disclosure provides a method for increasing the efficacy of a cancer therapy. In some embodiments, the method comprises administering a composition comprising an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to a gene, or a regulatory region thereof (e.g., a region upstream of the promoter), operably linked to a transcriptional modulator to transiently increase or decrease expression of the gene. In some embodiments, the method includes administration of the composition prior to or in combination with an additional cancer therapy, wherein the cancer therapy comprises a binding domain that specifically binds the gene. In some embodiments, the composition comprises a targeting domain for delivery of the site-specific transcriptional modulator to a specific cell. In some embodiments the cell is a tumor cell.

[0387] For example, delivery of a composition comprising an RNA molecule encoding a site-specific DNA binding molecule that specifically binds to MS4A1, or a regulatory region thereof (e.g., a region upstream of the promoter), operably linked to a transcriptional activator transiently increases expression of CD20 in cells that do not normally express CD20. In some embodiments, increasing expression of CD20 in cells that do not normally express CD20 increases the efficacy of CD20 based therapies (e g., CAR T therapy or anti-CD20 therapy).

[0388] Delivery of a composition comprising an RNA molecule encoding a sitespecific DNA binding molecule that specifically binds to an oncogene or a regulatory region thereof (e.g., a region upstream of the promoter), operably linked to a transcriptional repressor transiently decreases expression of the oncogene in cells. In some embodiments, decreasing expression of an oncogene in tumor cells decreases tumor growth. Exemplary oncogenes that can be targeted include, but are not limited to FOXR2, LM01 and LYLl. In some embodiments, the composition comprises a targeting domain for delivery of the site-specific transcriptional modulator to a specific cell. In some embodiments the cell is a tumor cell. In some embodiments, the targeting domain comprises binding domain that specifically binds to the oncogene. In some embodiments, the composition is administered alone. In some embodiments, the composition is administered in combination with at least one additional cancer therapy.

[0389] Exemplary diseases and disorders that can be treated using the methods and compositions of the invention include, but are not limited to, cancers, infectious diseases, and immunological diseases or disorders.

[0390] The following are non-limiting examples of cancers that can be treated or prevented by the disclosed methods: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytotna / malignant glioma, cervical cancer, childhood visual pathway tumor, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous cancer, cutaneous T- cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family of tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney (renal cell) cancer, Langerhans cell cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocvtoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (nonmelanoma), skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, supratentorial primitive neuroectodermal tumors and pineoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0391] In one embodiment, the present invention features methods for treating or preventing a viral infection or a disease or disorder associated therewith. In some embodiments, the virus is from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae (including SARS and SARS-CoV-2), Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. The virus can be human immunodeficiency virus (HIV), Chikungunya virus (CHIKV), dengue fever virus, papilloma viruses, for example, human papillomoa virus (HPV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, equine encephalitis viruses, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, Hanta virus (hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella-zoster, a.k.a., chickenpox), cytomegalovirus (CMV),for example human CMV, Epstein-Barr virus (EBV), flavivirus, foot and mouth disease virus, Lassa virus, arenavirus, or a cancer-causing virus. In some embodiments, the virus is a DNA virus or a retrovirus. In some embodiments, the virus is human immunodeficiency virus (HIV), human CMV, or Epstein-Barr virus (EBV).

[0392] 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 the disease or disorder, thereby preventing the disease or disorder.

[0393] 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.

[0394] 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.

[0395] 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 usedalone 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.

[0396] Administration of the compositions of the invention to a patient can be by any route, including but not limited to intravenous, intranodal, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via an aerosol, etc.), buccal (e.g., sub-lingual, etc.), topical (i.e., both skin and mucosal surfaces, including airway surfaces, etc.), intrathecal, intraarticular, intraplural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginal administration, by perfusion through a regional catheter, or by direct intralesional injection. In one embodiment, the compositions of the invention are administered by intravenous push or intravenous infusion given over a defined period (e.g., 0.5 to 2 hours). The compositions of the invention can be delivered by peristaltic means or in the form of a depot, although the most suitable route in any given case will depend, as is well known in the art, on such factors as the species, age, gender and overall condition of the subject, the nature and severity of the condition being treated and / or on the nature of the particular composition (i.e., dosage, formulation) that is being administered. In particular embodiments, the route of administration is via bolus or continuous infusion over a period of time, once or twice a week. In other particular embodiments, the route of administration is by subcutaneous injection given in at least one site (e.g., thigh, waist, buttocks, arm, etc.), optionally once or twice weekly. In one embodiment, the compositions, and / or methods of the invention are administered on an outpatient basis.

[0397] In one embodiment, 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.

[0398] The method comprises administering a combination of composition in any suitable ratio. For example, in one embodiment, the method comprises administering twoindividual 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.Pharmaceutical Compositions

[0399] The formulations of the pharmaceutical compositions (e.g., comprising at least one delivery vehicle) 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 (e.g., at least one delivery vehicle) 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.

[0400] 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.

[0401] Pharmaceutical compositions (e.g., comprising at least one delivery vehicle) 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.

[0402] 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 predeterminedamount 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.

[0403] 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.

[0404] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise at least one additional pharmaceutically active agent.

[0405] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0406] 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.

[0407] 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 notlimited to, a suspending, stabilizing, or dispersing agent. In one embodiment, 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.

[0408] 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.

[0409] In various embodiments, the targeted delivery vehicles may be administered to a subject such that the delivery vehicle contacts the targeted cell in vivo. In other embodiments, the cell may be contacted with the delivery vehicles ex vivo and then transferred back to a subject in need with adoptive cell transfer. In this embodiment, cells are removed from a patient and modified ex vivo by contacting them with the herein disclosed delivery vehicles.

[0410] 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. Such powders comprise particles wherein at least 98% of theparticles 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. 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. Dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0411] 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).

[0412] 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, a suspending, stabilizing, or dispersing agent. In one embodiment, 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.

[0413] 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, isotonicsodium 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.

[0414] 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.EXPERIMENTAL EXAMPLES

[0415] 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.

[0416] 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.Example 1 : mRNAs Encoding TALE Fusion Molecules

[0417] Described herein is a method using mRNAs encoding transcription activatorlike effectors (TALE) that are fused with activation or repressive domains for locus-specific gene transcription modulation. Delivery of mRNA via targeted lipid nanoparticles (LNP) will allow for temporary gene transcription modulation in specific cell subsets. TALEs use repetitive amino acid motifs that are tailored to recognize unique 20-mer DNA sequences throughout the genome. Since the DNA base pair recognition is changed by swapping out the amino acid motifs, any gene can be targeted without changing the size of the mRNA that would be loaded into the LNP for delivery. This allows for larger genes to be induced / restricted that would otherwise be unwieldy to use in direct mRNA form. Furthermore, TALEs have been shown to be better than CRISPR-Cas9 systems at targeting genomic regions that may be more condensed / inconducive to RNA transcription (Jaini et al., 2021, Nature Comm 12:606). This may be important in the context of latent HIV infection given the propensity for dormant and transcriptionally inactive viral DNA or cancers with repressed regulatory genes. Examples of two mRNA-delivered TALE that can specifically induce protein production at the HIV locus and the MS4A1 (CD20) locus are presented.

[0418] Two separate TALE recognition sequences in the conserved 5’ LTR of HIV genomic DNA were used to design the repeat motifs for HIV-specific TALEs. When fused with either VP64 (4 tandem repeats of the trans activator VP 16 protein from herpes simplex virus) or VPR (VP64, p65 from human NFKB, and Rta from Epstein Barr Virus), both of these HIV-specific TALEs turned on high levels of gene transcription and protein production at the HIV locus in a latent HIV-infected cell line at levels similar to treatment with the mitogenic phorbol 12-myristate 13-acetate (PMA) and ionomycin. The TALEs did not induce cell death whereas PMA / ionomycin is toxic to cells. Specific DNA recognition and targeted mRNA-LNP delivery reduces the risks of side effects while allowing for repeated modulation as needed with subsequent administration.

[0419] To extend these findings beyond the HIV context, a TALE recognizing upstream of the MS4A1 (also known as CD20; expressed by most B cells) promoter was designed and its fusion with the VPR activation domain induced surface expression of the CD20 protein in T cell lines that do not normally express CD20. Similar to the HIV-specific TALE, the CD20 TALE did not induce cell death. The clinical relevance of CD20 is evident inthe CD20 negative B cell non-Hodgkin lymphoma context which is not treatable using CD20- specific CAR-T cells or anti-CD20 monoclonal antibody therapy. Expression of CD20 could render these cancerous cells permissive to CAR-T or rituximab therapy. The present CD20 TALE data represents the first selective expression of CD20 without relying on the side effects of HD AC inhibitors, BCR inhibitors, or other inhibitors. As such, this method of transitory gene transcription modulation with TALEs has significant impact in quickly adapting and designing new treatments for various viral and oncogenic diseases. In the HIV context, this could be used as the “shock” method with less off-target effects than existing methods as the first step in eliminating the latent reservoir. In oncogenic settings, this invention could be used to render cancers permissive to existing treatments or systematically target cell regulation pathways.

[0420] Additional fusion domain optimizations and gene targets are contemplated. LNP delivery in in vitro cell lines and from ex vivo samples are contemplated.

[0421] A general schematic of TALE constructs is shown in Figure 1. Gene specific TALEs contain 18.5 repeat modules that each confer specificity towards a DNA nucleotide. Combined, a TALE with these repeat modules will confer specificity to a 20bp DNA sequence, thus enabling targeting towards a particular locus. The effector domain can be any domain used to modulate RNA transcription - such as activation (VP64, VPR, p300, p65-HSFl), repressive (KRAB), or knockout (TevI, FokI) used to modulate or eliminate RNA transcription. (Figure 1A and Figure IB) Testing versions of TALEs will contain fluorescent protein reporters in either N-terminal end or the C-terminal end. These fluorescent proteins will be separated by a T2A or P2A peptide sequence. (Figure 1C) General schematic of pre-clinical version of TALE without T2A / P2A and reporter domains. (Figure ID) General schematic of next-generation TALE with modified N / C terminal regions. The modifications only occur in the constant regions while the 18.5 repeat modules are unchanged. (Figure IE) General schematic of nextgeneration TALEs with modified N / C terminal regions and effector domain that is connected to Tat via a linker (including GGGGGS6). For sub-figures (Figure 1 A - Figure IE), the domain and construct widths are not to scale. (Figure IF) Example mRNA sequence following the construct shown in (Figure ID) to be used in mRNA LNPs. Axis scale and legend are specific to Figure IF only.

[0422] Schematic diagrams of specific targeting of TALES are provided in Figure 2.Figure 2A depicts specific targeting of various TALEs in the 5’ LTR region of integrated HIV- 1 DNA in JLat 10.6 cells. Figure 2B depicts specific targeting of various TALEs in the intergenic region of integrated HIV-1 DNA in JLat 10.6 cells. All basepair (bp) coordinates are relative to the proviral JLat 10.6 sequence.

[0423] Representative flow plots demonstrating HIV reactivation potential from different regions of Figure 2 are shown in Figure 3. Samples shown are JLat 10.6 cells which contain integrated HIV DNA with an GFP reporter. X-axis indicates GFP signal as a marker of HIV reactivation while Y-axis represents cell viability. TALE2 and TALE3 have the strongest reactivation potential.

[0424] As introduced in Figure 1, different generations of TALE mRNA constructs were electroporated into JLat 10.6 cells (Figure 4). The base TALE was the HIV-specific TALE2 and the effector domain was VP64. These data indicate that modifications / truncations to the N and C terminal constant regions do not impact the HIV reactivation potential as shown on the X-axis.

[0425] Various N / C terminal truncated TALE mRNAs were electroporated into different latent HIV-infected cell lines (Figure 5; each contains a GFP reporter as a measure of HIV reactivation). As positive controls, viral Tat mRNA and Tnfa (100 ng / ml) were used. Neg represents untreated cells. Figure 5 A depicts the efficiency of HIV reactivation as a percentage of GFP positive cells from gated live cells. Figure 5B depicts the strength of HIV reactivation, shown as the brightness of GFP signal. Error bars for Figure 5A and Figure 5B represent mean + / - standard error.

[0426] The next-generation TALE2 with p65-HSFl fused with Tat is able to reactivate HIV at higher levels than each individual component (TALE2-p65-HSFl and Tat) (Figure 6). mRNA encoding various components or fusion TALE-Tat were electroporated into JLat 6.3 and 8.4 cells. HIV reactivation was most potent in the fusion TALE-Tat condition.

[0427] TALEs can also target other loci, including in the human genome (Figure 7). Representative flow plots are shown for a mRNA encoding a CD20-specific TALE (N and C terminal truncated) fused with the p65-HSFl effector domain that was transfected in various HIV-infected cell lines. Strong CD20 signal is detected in all three cell lines.

[0428] JLat 10.6 cells were treated with 2 pg mRNA-LNP encoding either the HIV- specific TALE3 (unmodified) or viral protein Tat (Figure 8). TALE3 mRNA-LNP can potently reactivate proviral transcription above the baseline (untreated) control and at higher levels compared to viral Tat. A positive control with non-specific TNFa (100 ng / ml) treatment is included for comparison.

[0429] CD4-targeted mRNA LNP delivery of HIV-specific TALE reactivates provirus within 24 hours in ex vivo samples (Figure 9). Memory CD4+ T cells were isolated from three donors with HIV that were taking antiretroviral therapy. Cells were treated with 1 pg ibalizumab (anti-CD4) conjugated mRNA-LNP per million cells for 24 hours. mRNA-LNPs either encoded HIV specific TALE3 (unmodified) with VP64 activation domain or a negative control sequence (tdTomato). A positive control (16 pM PMA + 1 pM ionomycin) is included for comparison. In two of the donors, mRNA-LNPs encoding TALE3 increases HIV transcription, while mRNA-LNPs encoding tdTomato are not different from untreated conditions. The donor that did not have increased HIV transcription has a consensus sequence with mismatches to the TALE3 recognition site.Example 2: Targets for TALEExample 3 : Sequences

[0430] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0431] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

CLAIMSWhat is claimed is:

1. A composition for site-specific transient transcriptional modulation of a gene of interest, the composition comprising a delivery vehicle comprising at least one RNA molecule comprising or encoding a site-specific DNA binding molecule linked to a transcriptional modulator.

2. The composition of claim 1, wherein the site-specific DNA binding molecule comprises a transcription activator-like effector (TALE).

3. The composition of claim 1, wherein the transcriptional modulator comprises a transcriptional activation domain or a transcriptional repression domain.

4. The composition of claim 3, wherein the transcriptional activation domain is selected from the group consisting of: VP64, VPR, p300, and p65-HSFl.

5. The composition of any one of claims 1 to 4, wherein the nucleotide sequence comprises a sequence encoding SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47 retains the function of transcriptional activation.

6. The composition of any one of claims 1 to 5, wherein the nucleotide sequence comprises a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48 retains the function of transcriptional activation.

7. The composition of any one of claims 1 to 6, wherein composition comprises a fusion of the transcriptional modulator and Tat.

8. The composition of claim 7, wherein the Tat comprises an amino acid sequence of SEQ ID NO: 51 or a fragment or variant thereof.

9. The composition of claim 8, wherein the Tat comprises a nucleotide sequence of SEQ ID NO: 52 or a fragment or variant thereof.

10. The composition of claim 3, wherein the transcriptional repression domain comprises a Kriippel-associated box (KRAB) domain comprising a sequence as set forth in SEQ ID NO:49, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:49 retains the function of transcriptional repression.

11. The composition of claim 10, wherein the nucleotide sequence comprises the sequence as set forth in SEQ ID NO:50, or a fragment or variant thereof, wherein the fragment or variant of SEQ ID NO:50 retains the function of transcriptional repression.

12. The composition of any one of claims 1 to 11, wherein the site-specific DNA binding molecule specifically binds to a viral gene, an oncogene, or a human gene, or a regulatory region thereof.

13. The composition of claim 12, wherein the site-specific DNA binding molecule specifically binds to a viral latency gene, or a regulator region thereof.

14. The composition of claim 13, wherein the viral latency gene is an HIV gene, an EBV lytic gene, a human CMV gene, a Kaposi sarcoma-associated herpesvirus gene or hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), or a regulatory region thereof.

15. The composition of claim 12, wherein the TALE is an HIV-specific TALE and further wherein the activation domain is selected from the group consisting of VP64, VPR, p300 and p65-HSFL16. The composition of claim 15, wherein the RNA molecule comprises a nucleic acid sequence encoding SEQ ID NO:5, SEQ ID NO:7, SEQ ID NON, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47.

17. The composition of claim 15, wherein the RNA molecule comprises a nucleotide sequence comprising SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 or a fragment or variant thereof linked to a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48.

18. The composition of claim 15, wherein the RNA molecule further comprises a nucleotide sequence encoding Tat, or a fragment or variant thereof.

19. The composition of claim 18, wherein the Tat comprises SEQ ID NO: 51, or a fragment or variant thereof.

20. The composition of claim 18, wherein the RNA molecule comprises a nucleic acid sequence comprising SEQ ID NO: 52, or a fragment or variant thereof.

21. The composition of claim 15, wherein the RNA comprises a nucleic acid sequence encoding SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:83,SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 or SEQ ID NO:91 or a fragment or variant thereof.

22. The composition of claim 17, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90 or SEQ ID NO:92, or a fragment or variant thereof.

23. The composition of claim 12, wherein the TAEE is an HIV-specific TALE and further wherein the transcriptional repression domain comprises a Kriippel- associated box (KRAB) domain.

24. The composition of claim 23, wherein the RNA molecule comprises a nucleic acid sequence encoding SEQ ID NO:5, SEQ ID NO:7, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:49.

25. The composition of claim 24, wherein the RNA molecule comprises a nucleotide sequence comprising SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40 or a fragment or variant thereof linked to a sequence of SEQ ID NO:50.

26. The composition of claim 23, wherein the RNA comprises a nucleic acid sequence encoding SEQ ID NO:77, SEQ ID NO:79 or SEQ ID NO:81 or a fragment or variant thereof.

27. The composition of claim 26, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO:78, SEQ ID NO:80 or SEQ ID NO:82, or a fragment or variant thereof.

28. The composition of claim 12, wherein the site-specific DNA binding molecule specifically binds to CCR5, FOXP3, RORC, IL4, TOX, T0X2, MS4A1, F0XR2, LM01 or LYL1, or a regulatory region of CCR5, FOXP3, RORC, IL4, TOX, T0X2, MS4A1, F0XR2, LM01 or LYLE29. The composition of claim 28, wherein the TALE is a MS4A1 -specific TALE and further wherein the transcriptional activation domain is VPR, VP64, p300 or p65- HSF1.

30. The composition of claim 29, wherein the RNA comprises a nucleic acid sequence encoding SEQ ID NO:1 or SEQ ID NO:3 or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47.

31. The composition of claim 29, wherein the RNA comprises a nucleotide sequence comprising SEQ ID NO:2 or SEQ ID NO:4 or a fragment or variant thereof linked to a sequence of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:48.

32. The composition of claim 29, wherein the RNA comprises a nucleic acid sequence encoding SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:57 or a fragment or variant thereof.

33. The composition of claim 29, wherein the RNA comprises a nucleic acid sequence of SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO:58, or a fragment or variant thereof.

34. The composition of claim 38, wherein the TALE is an MS4A1 -specific TALE and further wherein the transcriptional repression domain comprises a Kriippel- associated box (KRAB) domain.

35. The composition of claim 23, wherein the RNA molecule comprises a nucleic acid sequence encoding SEQ ID NO: 1 or SEQ ID NO:3, or a fragment or variant thereof, linked to a nucleic acid sequence encoding SEQ ID NO:49.

36. The composition of claim 35, wherein the RNA molecule comprises a nucleotide sequence comprising SEQ ID NO:2, or SEQ ID NO:4 or a fragment or variant thereof linked to a sequence of SEQ ID NO:50.

37. The composition of any one of claims 1 to 36, wherein the composition comprises a lipid nanoparticle (LNP) encapsulating the RNA molecule.

38. The composition of any one of claims 1 to 37, wherein the RNA molecule comprises an mRNA molecule.

39. A method of transiently modulating the transcription of at least one gene in a subject in need thereof, the method comprising administering a composition of any one of claims 1 to 38 to the subject.

40. The method of claim 39, wherein the gene comprises a viral gene, an oncogene, or a human gene.

41. A method of inducing expression of latent viral genes in a subj ect, the method comprising administering to the subject a composition of any one of claims 1 to 38, wherein the composition comprises a site-specific DNA binding molecule that specifically binds to a viral latency gene, or a regulatory region thereof, operably linked to a transcriptional activation domain.

42. The method of claim 41, wherein the viral latency gene is an HIV gene, an EBV lytic gene, a human CMV gene, a Kaposi sarcoma-associated herpesvirus gene or hepatitis B virus (HBV) covalently closed circular DNA (cccDNA).

43. A method of reducing or preventing viral entry into cells in a subject, the method comprising administering to the subject a composition of claim 1, wherein the composition comprises a site-specific DNA binding molecule that specifically binds to CCR5, or a regulatory region thereof, operably linked to a transcriptional activation domain.

44. A method of transiently modulating expression of CD20 in a subject, the method comprising administering to the subject a composition of any one of claims 28-38, wherein the composition comprises a site-specific DNA binding molecule specific that specifically binds to MS4A1, or a regulatory region thereof, operably linked to a transcriptional modulation domain.

45. A method of improving T cell or NK cell functionality, the method comprising administering a composition of claim 1 to the subject.

46. The method of claim 31, wherein the composition comprises an RNA encoding a site-specific TALE that specifically binds to TOX or T0X2, or a regulatory region thereof, linked to a transcriptional repressor domain.

47. A method of increasing the efficacy of a vaccine or cancer therapy, the method comprising administering a composition of claim 1 prior to, concurrent with, or following administration of the vaccine or cancer therapy.

48. The method of claim 47, wherein the composition comprises an RNA encoding a site-specific TALE that specifically binds to IL4 or RORC, or a regulatory region thereof, linked to a transcriptional activation domain.

49. A method of inducing the formation of regulatory T cells in a subject in need thereof, the method comprising administering a composition of claim 1 to the subject.

50. The method of claim 49, wherein the composition comprises an RNA encoding a site-specific TALE that specifically binds to FOXP3, or a regulatory region thereof, linked to a transcriptional activation domain.

51. A method of suppressing tumor growth in a subject in need thereof, the method comprising administering a composition of claim 1 to the subject.

52. The method of claim 51, wherein the composition comprises an RNA encoding a site-specific TALE specific for binding to an oncogene, or a regulatory region thereof, linked to a transcriptional repression domain.

53. The method of claim 52, wherein the oncogene is selected from the group consisting ofFOXR2, LM01 and LYLl.

54. A plasmid for expression of an RNA molecule encoding a site-specific transient transcriptional modulation of a gene of interest, encoding a site-specific DNA binding molecule linked to a transcriptional modulator.

55. The plasmid of claim 54 comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:97 to SEQ ID NO: 146.

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