ARRDC1-mediated microvesicle-based delivery to the nervous system

Abstract

Methods, systems, compositions and strategies for the use of ARMM-mediated delivery of molecules (e.g., biological molecules, small molecules, proteins and nucleic acids (e.g., DNA, RNA), DNA plasmids, shRNA, mRNA) to cells of the nervous system (e.g., central nervous system and peripheral nervous system).

Description

Technical Field

[0001] Related Applications This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 63 / 038,461, filed on June 12, 2020, which is incorporated herein by reference.

Background Art

[0002] Delivery of molecules (e.g., biological molecules such as proteins and nucleic acids (e.g., DNA, RNA including DNA plasmids, shRNA, and mRNA), and small molecules) to cells of the central nervous system (「CNS」) and peripheral nervous system (PNS) is limited by several factors, including low target specificity and difficulty targeting terminally differentiated cells that are no longer dividing. Thus, there is a need to develop methods, compositions, and systems for effectively delivering therapeutic proteins, nucleic acids (such as RNA), and small molecules to the nervous system.

Summary of the Invention

[0003] This invention relates to the discovery that molecules such as proteins and nucleic acids containing ribonucleic acid (RNA), as well as small molecules, can be loaded onto microvesicles, specifically ARRDC1-mediated microvesicles (ARMMs), for delivery to cells of the nervous system, specifically the CNS and PNS. ARMMs can incorporate viral envelope proteins to enable the delivery of molecules to the nervous system. For example, vesicular stomatitis virus G protein (VSV-G) or rabies virus glycoprotein (RVG) can be co-expressed and manifested on the surface of ARMMS to target cells of the CNS and PNS. These proteins typically function to facilitate viral attachment and entry into cells. For example, VSV-G is known to mediate viral attachment to LDL receptors (LDLRs) or LDLR family members, and RVG is known to use nicotinic acetylcholine receptors and low-affinity nerve growth factor receptors for viral entry. These proteins have also been found to help ARMMs attach to cells containing cells of the nervous system.

[0004] Furthermore, the ARMM delivery systems described herein address many of the limitations of current delivery systems that hinder the safe and efficient delivery of proteins and nucleic acids (e.g., RNA, encompassing both protein-coding and non-coding RNAs) to CNS and PNS cells. Because ARMMs derive from endogenous budding pathways, they are less likely to induce a strong immune response, unlike viral delivery systems known to trigger inflammatory responses (Sen et al., “Cellular unfolded protein response against viruses used in gene therapy.” Front Microbiology. 2014;5:250,1-16.). In addition, ARMMs enable the specific packaging of any molecule (e.g., proteins or nucleic acids (e.g., DNA plasmids, mRNA, miRNA, or shRNA), or biological molecules such as small molecules). These molecules can then be delivered by fusion or uptake by specific recipient cells / tissues by incorporating antibodies or other types of molecules that recognize tissue-specific markers into the ARMM.

[0005] ARMMs are microvesicles distinct from exosomes and are produced by direct plasma membrane budding (DPMB), similar to 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 by its arrestin domain. ARMMs are described in detail, for example, by Lu et al., filed on February 6, 2013 (published on August 15, 2013, as International Publication No. 2013 / 119602), PCT application number PCT / US2013 / 024839, titled "Arrdc1-Mediated Microvesicles (ARMMs) and Uses Thereof," as well as in U.S. Patents 9,737,480, 9,816,080, 10,260,055, and International Publication No. 2018 / 067546, the full contents of which are incorporated herein by reference. The ARRDC1 / TSG101 interaction results in the relocation of TSG101 from endosomes to the plasma membrane, mediating the release of microvesicles containing TSG101, ARRDC1, and other cellular components as well as the molecule of interest.

[0006] The target molecule, whether a protein, nucleic acid, or small molecule present in nature or unnaturally, can associate with one or more ARMM proteins (e.g., ARRDC1), or can be modified to associate with TSG101 or ARRDC1. This association facilitates their incorporation into the ARMM, which can then be used to deliver the desired payload to target cells. For example, but not limited to, the payload RNA can be fused to the transactivation response (TAR) element, thereby associating with an ARRDC1 protein fused to an RNA-binding protein such as the Tat protein. Alternatively, the payload protein can be fused to one or more WW domains that associate with the PPXY (SEQ ID NO: 2) motif of ARRDC1. This association of the target molecule to an ARMM protein (e.g., ARRDC1) facilitates the loading of the molecule into an ARRDC1-containing ARMM. Alternatively, the molecule can be fused to an ARMM protein (e.g., TSG101 or ARRDC1) to load the payload into the ARMM. The molecule can be fused to an ARMM protein (e.g., TSG101 or ARRDC1) via a linker that can be cleaved upon delivery to the target cell.

[0007] Similarly, synthetic or native small molecules can be modified to associate with ARMM proteins (e.g., TSG101 or ARRDC1). This association can facilitate their incorporation into ARMM, allowing the molecules to be used for delivery to target cells. The incorporation of a cleavable linker may be used to release such molecules upon delivery to target cells. As a non-limiting example, small molecules can be linked to biotin, thereby allowing them to associate with ARRDC1 proteins fused to streptavidin. As another non-limiting example, small molecules can ligate to synthetic high-affinity ligands that specifically bind to mutant FKBP12, such as FKBP12(F36V) (Yang W, Rozamus LW, Narula S, Rollins CT, Yuan R, Andrade LJ, Ram MK, Phillips TB, van Schravendijk MR, Dalgarno D, Clackson T, Holt DA. Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket. J Med Chem. 2000 Mar 23;43(6):1135-42), which associate with the ARRDC1 protein fused to FKBP12(F36V). The association of small molecules to ARMM proteins (e.g., TSG101 or ARRDC1) facilitates the loading of small molecules into ARRDC1-containing ARMMs.

[0008] For example, ARMM's delivery platform enables multiple cis-acting structural elements of mRNA to function in association with intracellular and secreted therapeutic agents in nervous system cells, and these structural elements include, but are not limited to, (i) 5' cap structures, (ii) 5' untranslated regions (UTRs), (iii) codon-optimized coding sequences, (iv) 3' UTRs, (v) 3' poly-A tails consisting of a series of repeating adenine nucleotides, and (vi) inclusion of cis-acting dicoding elements within the RNA transcript that are recognized by specific RNA-binding proteins and induce specific cellular localization (e.g., to neuronal synapses) (see, for example, Chin A, Lecuyer E. RNA localization: Making its way to the center stage. Biochim Biophys Acta Gen Subj. 2017 Nov;1861(11 Pt B):2956-297).

[0009] As another example, the delivery platform for ARMM enables the targeting of multiple classes of protein and mRNA-based therapeutics to nervous system cells, and these therapeutics include, but are not limited to, transmembrane proteins, cytoplasmic proteins, nuclear proteins, mitochondrial proteins, endoplasmic reticulum proteins, Golgi proteins, peroxisome proteins, lysosomal proteins, and secretory proteins.

[0010] In relation to therapeutic agents for neurological disorders, targeted expression of single-chain variable fragment (scFv) antibodies, which consist of fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins linked by a short linker peptide, is also envisioned in this application. These scFv antibodies can selectively bind to specific antigens or can be manipulated to be multifunctional by adding other protein or nucleic acid-binding domains to the fusion, for example, in the case of bispecific scFv. Alternatively, mRNA encoding both the VH and VL chains may be used. Furthermore, single-domain antibodies (sdAbs) consisting of a single monomeric variable domain can be delivered to neurological cells as mRNA.

[0011] In relation to therapeutic agents for neurological disorders, targeted expression of antigenic peptides or neoantigens, which can occur through ARMM-mediated mRNA delivery, is also considered. The delivered mRNA is translated by ribosomes to produce neoantigen protein chains, which can then be processed by proteasomes to produce neoantigens. These neoantigens can associate with other membrane-bound proteins to present themselves, thereby enabling recognition by T cell receptors on T cells or other cells of the immune system.

[0012] In some aspects of the present invention, a lipid bilayer and an arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), molecule, and viral envelope protein containing the ARRDC1 protein are provided. In other aspects, the viral envelope protein is vesicular stomatitis virus G (VSV-G) or rabies virus glycoprotein (RVG).

[0013] In some aspects of the present invention, a recombinant expression construct encoding the ARRDC1 protein or a variant thereof under the control of a heterologous promoter, and microvesicle-producing cells containing a viral envelope protein are provided. In other aspects, the viral envelope protein is vesicular stomatitis virus G (VSV-G) or rabies virus glycoprotein (RVG).

[0014] In some aspects of the present invention, a method is provided for delivering molecules to target cells by bringing the target cells into contact with the microvesicles described herein. In other aspects, the cells are of the nervous system (NS), encompassing the central nervous system (CNS) and the peripheral nervous system (PNS). In yet another aspect, the target cells are neurons, astrocytes, oligodendrocytes, or microglia.

[0015] In some aspects of the present invention, a method is provided for treating a patient's disorder by administering to the patient microvesicles or microvesicle-producing cells as described herein. In other aspects of the present invention, the disorder is a disorder of the CNS system. In yet another aspect, the disorder affects the function of neurons, astrocytes, oligodendrocytes, or microglia. In yet another aspect of the present invention, the disorder is any of gain-of-function disorder, loss-of-function disorder, or repeat elongation.

[0016] Other advantages, features, and uses of the present invention will be apparent from the detailed description, drawings, non-limiting embodiments, and claims of certain exemplary, non-limiting embodiments. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows a non-limiting schematic diagram of an ARMM-based development workflow for neurological disorders involving the use of human induced pluripotent stem cell (iPSC) models for biological and therapeutic discovery and development. Such a workflow can be adapted to serve as a platform for the discovery and development of ARMM-based technologies and applications. In this schematic diagram, arm skin biopsies are used to obtain iPSC-derived terminated (no longer dividing) neurons for use in screening and identification of ARMM-based therapeutics.

[0018] [Figure 2] Figure 2 is a non-restrictive schematic diagram for screening ARMM-mediated payload delivery using a high-content single-cell level imaging assay. In this schematic diagram, imaging is achieved by automated confocal microscopy.

[0019] [Figure 3]Figure 3 is a non-limiting schematic diagram of a workflow for designing an ARMM-based technology for development, showing a representative type of payload and non-limiting CNS target cell types. The sequences shown: PSAP (SEQ ID NO: 1), PPXY (SEQ ID NO: 2), and GGUCUCUCUGGUUAGACCAGAUCUGAGCCUGGGAGCUCUCUGGCUAACUAGGGAACC (SEQ ID NO: 57)

[0020] [Figure 4] Figure 4 shows ARMM-mediated delivery and expression of GFP mRNA in human iPSC-derived neural progenitor cells.

[0021] [Figure 5A] Figure 5A shows ARMM-mediated delivery to GFP mRNA and expression of GFP mRNA in human iPSC-derived neural progenitor cells. [Figure 5B] Figure 5B shows ARMM-mediated delivery to GFP mRNA and expression of GFP mRNA in human neurons derived from 3D iPSC brain organoids.

[0022] [Figure 6] Figure 6 shows successful ARMM-mediated delivery of an mRNA payload and translation of GFP protein in human neurons using high-content imaging. GFP protein expression, MAP2 staining, and nuclear staining represented in that imaging. As shown, ARMM enables delivery of the payload to multiple intracellular regions of neurons, including axons, dendrites, and cell bodies.

[0023] [Figure 7] Figure 7 shows successful ARMM-mediated delivery of an mRNA payload and translation of GFP protein in human neurons. The sequences shown: PSAP (SEQ ID NO: 1), PPXY (SEQ ID NO: 2), and GGUCUCUCUGGUUAGACCAGAUCUGAGCCUGGGAGCUCUCUGGCUAACUAGGGAACC (SEQ ID NO: 57)

[0024] [Figure 8-1] Figure 8 shows a non-limiting illustrative schematic diagram of the use of ARMM to target representative gain-of-function and loss-of-function mechanisms in neurogenetic disorders. These schematics illustrate non-limiting examples of how ARMM can be used to tailor personalized medicine to the cells of a patient's nervous system (neuropathology figures adapted from van der Zee J, Van Broeckhoven C. Dementia in 2013: frontotemporal lobar degeneration-building on breakthroughs. Nat Rev Neurol. 2014 Feb;10(2):70-2). Sequences shown: PSAP (SEQ ID NO: 1), PPXY (SEQ ID NO: 2), and GGUCUCUCUGGUUAGACCAGAUCUGAGCCUGGGAGCUCUCUGGCUAACUAGGGAACC (SEQ ID NO: 57) [Figure 8-2] Figure 8 shows a non-limiting illustrative schematic diagram of the use of ARMM to target representative gain-of-function and loss-of-function mechanisms in neurogenetic disorders. These schematics illustrate non-limiting examples of how ARMM can be used to tailor personalized medicine to the cells of a patient's nervous system (neuropathology figures adapted from van der Zee J, Van Broeckhoven C. Dementia in 2013: frontotemporal lobar degeneration-building on breakthroughs. Nat Rev Neurol. 2014 Feb;10(2):70-2). Sequences shown: PSAP (SEQ ID NO: 1), PPXY (SEQ ID NO: 2), and GGUCUCUCUGGUUAGACCAGAUCUGAGCCUGGGAGCUCUCUGGCUAACUAGGGAACC (SEQ ID NO: 57)

[0025] [Figure 9]This figure shows a non-limiting example of the therapeutic use of CRISPR / dCas9 activation for enhancing the expression of the human GRN gene encoding progranulin, related to the treatment of possible frontotemporal dementia caused by loss-of-function mutations in GRN or decreased progranulin expression, and applicable to ARMM delivery technology.

[0026] [Figure 10] Figure 10 shows a non-limiting example of a method for ARMM optimization of nerve system cells for FMRP delivery to rescue neurons in patients with Fragile X syndrome.

[0027] [Figure 11] Figure 11 shows a non-limiting example of an ARMM optimization method for CNS cells for FMRP delivery to rescue neurons in patients with Fragile X syndrome.

[0028] [Figure 12A-B] Figures 12A and 12B show a non-restrictive schematic diagram of a fusion construct in which the RVG peptide is inserted into the second extracellular loop of TSPAN6 along with the HA tag (Figure 12A), and a non-restrictive illustrative Western blot showing the reliable detection of TSPAN6-RVG-HA in ARMM secreted from HEK293T cells (Figure 12B).

[0029] [Figure 13A-B] Figures 13A and 13B show a non-limiting example of a method for VSV-G insertion into ARMM (Figure 13A), and a non-limiting exemplary Western blot showing reliable detection of VSV-G in ARMM secreted from HEK293T cells (Figure 13B).

[0030] [Figure 14]Figure 14 shows a non-limiting example of ARRDC1-mediated delivery of a payload to cultured human iPSC-derived 3D brain organoids. Brain organoids were exposed to ARRDC1-GFP-VSVG ARMM for either 24 hours (top panel) or 48 hours (bottom panel). Insets show a high percentage of green fluorescent protein (GFP)-positive cells after dissociation and retrieval at 24 hours. [Modes for carrying out the invention]

[0031] definition As used in this application, the term "ARMM" refers to a microvesicle containing the ARRDC1 protein or a variant thereof and / or the TSG101 protein or a variant thereof. In some embodiments, the ARMM contains a payload, e.g., nucleic acids, proteins, or small molecules, that is expelled from a cell and present in the cytoplasm or associated with the cell membrane. In some embodiments, the ARMM is expelled from a transgenic cell containing a recombinant expression construct containing an transgene, and the ARMM contains a gene product, e.g., an RNA transcript and / or a protein encoded by the expression construct (e.g., ARRDC1-Tat fusion protein and TAR-payload RNA). In some embodiments, the ARMM is synthetically produced, for example, by contacting a lipid bilayer with the ARRDC1 protein or a variant thereof in a cell-free system where TSG101 or a variant thereof is present. In other embodiments, the ARMM is further synthetically produced by contacting the lipid bilayer with a HECT domain ligase and VPS4a. In some embodiments, the ARMM lacks late endosomal markers. Some ARMMs provided in this application do not contain or are negative for one or more exosome biomarkers. 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 in this application may contain exosome biomarkers. Therefore, some ARMMs may be negative for one or more exosome biomarkers but positive for one or more different exosome biomarkers.For example, such an ARMM may be negative for CD63 and Lamp-1 but may include PGK1 or GAPDH; or it may be negative for CD63, Lamp-1, CD9, and CD81 but positive for HLA-DRA. In some embodiments, the ARMM includes exosome biomarkers but at levels lower than those typically found in exosomes. For example, some ARMMs include one or more exosome biomarkers at levels less than approximately 1%, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the level of those biomarkers found in exosomes. To give an unspecified example, in some embodiments, the ARMM may be negative for CD63 and Lamp-1, include CD9 at a level less than approximately 5% of the level of CD9 typically found in exosomes, and be positive for ACTB. Exosome biomarkers other than those listed above are known to those skilled in the art, and the present invention is not limited in this respect.

[0032] As used in this application, the term “binding RNA” refers to ribonucleic acid (RNA) that binds to an RNA-binding protein, e.g., any RNA-binding protein known in the art and / or any RNA-binding protein described herein. In some embodiments, binding RNA is RNA that specifically binds to an RNA-binding protein. Binding RNA that “specifically binds” to an RNA-binding protein binds to the RNA-binding protein more readily and / or for a longer period of time with higher affinity, binding activity, than binding to another protein, such as a protein that does not bind to RNA or a protein that weakly binds to binding RNA. In some embodiments, binding RNA is a naturally occurring RNA or a non-natural variant thereof that binds to a particular RNA-binding protein. For example, binding RNA may be a TAR element, Rev response element, MS2 RNA, or any variant thereof that specifically binds to an RNA-binding protein. In some embodiments, binding RNA may be a transactivation response element (TAR element) or a variant thereof, which is an RNA stem-loop structure found at the 5' end of a nascent HIV-1 transcript that specifically binds to the transactivator (Tat) protein of transcription. In some embodiments, the binding RNA is a Rev response element (RRE) or a variant thereof that specifically binds to an accessory protein Rev (e.g., Rev derived from HIV-1). In some embodiments, the binding RNA is MS2 RNA that specifically binds to an MS2 phage coat protein. The binding RNA of this disclosure may be designed to specifically bind to a protein (e.g., an RNA-binding protein fused to ARRDC1) to facilitate the loading of the binding RNA (e.g., binding RNA fused to a payload RNA) into the ARMM.

[0033] As used in this application, the term "aptamer" 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 manipulated by in vitro selection, or alternatively by iterative rounds of the SELEX method (phylogenetic evolution of ligands by exponential enrichment), to bind to a variety of molecular targets, such as proteins, small molecules, macromolecules, metabolites, carbohydrates, metals, nucleic acids, cells, tissues, and organisms. Methods for manipulating aptamers that bind to various molecular targets such as proteins are known in the art, including U.S. Patents 6,376,19 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 Turek and Gold, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 This includes the DNA polymerases described in "Science. 1990," the entire contents of each of them being incorporated herein by reference.

[0034] As used in this application, the term “RNA-binding protein” refers to a polypeptide molecule that binds to binding RNA, e.g., binding RNA known in the art and / or any binding RNA described herein. In some embodiments, an RNA-binding protein is a protein that specifically binds to binding RNA. An RNA-binding protein that “specifically binds” to binding RNA binds to the binding RNA more readily and / or for a longer period of time with higher affinity, binding activity than one that binds to another RNA, such as a control RNA (e.g., RNA with a random nucleic acid sequence) or RNA that weakly binds to the RNA-binding protein. In some embodiments, an RNA-binding protein is a naturally occurring protein or a non-natural variant thereof that binds to a specific RNA. For example, in some embodiments, an RNA-binding protein may be a transcriptional transactivator (Tat) protein that specifically binds to a transactivation response element (TAR element). In some embodiments, an RNA-binding protein is a virion expression regulator (Rev) protein (e.g., Rev from HIV-1) or a variant thereof that specifically binds to a Rev response element (RRE). In some embodiments, an RNA-binding protein is a coat protein of an MS2 bacteriophage that specifically binds to MS2 RNA. RNA-binding proteins useful in this disclosure (e.g., binding proteins fused to ARRDC1) may be designed to specifically bind to binding RNA (e.g., binding RNA fused to payload RNA) in order to facilitate the loading of binding RNA into ARMM.

[0035] As used in this application, the terms “payload,” “payload protein,” “payload nucleic acid,” “payload DNA,” “payload RNA,” or “payload small molecule” refer to proteins, nucleic acids (including DNA or RNA, respectively), or small molecules that can be incorporated into the ARMM, for example, the liquid phase of the ARMM or the lipid bilayer of the ARMM. The 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. Patents 9,737,480, 9,816,080, 10,260,055, and International Publication 2018 / 067546, the entire contents of each of which are incorporated herein by reference.

[0036] The payload may be delivered to a subject, organ, tissue, or cell by association with or inclusion into an ARMM. In some embodiments, the payload is to be delivered to target cells in vitro, in vivo, or ex vivo. In some embodiments, the delivered payload is a bioactive drug, in other words, it has activity in cells, organs, tissues, and / or subjects. For example, a protein, nucleic acid (e.g., DNA or RNA), or small molecule that, when administered to a subject, has or is considered to be bioactive to that subject. In some embodiments, the delivered drug is a therapeutic agent.

[0037] As used in this application, the term “therapeutic agent” means any agent that, when administered to a subject, has a beneficial effect. In some embodiments, the payload is a small molecule, or a payload protein, or a nucleic acid such as DNA or RNA, associated with a small molecule. In some embodiments, the delivered payload is a diagnostic agent. In some embodiments, the delivered payload is a prophylactic agent. In some embodiments, the delivered payload is useful as an imaging agent. In some of these embodiments, the diagnostic or imaging agent is bioactive, and in others, it is not.

[0038] As used in this application, the term “central nervous system” or “CNS” refers to the nervous system, which consists of cells and tissues of the brain and spinal cord. The two main types of cells that make up the nervous system are neurons and glial cells. In some embodiments, neurons are excitatory, and in some embodiments, they are inhibitory. In some embodiments, the cells of the CNS include, but are not limited to, neuronal cells, glial cells, oligodendrocytes, astrocytes, microglia, cerebrospinal fluid (CSF), interstitial space, bone, cartilage, etc.

[0039] Where applicable in this application, the terms “peripheral nervous system” or “PNS” refer to all cells and tissues of the nervous system outside the cells and tissues of the brain and spinal cord (i.e., outside the CNS). The PNS consists of extra-CNS nerves and ganglia, which primarily contain two types of cells: sensory nerves that carry information to the CNS, and motor nerves that carry information from the CNS. The PNS also includes Schwann cells, which myelinize cells of the nervous system. Sensory nerve cells send information from organs or external stimuli to the CNS. Motor nerve cells carry information from the CNS to organs, muscles, and glands.

[0040] As used in this application, the terms “nervous system” or “NS” collectively refer to the CNS and PNS. Cells of the nervous system include neurons, astrocytes, oligodendrocytes, and microglia, which further interact with immune system cells, including vascular endothelial cells and T cells. While neurons, astrocytes, and oligodendrocytes are terminally differentiated cells, the CNS-specific niche contains neural stem cells and neurogenic cells that retain the ability to replicate via both symmetric and symmetric cell division to produce further stem cells, progenitor cells, and cells that will terminally differentiate into neurons and astrocytes. Microglia, which are resident immune system cells of the nervous system, can also proliferate.

[0041] The term "viral envelope protein" refers to proteins that typically function to assist viral attachment and entry into cells. In some embodiments, viral envelope proteins can be incorporated into ARMMS to enable targeting of cells in the CNS. Non-limiting examples of viral envelope proteins include vesicular stomatitis virus G protein (VSV-G) or rabies virus glycoprotein (RVG). VSV-G is known to mediate viral attachment to LDL receptors (LDLRs) or members of the LDLR family, while RVG is known to use nicotinic acetylcholine receptors and low-affinity nerve growth factor receptors for viral entry.

[0042] As used in this application, the term “linker” refers to a chemical part that links two molecules or parts, for example, the ARRDC1 protein and the Tat protein, the WW domain and the Tat protein, or the ARRDC1 protein and the Cas9 nuclease. Typically, a linker is located between or sandwiched between two groups, molecules, or other parts, and is linked to one each by a covalent bond, thus linking the two. In some embodiments, the linker comprises an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker comprises a nucleotide (e.g., DNA or RNA) or a plurality of nucleotides (e.g., a nucleic acid). In some embodiments, the linker is an organic molecule, a functional group, a polymer, or other chemical part. In some embodiments, the linker is a cleavable linker. For example, the linker comprises a bond that can be cleaved by exposure to UV light or hydrolytic enzymes, for example, a protease or esterase. In some embodiments, the linker is any sequence of amino acids 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 (e.g., a covalent bond, an amide bond, a disulfide bond, an ester bond, a carbon-carbon bond, a carbon heteroatom bond).

[0043] As used in this application, the term “animal” refers to any member of the animal kingdom. In some embodiments, the term “animal” refers to a human of any sex at any developmental stage. In some embodiments, the term “animal” refers to a non-human animal at any developmental stage. In certain embodiments, the non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, or pigs). Animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and nematodes. In some embodiments, an animal is a transgenic animal, a genetically modified animal, or a clone. In some embodiments, an animal is a transgenic non-human animal, a genetically modified non-human animal, or a non-human clone.

[0044] When used in this application, the terms “approximately” or “about” when applied to one or more values ​​of interest refer to values ​​similar to the given reference values. In certain embodiments, the terms “approximately” or “about” refer to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the given reference values, unless otherwise specified or evident from the context (e.g., if such a number exceeds 100% of the possible values).

[0045] As used in this application, when used with respect to two or more entities, e.g., chemical parts, molecules, and / or ARMMs, the term “associates with” means that the entities are physically associated or linked to one another, either directly or via one or more additional parts acting as linkers, to form a structure that is sufficiently stable to maintain a physically associated state under the conditions in which the structure is used, e.g., physiological conditions. ARMMs typically associate with active substances, e.g., nucleic acids, proteins, or small molecules, by mechanisms involving covalent bonds (e.g., via amide bonds) or non-covalent bonds (e.g., between ARRDC1 and the WW domain, or between the Tat protein and the TAR element). In certain embodiments, the drug to be delivered (e.g., payload protein, payload nucleic acid, or payload small molecule) is covalently linked to a molecule that non-covalently associates with a portion of the ARMM fused to the ARRDC1 protein or a variant thereof. In some embodiments, the linkage is by a linker, e.g., a cleavable linker. In some embodiments, entities (e.g., payload proteins, payload nucleic acids, or payload small molecules) are associated with ARMMs by inclusion within the ARMM, for example, by encapsulating the molecule within the ARMM. For example, in some embodiments, molecules present in the cytoplasm of an ARMM-producing cell (e.g., payload proteins, payload nucleic acids, or payload small molecules) are associated with ARMMs by encapsulating the cytoplasm within the ARMM at the time of ARMM budding. Similarly, membrane proteins or other molecules associated with the cell membrane of an ARMM-producing cell may be associated with ARMMs produced by the cell by inclusion within the ARMM membrane at the time of budding.

[0046] As used in this application, the term "bioactive" refers to any characteristic of a substance that is active in cells, organs, tissues and / or subjects. For example, a substance that has a biological effect on an organism when administered to that organism is considered bioactive. As one example, a payload RNA may be considered bioactive if it increases or decreases the expression of a gene product when administered to a subject or cell. As another example, a nuclease payload protein may be considered bioactive if it increases or decreases the expression of a gene product when administered to a subject.

[0047] As used in this application, the term “conserved” means a nucleotide residue or amino acid residue in a polynucleotide sequence or amino acid sequence that has not undergone modification at the same position in two or more related sequences being compared. Relatively conserved nucleotides or amino acids are those that are more conserved between related sequences than nucleotides or amino acids that appear elsewhere in the sequence. In some embodiments, two or more sequences are said to be “fully conserved” if they are 100% identical to each other. In some embodiments, two or more sequences are said to be “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 said to be “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 said to be “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 one another. In some embodiments, two or more sequences are said to be “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 one another.

[0048] As used in this application, the term “engineered” refers to a protein, nucleic acid, complex, substance, or entity designed, produced, prepared, synthesized, and / or manufactured by a human. Thus, an engineered product is a product that does not occur naturally. In some embodiments, an engineered protein or nucleic acid is a protein or nucleic acid designed to meet specific requirements or to have specific design features. For example, a payload RNA may be engineered to associate with ARRDC1 by fusing one or more WW domains to a Tat protein and fusing the payload RNA to a TAR element to facilitate the loading of the payload RNA into the ARMM. As another example, a payload RNA may be engineered to associate with ARRDC1 by fusing a Tat protein to ARRDC1 and fusing the payload RNA to a TAR element to facilitate the loading of the payload RNA into the ARMM. As yet another example, a payload protein may be engineered to associate with ARRDC1 by fusing one or more WW domains to a payload protein to facilitate the loading of the payload protein into the ARMM.

[0049] As used in this application, “expression” of a nucleic acid sequence means 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 RNA into a polypeptide or protein, and (4) post-translational modification of a polypeptide or protein.

[0050] As used in this application, “fusion protein” includes a first protein moiety that associates with a second protein moiety via a peptide bond, for example, the ARRCD1 protein or a variant thereof, or the TSG101 protein or a variant thereof. In certain embodiments, the fusion protein is encoded by a single fusion gene.

[0051] As used in this application, the term “gene” has its meaning as understood in the art. It will be understood by those skilled in the art that the term “gene” may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will be further understood that the definition of a gene includes reference to nucleic acids that do not code for proteins but rather code for functional RNA molecules such as gRNA, RNAi agents, ribozymes, tRNA, etc. For clarity, it should be noted that as used in this application, the term “gene” generally refers to a portion of nucleic acids that code for proteins, and that this term may optionally include regulatory sequences, as will be apparent to those skilled in the art from the context. This definition is not intended to exclude the application of the term “gene” to non-protein-coding expression units, but rather to clarify that in most cases, as used in this application, the term refers to protein-coding nucleic acids.

[0052] As used in this application, the terms “gene product” or “expression product” generally refer to RNA transcribed from a gene (before and / or after processing) or polypeptides encoded by RNA transcribed from a gene (before and / or after modification).

[0053] As used in this application, the term “green fluorescent protein” (GFP) originally refers to a protein isolated from the jellyfish Aequorea vicarnata, which fluoresces green when exposed to blue light, or to derivatives of such protein (e.g., enhanced or wavelength-shifted versions of that protein). The amino acid sequence of wild-type GFP is as follows: MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (Sequence ID 3)

[0054] Proteins homologous to Sequence ID No. 3 by 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% are also considered to be green fluorescent proteins.

[0055] As used in this application, the term “homologousness” 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 “homologous” to each other 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 “homologous” to each other 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 “homologousness” always 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 homologous if the polypeptides they encode are at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% identical with respect to at least one sequence of at least about 20 amino acids. In some embodiments, homologous nucleotide sequences are characterized by their ability to encode a sequence of at least 4-5 uniquely identified amino acids. For sequences to be considered homologous, both the identity and approximate spacing of these related amino acids must be considered. For nucleotide sequences less than 60 nucleotides in length, homology is determined by their ability to encode a sequence of at least 4-5 uniquely identified amino acids. According to the present invention, two protein sequences are considered homologous if the proteins are at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% identical with respect to at least one sequence of at least about 20 amino acids.

[0056] As used in this application, the term “identity” refers to the overall relevance between nucleic acids or proteins (e.g., DNA molecules, RNA molecules, and / or polypeptides). The calculation of percentage identity between two nucleic acid sequences can be performed, for example, by aligning the two sequences for the purpose of best comparison (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for best alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences 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. Then, the nucleotides at the corresponding nucleotide positions are compared. The molecules are identical at a given position when a position on the first sequence is occupied by the same nucleotide as the corresponding position on the second sequence. The percentage 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 the best alignment of the two sequences and the length of each gap. The comparison of two arrays and the determination of their percent identity can be accomplished using mathematical algorithms.For example, the percentage 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 percentage identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17). This is incorporated into the ALIGN program (version 2.0) and uses the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two nucleotide sequences can be determined using the GAP program of the GCG software package, using the NWSgapdna.CMP matrix. Commonly used methods for determining the percentage identity between sequences include, but are not limited to, those 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 coded into publicly available computer programs.Exemplary computer software for determining 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)).

[0057] As used in this application, the term "in vitro" refers to events occurring in an artificial environment rather than within living organisms (e.g., animals, plants, or microorganisms), such as in a test tube or reaction vessel, cell culture, or petri dish.

[0058] As used in this application, the term "in vivo" refers to an event occurring within a living organism (e.g., an animal, plant, or microorganism).

[0059] As used in this application, the term “isolated” means (1) a substance or entity that has been separated from at least some of the components to which it was originally produced (whether in nature or in an experimental environment), and / or (2) a substance or entity that has been produced, prepared, and / or manufactured by human hands. Isolated substances and / or entities 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 to which they were originally bound. In some embodiments, an isolated substance is 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 about 99% or more pure. As used in this application, a substance is “pure” if it substantially contains no other components.

[0060] As used in this application, the term “nucleic acid” in its broadest sense refers to compounds and / or substances that are or can be incorporated into an oligonucleotide chain via phosphodiester bonds. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain containing individual nucleotides. As used in this application, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to polymers of nucleotides (e.g., a chain consisting of at least two nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single-stranded and / or double-stranded DNA and / or complementary DNA (cDNA). Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms encompass nucleic acid analogs, i.e., analogs having something other than a phosphodiester backbone. For example, so-called “peptide nucleic acids,” which are known in the art and have peptide bonds in their backbone instead of phosphodiester bonds, are considered to be within the scope of this invention. The term “nucleotide sequence encoding an amino acid sequence” encompasses all nucleotide sequences that are degenerate versions of each other and / or all nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNA may contain introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, selectively purified, and chemically synthesized. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids may contain nucleoside analogs such as analogs with chemically modified bases or sugars, skeletal modifications, etc. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' direction. The term “nucleic acid segment” is used herein to refer to a nucleic acid sequence that is a portion 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 natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5- The following are or include: thiocytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biomodified bases (e.g., methylated bases); intercalation bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoamidite linkages). In some embodiments, the present invention specifically relates to “unmodified nucleic acids” meaning unchemically modified nucleic acids (e.g., polynucleotides and residues comprising nucleotides and / or nucleosides) in order to facilitate or achieve delivery.

[0061] As used in this application, the term “protein” refers to a sequence of at least two amino acids linked together by one or more peptide bonds. Proteins may include non-amino acid portions (e.g., glycoproteins) and / or may be processed or modified in other ways. Those skilled in the art will understand that a “protein” may be a complete protein chain produced by a cell (with or without a signal sequence) or a functional portion thereof. Those skilled in the art will further understand that a protein may, in some cases, include one or more protein chains linked, for example, by one or more disulfide bonds or by other means. Proteins may contain L-amino acids, d-amino acids, or both, and may contain any of the various amino acid modifications or analogues known in the art. Useful modifications include, for example, the addition, conjugation, functionalization, or other modifications (e.g., alpha-amidation) of chemical entities such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, amide groups, terminal acetyl groups, etc. In certain embodiments, protein modification results in a more stable protein (e.g., a longer half-life in vivo). These modifications may include protein cyclization, d-amino acid incorporation, and so on. None of these modifications should substantially hinder the desired biological activity of the protein. In certain embodiments, protein modification results in a more biologically active peptide. In some embodiments, the protein may include native amino acids, non-native amino acids, synthetic amino acids, amino acid analogs, and combinations thereof.

[0062] As used in this application, the terms “subject” or “patient” refer to any organism to which a composition according to the present invention may be administered for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, and humans) and / or plants. 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.

[0063] As used in this application, the term “therapeutably effective dose” means the amount of a delivered agent (e.g., nucleic acids, proteins, drugs, therapeutic agents, diagnostic agents, prophylactic agents, ARMMs, or ARMMs containing payload proteins or payload RNAs) that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, improve, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition.

[0064] As used in this application, the term “to treat” means to partially or completely prevent and / or reduce the occurrence of one or more symptoms or characteristics of a particular disease or condition. For example, “to treat” cancer may mean to inhibit the survival, growth, and / or spread of cancer. Treatment may be administered to subjects who do not show signs or symptoms of the disease, disorder, and / or condition, and / or subjects who show only early signs or symptoms of the disease or condition, for the purpose of reducing the risk of developing more serious consequences associated with the disease, disorder, and / or condition.

[0065] As used in this application, “vector” means any nucleic acid or nucleic acid-carrying particle, cell, or organism that can be used to transfer nucleic acids into a host cell. The term “vector” encompasses both viral and nonviral products and means for introducing nucleic acids into cells. A “vector” can be used in vitro, ex vivo, or in vivo. A vector capable of leading to the expression of an operablely linked gene is referred to here as an “expression vector.” Nonviral vectors encompass plasmids, cosmids, artificial chromosomes (e.g., bacterial or yeast artificial chromosomes) and may include, for example, liposomes, charged lipids (cytofectin), DNA-protein complexes, and biopolymers. Viral vectors encompass, for example, retroviruses, lentiviruses, adeno-associated viruses, poxviruses, baculoviruses, reoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, and adenovirus vectors. A vector may also include the entire genome sequence of a virus or a recombinant genome sequence. The vector may also contain a portion of the genome that includes a functional sequence for the production of a virus that can infect, enter, or be introduced into a cell in order to deliver nucleic acids within it.

[0066] As used in this application, the term “WW domain” refers to a protein domain having two basic residues at its C-terminus that mediate protein-protein interactions with a short proline-rich or proline-containing motif. It should be understood that the two basic residues of the WW domain (e.g., any two of H, R, and K) do not need to be at the true C-terminus of the WW protein domain. Rather, the two basic residues can be at the C-terminal portion of the WW protein domain (e.g., near the C-terminus of the WW protein domain). In some embodiments, the WW domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tryptophan (W) residues. In some embodiments, the WW domain contains at least two W residues. In some embodiments, the at least two W residues are separated by only 15–25 amino acids. In some embodiments, the at least two W residues are separated by only 19–23 amino acids. In some embodiments, the at least two W residues are separated by only 20–22 amino acids. WW domains, possessing two basic C-terminal amino acid residues, may have the ability to bind to short proline-rich or proline-containing motifs (i.e., the PPXY (SEQ ID NO: 2) motif). WW domains bind to a variety of distinct peptide ligands containing proline-rich core sequences, such as PPXY (SEQ ID NO: 2) found in AARDC1. 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 triple-stranded antiparallel β-sheets with two ligand-binding grooves.

[0067] 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 coexist with several other interacting domains, including membrane targeting domains such as C2 in NEDD4 family proteins, the phosphotyrosine-binding (PTB) domain of FE65 protein, the FF domains of CA150 and FBPIl, and the plextrin homology (PH) domain of PLEKHA5. WW domains are also ligated to various catalytic domains, including the HECT E3 protein-ubiquitin ligase domain of NEDD4 family proteins, the rotomerase or peptidylprolyl (proly) isomerase domain of Pin1, and the Rho GAP domains of ArhGAP9 and ArhGAP12.

[0068] In this disclosure, the WW domain may be a WW domain that naturally has two basic amino acids at its C-terminus. In some embodiments, the WW domain or WW domain variant may be derived from the human ubiquitin ligases WWP1, WWP2, Nedd4-1, Nedd4-2, Smurf1, Smurf2, ITCH, NEDL1, or NEDL2. Exemplary amino acid sequences of WW domain-containing proteins (WW domains are underlined) are listed below. It should be understood that any WW domain or WW domain variant of an exemplary protein may be used in, but is not intended to limit, the inventions described herein.

[0069] Human WWP1 amino acid sequence (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). MATASPRSDT SNNHSGRLQL QVTVSSAKLK RKKNWFGTAI YTEVVVDGEI 50 TKTAKSSSSS NPKWDEQLTV NVTPQTTLEF QVWSHRTLKA DALLGKATID 100 LKQALLIHNR KLERVKEQLK LSLENKNGIA QTGELTVVLD GLVIEQENIT 150 NCSSSPTIEI QENGDALHEN GEPSARTTAR LAVEGTNGID NHVPTSTLVQ 200 NSCCSYVVNG DNTPSSPSQV AARPKNTPAP KPLASEPADD TVNGESSSFA 250 PTDNASVTGT PVVSEENALS PNCTSTTVED PPVQEILTSS ENNECIPSTS 300 AELESEARSI LEPDTSNSRS SSAFEAAKSR QPDGCMDPVR QQSGNANT ET 350 LPSGWEQRKD PHGRTYYVDH NTRTTTWERP QPLPPGWERR VDDRRRVYYV 400 DHNTRTTTWQ RPTM ESVRNF EQWQSQRNQL QGAMQQFNQR YLYSASMLAA 450 ENDPY GPLPP GWEKRVDSTD RVYFVNHNTK TTQWEDPRT Q GLQNE EPLPE 500 GWEIRYTREG VRYFVDHNTR TTTFKDPRN G KSSVTKGGPQ IAYERGFRWK 550 LAHFRYLCQS NALPSHVKIN VSRQTLFEDS FQQIMALKPY DLRRRLYVIF 600 RGEEGLDYGG LAREWFFLLS HEVLNPMYCL FEYAGKNNYC LQINPASTIN 650 PDHLSYFCFI GRFIAMALFH GKFIDTGFSL PFYKRMLSKK LTIKDLESID 700 TEFYNSLIWI RDNNIEECGL EMYFSVDMEI LGKVTSHDLK LGGSNILVTE 750 ENKDEYIGLM TEWRFSRGVQ EQTKAFLDGF NEVVPLQWLQ YFDEKELEVM 800 LCGMQEVDLA DWQRNTVYRH YTRNSKQIIW FWQFVKETDN EVRMRLLQFV 850 TGTCRLPLGG FAELMGSNGP QKFCIEKVGK DTWLPRSHTC FNRLDLPPYK 900 SYEQLKEKLL FAIEETEGFG QE (Sequence ID 4) 922 WWI (349-382): ETLPSGWEQRKDPHGRTYYVDHNTRTTTWERPQP(Sequence ID 5) WW2 (381-414): QPLPPGWERRVDDRRRVYYVDHNTRTTTWQRPTM (Sequence ID 6) WW3 (456-489): ENDPYGPLPPGWEKRVDSTDRVYFVNHNTKTTQWEDPRT(Sequence ID 7) WW4 (496-529): EPLPEGWEIRYTREGVRYFVDHNTRTTTFKDPRN (Sequence ID 8)

[0070] 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). MASASSSRAG VALPFEKSQL TLKVVSAKPK VHNRQPRINS YVEVAVDGLP 50 SETKKTGKRI GSSELLWNEI IILNVTAQSH LDLKVWSCHT LRNELLGTAS 100 VNLSNVLKNN GGKMENMQLT LNLQTENKGS VVSGGELTIF LDGPTVDLGN 150 VPNGSALTDG SQLPSRDSSG TAVAPENRHQ PPSTNCFGGR SRTHRHSGAS 200 ARTTPATGEQ SPGARSRHRQ PVKNSGHSGL ANGTVNDEPT TATDPEEPSV 250 VGVTSPPAAP LSVTPNPNTT SLPAPATPAE GEEPSTSGTQ QLPAAAQAP D 300 ALPAGWEQRE LPNGRVYYVD HNTKTTTWER PLPPGWEKRT DPRGRFYYVD 350 HNTRTTTWQR PTA EYVRNYE QWQSQRNQLQ GAMQHFSQRF LYQSSSASTD 400 HDPL GPLPPG WEKRQDNGRV YYVNHNTRTT QWEDPRT QGM IQE PALPPGW 450 EMKYTSEGVR YFVDHNTRTT TFKDPRP GFE SGTKQGSPGA YDRSFRWKYH 500 QFRFLCHSNA LPSHVKISVS RQTLFEDSFQ QIMNMKPYDL RRRLYIIMRG 550 EEGLDYGGIA REWFFLLSHE VLNPMYCLFE YAGKNNYCLQ INPASSINPD 600 HLTYFRFIGR FIAMALYHGK FIDTGFTLPF YKRMLNKRPT LKDLESIDPE 650 FYNSIVWIKE NNLEECGLEL YFIQDMEILG KVTTHELKEG GESIRVTEEN 700 KEEYIMLLTD WRFTRGVEEQ TKAFLDGFNE VAPLEWLRYF DEKELELMLC 750 GMQEIDMSDW QKSTIYRHYT KNSKQIQWFW QVVKEMDNEK RIRLLQFVTG 800 TCRLPVGGFA ELIGSNGPQK FCIDKVGKET WLPRSHTCFN RLDLPPYKSY 850 EQLREKLLYA IEETEGFGQE (SEQ ID NO: 9) 870 WW1 (300~333): DALPAGWEQRELPNGRVYYVDHNTKTTTWERPLP (SEQ ID NO: 10) WW2 (330~363): PLPPGWEKRT DPRGRFYYVDHNTRTTTWQRPTA(Sequence ID 11) WW3 (405-437): HDPLGPLPPGWEKRQDNGRVYYVNHNTRTTQWEDPRT(Sequence ID 12) WW4 (444-477): PALPPGWEMKYTSEGVRYFVDHNTRTTTFKDPRP (Sequence ID 13)

[0071] 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). MAQSLRLHFA ARRSNTYPLS ETSGDDLDSH VHMCFKRPTR ISTSNVVQMK 50 LTPRQTALAP LIKENVQSQE RSSVPSSENV NKKSSCLQIS LQPTRYSGYL 100 QSSNVLADSD DASFTCILKD GIYSSAVVDN ELNAVNDGHL VSSPAICSGS 150 LSNFSTSDNG SYSSNGSDFG SCASITSGGS YTNSVISDSS SYTFPPSDDT 200 FLGGNLPSDS TSNRSVPNRN TTPCEIFSRS TSTDPFVQDD LEHGLEIMKL 250 PVSRNTKIPL KRYSSLVIFP RSPSTTRPTS PTSLCTLLSK GSYQTSHQFI 300 ISPSEIAHNE DGTSAKGFLS TAVNGLRLSK TICTPGEVRD IRPLHRKGSL 350 QKKIVLSNNT PRQTVCEKSS EGYSCVSVHF TQRKAATLDC ETTNGDCKPE 400 MSEIKLNSDS EYIKLMHRTS ACLPSSQNVD CQININGELE RPHSQMNKNH 450 GILRRSISLG GAYPNISCLS SLKHNCSKGG PSQLLIKFAS GNEGKVDNLS 500 RDSNRDCTNE LSNSCKTRDD FLGQVDVPLY PLPTENPRLE RPYTFKDFVL 550 HPRSHKSRVK GYLRLKMTYL PKTSGSEDDN AEQAEELEPG WVVLDQPDAA 600 CHLQQQQEP S PLPPGWEERQ DILGRTYYVN HESRRTQWKR PTP QDNLTDA 650 ENGNIQLQAQ RAFTTRRQIS EETESVDNRE SSENWEIIRE DEATMYSNQA 700 FPPPPSSNL DVPTHLAEEL NARLTIFGNS AVSQPASSSN HSSRRGSLQA 750 YTFEEQPTLP VLLPTS SGLP PGWEEKQDER GRSYYVDHNS RTTTWTKPTV 800 QATVETSQLT SSQSSAGPQS QASTSDSGQQ VTQPSEIEQ G FLPKGWEVRH 850 APNGRPFFID HNTKTTTWED PRL KIPAHLR GKTSLDTSND L GPLPPGWEE 900 RTHTDGRIFY INHNIKRTQW EDPRL ENVAI TGPAVPYSRD YKRKYEFFRR 950 KLKKQNDIPN KFEMKLRRAT VLEDSYRRIM GVKRADFLKA RLWIEFDGEK 1000 GLDYGGVARE WFFLISKEMF NPYYGLFEYS ATDNYTLQIN PNSGLCNEDH 1050 LSYFKFIGRV AGMAVYHGKL LDGFFIRPFY KMMLHKPITL HDMESVDSEY 1100 YNSLRWILEN DPTELDLRFI IDEELFGQTH QHELKNGGSE IVVTNKNKKE 1150 YIYLVIQWRF VNRIQKQMAA FKEGFFELIP QDLIKIFDEN ELELLMCGLG 1200 DVDVNDWREH TKYKNGYSAN HQVIQWFWKA VLMMDSEKRI RLLQFVTGTS 1250 RVPMNGFAEL YGSNGPQSFT VEQWGTPEKL PRAHTCFNRL DLPPYESFEE 1300 LWDKLQMAIE NTQGFDGVD(Sequence ID 14) 1319 WWI (610-643): SPLPPGWEERQDILGRTYYVNHESRRTQWKRPTP(Sequence ID 15) WW2 (767-800): SGLPPGWEEKQDERGRSYYVDHNSRTTTWTKPTV(Sequence ID 16) WW3 (840-873): GFLPKGWEVRHAPNGRPFFIDHNTKTTTWEDPRL (Sequence ID 17) WW4 (892-925): GPLPPGWEERTHTDGRIFYINHNIKRTQWEDPRL(Sequence ID 18)

[0072] 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). MATGLGEPVYGLSEDEGESRILRVKVVSGIDLAKKDIFGASDPYVKLSLYVADENRELALVQTKTIKKTLNPKWNEEFYFRVNPSNHRLLFEVFDENRLTRDDFLGQVDVPLSHLPTEDPTMERPYTFKDFLLRPRSHKSRVKGFLRLKMAYMPKNGGQDEENSDQRDDMEHGWEVVDSNDSASQHQEELPPPPLPP GWEEKVDNLGRTYYVNHNNRTTQWHRPSLMDVSSESDNNIRQINQEAAHRRFRSRRHISEDLEPEPSEGGDVPEPWETISEEVNIAGDSLGLALPPPPASPGSRTSPQELSEELSRRLQITPDSNGEQFSSLIQREPSSRLRSCSVTDAVAEQGHLPPPSVAYVHTTPGL PSGWEERKDAKGRTYYVNHNNRTTTWTRP IMQLAEDGASGSATNSNNHLIEPQIRRPRSLSSPTVTLSAPLEGAKDSPVRRAVKDTLSNPQSPQPSPYNSPKPQHKVTQSFL PPGWEMRIAPNGRPFFIDHNTKTTTWEDPRL KFPVHMRSKTSLNPNDLGPL PPGWEERIHLDGRTFYIDHNSKITQWEDPRL QNPAITGPAVPYSREFKQKYDYFRKKLKKPADIPNRFEMKLHRNNIFEESYRRIMSVKRPDVLKARLWIEFESEKGLDYGGVAREWFFLLSKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFTFIGRVAGLAVFHGKLLDGFFIRPFYKMMLGKQITLNDMESVDSEYYNSLKWILENDPTELDLMFCIDEENFGQTYQVDLKPNGSEIMVTNENKREYIDLVIQWRFVNRVQKQMNAFLEGFTELLPIDLIKIFDENELELLMCGLGDVDVNDWRQHSIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQLFTIEQWGSPEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVENAQGFEGVD(Sequence ID 19) WWI (198-224): GWEEKVDNLGRTYYVNHNNRTTQWHRP (Sequence ID 20) WW2 (368-396): PSGWEERKDAKGRTYYVNHNNRTTTWTRP (Sequence ID 21) WW3 (480-510): PPGWEMRIAPNGRPFFIDHNTKTTTWEDPRL(Sequence ID 22) WW4 (531-561): PPGWEERIHLDGRTFYIDHNSKITQWEDPRL (Sequence ID 23)

[0073] 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). MSNPGTRRNG SSIKIRLTVL CAKNLAKKDF FRLPDPFAKI VVDGSGQCHS 50 TDTVKNTLDP KWNQHYDLYV GKTDSITISV WNHKKIHKKQ GAGFLGCVRL 100 LSNAISRLKD TGYQRLDLCK LNPSDDTDAVR GQIVVSLQTR DRIGTGGSVV 150 DCRGLLENEG TVYEDSGPGR PLSCFMEEPA PYTDSTGAAA GGGNCRFVES 200 PSQDQRLQAQ RLRNPDVRGS LQTPQNRPHG HQS PELPEGY EQRTTVQGQV 250 YFLHTQTGVS TWHDPRI PSP SGTIPGGDAA FLYEFLLQGH TSEPRDLNSV 300 NCDEL GPLPP GWEVRSTVSG RIYFVDHNNR TTQFTDPRL H HIMNHQCQLK 350 EPSQPLPLPS EGSLEDEELP AQRYERDLVQ KLKVLRHELS LQQPQAGHCR 400 IEVSREEIFE ESYRQIMKMR PKDLKKRLMV KFRGEEGLDY GGVAREWLYL 450 LCHEMLNPYY GLFQYSTDNI YMLQINPDSS INPDHLSYFH FVGRIMGLAV 500 FHGHYINGGF TVPFYKQLLG KPIQLSDLES VDPELHKSLV WILENDITPV 550 LDHTFCVEHN AFGRILQHEL KPNGRNVPVT EENKKEYVRL YVNWRFMRGI 600 EAQFLALQKG FNELIPQHLL KPFDQKELEL IIGGLDKIDL NDWKSNTRLK 650 HCVADSNIVR WFWQAVETFD EERRARLLQF VTGSTRVPLQ GFKALQGSTG 700 AAGPRLFTIH LIDANTDNLP KAHTCFNRID IPPYESYEKL YEKLLTAVEE 750 TCGFAVE (Sequence ID 24) 757 WWI (234-267): PELPEGYEQRTTVQGQVYFLHTQTGVSTWHDPRI (Sequence ID 25) WW2 (306-339): GPLPPGWEVRSTVSGRIYFVDHNNRTTQFTDPRL(Sequence ID 26)

[0074] 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). MSNPGGRRNG PVKLRLTVLC AKNLVKKDFF RLPDPFAKVV VDGSGQCHST 50 DTVKNTLDPK WNQHYDLYIG KSDSVTISVW NHKKIHKKQG AGFLGCVRLL 100 SNAINRLKDT GYQRLDLCKL GPNDNDTVRG QIVVSLQSRD RIGTGGQVVD 150 CSRLFD NDLP DGWEERRTAS GRIQYLNHIT RTTQWERPTR PASEYSSPGR 200 PLSCFVDENT PISGTNGATC GQSSDPRLAE RRVRSQRHRN YMSRTHLHTP 250 PDLPEGYEQR TTQQGQVYFL HTQTGVSTWH DPRV PRDLSN INCEEL GPLP 300 PGWEIRNTAT GRVYFVDHNN RTTQFTDPRL SANLHLVLNR QNQLKDQQQQ 350 QVVSLCPDDT ECLTVPRYKR DLVQKLKILR QELSQQQPQA GHCRIEVSRE 400 EIFEESYRQV MKMRPKDLWK RLMIKFRGEE GLDYGGVARE WLYLLSHEML 450 NPYYGLFQYS RDDIYTLQIN PDSAVNPEHL SYFHFVGRIM GMAVFHGHYI 500 DGGFTLPFYK QLLGKSITLD DMELVDPDLH NSLVWILEND ITGVLDHTFC 550 VEHNAYGEII QHELKPNGKS IPVNEENKKE YVRLYVNWRF LRGIEAQFLA 600 LQKGFNEVIP QHLLKTFDEK ELELIICGLG KIDVNDWKVN TRLKHCTPDS 650 NIVKWFWKAV EFFDEERRAR LLQFVTGSSR VPLQGFKALQ GAAGPRLFTI 700 HQIDACTNNL PKAHTCFNRI DIPPYESYEK LYEKLLTAIE ETCGFAVE 748 (Sequence ID 27) WWI (157-190): NDLPDGWEERRTASGRIQYLNHITRTTQWERPTR (Sequence ID 28) WW2 (251-284): PDLPEGYEQRTTQQGQVYFLHTQTGVSTWHDPRV (Sequence ID 29) WW3 (297-330): GPLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRL(Sequence ID 30)

[0075] 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). MSDSGSQLGS MGSLTMKSQL QITVISAKLK ENKKNWFGPS PYVEVTVDGQ 50 SKKTEKCNNT NSPKWKQPLT VIVTPVSKLH FRVWSHQTLK SDVLLGTAAL 100 DIYETLKSNN MKLEEVVVTL QLGGDKEPTE TIGDLSICLD GLQLESEVVT 150 NGETTCSENG VSLCLPRLEC NSAISAHCNL CLPGLSDSPI SASRVAGFTG 200 ASQNDDGSRS KDETRVSTNG SDDPEDAGAG ENRRVSGNNS PSLSNGGFKP 250 SRPPRPSRPP PPTPRPPASV NGSPSATSES DGSSTGSLPP TNTNTNTSEG 300 ATSGLIIPLT ISGGSGPRPL NPVTQ APLPP GWEQRVDQHG RVYYVDHVEK 350 RTTWDRPEPL PPGWERRVDN MGRIYYVDHF TRTTTWQRPT L ESVRNYEQW 400 QLQRSQLQGA MQQFNQRFIY GNQDLFATSQ SKEFDPL GPL TohokuPGWEKRTDS 450 NGRVYFVNHN TRITQWEDPR S QGQLNE KPL PEGWEMRFTV DGIPYFVDHN 500 RRTTTYIDPR T GKSALDNGP QIAYVRDFKA KVQYFRFWCQ QLAMPQHIKI 550 TVTRKTLFED SFQQIMSFSP QDLRRRLWVI FPGEEGLDYG GVAREWFFLL 600 SHEVLNPMYC LFEYAGKDNY CLQINPASYI NPDHLKYFRF IGRFIAMALF 650 HGKFIDTGFS LPFYKRILNK PVGLKDLESI DPEFYNSLIW VKENNIEECD 700 LEMYFSVDKE ILGEIKSHDL KPNGGNILVT EENKEEYIRM VAEWRLSRGV 750 EEQTQAFFEG FNEILPQQYL QYFDAKELEV LLCGMQEIDL NDWQRHAIYR 800 HYARTSKQIM WFWQFVKEID NEKRMRLLQF VTGTCRLPVG GFADLMGSNG 850 PQKFCIEKVG KENWLPRSHT CFNRLDLPPY KSYEQLKEKL LFAIEETEGF 900 GQE (Sequence ID 31) 903 ITCH WW1 (326-359): APLPPGWEQRVDQHGRVYYVDHVEKRTTWDRPEP(Sequence ID 32) ITCH WW2 (358~391): EPLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTL(Sequence ID 33) ITCH WW3 (438~471): GPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRS(Sequence ID 34) ITCH WW4 (478~511): KPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRT(Sequence ID 35)

[0076] 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). MLLHLCSVKN LYQNRFLGLA AMASPSRNSQ SRRRCKEPLR YSYNPDQFHN 50 MDLRGGPHDG VTIPRSTSDT DLVTSDSRST LMVSSSYYSI GHSQDLVIHW 100 DIKEEVDAGD WIGMYLIDEV LSENFLDYKN RGVNGSHRGQ IIWKIDASSY 150 FVEPETKICF KYYHGVSGAL RATTPSVTVK NSAAPIFKSI GADETVQGQG 200 SRRLISFSLS DFQAMGLKKG MFFNPDPYLK ISIQPGKHSI FPALPHHGQE 250 RRSKIIGNTV NPIWQAEQFS FVSLPTDVLE IEVKDKFAKS RPIIKRFLGK 300 LSMPVQRLLE RHAIGDRVVS YTLGRRLPTD HVSGQLQFRF EITSSIHPDD 350 EEISLSTEPE SAQIQDSPMN NLMESGSGEP RSEAPESSES WKPEQLGEGS 400 VPDGPGNQSI ELSRPAEEAA VITEAGDQGM VSVGPEGAGE LLAQVQKDIQ 450 PAPSAEELAE QLDLGEEASA LLLEDGEAPA STKEEPLEEE ATTQSRAGRE 500 EEEKEQEEEG DVSTLEQGEG RLQLRASVKR KSRPCSLPVS ELETVIASAC 550 GDPETPRTHY IRIHTLLHSM PSAQGGSAAE EEDGAEEEST LKDSSEKDGL 600 SEVDTVAADP SALEEDREEP EGATPGTAHP GHSGGHFPSL ANGAAQDGDT 650 HPSTGSESDS SPRQGGDHSC EGCDASCCSP SCYSSSCYST SCYSSSCYSA 700 SCYSPSCYNG NRFASHTRFS SVDSAKISES TVFSSQDDEE EENSAFESVP 750 DSMQSPELDP ESTNGAGPWQ DELAAPSGHV ERSPEGLESP VAGPSNRREG 800 ECPILHNSQP VSQLPSLRPE HHHYPTIDE P LPPNWEARID SHGRVFYVDH 850 VNRTTTWQRP TA AATPDGMR RSGSIQQMEQ LNRRYQNIQR TIATERSEED 900 SGSQSCEQAP AGGGGGGGSD SEAESSQSSL DLRREGSLSP VNSQKITLLL 950 QSPAVKFITN PEFFTVLHAN YSAYRVFTSS TCLKHMILKV RRDARNFERY 1000 QHNRDLVNFI NMFADTR LEL PRGWEIKTDQ QGKSFFVDHN SRATTFIDP R 1050 I PLQNGRLPN HLTHRQHLQR LRSYSAGEAS EVSRNRGASL LARPGHSLVA 1100 AIRSQHQHES LPLAYNDKIV AFLRQPNIFE MLQERQPSLA RNHTLREKIH 1150 YIRTEGNHGL EKLSCDADLV ILLSLFEEEI MSYVPLQAAF HPGYSFSPRC 1200 SPCSSPQNSP GLQRASARAP SPYRRDFEAK LRNFYRKLEA KGFGQGPGKI 1250 KLIIRRDHLL EGTFNQVMAY SRKELQRNKL YVTFVGEEGL DYSGPSREFF 1300 FLLSQELFNP YYGLFEYSAN DTYTVQISPM SAFVENHLEW FRFSGRILGL 1350 ALIHQYLLDA FFTRPFYKAL LRLPCDLSDL EYLDEEFHQS LQWMKDNNIT 1400 DILDLTFTVN EEVFGQVTER ELKSGGANTQ VTEKNKKEYI ERMVKWRVER 1450 GVVQQTEALV RGFYEVVDSR LVSVFDAREL ELVIAGTAEI DLNDWRNNTE 1500 YRGGYHDGHL VIRWFWAAVE RFNNEQRLRL LQFVTGTSSV PYEGFAALRG 1550 SNGLRRFCIE KWGKITSLPR AHTCFNRLDL PPYPSYSMLY EKLLTAVEET 1600 STFGLE (SEQ ID NO: 36) 1606 WWI (829-862): PLPPNWEARIDSHGRVFYVDHVNRTTTWQRPTA (Sequence ID 37) WW2 (1018-1051): LELPRGWEIKTDQQGKSFFVDHNSRATTFIDPRI (Sequence ID 38)

[0077] 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). MASSAREHLL FVRRRNPQMR YTLSPENLQS LAAQSSMPEN MTLQRANSDT 50 DLVTSESRSS LTASMYEYTL GQAQNLIIFW DIKEEVDPSD WIGLYHIDEN 100 SPANFWDSKN RGVTGTQKGQ IVWRIEPGPY FMEPEIKICF KYYHGISGAL 150 RATTPCITVK NPAVMMGAEG MEGGASGNLH SRKLVSFTLS DLRAVGLKKG 200 MFFNPDPYLK MSIQPGKKSS FPTCAHHGQE RRSTIISNTT NPIWHREKYS 250 FFALLTDVLE IEIKDKFAKS RPIIKRFLGK LTIPVQRLLE RQAIGDQMLS 300 YNLGRRLPAD HVSGYLQFKV EVTSSVHEDA SPEAVGTILG VNSVNGDLGS 350 PSDDEDMPGS HHDSQVCSNG PVSEDSAADG TPKHSFRTSS TLEIDTEELT 400 STSSRTSPPR GRQDSLNDYL DAIEHNGHSR PGTATCSERS MGASPKLRSS 450 FPTDTRLNAM LHIDSDEEDH EFQQDLGYPS SLEEEGGLIM FSRASRADDG 500 SLTSQTKLED NPVENEEAST HEAASFEDKP ENLPELAESS LPAGPAPEEG 550 EGGPEPQPSA DQGSAELCGS QEVDQPTSGA DTGTSDASGG SRRAVSETES 600 LDQGSEPSQV SSETEPSDPA RTESVSEAST RPEGESDLEC ADSSCNESVT 650 TQLSSVDTRC SSLESARFPE TPAFSSQEEE DGACAAEPTS SGPAEGSQES 700 VCTAGSLPVV QVPSGEDEGP GAESATVPDQ EELGEVWQRR GSLEGAAAAA 750 ESPPQEEGSA GEAQGTCEGA TAQEEGATGG SQANGHQPLR SLPSVRQDVS 800 RYQRVD EALP PNWEARIDSH GRIFYVDHVN RTTTWQRPTA PPAPQVLQRS 850 NSIQQMEQLN RRYQSIRRTM TNERPEENTN AIDGAGEEAD FHQASADFRR 900 ENILPHSTSR SRITLLLQSP PVKFLISPEF FTVLHSNPSA YRMFTNNTCL 950 KHMITKVRRD THHFERYQHN RDLVGFLNMF ANKQ LELPRG WEMKHDHQGK 1000 AFFVDHNSRT TTFIDPRL PL QSSRPTSALV HRQHLTRQRS HSAGEVGEDS 1050 RHAGPPVLPR PSSTFNTVSR PQYQDMVPVA YNDKIVAFLR QPNIFEILQE 1100 RQPDLTRNHS LREKIQFIRT EGTPGLVRLS SDADLVMLLS LFEEEIMSYV 1150 PPHALLHPSY CQSPRGSPVS SPQNSPGTQR ANARAPAPYK RDFEAKLRNF 1200 YRKLETKGYG QGPGKLKLII RRDHLLEDAF NQIMGYSRKD LQRNKLYVTF 1250 VGEEGLDYSG PSREFFFLVS RELFNPYYGL FEYSANDTYT VQISPMSAFV 1300 DNHHEWFRFS GRILGLALIH QYLLDAFFTR PFYKALLRIL CDLSDLEYLD 1350 EEFHQSLQWM KDNDIHDILD LTFTVNEEVF GQITERELKP GGANIPVTEK 1400 NKKEYIERMV KWRIERGVVQ QTESLVRGFY EVVDARLVSV FDARELELVI 1450 AGTAEIDLSD WRNNTEYRGG YHDNHIVIRW FWAAVERFNN EQRLRLLQFV 1500 TGTSSIPYEG FASLRGSNGP RRFCVEKWGK ITALPRAHTC FNRLDLPPYP 1550 SFSMLYEKLL TAVEETSTFG LE (Sequence ID 39) 1572 WWI (807-840): EALPPNWEARIDSHGRIFYVDHVNRTTTWQRPTA(Sequence ID 40) WW2 (985-1018): LELPRGWEMKHDHQGKAFFVDHNSRTTTFIDPRL(Sequence ID 41)

[0078] In some embodiments, a WW domain consists essentially of a WW domain or a WW domain variant. To consist essentially of a 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.

[0079] Alternatively, the WW domain may be a modified WW domain that includes two basic amino acids at the C-terminus of the domain. The technique is publicly known in the art and is described, for example, in Sambrook et al., (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press. Therefore, those skilled in the art can immediately modify an existing WW domain that normally does not have two basic C-terminal residues by including two basic residues at the C-terminus.

[0080] Basic amino acids are amino acids that possess a side-chain functional group with a pKa greater than 7, and include 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 a WW domain may be the same basic amino acid or may be different basic amino acids. In one embodiment, the two basic amino acids are two arginines.

[0081] The term WW domain also encompasses variants of the WW domain, any such variant having two basic amino acids at its C-terminus and retaining the WW domain's ability to bind to the PPXY(SEQ ID NO: 2) motif. Such WW domain variants refer to WW domains that retain the ability to bind to the PPXY(SEQ ID NO: 2) motif (i.e., the PPXY(SEQ ID NO: 2) motif of ARRDC1) and have undergone mutations including point mutations, insertion mutations, and / or deletion mutations in one or more amino acids, but still retain the ability to bind to the PPXY(SEQ ID NO: 2) motif. Thus, variants or derivatives encompass deletions, insertions, and additions including shortenings and fragments, e.g., conservative substitutions, site-specific variants, and allele variants, as well as modifications and post-translational modifications including one or more non-aminoacyl groups (e.g., sugars, lipids, etc.) covalently linked to the peptide. In creating such changes, substitutions of similar amino acid residues may be made based on the relative similarity of the side-chain substituents, such as their size, charge, hydrophobicity, hydrophilicity, and similarity, and such substitutions can be assayed by conventional tests for their effects on the peptide's function.

[0082] The WW domain can be part of a longer protein. Therefore, in various different embodiments, a protein may contain, consist of, or essentially consist of a WW domain, as defined herein. A polypeptide may be a protein that incorporates a WW domain as a functional domain within its protein sequence.

[0083] Detailed description of certain aspects of the present invention This disclosure relates, at least in part, to the discovery that a payload RNA encoding GFP associated with the ARRDC1 protein can be loaded into an ARMM and delivered to cells of the nervous system, including neurons in the central nervous system (CNS). The uptake of these ARMMs and payloads is enhanced by the presence of a viral envelope protein containing VSV-G, but is not limited to this. Different types of payloads, such as payload nucleic acids containing payload proteins and payload RNAs, can be loaded into such ARMMs for delivery to cells in the CNS. Various types of payload proteins, payload nucleic acids, payload RNAs, payload proteins, payload nucleic acids, and payload RNAs are known in the art and include those described in U.S. Patents 9,737,480, 9,816,080, 10,260,055, and International Publication 2018 / 067546, the entire contents of each of which are incorporated herein by reference. ARMMs Arrestin domain-containing protein 1-mediated microvesicles (ARMMs) are extracellular vesicles (EVs) distinct from exosomes. ARMM budding requires Arrestin domain-containing protein 1 (ARRDC1), localized on the cytosolic side of the plasma membrane, which, via a tetrapeptide motif, recruits the ESCRT-I complex protein TSG101 to the cell surface, initiating outward membrane budding. Thus, in contrast to exosomes, ARMM biosynthesis occurs in the plasma membrane. ARMMs exhibit several additional characteristics that could make them potentially ideal vehicles for therapeutic delivery. ARRDC1 is not only required but also sufficient to drive ARMM budding. In fact, simple overexpression of the ARRDC1 protein increases ARMM production in cells. This allows for controlled production of ARMMs using modern biological manufacturing methods. Furthermore, this suggests that endogenous proteins such as cell surface receptors can be actively recruited to ARMMs and delivered to receptor cells to initiate intercellular communication, and that exogenous payload molecules can similarly be packaged and delivered via ARMMs.

[0084] ARRDC1 ARRDC1 is a protein that contains the PSAP (SEQ ID NO: 1) and PPXY (SEQ ID NO: 2) motifs at its C-terminus and interacts with TSG101 as described in this application. It should be understood that the PSAP (SEQ ID NO: 1) and PPXY (SEQ ID NO: 2) motifs do not need to be at the true C-terminus of ARRDC1. Rather, they may be at the C-terminal portion of the ARRDC1 protein (e.g., near the C-terminus of ARRDC1). This disclosure also intends variants of ARRDC1, such as ARRDC1 proteins and / or fragments of ARRDC1 proteins, that have some degree of identity with 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 that contains the PSAP (SEQ ID NO: 1) and PPXY (SEQ ID NO: 2) motifs 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 any one amino acid sequence of SEQ ID NOs. 42–44, contains the PSAP (SEQ ID NO: 1) motif and the PPXY (SEQ ID NO: 2) motif, and interacts with TSG101.In some embodiments, the ARRDC1 protein is 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, and at least It also has 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 consecutive amino acids, contains the PSAP (SEQ ID NO: 1) motif and the PPXY (SEQ ID NO: 2) motif, and interacts with TSG101. In some embodiments, the ARRDC1 protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences described in SEQ ID NOs: 42-44, contains the PSAP (SEQ ID NO: 1) motif and the PPXY (SEQ ID NO: 2) motif, and interacts with TSG101. In some embodiments, the ARRDC1 protein contains any one of the amino acid sequences described in SEQ ID NOs: 42-44. Exemplary, non-limiting ARRDC1 protein sequences are provided herein, and additional suitable ARRDC1 protein variants conforming to aspects of the present invention are known in the art. It will be understood by those skilled in the art 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):

[0085] >gi|22748653|ref|NP_689498.1|Arrestin Domain-Containing Protein 1[Homo sapiens] [ka] (Sequence ID 42)

[0086] >gi|244798004|ref|NP_001155957.1|Arrestin domain-containing protein 1 isoform a[Mus musculus] [ka] [ka] (Sequence No. 43)

[0087] >gi|244798112|ref|NP_848495.2|Arrestin domain-containing protein 1 isoform b[Mus musculus] [ka] (Sequence ID 44)

[0088] TSG101 In certain embodiments, the microvesicles of the present invention further include TSG101 (tumor susceptibility gene 101), which belongs to a group of obviously inactive homologs of ubiquitin-conjugating enzymes. The protein contains a coiled-coil domain, which interacts with stasmin, a cytosolic lin protein associated with tumorigenesis. TSG101 is a protein containing a UEV domain, which interacts with ARRDC1. As referred to herein, UEV refers to a ubiquitin E2 variant domain consisting of approximately 145 amino acids. The structure of the domain contains an α / β folding structure similar to that of the canonical E2 enzyme, but has an additional N-terminal helix and lacks two further C-terminal helices. The UEV, often found in the TSG101 / Vps23 protein, interacts with ubiquitin molecules and is essential for the transport of numerous ubiquitinated payloads into the multivesicle endoplasmic reticulum (MVB). Furthermore, the UEV domain can bind to Pro-Thr / Ser-Ala-Pro peptide ligands, a fact utilized by viruses such as HIV. Therefore, the TSG101 UEV domain binds to the PTAP tetrapeptide motif in the viral Gag protein involved in viral budding. This disclosure also intends to present variants of TSG101, such as TSG101 proteins and / or fragments of TSG101 proteins, that have some degree of identity with the TSG101 protein (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity) and can interact with ARRDC1. Thus, the TSG101 protein may be a protein containing a UEV domain and interacting with ARRDC1. In some embodiments, the TSG101 protein is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to any one of the amino acid sequences 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 identical consecutive amino acids, contains 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 described in SEQ ID NOs.45-47, and contains a UEV domain. In some embodiments, the ARRDC1 protein contains any one of the amino acid sequences described in SEQ ID NOs.45-47. Exemplary, non-limiting TSG101 protein sequences are provided herein, and additional suitable TSG101 protein sequences, isoforms, and variants are known in the art. It will be understood by those skilled in the art that the present invention is not limited in this respect. Exemplary TSG101 sequences include the following sequences (the UEV domain in these sequences includes amino acids 1-145, which are underlined in the following sequences):

[0089] >gi|5454140|ref|NP_006283.1|Tumor susceptibility gene 101 protein [Homo sapiens] MAVSESQLKKMVSKYKYRDLTVRETVNVITLYKDLKPVLDSYVFNDGSSRELMNLTGTIPVPYRGNTYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHEWKHPQSDLLGLIQVMIVVFGDEPPVFSRP ISASYPPYQATGPPNTSYMPGMPGGISPYPSGYPPNPSGYPGCPYPPGGPYPATTSSQYPSQPPVTTVGPSRDGTISEDTIRASLISAVSDKLRWRMKEEMDRAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDTIFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY(Sequence ID 45)

[0090] >gi|11230780|ref|NP_068684.1|Tumor susceptibility gene 101 protein [Mus musculus] MAVSESQLKKMMSKYKYRDLTVRQTVNVIAMYKDLKPVLDSYVFNDGSSRELVNLTGTIPVRYRGNIYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHDWKHPRSELLELIQIMIVIFGEEPPVFSRP TVSASYPPYTATGPPNTSYMPGMPSGISAYPSGYPPNPSGYPGCPYPPAGPYPATTSSQYPSQPPVTTVGPSRDGTISEDTIRASLISAVSDKLRWRMKEEMDGAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDTIFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY (Sequence ID 46)

[0091] >gi|48374087|ref|NP_853659.2|Tumor susceptibility gene 101 protein [Rattus norvegicus] MAVSESQLKKMMSKYKYRDLTVRQTVNVIAMYKDLKPVLDSYVFNDGSSRELVNLTGTIPVRYRGNIYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHDWKHPRSELLELIQIMIVIFGEEPPVFSRPTVSASYPPYTAAGPPNTSYLPSMPSGISAYPSGYPPNPSGYPGCPYPPAGPYPATTSSQYPSQPPVTTAGPSRDGTISEDTIRASLISAVSDKLRWRMKEEMDGAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDTIFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY (Sequence ID 47)

[0092] The structure of the UEV domain is known to those skilled in the art (see, for example, Owen Pornillos et al., Structure and functional interactions of the Tsg101 UEV domain, EMBO J.2002 May 15;21(10):2397-2406, whose entire contents are incorporated into this application by reference).

[0093] Expression construct Some aspects of the present invention provide expression constructs for encoding gene products that induce or promote ARMM production in cells having such constructs. In some aspects, the expression constructs described herein encode fusion proteins described herein, such as ARRDC1 fusion protein and TSG101 fusion protein. In some aspects, the expression construct encodes the ARRDC1 protein or a variant thereof, and / or the TSG101 protein or a variant thereof. In some aspects, overexpression of either or both of these gene products in cells increases ARMM production by the cells, thus transforming the cells into microvesicle-producing cells. In some aspects, such an expression construct includes at least one restriction or recombination site at either the C-terminus or N-terminus of the encoded ARRDC1 or a variant thereof, from which in-frame cloning of the protein sequence can be fused. As another example, the expression construct includes at least one restriction or recombination site that allows in-frame cloning of the protein sequence to be fused at either the C-terminus or N-terminus of one or more encoded WW domains.

[0094] In some embodiments, the expression construct comprises (a) a nucleotide sequence encoding the ARRDC1 protein or a variant thereof operably linked to a heterologous promoter, and (b) a restriction or recombination site located adjacent to the nucleotide sequence encoding ARRDC1, enabling in-frame insertion of a nucleotide sequence encoding a payload protein, or an RNA-binding protein or RNA-binding protein variant sequence, into 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 inductive promoter. Some aspects of the present invention provide an expression construct comprising (a) a nucleotide sequence encoding the TSG101 protein or a variant thereof operably linked to a heterologous promoter, and (b) a restriction or recombination site located adjacent to the nucleotide sequence encoding TSG101, enabling in-frame insertion of a nucleotide sequence encoding a payload protein, or an RNA-binding protein, a DNA-binding protein, or a variant sequence thereof, into 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 inductive promoter.

[0095] Some aspects of the present invention provide an expression construct comprising (a) a nucleotide sequence encoding a WW domain or a 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, enabling in-frame insertion of a payload protein or RNA-binding protein, or a protein variant thereof, into the nucleotide sequence encoding the WW domain. In some embodiments, the heterologous promoter may be a constitutive promoter, and in some embodiments, the heterologous promoter may be an inductive promoter. The expression construct may encode a payload protein or RNA-binding protein fused to at least one WW domain. In some embodiments, the expression construct encodes a payload protein or RNA-binding protein or a variant thereof fused to at least one WW domain or a variant thereof. Any of the expression constructs described herein may encode any WW domain or a variant thereof. In some embodiments, the heterologous promoter may be a constitutive promoter, and in some embodiments, the heterologous promoter may be an inductive promoter.

[0096] The expression constructs described herein may include any nucleic acid sequence capable of encoding a WW domain or a variant thereof. For example, nucleic acid sequences encoding a WW domain or a WW domain variant may be derived from human ubiquitin ligases WWP1, WWP2, Nedd4-1, Nedd4-2, Smurf1, Smurf2, ITCH, NEDL1, or NEDL2. Exemplary nucleic acid sequences of WW domain-containing proteins are listed below. It should be understood that any nucleic acid encoding a WW domain or a WW domain variant of an exemplary protein may be used in the inventions described herein, but is not intended to limit them.

[0097] Human WWP1 nucleic acid sequence (uniprot.org / uniprot / Q9H0M0).

[0098] Human WWP2 nucleic acid sequence (uniprot.org / uniprot / O00308).

[0099] Human Nedd4-1 nucleic acid sequence (uniprot.org / uniprot / P46934).

[0100] Human Nedd4-2 nucleic acid sequence (>gi|345478679|ref|NM_015277.5|d mRNA, a developmentally downregulated 4-like E3 ubiquitin protein ligase (NEDD4L) transcript variant expressed in Homo sapiens neural progenitor cells).

[0101] Human Smurf1 nucleic acid sequence (uniprot.org / uniprot / Q9HCE7).

[0102] Human Smurf2 nucleic acid sequence (uniprot.org / uniprot / Q9HAU4).

[0103] Human ITCH nucleic acid sequence (uniprot.org / uniprot / Q96J02).

[0104] Human NEDL1 nucleic acid sequence (uniprot.org / uniprot / Q76N89).

[0105] Human NEDL2 nucleic acid sequence (uniprot.org / uniprot / Q9P2P5).

[0106] Some aspects of the present invention provide expression constructs that encode any of the proteins, nucleic acids such as RNA, or fusions thereof as described herein.

[0107] Nucleic acids encoding any of the proteins and / or nucleic acids (including RNA) described herein may be among a large number of nucleic acid “vectors” known in the art. As used herein, “vector” means any nucleic acid or nucleic acid-carrying particle, cell, or organism that can be used to transfer nucleic acid into a host cell. The term “vector” encompasses both viral and nonviral products and means for introducing nucleic acid into a cell. A “vector” can be used in vitro, ex vivo, or in vivo. Nonviral vectors include plasmids, cosmids, artificial chromosomes (e.g., bacterial or yeast artificial chromosomes), and may include, for example, liposomes, charged lipids (cytofectin), DNA-protein complexes, and biopolymers. Viral vectors include, for example, retroviruses, lentiviruses, adeno-associated viruses, poxviruses, baculoviruses, reoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, and adenovirus vectors. A vector may also include the entire genome sequence of a virus or a recombinant genome sequence. The vector may also contain a portion of the genome that includes a functional sequence for the production of a virus that can infect, enter, or be introduced into a cell in order to deliver nucleic acids within it.

[0108] The expression of any protein and / or nucleic acid (including RNA) described herein may be controlled by any regulatory sequence (e.g., promoter sequence) known in the art. Regulatory sequences described herein are nucleic acid sequences that control the expression of a nucleic acid sequence. Regulatory or regulatory sequences may include sequences responsible for expressing a particular nucleic acid, or other sequences such as heterogeneous synthetic or partially synthetic sequences. These sequences may be of eukaryotic, prokaryotic, or viral origin, stimulating or repressing gene transcription in a specific or nonspecific manner, and in an inducible or non-inducible manner. Regulatory or regulatory regions may include origins of replication, RNA splice sites, introns, chimeric or hybrid introns, promoters, enhancers, transcription termination sequences, poly(A) sites, locus control regions, and signal sequences that direct polypeptides into the secretory pathways of target cells. Heterogeneous regulatory regions are regulatory regions that do not naturally associate with the expressed nucleic acid to which they are linked. Heterogeneous regulatory regions include regulatory regions from different species, regulatory regions from different genes, hybrid regulatory sequences, and regulatory sequences that do not occur naturally but are designed by those skilled in the art.

[0109] The term "operably linked" refers to the arrangement of sequences or regions configured such that their components perform their normal or intended functions. Therefore, regulatory or control sequences operably linked to a coding sequence can influence the expression of the coding sequence. Regulatory or control sequences do not need to be contiguous with the coding sequence, as long as they function to lead to proper expression or polypeptide production. Thus, for example, an intervening sequence that is not translated but is transcribed may exist between the promoter sequence and the coding sequence, and the promoter sequence can still be considered operably linked to the coding sequence. As described herein, a promoter sequence is a DNA regulatory region located slightly off-5' of a gene that acts as a binding site for RNA polymerase. A promoter sequence can bind to RNA polymerase in a cell and / or initiate transcription of a downstream (3' direction) coding sequence. A promoter sequence can be a promoter capable of initiating transcription in prokaryotes or eukaryotes. Some, not limited to, examples of eukaryotic promoters include the cytomegalovirus (CMV) promoter, the chicken β-actin (CBA) promoter, and hybrid forms of the CBA promoter (CBh).

[0110] Cells that produce microvesicles containing payloads The microvesicle-producing cells of the present invention may be cells comprising any of the expression constructs described herein, any of the fusion proteins, or payloads of molecules (e.g., biological molecules, small molecules, proteins, and nucleic acids (e.g., DNA, RNA), DNA plasmids, siRNA, mRNA). For example, the microvesicle-producing cells of the present invention may comprise one or more recombinant expression constructs encoding (1) the ARRDC1 protein or a PSAP (SEQ ID NO: 1) motif-containing variant, and (2) an RNA-binding protein (e.g., Tat protein) that associates with the ARRDC1 protein or a PSAP (SEQ ID NO: 1) motif-containing variant. In some embodiments, the microvesicle-producing cells may comprise, under the control of a heterologous promoter, one or more recombinant expression constructs encoding (1) the ARRDC1 protein or a PSAP (SEQ ID NO: 1) motif-containing variant, and (2) a payload protein such as an RNA-binding protein or a variant thereof fused to at least one WW domain. In certain embodiments, the expression construct of the microvesicle-producing cell encodes a payload protein having one or more WW domains or variants thereof. In certain embodiments, the expression construct in microvesicle-producing cells encodes RNA-binding proteins, such as RNA that associates with (e.g., specifically binds to) therapeutic RNA.

[0111] Any of the expression constructs described herein can be stably inserted into the cellular genome. In some embodiments, the expression construct is maintained within the cell but not inserted into the cellular genome. In some embodiments, the expression construct resides on a vector, such as a plasmid vector, cosmid vector, viral vector, or artificial chromosome. In some embodiments, the expression construct further comprises additional sequences or elements that facilitate the maintenance and / or replication of the expression construct in microvesicle-producing cells or that improve the expression of the fusion protein in the cell. Such additional sequences or elements may include, for example, origins of replication, antibiotic resistance cassettes, polyA sequences, and / or transcription insulators. Several expression constructs suitable for generating microvesicle-producing cells according to aspects of the present invention are described elsewhere in this application. Methods and reagents for generating additional expression constructs suitable for generating microvesicle-producing cells according to aspects of the present invention will be apparent to those skilled in the art based on this disclosure. In some embodiments, the microvesicle-producing cells are mammalian cells, such as mouse cells, rat cells, hamster cells, rodent cells, or non-human primate cells. In some embodiments, the microvesicle-producing cells are human cells.

[0112] Those skilled in the art may use prior art, such as molecular or cell biology, virology, microbiology, and recombinant DNA techniques. Illustrative techniques are described in detail in the literature. For example, the present invention may be manufactured and used in accordance with the following general text. 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(DNGlover ed.1985),Oligonucleotide Synthesis(MJGaited.1984),Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. (1985)), Transcription And Translation Hames & Higgins, eds. (1984); Animal Cell Culture (RI. Freshney, ed. (1986)), Immobilized Cells And Enzymes (IRL Press, (1986)), Gennaro et al., (eds.) Remington's Pharmaceutical Sciences, 18th edition;B.Perbal,A Practical Guide To Molecular Cloning (1984), FMAusubel et al.,(eds.),Current Protocols in Molecular Biology,John Wiley & Sons,Inc.(updates through 2001),Coligan et al.,(eds.),Current Protocols in Immunology,John Wiley & Sons,Inc.(updates through 2001),W.Paul et al.,(eds.)Fundamental Immunology,Raven Press;E.J. Murray et al., (ed.) Methods in Molecular Biology: Gene Transfer and Expression Protocols, The Humana Press Inc. (1991) (especially vol. 7), and JECelis et al., Cell Biology: A Laboratory Handbook, Academic Press (1994). .

[0113] Delivery of ARMM containing payload molecules The microvesicles of the present invention (e.g., ARMMs including either an expression construct and / or a payload of molecules (e.g., biological molecules, small molecules, proteins, and nucleic acids (e.g., DNA, RNA), DNA plasmids, siRNA, mRNA)) may further have a targeting moiety. The targeting moiety is used to target the delivery of the ARMM to a specific cell type, which may result in the release of the contents of the ARMM into the cytoplasm of the specific targeted cell type. The targeting moiety may be a viral envelope protein that normally functions to assist in viral attachment and entry into cells. The viral envelope protein may enable targeting of cells in the CNS. The viral envelope protein includes, but is not limited to, vesicular stomatitis virus G protein (VSV-G, Genbank accession number and version number: AJ 318514.1) or rabies virus glycoprotein (RVG, Genbank accession number and version number: M 38452.1). The VSV-G protein facilitates viral entry by mediating viral attachment to LDL receptors (LDLRs) or LDLR family members present on target cells. Following binding, the VSV-G-LDLR complex rapidly undergoes endocytosis, proceeding to mediate the fusion of the viral envelope with the endosomal membrane. VSV-G enters cells via partially clathrin-coated vesicles, which contain more clathrin and clathrin adapters 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 very broad targeting. RVG is a trimer 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) facilitate binding (e.g., targeting) to cells in the nervous system, such as the CNS and PNS.

[0114] The targeting moiety can selectively bind to antigens on target nervous system cells. For example, the targeting moiety may be a membrane-bound immunoglobulin, integrin, receptor, receptor ligand, aptamer, small molecule, or a variant thereof. Numerous cell surface proteins may also be included in the ARMM to facilitate the binding of the ARMM to target cells and / or to facilitate the uptake of the ARMM into target nervous system cells. Suitable integrins, receptor tyrosine kinases, G protein-coupled receptors, and membrane-bound immunoglobulins for use in embodiments of the present invention will be apparent to those skilled in the art. 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 / smoothed receptor, CXCR4, CCR5, or beta-adrenergic receptor.

[0115] Further molecules, such as synthetic small molecules or natural products, can be modified to associate with ARMM proteins (e.g., TSG101 or ARRDC1) for targeting purposes. This association can facilitate their incorporation into ARMM, allowing the molecules to be used for delivery to target cells. By incorporating a cleavable linker, the small molecules can be released upon delivery to target cells. As a non-limiting example, small molecules can be linked to biotin, thereby allowing them to associate with the ARRDC1 protein fused to streptavidin. As another non-limiting example, small molecules can ligate to synthetic high-affinity ligands that specifically bind to mutant FKBP12, such as FKBP12(F36V) (Yang W, Rozamus LW, Narula S, Rollins CT, Yuan R, Andrade LJ, Ram MK, Phillips TB, van Schravendijk MR, Dalgarno D, Clackson T, Holt DA. Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket. J Med Chem. 2000 Mar 23;43(6):1135-42), which associate with the ARRDC1 protein fused to FKBP12(F36V). The association of small molecules to ARMM proteins (e.g., TSG101 or ARRDC1) facilitates the loading of small molecules into ARRDC1-containing ARMMs.

[0116] Some aspects of the present invention relate to the recognition that ARMM is taken up by target cells (e.g., CNS cells), and that ARMM uptake results in the release of the contents of the ARMM into the cytoplasm of the target cells (e.g., CNS cells). In some aspects, the payload is a drug that causes a desired change in the target cells, such as a change in cell viability, proliferation rate, differentiation stage, cellular identity, chromatin state, transcription rate of one or more genes, transcription profile, or post-transcriptional changes of gene compression. It will be understood by those skilled in the art that the drug to be delivered will be selected according to the desired effect on the target cells.

[0117] In some embodiments, cells derived from the target are obtained, and the payload is delivered to the cells ex vivo by a system or method provided herein. In some embodiments, the treated cells are selected as cells in which a desired gene is expressed or repressed. In some embodiments, the treated cells having the desired payload protein or payload RNA are returned to the target from which they were obtained.

[0118] In some embodiments, an ARMM comprising any fusion protein, any binding RNA, any payload RNA, and / or any binding RNA fused to any payload RNA as described herein further encompasses a detectable label. Such an ARMM allows for the labeling of target cells without genetic manipulation. Suitable detectable labels 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 that catalyze reactions resulting in detectable reaction products. Suitable detectable labels according to some aspects of the present invention further encompass membrane-bound antigens, such as membrane-bound ligands that can be detected by commonly available antibodies or antigen-binding agents.

[0119] In some embodiments, an ARMM is provided comprising a payload RNA encoding a transcription factor, transcriptional repressor, fluorescent protein, kinase, phosphatase, protease, ligase, chromatin modulator, or recombinase. In some embodiments, an ARMM is provided comprising a payload RNA (e.g., siRNA) that inhibits the expression of a transcription factor, transcriptional repressor, fluorescent protein, kinase, phosphatase, protease, ligase, chromatin modulator, or recombinase. In some embodiments, the payload RNA is therapeutic RNA. In some embodiments, the payload RNA is RNA that affects a change in the state or identity of a target cell. For example, in some embodiments, the payload RNA encodes a reprogramming factor. Appropriate transcription factors, transcriptional repressors, fluorescent proteins, kinases, phosphatases, proteases, ligases, chromatin modulators, recombinases, and reprogramming factors may be encoded by a payload RNA that associates with binding RNA to facilitate their incorporation into the ARMM, and their functions may be tested by any method known to those skilled in the art; the present invention is not limited in this respect.

[0120] Methods for isolating ARMMs as described herein are also provided. One exemplary method comprises collecting the culture medium or supernatant of 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 ARMMs, e.g., cells expressing recombinant ARMM proteins, e.g., recombinant ARRDC1 or TSG101 proteins such as RNA-binding proteins (e.g., Tat protein) or variants of ARRDC1 or TSG101 proteins fused to them. In some embodiments, the supernatant is pre-cleared of cell debris by centrifugation, e.g., two consecutive centrifuges at increasing G values ​​(e.g., 500G and 2000G). In some embodiments, the method comprises passing the supernatant through a 0.2 μm filter to remove all large debris and whole cells of the cell debris. In some embodiments, the supernatant is subjected to ultracentrifugation at 120,000 G for, for example, 2 hours, depending on the volume of the centrifuge. The resulting pellet contains microvesicles. In some embodiments, exosomes are depleted from the microvesicle pellet by staining and / or sorting (e.g., by FACS or MACS) using exosome markers described herein. The isolated or concentrated ARMM can be suspended in culture medium or a suitable buffer as described herein.

[0121] Methods for microvesicle-mediated delivery of payloads to nerve cells Some aspects of the present invention provide a method for delivering a drug to target cells of the nervous system. Target cells can be brought into contact with ARMM in various ways. For example, target cells can be brought into contact directly with ARMM as described herein or with ARMM isolated from microvesicle-producing cells. Contact can be made by administering ARMM to target cells on a culture dish in vitro (e.g., parenterally or parenterally) or by administering ARMM to a subject in vivo. In some embodiments, ARMM is produced from cells obtained from a subject. In some embodiments, ARMM produced from cells obtained from a subject is administered to the subject from which the ARMM-producing cells were obtained. In some embodiments, ARMM produced from cells obtained from a subject is administered to a subject different from the subject from which the ARMM-producing cells were obtained. For example, cells may be obtained from a subject and engineered to express one or more of the constructs provided herein (e.g., they may be engineered to express binding RNA, ARRDC1 protein, ARRDC1 protein fused to an RNA-binding protein, and / or payload RNA associated with an RNA-binding protein fused to a WW domain). Cells obtained from a subject and engineered to express one or more of the constructs provided herein may be administered to the same subject from which the cells were obtained or to a different subject. Alternatively, cells obtained from a subject and engineered to express one or more of the constructs provided herein may produce ARMM, which may be isolated and administered to the same form of subject from which the cells were obtained or to a different subject from which the cells were obtained.

[0122] Alternatively, target cells of the nervous system can be brought into contact with the microvesicle-producing cells described herein, for example, by co-culturing the target cells and microvesicle-producing cells in vitro, or by administering the microvesicle-producing cells to a subject having the target cells in vivo. Thus, this method may include bringing the target cells into contact with microvesicles, for example, ARMMs containing any of the delivered payload proteins described herein. The target cells can be brought into contact with the microvesicle-producing cells described herein, or with isolated microvesicles having a lipid bilayer and the ARRDC1 protein or a variant thereof, a payload, and a viral envelope protein.

[0123] It should be understood that target cells of the nervous system can be of any origin, e.g., of biological origin. In some embodiments, the target cells are mammalian cells. 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 cells are stem cells. It should also be understood that the target cells can be any cell type of the nervous system. In other cases, the target cells can be any differentiated cell type found in the subject. In some embodiments, the target cells are in vitro cells, and the method includes administering microvesicles to in vitro cells or co-culturing target cells with in vitro microvesicle-producing cells. In some embodiments, the target cells are cells in the subject, and the method includes administering microvesicles or microvesicle-producing cells to the subject. In some embodiments, the subject is a mammalian subject, e.g., rodents, mice, rats, hamsters, or non-human primates. In some embodiments, the subject is a human subject.

[0124] In some embodiments, the target cells are pathological cells. In some embodiments, the target cells are cancer cells. In some embodiments, the microvesicles are bound to a conjugate agent that selectively binds to an antigen on the surface of the target cells. In some embodiments, the antigen on the target cells is a cell surface antigen. In some embodiments, the conjugate agent is a membrane-bound immunoglobulin, integrin, receptor, or receptor ligand. Suitable surface antigens of target cells (e.g., cells of the nervous system), for example, specific target cell types, such as cancer cells, are known to those skilled in the art, as are suitable conjugate agents that specifically bind to such antigens. Membrane-bound conjugate agents, such as membrane-bound immunoglobulins, and methods for producing membrane-bound antibodies or antibody fragments that specifically bind to surface antigens expressed on the surface of cancer cells are also known to those skilled in the art. The choice of conjugate agent will naturally depend on the identity or type of the target cells. The various types of cell surface antigens specifically expressed on nervous system cells that can be targeted by ARMMs, including membrane-bound conjugate agents, will be obvious to those skilled in the art. It will be understood that the present invention is not limited in this respect.

[0125] In some embodiments, target cells of the nervous system encompass disease targets. For example, non-limiting examples of gene targets for ARMM therapeutics for Alzheimer's disease (familial and late-onset) and related indications include APP, PSEN1, PSEN2, APOE(e2), APOE(e3), APOE(e4), ADAMTS4, HESX1, HS3ST1, HLA-DQB1, NYAP1, CNTNAP2, ECHDC3, ADAM10, APH1B, KAT8, ABI3, SCIMP, ACE, ALPK2, B HMG1, ADAMTS1, IQCK1, CLU, SORL1, ABCA7, TREM2, CD33, MS4A6A, CR1, EPHA1, HLA-DRB1, HLA-DRB5, IL1RAP, INPP5D, PL Includes CG2, CD2AP, BIN1, RIN3, SLC24A4, PICALM, PTK2B, CASS4, ABI3, FERMT2, SPI1, MEF2C, ZCWPW1, NME8, CR1, PICALM. Non-limiting gene targets for ARMM therapeutics for frontotemporal dementia / amyotrophic lateral sclerosis spectrum disorders and related indications include MAPT, GRN, C9ORF72, SOD1, FUS, UBQLN2, CHCHD10, SQSTM1, VCP, CHMP2B, TBK1, CTSD, CTSF, TRKA, ERBB4, EWSR1, TAF15, HNRNPA1, HNRNPA2B1, ATXN2, OPTN, ANG, SETX, DAO, PFN1, ALS2, VAPB, SIGMAR1, MATR3, NEK1, PFN1, and TIA1TUBA4A. Non-limiting gene targets for ARMM therapeutics for Parkinson's disease and related indications include SNCA, LRRK2, PARK7, PINK1, PRKN, DJ-1, VPS35, UCHL1, ATP13A2, and GBA1.Non-limiting examples of gene targets for ARMM therapy for gene targeting of other neurological diseases include: SMN1, SMN2, HTT, DMPK, FMR1, MECP2, CIC, TCF4, CNTNAP2, STXBP1, SHANK2, TSC1, TSC2, SPG11, SEPT9, PANK2, PLA2G6, C19orf12, FTL, MR1, SLC2A1, DRD2, GCH1, GCDH, PRKRA, SGCE, THAP1, TOR1A, TAF1, TIMM8A This includes ACTB, SLC6A3, DYNC1H1, YARS, MPZ, NEFL, PMP22, ARHGEF10, LITAF, EGR2, MFN2, RAB7A, LMNA, GARS, HSPB1, GDAP1, HSPB8, DNM2, SH3TC2, MTMR2, SBF2, NDRG1, PRX, FGD4, FIG4, GJB1, PRPS1, CTDP1, GAN, BSCL2, WNK1, IKBKAP, NTRK1, NGF, SPTLC1, and TH. Non-limiting examples of gene targets for ARMM therapeutics for pain disorders and related indications include SCN9A and FAAH.

[0126] Pharmaceutical composition Other aspects of this disclosure relate to pharmaceutical compositions comprising either ARMM or microvesicle (e.g., ARMM)-producing cells provided herein. Where used herein, the term “pharmaceutical composition” refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, extension of half-life, or as other therapeutic compounds).

[0127] As used in this application, 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 stearate or zinc stearate, or stearic acid), or solvent encapsulation material, that is involved in the transport or delivery of a compound from one site of the body (e.g., a site of delivery) to another site (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the target tissue (e.g., physiologically compatible, sterile, physiological pH, etc.). Some examples of substances that can function as pharmaceutically acceptable carriers include: (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) tragacanth powder; (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 soybean oil; (10) pro (11) Glycols such as pyrene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG), esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer, polyester, polycarbonate and / or polyanhydride, bulking agents such as polypeptides and amino acids, serum components such as serum albumin, HDL and LDL, and C2-C2 ethanol. 12This includes alcohols and (24) other non-toxic, suitable substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulations. The terms “excipient,” “carrier,” and “pharmaceutically acceptable carrier” are used interchangeably in this application.

[0128] In some embodiments, the pharmaceutical composition is formulated to deliver, for example, a payload protein or payload RNA (e.g., a payload RNA expressing a tumor suppressor) to cells for delivery to a target. Suitable routes for administering the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, percutaneous, intradermal, intrafocal, intra-articular, intraperitoneal, intrabladderal, transmucosal, gingival, intradermal, intracochlear, intratympanic, intra-organ, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periorbital, intratumoral, intracerebral, and intraventricular administration.

[0129] In some embodiments, the pharmaceutical compositions described herein are administered topically to a disease site (e.g., cells of the nervous system). In some embodiments, the pharmaceutical compositions described herein are administered to a target by injection, by catheter, by suppository, or by implant, the implant comprising a membrane such as a ciarastic membrane, or a porous, non-porous, or gelatinous material containing fibers.

[0130] In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a composition suitable for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, the pharmaceutical composition for administration by injection is a solution in sterile isotonic aqueous buffer. If necessary, the drug may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. When the drug is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0131] For systemic administration, this pharmaceutical composition may be a liquid, such as sterile saline, Ringer's lactate solution, or Hanks' solution. Furthermore, this pharmaceutical composition may be in solid form and may be redissolved or suspended immediately before use. A lyophilized form is also intended.

[0132] The pharmaceutical compositions described herein may be administered or packaged, for example, as unit doses. When used in reference to the pharmaceutical compositions of this disclosure, the term "unit dose" refers to a physically distinct unit suitable as the unit dose in question, each unit comprising a predetermined amount of active material calculated to produce a desired therapeutic effect in conjunction with the necessary diluent, i.e., carrier or vehicle.

[0133] Furthermore, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) a container for containing the ARMM or microvesicle-producing cells of the present invention, and (b) a second container for containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent can be used, for example, for the reconstitution or dilution of the ARMM or microvesicle-producing cells of the present invention. Optionally, such container may be associated with a notice in the format prescribed by a government agency regulating the manufacture, use or sale of pharmaceutical or biological products, the notice indicating approval by such agency for manufacture, use or sale for human administration.

[0134] In another embodiment, the product includes materials useful for treating the diseases described above. In some embodiments, the product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, such as glass or plastic. In some embodiments, the container may hold 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 that can be punctured by a subcutaneous injection needle. The activator 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 a selected disease. The product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other elements desirable from a commercial and user perspective, and may include other buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use.

[0135] Kits, vectors, cells Several embodiments of this disclosure provide a kit comprising a nucleic acid construct comprising a nucleotide sequence encoding 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 one of the proteins, fusion proteins, and / or RNAs provided herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives the expression of one of the proteins, fusion proteins, and / or RNAs provided herein.

[0136] Several aspects of this disclosure provide microvesicle (e.g., ARMM) producing cells comprising any of the proteins, fusion proteins, and / or RNAs provided herein. In some embodiments, the cells 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 vector comprises a virus-targeting protein.

[0137] However, those skilled in the art will understand that additional proteins, fusion proteins, and RNAs are apparent based on this disclosure and knowledge in the art.

[0138] The functions and advantages of these and other embodiments of the present invention will be better understood from the examples below. The following examples are intended to illustrate the benefits of the present invention and to describe specific embodiments, and are not intended to exemplify the entire scope of the invention. It will be understood that the examples are not intended to limit the scope of the invention. [Examples]

[0139] Example 1: Development of an ARMM platform for CNS disorders in human induced pluripotent stem cell (iPSC) models for biological and therapeutic discovery / development. A methodology has been developed in which fibroblasts are isolated from a subject by skin biopsy (although other isolation techniques may be used), and the cells are reprogrammed into induced pluripotent stem cells (iPSCs) and induced to differentiate into neural progenitor cells (Figure 1). iPSCs are skin or blood-derived cells reprogrammed into an embryonic pluripotency state. This embryonic state allows the cells to differentiate into additional cell types as needed, providing a virtually unlimited source of any type of cell needed for therapeutic or research purposes (for example, iPSCs can be differentiated into neurons to treat or study neurological disorders). Neural progenitor cells are CNS progenitor cells that give rise to many, if not all, of the glial and neuronal cell types present in the CNS. Neural progenitor cells do not produce non-neuronal cells, such as immune system cells, which are also present in the CNS. The cells are differentiated into neurons in vitro, banked for tau protein expression using PHF1 (phosphorylated tau protein) and K9JA (total tau protein), and characterized against a control group of neurons of the same species (in this case, human). Next, ARRDC1-mediated microvesicles (ARMMs) containing target probes and therapeutic leads (e.g., small molecules, proteins, nucleic acids) are screened against control cells and target neurons.

[0140] Imaging systems have also been developed to analyze the results of ARMM-mediated payload screens. Automated confocal microscopy was used in the assay to analyze high-content, single-cell level imaging (Figure 2). Laser line scanning confocal technique was used for ultra-wide fields with a next-generation sCMOS detector (5.5 Mp). Human neural progenitor cells, neurons, and glial cells were analyzed using 4-channel imaging with high-density 96-well plates.

[0141] Example 2: Payload and target cell type for platform development A platform is being developed for using ARRDC1-mediated microvesicles (ARMMs) to deliver molecular payloads (e.g., biological molecules such as proteins, nucleic acids (e.g., DNA, RNA, DNA plasmids, siRNA, mRNA)), editing complexes, and small molecules to various nervous system cell types, such as CNS cells. ARMMs carry one of these molecules as a payload and are used to deliver the payload to nervous system cells, such as CNS cells (Figure 3). For example, a molecule can be directly ligated to the ARRDC1 protein, a molecule can associate with the ARRDC1 protein by fusing one or more WW domains to the molecule, thereby enabling the molecule to associate with the ARRDC1 PPXY (SEQ ID NO: 2) motif, or a molecule can associate with the ARRDC1-Tat fusion protein for the delivery of TAR payload RNA. The platform is intended to target a variety of molecular payloads, including, but not limited to, proteins, peptides, DNA, RNA (e.g., mRNA, siRNA, shRNA, miRNA, ribozymes), antibody fragments, signaling proteins, editing complexes (e.g., CRISPR / Cas9, its variants), and small molecules. Furthermore, the platform targets a variety of cells in the nervous system, including, but not limited to, neurons, glia, oligodendrocytes, astrocytes, and microglia, as well as cells of the CNS.

[0142] Example 3: Use of viral envelope proteins on CNS target cells The viral envelope protein VSV-G was analyzed to investigate whether it could be used to enhance the uptake of ARMM containing the molecular payload. For delivery to neural progenitor cells, ARMM was added at various concentrations as four experimental sets: blank, ARRDC1, ARRDC1-GFP, and ARRDC1-GFP-VSV-G (as shown at the top of the plate) and incubated for 24 hours (24h) (Figure 4). The experiment was performed with two replicas; replica 1 did not undergo washout, while replica 2 underwent washout at 3 hours. Both plates were imaged after incubation. As shown, the use of the VSV-G protein increased GFP delivery and expression across both replicas and concentrations. Specifically, when looking at GFP expression in iPSC-derived neural progenitor cells in the case of the ARRDC1-GFP-VSV-G delivery system washed out at 3 hours, a strong, nearly uniform punctate signal was observed throughout the cells, and this system did not exhibit visible toxicity (Figure 5A). Furthermore, imaging demonstrated the intracellular localization of GFP delivered by ARMM utilizing the VSV-G protein in human neurons isolated from iPSC-derived brain organoids (Figure 5B).

[0143] Example 4: ARMM-mediated delivery of mRNA payload in human neurons The success of payload delivery and protein translation was further evaluated (Figure 6). ARRDC1-Tat (control) and ARRDC1-Tat-V (1 × 10⁶) with TAR-GFP mRNA as the payload RNA payload cargo. 10 Particles / milliliter (particles / mL) were differentiated for 5 weeks and then introduced into neurons. Cells were fixed 24 hours after ARMM exposure and imaged using immunocytochemistry techniques. As shown, ARMM successfully delivered its payload to the cells and was subsequently successfully translated into protein (Figures 6-7).

[0144] Example 5: Targeting multiple neurogenetic diseases using ARMM ARMM-mediated delivery technology can be adapted to different targets for both gain-of-function disorders (e.g., resulting from mutations or dysfunction of MAPT, SNCA, HTT, ATXN2, etc.) and loss-of-function disorders (e.g., resulting from mutations or dysfunction of GRN, GBA1, FMR1, MECP2, TCF4, etc.), as well as repeat elongation (e.g., resulting from mutations in C9orf72, etc.) (Figure 8, Source: van der Zee & Van Broeckhoven, Nat. Rev. Neurol. 2014). Molecules for use as possible payloads include, but are not limited to, shRNA, miRNA, ribozymes, scFv PROTACs, editing factors (e.g., nucleic acid editing factors (e.g., CRISPR / Cas9, its variants)), and mRNA. Experiments can be conducted on patient-derived iPSC cells to optimize shRNA / antisense RNA delivery to overcome gain-of-function accumulation of defective tau and dipeptide repeats by ARMM-mediated molecular delivery, such as C9orf72 or dCas9 modified with transcription activators (CRISPRa) or repressors (CRISPRi).

[0145] Figure 9 shows an example of using a CRISPR editor compatible with ARMM delivery technology. This schematic diagram illustrates the use of dCas9 modified with a transcription activator (CRISPRa) that has a guide RNA targeting the GRN locus to enhance GRN gene transcription and overcome the loss of function of progranulin (PGRN) production caused by GRN mutations. This graph shows the amount of PGRN per 110 μg of total protein (ng / ml) when using a dummy sample, a dCas9-VPR sample, and dCas9-VPR+2 GRN guide RNA, as determined by PGRN ELISA (R & D Quantikine).

[0146] Based on our development of a patient-derived iPSC model, we can also deliver FMRP to rescue neurons in patients with fragile X syndrome using a CNS-optimized ARMM (Figures 10-11). For example (Source: Sheridan SD, Theriault KM, Reis SA, Zhou F, Madison JM, Daheron L, Loring JF, Haggarty SJ. Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome. PLoS One. 2011;6(10):e26203), this graph shows the percentage of CpG methylation for each FMR1 CpG site with either complete mutations (848-iPS1-NP, 848-iPS3-NP, 131-iPS1-NP), pre-mutation (131-iPS3-NP), or healthy control (8330-iPS8-NP), along with the provided FMR1 promoter map. Increased CpG methylation results in epigenetic silencing of FMR1. This image shows NESTIN and SOX1 staining for each sample, indicating that the cells are neural progenitor cells. Western blot shows the amount of FMRP produced for each sample, using β-actin as a control. Figure 11 shows the differentiation of cells over 18 days, resulting in postmittal neurons and glial cells. These cells were fixed and immunostained as shown in the image. Observable phenotypic differences between the control and the fragile X patient lineage with reduced FMRR expression allow for a screening assay using high-content imaging to optimize ARMM-based therapies.Similar screening methods for optimizing ARMM-based treatment are expected to be effective for Rett syndrome, caused by mutations in MECP2 and similar iPSC models developed by the inventors, in conjunction with other rare neurodevelopmental disorders (e.g., Mellios N, Feldman DA, Sheridan SD, Ip JPK, Kwok S, Amoah SK, Rosen B, Rodriguez BA, Crawford B, Swaminathan R, Chou S, Li Y, Ziats M, Ernst C, Jaenisch R, Haggarty SJ, Sur M. MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling. Mol Psychiatry. 2018 Apr;23(4):1051-1065).

[0147] Example 6: Insertion of RVG into ARMM Using proteomics studies of ARMMs derived from multiple human cells, we identified several proteins enriched in ARMM vesicles. Among these proteins were tetraspanins such as TSPAN14 and TSPAN6. These proteins possess multiple extracellular loop regions that allow for the insertion of "homing peptides" or other targeting moieties, thereby enabling the potential targeting of ARMMs to specific cells / tissues. We developed a fusion construct in which a rabies virus glycoprotein (RVG) peptide, along with an HA tag, was inserted into the second extracellular loop of TSPAN6 (Figure 12A). Western blotting was performed on whole cell lysates and ARMMs using antibodies against the following targets: ARRDC1 / GFP, TSPAN6 / RVG / HA, CD9, and vinculin. Following transfection into HEK293 cells, Western blot analysis showed that TSPAN6-RVG-HA was reliably detected in ARMMs secreted from HEK293 T cells (Figure 12B).

[0148] Example 7: Insertion of VSV-G into ARMM According to Table 1, HEK293T cells (2 × 10⁶) in the presence or absence of VSV-G. 6 Cells / plates were transfected with ARRDC1(A1) or ARRDC1 together with TAR-GFP. Table 1. Transfection Information [Table 1] Extracellular vesicles were isolated using ultracentrifugation (Figure 13A). Culture medium was collected 48 and 72 hours after transfection. The medium was centrifuged at 3,000 g for 10 minutes and passed through a 0.22 μm filter. The supernatant was collected and centrifuged at 10,000 g for 10 minutes. The obtained supernatant was ultracentrifuged at 320,000 rpm for 2 hours. ARMM was then resuspended in phosphate-buffered saline (PBS). Western blotting was performed on the total cell lysates and ARMM using antibodies against the following targets: unpurified ARRDC1 serum, VSV-G, CD9, and vinculin (Figure 13B). The results indicate that VSV-G was reliably detected in ARMM. Furthermore, VSV-G appeared to increase ARMM production, as indicated by the increased amount of ARRDC1 in the extracellular vesicle preparation.

[0149] Example 8: ARRDC1-mediated delivery of payload to cultured human iPSC-derived 3D brain organoids Starting from human induced pluripotent stem cells (iPSCs), brain tissue-like neurons obtained from both the deep and superficial cortical layers in 3D culture were differentiated together with astrocytes according to a publicly available method (Pasca AM, Sloan SA, Clarke LE, Tian Y, Makinson CD, Huber N, Kim CH, Park JY, O'Rourke NA, Nguyen KD, Smith SJ, Huguenard JR, Geschwind DH, Barres BA, Pasca SP. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nature Methods. 2015 Jul;12(7):671-8.). Brain organoids were simultaneously matured for 9 months in a maturation medium (basal nerve A, GlutaMax, vitamin A-free B-27 supplement, penicillin / streptomycin), after which single organoids were isolated and subsequently exposed to ARRDC1-GFP-VSVG extracellular vesicles (14.3 mL) for 24 or 48 hours in a 96-well plate. The single organoids were then dissociated with Accutase, the resulting cells were collected, and attached to a 96-well plate coated with laminin / poly-L-ornithine for 24 hours. The cells were then imaged under transmitted light or GFP fluorescence was detected using an automated confocal microscope (IN Cell Analyzer 6000).

[0150] As shown in Figure 14, delivery of the target protein (GFP) was detectable by fluorescence imaging in most cells in both 24-hour and 48-hour incubations with ARRDC1-GFP-VSVG extracellular vesicles. Longer exposures up to 48 hours, as tested, did not cause apparent toxicity, as measured by the ability to retrieve viable neurons attached to laminin / poly-L-ornithine-coated 96-well plates. Based on the number of GFP-positive cells, these data demonstrate the ability of ARMM particles to deliver payloads not only to the outer cell layer but beyond. Overall, these results further demonstrate the effectiveness of using ARRDC1-mediated microvesicles for intracellular delivery of therapeutic macromolecules to human nervous system cells.

[0151] reference All publications, patents, and sequence database entries referred to in this application that encompass the items listed above are incorporated hereby by reference as a whole, as is indicated specifically and individually that each individual publication or patent is incorporated by reference. In case of conflict, the application that encompasses either definition of this application shall prevail.

[0152] Equivalents and range Those skilled in the art will be able to understand, or verify without using anything more than routine experimentation, many equivalents of the particular aspects of the invention described herein. The scope of the invention is not intended to be limited to the foregoing, but rather as defined by the appended claims.

[0153] Unless otherwise indicated or otherwise obvious from the context, articles such as “a,” “an,” and “the” in a claim may mean one or more than one. Unless otherwise indicated or otherwise obvious from the context, a claim or statement containing “or” among one or more members of a group is considered satisfied if one member, more than one member, or all members of that group are present in, used in, or otherwise related to a given product or process. The present invention encompasses embodiments in which exactly one member of a group is present in, used in, or otherwise related to a given product or process. The present invention also encompasses embodiments in which more than one or all members of a group are present in, used in, or otherwise related to a given product or process.

[0154] Furthermore, it should be understood that the present invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims or relevant parts of the specification that are introduced into other claims. For example, any claim dependent on another claim may be modified to encompass one or more limitations found in any other claim dependent on the same basic claim. Furthermore, where a claim describes a composition, it should be understood that, unless otherwise indicated or it is not obvious to a person skilled in the art that such a description would result in a contradiction or inconsistency, methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods disclosed herein or other methods known in the art are included.

[0155] Where elements are presented as a list, for example in Markush group format, each subgroup of the elements is also disclosed, and it should be understood that any element(s) may be removed from the group. It should also be noted that "includes" is intended to be open, allowing for the inclusion of additional elements or steps. In general, where the present invention or an aspect of the present invention is referred to in terms of including certain elements, features, steps, etc., it should be understood that a particular aspect of the present invention or an aspect of the present invention consists of, or is essentially composed of, such elements, features, steps, etc. For the sake of brevity, these aspects are not specifically described in this application. Therefore, for each aspect of the present invention that includes one or more elements, features, steps, etc., the present invention also provides aspects consisting of, or being essentially composed of, those elements, features, steps, etc.

[0156] Where a range is given, the endpoints are encompassed. Furthermore, unless otherwise indicated or otherwise obvious from the context and / or the understanding of those skilled in the art, unless the context explicitly states otherwise, it should be understood that, in different embodiments of the invention, any particular value within the claimed range can take up to one-tenth of the lower limit unit of the range. Unless otherwise indicated or otherwise obvious from the context and / or the understanding of those skilled in the art, it should also be understood that any value expressed as a range can take up to one-tenth of the subrange of a given range, and the endpoints of a subrange are expressed with the same precision as one-tenth of the lower limit unit of the range.

[0157] In addition, it should be understood that any particular aspect of the present invention may be expressly excluded from any one or more claims. Where a range is given, any value within that range may be expressly excluded from any one or more claims. Any aspect, element, feature, application, or aspect of any composition and / or method of the present invention may be excluded from any one or more claims. For the sake of brevity, not all aspects in which one or more elements, features, applications, or aspects are excluded are expressly defined in this application.

Claims

1. 1. An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), comprising: (i) a lipid bilayer and an ARRDC1 protein, or a variant thereof; (ii) a molecule, and (iii) Viral envelope proteins The microvesicle comprising:

2. The microvesicle of claim 1, further comprising a TSG101 protein.

3. 3. The microvesicle according to claim 1 or 2, wherein the viral envelope protein is vesicular stomatitis virus G (VSV-G) or rabies virus glycoprotein (RVG).

4. The microvesicle according to any one of claims 1 to 3, further comprising a payload.

5. The microvesicle of claim 4, wherein the ARRDC1 protein is associated with a payload.

6. The microvesicle of claim 4 or 5, wherein the ARRDC1 protein is associated with the payload via a cleavable linker.

7. The microvesicle according to any one of claims 4 to 6, wherein the payload further comprises a detectable label.

8. The microvesicle according to any one of claims 4 to 7, wherein the payload is a therapeutic drug.

9. The microvesicle according to any one of claims 4 to 8, wherein the payload is a protein.

10. The microvesicle according to any one of claims 4 to 8, wherein the payload is a nucleic acid.

11. The microvesicle according to any one of claims 4 to 8, wherein the payload is a small molecule.

12. 1. A microvesicle-producing cell, comprising: A recombinant expression construct encoding the ARRDC1 protein or a variant thereof under the control of a heterologous promoter, and Viral Envelope Proteins The microvesicle-producing cell comprising:

13. The microvesicle-producing cell according to claim 12, wherein the viral envelope protein is VSV-G or RVG.

14. The microvesicle-producing cell of claim 12 or 13, wherein the recombinant expression construct further encodes a protein payload covalently linked to the ARRDC1 protein via a cleavable linker.

15. The microvesicle-producing cell according to any one of claims 12 to 14, further comprising a therapeutic agent.

16. A method for delivering a molecule to a target cell in vitro or ex vivo, the method comprising contacting the target cell with a microvesicle according to any one of claims 1 to 11.

17. The method of claim 16, wherein the target cell is a cell of the nervous system (NS).

18. A pharmaceutical composition for treating a disorder in a subject, comprising a microvesicle according to any one of claims 1 to 11 or a microvesicle-producing cell according to any one of claims 12 to 15.

19. 19. The pharmaceutical composition of claim 18, wherein the disorder is a disorder of the central nervous system (CNS).

20. A kit comprising a microvesicle according to any one of claims 1 to 11 or a microvesicle-producing cell according to any one of claims 12 to 15.

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