ARRDC1-mediated microvesicle-based delivery of therapeutic agents to ocular cells and tissues

Abstract

The present invention provides methods, systems, and compositions for ARMM-mediated delivery of molecules of interest (e.g., therapeutic agents) to ocular cells and tissues. The present invention further relates to compositions and methods for producing, testing, and administering ARRDC1-mediated microvesicles ("ARMM") to internal structures of the eye. More particularly, the present invention provides compositions and methods for producing, testing, and administering ARMM particles containing one or more therapeutic agents (e.g., biomolecules, 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)). Methods of administering therapeutic agents associated with ARMM are also provided, including, but not limited to, methods of treating or contacting ocular cells and tissues with one or more dosing regimens. In particular, the present invention provides methods of administering therapeutic agents via ARMM to cells and tissues, including the retina, or to the subretinal space. Additionally, the present invention relates to methods for producing (eg, culturing, clarifying, isolating, and concentrating) the compositions of the present invention from stable producer cell lines and from cell cultures.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of U.S. Provisional Patent Application No. 63 / 412,161, filed September 30, 2022, U.S. Provisional Patent Application No. 63 / 533,841, filed August 21, 2023, and U.S. Provisional Patent Application No. 63 / 538,152, filed September 13, 2023, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present invention provides methods, systems, and compositions for ARMM-mediated delivery of molecules of interest (e.g., therapeutic agents) to ocular cells and tissues. The present invention further relates to compositions and methods for producing, testing, and administering ARRDC1-mediated microvesicles ("ARMM") to internal structures of the eye. More particularly, the present invention provides compositions and methods for producing, testing, and administering ARMM particles containing one or more therapeutic agents (e.g., biomolecules, 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)). Methods of administering therapeutic agents associated with ARMM are also provided, including, but not limited to, methods of treating or contacting ocular cells and tissues with one or more dosing regimens. In particular, the present invention provides methods of administering therapeutic agents via ARMM to cells and tissues, including the retina, or to the subretinal space. Additionally, the present invention relates to methods for producing (eg, culturing, clarifying, isolating, and concentrating) the compositions of the present invention from stable producer cell lines and from cell cultures. [Background technology]

[0003] Vision is unarguably a crucial human sense. It is perhaps the most important, based on the part of the brain dedicated to processing visual information. Against this background, it is important to consider that approximately 12 million people aged 40 or older in the United States have visual impairment, of which approximately 1 million are blind, 3 million have corrected visual impairment, and 8 million have visual impairment due to uncorrected refractive error. (Flaxman AD, et al., “Prevalence of visual acuity loss or blindness in the US,” JAMA Ophthalmology, 139(7):717-723 (2021)). More specifically, statistics from 2012 in the United States indicate that approximately 4.2 million people aged 40 or older currently suffer from uncorrectable visual impairment (i.e., best-corrected visual acuity in the better eye ≤ 20 / 40), of which 1.02 million are blind (i.e., best-corrected visual acuity in the better eye ≤ 20 / 200). The number of people projected to become blind will more than double to approximately 8.96 million by 2050 due to the increasing prevalence of diabetes and other chronic diseases and the rapidly aging population.

[0004] Additionally, approximately 6.8% of children under the age of 18 in the United States have been diagnosed with an eye and vision condition. Nearly 3 percent of children under the age of 18 in the United States are blind or have a visual impairment, defined as difficulty seeing even with glasses or contact lenses. Visual impairment in the United States is one of the top 10 disabilities among adults aged 18 and over and one of the most prevalent disabling conditions among children.

[0005] Over 70% of survey respondents to the U.S. National Eye Health Education Program ("NEHEP") 2005 Public Knowledge, Attitudes, and Practices Survey indicated that a decline in their vision would have the greatest impact on their daily lives. However, less than 11% of respondents were aware that there are no widely accepted early warning signs of glaucoma and diabetic retinopathy.

[0006] Poor vision causes substantial social and economic harm to millions of people, including severe suffering, disability, lost productivity, and impaired quality of life. The annual economic impact of major vision problems among adults aged 40 and over is estimated to exceed $145 billion.

[0007] Although the causes of vision-related diseases and blindness are numerous and varied, many ophthalmic diseases are now known or suspected to be the result of one or more genetic abnormalities. Research has shown that approximately 10 percent of the 10,000 suspected human genetic disorders are believed to have ocular manifestations. Genetic eye diseases can be inherited according to various genetic patterns, including autosomal dominant, autosomal recessive, X-linked dominant (rarely) or recessive, multifactorial, and cytoplasmic inheritance. Research into the molecular basis of ophthalmic diseases has increased in recent years, resulting in many important discoveries in molecular ophthalmology and the characterization of the genetic basis of many ophthalmic disorders. Research on retinal diseases has revealed many genes and signaling pathways that are promising targets for gene therapy or other treatments.

[0008] Many of these discoveries have led to more general advances in molecular genetics and the art's understanding of the molecular mechanisms of certain non-ophthalmological diseases. For example, the identification and cloning of the retinoblastoma gene (RB1) led to the discovery of a tumor suppressor gene. In another example, research into the causes of retinitis pigmentosa led to the identification of point mutations that encode altered, retinal-degrading photoreceptor proteins, providing promising targets for future therapies.

[0009] Furthermore, some of these discoveries are incorporated into gene therapy attempts aimed at improving eye diseases.For example, in gene replacement, the so-called classical gene therapy technique, functional copies of genes are usually delivered to target cells by non-integrating adeno-associated virus (" AAV ") vectors to supply missing genes or restore defective gene function.Ideally, gene replacement strategies are preferably utilized to combat autosomal recessive and X-linked genetic diseases (for example, certain types of hereditary retinal dystrophy (" IRD ")).

[0010] Briefly, IRDs can result from one or more of a wide range of genetic mutations. In fact, approximately 250 genetic mutations are known to be involved in various types of IRDs. Genes responsible for IRDs are primarily expressed in photoreceptor cells and, to a lesser extent, in retinal pigment epithelial cells ("RPE"). Many IRDs result from dysregulation of genes encoding structural proteins. Expression of these genes must be carefully regulated to maintain the appropriate ratio with other cellular components to restore function. Design of successful gene replacement compositions and methods therefore requires careful consideration of intended expression in target cells.

[0011] To date, the only existing successful application of AAV-mediated gene replacement, the drug voletigene neparvovec-rzyl, has been approved in the United States and Europe for the treatment of Leber congenital amaurosis type 2 ("LCA2"), which is considered the most severe type of IRD.

[0012] Despite the success of this AAV-mediated gene replacement therapy in ameliorating LCA2 disease, the general use of gene replacement for other types of IRDs and other genetic retinal disorders remains challenging. One major difficulty relates to the effectiveness of gene replacement strategies to counter gain-of-function mutations in autosomal dominant IRDs compared with related loss-of-function disorders (e.g., LCA2), because gain-of-function mutations result in continued expression of the pathogenic mutant protein.

[0013] Furthermore, the limited packaging capacity of AAV-mediated vectors (approximately 4.5 kb) can limit gene replacement therapies that utilize these vectors. Others have investigated alternative packaging systems for gene replacement, such as lentiviruses, non-viral vectors (e.g., plasmids), DNA nanoparticles, delivery of antisense oligonucleotides, and nucleic acid sequences.

[0014] Despite advances in understanding the molecular basis of many eye diseases, there remains a need in the art for additional therapeutic agents and associated delivery platforms for treating eye diseases, particularly diseases of the retina, and more particularly diseases affecting its ganglion cell layer, photoreceptors, and retinal pigment epithelium ("RPE"). Yet, the search for treatments for eye diseases can be difficult given the unique anatomical structure and natural defense mechanisms of the eye (e.g., the lack of efferent lymphatic vessels, the incomplete immune privilege of the eye, the tight blood-ocular barrier in retinal cells, and the like). Summary of the Invention

[0015] The present invention relates to the discovery that molecules, e.g., proteins and nucleic acids, including ribonucleic acid (RNA), and small molecules, can be loaded into microvesicles, specifically ARRDC1-mediated microvesicles (ARMMs), for delivery to cells of the eye, specifically the retina, and more specifically ganglion cells and retinal pigment epithelial cells ("RPE").

[0016] In certain embodiments, ARMM can incorporate viral envelope proteins to enable delivery of molecules to the retina. For example, to target retinal cells (e.g., RPE cells), vesicular stomatitis virus G protein (VSV-G) or rabies virus glycoprotein (RVG) can be co-expressed and present on the surface of ARMM. These proteins usually function to assist virus attachment and virus entry into cells. For example, VSV-G mediates virus attachment to LDL receptor ("LDLR") or LDLR family members, and RVG is known to use nicotinic acetylcholine receptors and low-affinity nerve growth factor receptors for virus entry. It has also been shown that these proteins can assist ARMM attachment to cells, including retinal cells.

[0017] Furthermore, the ARMM delivery system described herein addresses many of the limitations of current delivery systems that prevent the safe and efficient delivery of proteins and nucleic acids (e.g., RNA, including both protein-coding and non-coding RNA) to the retina. Because ARMMs are derived from the endogenous budding pathway, they are unlikely to provoke a strong immune response, unlike viral delivery systems, which are known to provoke inflammatory responses. (See Sen et al., "Cellular unfolded protein response against viruses used in gene therapy," Front Microbiology, 5:250, 1-16 (2014)). Furthermore, ARMMs allow for the specific packaging of many types and classes of potentially therapeutic molecules (e.g., biomolecules, e.g., proteins or nucleic acids (e.g., DNA, plasmids, mRNA, miRNA, or shRNA), or small molecules). Although the present invention is not limited to any particular mechanism, in certain embodiments it is contemplated that antibodies or other types of targeting or directional determining molecules may be incorporated into or onto the ARMM to recognize tissue-specific markers, thereby allowing the ARMM to be delivered by fusion with or uptake by specific recipient cells and tissues.

[0018] ARMMs are microvesicles distinct from exosomes, which are generated by direct plasma membrane budding ("DPMB"), such as budding viruses. DPMBs are driven by the specific interaction of TSG101 with the tetrapeptide PSAP (SEQ ID NO: 1) motif of the arrestin domain-containing protein ARRDC1 accessory protein, which is localized to the plasma membrane via its arrestin domain. ARMMs are described in detail, for example, in PCT Application No. PCT / US2013 / 024839, entitled "Arrdc1-Mediated Microvesicles (ARMMs) and Uses Thereof," filed by Lu, Q. et al. on February 6, 2013 (published on August 15, 2013 as WO 2013 / 119602), and U.S. Patent Nos. 9,737,480, 9,816,080, and 10,260,055; and PCT Publication No. WO 2018 / 067546, the entire contents of which are hereby incorporated by reference in their entireties. ARRDC1 / TSG101 interaction results in the translocation of TSG101 from the endosome to the plasma membrane, mediating the release of microvesicles containing TSG101, ARRDC1, and other cellular components, as well as the molecule of interest.

[0019] Naturally occurring or non-naturally occurring molecules of interest include, but are not limited to, proteins, nucleic acids, and small molecules that can be preferably linked to one or more ARMM-associated proteins (e.g., ARRDC1), or more specifically, modified to link to TSG101 or ARRDC1 or specific motifs therein. These links facilitate the incorporation of the molecule into ARMM, which can then be used to deliver the desired payload (molecule of interest) into target cells. For example, but not limited to, a payload RNA can be linked to an ARRDC1 protein fused to an RNA-binding protein such as a Tat protein (e.g., bovine TAT protein) by fusing it to a transactivation response (TAR) element. Alternatively, the payload protein can be fused to one or more WW domains that link to the PPXY (SEQ ID NO: 2) motif of ARRDC1. Linking a molecule to an ARMM-associated protein (e.g., ARRDC1) facilitates loading of the molecule into an ARMM containing ARRDC1. Alternatively, the molecule can be fused to an ARMM protein (e.g., TSG101 or ARRDC1) and the payload can be loaded into the ARMM. The molecule can be fused to the ARMM protein (e.g., TSG101 or ARRDC1) via a linker, which can be cleaved once delivered to a target cell.

[0020] For example, the ARMM delivery platform allows multiple cis-acting structural elements of mRNA to function in the context of intracellular and secreted therapeutics for target cells, including, but not limited to, (i) a 5' cap structure, (ii) a 5' untranslated region (UTR), (iii) a codon-optimized coding sequence, (iv) a 3' UTR, (v) a 3' poly-A tail consisting of a stretch of repeated adenine nucleotides, and (vi) the insertion of a cis-acting zipcode element within the RNA transcript that is recognized by specific RNA-binding proteins, resulting in specific cellular localization (e.g., to neuronal synapses). (See, e.g., Chin A., Lecuyer E., "RNA localization: Making its way to the center stage," Biochim. Biophys. Acta. Gen Subj., 1861(11 Pt B):2956-2970 (2017)).

[0021] As another example, the delivery platform for ARMM allows for the targeting of multiple classes of protein and mRNA-based therapeutics to target cells (e.g., cells of the retina). Suitable therapeutic agents for use in the compositions and methods of the invention include, but are not limited to, transmembrane proteins, cytoplasmic proteins, nuclear proteins, mitochondrial proteins, endoplasmic reticulum proteins, Golgi apparatus proteins, peroxisomal proteins, lysosomal proteins, and secreted proteins.

[0022] Also contemplated herein in the context of therapeutic agents for vision-related disorders is the targeted expression of single-chain variable fragment (scFv) antibodies composed of a fusion protein of the variable regions of immunoglobulin heavy (VH) and light (VL) chains linked with a short linker peptide. These scFv antibodies can selectively bind to specific antigens, or they can be engineered to be multifunctional by adding a fusion protein binding domain or a nucleic acid binding domain, as in the case of bispecific scFvs. Alternatively, mRNA encoding both the VH and VL chains can be used. Furthermore, single-domain antibodies (sdAbs), consisting of one monomeric variable domain, can be delivered to ocular cells and tissues as mRNA. In further embodiments, various other truncated antibodies and functional fragments thereof can be utilized.

[0023] In the context of therapeutics for visual impairment, the targeted expression of antigenic peptides, or neoantigens, that can occur using ARMM-mediated delivery of mRNA is also contemplated. The delivered mRNA is translated by ribosomes to produce neoantigen protein chains, which are processed by proteasomes to produce neoantigens. These neoantigens can present themselves in association with other membrane-bound proteins, allowing them to be recognized by T cell receptors on T cells or other immune system cells.

[0024] In some embodiments, the present invention provides arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMMs) containing a lipid bilayer and an ARRDC1 protein, a molecule (e.g., a therapeutic agent), and optionally a viral envelope protein. In some embodiments, the viral envelope protein is vesicular stomatitis virus G (VSV-G) or rabies virus glycoprotein (RVG).

[0025] In some embodiments of the present invention, a microvesicle-producing cell is provided, which contains a recombinant expression construct encoding an ARRDC1 protein or a variant thereof under the control of a heterologous promoter, and optionally a viral envelope protein. In other embodiments, the viral envelope protein is vesicular stomatitis virus G (VSV-G) or rabies virus glycoprotein (RVG).

[0026] In some further aspects of the present invention, methods are provided for delivering molecules (e.g., one or more therapeutic agents) to a target cell type, tissue, or structure by contacting the target with microvesicles as described herein. In other aspects, the tissue and structure includes the retina. In some aspects, the tissue or structure includes elements of the retina, such as the retinal pigment epithelium and the retinal neural layer (e.g., retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, photoreceptor cells, and the like).

[0027] In some aspects of the present invention, methods are provided for treating a disorder in a patient by administering to the patient the microvesicles or microvesicle-producing cells described herein. In still other aspects, the disorder affects the function of the eye. In still other aspects, the disorder affects the function of one or more of the retina, sclera, choroid, iris, lens, cornea, vitreous humor, macula, optic nerve, and similar structures, tissues, and cells of the eye. In other aspects of the present invention, the disorder is either a gain-of-function disorder, a loss-of-function disorder, or a repeat expansion disease. In still other aspects of the present invention, the disorder results from one or more substitutions (e.g., missense or nonsense), insertions, deletions, deletion-insertions, duplications, inversions, frameshifts, or repeat expansions.

[0028] In some embodiments, the time-dependent uptake of ARMM in the neural retina is obtained.In some embodiments, the ocular-associated ARMM composition is effectively taken up by one or more of the target rod cells, cone cells, and retinal pigment epithelium.In certain other embodiments, the ocular-associated ARMM composition of the present invention does not cause harmful cytotoxicity, leukocyte or macrophage infiltration, or cell death in the cells, tissues, and structures of the recipient's eye.

[0029] Other advantages, features, and uses of the present invention will become apparent from the detailed description of certain exemplary, non-limiting embodiments, the drawings, the non-limiting, proven examples, and the claims. [Brief explanation of the drawings]

[0030] [Figure 1]Figure 1A shows a representative embodiment of functional delivery of Cre recombinase enzyme to the retinal pigment epithelium and photoreceptors after subretinal injection in Ai14 mice. More specifically, Figure 1A shows a schematic representation of Cre activity reported by a tdTomato-based expression cassette at the Rosa26 locus in Ai14 mice. Figure 1B shows a typical layer structure of the vertebrate eye, as revealed by a cross-section. As indicated, different cell types reside in specific layers. In a preferred method, the ARMM composition administered using subretinal injection is located between one or more of the retinal pigment epithelium cell layer, photoreceptor cell layer, ganglion cell layer, inner nuclear layer, and / or outer nuclear layer. Figure 1C shows representative confocal micrographs of cross-sections obtained from an approximately 2-month-old uninjected Ai14 mouse (upper panel) and a littermate (lower panel) subretinal-injected with ARMM containing Cre as a fusion protein with ARRDC1 (A1-Cre) as a payload and harvested 10 days after injection. Sections were stained with Hoechst to mark the nuclear layer and with a primary antibody against CRALBP to mark Müller glia in the neural retina and RPE cell layers. Arrows in the inset image indicate tdTomato-positive photoreceptor nuclei in the outer nuclear layer. Further detailed enlargements of the outer nuclear layer and retinal pigment epithelium are shown in the inset image. Representative images are shown, selected from sections of five independent animals from two different litter groups administered two different lots of the A1-cre test substance on separate days. (Scale: 50 microns.) [Figure 2] FIG. 1 shows the results of Example 2, in which green fluorescent protein-loaded ARMM (ARRDC1-GFP-ARMM) (SEQ ID NO: 50 and SEQ ID NO: 51) was subretinal injected into Goettingen minipigs. [Figure 3] FIG. 1 is a cross-sectional view of an eyeball obtained from an animal in Example 2. [Figure 4]FIG. 1 shows the results of Example 3, in which mcherry-loaded ARMM (ARRDC1-mCherry) (SEQ ID NO: 52 and SEQ ID NO: 53) was subretinal injected into non-human primates, specifically African green monkeys (Chlorocebus sabaeus) from St. Kitts. [Figure 5] FIG. 1 is a cross-sectional view of an eyeball obtained from an animal in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0031] definition As used herein, the term "eye" refers to the organ of vision. The eye has several components, including but not limited to the cornea, iris, pupil, lens, retina, macula, optic nerve, choroid, and vitreous body.

[0032] As used herein, "ophthalmic" may refer to the retina, fundus, optic disc, macula, iris, pupil, lens, blood vessels, vitreous, or other anatomical components associated with the eye.

[0033] As used herein, the term "retina" refers to the classically defined "neural retina," a nerve layer covering the back of the eye that contains cells (e.g., photoreceptors, amacrine cells, bipolar cells, horizontal cells, and ganglion cells) that are light-sensitive and generate nerve impulses that travel via the optic nerve to the brain, where visual images are formed. The retina receives light and converts it into neural signals. Supporting cells, such as the retinal pigment epithelium ("RPE"), which are directly involved in visual perception and provide support to the neural retina, are also included in the term "retina."

[0034] The term "central retina," as used herein, refers to the outer macula and / or inner macula and / or fovea. The term "central retinal cell type," as used herein, refers to cell types of the central retina, such as cone photoreceptors, rod photoreceptors, retinal ganglion cells, etc., and also to cells that directly support visual perception, such as the retinal pigment epithelium, which is anatomically juxtaposed to the photoreceptors of the central retina.

[0035] As used herein, the term "macula lutea (macula)" refers to the region of the central retina that has a higher relative concentration of cone photoreceptors than rod photoreceptors compared to the peripheral retina. As used herein, the term "outer macula" may also be described as "peripheral macula." As used herein, the term "inner macula" may also be described as "central macula."

[0036] As used herein, the term "fovea" refers to a small region in the central retina of a primate, identifiable as a small depression approximately equal to or less than 0.5 mm in diameter, which contains exclusively cone cells compared to the peripheral retina and macula.

[0037] As used herein, the term "subretinal space" refers to a location in the retina between photoreceptor cells and retinal pigment epithelial cells. The subretinal space may be a latent space, for example, before any subretinal injection. In some embodiments, an amount (e.g., a therapeutic amount) of the ARMM-mediated composition of the present invention can be placed in the subretinal space (e.g., via subretinal injection). In this case, the ARMM-mediated composition is "in contact with the subretinal space." Cells "in contact with the subretinal space" include cells facing the subretinal space, such as RPE and photoreceptor cells.

[0038] As used herein, the term "bleb" refers to a fluid cavity within the subretinal space of the eye. The blebs of the present invention can be created by a single fluid injection into one cavity, by multiple injections of one or more fluids into the same cavity, or by multiple injections into multiple cavities that, in varying locations, create a total fluid cavity useful for providing a therapeutic effect throughout a desired portion of the subretinal space.

[0039] As used herein, the term "optic nerve" refers to the connection between the eye and the brain. Each optic nerve is part of the second pair of cranial nerves. The optic nerve carries impulses formed by the retina to the visual cortex of the brain, where the brain interprets the impulses as an image.

[0040] As used herein, the term "choroid" refers to the vascular layer of the eye, which contains connective tissue and lies between the retinal pigment epithelium and the sclera.

[0041] The term "ARRDC1-mediated microvesicles" or "ARMM", as used herein, refers to microvesicles comprising an ARRDC1 protein or a variant thereof, and / or a TSG101 protein or a variant thereof. ARMMs are described in detail, for example, in PCT Application No. PCT / US2013 / 024839, entitled "Arrdc1-Mediated Microvesicles (ARMMs) and Uses Thereof," filed February 6, 2013 (published August 15, 2013 as WO 2013 / 119602), by Lu et al., as well as in U.S. Pat. Nos. 9,737,480, 9,816,080, 10,260,055, 10,945,954, 11,001,817, and PCT Publications WO 2018 / 067546, WO 2021 / 0662196, and WO 2021 / 252924, the entire contents of which are hereby incorporated by reference in their entireties. In some embodiments, the ARMM is released from a cell (e.g., a producer cell) and comprises a drug (payload), e.g., a nucleic acid, a protein, or a small molecule, present in the cytoplasm or associated with the cell membrane. Typical payloads include, but are not limited to, a nucleic acid, a protein, or a small molecule present in the cytoplasm or associated with the cell membrane. In some embodiments, the ARMM is released from a cell (e.g., a transgenic cell) and comprises a drug, e.g., a nucleic acid, a protein, or a small molecule, present in the cytoplasm or associated with the cell membrane. In some embodiments, the ARMM is released from a transgenic cell harboring a recombinant expression construct comprising a transgene, and the ARMM comprises a gene product, e.g., an RNA transcript and / or protein (e.g., an ARRDC1-Tat fusion protein and a TAR-payload RNA), encoded by the expression construct. In some embodiments, the ARMM is synthetically produced, e.g., by contacting a lipid bilayer with an ARRDC1 protein or a variant thereof in a cell-free system in the presence of TSG101 or a variant thereof.In other embodiments, the ARMM is synthetically produced by contacting a lipid bilayer with a HECT domain ligase and VPS4a. In some embodiments, the ARMM lacks late endosomal markers. Some of the ARMMs provided herein do not contain one or more exosome biomarkers or are negative for such markers. Exosome biomarkers are known to those skilled in the art and include, but are not limited to, CD63, Lamp-1, Lamp-2, CD9, HSPA8, GAPDH, CD81, SDCBP, PDCD6IP, ENO1, ANXA2, ACTB, YWHAZ, HSP90AA1, ANXA5, EEF1A1, YWHAE, PPIA, MSN, CFL1, ALDOA, PGK1, EEF2, ANXA1, PKM2, HLA-DRA, and YWHAB. Certain ARMMs provided herein may contain exosome biomarkers. Thus, some ARMM may be negative for one or more other exosomal biomarkers but positive for one or more different exosomal biomarkers. For example, such ARMM may be negative for CD63 and Lamp-1 but contain PGK1 or GAPDH, or may be negative for CD63, Lamp-1, CD9, and CD81 but positive for HLA-DRA. In some embodiments, ARMM contain exosomal biomarkers, but at levels lower than those found in exosomes. For example, some ARMM contain one or more exosomal biomarkers at levels less than about 1%, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 40%, or less than about 50% of the levels of such biomarkers found in exosomes. As a non-limiting example, in some embodiments, ARMM may be negative for CD63 and Lamp-1, contain CD9 at levels less than about 5% of the levels of CD9 typically found in exosomes, and be positive for ACTB. Exosome biomarkers other than those listed above are known in the art, and the invention is not limited in this respect.

[0042] The term "cargo protein," as used herein, refers to a protein that can be incorporated into an ARMM, for example, into the liquid phase of the ARMM or into the lipid bilayer of the ARMM. The term "cargo protein to be delivered" refers to any protein that can be delivered to a subject, organ, tissue, or cell via its association with or encapsulation in the ARMM. In some embodiments, the cargo protein is delivered to target cells in vitro, in vivo, or ex vivo. In some embodiments, the cargo protein to be delivered is a biologically active agent, i.e., the cargo protein to be delivered has activity in cells, organs, tissues, and / or subjects. For example, a protein that, when administered to a subject, has a biological effect on the subject, is considered to be biologically active. In certain embodiments, the cargo protein is a nuclease or a variant thereof (e.g., a Cas9 protein or a variant thereof). In certain embodiments, the nuclease can be a Cas9 nuclease, a TALE nuclease, a zinc finger nuclease, or any variant thereof. Nucleases, including Cas9 proteins and variants thereof, are described in more detail elsewhere herein. In some embodiments, the Cas9 protein or its variants are linked to nucleic acids. For example, the cargo protein can be a Cas9 protein linked to gRNA. In some embodiments, the cargo protein to be delivered is a therapeutic drug.

[0043] As used herein, the term "therapeutic agent" refers to any agent that produces a beneficial effect when administered to a subject. In some embodiments, a therapeutic agent comprises a small molecule, a protein (or peptide), one or more nucleic acids, or an agent linked to a small molecule. In some embodiments, the payload to be delivered is a diagnostic agent. In some embodiments, the agent to be delivered is a prophylactic agent. In some embodiments, the agent to be delivered is useful as an imaging agent. In some of these embodiments, the diagnostic or imaging agent is biologically active; in other embodiments, it is not biologically active. In some embodiments, a therapeutic agent comprises an agent that reduces (knocks down) the expression of one or more genes in an organism (e.g., a subject). In other embodiments, a therapeutic agent comprises an agent that inactivates or eliminates (knocks out) one or more specific genes in an organism (e.g., a subject). In some embodiments, the therapeutic agent delivered to a cell is a transcription factor, a tumor suppressor, a developmental regulator, a growth factor, a metastasis suppressor, a pro-apoptotic protein, a nuclease, or a recombinase.

[0044] As used herein, the term "therapeutic effect" refers to an outcome of treatment that is deemed desirable and advantageous. Therapeutic effects include, directly or indirectly, the arrest, reduction, or elimination of symptoms of a disease. Therapeutic effects also include, directly or indirectly, the arrest, reduction, or elimination of the progression of symptoms of a disease.

[0045] As used herein, the term "transcription factor" refers to a DNA-binding protein that regulates the transcription of DNA into RNA, for example, by activating or repressing transcription. Some transcription factors affect transcription regulation alone, while others act in concert with other proteins. Some transcription factors can both activate and repress transcription under certain conditions. Typically, transcription factors bind one or more specific target sequences that closely resemble specific consensus sequences within the regulatory region of a target gene. Transcription factors can regulate the transcription of target genes alone or in complexes with other molecules. Examples of transcription factors include, but are not limited to, Sp1, NF1, CCAAT, GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix, SREBP, p53, CREB, AP-1, Mef2, STAT, R-SMAD, NF-κB, Notch, TUBBY, and NFAT.

[0046] The term "binding RNA," as used herein, refers to a ribonucleic acid (RNA) that binds to an RNA-binding protein, e.g., any RNA-binding protein known in the art and / or described herein. In some embodiments, the binding RNA is an RNA that specifically binds to an RNA-binding protein. A binding RNA that "specifically binds" to an RNA-binding protein binds to the RNA-binding protein with greater affinity, avidity, more readily, and / or for a longer period than its binding to another protein, e.g., a protein that does not bind RNA or a protein that binds weakly to the binding RNA. In some embodiments, the binding RNA is a naturally occurring RNA or a non-naturally occurring variant thereof that binds to a specific RNA-binding protein. For example, the binding RNA can be a TAR element, a Rev response element, MS2 RNA, or any variant thereof that specifically binds an RNA-binding protein. In some embodiments, the binding RNA can be a transactivation response element (TAR element) or a variant thereof, which is an RNA stem-loop structure found at the 5' end of nascent HIV-1 transcripts and specifically binds to the transcriptional transactivator (Tat) protein. In some embodiments, the binding RNA is a Rev response element (RRE) or a variant thereof, which specifically binds to the accessory protein Rev (e.g., Rev from HIV-1). In some embodiments, the binding RNA is MS2 RNA, which specifically binds to the MS2 phage coat protein. The binding RNA of the present disclosure can be designed to specifically bind a protein (e.g., an RNA-binding protein fused to ARRDC1) and facilitate loading of the binding RNA (e.g., a binding RNA fused to a payload RNA) into ARMM.

[0047] The term "aptamer," as used herein, refers to a nucleic acid (e.g., RNA, DNA) that binds to a specific target molecule, such as an RNA-binding protein. In some embodiments, nucleic acid (e.g., DNA or RNA) aptamers are engineered to bind to various molecular targets, such as proteins, small molecules, macromolecules, metabolites, carbohydrates, metals, nucleic acids, cells, tissues, and organisms, through repeated in vitro selection, or alternatively, through SELEX (systematic evolution of ligands by exponential enrichment) methods.Methods for engineering aptamers to bind to various molecular targets, such as proteins, are known in the art and include U.S. Pat. Nos. 637,619 and 9,061,043; Shui, B., et al., “RNA aptamers that functionally interact with green fluorescent protein and its derivatives,” Nucleic Acids Res., Mar; 40(5): e39 (2012); Trujillo, UH, et al., “DNA and RNA aptamers: from tools for basic research towards therapeutic applications,” Comb. Chem. High Throughput Screen, 9(8):619-32 (2006); Srisawat, C., et al., “Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins,” RNA, 7:632-641 (2001); and Tuerk and Gold, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase,” Science, (1990), the entire contents of each of which are hereby incorporated by reference in their entireties.

[0048] The term "RNA-binding protein," as used herein, refers to a polypeptide molecule that binds to a binding RNA, e.g., any of the binding RNAs known in the art and / or described herein. In some embodiments, the RNA-binding protein is a protein that specifically binds to a binding RNA. An RNA-binding protein that "specifically binds" to a binding RNA binds to the binding RNA with greater affinity, avidity, more readily, and / or for a longer period than its binding to another RNA, e.g., a control RNA (e.g., an RNA having a random nucleic acid sequence) or an RNA that binds weakly to the RNA-binding protein. In some embodiments, the RNA-binding protein is a naturally occurring protein or a non-naturally occurring variant thereof that binds to a specific RNA. For example, in some embodiments, the RNA-binding protein can be a transcriptional transactivator (Tat) protein that specifically binds a transactivation response element (TAR element). In some embodiments, the Tat protein is derived from bovine. In some embodiments, the RNA-binding protein is a regulator of virion-expressed (Rev) protein (e.g., Rev from HIV-1) or a variant thereof that specifically binds to a Rev-response element (RRE). In some embodiments, the RNA-binding protein is a coat protein of MS2 bacteriophage that specifically binds to MS2 RNA. RNA-binding proteins (e.g., binding proteins fused to ARRDC1) useful in the present disclosure can be designed to specifically bind binding RNAs (e.g., binding RNAs fused to payload RNAs) and facilitate loading of the binding RNAs into ARMM.

[0049] The terms "payload," "payload protein," "payload nucleic acid," "payload DNA," "payload RNA," or "payload small molecule," as used herein, refer to a protein, a nucleic acid, including DNA or RNA, or a small molecule, respectively, that can be incorporated into an ARMM, for example, into the liquid phase of the ARMM or into the lipid bilayer of the ARMM. Types of payload proteins, payload nucleic acids, payload DNA, payload RNA, and payload small molecules are known in the art and include those described in U.S. Pat. No. 9,737,480, U.S. Pat. No. 9,816,080, U.S. Pat. No. 10,260,055, and PCT Publication WO 2018 / 067546, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0050] The payload can be delivered to a subject, organ, tissue, or cell via its association with or encapsulation in the ARMM. In some embodiments, the payload is delivered to target cells in vitro, in vivo, or ex vivo. In some embodiments, the payload to be delivered is a biologically active agent, i.e., the payload to be delivered has activity in cells, organs, tissues, and / or subjects. For example, a protein, a nucleic acid (e.g., DNA or RNA), or a small molecule that, when administered to a subject, produces a biological effect on the subject, is biologically active. In some embodiments, the payload to be delivered is a therapeutic drug.

[0051] The term "viral envelope protein" refers to a protein that normally functions to aid in the attachment and entry of viruses into cells. In some embodiments, viral envelope proteins can be incorporated into ARMMs to target ocular cells. Non-limiting examples of viral envelope proteins include vesicular stomatitis virus G protein (VSV-G) or rabies virus glycoprotein (RVG). VSV-G mediates viral attachment to LDL receptors (LDLRs) or LDLR family members, and RVG is known to utilize nicotinic acetylcholine receptors and low-affinity nerve growth factor receptors for viral entry.

[0052] The term "linker," as used herein, refers to a chemical moiety that links two molecules or moieties, e.g., an ARRDC1 protein and a Tat protein, a WW domain and a Tat protein, or an ARRDC1 protein and a Cas9 nuclease. Typically, the linker is located between or adjacent to two groups, molecules, or other moieties, and the linker connects them by covalently binding each other. In some embodiments, the linker comprises one amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker comprises one nucleotide (e.g., DNA or RNA) or multiple nucleotides (e.g., nucleic acids). In some embodiments, the linker is an organic molecule, a functional group, a polymer, or one or more other chemical moieties. In some embodiments, the linker is a cleavable linker, e.g., the linker comprises a bond that can be cleaved, e.g., upon exposure to UV light or a hydrolytic enzyme, e.g., a protease or esterase. In some embodiments, the linker is of any length having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids). In other embodiments, the linker is a chemical bond (eg, a covalent bond, an amide bond, a disulfide bond, an ester bond, a carbon-carbon bond, a carbon-heteroatom bond, and the like).

[0053] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, the term "animal" refers to humans of either sex at any stage of development. In some embodiments, the term "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). Animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone. In some embodiments, the animal is a transgenic non-human animal, a genetically engineered non-human animal, or a non-human clone.

[0054] As used herein, the terms "associated," "conjugated," "linked," "attached," and the like, when used in reference to two or more entities, e.g., chemical moieties, molecules, and / or ARMMs, mean that the entities are physically associated or bound to one another, either directly or through one or more additional moieties that function as linkers, to form a structure that is sufficiently stable to maintain the entities in a physically associated state under the conditions in which the structure is used, e.g., physiological conditions. ARMM microvesicles are typically associated with agents, e.g., nucleic acids, proteins, or small molecules, by mechanisms involving covalent (e.g., via an amide bond) or non-covalent associations (e.g., between ARRDC1 and the WW domain, or between the Tat protein and the TAR element). In certain embodiments, the agent (e.g., a therapeutic agent, a payload protein, a payload nucleic acid, or a payload small molecule) is covalently bound to a molecule that is non-covalently associated with a portion of the ARMM, and is fused to an ARRCD1 protein, a TSG101 protein or a variant thereof, or a protein or a variant thereof that is covalently bound to the lipid bilayer by a bond (e.g., an amide bond). In some embodiments, the association is via a linker, e.g., a cleavable linker. In some embodiments, the entity (e.g., a payload protein, a payload nucleic acid, or a payload small molecule) is associated with the ARMM by inclusion in the ARMM, e.g., by encapsulation of the molecule within the ARMM. For example, in some embodiments, a molecule (e.g., a therapeutic agent, a payload protein, a payload nucleic acid, or a payload small molecule) present in the cytoplasm of an ARMM-producing cell is associated with the ARMM by encapsulating the agent-bearing cytoplasm within the ARMM upon budding of the ARMM. Similarly, membrane proteins or other molecules associated with the plasma membrane of ARMM-producing cells can associate with the ARMM produced by the cells by encapsulating it in the membrane of the ARMM upon budding.

[0055] As used herein, the term "biologically active" refers to any characteristic of a substance that has activity in a cell, organ, tissue, and / or subject. For example, a substance is biologically active if, when administered to an organism, it produces or causes a biological effect on the organism. As an example, a payload RNA can be considered biologically active if, when administered to a subject or cell, it increases or decreases the expression of a gene product. As another example, a nuclease payload protein can be considered biologically active if, when administered to a subject, it increases or decreases the expression of a gene product.

[0056] As used herein, the term "conserved" refers to a nucleotide or amino acid residue in a polynucleotide or amino acid sequence that is present unaltered in the same position in two or more related sequences being compared. Relatively conserved nucleotides or amino acids are those that are conserved among more related sequences than are present elsewhere in the sequence. In some embodiments, two or more sequences are described as "completely conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are described as "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are described as "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other. In some embodiments, two or more sequences are described as "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are described as "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other.

[0057] The term "engineered," as used herein, refers to a protein, nucleic acid, complex, substance, or entity that is designed, produced, prepared, synthesized, and / or manufactured by humans. Thus, an engineered product is a product that does not occur in nature. In some embodiments, an engineered protein or nucleic acid is a protein or nucleic acid designed to fulfill a need or have a desired characteristic. For example, a payload RNA can be engineered to bind to ARRDC1 by fusing one or more WW domains to the Tat protein and fusing the payload RNA to a TAR element to facilitate loading of the payload RNA into ARMM. As another example, a payload RNA can be engineered to bind to ARRDC1 by fusing the Tat protein to ARRDC1 and fusing the payload RNA to a TAR element to facilitate loading of the payload RNA into ARMM. As another example, a payload protein can be engineered to bind to ARRDC1 by fusing one or more WW domains to the payload protein to facilitate loading of the payload protein into ARMM.

[0058] As used herein, the term "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA transcript from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing), (3) translation of the RNA transcript into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0059] The term "operably linked," as used herein, refers to the arrangement of sequences or regions in which the components are configured to perform their normal or intended function. Thus, regulatory or control sequences operably linked to a coding sequence can affect the expression of the coding sequence. Regulatory or control sequences need not be contiguous with the coding sequence, so long as they function to direct proper expression or polypeptide production. Thus, for example, intervening sequences that are not translated but are transcribed can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered operably linked to the coding sequence. The promoter sequence described herein is a DNA regulatory region located a short distance from the 5' end of a gene that acts as a binding site for RNA polymerase. The promoter sequence can bind RNA polymerase in a cell and / or initiate transcription of a downstream (3' direction) coding sequence. The promoter sequence can be a promoter that can initiate transcription in prokaryotes or eukaryotes. Some non-limiting examples of eukaryotic promoters include the cytomegalovirus (CMV) promoter, the chicken beta-actin (CBA) promoter, and a hybrid form of the CBA promoter (CBh).

[0060] As used herein, a "fusion protein" includes a first protein portion, such as an ARRCD1 protein or a variant thereof, or a TSG101 protein or a variant thereof, linked to a second protein portion, such as a protein to be delivered to a target cell, via a peptide bond. In certain embodiments, the fusion protein is encoded by a single fusion gene.

[0061] As used herein, the term "gene" has its meaning as understood in the art. Those skilled in the art will understand that the term "gene" can include gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will be further understood that the definition of gene includes reference to nucleic acids that do not encode proteins, but rather encode functional RNA molecules such as gRNAs, RNAi agents, ribozymes, tRNAs, etc. Note that as used in this application, the term "gene" generally refers to a portion of a nucleic acid that encodes a protein. This term may optionally encompass regulatory sequences, as will be clear to those skilled in the art from the context. This definition is not intended to exclude the application of the term "gene" to expression units that do not encode proteins, but rather to clarify that in most cases, the term as used herein refers to a nucleic acid that encodes a protein.

[0062] As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0063] As used herein, the term "green fluorescent protein" ("GFP") refers to a protein originally isolated from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light, or a derivative of such a protein (e.g., a hypersensitive protein or a wavelength-shifted protein). The amino acid sequence of wild-type GFP is:

[0064] [ka]

[0065] Proteins that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to SEQ ID NO:3 are also considered to be green fluorescent proteins.

[0066] As used herein, the term "homology" refers to the overall relationship between nucleic acids (e.g., DNA molecules and / or RNA molecules) or polypeptides. In some embodiments, nucleic acids or proteins are considered to be "homologous" to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical. In some embodiments, nucleic acids or proteins are considered to be "homologous" to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical. The term "homologous" necessarily refers to a comparison between at least two sequences (nucleotide sequences or amino acid sequences). According to the present invention, two nucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, or at least about 90% identical over at least a stretch of at least about 20 amino acids. In some embodiments, homologous nucleotide sequences are characterized by their ability to encode a stretch of at least 4-5 uniquely specified amino acids. Both the identity and the approximate spacing of these amino acids compared to each other must be considered in order for sequences to be considered homologous. For nucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode a stretch of at least 4-5 uniquely specified amino acids. According to the present invention, two protein sequences are considered to be homologous if the proteins are at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, or at least about 90% identical over at least a stretch of at least about 20 amino acids.

[0067] As used herein, the term "identity" refers to the overall relationship of nucleic acids or proteins (e.g., DNA molecules, RNA molecules, and / or polypeptides). The percent identity of two nucleic acid sequences can be calculated, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Nucleotides at corresponding nucleotide positions are compared in this manner. If a position in the first sequence contains a nucleotide identical to the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Commonly used methods for determining percent identity between sequences include, but are not limited to, the method disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference.Techniques for determining identity are codified in publicly available computer programs. Typical computer software for determining the homology between two sequences includes, but is not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, SF, et al., J. Molec. Biol., 215, 403 (1990)).

[0068] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reactor, in cell culture, in a Petri dish, etc., rather than in an organism (e.g., an animal, plant, or microorganism).

[0069] As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, plant, or microorganism).

[0070] As used herein, the term "ex vivo" refers to an event outside of a living organism, and is therefore understood to refer to a medical procedure in which an organ, cell, or tissue is removed from a living organism for treatment or procedure and then returned to the same or another living organism. In certain embodiments, ex vivo treatment involves inducing one or more genetic modifications in a patient's cells outside the patient's body to achieve a therapeutic effect therein, and then transferring (e.g., transplanting) the cells back into the patient.

[0071] As used herein, the term "isolated" refers to a substance or entity that (1) has been separated from at least some of the components with which it was associated when originally produced (in nature or in an experimental setting) and / or (2) has been produced, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which it was originally associated. In some embodiments, an isolated substance is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0072] As used herein, the term "nucleic acid" in its broadest sense refers to a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleotides. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a stretch of at least two nucleotides). In some embodiments, "nucleic acid" encompasses RNA, as well as single- and / or double-stranded DNA and / or complementary DNA (cDNA). Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that encode degenerate and / or identical amino acid sequences. Nucleotide sequences encoding proteins and / or RNA may contain introns. Nucleic acids can be purified from natural sources, produced and optionally purified using recombinant expression systems, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, e.g., analogs having chemically modified bases or sugars, backbone modifications, etc. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated. The term "nucleic acid segment" is used herein to refer to a nucleic acid sequence that is part of a longer nucleic acid sequence. In many embodiments, a nucleic acid segment contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more residues.In some embodiments, nucleic acids are selected from naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-amino The nucleic acid may be or contain adenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages). In some embodiments, the present invention is specifically directed to "unmodified nucleic acids," meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified to facilitate or achieve delivery.

[0073] As used herein, the term "protein" refers to a stretch of at least two amino acids linked together by one or more peptide bonds. A protein may contain moieties other than amino acids (e.g., may be a glycoprotein) and / or may be processed or modified in other ways. Those of skill in the art will understand that a "protein" may be an entire protein chain produced by a cell (with or without a signal sequence) or a functional portion thereof. Those of skill in the art will further understand that a protein may sometimes include two or more protein chains linked, for example, by one or more disulfide bonds or associated by other means. Proteins may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, the addition of chemical entities, such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, amide groups, terminal acetyl groups, linkers, etc., for conjugation, functionalization, or other modification (e.g., alpha amidation). In certain embodiments, protein modifications result in more stable proteins (e.g., increased in vivo half-life). These modifications include cyclization of the protein, incorporation of D-amino acids, etc. None of the modifications substantially interfere with the desired biological activity of the protein. In certain embodiments, protein modifications result in more biologically active proteins. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, amino acid analogs, and combinations thereof.

[0074] As used herein, the term "subject" or "patient" refers to any living organism to which a composition according to the present invention can be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, livestock and farm animals, pets, non-human primates, and humans). In some embodiments, the subject is a patient who has or is suspected of having a disease or disorder. In other embodiments, the subject is a healthy volunteer.

[0075] As used herein, the terms "disease" and "disorder" refer to any condition, pathological state, or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0076] As used herein, the terms "retinal disease" and "retinal disorder," as well as equivalent terms, refer to any disease, disorder, or symptom associated with the retina, including, but not limited to, retinal degenerative diseases (e.g., retinitis pigmentosa, age-related macular degeneration, etc.), retinopathies (e.g., diabetic retinopathy, proliferative retinopathy, simple retinopathy, etc.), and the like. As used herein, the compositions and methods of the present invention can prevent, treat, or inhibit the progression of diseases or disorders, including, but not limited to, retinal degenerative diseases, age-related macular degeneration, myopic maculopathy, macular dystrophy, diabetic retinopathy, uveitis, and the like. Examples of disorders or symptoms include disorders in visual acuity, contrast sensitivity, light-dark adaptation, color vision, and the like, and associated symptoms.

[0077] As used herein, "retinal degenerative disease" refers to any disease caused by degeneration of the retina, including, but not limited to, retinitis pigmentosa, MERTK retinitis pigmentosa, age-related macular degeneration, and the like.

[0078] The term "improvement of vision," as used herein, refers to the improvement or restoration of vision-related abilities (e.g., visual acuity, color vision, contrast sensitivity, light-dark adaptation, etc.). For example, with regard to visual acuity, visual acuity can be measured by a Snellen chart or E-chart, in addition to visual acuity tests using Landolt rings, and can be expressed in decimal or fractional visual acuity. These can also be expressed as log MAR visual acuity. In mice, visual acuity can be measured using visual stimuli that manipulate the spatial frequency of light and dark stripes. Visual acuity can also be determined by measuring visual evoked potentials.

[0079] As used herein, the term "treating" or "treatment" refers to partially or completely preventing, altering, and / or reducing the occurrence of one or more symptoms or characteristics of a particular disease or adverse condition. In one sense of the present invention, treatment can be performed to prevent or ameliorate a pathological condition in a subject. The therapeutic effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and achieving remission or improving prognosis. Treatment can also be administered to a subject who does not exhibit signs or symptoms of a disease, disorder, or condition, or who exhibits only early signs or symptoms of a disease or condition, with the aim of reducing the risk of developing or progressing to more severe effects associated with the disease, disorder, or condition. Treatment may prevent the onset of a disorder or a symptom of a disorder in a subject. Treatment can prevent physical impairment (e.g., decreased vision, visual acuity, low vision, blindness) caused by a disorder (e.g., inherited retinal dystrophies, Stargardt macular dystrophy, choroideremia, Usher syndrome 1b or 1c, MERTK retinitis pigmentosa, macular edema, and the like) by preventing or reversing its progression.

[0080] As used herein, the term "therapeutically effective amount" refers to an amount of an agent or payload (e.g., a nucleic acid, protein, drug, therapeutic agent, diagnostic agent, prophylactic agent, ARMM, or ARMM containing a payload protein or payload RNA) to be delivered that is sufficient to treat, ameliorate symptoms, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. A therapeutically effective amount can be initially determined from preliminary in vitro tests and / or animal models. A therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximum efficacy based on the above methods and other well-known methods is within the ability of one skilled in the art. General principles for determining therapeutic efficacy can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference in its entirety.

[0081] As used herein, the term "targeting ligand" refers to a ligand or functional portion thereof that binds to a "target receptor" that distinguishes the targeted cell from other cells. The ligand can bind by expression or selective expression on the target cell of a ligand receptor that is accessible for ligand binding.Examples of such ligands include GE11 peptide, anti-EGFR nanobodies, cRGD (cyclo(RGDfC)), KE108 peptide, octreotide, prostate-specific membrane antigen (PSMA) aptamers, TRC105, chimeric monoclonal antibodies, tumor-specific monoclonal or polyclonal antibodies (e.g., rituximab, trastuzumab, bevacizumab, alemtuzumab, panitumumab, etc., and biological equivalents and portions thereof), arginylglycylaspartic acid ("RGD"), DARPins, R NA aptamers, DNA aptamers, inteins, exteins, viral and non-viral derived cell-cell fusion proteins (“fusogens”) (e.g., VSV-G, syncytin-1, syncytin-2, HAP2, SNAREs (e.g., VAMP1, 2, 3, 4, 7, 8)), membrane proteins such as tetraspanins (“TM4SF proteins”) (e.g., TSPAN1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 6, 27, 28, 29, 30, 31, 32, and 33, and the like), peptide ligands identified from library screening, tumor-specific peptides, tumor-specific aptamers, Fab or scFv (i.e., single-chain variable region) fragments of antibodies, such as Fab fragments of antibodies against EphA2 or other proteins specifically expressed or uniquely accessible on metastatic cancer cells, growth factors such as EGF, FGF, insulin and insulin-like growth factors and cognate polypeptides, somatostatin and its analogs, transferrin, lipoprotein complexes, Arg-Gly-Asp containing peptides, microtubule-binding sequences (MTAS), various galectins, δ-opioid receptor ligands, cholecystokinin A receptor ligands, ligands specific for the angiotensin AT1 or AT2 receptors, peroxisome proliferator-activated receptor γ ligands, and other molecules that specifically bind to receptors selectively expressed on the surface of target cells or infectious organisms, and fragments of any of these molecules.

[0082] The term "target receptor," as used herein, refers to a receptor expressed by a cell that can bind a cell-targeting ligand. The receptor can be expressed on the surface of the cell. The receptor can be a transmembrane receptor. Examples of such target receptors include, but are not limited to, EGFR, α v These include β3 integrin, somatostatin receptor, folate receptor, prostate-specific membrane antigen, CD105, mannose receptor, estrogen receptor, GM1 ganglioside, and the like.

[0083] In some embodiments, a cell membrane-penetrating peptide may be attached to one or more PEG terminal groups instead of or in addition to a targeting ligand. As used herein, the terms "cell membrane-penetrating peptide" ("CPP"), "protein transduction domain" ("PTD"), or "membrane translocation sequence" refer to a short peptide (e.g., 4 to about 40 amino acids) that translocates through a cell membrane to access the interior of the cell and delivers various cargoes, including covalently and non-covalently conjugated proteins and oligonucleotides, into the cell. In preferred embodiments, the CPP comprises 1) a relatively abundant positively charged amino acid (e.g., lysine or arginine), 2) an amino acid sequence containing an alternating pattern of polar charged amino acids and non-polar hydrophobic amino acids, or 3) an amino acid sequence containing a hydrophobic peptide (e.g., mostly non-polar residues with a low net charge, or hydrophobic amino acid groups).(For example, U.S. Patent Application Publication No. 2022 / 0177494, Oliveira, EC, et al., “Predicting cell-penetrating peptides using machine learning algorithms and navigating in their chemical space,” Scientific Reports., 11(1):7628 (2021), Derakhshankhah, H., and Jafari, S., “Cell penetrating peptides: A concise review with emphasis on biomedical applications,” Biomedicine & Pharmacotherapy., 108:1090-1096 (2018), Milletti, F., “Cell-penetrating peptides: classes, origin, and current landscape,” Drug Discovery Today, 17(15-16):850-860 (2012), Stalmans, S., et al., “Chemical-functional diversity in cell-penetrating peptides,” PLOS ONE, 8(8):e71752 (2013); Wagstaff, KM, and Jans, DA, “Protein transduction: cell penetrating peptides and their therapeutic applications,” Current Medicinal Chemistry, 13(12):1371-1387 (2006), the entire contents of each of which are hereby incorporated by reference in their entirety.Examples of CPP peptides include, but are not limited to, TAT cell membrane permeable peptide, MAP, penetratin or antennapedia PTD, penetratin-Arg, antitrypsin (358-374), temporin L, maurocalcin, pVEC (cadherin-5), calcitonin, neuromedulin, penetratin, TAT-HA2 fusion peptide, TAT (47-57), SynB1, SynB3, PTD-4, PTD-5, FHV Coat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), HIV-1 Tat(48-60) or D-Tat, R9-Tat, transportan, SBP or human P1, FBP, MPG(δNLS), Pep-1 or Pep-1-cysteamine, Pep-2, cyclic sequences, polyarginine (R×N (4 < N < 17) chimera), polylysine (K×N (4 < N < 17) chimera), (RAca)6R, (RAbu)6R, (RG)6R, (RM)6R, (RT)6R, (RS)6R, R10, (RA)6R, and R7.

[0084] As used herein, "vector" refers to any nucleic acid, or particle, cell, or organism carrying a nucleic acid, that can be used to transfer a nucleic acid into a host cell. The term "vector" includes both viral and non-viral products and means for introducing a nucleic acid into a cell. "Vectors" can be used in vitro, ex vivo, or in vivo. Vectors capable of directing the expression of an operably linked gene are referred to herein as "expression vectors." Non-viral vectors include, for example, plasmids, cosmids, artificial chromosomes (e.g., bacterial artificial chromosomes or yeast artificial chromosomes), liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. Viral vectors include, but are not limited to, retroviral, lentiviral, adeno-associated virus, poxvirus, baculovirus, reovirus, vaccinia virus, herpes simplex virus, Epstein-Barr virus, and adenoviral vectors. Vectors can also include entire viral genomic sequences or recombinant genomic sequences. A vector may also include a portion of a genome containing functional sequences to give rise to a virus that can infect, enter, or transduce a cell to deliver nucleic acid into the cell.

[0085] The term "WW domain," as used herein, refers to a protein domain having two basic residues at its C-terminus that mediate protein-protein interactions with short proline-rich or proline-containing motifs. It should be understood that the two basic residues (e.g., any two of H, R, and K) of a WW domain need not be at the absolute C-terminus of the WW protein domain. Rather, the two basic residues may be located in the C-terminal portion of the WW protein domain (e.g., the C-terminal half of the WW protein domain). In some embodiments, a WW domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tryptophan (W) residues. In some embodiments, a WW domain contains at least two W residues. In some embodiments, the at least two W residues are 15-25 amino acids apart. In some embodiments, the at least two W residues are 19-23 amino acids apart. In some embodiments, the at least two W residues are 20-22 amino acids apart. WW domains, which have two basic C-terminal amino acid residues, can have the ability to bind short proline-rich or proline-containing motifs (e.g., the PPXY (SEQ ID NO: 2) motif). WW domains bind a variety of different peptide ligands, including those with a core proline-rich sequence, such as the PPXY (SEQ ID NO: 2) motif found in ARRDC1. WW domains can be 30-40 amino acid protein-interaction domains with two signature tryptophan residues separated by 20-22 amino acids. The three-dimensional structure of WW domains indicates that they generally fold into a three-stranded antiparallel β-sheet with two ligand-binding grooves.

[0086] WW domains are found in many eukaryotes and are present in approximately 50 human proteins (Bork, P. & Sudol, M., "The WW domain: a signaling site in dystrophin?" Trends Biochem Sci., 19, 531-533 (1994)). WW domains can occur with several other interaction domains, including membrane targeting domains, such as the C2 domain of NEDD4 family proteins, the phosphotyrosine-binding (PTB) domain of FE65 proteins, the FF domain of CA150 and FBP1l, and the pleckstrin homology (PH) domain of PLEKHA5. WW domains are also linked to various catalytic domains, including the HECT E3 protein-ubiquitin ligase domain of NEDD4 family proteins, the rotomerase domain or peptidylprolyl isomerase domain of Pinl, and the Rho GAP domain of ArhGAP9 and ArhGAP12.

[0087] The WW domain may be a WW domain that naturally has two basic amino acids at the C-terminus. In some embodiments, the WW domain or WW domain variant may be derived from human ubiquitin ligase WWP1, WWP2, Nedd4-1, Nedd4-2, Smurf1, Smurf2, ITCH, NEDL1, or NEDL2. Exemplary amino acid sequences of WW domain-containing proteins (underlined WW domains) are listed below. It should be understood that any of the WW domains or WW domain variants of representative proteins can be used in the present invention and described herein, and are not meant to be limiting.

[0088] Amino acid sequence of human WWP1 (uniprot.org / uniprot / Q9H0M0). The four underlined WW domains correspond to amino acids 349–382 (WW1), 381–414 (WW2), 456–489 (WW3), and 496–529 (WW4).

[0089] [ka]

[0090] WW1 (349-382): ETLPSGWEQRKDPHGRTYYVDHNTRTTTWERPQP (SEQ ID NO: 5).

[0091] WW2 (381-414): QPLPPGWERRVDDRRRVYYVDHNTRTTTWQRPTM (SEQ ID NO: 6).

[0092] WW3 (456-489): ENDPYGPLPPGWEKRVDSTDRVYFVNHNTKTTQWEDPRT (SEQ ID NO: 7).

[0093] WW4 (496-529): EPLPEGWEIRYTREGVRYFVDHNTRTTTFKDPRN (SEQ ID NO: 8).

[0094] Human WWP2 amino acid sequence (uniprot.org / uniprot / O00308). The four underlined WW domains correspond to amino acids 300–333 (WW1), 330–363 (WW2), 405–437 (WW3), and 444–547 (WW4).

[0095] [ka]

[0096] WW1 (300-333): DALPAGWEQRELPNGRVYYVDHNTKTTTWERPLP (SEQ ID NO: 10).

[0097] WW2 (330-363): PLPPGWEKRTDPRGRFYYVDHNTRTTTWQRPTA (SEQ ID NO: 11).

[0098] WW3 (405-437): HDPLGPLPPGWEKRQDNGRVYYVNHNTRTTQWEDPRT (SEQ ID NO: 12).

[0099] WW4 (444-477): PALPPGWEMKYTSEGVRYFVDHNTRTTTFKDPRP (SEQ ID NO: 13).

[0100] Human Nedd4-1 amino acid sequence (uniprot.org / uniprot / P46934). The four underlined WW domains correspond to amino acids 610–643 (WW1), 767–800 (WW2), 840–873 (WW3), and 892–925 (WW4).

[0101] [ka]

[0102] WW1 (610-643): SPLPPGWEERQDILGRTYYVNHESRRTQWKRPTP (SEQ ID NO: 15).

[0103] WW2 (767-800): SGLPPGWEEKQDERGRSYYVDHNSRTTTWTKPTV (SEQ ID NO: 16).

[0104] WW3 (840-873): GFLPKGWEVRHAPNGRPFFIDHNTKTTTWEDPRL (SEQ ID NO: 17).

[0105] WW4 (892-925): GPLPPGWEERTHTDGRIFYINHNIKRTQWEDPRL (SEQ ID NO: 18).

[0106] 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).

[0107] [ka]

[0108] WW1 (198-224): GWEEKVDNLGRTYYVNHNNRTTQWHRP (SEQ ID NO: 20).

[0109] WW2 (368-396): PSGWEERKDAKGRTYYVNHNNRTTTWTRP (SEQ ID NO: 21).

[0110] WW3 (480-510): PPGWEMRIAPNGRPFFIDHNTKTTTWEDPRL (SEQ ID NO: 22).

[0111] WW4 (531-561): PPGWEERIHLDGRTFYIDHNSKITQWEDPRL (SEQ ID NO: 23).

[0112] 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).

[0113] [ka]

[0114] WW1 (234-267): PELPEGYEQRTTVQGQVYFLHTQTGVSTWHDPRI (SEQ ID NO: 25).

[0115] WW2 (306-339): GPLPPGWEVRSTVSGRIYFVDHNNRTTQFTDPRL (SEQ ID NO: 26).

[0116] 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).

[0117] [ka]

[0118] WW1 (157-190): NDLPDGWEERRTASGRIQYLNHITRTTQWERPTR (SEQ ID NO: 28).

[0119] WW2 (251-284): PDLPEGYEQRTTQQGQVYFLHTQTGVSTWHDPRV (SEQ ID NO: 29).

[0120] WW3 (297-330): GPLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRL (SEQ ID NO: 30).

[0121] 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).

[0122] [ka]

[0123] ITCH WW1(326~359): APLPPGWEQRVDQHGRVYYVDHVEKRTTWDRPEP (SEQ ID NO: 32).

[0124] ITCH WW2(358~391): EPLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTL (SEQ ID NO: 33).

[0125] ITCH WW3(438~471): GPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRS (SEQ ID NO: 34).

[0126] ITCH WW4(478~511): KPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRT (SEQ ID NO: 35).

[0127] 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).

[0128] [ka]

[0129] WW1 (829-862): PLPPNWEARIDSHGRVFYVDHVNRTTTWQRPTA (SEQ ID NO: 37).

[0130] WW2 (1018-1051): LELPRGWEIKTDQQGKSFFVDHNSRATTFIDPRI (SEQ ID NO: 38).

[0131] 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).

[0132] [ka]

[0133] WW1 (807-840): EALPPNWEARIDSHGRIFYVDHVNRTTTWQRPTA (SEQ ID NO: 40).

[0134] WW2 (985-1018): LELPRGWEMKHDHQGKAFFVDHNSRTTTFIDPRL (SEQ ID NO: 41).

[0135] In some embodiments, the WW domain consists essentially of a WW domain or WW domain variant, meaning that the domain, peptide, or polypeptide consists essentially of an amino acid sequence where such amino acid sequence is present in the domain, peptide, or polypeptide with only a few additional amino acid residues, e.g., about 1 to about 10 or so additional residues, typically 1 to about 5 additional residues.

[0136] 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, for example, in Sambrook et al., Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (2001). Thus, one skilled in the art can easily modify an existing WW domain that does not normally have two C-terminal basic residues to include two basic residues at the C-terminus.

[0137] Basic amino acids are amino acids with a side chain functional group with a pKa greater than 7, including lysine, arginine, and histidine, as well as basic amino acids not included in the 20 α-amino acids commonly found in proteins. The two basic amino acids at the C-terminus of the WW domain may be the same or different. In one embodiment, the two basic amino acids are two arginines.

[0138] The term WW domain also includes any variant of the WW domain, provided that such variant has two basic amino acids at its C-terminus and maintains the ability of the WW domain to bind to the PPXY (SEQ ID NO: 2) motif. Such a WW domain variant refers to a WW domain that retains its ability to bind to 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 mutations, insertion mutations, and / or deletion mutations, but still retains the ability to bind to the PPXY (SEQ ID NO: 2) motif. Variants or derivatives therefore include deletions, including truncations and fragments; insertions and additions, e.g., conservative substitutions, site-directed mutations, and allelic variants; and modifications, including one or more non-aminoacyl groups (e.g., sugars, lipids, etc.) covalently attached to the peptide and post-translational modifications. In making such changes, substitutions of similar amino acid residues can be made on the basis of the relative similarity of the side-chain substituents, e.g., their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions can be assayed for their effect on the function of the peptide by routine testing.

[0139] A WW domain can be part of a longer protein. Thus, in various different embodiments, a protein comprises, consists of, or consists essentially of a WW domain, as defined herein. A polypeptide can be a protein that includes a WW domain as a functional domain within the protein sequence.

[0140] The term "Cas9" or "Cas9 protein" or "Cas9 polypeptide" refers to a Cas9 protein and RNA-guided nucleases, including fusion proteins containing such Cas9 proteins and variants thereof (e.g., proteins comprising an active, inactive, or modified DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). In some embodiments, fusion proteins include fusion proteins that modify the epigenome or control transcriptional activity. Variants include deletions or additions, e.g., the addition of one, two, or more nuclear localization sequences (e.g., those derived from SV40 and others known in the art), e.g., the addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 such sequences, or ranges between and including any two of the foregoing values.

[0141] In some embodiments, the Cas9 polypeptide is a Cas9 protein found in type II CRISPR-associated systems. Suitable Cas9 polypeptides that may be used in certain embodiments of the invention include, but are not limited to, Cas9 proteins from Streptococcus pyogenes (Sp. Cas9), Francisella novicida, Staphylococcus aureus, Streptococcus thermophiles, Neisseria meningitidis, and variants thereof.

[0142] Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (e.g., viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, accurate processing of the pre-crRNA requires a transcoded small RNA (tracrRNA), endogenous RNase 3 (mc), and the Cas9 protein. The tracrRNA serves as a guide for processing of the pre-crRNA by RNase 3. Cas9 / crRNA / tracrRNA then cleaves linear or circular dsDNA targets complementary to the spacer by endogenous nucleolytic degradation. The target strand that is not complementary to the crRNA is first cleaved by endonucleasis, and then trimmed at 3'-5' by exonucleolysis. In nature, DNA binding and cleavage typically require both proteins and RNAs. However, single guide RNA ("sgRNA" or simply "gRNA") can be engineered to incorporate the characteristics of both crRNA and tracrRNA into one RNA species. (See, for example, M., et al., Science, 337:816-821 (2012), the entire contents of which are hereby incorporated by reference.) Cas9 recognizes a short motif (PAM or protospacer adjacent motif) within the CRISPR repeat sequence to help distinguish between self and non-self. The sequence and structure of Cas9 nuclease are well known to those skilled in the art.(See, e.g., Ferretti et al., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Proc. Natl. Acad. Sci. USA, 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 include Cas9 sequences from organisms and loci disclosed in the art.(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, AC, 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.

[0143] In some embodiments, the Cas9 polypeptide is wild-type Cas9, a nickase, or comprises a nuclease-inactivated (nuclease-"dCas9," short for "dead" Cas9) protein.

[0144] Methods for generating a Cas9 protein (or variants thereof) with 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 these documents are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, 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, the 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." Cas9 variants have homology to Cas9 or variants 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% identical 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% identical to a corresponding variant of wild-type Cas9. In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, SEQ ID NO: 1 (nucleotide), SEQ ID NO: 22 (amino acid)).

[0145] [ka]

[0146] [ka]

[0147] [ka]

[0148] [ka] (Single underline: HNH domain, double underline: RuvC domain)

[0149] In some embodiments, wild-type Cas9 corresponds to or comprises SEQ ID NO: 3 (nucleotide) and / or SEQ ID NO: 4 (amino acid).

[0150] [ka]

[0151] [ka]

[0152] [ka]

[0153] [ka] (Single underline: HNH domain, double underline: RuvC domain)

[0154] In some embodiments, the dCas9 corresponds to, or partially or completely comprises, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises a D10A and / or H820A mutation. dCas9 (D10A and H840A) is as follows:

[0155] [ka] (Single underline: HNH domain, double underline: RuvC domain)

[0156] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H820A, which, for example, result in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, dCas9 variants or homologs (e.g., variants of SEQ ID NO: 5) are provided that 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% identical to SEQ ID NO: 5. In some embodiments, variants of Cas9 (e.g., variants of SEQ ID NO: 5) are provided that have amino acid sequences that are about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more amino acids shorter or longer than SEQ ID NO: 5.

[0157] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the sequences provided above. In other embodiments, however, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, the Cas9 fusion proteins provided herein comprise a Cas9 fragment, where 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 the nuclease domain or no nuclease domain at all. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and further suitable sequences of Cas9 domains and fragments will be apparent to those skilled 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), a base editor, a prime editor, a recombinase domain (e.g., Hin, Gin, or Tn3), a deaminase (e.g., cytidine deaminase or adenosine deaminase), or an epigenetic modifier domain (e.g., TET1, p300).

[0158] In some embodiments, Cas9 is used to treat or inhibit the growth of Corynebacterium ulcerans (NCBI Reference Numbers: NC_015683.1, NC_017317.1), Corynebacterium diphtheria (NCBI Reference Numbers: NC_016782.1, NC_016786.1), Spiroplasma syrphidicola (NCBI Reference Number: NC_021284.1), Prevotella intermedia (NCBI Reference Number: NC_017861.1), Spiroplasma taiwanense (NCBI Reference Number: NC_021846.1), Streptococcus iniae, or other pathogenic bacteria. iniae (NCBI Reference Number: NC_021314.1), Belliella baltica (NCBI Reference Number: NC_018010.1), Psychroflexus torquisl (NCBI Reference Number: NC_018721.1), Streptococcus thermophilus (NCBI Reference Number: YP_820832.1), Listeria innocua (NCBI Reference Number: NP472073.1), Campylobacter jejuni (NCBI Reference Number: YP_02344900.1), or Neisseria meningitidis This refers to Cas9 derived from C. meningitidis (NCBI reference number: YP_02342100.1).

[0159] The term "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively.

[0160] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used interchangeably herein to refer to a nuclease complexed with (e.g., bound to or associated with) one or more RNA molecules that are not the target of cleavage. In some embodiments, when an RNA-programmable nuclease is complexed with RNA, it may be described as a nuclease:RNA complex. RNA-programmable nucleases include Cas9 nucleases. Typically, the bound RNA is described as a guide RNA (gRNA). A gRNA may exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule may be described as a single guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that has homology to the target nucleic acid (e.g., directs binding of the Cas9 complex to the target) and (2) a domain that binds the Cas9 protein. The gRNA comprises a nucleotide sequence complementary to a target site, which nucleotide sequence mediates binding of a nuclease / RNA complex to said target site and provides sequence specificity for the nuclease:RNA complex.

[0161] The term "recombinase," as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, resulting in the excision, integration, inversion, or exchange (e.g., transposition) 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, but are not limited to, 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, but are not limited to, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The names serine and tyrosine recombinases are derived from the conserved nucleophilic amino acid residues that the recombinases use to attack DNA and covalently bind to it during strand exchange. Recombinases have many uses, including the generation of gene knockouts / knockins 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 of which are hereby incorporated by reference in their entirety. The recombinases provided herein are not exclusive examples of recombinases that may be used in embodiments of the present invention. The methods and compositions of the present invention can be expanded by searching databases for new orthogonal recombinases or by designing synthetic recombinases with defined DNA specificity. (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 of which are hereby incorporated by reference in their entirety. Other examples of recombinases useful in the methods and compositions described herein will be known to those of skill in the art, and it is expected that any new recombinases discovered or created can be used in different embodiments of the present invention. In some embodiments, the recombinase (or its catalytic domain) is fused to a Cas9 protein (e.g., dCas9).

[0162] The terms "recombining" and "recombination" in the context of nucleic acid modification (e.g., genomic modification) are used to refer to a process in 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, among other things, the insertion, inversion, excision, or transposition of nucleic acid sequences within or between one or more nucleic acid molecules.

[0163] As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or fewer percent in either direction (greater or lesser) of the stated reference value, unless otherwise stated or otherwise clear from the context (e.g., when such number exceeds 100% of the possible value).

[0164] General Description of the Invention ARMM-related compositions and methods for producing and using the particles are known in the art and are described in, but not limited to, U.S. Patent No. 9,737,480, U.S. Patent No. 9,816,080, U.S. Patent No. 10,260,055, PCT Publication WO 2018 / 067546, and U.S. Patent Application Publication Nos. 2022 / 0119785, 2022 / 0170013, 2022 / 0220462, and 2022 / 0282275, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0165] In one aspect of the present invention, compositions and methods are provided for ARMM-mediated delivery of therapeutic agents and payloads to ocular cells, particularly the retina. The ARMM-based compositions and methods of the present invention are expected to offer certain advantages over viral vector administration, including, but not limited to, transient expression, evasive immune privilege, and lack of repeat replication, among other advantages. ARMM containing an ARRDC1-Cre fusion protein has been functionally administered to the eyes (e.g., the subretinal space) of Ai14 Cre reporter mice. Briefly, in the Ai14 system, expression of the fluorescent protein tdTomato is activated only by exogenously derived functional Cre recombinase protein. In Ai14 animals, Cre-dependent expression of TdTomato in target cells demonstrates uptake of A1-cre-loaded ARMM particles by these cells. A preferred embodiment of the present invention therefore provides for the delivery of nuclear cargo capable of editing the nuclear genome of target cells.

[0166] Representative genetic eye diseases In a preferred embodiment, the compositions and methods of the present invention are intended to treat, alleviate, improve, or cure diseases or conditions caused by one or more abnormalities in the patient's genome.Non-limiting examples of the gene targets of the therapeutic agent delivered by ARMM 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's congenital amaurosis ("LCA")); and TULP1, LRAT, IMPDH1 (i.e., potential LCA-like phenotypes). In further embodiments, the GUCY2D gene (i.e., autosomal dominant cone-rod dystrophy), the RPE65 gene (i.e., retinitis pigmentosa), and the AIPL1 gene (i.e., cone-rod dystrophy and retinitis pigmentosa) are also believed to be associated with additional conditions.

[0167] In still further embodiments, the compositions and methods of the present invention are directed to, but are not limited to, BEST1 (i.e., vitelliform macular dystrophy, age-related macular degeneration, autosomal dominant vitreoretinochoroidopathy, and retinitis pigmentosa, microphthalmia, autosomal recessive 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); PDE6 B (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 punctata albedo, retinitis pigmentosa); and WDR19 (i.e., craniectodermal dysplasia, asphyxiating thoracic dystrophy, nephronophthisis, retinitis pigmentosa, Senior-Loken syndrome).

[0168] Further embodiments are directed to delivering payloads and therapeutic agents associated with the ARMM to cells and tissues of the retinal pigment epithelium to treat diseases and conditions including, but not limited to, age-related macular degeneration, Best vitelliform macular dystrophy, Thorsby fundus dystrophy, choroidal neovascularization, diabetic macular edema, MERTK retinitis pigmentosa, RPE65 Leber congenital amaurosis, and bestrophinopathy (e.g., autosomal dominant vitreoretinochoroidopathy). In other embodiments, the ARMM is 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), Stargardt macular dystrophy, color vision deficiency, Usher syndrome 1c, and cone-rod dystrophies. In still further embodiments, the ARMM is intended to deliver payloads and therapeutic agents associated with the ARMM to retinal pigment epithelial cells and photoreceptors to treat diseases and conditions including, but not limited to, Usher syndrome 1b, choroideremia, Bardet-Biedl syndrome, and retinitis pigmentosa.

[0169] Thus, in some cases, the compositions described herein are administered to a subject having or suspected of having an eye disease (e.g., as described herein).

[0170] In some cases, the compositions described herein are administered to a subject who has or is suspected of having Stargardt disease.In some cases, the Stargardt disease is Stargardt disease-1, Stargardt disease-3, Stargardt disease-4, or fundus flava (FFM).In some cases, the subject who has or is suspected of having Stargardt disease has one or more genetic mutations in ABCA4 gene, ELOVL4 gene, BEST1 gene, and / or PRPH2 gene.

[0171] In some cases, the compositions described herein are administered to a subject who has or is suspected of having 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 who has or is suspected of having Usher syndrome has one or more genetic mutations in MYO7A, USH1C, CDH23, PCDH15, USH2A, ADGRV1, WHRN, and / or CLRN1 genes.

[0172] In some cases, the compositions described herein are administered to patients who have one or more of the following genetic variants compared to the human retina-specific phospholipid-transporting ATPase ABCA4 (UniProt ID P78363) encoded by human ABCA4:

[0173] [Table 1-1]

[0174] [Table 1-2]

[0175] [Table 1-3]

[0176] [Table 1-4]

[0177] In some cases, the compositions described herein are administered to patients who have one or more of the following genetic variants compared to human non-classical myosin-VIIa (UniProt ID Q13402) encoded by human MYO7A:

[0178] [Table 2]

[0179] In some cases, the compositions described herein are administered to patients who have one or more of the following genetic variants compared to human cadherin-23 (UniProt ID Q9H251), which is encoded by human CDH23:

[0180] [Table 3]

[0181] In some cases, the compositions described herein are administered to patients who have one or more of the following genetic variants compared to human protocadherin-15 (UniProt ID Q96QU1), which is encoded by human PCDH15:

[0182] [Table 4]

[0183] In some cases, the compositions described herein are administered to patients who have one or more of the following genetic variants compared to human usherin (UniProt ID O75445), which is encoded by human USH2A:

[0184] [Table 5]

[0185] In some cases, the compositions described herein are administered to patients who have one or more of the following genetic variants compared to human adhesion G protein-coupled receptor V1 (UniProt ID Q8WXG9), encoded by human ADGRV1:

[0186] [Table 6]

[0187] In some cases, the compositions described herein are administered to patients who have one or more of the following genetic variants compared to human clarin-1 (UniProt ID P58418), which is encoded by human CLRN1:

[0188] [Table 7]

[0189] 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 regulate their 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 a portion thereof (e.g., an RNA-guided genome editor or a portion thereof described herein, e.g., a nuclease, base editor, prime editor, or a portion thereof).

[0190] General Pharmaceutical Formulations and Compositions A further aspect of the present disclosure relates to a pharmaceutical composition comprising any of the ARMM 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 includes additional agents (e.g., for specific delivery, increased half-life, potency, efficacy, biological effect, and the like, as well as other therapeutic agents and compounds).

[0191] As used herein, 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, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound from one site in the body (e.g., a delivery site) to another site (e.g., an organ, tissue, system, or part of the body). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the cells and tissues of the subject (e.g., physiologically compatible, sterile, at physiological pH, etc.). Some examples of materials that can function 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 carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate, (4) powdered tragacanth, (5) malt, (6) gelatin, (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, (8) excipients, such as cocoa butter and suppository wax, (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and corn starch. 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) buffers, 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 buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, (22) C2-C 12Included are alcohols, such as ethanol, and (24) other non-toxic, compatible substances utilized in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, antioxidants, and the like may also optionally be present in the formulation. The terms "excipient," "carrier," "pharmaceutically acceptable carrier," and similar terms are used interchangeably herein.

[0192] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, for example, for delivery of a therapeutic agent, payload protein, or payload nucleic acid to a cell. Suitable routes of administration of the pharmaceutical compositions described herein include, but are not limited to, subretinal, suprachoroidal, intravitreal, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration (see, e.g., Hartman, RR, and Kompella, UB, "Intravitreal, Subretinal, and Suprachoroidal Injections: Evolution of Microneedles for Drug Delivery," J. Ocul. Pharmacol. Ther., 34(1-2):141-153 (2018)).

[0193] In some embodiments, the pharmaceutical compositions described herein are administered locally to the affected site (e.g., to cells of the eye). In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, catheter, suppository, or implant, where the implant is made of a porous, non-porous, or gelatinous material, including a membrane, such as a silastic membrane, or a fiber.

[0194] In some embodiments, the composition is formulated according to conventional procedures and is suitable for injection, intravenous administration, or subcutaneous administration to a subject, such as a human. In some embodiments, the composition for administration by injection comprises a solution in a sterile isotonic aqueous buffer. If necessary, the formulation may also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the injection site. When the formulation is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration. When the formulation is administered by infusion, the formulation can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline.

[0195] The formulation (pharmaceutical composition) for systemic administration can be liquid, such as sterile saline, lactated Ringer's solution, or Hank's solution.In addition, the pharmaceutical composition can be in solid form, and can be redissolved or suspended immediately before use.Certain formulations in lyophilized form are also contemplated.

[0196] The compositions described herein can be administered or packaged as unit doses. The term "unit dose", when used in reference to the pharmaceutical compositions of the present disclosure, refers to a physically discrete unit suitable as a single dosage for a subject, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, together with the required diluent, i.e., carrier or vehicle.

[0197] Furthermore, the composition can be provided as a kit comprising 1) a container containing the ARMM or microvesicle-producing cells of the present invention, and 2) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent can be used, for example, to reconstitute or dilute the ARMM or microvesicle-producing cells of the present invention. Optionally, such a container can be accompanied by a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the government agency for manufacture, use, or sale for human administration. In this regard, national 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 UK Medicines and Healthcare Products Regulatory Agency, the National Medical Products Administration, and the like.

[0198] Another aspect includes an article of manufacture containing materials useful for treating the diseases described herein. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, such as glass or plastic. It is further understood that suitable containers comprise materials that are sufficiently non-reactive to protect the contents within the container. In some embodiments, the container holds a composition effective for treating the diseases described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or vial with a stopper pierceable by a needle for subdermal injection. The active agent in the composition is a compound of the present invention. In some embodiments, a label on or associated with the container indicates that the composition is used to treat one or more selected diseases. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with directions and labeling for acceptable, approved, or permitted uses.

[0199] In some embodiments, treatments are contemplated in which one therapeutic agent is administered using ARMM-mediated delivery of the agent to a target cell, tissue, system, or mammalian subject.

[0200] In some other embodiments, treatments are contemplated in which two therapeutic agents are administered using ARMM-mediated delivery of each agent to a target cell, tissue, system, or mammalian subject.

[0201] Yet some other embodiments contemplate treatments in which three or more therapeutic agents are administered using ARMM-mediated delivery of each agent to a target cell, tissue, system, or mammalian subject.

[0202] In 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, it is contemplated that each (i.e., two or more) agent is provided within a single ARRDC1-mediated microvesicle (ARMM) particle for administration.

[0203] Similarly, in the compositions and methods described herein, where two or more respective therapeutic agents are administered (e.g., co-administered) to a target cell, tissue, system, or subject, it is further contemplated that each (i.e., two or more) agent is provided within a different ARRDC1-mediated microvesicle (ARMM) particle for administration.

[0204] Kits, vectors, and cells Some aspects of the present disclosure provide kits that include a nucleic acid construct comprising a nucleotide sequence encoding any one or more of the proteins (e.g., ARRDC1 and TSG101), fusion proteins, and / or nucleic acids provided herein. In some embodiments, the nucleotide sequence encodes any one of the proteins, fusion proteins, and / or RNAs provided herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of any one of the proteins, fusion proteins, and / or RNAs provided herein.

[0205] Some aspects of the present disclosure provide microvesicle (e.g., ARMM)-producing cells comprising any of the proteins, fusion proteins, and nucleic acids (e.g., RNA) provided herein. In some embodiments, the cells specifically comprise nucleotides encoding 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 vectors comprise one or more cell-targeting or cell-entry proteins (e.g., viral and / or human fusogens).

[0206] However, it should be understood that additional proteins, fusion proteins, and RNAs will become apparent to those skilled in the art based on this disclosure and knowledge in the art.

[0207] The function and advantages of these and other embodiments of the present invention will be more fully understood from the following examples, which are intended to illustrate the benefits of the present invention and to describe specific embodiments, but are not intended to exemplify the full scope of the invention.

[0208] Therefore, it will be understood that the examples do not limit the scope of the invention.

[0209] Detailed Description of Certain Embodiments of the Invention The present invention provides methods, systems, and compositions for ARRDC1-mediated microvesicle ("ARMM") 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 for producing, testing, and administering ARMM to one or more internal structures of the eye. More particularly, the present invention provides compositions and methods for producing, testing, and administering ARMM comprising one or more therapeutic agents (e.g., biomolecules, 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)). Methods of administering therapeutic agents associated with ARMM are also provided, including, but not limited to, methods of treating or contacting ocular cells and tissues with one or more typical dosing regimens (e.g., 1) in vivo administration of ARMM to a patient, 2) ex vivo administration of ARMM to target cells and transplantation of ARMM-treated cells into a patient, and 3) in vivo and ex vivo regimens. In particular, the present invention provides methods of administering therapeutic agents via ARMM to cells and tissues, including the retina, or to the subretinal space. Additionally, the present invention relates to methods of producing (e.g., culturing, clarifying, separating, and concentrating) compositions of the invention obtained from stable producer cell lines and / or from transient cell cultures.

[0210] More particularly, the present disclosure relates to the discovery that therapeutic agents linked to ARRDC1 protein can be loaded into ARMM and delivered to cells and tissues, including the retina. In some embodiments, the uptake of these ARMM and their associated payloads is enhanced by the presence of a portion of viral envelope proteins, including but not limited to VSV-G, on the surface of the ARMM. Different therapeutic agents, for example, proteins and nucleic acids, including various RNAs, can be loaded into such ARMM for delivery to retinal cells. In preferred embodiments, various types of endonucleases (e.g., gRNA-linked CRISPR / Cas9), gRNA-linked base editors and prime editors, and effector proteins are expected to be suitable for delivery to ocular cells and tissues, more particularly retinal cells and the subretinal space, using the compositions and methods of the present invention.

[0211] ARMM Arrestin domain-containing protein 1-mediated microvesicles ("ARMMs") are extracellular vesicles ("EVs") distinct from exosomes. ARMM budding requires ARRDC1, which is localized to the cytoplasmic side of the plasma membrane and recruits the ESCRT-I complex protein TSG101 to the cell surface via a tetrapeptide motif to initiate outward membrane budding. Thus, in contrast to exosomes, ARMM biogenesis 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 ARMM budding. Overexpression of ARRDC1 protein increases ARMM production in cells, enabling controlled production of ARMM using modern biological production methods. Furthermore, endogenous proteins, such as cell surface receptors, can be actively recruited into ARMMs and delivered to recipient cells to initiate cell-to-cell communication, suggesting that exogenous payload molecules can similarly be packaged and delivered via ARMMs.

[0212] ARRDC1 In a preferred embodiment, ARRDC1 is a protein comprising a PSAP (SEQ ID NO: 1) motif and a PPXY (SEQ ID NO: 2) motif at its C-terminus, and interacts with TSG101 as shown herein. It should be understood that the PSAP (SEQ ID NO: 1) motif and the PPXY (SEQ ID NO: 2) motif do not necessarily have to be at the absolute C-terminus of ARRDC1. Rather, these motifs may be located in the C-terminal portion of the ARRDC1 protein (e.g., the C-terminal half of ARRDC1). The present disclosure also relates to variants of ARRDC1, such as fragments of the ARRDC1 protein and / or ARRDC1 proteins that have a certain degree of identity to the ARRDC1 protein (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity) and can interact with TSG101. Thus, the ARRDC1 protein may be a protein comprising 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, contains 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 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, at least 430, at least 440, at least 450, at least 460, at least 470, at least 480, at least 490, at least 500, at least 510, at least 520, at least 530, at least 540, at least 550, at least 560, at least 570, at least 580, at least 590, at least 600, at least 610, at least 620, at least 630, at least 640, at least 650, at least 660, at least 670, at least 680, at least 690, at least 700, at least 710, at least 720, at least 73 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, contain a PSAP (SEQ ID NO: 1) motif and a PPXY (SEQ ID NO: 2) motif, and interact 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 TSGlOl. 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 ARRDC1 protein variants suitable according to aspects of the present invention are known in the art. Those skilled in the art will understand that the present 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):

[0213] >gi|22748653|ref|NP_689498.1|Arrestin domain-containing protein 1 [Homo sapiens]

[0214] [ka]

[0215] >gi|244798004|ref|NP_001155957.1|Arrestin domain-containing protein 1 isoform a [Mus musculus]

[0216] [ka]

[0217] >gi|244798112|ref|NP_848495.2|Arrestin domain-containing protein 1 isoform b [Mus musculus]

[0218] [ka]

[0219] TSG101 In certain embodiments, the microvesicles of the present invention further comprise TSG101 (tumor susceptibility gene 101), which belongs to a group of putative inactive homologs of ubiquitin-conjugating enzymes. This protein has a coiled-coil domain that interacts with stathmin, a cytoplasmic phosphoprotein involved in tumorigenesis. TSG101 is a protein that contains a UEV domain and interacts with ARRDC1. As described herein, UEV refers to the approximately 145 amino acid ubiquitin E2 variant domain. The domain structure contains an α / β fold similar to that of standard E2 enzymes, but has an additional N-terminal helix and lacks two C-terminal helices. As often seen in the TSG101 / Vps23 protein, UEV interacts with ubiquitin molecules and is essential for the transport of many ubiquitinated payloads to multivesicular bodies (MVBs). Furthermore, the UEV domain can bind to the Pro-Thr / Ser-Ala-Pro peptide ligand, which is utilized by viruses such as HIV. Thus, the TSG101 UEV domain binds to the PTAP tetrapeptide motif in the viral Gag protein, which is involved in viral budding. The present disclosure also contemplates variants of TSG101, such as fragments of the TSG101 protein and / or TSG101 proteins that have a degree of identity to the TSG101 protein (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity) and can interact with ARRDC1. Thus, the TSG101 protein can be a protein that includes 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, contains 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 integers therebetween, 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; additional suitable TSG101 protein sequences, isoforms, and variants are known in the art. Those skilled in the art will understand that the present invention is not limited in this respect. Exemplary TSG101 sequences include the following (the UEV domain within these sequences comprises amino acids 1-145 and is underlined in the sequences below):

[0220] >gi|5454140|ref|NP_006283.1|Tumor susceptibility gene 101 protein [Homo sapiens]

[0221] [ka]

[0222] >gi|11230780|ref|NP_068684.1|Tumor susceptibility gene 101 protein [Mus musculus]

[0223] [ka]

[0224] >gi|48374087|ref|NP_853659.2|Tumor susceptibility gene 101 protein [Rattus norvegicus]

[0225] [ka]

[0226] The structure of the UEV domain is known to those skilled 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).

[0227] Expression constructs Some aspects of the present invention provide expression constructs for encoding one or more gene products, which induce or promote the production of ARMM in cells harboring such constructs. In some embodiments, the expression constructs described herein encode the fusion proteins described herein, such as ARRDC1 fusion proteins and TSG101 fusion proteins. In some embodiments, the expression constructs encode ARRDC1 protein or a variant thereof, and / or TSG101 protein or a variant thereof. In some embodiments, overexpression of either or both gene products in a cell increases the production of ARMM in the cell, thus converting the cell into a microvesicle-producing cell. In some embodiments, such expression constructs contain at least one restriction or recombination site at either the C-terminus or N-terminus of the encoding ARRDC1 or its variant, allowing for in-frame cloning of a protein sequence to be fused. As another example, the expression constructs contain at least one restriction or recombination site at either the C-terminus or N-terminus of one or more encoding WW domains, allowing for in-frame cloning of a protein sequence to be fused.

[0228] 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 or recombination site located adjacent to the nucleotide sequence encoding ARRDC1, the restriction or recombination site permitting insertion of a nucleotide sequence encoding a payload protein, or an RNA-binding protein sequence or an RNA-binding protein variant sequence in frame with the nucleotide sequence encoding ARRDC1. In some embodiments, the heterologous promoter may be a constitutive promoter, and in some embodiments, the heterologous promoter may be an inducible promoter. Some aspects of the present invention provide expression constructs comprising (a) a nucleotide sequence encoding a TSG101 protein or variant thereof operably linked to a heterologous promoter, and (b) a restriction or recombination site located adjacent to the nucleotide sequence encoding TSG101, the restriction or recombination site permitting insertion of a nucleotide sequence encoding a payload protein, or an RNA-binding protein, a DNA-binding protein, or a variant thereof in frame with the nucleotide sequence encoding TSG101. In some embodiments, the heterologous promoter may be a constitutive promoter, and in some embodiments, the heterologous promoter may be an inducible promoter.

[0229] Some aspects of the present invention provide expression constructs comprising: (a) a nucleotide sequence encoding a WW domain or variant thereof operably linked to a heterologous promoter; and (b) a restriction or recombination site located adjacent to the nucleotide sequence encoding the WW domain, the restriction or recombination site permitting insertion of a payload protein or an RNA-binding protein or a protein variant thereof in frame with the nucleotide sequence encoding the WW domain. In some embodiments, the heterologous promoter may be a constitutive promoter; in some embodiments, the heterologous promoter may be an inducible promoter. The expression construct may encode a payload protein or an RNA-binding protein fused to at least one WW domain. In some embodiments, the expression construct encodes a payload protein or an RNA-binding protein or a 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.

[0230] The expression constructs described herein can include any nucleic acid sequence capable of encoding a WW domain or its variant. For example, the nucleic acid sequence encoding a WW domain or a WW domain variant can be derived from human ubiquitin ligase WWP1, WWP2, Nedd4-1, Nedd4-2, Smurf1, Smurf2, ITCH, NEDL1, or NEDL2. Exemplary nucleic acid sequences of proteins having WW domains are listed below. It is understood that any nucleic acid encoding a WW domain or a WW domain variant of an exemplary protein can be used in the present invention, and is not limited to the nucleic acid sequences described herein.

[0231] Human WWP1 nucleic acid sequence (uniprot.org / uniprot / Q9H0M0).

[0232] [ka]

[0233] [ka]

[0234] Human WWP2 nucleic acid sequence (uniprot.org / uniprot / O00308).

[0235] [ka]

[0236] [ka]

[0237] Human Nedd4-1 nucleic acid sequence (uniprot.org / uniprot / P46934).

[0238] [ka]

[0239] [ka]

[0240] Human Nedd4-2 nucleic acid sequence (>gi|345478679|ref|NM_015277.5|Developmentally downregulated 4-like E3 ubiquitin protein ligase (NEDD4L) expressed by Homo sapiens neural progenitor cells, transcript variant d, mRNA).

[0241] [ka]

[0242] Human Smurf1 nucleic acid sequence (uniprot.org / uniprot / Q9HCE7).

[0243] [ka]

[0244] Human Smurf2 nucleic acid sequence (uniprot.org / uniprot / Q9HAU4).

[0245] [ka]

[0246] Human ITCH nucleic acid sequence (uniprot.org / uniprot / Q96J02).

[0247] [ka]

[0248] Human NEDL1 nucleic acid sequence (uniprot.org / uniprot / Q76N89).

[0249] [ka]

[0250] [ka]

[0251] Human NEDL2 nucleic acid sequence (uniprot.org / uniprot / Q9P2P5).

[0252] [ka]

[0253] Some aspects of the invention provide expression constructs that encode any of the proteins, nucleic acids such as RNA, or fusions thereof described herein.

[0254] The nucleic acids encoding any of the proteins and / or nucleic acids (including RNA) described herein can be within any number of nucleic acid vectors known in the art. Vectors suitable for use in the compositions and methods of the invention include both viral and non-viral products, as well as additional means for introducing nucleic acids into cells.

[0255] The expression of any of the proteins and / or nucleic acids (including RNA) described herein can be controlled by any regulatory sequence (e.g., promoter sequence) known in the art. The regulatory sequences described herein are nucleic acid sequences that regulate the expression of a nucleic acid sequence. Regulatory or control sequences may include sequences involved in the expression of a particular nucleic acid or may include other sequences, such as heterologous, synthetic, or partially synthetic sequences. Sequences may be of eukaryotic, prokaryotic, or viral origin that stimulate or repress gene transcription 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, transcription termination sequences, polyA sites, locus control regions, and signal sequences that direct a polypeptide into the secretory pathway of a target cell. A heterologous regulatory region is a regulatory region that is not naturally associated with the nucleic acid to which it is linked and which is to be expressed. Heterologous regulatory regions include regulatory regions derived from different species, regulatory regions derived from different genes, hybrid regulatory sequences, and non-naturally occurring regulatory sequences, which may be designed by one of skill in the art.

[0256] Typical cells producing payload-containing ARMM The microvesicle-producing cells of the present invention may be cells containing any of the expression constructs, any of the fusion proteins, or any of the molecular payloads (e.g., biomolecules, small molecules, proteins, and nucleic acids described herein). For example, the microvesicle-producing cells of the present invention may contain one or more recombinant expression constructs encoding (1) the ARRDC1 protein or a variant thereof having a PSAP (SEQ ID NO: 1) motif, and (2) an RNA-binding protein (e.g., Tat protein) that binds to the ARRDC1 protein or a variant thereof having a PSAP (SEQ ID NO: 1) motif. In some embodiments, the microvesicle-producing cells may contain one or more recombinant expression constructs under the control of a heterologous promoter that encode (1) the ARRDC1 protein or a variant thereof having a PSAP (SEQ ID NO: 1) motif, and (2) a payload protein, e.g., an RNA-binding protein fused to at least one WW domain, or a variant thereof. In certain embodiments, the expression construct in the microvesicle-producing cell encodes a payload protein or variant thereof having one or more WW domains. In certain embodiments, the expression construct in the microvesicle-producing cell encodes an RNA binding protein, e.g., an RNA that associates with (e.g., specifically binds to) a therapeutic RNA.

[0257] Any of the expression constructs described herein can be stably inserted into the genome of a cell. In some embodiments, the expression construct is maintained within the cell but is not inserted into the genome of the cell. In some embodiments, the expression construct is in a vector, e.g., 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 maintenance and / or replication of the expression construct in the microvesicle-producing cell or that enhance expression of the fusion protein in the cell. Such additional sequences or elements include, for example, an origin of replication, an antibiotic resistance cassette, a polyA sequence, and / or a transcriptional isolator. Some expression constructs suitable for generating microvesicle-producing cells according to aspects of the invention are described elsewhere herein. Methods and reagents for generating additional expression constructs suitable for generating microvesicle-producing cells according to aspects of the invention will be apparent to those skilled in the art based on this disclosure. In some embodiments, the microvesicle-producing cell is a mammalian cell, e.g., a mouse cell, a rat cell, a hamster cell, a rodent cell, or a non-human primate cell. In some embodiments, the microvesicle-producing cells are human cells.

[0258] Those skilled in the art have access to conventional techniques, such as molecular or cell biology, virology, microbiology, and recombinant DNA techniques, and typical techniques are explained fully in the literature. For example, the present invention can be practiced and used with the aid of the following general texts: 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 (R.I. 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), FM Ausubel et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (updates through 2001), Colligan et al., (eds.), Current Protocols in Immunology, John Wiley & Sons, Inc.(updates through 2001), W. Paul et al., (eds.) Fundamental Immunology, Raven Press; EJ Murray et al., (ed.) Methods in Molecular Biology: Gene Transfer and Expression Protocols, The Humana Press Inc. (1991) (especially vol.7), and JE Celis et al., Cell Biology: A Laboratory Handbook, Academic Press (1994). .

[0259] Delivery of ARMM containing payload molecules The microvesicles of the present invention (e.g., ARMM containing any of the expression constructs and / or any of the molecular (e.g., therapeutic drugs, biomolecules, small molecules, proteins, and nucleic acid (e.g., DNA, RNA), DNA plasmids, siRNA, shRNA, mRNA) payloads) may optionally further comprise a targeting moiety. The targeting moiety can be used to target the delivery of the ARMM to a specific cell type, resulting in release of the contents of the ARMM into the cytoplasm of the specific target cell type. The targeting moiety may be a viral envelope protein or portion thereof, which normally functions to aid in viral attachment and entry into cells. The viral envelope protein may enable 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). The VSV-G protein promotes viral entry by mediating viral attachment to the LDL receptor (LDLR) or LDLR family members present on target cells. After binding, the VSV-G-LDLR complex is rapidly endocytosed and subsequently mediates fusion of the viral envelope with the endosomal membrane. VSV-G enters cells via partially clathrin-coated vesicles, and virus-containing vesicles contain more clathrin and clathrin adaptors than conventional vesicles. VSV-G is a common coat protein in vector expression systems used to introduce genetic material into in vitro systems or animal models, primarily due to its extremely broad tropism. RVG is a trimeric, surface-exposed viral coat protein known to use nicotinic acetylcholine receptors and low-affinity nerve growth factor receptors for viral entry. In some embodiments, viral envelope proteins (e.g., VSV-G, RVG) promote the binding (e.g., targeting) of ARMM to target cells.

[0260] The targeting moiety can selectively bind to a surface antigen of a target cell. For example, the targeting moiety can be a membrane-bound immunoglobulin, integrin, receptor, receptor ligand, aptamer, small molecule, or variant thereof. Any number of cell surface proteins can also be included in the ARMM to promote binding of the ARMM to the target cell and / or promote uptake of the ARMM into the target cell. Integrins, receptor tyrosine kinases, G protein-coupled receptors, and membrane-bound immunoglobulins suitable for use with embodiments of the present invention will be apparent to those skilled in the art, and the present invention is not limited in this respect. For example, in some embodiments, the integrin is α1β1, α2β1, α4β1, α5β1, α6β1, αLβ2, αMβ2, αIIbβ3, αVβ3, αVβ5, αVβ6, or α6β4 integrin. In some embodiments, the receptor tyrosine kinase is 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, secretin receptor, metabotropic glutamate / pheromone receptor, cyclic AMP receptor, frizzled / smoothened receptor, CXCR4, CCR5, or beta adrenergic receptor.

[0261] Additional molecules, such as synthetic small molecules or natural products, can be modified to be linked to ARMM proteins (for example, TSG101 or ARRDC1) for targeting purposes.This linkage can promote their incorporation into ARMM, and can then be used to increase the delivery of ARMM to target cells.The incorporation of a cleavable linker can be used to release small molecules when delivered into or to target cells.As a non-limiting example, small molecules can be linked to biotin, so that they can be linked to ARRDC1 proteins that are fused to streptavidin. As another non-limiting example, a small molecule can be linked to a 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 binds to an ARRDC1 protein fused to FKBP12(F36V). Linking the small molecule to an ARMM protein (e.g., TSG101 or ARRDC1) facilitates loading of the small molecule into an ARRDC1-containing ARMM.

[0262] Some aspects of the present invention relate to the recognition that the ARMM is taken up by target cells (e.g., cells of the retina, including retinal pigment epithelial cells, photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, and / or ganglion cells), and that uptake of the ARMM results in the release of the contents of the ARMM into the cytoplasm of the target cell. In some embodiments, the payload is an agent that brings about a desired change in the target cell, such as cell survival, a change in 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 transcription profile, or a post-transcriptional change in gene compaction of the target cell, and the like. Those skilled in the art will understand that the agent to be delivered will be selected according to the desired effect in the target cell (e.g., a base editor).

[0263] In some embodiments, cells of a subject are obtained and a payload is delivered to the cells ex vivo using the systems or methods provided herein. In some embodiments, the treated cells are selected for cells in which a desired gene is expressed or repressed. In some embodiments, the treated cells with the desired payload are returned to the subject from which the cells were obtained.

[0264] In some embodiments, the ARMM further comprises a detectable label. In certain embodiments, the detectably labeled ARMM allows target cells to be labeled 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, such as membrane-bound or cytosolic enzymes, which catalyze a reaction that produces a detectable reaction product. Suitable detectable labels according to some aspects of the present invention further include membrane-bound antigens, such as membrane-bound ligands, which can be detected with commonly available antibodies or antigen-binding agents. Detectably labeled ARMMs are used in a variety of diagnostic and analytical methods and applications.

[0265] In some embodiments, ARMMs are provided that include payload RNAs encoding transcription factors, transcription repressors, fluorescent proteins, kinases, phosphatases, proteases, ligases, chromatin modulators, recombinases, and the like. In some embodiments, ARMMs are provided that include payload RNAs that inhibit expression of transcription factors, transcription repressors, fluorescent proteins, kinases, phosphatases, proteases, ligases, chromatin modulators, or recombinases. In some embodiments, the payload RNA is a therapeutic RNA. In some embodiments, the payload RNA is an RNA that causes 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, transcription repressors, fluorescent proteins, kinases, phosphatases, proteases, ligases, chromatin modulators, recombinases, and reprogramming factors can be encoded by payload RNAs that associate with binding RNAs to promote their incorporation into the ARMM, and their function can be tested by any method known to those of skill in the art; the invention is not limited in this respect.

[0266] Methods for isolating the ARMM described herein are also provided. One exemplary method involves utilizing conventional techniques to collect culture medium or supernatant from a cell culture containing microvesicle-producing cells. In some embodiments, the cell culture comprises cells obtained from a subject, e.g., cells suspected of exhibiting a pathological phenotype, e.g., a hyperproliferative phenotype. In some embodiments, the cell culture comprises genetically engineered cells that produce ARMM, e.g., cells expressing a recombinant protein, e.g., a recombinant ARRDC1 or TSG101 protein, e.g., an ARRDC1 or TSG101 protein optionally fused to an RNA-binding protein (e.g., Tat protein) or a variant thereof. In some embodiments, the supernatant is precleared of cellular debris by centrifugation, e.g., by two successive centrifugations at increasing G-values ​​(e.g., 500G and 2000G). In some embodiments, the method includes passing the supernatant through a 0.2 μm filter to eliminate all large cellular debris and whole cells. In some embodiments, the supernatant is subjected to ultracentrifugation, for example, at 120,000 G for 2 hours, depending on the volume of the centrifugal mass. The resulting pellet contains microvesicles. In some embodiments, exosomes are depleted from the microvesicle pellet by staining and / or sorting (for example, by FACS or MACS) using the exosome markers described herein. The isolated or enriched ARMM can be suspended in culture medium or suitable buffer as described herein.

[0267] Methods for ARMM-mediated delivery of payloads to cells Some aspects of the present invention provide methods for delivering agents (e.g., one or more therapeutic agents) to target cells in the eye. The target cells can be contacted with the ARMM in various ways. For example, the target cells can be directly contacted with the ARMM described herein or with ARMM isolated from microvesicle-producing cells. The contact can be in vitro by administering the ARMM to the target cells in a culture dish, or in vivo by administering the ARMM to a subject. In some embodiments, the ARMM is produced from cells obtained from a subject. In some embodiments, the ARMM produced from cells obtained from a particular subject is administered to the same subject. Conversely, in some other embodiments, the ARMM produced from cells obtained from a subject is administered to a different subject. As an example, cells can 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 linked to 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, a guide sequence and a regulatory sequence, and the like).

[0268] Alternatively, target cells may be contacted with the microvesicle-producing cells described herein, for example, by co-culturing the target cells and the microvesicle-producing cells in vitro, or by administering the microvesicle-producing cells to a subject having the target cells in vivo.Thus, the method may include contacting the target cells with microvesicles, for example, with an ARMM containing any of the payloads to be delivered described herein.Target cells may be contacted with the microvesicle-producing cells described herein, or with isolated microvesicles having a lipid bilayer, an ARRDC1 protein or a variant thereof, a payload (e.g., a therapeutic agent), and optionally a viral envelope protein.

[0269] It should be understood that the target cell can be of any origin, e.g., from an organism. In some embodiments, the target cell is a mammalian cell. Some non-limiting examples of mammalian cells include, but are not limited to, mouse cells, rat cells, hamster cells, rodent cells, and non-human primate cells. In some embodiments, the target cell is a human cell. It should also be understood that the target cell can be of any cell type, and may preferentially be an ocular cell. In other cases, the target cell can be any differentiated cell type found in a subject. In some embodiments, the target cell is an in vitro cell, and the method comprises administering microvesicles to the cell in vitro or co-culturing the target cell with microvesicle-producing cells in vitro. In some embodiments, the target cell is a cell within a subject, and the method comprises administering microvesicles or microvesicle-producing cells to the subject. In some embodiments, the subject is a mammalian subject, e.g., a rodent, mouse, rat, hamster, or non-human primate. In some embodiments, the subject is a human subject.

[0270] In some embodiments, the target cells are pathological cells. In some embodiments, the target cells are cancer cells. In some embodiments, the microvesicles are associated with a binding agent that selectively binds an antigen on the surface of the target cells. In some embodiments, the compositions and methods of the present invention comprise one or more targeting ligands that bind (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, integrin, receptor, receptor ligand, or lectin, among other suitable candidate molecules and moieties. Suitable surface antigens of target cells (e.g., ocular cells) 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, e.g., membrane-bound immunoglobulins, membrane-bound antibodies, or antibody fragments that specifically bind surface antigens expressed on the surface of cells, are also known to those of skill in the art. The choice of binding agent will, of course, depend on the identity or type of the target cell. Those skilled in the art will recognize cell surface antigens that are specifically expressed on various types of tissues and cells that can be targeted by ARMMs, including membrane-bound binding agents, and it will be understood that the invention is not limited in this respect. [Example]

[0271] Example 1: Production of ARMM Expi293™ cells (Thermo Fisher Scientific, Waltham, MA) were seeded in 10 ml of Expi293™ Expression Medium (Thermo Fisher Scientific) at a density of 36 / ml. For each 10 ml of cells, 5 μg of ARRDC1-Cre plasmid (SEQ ID NO: 48) and 5 μg of VSVG plasmid (SEQ ID NO: 49), both shown below, were mixed with 600 μl of OptiMEM™ (Thermo Fisher Scientific) for less than 5 minutes. 33 μl of expifectamine was mixed with 567 μl of OptiMEM™ and then mixed with the DNA and OptiMEM™ mixture. The sample was left at room temperature for 10 minutes and then added to the Expi™ cells for approximately 16–18 hours. After incubation, the medium was changed the next morning, and the cells were centrifuged at 500 × g for 5 minutes and replated in a final volume of 40 ml of medium. The cells were incubated for an additional 48 hours.

[0272] After 48 hours of incubation, the cells were placed in a 50 ml centrifuge tube and centrifuged at 500 x g for 5 minutes. The supernatant was removed and centrifuged again at 2000 x g for 10 minutes. The supernatant was passed through a 0.22 μM vacuum filter. The supernatant was placed in a 38.5 ml ultracentrifuge tube (Beckman-Coulter Life Sciences, Brea, CA) and spun at 174,900 x g for 2 hours at 4°C. The supernatant was removed, and the pellet was resuspended in PBS and quantified by nanoparticle tracking analysis.

[0273] ARRDC1-Cre sequence

[0274] [ka]

[0275] [ka]

[0276] VSVG array

[0277] [ka]

[0278] Nanoparticle Tracking Analysis ARMM was analyzed and quantified by a ZetaView® instrument (Particle Metrix GmbH, Inning am Ammersee, Germany). Samples containing vesicles were diluted with phosphate-buffered saline (PBS). After dilution, the samples were subjected to nanoparticle tracking analysis.

[0279] [Example 2] Evaluation of subretinal injection of ARMM in Goettingen minipigs Briefly, the intraocular biodistribution of ARMM via subretinal delivery was evaluated in a Göttingen minipig model. Adult Göttingen minipigs were subretinal injected with green fluorescent protein-loaded ARMM (ARRDC1-GFP-ARMM). (SEQ ID NO: 50 and SEQ ID NO: 51). Dose extrapolation from a previous mouse subretinal study was comparable to the potential human dose, given the comparable size, physiology, and anatomy of pig and human eyes. For histological and immunohistochemical evaluation, a time-course analysis was performed by harvesting eyes 6, 12, and 24 hours after subretinal injection of ARMM. GFP staining was used as a surrogate for ARMM uptake. Co-staining of GFP with the following cell type-specific markers was examined: RPE65 for retinal pigment epithelium (RPE), rhodopsin for rod photoreceptors, and L / M opsin for cone photoreceptors, respectively. Wide-field color fundus imaging and confocal scanning laser ophthalmoscopy (cSLO) imaging were also performed as in vivo readouts for GFP signal. Eyes from uninjected animals were used as negative controls for GFP immunohistochemistry. Eye sections were evaluated for colocalization of GFP with cell-type-specific markers and histopathological examination. Detailed data on GFP staining in ocular structures are presented as a function of time.

[0280] ARRDC1-GFP

[0281] [ka]

[0282] ARRDC1-GFP

[0283] [ka] (ARRDC1: underlined, linker: double underlined, GFP: dashed underline).

[0284] Surgical administration procedure Test animals were anesthetized and placed in the lateral decubitus position. Topical proparacaine was applied to the eye. The conjunctival fornix was flushed with a 1:50 dilution of betadine / saline, and the eyelid margin was wiped with undiluted 5% betadine solution. One eye was draped and a wire speculum was placed. A lateral canthotomy was performed using Stevens tendon scissors. A caliper was used to mark spots on the superior and inferior temporal sclera approximately 3 mm posterior to the limbus. A bipolar cautery was used to cauterize the sclera below the marked spots, followed by topical administration of undiluted 5% betadine solution. While the eye was held in place using intrascleral forceps, a vitreoretinal microblade and bulbed cannula were inserted into each of the marked spots and pushed through the conjunctiva and sclera into the vitreous humor. The trocar was placed facing the posterior axis of the globe and removed, leaving a scleral port. A 31 g needle was inserted tangentially through the limbus into the anterior chamber to remove 100 μL of aqueous humor. A surgical direct contact lens was placed on the cornea with sterile coupling gel. To facilitate direct visualization of the posterior segment via microscope, an intraocular illumination probe was inserted through one of the scleral ports. A subretinal injection cannula was inserted through the second port and advanced to the central vitreous. A small-diameter injection cannula was then advanced until it contacted the retinal surface. ARMM particles (i.e., ARRDC1-GFP) were then slowly delivered to induce and fill a subretinal bleb. Once adequate bleb formation was observed, injection was continued until the entire dosage volume (e.g., 50 μL / bleb) was delivered to the subretinal space. If no bleb formation was observed, a smaller diameter injection cannula was repositioned at the same location or a different location depending on the surgeon's preference, and the injection was attempted again. Subretinal images were taken to capture the location of the bleb.

[0285] Once the injection dose was delivered, the injection cannula and intraocular illumination probe were removed from the scleral port, and the contact lens was removed from the cornea. The scleral port was then removed. The lateral canthotomy site was closed using 7-0 Vicryl suture. All time functions were based on the last injection in the last medicated eye. The same injection procedure was performed on the contralateral eye.

[0286] In one embodiment demonstrating the technology in this example, four female and four male naive Göttingen minipig test animals (Marshall Bioresources, North Road, NY) at least four months old at the time of the study were divided equally into four groups of two and subretinally dosed with ARRDC1-GFP-ARMM according to Table 1 below.

[0287] [Table 8]

[0288] Pre-surgical procedures during survival were performed in accordance with standard operating procedures for the study facility. Study animals were housed, fed, watered, identified, and handled according to standard USDA protocols and methods before and during the study.

[0289] Postoperative procedures Postoperative procedures were performed according to standard operating procedures for the study facility. Incision site verification and pain scoring were not required after this minimally invasive procedure. Ophthalmological endpoints were adjudicated by an ophthalmologist.

[0290] Fundus examination was performed by an ophthalmologist via indirect ophthalmoscopy and slit-lamp biomicroscopy. Uveitis was scored at the time of eye examination. Wide-field color fundus imaging was performed in anesthetized animals using a Clarity RetCam Shuttle (MediMark, Grenoble, France). Confocal scanning laser ophthalmoscopy (cSLO) imaging and green fluorescent protein (GFP) imaging were performed in anesthetized animals using a Spectralis HRA / OCT system (Heidelberg Engineering Inc., Franklin, MA).

[0291] Euthanasia procedure Euthanasia was performed by administration of euthanasia solution, under sedation as necessary, followed by facility-approved terminal procedures as required. Sacrificial procedures are outlined in Table 2 below.

[0292] [Table 9]

[0293] "Histological processing" included embedding in paraffin, sectioning, mounting on glass slides, and staining with hematoxylin and eosin.

[0294] Samples from control eyes (i.e., Group 1) were collected separately from animals receiving the test substance. Eyes from all animals were collected for ocular histology. After sacrifice, eyes were permanently marked with a 12 o'clock orientation. Eyes were enucleated along with the proximal optic nerve. After enucleation, the orientation was remarked. Right and left eyes were individually labeled and stored in Davidson's fixative for 24-48 hours, followed by storage in 70% ethanol for up to an additional 72 hours.

[0295] Ocular histology was performed on trimmed eyes with a single sagittal cut toward the optic nerve. Typically, left eyes were trimmed with a single sagittal cut at the optic nerve in a plane containing the optic nerve head. Typically, right eyes were trimmed with a single cut superior to the optic nerve in a plane containing both optic nerve heads. The bisected globe containing the injected bleb was placed cut-side down in a cassette. The lens and transverse sections of the optic nerve were placed in separate blocks. If necessary, eyes were step-sectioned to include sections of the subretinal injected bleb. Sections for each block were collected as follows: Level 1 (approximately within the eye, until the optic disc was 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 obtained approximately 500 microns past level 1), level 3 (sections obtained approximately 500 microns past level 2), level 4 (sections obtained approximately 500 microns past level 3), level 5 (sections obtained approximately 500 microns past level 4), level 6 (approximately until the entire surface of the tissue is obtained, collected after full exposure of the second block), level 7 (sections obtained approximately 500 microns past level 6), level 8 (sections obtained approximately 500 microns past level 7), level 9 (sections obtained approximately 500 microns past level 8), and level 10 (sections obtained approximately 500 microns past level 9). Each level consisted of one slide stained with hematoxylin and eosin and eight unstained slides. Blocks containing only the lens and optic nerve were initially collected with hematoxylin and eosin alone.

[0296] The collected tissue was then histopathologically evaluated by light microscopy using standard procedures and protocols, using dyes and other molecules to highlight cells and tissue regions of interest (e.g., RPE65 (retinal pigment epithelial cells), L / M opsin (cone outer segments), rhodopsin (rod outer segments), and the like).

[0297] Tissues were then selected for immunohistochemical analysis. These tissues were stored in Davidson's fixative or other designated fixative for 24-48 hours. Tissues may also be kept in 70% ethanol for up to an additional 72 hours before processing into paraffin.

[0298] The results of this example are shown in FIGS.

[0299] [Example 3] Evaluation of subretinal injection of ARMM in non-human primates In this example, non-human primates, specifically African green monkeys (Chlorocebus sabaeus) from St. Kitts, were selected for subsequent use in a subretinal injection dosing study of ARRDC1-mCherry-ARMM (SEQ ID NO: 52 and SEQ ID NO: 53) using baseline health assessment techniques, including complete blood count (CBC), general health, and ocular health via tonometry, slit-lamp biomicroscopy, fundusoscopy, and color and fluorescent fundus imaging. 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 mydriasis was performed with topical 10% phenylephrine, 1% tropicamide, and / or 1% cyclopentolate. Animals with normal findings were enrolled in the dosing study and assigned to treatment groups as described in Table 3.

[0300] [Table 10]

[0301] ARRDC1-mCherry

[0302] [ka]

[0303] [ka] (ARRDC1: underlined, linker: double underlined, mCherry: dashed underline)

[0304] Surgical Medication Procedures One day before subretinal dosing, topical 1% atropine gel was administered to each eye. Following this procedure, on the day of dosing, topical 10% phenylephrine, 1% tropicamide, and / or 1% cyclopentolate were administered to the incompletely dilated eye. Test animals received a single subretinal injection in both eyes (OU) according to the treatment assignments in Table 3. After placement of an eye speculum, proparacaine hydrochloride 0.5% was administered dropwise. After 30 seconds, 5% Betadine® solution was administered, followed by a rinse with sterile saline. A sterile eye drape and eye speculum were then placed on each animal. A 25-gauge vitrectomy port, Alcon® Surgery Valved Entry System 1-CT (Alcon Surgery Inc., Fort Worth, TX), was placed through the port introducer device in the superior temporal quadrant at the level of the ora serrata. A second vitrectomy port was placed in the inferior temporal quadrant at the level of the ora serrata. After placement, a vitrectomy contact lens was placed on the center of the cornea using 0.9% saline as a coupling agent. Once temporarily positioned by the surgeon, a 25-gauge light pipe was inserted into the vitreous cavity through the left vitrectomy port to provide intraocular illumination while maintaining the tip within the anterior vitreous. A subretinal cannula, MedOne Surgical 25 / 38g, part number 3237 (MedOne Surgical Inc., Sarasota, FL), attached to a MedOne microfluidic injector, part number 3275, interfaced with an Alcon Constellation Vision System, was introduced through the second vitrectomy port. Aiming for a point within the superior temporal region approximately two papillary diameters above the fovea, a 38-gauge flexible microtip was advanced until it gently touched the retinal surface. When slight whitening of the retinal surface at the point of contact was observed, the test substance was injected with sufficient pressure to raise the bleb, which was then adjusted to maintain a pressure between 4 and 6 mmHg. The threshold pressure was set at 14 mmHg. Once the first bleb appeared, the target volume (Table 1) of test substance was administered. The surgical instruments were removed after injection, and the sclerotomy was allowed to self-close.Postoperative fundus imaging was performed to record the location and diameter of the subretinal bleb, after which topical antibiotic ointment was applied to the eye.

[0305] Postoperative procedures Intraocular pressure (IOP) was measured in the OU using a Tonovet Plastonometer (iCare Inc., Raleigh, NC) set to the cat(c) calibration setting. Three measurements were obtained from each eye, and the mean IOP was calculated.

[0306] Both eyes were examined by slit-lamp biomicroscopy and retinoscopy using a 90-diopter lens. Using a non-human primate ocular scoring system, scoring was applied to the qualitative clinical findings of the eye, and a summary score was derived from the examination components.

[0307] Detection of GFP expression in the OU using color imaging of the anterior segment and fundus, as well as fluorescent fundus imaging, was performed in a 50° field centered on the macula. Additional peripheral images were obtained 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 photographs were captured at a shutter speed (Tv) of 1 / 25 s, ISO 400, and flash 18. Monochrome and color fluorescent images were captured with inset excitation and barrier filters (587 nm excitation filter / 610 nm barrier filter), Tv 1 / 5 s, ISO 3200, and flash 300. Quantitative analysis was applied where appropriate to evaluate the fluorescent photographs using a scoring system that defines the degree of mCherry expression, where the scores are 0=none, 1=very slight, 2=slight, 3=moderate, 4=bright, and 5=intense in the foveal, peripheral, and perivascular regions of the eye, areas where blebbing is observed after medication.

[0308] The head, trunk, limbs, and integument of each test animal were evaluated, auscultated, and vitals assessed, including body temperature, and heart and respiratory rates measured manually over a 15-second period.

[0309] 0.5 mL of blood was transferred to a K2EDTA lavender-top vacutainer, gently inverted several times, and kept on ice for complete blood count with differential analysis on an Abaxis VetScan HM5 hematology system (Abaxis, Inc., Union City, CA).

[0310] Euthanasia procedure Test animals were euthanized at the end of the study with ketamine (8 mg / kg, IM) and xylazine (1.6 mg / kg, IM), followed by intravenous administration of sodium pentobarbital (100 mg / kg) over approximately 10 seconds to effect. After the corneal reflex disappeared and before sample collection, the animals were exsanguinated by incising the caudal vena cava, after which the diaphragm was immediately punctured to relieve the intrathoracic negative pressure.

[0311] A postmortem veterinary examination of the carcass, external body orifices, abdominal, thoracic, and cranial cavities, organs, and organs, including the external characteristics of the tissues, was performed to identify and record any gross abnormalities or pathology.

[0312] After placing a suture at the 12 o'clock position on the limbus, both eyes were enucleated, trimmed of excess orbital tissue, and fixed by immersion in Davidson for 24 hours, after which they were transferred to phosphate-buffered saline with 0.05% sodium azide.

[0313] Fixed eyeballs were embedded in paraffin and sectioned into 20 horizontal sections spanning the peripheral and macular regions of the retina and stained with hematoxylin and eosin for analysis by a qualified veterinary pathologist. Prior to treatment, the eyeballs were trimmed in a sagittal fashion into three sections, with the middle section containing the pupil-optic nerve.

[0314] The eye specimens were sectioned with a microtome as follows: the nasal and temporal hemispheres (calottes) and the optic nerve at three levels with a minimum of 80 μm between levels. One slide was collected at each level. The pupil-optic nerve was sectioned at five levels with a minimum of 80 μm between levels across the macular and peripheral regions of the retina, with 10 slides collected at each level. One slide from each level was stained with hematoxylin and eosin.

[0315] Using purposeful, existing tissue samples, a monoplex immunofluorescence assay for mCherry was optimized for specificity and sensitivity, providing a clear signal:noise distinction. One slide of each pupil-optic nerve section was selected for mCherry / cell marker duplex immunofluorescence. All staining and assays were performed according to standard laboratory procedures and quality checked by microscopy.

[0316] 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 a 20x objective (original magnification 49x) and quality checked to ensure complete images were captured and that no noticeable scanning artifacts were present.

[0317] The results of this example are shown in FIGS.

[0318] References All publications, patents, and sequence database entries referred to herein, including those 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 will control, including all definitions herein.

[0319] Equivalents and Scope 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 limited to the above description, but rather is as set forth in the appended claims.

[0320] In the claims, articles such as "a," "an," and "the" can mean one or more than one, unless indicated otherwise or clear from the context. Unless indicated otherwise or clear from the context, a claim or description containing "or" between one or more members of a group is considered satisfied if one, more than one, or all of the group members are present in, utilized in, or otherwise relevant to a given product or process. The invention includes embodiments in which exactly one member of a group is present in, utilized in, or otherwise relevant to a given product or process. The invention also includes embodiments in which two or more or all of the group members are present in, utilized in, or otherwise relevant to a given product or process.

[0321] Furthermore, although terms such as "first," "second," etc. may be used herein to describe various elements, these elements are not limited by these terms. These terms are used only to distinguish one element from another. Thus, a "first" element discussed below could also be described as a "second" element without departing from the teachings of the present disclosure. The order of operations (or acts / steps) is not limited to the order shown in the claims or drawings unless specifically indicated otherwise.

[0322] Furthermore, it is understood that the present 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 the relevant portion of this specification are introduced into another claim. For example, any claim that is dependent on another claim can be amended to include one or more limitations set forth in any other claim that is dependent on the same base claim. Furthermore, when a claim recites a composition, it is understood that it includes methods of using the composition for any purpose disclosed herein, and methods of making the composition according to the manufacturing methods disclosed herein or other methods known in the art, unless otherwise indicated or unless a contradiction or inconsistency would arise.

[0323] Where elements are presented as a list, e.g., in Markush group format, it is understood that each subgroup of elements is also disclosed, and that any element can be excluded from the group. Note that the term "comprising" is intended to be open, allowing for the inclusion of additional elements or steps. Generally, where the invention, or aspects of the invention, are described as including certain elements, features, steps, etc., it should be understood that certain embodiments of the invention or aspects of the invention consist of or consist essentially of such elements, features, steps, etc. For purposes of simplicity, these embodiments have not been specifically set forth in these exact words herein. Thus, for each embodiment of the invention that includes one or more elements, features, steps, etc., the invention also provides embodiments that consist of or consist essentially of these elements, features, steps, etc.

[0324] When ranges are stated, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and / or the understanding of one skilled in the art, it is understood that values ​​expressed as ranges can refer to any specific value within the stated range in different embodiments of the present invention, up to one-tenth of the lower limit of that range, unless otherwise clearly indicated from the context. Unless otherwise indicated or otherwise apparent from the context and / or the understanding of one skilled in the art, values ​​expressed as ranges can refer to any subrange within the given range, where the endpoints of the subrange are expressed to the same degree of accuracy as one-tenth of the lower limit of that range.

[0325] Furthermore, it is understood that any embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where a range is given, any value within that range may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, use, or aspect of the compositions and / or methods of the present invention may be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects are excluded are not explicitly set forth herein.

Claims

1. Arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMM), (i) a lipid bilayer and the ARRDC1 protein; (ii) a molecule, and (iii) viral envelope proteins The microvesicles comprising:

2. 2. The microvesicles of claim 1, wherein the viral envelope protein is vesicular stomatitis virus G (VSV-G).

3. 2. The microvesicles of claim 1, wherein the viral envelope protein is rabies virus glycoprotein (RVG).

4. a recombinant expression construct encoding the ARRDC1 protein or a variant thereof under the control of a heterologous promoter; and viral envelope proteins A microvesicle-producing cell comprising:

5. The microvesicle-producing cell of claim 4, wherein the viral envelope protein is VSV-G.

6. 5. The microvesicle-producing cell of claim 4, wherein the viral envelope protein is RVG.

7. 10. A method of delivering a molecule to a target cell, the method comprising contacting the target cell with the microvesicles of any one of claims 1 to 3.

8. The method of claim 7 , wherein the target cell is an ocular cell.

9. The method of claim 8 , wherein the ocular cell is a retinal cell.

10. 10. A method of treating a disorder in a patient, comprising administering to the patient microvesicles according to any one of claims 1 to 3.

11. 10. A method of treating a disorder in a patient, the method comprising administering to the patient microvesicle-producing cells according to any one of claims 4 to 6.

12. 12. The method of claim 10 or 11, wherein the disorder is an eye disorder.

13. Arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMM), (i) a lipid bilayer and an ARRDC1 protein; and (ii) therapeutic agents The microvesicles comprising:

14. 12. The method of claim 10 or 11, wherein the patient is a mammal.

15. 15. The method of claim 14, wherein the mammal is a primate.

16. 16. The method of claim 15, wherein the primate is a human.

17. Arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMM) for use in drugs.

18. Arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMM) for use in the treatment of ocular diseases.

19. 19. The use of the arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMM) of claim 18, wherein the disease is Stargardt's macular dystrophy or age-related macular degeneration.

20. A composition substantially as shown and described.

21. A process substantially as shown and described.

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