VESIGEN THERAPEUTICS INC (US)
CHINCHORE YASHODHAN (US)
| Claims What is claimed is: 1. An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), comprising: (i) a lipid bilayer and an ARRDC1 protein, (ii) a molecule, and (iii) a viral envelope protein. 2. The microvesicle of claim 1, wherein said viral envelope protein is vesicular stomatitis virus G (VSV-G). 3. The microvesicle of claim 1, wherein said viral envelope protein is rabies virus glycoprotein (RVG). 4. A microvesicle-producing cell comprising: a recombinant expression construct encoding an ARRDC1 protein or a variant thereof under the control of a heterologous promoter, and a viral envelope protein. 5. The microvesicle-producing cell of claim 4, wherein said viral envelope protein is VSV-G. 6. The microvesicle-producing cell of claim 4, wherein said viral envelope protein is RVG. 7. A method of delivering a molecule to a target cell, said method comprising contacting the target cell with the microvesicle of any of claims 1-3. 8. The method of claim 7, wherein said target cell is a cell of the eye. 9. The method of claim 8, wherein said cell of the eye is a cell of the retina. 10. A method of treating a disorder in a patient, the method consisting of administering to the patient a microvesicle of any of claims 1-3. 11. A method of treating a disorder in a patient, the method consisting of administering to the patient a microvesicle-producing cell of any of claims 4-6. 12. The method of claims 10 or 11, wherein said disorder is a disorder of the eye. 13. An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), comprising: (i) a lipid bilayer and an ARRDC1 protein, and (ii) a therapeutic agent. 14. The method of claims 10 or 11, wherein said patient is a mammal. 15. The method of claim 14, wherein said mammal is a primate. 16. The method of claim 15, wherein said primate is a human. 17. An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM) for use a medicament. 18. An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM) for use in the treatment of diseases of the eye. 19. The use of said arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM) of claim 18, wherein said diseases are Stargardt Macular Dystrophy or Age-Related Macular Degeneration. 20. A composition substantially as shown and described. 21. A method substantially as shown and described. |
[0094] Human Nedd4-2 amino acid sequence (>gi|21361472|ref|NP_056092.2| E3 ubiquitin-protein ligase NEDD4-like isoform 3 [Homo sapiens]). The four underlined WW domains correspond to amino acids 198 – 224 (WW1), 368 – 396 (WW2), 480 – 510 (WW3), and 531 – 561 (WW4).
[0095] Human Smurf1 amino acid sequence (uniprot.org/uniprot/ Q9HCE7). The two underlined WW domains correspond to amino acids 234 – 267 (WW1) and 306 – 339 (WW2).
[0096] Human Smurf2 amino acid sequence (uniprot.org/uniprot/Q9HAU4). The three underlined WW domains correspond to amino acids 157 – 190 (WW1), 251 – 284 (WW2), and 297 – 330 (WW3). [0097] Human ITCH amino acid sequence (uniprot.org/uniprot/Q96J02). The four underlined WW domains correspond to amino acids 326 – 359 (WW1), 358 – 391 (WW2), 438 – 471 (WW3), and 478 – 511 (WW4). [0098] Human NEDL1 amino acid sequence (uniprot.org/uniprot/Q76N89). The two underlined WW domains correspond to amino acids 829 – 862 (WW1), and 1018 – 1051 (WW2).
[0099] Human NEDL2 amino acid sequence (uniprot.org/uniprot/ Q9P2P5). The two underlined WW domains correspond to amino acids 807 – 840 (WW1) and 985 – 1018 (WW2).
[00100] In some embodiments, the WW domain consists essentially of a WW domain or WW domain variant. Consists essentially of means that a domain, peptide, or polypeptide consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example, from about 1 to about 10 or so additional residues, typically from 1 to about 5 additional residues in the domain, peptide, or polypeptide. [00101] Alternatively, the WW domain may be a WW domain that has been modified to include two basic amino acids at the C-terminus of the domain. Techniques are known in the art and are described in the art, for example, in Sambrook et al., Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (2001). Thus, a skilled person could readily modify an existing WW domain that does not normally have two C- terminal basic residues so as to include two basic residues at the C-terminus. [00102] Basic amino acids are amino acids that possess a side-chain functional group that has a pKa of greater than 7 and includes lysine, arginine, and histidine, as well as basic amino acids that are not included in the twenty α-amino acids commonly included in proteins. The two basic amino acids at the C-terminus of the WW domain may be the same basic amino acid or different basic amino acids. In one embodiment, the two basic amino acids are two arginines. [00103] The term WW domain also includes variants of a WW domain provided that any such variant possesses two basic amino acids at its C-terminus and maintains the ability of the WW domain to associate with the PPXY (SEQ ID NO: 2) motif. A variant of such a WW domain refers to a WW domain that retains the ability of the variant to associate with the PPXY (SEQ ID NO: 2) motif (i.e., the PPXY (SEQ ID NO:2) motif of ARRDC1 and that has been mutated at one or more amino acids, including point, insertion, and/or deletion mutations, but still retains the ability to associate with the PPXY (SEQ ID NO: 2) motif. A variant or derivative, therefore, includes deletions, including truncations and fragments; insertions and additions, for example, conservative substitutions, site-directed mutants and allelic variants; and modifications, including one or more non-amino acyl groups (e.g., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. [00104] The WW domain may be part of a longer protein. Thus, the protein, in various different embodiments, comprises the WW domain, consists of the WW domain, or consists essentially of the WW domain, as defined herein. The polypeptide may be a protein that includes a WW domain as a functional domain within the protein sequence. [00105] The term “Cas9,” or “Cas9 protein,” or “Cas9 polypeptide” refers to an RNA- guided nuclease comprising a Cas9 protein as well as fusion proteins containing such Cas9 proteins and variants thereof (e.g., a protein comprising an active, inactive, or altered DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). In some embodiments, the fused proteins may include those that modify the epigenome or control transcriptional activity. The variants may include deletions or additions, such as, e.g., addition of one, two, or more nuclear localization sequences (such as from SV40 and others known in the art), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such sequences or a range between and including any two of the foregoing values. [00106] In some embodiments, the Cas9 polypeptide is a Cas9 protein found in a type II CRISPR-associated system. Suitable Cas9 polypeptides that may be used in certain embodiments of the present invention include, but are not limited to, Cas9 protein from Streptococcus pyogenes (Sp. Cas9), F. novicida, S. aureus, S. thermophiles, N. meningitidis, and variants thereof. [00107] A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (e.g., viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (mc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3'-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA,”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. (See, e.g., Jinek M., et al., Science, 337:816-821 (2012), the entire contents of which is hereby incorporated by reference). Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art. (See, e.g., Ferretti et al., “Complete genome sequence of an M1 strain of Streptococcus pyogenes,” Proc. Natl. Acad. Sci. U.S.A., 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,” Deltcheva E., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,” Nature, 471:602-607 (2011); and Jinek, M., et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in the art. (See, e.g., Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems,” RNA Biology, 10:5, 726-737 (2013); Karvelis, G., et al., “Harnessing the natural diversity and in vitro evolution of Cas9 to expand the genome editing toolbox,” Current Opinion in Microbiology, 37:88-94 (2017); Komor, A. C., et al., “CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes,” Cell, 168:20-36 (2017); and Murovec, J., et al., “New variants of CRISPR RNA-guided genome editing enzymes,” Plant Biotechnol. J., 15:917-26 (2017), the entire contents of each of which are incorporated herein by reference). [00108] In some embodiments, the Cas9 polypeptide is a wild-type Cas9, a nickase, or comprises a nuclease inactivated (“dCas9” for nuclease-"dead" Cas9) protein. [00109] Methods for generating a Cas9 protein (or a variant thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science.337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression,” Cell, 28;152(5):1173-83 (2013), the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H841A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science, 337:816-821 (2012); Qi et al., Cell, 28;152(5):1173-83 (2013)). In some embodiments, proteins comprising variants of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or variants thereof are referred to as "Cas9 variants." A Cas9 variant shares homology to Cas9, or a variant thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to wild type Cas9. In some embodiments, the Cas9 variant comprises a variant of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to the corresponding variant of wild type Cas9. In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, SEQ ID NO:1 (nucleotide); SEQ ID NO:2 (amino acid)). In some embodiments, wild type Cas9 corresponds to, or comprises SEQ ID NO:3 (nucleotide) and/or SEQ ID NO: 4 (amino acid): In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and/or H820A mutation. dCas9 (D10A and H840A): [00110] In other embodiments, dCas9 variants having mutations other than D10A and H820A are provided, which e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 (e.g., variants of SEQ ID NO: 5) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to SEQ ID NO:5. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 5) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 5, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more. [00111] In some embodiments, Cas9 fusion proteins as provided herein comprise the full- length amino acid of a Cas9 protein, e.g., one of the sequences provided above. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art. In some of these embodiments, the fusion protein comprises a transcriptional activator (e.g., VP64), a transcriptional repressor (e.g., KRAB, SID), a nuclease domain (e.g., FokI), base editors, prime editors, a recombinase domain (e.g., Hin, Gin, or Tn3), a deaminase (e.g., a cytidine deaminase or an adenosine deaminase) or an epigenetic modifier domain (e.g., TET1, p300). [00112] In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP 472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria meningitidis (NCBI Ref: YP_002342100.1). [00113] The term "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. [00114] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA molecules that are not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. RNA-programmable nucleases include Cas9 nucleases. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though "gRNA" is used interchangeably to refer to guide RNAs that exist as either single molecules or as two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site and provides the sequence specificity of the nuclease:RNA complex. [00115] The term "recombinase," as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Examples of serine recombinases include, without limitation, Hin, Gin, Tn3, β-six, CinH, ParA, γδ, Bxb1, φC31, TP901, TG1, φBT1, R4, φRV1, φFC1, MR11, A118, U153, and gp29. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange. Recombinases have numerous applications, including the creation of gene knockouts/knock-ins and gene therapy applications. (See, e.g., Brown et al., “Serine recombinases as tools for genome engineering,” Methods, 53(4):372-379 (2011); Hirano et al., “Site-specific recombinases as tools for heterologous gene integration,” Appl. Microbiol. Biotechnol., 92(2):227-239 (2011); Chavez and Calos, “Therapeutic applications of the ΦC31 integrase system,” Curr. Gene Ther., 11(5):375-381 (2011); Turan and Bode, “Site- specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications,” FASEB J., 25(12):4088-4107 (2011); Venken and Bellen, “Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and ΦC31 integrase,” Methods Mol. Biol., 859:203-228 (2012); Murphy, “Phage recombinases and their applications,” Adv. Virus Res., 83:367-414 (2012); Zhang et al., “Conditional gene manipulation: Cre-ating a new biological era,” J. Zhejiang Univ. Sci. B., 13(7):511-524 (2012); Karpenshif and Bernstein, “From yeast to mammals: recent advances in genetic control of homologous recombination,” DNA Repair (Amst), 1;11(10):781-788 (2012); the entire contents of each are hereby incorporated by reference in their entirety). The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the invention. The methods and compositions of the invention can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities. (See, e.g., Groth et al., “Phage integrases: biology and applications,” J. Mol. Biol., 335, 667-678 (2004); Gordley et al., “Synthesis of programmable integrases,” Proc. Natl. Acad. Sci. USA., 106, 5053-5058 (2009); the entire contents of each are hereby incorporated by reference in their entirety). Other examples of recombinases that are useful in the methods and compositions described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the invention. In some embodiments, a recombinase (or catalytic domain thereof) is fused to a Cas9 protein (e.g., dCas9). [00116] The terms “recombine” and “recombination,” in the context of a nucleic acid modification (e.g., a genomic modification), are used to refer to the process by which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a recombinase protein. Recombination can result in, inter alia, the insertion, inversion, excision, or translocation of a nucleic acid sequence, e.g., in or between one or more nucleic acid molecules. [00117] As used herein, the terms “approximately,” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (for example, when such number would exceed 100% of a possible value). General Description of the Invention [00118] ARMMs related compositions and methods for producing and using said particles are known in the art, and are described in, but not limited to, U.S. Pat. Nos.9,737,480; 9,816,080; 10,260,055; PCT Application Publication WO2018/067546; and U.S. Pat. Pub. Nos.20220119785; 20220170013; 20220220462; and 20220282275; the entire contents of each of which are hereby incorporated by reference in their entirety. [00119] In one aspect of the instant invention, compositions and methods are provided for ARMMs-mediated delivery of therapeutic agents and payloads to cells of the eye, and in particular, to the retina. It is contemplated that the ARMMs based compositions and methods of the present invention provide certain advantages as compared to the administration of viral vectors including, but not limited to, transient expression, circumventing immune privilege, repeat disability, among other advantages. ARMMs comprising an ARRDC1-Cre fusion protein have been functionally administered into the eyes (e.g., subretinal space) of Ai14 Cre reporter mice. Briefly, in the Ai14 system, the expression of the fluorescent protein tdTomato, is activated only by exogenously derived functional Cre recombinase protein. In Ai14 animals, Cre-dependent TdTomato expression in target cells demonstrates uptake of A1-cre loaded ARMM particles by those cells. Preferred embodiments of the invention thusly provide delivery of nuclear cargos capable of editing the nuclear genome of target cells. Representative Genetic Diseases of the Eye [00120] In preferred embodiments, the compositions and methods of the present invention are directed to treating, mitigating, ameliorating, or curing a disease or condition caused by one or more abnormalities in a patient’s genome. A non-limiting example of genetic targets for ARMM delivered therapeutics include, but are not limited to: CFH (i.e. age-related macular degeneration), ABCA4 (i.e., Stargardt disease, cone-rod dystrophy, age-related macular degeneration, and retinitis pigmentosa); ELOVL4 (i.e., Stargardt disease); USH2A (i.e., autosomal recessive retinitis pigmentosa, and Usher Syndrome); RPGR and RP2 (i.e., x- linked retinitis pigmentosa); GUCY2D, A1PL1, RDH12, RPGR1P1, and CEP290 (i.e., Leber Congenital Amaurosis (“LCA”)); and TULP1, LRAT, IMPDH1 (i.e., potential LCA-like phenotypes). In additional embodiments, the GUCY2D (i.e., autosomal dominant cone-rod dystrophy), RPE65 (i.e., retinitis pigmentosa), and AIPL1 (i.e., cone-rod dystrophy and retinitis pigmentosa) genes are also thought to be associated with additional conditions. [00121] In still further embodiments, compositions and methods of the present invention are contemplated for treating or ameliorating one or more conditions associated with genes, including, but not limited to: BEST1 (i.e., vitelliform macular dystrophy, age-related macular degeneration, autosomal dominant vitreoretinochoroidopathy, and retinitis pigmentosa, nanophthalmos, autosomal recessive, and bestrophinopathy (ARB)); CLRN1 (i.e., Usher syndrome type IIIA (USH3A), and retinitis pigmentosa); CRB1 (i.e., Leber Congenital Amaurosis, cone-rod dystrophy, retinitis pigmentosa); CRX (i.e., Leber Congenital Amaurosis, cone-rod dystrophy, retinitis pigmentosa); PDE6B (i.e., autosomal dominant congenital stationary night blindness, retinitis pigmentosa); PRPH2 (i.e., vitelliform macular dystrophy, cone-rod dystrophy, retinitis pigmentosa); RHO (i.e., autosomal dominant congenital stationary night blindness, autosomal dominant retinitis pigmentosa); RPE65 (i.e., Leber Congenital Amaurosis, fundus albipunctatus, retinitis pigmentosa); and WDR19 (i.e., cranioectodermal dysplasia, asphyxiating thoracic dystrophy, nephronophthisis, retinitis pigmentosa, Senior-Løken syndrome). [00122] Additional embodiments are directed to delivering ARMMs associated payloads and therapeutic agents to retinal pigment epithelium cells and tissues to treat diseases and conditions including, but not limited to, age-related macular degeneration, Best Vitelliform Macular Dystrophy, Sorsby’s Fundus Dystrophy, choroidal neovascularization, diabetic macular edema, MERTK retinitis pigmentosa, RPE65 Leber’s Congenital Amaurosis, and Bestrophinopathies (e.g., autosomal dominant vitreoretinochoroidopathy). In other embodiments, ARMMs are directed to delivering payloads and therapeutic agents to photoreceptor cells to treat diseases and conditions including, but not limited to, retinitis pigmentosa (e.g., autosomal dominant, X-linked, and/or recessive type), Stargardt Macular Dystrophy, Achromatopsia, Usher Syndrome 1c, and Cone-Rod Dystrophy. In still further embodiments, to delivering ARMMs associated payloads and therapeutic agents to retinal pigment epithelium cells and photoreceptors to treat diseases and conditions including, but not limited to, Usher Syndrome 1b, Choroideremia, Bardet-Biedl Syndrome, and retinitis pigmentosa. [00123] Thus, in some cases, the compositions described here are administered to a subject having or suspected to have a disease of the eye (e.g., as described herein). [00124] In some cases, the compositions described herein are administered to a subject having or suspected to have Stargardt disease. In some cases, the Stargardt disease is Stargardt disease-1, Stargardt disease-3, Stargardt disease-4, or fundulus flavimaculatus (FFM). In some cases, the subject having or suspected to have Stargardt disease has one or more genetic variations in the ABCA4 gene, ELOVL4 gene, BEST1 gene, and/or PRPH2 genes. [00125] In some cases, the compositions described herein are administered to a subject having or suspected to have Usher Syndrome. In some cases, the Usher Syndrome is Usher Syndrome Type 1, Usher Syndrome Type 2, or Usher Syndrome Type 3. In some cases, the subject having or suspected to have Usher Syndrome has one or more genetic variations in the MYO7A, USH1C, CDH23, PCDH15, USH2A, ADGRV1, WHRN, and/or CLRN1 genes. [00126] In some cases, the compositions described herein are administered to a patient having one or more of the following genetic variants relative to human Retinal-specific phospholipid-transporting ATPase ABCA4 (UniProt ID P78363), encoded by human ABCA4: [00127] In some cases, the compositions described herein are administered to a patient having one or more of the following genetic variants relative to human Unconventional myosin-VIIa (UniProt ID Q13402), encoded by human MYO7A: [00128] In some cases, the compositions described herein are administered to a patient having one or more of the following genetic variants relative to human Cadherin-23 (UniProt ID Q9H251), encoded by human CDH23: [00129] In some cases, the compositions described herein are administered to a patient having one or more of the following genetic variants relative to human Protocadherin-15 (UniProt ID Q96QU1), encoded by human PCDH15: [00130] In some cases, the compositions described herein are administered to a patient having one or more of the following genetic variants relative to human Usherin (UniProt ID O75445), encoded by human USH2A: [00131] In some cases, the compositions described herein are administered to a patient having one or more of the following genetic variants relative to human Adhesion G-protein coupled receptor V1 (UniProt ID Q8WXG9), encoded by human ADGRV1: [00132] In some cases, the compositions described herein are administered to a patient having one or more of the following genetic variants relative to human Clarin-1 (UniProt ID P58418), encoded by human CLRN1: [00133] In some cases, the composition administered to the subject comprises a therapeutic payload that targets one or more of the genetic variants described herein. In some cases, the therapeutic payload targets one or more of the genetic variants described herein to modulate its expression (e.g., by overexpression or inhibition). In some cases, the therapeutic payload targets one or more of the genetic variants described herein for genome editing (e.g., of the genetic variant). In some cases, the therapeutic payload is a genome editor or portion thereof (e.g., as described herein, e.g., an RNA guided genome editor or portion thereof, e.g., a nuclease, a base editor, a prime editor, or a portion thereof). General Pharmaceutical Formulations and Compositions [00134] Additional aspects of the present disclosure relate to pharmaceutical compositions comprising any of the ARMMs or microvesicle (e.g., ARMM) producing cells provided herein. The term “pharmaceutical composition,” as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increasing half-life, potency, efficacy, biological effect, and the like, as well as other therapeutic agents and compounds). [00135] As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue, system, or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the cell and tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically- acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C 2 -C 12 alcohols, such as ethanol; and (24) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, and the like, can also optionally be present in a formulation. Terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” and the like, are used interchangeably herein. [00136] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for delivering a therapeutic agent, payload protein, or payload nucleic acid to a cell. Suitable routes of administrating of the pharmaceutical compositions described herein include, without limitation: subretinal, suprachoroidal, intrvitereal, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. (See, e.g., Hartman, R.R., and Kompella, U.B., “Intravitreal, Subretinal, and Suprachoroidal Injections: Evolution of Microneedles for Drug Delivery,” J. Ocul. Pharmacol. Ther., 34(1-2):141–153 (2018)). [00137] In some embodiments, a pharmaceutical composition described herein is administered locally to a diseased site (e.g., cell of the eye). In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. [00138] In some embodiments, the compositions are formulated in accordance with routine procedures and is a composition adapted for injection, intravenous, or subcutaneous administration to a subject, e.g., a human. In some embodiments, the compositions for administration by injection comprise a solution in a sterile isotonic aqueous buffer. Where necessary, the formulations can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Where the formulations are administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. Where the formulations are to be administered by infusion, they can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. [00139] A formulation (a pharmaceutical composition) for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer’s solution, or Hank’s solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms of certain formulations are also contemplated. [00140] The compositions described herein may be administered or packaged as a unit dose. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle. [00141] Further, the compositions can be provided as kits comprising: 1) a container containing an ARMM or microvesicle producing cell of the invention; and 2) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used, e.g., for reconstitution or dilution of the ARMM or microvesicle producing cell of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. In this regard, state-specific and regional regulatory agencies are understood to include, but are not limited to, the U.S. Food and Drug Administration, the U.S. Department of Agriculture, the European Medicines Agency, the United Kingdom Medicines and Healthcare Products Regulatory Agency, the National Medical Products Administration, and the like. [00142] In another aspect, an article of manufacture containing materials useful for treating the diseases described herein is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from various materials such as glass or plastic. Suitable containers are further understood to include materials that are sufficiently non-reactive and protective of the contents therein. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating one or more diseases of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions and indications for acceptable, recognized, or permitted use(s). [00143] In some embodiments, treatments are contemplated wherein one therapeutic agent is administered using ARMM-mediated delivery of an agent to target cells, tissues, systems, or mammalian subjects. [00144] In some other embodiments, treatments are contemplated wherein two therapeutic agents are administered using ARMM-mediated delivery of the respective agents to target cells, tissues, systems, or mammalian subjects. [00145] In still some other embodiments, treatments are contemplated wherein three or more therapeutic agents are administered using ARMM-mediated delivery of the respective agents to target cells, tissues, systems, or mammalian subjects. [00146] It is contemplated that within the compositions and methods described herein, when two or more respective therapeutic agents are administered (e.g., co-administered) to a target, cell, tissue, system, or subject that the respective (i.e., two or more) agents are provided in a single ARRDC1-mediated microvesicle (ARMM) particle for administration. [00147] It is likewise further contemplated that within the compositions and methods described herein, when two or more respective therapeutic agents are administered (e.g., concomitantly administered) to a target cell, tissue, system, or subject that the respective (i.e., two or more) agents are provided respectively in different ARRDC1-mediated microvesicles (ARMMs) particles for administration. Kits, Vectors, and Cells [00148] Some aspects of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding one or more of any of the proteins (e.g., ARRDC1, and TSG101), fusion proteins and/or nucleic acids provided herein. In some embodiments, the nucleotide sequence encodes any of the proteins, fusion proteins, and/or RNAs provided herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives the expression of any of the proteins, fusion proteins, and/or RNAs provided herein. [00149] Some aspects of this disclosure provide microvesicle (e.g., ARMM) producing cells comprising any of the proteins, fusion proteins, and nucleic acids (e.g., RNAs) provided herein. In some embodiments, the cells specifically comprise a nucleotide that encodes any of the proteins, fusion proteins, and/or RNAs provided herein. In some embodiments, the cells comprise any of the nucleotides or vectors provided herein. In some embodiments, the vector comprises one or more cell targeting or cell entry proteins (e.g., viral and/or human fusogen(s)). [00150] It should be appreciated, however, that additional proteins, fusion proteins, and RNAs would be apparent to the skilled artisan based on the present disclosure and knowledge in the art. [00151] The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments but are not intended to exemplify the full scope of the invention. [00152] Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention. Detailed Description of Certain Embodiments of the Invention [00153] The present invention provides methods, systems, and compositions for ARRDC1-mediated microvesicles (“ARMMs”) delivery of molecules of interest (e.g., therapeutic agents) to cells and tissues of the eye. The present invention further relates to compositions and methods of producing, testing, and administering ARMMs to one or more internal structures of the eye. More particularly, the present invention provides compositions and methods of producing, testing, and administering ARMMs comprising one or more therapeutic agents (e.g., biological molecules including, but not limited to, CRISPR/Cas9, and other similar endonucleases, base editors, small molecules, proteins, and nucleic acids (e.g., DNA, RNA, siRNA, mRNA, miRNA, and the like)). Also provided are methods of administering therapeutic agents associated with ARMMs, including, but not limited to, methods of treating or contacting cells and tissues of the eye in one or more exemplary dosing regimens (e.g., 1) in vivo administration of ARMMs to a patient; 2) ex vivo administration of ARMMs to target cells and implantation of the ARMMs treated cells to a/the patient; and 3) in vivo and ex vivo regimens). In particular, the present invention provides methods of administering therapeutic agents via ARMMs to the cells and tissues that comprise the retina or into the subretinal space. Additionally, the present invention relates to methods of manufacturing (e.g., culturing, clarifying, separating, and concentrating) the inventive compositions obtained from stable producer cell lines and/or from transient cell cultures. [00154] More particularly, the instant disclosure relates to the discovery that therapeutic agents associated with an ARRDC1 protein can be loaded into ARMMs and delivered to cells and tissues comprising the retina. In some embodiments, the uptake of these ARMMs and associated their payload(s) is enhanced by the presence of portions of viral envelope proteins, including, but not limited to, VSV-G on the surface of ARMMs. Different therapeutic agents, such as proteins and nucleic acids, including various RNAs, can be loaded in such ARMMs for delivery to retina cells. In preferred embodiments, various types of endonucleases (e.g., CRISPR/Cas9 with associated gRNA), base and prime editors with associated gRNA, and effector proteins, are contemplated as being suitable for delivery using the compositions and methods of the present invention to the cells and tissues of the eye, and more particularly, to the cells of the retina and subretinal space. ARMMs [00155] Arrestin Domain Containing Protein 1 mediated microvesicles (“ARMMs”) are extracellular vesicles (“EVs”) that are distinct from exosomes. The budding of ARMMs requires ARRDC1, which is localized to the cytosolic side of the plasma membrane and, through a tetrapeptide motif, recruits the ESCRT-I complex protein TSG101 to the cell surface to initiate the outward membrane budding. Thus, in contrast to exosomes, the biogenesis of ARMMs occurs at the plasma membrane. ARMMs exhibit several additional features that make them potentially ideal vehicles for therapeutic delivery. ARRDC1 is not only necessary but also sufficient to drive ARMMs budding. Overexpression of the ARRDC1 protein increases the production of ARMMs in cells. This allows controlled production of ARMMs using modern biological manufacturing methods. Moreover, endogenous proteins such as cell surface receptors are actively recruited into ARMMs and can be delivered into recipient cells to initiate intercellular communication, suggesting that the exogenous payload molecules may be similarly packaged and delivered via ARMMs. ARRDC1 [00156] In preferred embodiments, ARRDC1 is a protein that comprises a PSAP (SEQ ID NO: 1) motif and a PPXY (SEQ ID NO: 2) motif in its C-terminus, and interacts with TSG101 as shown herein. It should be appreciated that the PSAP (SEQ ID NO: 1) motif and the PPXY (SEQ ID NO: 2) motif are not required to be at the absolute C-terminal end of the ARRDC1. Rather, they may be at a C-terminal portion of the ARRDC1 protein (e.g., the C- terminal half of the ARRDC1). The disclosure also contemplates variants of ARRDC1, such as fragments of ARRDC1 and/or ARRDC1 proteins that have a degree of identity (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity) to an ARRDC1 protein and are capable if interacting with TSG101. Accordingly, an ARRDC1 protein may be a protein that comprises a PSAP (SEQ ID NO: 1) motif and a PPXY (SEQ ID NO: 2) motif and interacts with TSG101. In some embodiments, the ARRDC1 protein is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-44, comprises a PSAP (SEQ ID NO: 1) motif and a PPXY (SEQ ID NO: 2) motif, and interacts with TSG101. In some embodiments, the ARRDC1 protein has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, at least 390, at least 400, at least 410, at least 420, or at least 430 identical contiguous amino acids of any one of SEQ ID NOs: 42-44, comprises a PSAP (SEQ ID NO: 1) motif and a PPXY (SEQ ID NO: 2) motif, and interacts with TSG101. In some embodiments, the ARRDC1 protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 42-44 comprises a PSAP (SEQ ID NO: 1) motif and a PPXY (SEQ ID NO: 2) motif, and interacts with TSG101. In some embodiments, the ARRDC1 protein comprises any one of the amino acid sequences set forth in SEQ ID NOs: 42-44. Exemplary, non-limiting ARRDC1 protein sequences are provided herein, and additional, suitable ARRDC1 protein variants according to aspects of this invention are known in the art. It will be appreciated by those of skill in the art that this invention is not limited in this respect. Exemplary ARRDC1 sequences include the following (PSAP (SEQ ID NO: 1) and PPXY (SEQ ID NO: 2) motifs are marked): [00157] >gi|22748653|ref|NP_689498.1| arrestin domain-containing protein 1 [Homo sapiens] [00158] >gi|244798004|ref|NP_001155957.1| arrestin domain-containing protein 1 isoform a [Mus musculus] [00159] >gi|244798112|ref|NP_848495.2| arrestin domain-containing protein 1 isoform b [Mus musculus] TSG101 [00160] In certain embodiments, the inventive microvesicles further comprise TSG101 (tumor susceptibility gene 101) TSG101 belongs to a group of putative inactive homologs of ubiquitin-conjugating enzymes. The protein contains a coiled-coil domain that interacts with stathmin, a cytosolic phosphoprotein implicated in tumorigenesis. TSG101 is a protein that comprises a UEV domain and interacts with ARRDC1. As referred to herein, UEV refers to the Ubiquitin E2 variant domain of approximately 145 amino acids. The structure of the domain contains a α/β fold similar to the canonical E2 enzyme but has an additional N- terminal helix and further lacks the two C-terminal helices. Often found in TSG101/Vps23 proteins, the UEV interacts with a ubiquitin molecule and is essential for the trafficking of a number of ubiquitylated payloads to multivesicular bodies (MVBs). Furthermore, the UEV domain can bind to Pro-Thr/Ser-Ala-Pro peptide ligands, a fact exploited by viruses such as HIV. Thus, the TSG101 UEV domain binds to the PTAP tetrapeptide motif in the viral Gag protein that is involved in viral budding. The disclosure also contemplates variants of TSG101, such as fragments of TSG101 and/or TSG101 proteins that have a degree of identity (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity) to a TSG101 protein and are capable of interacting with ARRDC1. Accordingly, a TSG101 protein may be a protein that comprises a UEV domain and interacts with ARRDC1. In some embodiments, the TSG101 protein is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 45-47, comprises a UEV domain, and interacts with ARRDC1. In some embodiments, the TSG101 protein has at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 310, at least 320, at least 330, at least 340, at least 350, at least 360, at least 370, at least 380, or at least 390, and any integer between the numbers, identical contiguous amino acids of any one of SEQ ID NOs: 45-47, comprises a UEV domain and interacts with ARRDC1. In some embodiments, the TSG101 protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 45-47 and comprises a UEV domain. In some embodiments, the ARRDC1 protein comprises any one of the amino acid sequences set forth in SEQ ID NOs: 45-47. Exemplary, non-limiting TSG101 protein sequences are provided herein, and additional, suitable TSG101 protein sequences, isoforms, and variants are known in the art. It will be appreciated by those of skill in the art that this invention is not limited in this respect. Exemplary TSG101 sequences include the following sequences (the UEV domain in these sequences includes amino acids 1-145 and is underlined in the sequences below): [00161] >gi|5454140|ref|NP_006283.1| tumor susceptibility gene 101 protein [Homo sapiens] [00162] >gi|11230780|ref|NP_068684.1| tumor susceptibility gene 101 protein [Mus musculus] [00163] >gi|48374087|ref|NP_853659.2| tumor susceptibility gene 101 protein [Rattus norvegicus] [00164] The structure of UEV domains is known to those of skill in the art. (See, e.g., Owen Pornillos et al., Structure and functional interactions of the Tsg101 UEV domain, EMBO J., 21(10): 2397–2406 (2002), the entire contents of which are incorporated herein by reference). Expression Constructs [00165] Some aspects of this invention provide expression constructs for encoding a gene product or gene products that induce or facilitate the generation of ARMMs in cells harboring such a construct. In some embodiments, the expression constructs described herein encode a fusion protein as described herein, such as ARRDC1 fusion proteins and TSG101 fusion proteins. In some embodiments, the expression constructs encode an ARRDC1 protein, or variant thereof, and/or a TSG101 protein, or variant thereof. In some embodiments, overexpression of either or both gene products in a cell increases the production of ARMMs in the cell, thus turning the cell into a microvesicle producing cell. In some embodiments, such an expression construct comprises at least one restriction or recombination site that allows in-frame cloning of a protein sequence to be fused, either at the C-terminus, or at the N-terminus of the encoded ARRDC1, or variant thereof. As another example, an expression construct comprises at least one restriction or recombination site that allows in-frame cloning of a protein sequence to be fused either at the C-terminus or at the N-terminus of one or more encoded WW domains. [00166] In some embodiments, the expression construct comprises (a) a nucleotide sequence encoding an ARRDC1 protein, or variant thereof, operably linked to a heterologous promoter, and (b) a restriction site or a recombination site positioned adjacent to the ARRDC1-encoding nucleotide sequence allowing for the insertion of a nucleotide sequencing encoding a payload protein, or an RNA binding protein or RNA binding protein variant sequence, in frame with the ARRDC1-encoding nucleotide sequence. In some embodiments, the heterologous promoter may be a constitutive promoter, in some embodiments, the heterologous promoter may be an inducible promoter. Some aspects of this invention provide an expression construct comprising (a) a nucleotide sequence encoding a TSG101 protein, or variant thereof, operably linked to a heterologous promoter, and (b) a restriction site or a recombination site positioned adjacent to the TSG101-encoding nucleotide sequence allowing for the insertion of a nucleotide sequencing encoding a payload protein, or an RNA binding protein, DNA binding protein, or variant sequence thereof, in frame with the TSG101-encoding nucleotide sequence. In some embodiments, the heterologous promoter may be a constitutive promoter, in some embodiments, the heterologous promoter may be an inducible promoter. [00167] Some aspects of this invention provide an expression construct comprising (a) a nucleotide sequence encoding a WW domain, or variant thereof, operably linked to a heterologous promoter, and (b) a restriction site or a recombination site positioned adjacent to the WW domain-encoding nucleotide sequence allowing for the insertion of a payload protein or RNA binding protein, or a protein variant sequence thereof in frame with the WW domain- encoding nucleotide sequence. In some embodiments, the heterologous promoter may be a constitutive promoter, in some embodiments, the heterologous promoter may be an inducible promoter. The expression constructs may encode a payload protein, or an RNA binding protein fused to at least one WW domain. In some embodiments, the expression constructs encode a payload protein or an RNA binding protein, or variant thereof, fused to at least one WW domain, or variant thereof. Any of the expression constructs, described herein, may encode any WW domain or variant thereof. In some embodiments, the heterologous promoter may be a constitutive promoter, in some embodiments, the heterologous promoter may be an inducible promoter. [00168] The expression constructs, described herein, may comprise any nucleic acid sequence capable of encoding a WW domain or variant thereof. For example, a nucleic acid sequence encoding a WW domain or WW domain variant may be from the human ubiquitin ligase WWP1, WWP2, Nedd4-1, Nedd4-2, Smurf1, Smurf2, ITCH, NEDL1, or NEDL2. Exemplary nucleic acid sequences of WW domain containing proteins are listed below. It should be appreciated that any of the nucleic acids encoding WW domains or WW domain variants of the exemplary proteins may be used in the invention, described herein, and are not meant to be limiting. [00169] Human WWP1 nucleic acid sequence (uniprot.org/uniprot/Q9H0M0). [00170] Human WWP2 nucleic acid sequence (uniprot.org/uniprot/ O00308).
[00171] Human Nedd4-1 nucleic acid sequence (uniprot.org/uniprot/ P46934).
[00172] Human Nedd4-2 nucleic acid sequence (>gi|345478679|ref|NM_015277.5| Homo sapiens neural precursor cell expressed, developmentally down-regulated 4-like, E3 ubiquitin protein ligase (NEDD4L), transcript variant d, mRNA).
[00173] Human Smurf1 nucleic acid sequence (uniprot.org/uniprot/ Q9HCE7).
[00174] Human Smurf2 nucleic acid sequence (uniprot.org/uniprot/Q9HAU4). [00175] Human ITCH nucleic acid sequence (uniprot.org/uniprot/Q96J02). [00176] Human NEDL1 nucleic acid sequence (uniprot.org/uniprot/Q76N89). [00177] Human NEDL2 nucleic acid sequence (uniprot.org/uniprot/ Q9P2P5). [00178] Some aspects of this invention provide expression constructs that encode any of the proteins, nucleic acids, such as RNAs, or fusions thereof described herein. [00179] Nucleic acids encoding any of the proteins and/or nucleic acid (including RNA) described herein, may be in any number of nucleic acid vectors known in the art. Vectors suitable for use in the compositions and methods of the present invention include both viral and nonviral products and additional means for introducing nucleic acid(s) into cells. [00180] Expression of any of the proteins and/or nucleic acid (including RNA) described herein, may be controlled by any regulatory sequence (e.g., a promoter sequence) known in the art. Regulatory sequences, as described herein, are nucleic acid sequences that regulate the expression of a nucleic acid sequence. A regulatory or control sequence may include sequences that are responsible for expressing a particular nucleic acid or may include other sequences, such as heterologous, synthetic, or partially synthetic sequences. The sequences can be of eukaryotic, prokaryotic, or viral origin that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory or control regions may include origins of replication, RNA splice sites, introns, chimeric or hybrid introns, promoters, enhancers, transcriptional termination sequences, poly A sites, locus control regions, signal sequences that direct the polypeptide into the secretory pathways of the target cell. A heterologous regulatory region is a regulatory region not naturally associated with the expressed nucleic acid it is linked to. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences that do not occur in nature, but which are designed by one of ordinary skill in the art. Exemplary Cells Producing ARMMs Containing Payload [00181] A microvesicle-producing cell of the present invention may be a cell containing any of the expression constructs, any of the fusion proteins, or any of the payloads of molecules (e.g., biological molecules, small molecules, proteins, and nucleic acids described herein. For example, an inventive microvesicle-producing cell may contain one or more recombinant expression constructs encoding (1) an ARRDC1 protein, or PSAP (SEQ ID NO: 1) motif-containing variant thereof and (2) an RNA binding protein (e.g., a Tat protein), that is associated with the ARRDC1 protein, or PSAP (SEQ ID NO: 1) motif-containing variant thereof. In some embodiments, a microvesicle-producing cell may contain one or more recombinant expression constructs encoding (1) an ARRDC1 protein, or PSAP (SEQ ID NO: 1) motif-containing variant thereof, and (2) a payload protein, such as an RNA binding protein fused to at least one WW domain, or variant thereof, under the control of a heterologous promoter. In certain embodiments, the expression construct in the microvesicle producing cell encodes a payload protein with one or more WW domains or variants thereof. In certain embodiments, an expression construct in the microvesicle producing cell encodes an RNA that associates with (e.g., binds specifically) an RNA binding protein, for example a therapeutic RNA. [00182] Any of the expression constructs, described herein, may be stably inserted into the genome of the cell. In some embodiments, the expression construct is maintained in the cell, but not inserted into the genome of the cell. In some embodiments, the expression construct is in a vector, for example, a plasmid vector, a cosmid vector, a viral vector, or an artificial chromosome. In some embodiments, the expression construct further comprises additional sequences or elements that facilitate the maintenance and/or the replication of the expression construct in the microvesicle-producing cell, or that improve the expression of the fusion protein in the cell. Such additional sequences or elements may include, for example, an origin of replication, an antibiotic resistance cassette, a polyA sequence, and/or a transcriptional isolator. Some expression constructs suitable for the generation of microvesicle producing cells according to aspects of this invention are described elsewhere herein. Methods and reagents for the generation of additional expression constructs suitable for the generation of microvesicle producing cells according to aspects of this invention will be apparent to those of skill in the art based on the present disclosure. In some embodiments, the microvesicle producing cell is a mammalian cell, for example, a mouse cell, a rat cell, a hamster cell, a rodent cell, or a nonhuman primate cell. In some embodiments, the microvesicle producing cell is a human cell. [00183] One skilled in the art may employ conventional techniques, such as molecular or cell biology, virology, microbiology, and recombinant DNA techniques. Exemplary techniques are explained fully in the literature. For example, one may rely on the following general texts to make and use the invention: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and Sambrook et al., Third Edition (2001); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed.1985); Oligonucleotide Synthesis (M.J. Gaited.1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription and Translation Hames & Higgins, eds. (1984); Animal Cell Culture (RI. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); Gennaro et al., (eds.) Remington's Pharmaceutical Sciences, 18th edition; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (updates through 2001), Coligan et al., (eds.), Current Protocols in Immunology, John Wiley & Sons, Inc.(updates through 2001); W. Paul et al., (eds.) Fundamental Immunology, Raven Press; E.J. Murray et al., (ed.) Methods in Molecular Biology: Gene Transfer and Expression Protocols, The Humana Press Inc. (1991)(especially vol.7); and J.E. Celis et al., Cell Biology: A Laboratory Handbook, Academic Press (1994). Delivery of ARMMs Containing Payload Molecules [00184] The inventive microvesicles (e.g., ARMMs containing any of the expression constructs and/or any of the payload of molecules (e.g., therapeutic agents, biological molecules, small molecules, proteins, and nucleic acids (e.g., DNA, RNA), DNA plasmids, siRNA, shRNA, mRNA)), may optionally further comprise a targeting moiety. The targeting moiety may be used to target the delivery of ARMMs to specific cell types, resulting in the release of the contents of the ARMM into the cytoplasm of the specific targeted cell type. A targeting moiety may be a viral envelope protein, or portion thereof, that normally functions to aid viral attachment and entry into cells. The viral envelope protein may allow for the targeting of cells of the CNS. Viral envelope proteins include, but are not limited to, vesicular stomatitis virus G protein (VSV-G; Genbank Accession and Version Number: AJ318514.1) or rabies virus glycoprotein (RVG; Genbank Accession and Version Number: M38452.1). VSV-G protein, facilitates viral entry by mediating viral attachment to an LDL receptor (LDLR) or an LDLR family member present on a target cell. Subsequent to binding, the VSV-G-LDLR complex is rapidly endocytosed and proceeds to mediate the fusion of the viral envelope with the endosomal membrane. VSV-G enters the cell through partially clathrin-coated vesicles; virus-containing vesicles contain more clathrin and clathrin adaptor than conventional vesicles. VSV-G is a common coat protein for vector expression systems used to introduce genetic material into in vitro systems or animal models, mainly because of its extremely broad tropism. RVG is a trimeric and surface-exposed viral coat protein known to use the nicotinic acetylcholine receptor and the low affinity nerve growth factor receptor for viral entry. In some embodiments, a viral envelope protein (e.g., VSV-G, RVG) facilitates binding (e.g., targeting) of the ARMMs to targeted cells. [00185] A targeting moiety may selectively bind a surface antigen of the target cell. For example, the targeting moiety may be a membrane-bound immunoglobulin, an integrin, a receptor, a receptor ligand, an aptamer, a small molecule, or a variant thereof. Any number of cell surface proteins may also be included in an ARMM to facilitate the binding of an ARMM to a target cell and/or to facilitate the uptake of an ARMM into a target cell. Integrins, receptor tyrosine kinases, G-protein coupled receptors, and membrane-bound immunoglobulins suitable for use with embodiments of this invention will be apparent to those of skill in the art and the invention is not limited in this respect. For example, in some embodiments, the integrin is an α1β1, α2β1, α4β1, α5β1, α6β1, αLβ2, αMβ2, αIIbβ3, αVβ3, αVβ5, αVβ6, or a α6β4 integrin. In some embodiments, the receptor tyrosine kinase is a an EGF receptor (ErbB family), insulin receptor, PDGF receptor, FGF receptor, VEGF receptor, HGF receptor, Trk receptor, Eph receptor, AXL receptor, LTK receptor, TIE receptor, ROR receptor, DDR receptor, RET receptor, KLG receptor, RYK receptor, or MuSK receptor. In some embodiments, the G-protein coupled receptor is a rhodopsin-like receptor, the secretin receptor, metabotropic glutamate/pheromone receptor, cyclic AMP receptor, frizzled/smoothened receptor, CXCR4, CCR5, or beta-adrenergic receptor. [00186] Additional molecules, such as synthetic small molecules or natural products, can be modified to associate with an ARMM protein (e.g., TSG101 or ARRDC1) for the purpose of targeting. This association can facilitate their incorporation with ARMMs, which in turn can be used to augment delivery of ARMMs to target cells. The incorporation of a cleavable linker may be used to allow the small molecule to be released upon delivery in or to a target cell. As a non-limiting example, a small molecule can be linked to biotin, thereby allowing it to associate with an ARRDC1 protein which is fused to a streptavidin. As another non- limiting example, a small molecule can be linked to synthetic high affinity ligand that specifically binds to a mutant form of FKBP12 such as FKBP12(F36V) (Yang, W., et al., “Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket” J. Med. Chem., 43(6):1135-1142 (2000)), which will associate with an ARRDC1 protein which is fused to FKBP12(F36V). The association of the small molecule to an ARMM protein (e.g., TSG101 or ARRDC1), facilitates the loading of the small molecule into the ARRDC1-containing ARMM. [00187] Some aspects of this invention relate to the recognition that ARMMs are taken up by target cells (e.g., cells of the retina, including, retinal pigmented epithelium cells, photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, and/or ganglion cells) and ARMM uptake results in the release of the contents of the ARMM into the cytoplasm of the target cells. In some embodiments, the payload is an agent that affects a desired change in the target cell, for example, a change in cell survival, proliferation rate, a change in differentiation stage, a change in cell identity, a change in chromatin state, a change in the transcription rate of one or more genes, a change in the transcriptional profile, or a post- transcriptional change in gene compression of the target cell, and the like. It will be understood by those of skill in the art, that the agent to be delivered will be chosen according to the desired effect in the target cell (e.g., a base editor). [00188] In some embodiments, cells from a subject are obtained and a payload is delivered to the cells by a system or method provided herein ex vivo. In some embodiments, the treated cells are selected for those cells in which a desired gene is expressed or repressed. In some embodiments, treated cells carrying a desired payload are returned to the subject they were obtained from. [00189] In some embodiments, the ARMMs further comprise a detectable label. In certain embodiments, detectably labeled ARMMs allow for the labeling of target cells without genetic manipulation. Detectable labels suitable for direct delivery to target cells are known in the art, and include, but are not limited to, fluorescent proteins, fluorescent dyes, membrane-bound dyes, and enzymes, for example, membrane-bound or cytosolic enzymes, catalyzing the reaction resulting in a detectable reaction product. Detectable labels suitable according to some aspects of this invention further include membrane-bound antigens, for example, membrane-bound ligands that can be detected with commonly available antibodies or antigen binding agents. Detectably labeled ARRMs find use in various diagnostic and analytical methods and applications. [00190] In some embodiments, ARMMs are provided that comprise a payload RNA that encodes a transcription factor, a transcriptional repressor, a fluorescent protein, a kinase, a phosphatase, a protease, a ligase, a chromatin modulator, a recombinase, and the like. In some embodiments, ARMMs are provided that comprise a payload RNA that inhibits the expression of a transcription factor, a transcriptional repressor, a fluorescent protein, a kinase, a phosphatase, a protease, a ligase, a chromatin modulator, or a recombinase. In some embodiments, the payload RNA is a therapeutic RNA. In some embodiments, the payload RNA is an RNA that affects a change in the state or identity of a target cell. For example, in some embodiments, the payload RNA encodes a reprogramming factor. Suitable transcription factors, transcriptional repressors, fluorescent proteins, kinases, phosphatases, proteases, ligases, chromatin modulators, recombinases, and reprogramming factors may be encoded by a payload RNA that is associated with a binding RNA to facilitate their incorporation into ARMMs and their function may be tested by any methods that are known to those skilled in the art, and the invention is not limited in this respect. [00191] Methods for isolating the ARMMs described herein are also provided. One exemplary method includes employing conventional techniques of collecting culture medium, or supernatant, from a cell culture comprising microvesicle-producing cells. In some embodiments, the cell culture comprises cells obtained from a subject, for example, cells suspected to exhibit a pathological phenotype, for example, a hyperproliferative phenotype. In some embodiments, the cell culture comprises genetically engineered cells producing ARMMs, for example, cells expressing a recombinant protein, for example, a recombinant ARRDC1 or TSG101 protein, such as an ARRDC1 or TSG101 protein, optionally fused to an RNA binding protein (e.g., a Tat protein) or variant thereof. In some embodiments, the supernatant is pre-cleared of cellular debris by centrifugation, for example, by two consecutive centrifugations of increasing G value (e.g., 500G and 2000G). In some embodiments, the method comprises passing the supernatant through a 0.2 µm filter, eliminating all large pieces of cell debris and whole cells. In some embodiments, the supernatant is subjected to ultracentrifugation, for example, at 120,000G for 2 hours, depending on the volume of centrifugate. The pellet obtained comprises microvesicles. In some embodiments, exosomes are depleted from the microvesicle pellet by staining and/or sorting (e.g., by FACS or MACS) using an exosome marker as described herein. Isolated or enriched ARMMs can be suspended in culture media or a suitable buffer, as described herein. Methods of ARMMs-Mediated Delivery of Payload to Cells [00192] Some aspects of this invention provide a method of delivering an agent (e.g., a therapeutic agent or agents) to a target cell of the eye. The target cell can be contacted with an ARMM in different ways. For example, a target cell may be contacted directly with an ARMM as described herein, or with an isolated ARMM from a microvesicle producing cell. The contacting can be done in vitro by administering the ARMM to the target cell in a culture dish, or in vivo by administering the ARMM to a subject. In some embodiments, the ARMMs are produced from cells obtained from a subject. In some embodiments, ARMMs that are produced from a cell obtained from a particular subject are administered to the same subject. Conversely, in some other embodiments, ARMMs produced from a cell that was obtained from the subject are administered to a different subject. As one example, a cell may be obtained from a subject and engineered to express one or more of the constructs provided herein (e.g., engineered to express a payload RNA associated with a binding RNA, an ARRDC1 protein, an ARRDC1 protein fused to an RNA binding protein, an RNA binding protein fused to a WW domain, a Cas9 protein, a base editor, guide and regulator sequences, and the like). [00193] Alternatively, a target cell can be contacted with a microvesicle producing cell as described herein, for example, contacted in vitro by co-culturing the target cell and the microvesicle producing cell, or contacted in vivo by administering a microvesicle producing cell to a subject harboring the target cell. Accordingly, the method may include contacting the target cell with a microvesicle, for example, an ARMM containing any of the payloads to be delivered, as described herein. The target cell may be contacted with a microvesicle- producing cell, as described herein, or with an isolated microvesicle that has a lipid bilayer, an ARRDC1 protein or variant thereof, a payload (e.g., therapeutic agent(s)), and optionally a viral envelope protein. [00194] It should be appreciated that the target cell may be of any origin, for example, from an organism. In some embodiments, the target cell is a mammalian cell. Some non- limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, a hamster cell, a rodent cell, and a nonhuman primate cell. In some embodiments, the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type, and preferentially, cells of the eye. In other cases, the target cell may be any differentiated cell type found in a subject. In some embodiments, the target cell is a cell in vitro, and the method includes administering the microvesicle to the cell in vitro, or co-culturing the target cell with the microvesicle-producing cell in vitro. In some embodiments, the target cell is a cell in a subject, and the method comprises administering the microvesicle or the microvesicle-producing cell to the subject. In some embodiments, the subject is a mammalian subject, for example, a rodent, a mouse, a rat, a hamster, or a non-human primate. In some embodiments, the subject is a human subject. [00195] In some embodiments, the target cell is a pathological cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the microvesicle is associated with a binding agent that selectively binds an antigen on the surface of the target cell. In some embodiments, the compositions and methods of the present invention comprise one or more targeting ligands that associate (e.g., bind) to one or more targeting receptors. In some embodiments, the antigen of the target cell is a cell surface antigen. In some embodiments, the binding agent is a membrane-bound immunoglobulin, an integrin, a receptor, a receptor ligand, or a lectin, among other suitable candidate molecules and moieties. Suitable surface antigens of target cells (e.g., cells of the eye) are known to those of skill in the art as are suitable binding agents that specifically bind such antigens. Methods for producing membrane-bound binding agents, for example, membrane-bound immunoglobulins, membrane-bound antibodies, or antibody fragments that specifically bind a surface antigen expressed on the surface of cells, are also known to those of skill in the art. The choice of the binding agent will depend, of course, on the identity or the type of target cell. Cell surface antigens specifically expressed on various types of optical tissues and cells that can be targeted by ARMMs comprising membrane-bound binding agents will be apparent to those of skill in the art. It will be appreciated that the present invention is not limited in this respect. EXAMPLES Example 1 ARMMs Production [00196] Expi293 TM cells (Thermo Fisher Scientific, Waltham, MA) were seeded in Expi293 TM expression medium (Thermo Fisher Scientific) at a density of 36/ml in 10ml. For each 10ml of cells, 5µg of ARRDC1-Cre plasmid (SEQ ID NO: 48) and 5µg of VSVG plasmid (SEQ ID NO: 49), both below, were mixed with 600µl OptiMEM TM (Thermo Fisher Scientific) for less than five (5) minutes.33µl of expifectamine was mixed with 567µl of OptiMEM TM and then mixed with DNA and OptiMEM TM mixture. Samples were left at room temperature for 10 minutes and added to Expi TM cells for about 16-18 hours. After the incubation the media was changed the following morning and cells were centrifuged at 500 x g for five (5) minutes and reseeded in a final volume of 40ml medium. Cells were incubated for an additional 48 hours. [00197] After the 48 hour incubation, cells were placed in a 50ml centrifuge tubes and centrifuged for five (5) minutes at 500 x g. The supernatant was removed and centrifuged again at 2000 x g for 10 minutes. The supernatant was put through 0.22µM vacuum filter. The supernatant was placed in 38.5ml ultracentrifuge tubes (Beckman-Coulter Life Sciences, Brea, CA) and spun at 174,900 x g for two (2) hours at 4°C. The supernatant was removed, and the pellet was resuspended in PBS and quantified by nanoparticle tracking analysis. Nanoparticle Tracking Analysis [00198] ARMMs were analyzed and quantitated by the ZetaView ® instrument (Particle Metrix GmbH, Inning am Ammersee, Germany). Samples containing vesicles were diluted with phosphate-buffered saline (PBS). The samples were subject to nanoparticle tracking analysis after dilution. Example 2 Evaluation in Subretinal Injection of ARMMs into Göttingen Minipigs [00199] Briefly, ocular biodistribution of ARMMs was evaluated in a Göttingen minipig model via subretinal delivery. Adult Göttingen minipigs were subretinally injected with ARMMs that were loaded with green fluorescent protein (ARRDC1-GFP-ARMMs). (SEQ ID NO: 50) and SEQ ID NO: 51). The dose was extrapolated from previously conducted mouse subretinal studies and was equivalent to a potential human dose, given the comparable size, physiology, and anatomy of pig and human eyes. For histological and immunohistochemical assessment, a time-course analysis was conducted by harvesting the eyes at 6 hours, 12 hours, and 24 hours after subretinal injection of ARMMs. GFP staining was used as a proxy for ARMMs uptake. Co-staining of GFP with the following cell type- specific markers were examined: RPE65 for Retinal Pigment Epithelium (RPE) and Rhodopsin for Rod and L/M Opsin for Cone photoreceptors, respectively. Wide-field color fundus imaging and confocal scanning laser ophthalmoscopy (cSLO) imaging was also taken as in-life readouts for the GFP signal. Eyes from uninjected animals were used as a negative control for GFP immunohistochemistry. The eye sections were assessed for colocalization of GFP with the cell type-specific markers and histopathological examination. Detailed data of GFP staining in ocular structures are presented as a function of time. Surgical Dosing Procedures [00200] Test animals were anesthetized and placed in lateral recumbency. Topical Proparacaine was applied to the eye. The conjunctival fornices were flushed with a 1:50 dilution of betadine solution/saline and the eyelid margins swabbed with undiluted 5% betadine solution. One eye was draped and a wire eyelid speculum is placed. A lateral canthotomy was performed using Stevens tenotomy scissors. A caliper was used to mark spots approximately 3 mm posterior to the limbus on the superotemporal and inferotemporal sclera. Bipolar cautery was used to cauterize the sclera under the marked spots, followed by topical application of undiluted 5% betadine solution. Scleral fixation forceps were used to fix the globe position while a microvitreoretinal blade with a valved cannula being inserted at each marked spot, pushed through the conjunctiva and sclera, and advanced into the vitreous humor. The trocar was positioned to face the posterior axis of the globe, and retracted to leave the scleral port in place. A 31 g needle was inserted tangentially through the limbus and into the anterior chamber to remove 100 µL aqueous humor. A direct contact surgical lens was placed on the cornea with sterile coupling gel. An endoilluminator probe was inserted through one of the scleral ports to facilitate direct visualization of the posterior segment through the microscope. A subretinal injection cannula was inserted through the second port and advanced into the mid-vitreous. The small diameter injection cannula was then advanced until it contacted the retinal surface. The ARMMs particles (i.e., ARRDC1-GFP) are then slowly delivered to induce and fill subretinal blebs. Upon appropriate bleb formation being visualized, the injection was continued to deliver the entire dose volume (e.g., 50 µL/bleb) into the subretinal space. If bleb formation was not visualized, the small diameter injection cannula was repositioned and the injection was attempted again at the same location or an alternative location depending on the surgeon’s preference. Subretinal images were taken were taken to capture the location of blebs. [00201] Once the injection dose(s) were delivered, the injection cannula and endoilluminator probe were removed from the scleral ports, and the contact lens was removed from the cornea. The scleral ports were then removed. The lateral canthotomy site was closed using a 7-0 Vicryl suture. Any timed functions are based on the last injection of the last eye dosed. An identical injection procedure was performed on the contralateral eye. [00202] In one embodiment illustrating the techniques in this Example, four female and four male naïve Göttingen minipig test animals (Marshall Bioresources, North Rose, New York), at least four months of age at the time of study, were divided equally into four group of two were dosed subrationally with ARRDC1-GFP-ARMMS according to Table 1 below. Table 1 Subretinal Injection of ARRDC1-GFP-ARMMs into Göttingen Minipigs [00203] Pre-operative in-life procedures were performed in accordance with standard operating procedures for the test facility. Test animals were housed, fed, watered, identified, and handled according to standard USDA protocols and methods before and during the study. Postoperative Procedures [00204] Post-operative procedures were conducted in accordance with standard operating procedures for the test facility. Incision site checks and pain scoring were not required following this minimally invasive procedure. Ophthalmology endpoints were interpreted by an ophthalmologist. [00205] The ophthalmoscopic examinations were conducted by an ophthalmologist via indirect ophthalmoscopy and slit-lamp biomicroscopy. Uveitis Scoring was performed, at the time of eye examinations. Wide-field, color fundus imaging was conducted on anesthetized animals using the Clarity RetCam Shuttle (MediMark, Grenoble, France). Confocal scanning laser ophthalmoscopy (cSLO) imaging with green fluorescent protein (GFP) imaging was conducted on anesthetized animals using the Spectralis HRA/OCT System (Heidelberg Engineering Inc., Franklin, MA). Terminal Procedures [00206] Euthanasia will be by euthanasia solution administration, under sedation, if necessary, followed by a facility approved final procedure if required. Terminal procedures are summarized in Table 2 below. Table 2 Terminal Procedures [00207] “Histology Processing” comprised embedding in paraffin, sectioning, mounting on glass slides, and staining with hematoxylin and eosin. [00208] Samples from control eyes (i.e., Group 1) were collected separately from those administered test article. Eyes from all animals were collected for ocular histology. Following termination, eyes were permanently marked for orientation at the 12 o’clock position. Eyes with proximal optic nerves were enucleated. After enucleation, orientation was remarked. The right and left eyes were labelled separately and stored in Davidson’s fixative for 24-48 hours and subsequently stored in 70% ethanol for up to an additional 72 hours. [00209] Ocular histology was conducted on trim eyes with a single cut sagittal to the optic nerve. Standardly the left eye was trimmed with a single cut sagittal at the optic nerve at a plane that includes the optic disc. Standardly the right eye was trimmed with a single cut superior to the optic nerve at a plane that includes both optic disc. The halved globe containing the injection blebs were submitted with the cut side down in the cassette. The lens and a transverse section of the optic nerve were submitted in a separate block. The eyes were step sectioned as necessary to include sections through the subretinal injection blebs. Sections from each block were collected as follows: Level 1 (rough into the eye until the optic disc is exposed and captured (Booler HS, et al., “Scientific and Regulatory Policy Committee Points to Consider: Fixation, Trimming, and Sectioning of Nonrodent Eyes and Ocular Tissues for Examination in Ocular and General Toxicity Studies. Toxicol Pathol., 50(2):235-251 [2022])); Level 2 (sections taken approximately 500 microns past Level 1); Level 3 (sections taken approximately 500 microns past Level 2); Level 4 (sections taken approximately 500 microns past Level 3); Level 5 (sections taken approximately 500 microns past Level 4); Level 6 (rough the second block to obtain a full face of the tissue, collected after full exposure); Level 7 (sections taken approximately 500 microns past Level 6); Level 8 (sections taken approximately 500 microns past Level 7); Level 9 (sections taken approximately 500 microns past Level 8); and Level 10 (sections taken approximately 500 microns past Level 9). Each level consists of one hematoxylin and eosin stained and eight unstained slides. The block only containing the lens and optic nerve has only hematoxylin and eosin collected initially. [00210] Collected tissues were then evaluated histopathologically by light microscopy using stains and other molecules to highlight moieties on cells and tissues of interest (e.g., RPE65 (retinal pigmented epithelium cells), L/M opsin (cone outer segments), Rhodopsin (rod outer segments), and the like) using standard procedures and protocols. [00211] Additionally, tissues were selected for immunohistochemical analysis. These tissues were preserved in Davidson’s fixative, or other designated fixatives for 24-48 hours. Tissues may also be held in 70% ethanol for up to an additional 72 hours prior to processing to paraffin. [00212] The results from this Example are shown in FIG.2 and FIG.3. Example 3 Evaluation in Subretinal Injection of ARMMs into Nonhuman Primates [00213] In this example, nonhuman primates, specifically, St. Kitt’s African Green monkeys (Chlorocebus sabaeus) are selected using baseline health assessment techniques including, complete blood count (CBC), general well-being, and ocular health by tonometry, slit lamp biomicroscopy, fundoscopy, color and fluorescence fundus imaging for subsequent use in subretinal injection administration study with ARRDC1-mCherry-ARMMs (SEQ ID NO: 52 and SEQ ID NO: 53). Baseline screening and all subsequent procedures were performed under sedation with intramuscular ketamine (8 mg/kg) and xylazine (1.6 mg/kg) to effect, and pupil dilation with topical 10% phenylephrine, 1% tropicamide and/or 1% cyclopentolate. Animals with normal findings were enrolled in the administration study and assigned to treatment groups as described in Table 3. Table 3 Nonhuman Primate Groups
Surgical Dosing Procedures [00214] Topical 1% atropine gel was administered to each eye one day prior to subretinal dosing. This procedure was followed with topical 10% phenylephrine, 1% tropicamide and/or 1% cyclopentolate on the day of dosing in eyes not fully dilated. Test animals received one subretinal injection in both eyes oculus uterque (OU) in accordance with the treatment assignments in Table 3. After eye speculum placement, a drop of proparacaine hydrochloride 0.5% was administered. After 30 seconds, a 5% Betadine ® solution was administered, followed by a sterile saline rinse. A sterile eye drape and lid speculum was then placed on the respective animals. A 25-gauge vitrectomy port, Alcon ® Surgery Valved Entry System 1-CT (Alcon Surgery Inc., Fort Worth, TX) was placed via port introducer device at the level of the ora serrata in the superotemporal quadrant. A second vitrectomy port was placed at the level of the ora serrata in the inferotemporal quadrant. After vitrectomy port placement, a contact vitrectomy lens was placed and centered on the cornea, employing 0.9% saline as a coupling agent. With the surgeon positioned temporally, a 25-gauge light pipe was inserted through the vitrectomy port on the left into the vitreous cavity for intraocular illumination, keeping the tip in the anterior vitreous. A subretinal cannula, MedOne Surgical 25/38g, part number 3237, (MedOne Surgical Inc., Sarasota, FL) attached to a MedOne microfluid injector, part number 3275, coupled to an Alcon Constellation Vision System was introduced through the second vitrectomy port. The 38-gauge flexible microtip was advanced to gently touch the retinal surface, targeting a point in the superotemporal region within approximately 2 disc diameters superior to the fovea. Upon observing slight blanching of the retinal surface at the point of contact, test article was injected at a pressure sufficient to elevate the bleb then adjusted to maintain within 4-6 mmHg. Pressure threshold was set at 14 mmHg. When an initial bleb raised, a target volume (Table 1) of test article was administered. The surgical instruments were removed following injection and the sclerotomies self-sealed. A topical antibiotic ointment was instilled in the eye after post-operative fundus imaging to document subretinal bleb location and dimension. Postoperative Procedures [00215] Intraocular pressure (IOP) was measured OU using a Tonovet Plus tonometer (iCare Inc., Raleigh, NC) set to the cat (c) calibration setting. Three measures were taken from each eye and the mean IOP calculated. [00216] Eyes oculus uterque were examined by slit lamp biomicroscopy and retinoscopy using a 90-diopter lens. Scoring was applied to qualitative clinical ophthalmic findings using a nonhuman primate ophthalmic scoring system and summary score derived from exam components. [00217] Color anterior and fundus imaging and fluorescent fundus imaging was used to detect GFP expression performed OU with a 50° field of view centered on the macula with additional peripheral images acquired in each quadrant using a Topcon TRC-50EX (Topcon Medical Systems, Inc., Paramus, NJ) retinal camera with Canon 6D digital imaging hardware and New Vision Fundus Image Analysis System software (URAL Telekom, Antalya, Turkey). Color fundus photos were captured with shutter speed (Tv) 1/25 sec, ISO 400 and flash 18. Monochromatic and color fluorescent images were captured with exciter and barrier filters engaged (587 nm exciter/610 nm barrier filter), Tv 1/5 sec, ISO 3200 and flash 300. Fluorescence photographs were evaluated using a scoring system to define extent of mCherry expression with quantitative analysis applied as appropriate where a score of 0 = absent; 1 = trace; 2 = slight; 3 = moderate; 4 = bright and 5 = intense in the areas where bleb formation is seen post-dosing, foveal, peripheral, and perivascular regions of the eye. [00218] The head, torso, limbs, and integument of each test animal were evaluated, auscultation performed, and vitals assessed, including temperature, and heart and respiratory rate measured manually over a 15 second interval. [00219] 0.5 mL blood, was transferred to K2EDTA lavender top vacutainers, gently inverted several times and retained on ice for complete blood count with differential analysis on an Abaxis VetScan HM5 hematology system (Abaxis, Inc., Union City, CA). Terminal Procedures [00220] Test animals were euthanized at study terminus with ketamine (8 mg/kg IM) and xylazine (1.6 mg/kg IM) followed by sodium pentobarbital (100 mg/kg) administered intravascularly over a period of ~10 seconds to effect. After loss of corneal reflex, and prior to sample collection, animals were exsanguinated by incising the caudal vena cava followed by immediate perforation of the diaphragm relieving negative intrathoracic pressure. [00221] Post-mortem veterinary examination of organs including external features of the carcass, external body orifices, abdominal, thoracic, and cranial cavities, organs, and tissues were performed to identify and document any gross abnormality or pathology. [00222] After placing a suture in the limbus at the 12 o’clock position both globes oculus uterque were enucleated, excess orbital tissue trimmed, immersion fixed in Davidson’s for 24 hours before being transferred to phosphate buffered saline with 0.05% sodium azide. [00223] Fixed globes were embedded in paraffin and sectioned in twenty stepped horizontal sections spanning peripheral and macular retinal regions and stained with hematoxylin and eosin for analysis by a board-certified veterinary pathologist. Globes were trimmed in a sagittal fashion in three sections prior to processing, with the mid-section encompassing the pupil-optic nerve. [00224] Ocular specimens were sectioned with a microtome as follows: nasal and temporal calottes, and optic nerve three levels with a minimum of 80 µm between levels. One slide was collected at each level. The pupil-optic nerve was sectioned in five levels with a minimum of 80 µm in between each level, spanning macular and peripheral retinal regions with ten slides collected at each level. One slide from each level will be stained with hematoxylin and eosin. [00225] A fit for purpose, monoplex, immunofluorescence assay for mCherry was optimized for specificity and sensitivity with clear signal:noise differentiation using existing tissue samples. One slide from each eye pupil-optic nerve section was selected for mCherry/cell marker duplex immunofluorescence. All stains and assays were performed according to routine laboratory methods and microscopically quality checked. [00226] Digital whole slide images of all stained glass microscope slides were generated using a 3DHistech P150 digital slide scanner (3DHISTECH Ltd, Budapest, Hungary). Slides were scanned using the 20x objective (49x native magnification) and quality checked to ensure complete image capture and lack of significant scanning artifacts. [00227] The results from this Example are shown in FIG.4 and FIG.5. References [00228] All publications, patents and sequence database entries mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. Equivalents and Scope [00229] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims. [00230] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all the group members are present in, employed in, or otherwise relevant to a given product or process. [00231] Additionally, although the terms “first,” “second,” etc., may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts/steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise. [00232] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. [00233] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus, for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc. [00234] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range. [00235] In addition, it is to be understood that any embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.
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