Enhancement of the delivery of biopharmaceuticals via receptor binding

Abstract

The present disclosure provides methods and compositions for targeting lipid bilayer particles, such as secreted extracellular vesicles, and cargo entities contained therein, to recipient cells.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 341,914, filed May 13, 2022, which is hereby incorporated by reference in its entirety.

[0002] Government Support Statement This invention was made with government support under grant number P30 AI117943 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0003] Technical Field The present disclosure generally relates to methods and compositions for loading cargo entities into secreted lipid bilayer particles (e.g., cell - derived membrane particles such as extracellular vesicles).

Background Art

[0004] The rapidly growing field of nucleic acid therapy has attracted much attention because it has the potential to treat various diseases, disorders, and conditions that are difficult to address with other therapies. However, as pointed out in a recent review, "while nucleic acid therapeutics have the potential to expand the range of treatable diseases, their widespread use is limited by multiple delivery challenges" (Gupta et al., “Nucleic acid delivery for therapeutic applications” in Adv. Drug Delivery Reviews 178:113 - 834, 2021).

[0005] The following discussion is provided only to assist the reader in understanding the present disclosure and does not admit to describing or constituting prior art.

[0006] Secreted extracellular vesicles (EVs), such as exosomes and microvesicles, are nanometer-sized lipid vesicles produced by many types of cells and transmit proteins, nucleic acids, and other molecules between cells in the human body and other animals. There are also many known viruses that use EVs to deliver genomes to other cells (such as enveloped viruses). EVs hold the potential for a wide variety of therapeutic applications and are an attractive platform for delivering a wide variety of therapeutic agents. For example, it has already been shown that targeted exosomes are effective in delivering RNA to mouse neurons and tumor cells. Membrane particles derived from other cells can also be used for the same purpose.

[0007] It is difficult to control the composition of EVs both in the lumen and in the membrane. There are various passive approaches for both, but an excellent manipulation protocol of EV composition is required to achieve the therapeutic potential of EVs. The disclosed technology aims to address these limitations of the current technology.

Summary of the Invention

[0008] The present disclosure provides a surprising new technique for achieving the delivery of cargo (e.g., nucleic acid cargo, polypeptide cargo, nucleocapsid cargo, and combinations thereof) to recipient cells or cell populations using engineered lipid bilayer particles.

[0009] In particular, the present disclosure provides the insight that a combination of a specific affinity agent polypeptide and a fusion promoter polypeptide on the surface of lipid bilayer particles can confer surprisingly improved delivery properties to such particles, such as achieving more efficient delivery and / or delivery to specific cell types.

[0010] The present disclosure identifies the cause of one or more problems associated with many conventional strategies for payload delivery (e.g., nucleic acid payload delivery), particularly those technologies intended to achieve in vivo delivery. In particular, the present disclosure identifies the cause of specific problems associated with conventional strategies that utilize a viral fusogen (e.g., VSV-G) or a variant thereof to achieve payload delivery; the present disclosure demonstrates that combining a fusogen entity polypeptide (e.g., as described herein, a fusogen entity polypeptide (e.g., a viral fusogen entity polypeptide) with a targeting chimeric polypeptide) solves such identified problems and / or achieves otherwise particularly beneficial results (e.g., particularly accurate and / or efficient payload delivery).

[0011] The present disclosure particularly highly evaluates the challenges associated with the delivery of an effective payload (e.g., a nucleic acid payload) to specific immune cells, such as T cells. The present disclosure documents the particular effectiveness of the provided technology in the delivery of a payload (e.g., a nucleic acid payload) to T cells, such as activated T cells.

[0012] In particular, the present disclosure provides engineered lipid bilayer particles and preparations thereof that include on their surface both a fusogen entity polypeptide and a targeting chimeric polypeptide as described herein. In some embodiments, such provided particles and / or preparations are characterized by specific payload delivery attributes. In some embodiments, such provided particles and / or preparations achieve payload delivery (e.g., specific payload delivery and / or enhanced payload delivery) to a particular cell(s) or cell population of interest; in some embodiments, such delivery is in vivo. The present disclosure provides, in particular, that a specific combination of a fusogen entity polypeptide and a targeting chimeric polypeptide as described herein can drive a specific function.

[0013] In some embodiments, the technology provided is useful for the delivery of viral vectors (e.g., lentiviral cores, adeno-associated virus particles) within lipid bilayer particles and / or virus-like particles. Alternatively or additionally, in some embodiments, the technology provided is useful for the delivery of non-viral vectors (e.g., nucleic acid payloads not packaged within a protein core or capsid structure).

[0014] In particular, the present disclosure relates to the problems associated with in vivo gene transfer into cells, including specific gene transfer into certain immune system cells (e.g., T cells), and more particularly, the problem of in vivo delivery of cargo (e.g., payload) to specifically and efficiently target certain recipient cells of interest (e.g., T cells). In vitro delivery of cargo (e.g., payload) to target cells, particularly T cells, is partially satisfied by several methods, but there remain unmet needs for specific in vivo delivery, non-toxic in vivo and in vitro delivery, and more efficient in vitro delivery to cells.

[0015] In particular, in some embodiments, the present disclosure provides technologies (e.g., systems, engineered lipid bilayer parties, production cells, manufacturing and delivery methods) that mediate the fusion of engineered lipid bilayer particles with recipient cells (e.g., to deliver cargo).

[0016] Particularly useful applications of the technology provided include, for example, CAR T cell therapy, such as the production of CAR T cells for cancer treatment, immune system disorders, and other applications.

[0017] In particular, in some embodiments, the present disclosure provides technologies that enhance the delivery of a specific cargo (e.g., payload) and / or delivery to a specific recipient cell or cell population, including delivery to specific immune cells or cell populations, particularly delivery to T cells or T cell populations.

[0018] In some embodiments, the present disclosure achieves specificity and / or efficiency of payload delivery through the combined activity of a fusogenic entity polypeptide and a targeting chimeric polypeptide. In some embodiments, the techniques provided achieve delivery that exhibits higher specificity and / or efficiency when compared to a particular reference; in some embodiments, such a reference can be a sufficiently comparable system that includes one or the other of the fusogenic entity polypeptide and the targeting chimeric polypeptide, but not both. In many embodiments, a suitable reference can be a sufficiently comparable system that includes the fusogenic entity polypeptide but not the targeting chimeric polypeptide. Alternatively or additionally, in some embodiments, a suitable reference can be a sufficiently comparable system that includes a particular viral fusogenic entity polypeptide (e.g., VSV-G or a variant thereof) and lacks, for example, the targeting chimeric polypeptides described herein. In some embodiments, a suitable reference does not utilize the same affinity agent polypeptide, even if it includes at least one surface agent having some affinity for the surface of the recipient cell or population thereof.

[0019] The present disclosure provides targeting chimeric polypeptides, fusogenic polypeptides, and systems and methods for using them to target cargo entities to lipid bilayer particles such as cell-derived membrane particles including, but not limited to, extracellular vesicles. The present disclosure also provides methods for manufacturing engineered producer cells, methods for manufacturing preparations of lipid bilayer particles, methods for delivering cargo entities to recipient cells, and cargo entities or recipient cells comprising the cargo entities, wherein these recipient cells can further comprise a targeting chimeric polypeptide and a fusogenic entity polypeptide (e.g., received by fusion of the recipient cell membrane and the lipid bilayer particle as described herein), and these recipient cells further have a nucleus.

[0020] In one aspect, the present disclosure provides a targeting chimeric polypeptide comprising (a) a targeting ligand (e.g., a targeting ligand present on the surface of a recipient cell of interest; specifically, including human cells and / or immune cells such as T cells, and more particularly including CD2 and / or CD5, e.g., human CD2 and / or human CD5), where the targeting domain comprises an antibody agent such as Fab, Fab’, F(ab’) 2 , Fd, scFv, disulfide-bonded Fv (sdFv), de novo designed binding molecule, affibody, DARPIN, nanobody, variable lymphocyte receptor (VLR), camelid antibody, etc.; and optionally (b) a transmembrane domain. In some embodiments, the targeting domain is scFv.

[0021] In some embodiments, the transmembrane domain comprises AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKP (SEQ ID NO: 18), a variant amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 18, or a functional fragment thereof.

[0022] In some embodiments, the targeting domain comprises the amino acid sequence of NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSGGGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSS (SEQ ID NO: 20).

[0023] In some embodiments, the targeting domain comprises the amino acid sequence of CPSQCSCSGTEVHCQRKSLASVPAGIPTTTRVLYLHVNEITKFEPGVFDRLVNLQQLYLGGNQLSALPDGVFDRLTQLTRLDLYNNQLTVLPAGVFDRLVNLQTLDLHNNQLKSIPRGAFDNLKSLTHIWLFGNPWDCACSDILYLSGWLGQHAGKEQGQAVCSGTNTPVRAVTEASTSPSKCP (SEQ ID NO: 24).

[0024] In some embodiments, the targeting chimeric polypeptide may further comprise a first cargo entity linked to the transmembrane domain via a linker. In some embodiments, the linker is SEQ ID NO: 10 (TSGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG), and SEQ ID NO: 17 (ESKYGPPAPPAP); or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 14, 15, 16, or 17.

[0025] In another aspect, the disclosure provides a lipid bilayer particle (e.g., a cell-derived membrane particle (CDMP)) comprising a targeting chimeric polypeptide and / or a fusion entity polypeptide disclosed herein.

[0026] In some embodiments, the lipid bilayer particle is a CDMP. In some embodiments, the CDMP is selected from extracellular vesicles, virus particles, virus-like particles (VLPs), apoptotic bodies, platelet-like particles, and combinations thereof. In some embodiments, the extracellular vesicle is an exosome, a microvesicle, and / or a combination thereof.

[0027] In some embodiments, the lipid bilayer particle (e.g., CDMP) comprises a fusogenic entity polypeptide. In some embodiments, the fusogenic entity polypeptide is a viral polypeptide (e.g., a glycoprotein). In some embodiments, the viral glycoprotein is selected from the glycoproteins selected from the group consisting of lentiviral glycoprotein, or vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowlpox virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein. In another aspect, the present disclosure provides a lipid bilayer particle comprising a glycoprotein selected from the group consisting of vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowlpox virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, lymphocytic choriomeningitis virus (LCMV) glycoprotein, and any combination thereof. The expression of such a glycoprotein or combination of glycoproteins (e.g., measles virus glycoprotein H and measles virus glycoprotein F) can be an embodiment independent of (i.e., not including) the targeting chimeric polypeptides disclosed herein, as these glycoproteins provide novel utility with respect to the binding and fusion of lipid bilayer particles to recipient cells independently.

[0028] In some embodiments, the fusogenic entity polypeptide is a non-viral polypeptide as described herein.

[0029] In some embodiments, the lipid bilayer particles contain cargo entities as described herein.

[0030] In some embodiments, the disclosed lipid bilayer particles (e.g., CDMP) can include a chimeric loading polypeptide that includes a cargo loading domain that includes an abscisic acid-insensitive 1 (ABI1) sequence, and optionally can further include a cargo entity. In some embodiments, the chimeric loading polypeptide includes a cargo loading domain that includes an abscisic acid-sensitive 1 (ABI1) sequence and a cargo entity. In some embodiments, the chimeric loading polypeptide further includes a linker that links the cargo entity and the cargo loading domain. In some embodiments, the linker of the chimeric loading polypeptide includes SEQ ID NO: 10 (TSGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG), and SEQ ID NO: 17 (amino acid sequence from (ESKYGPPAPPAP); or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 14, 15, 16, or 17).

[0031] In some embodiments, the cargo loading domain of the chimeric loading polypeptide is a truncated variant of a wild-type protein that includes an extracellular vesicle targeting domain. In some embodiments, the cargo loading domain of the chimeric loading polypeptide includes residues 126-423 of wild-type ABI1.In some embodiments, the cargo loading domain of the chimeric loading polypeptide comprises MTRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO: 6), VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO: 7), a variant amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 7, or a functional fragment of SEQ ID NO: 6, SEQ ID NO: 7, or a variant amino acid sequence thereof.

[0032] In some embodiments, the cargo entity of the chimeric loading polypeptide is a cytoplasmic cargo molecule. In some embodiments, the cargo entity of the chimeric loading polypeptide is a membrane-bound cargo entity.

[0033] In some embodiments, the first cargo entity is an ABA-binding sequence or comprises an ABA-binding sequence. In some embodiments, the first cargo entity is or comprises an ABA-binding sequence comprising a pyrabactin resistance 1-like (PYL1) sequence. In some embodiments, the PYL1 sequence comprises residues 33-209 of wild-type PYL1.

[0034] In some embodiments, the PYL1 sequence is MGGGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 2), TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 3), or a variant amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3, or a functional fragment of SEQ ID NO: 2, SEQ ID NO: 3, or a variant amino acid sequence thereof.

[0035] In some embodiments, the lipid bilayer particles disclosed herein may further comprise abscisic acid (ABA).

[0036] In some embodiments, the lipid bilayer particle contains or includes therein a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, a prime editor, a nuclease (e.g., TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex. In some embodiments, the cargo entity is selected from the group consisting of a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinase, a base editor, a prime editor, a nuclease (e.g., TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, and a ribonucleoprotein complex.

[0037] In another aspect, the disclosure provides a nucleic acid encoding a chimeric targeting polypeptide and / or a fusion factor entity polypeptide disclosed herein.

[0038] In another aspect, the present disclosure provides a production cell comprising a targeting chimeric polypeptide and / or a fusion factor entity polypeptide disclosed herein, a lipid bilayer particle disclosed herein, or a nucleic acid disclosed herein. In some embodiments, the production cell is a mammalian cell. In some embodiments, the mammalian cell is optionally selected from HEK293, HEK293FT, mesenchymal stem cells, megakaryocytes, induced pluripotent stem cells (iPSCs), T cells, red blood cells, erythroid precursors, and iPSC-derived versions of any of the foregoing cells. In another aspect, the present disclosure provides a method for producing lipid bilayer particles, the method comprising culturing a production cell comprising a targeting chimeric polypeptide and / or a fusion factor entity polypeptide disclosed herein, a lipid bilayer particle (e.g., CDMP) disclosed herein, or a nucleic acid disclosed herein, and harvesting the lipid bilayer particles (e.g., CDMP) produced by the cells.

[0039] In another aspect, the present disclosure provides a method for targeted delivery of a cargo entity to a recipient cell (e.g., an immune cell such as a lymphocyte), the method comprising administering to an individual a lipid bilayer particle disclosed herein, wherein the lipid bilayer particle comprises the cargo entity.

[0040] In some embodiments, the cargo entity comprises a viral nucleocapsid, a synthetic nucleic acid, a transcription factor, a recombinant enzyme, a base editor, a prime editor, a nuclease (e.g., TALEN, ZFN, etc.), a kinase, a kinase inhibitor, an activator or inhibitor of receptor signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.

[0041] In some embodiments, the cargo entity comprises a nucleic acid sequence encoding a chimeric antigen receptor.

[0042] In another aspect, the present disclosure provides a method for targeted delivery of cargo entities to recipient cells (e.g., immune cells such as lymphocytes), comprising obtaining a population of recipient cells (e.g., lymphocytes) from an individual and contacting the population of recipient cells (e.g., lymphocytes) with lipid bilayer particles disclosed herein ex vivo, wherein the lipid bilayer particles contain the cargo entities.

[0043] In some embodiments, the population of lymphocytes is obtained via apheresis.

[0044] In some embodiments, the ex vivo method may further comprise administering the population of recipient cells (e.g., lymphocytes) to the individual after the recipient cells (e.g., lymphocytes) have contacted the lipid bilayer particles (e.g., as if the lipid bilayer particles have fused with the recipient cells).

[0045] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objectives, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and the detailed description of the disclosure.

[0046] The drawings include the following figures.

Brief Description of the Drawings

[0047]

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DETAILED DESCRIPTION OF THE INVENTION

[0048] The present disclosure provides surprising insights and useful techniques for delivering cargo entities to recipient cells of interest. In particular, the present disclosure evaluates that extracellular vesicles displaying a fusion protein comprising a targeting domain and a transmembrane domain (e.g., the PDGFR transmembrane domain) exhibit particularly efficient and / or effective vesicle targeting to specific recipient cells. Exemplary useful such fusion proteins are described in PCT Publication WO2019 / 199941 (entitled "Extracellular Vesicles Comprising Membrane Proteins Based on Targeting Affinity Domains", published on October 17, 2019, the content of which is incorporated herein by reference in its entirety), and such fusion proteins have been demonstrated to be able to mediate vesicle uptake by recipient cells when displayed on various vesicle types (e.g., exosomes, microvesicles). The present disclosure provides surprising further developments regarding such fusion proteins, for example, on various lipid bilayer particles, their combinations with fusion factor entity polypeptides as described herein achieve significantly efficient and / or effective target delivery of cargo to specific recipient cells of interest, including certain human cells and / or immune cells, particularly T cells (e.g., human T cells), thereby providing unique and important value in the art.

[0049] One of ordinary skill in the art, upon reading this specification, would understand that when a cargo entity is delivered to recipient cells in accordance with the present teachings, such cargo entities in some embodiments can modify (e.g., genetically modify) recipient cells such that they are useful in many applications, including various therapeutic applications. The cargo entity can be delivered to specific recipient cells by lipid bilayer particles that include cell-derived membrane particles (CDMPs) (e.g., extracellular vesicles (EVs)).

[0050] As described herein, particularly useful embodiments of the provided technology are those that effect genetic modification of T cells. Genetic modification of T cells enables applications ranging from cancer immunotherapy to HIV treatment, yet delivery of therapeutics targeted to T cells remains challenging. Extracellular vesicles (EVs) and other cell-derived membrane particles (CDMPs) are nanoscale particles secreted by all cells that naturally encapsulate and traffic nucleic acid–protein complexes such as proteins, nucleic acids, and viral nucleocapsids (e.g., enveloped viruses), and are an attractive and clinically relevant platform for engineering biocompatible delivery vehicles. The present disclosure provides a series of techniques for genetically engineering producer cells to produce multifunctional lipid bilayer particles (e.g., CDMPs such as EVs). The present disclosure further provides high-affinity targeting domains (e.g., targeting chimeric polypeptides) and / or fusogenic entity polypeptides (e.g., glycoproteins) displayed on the surface of lipid bilayer particles to achieve specific and efficient binding to recipient cells (e.g., immune cells such as T cells). The fusogenic entity polypeptide (e.g., glycoprotein) can increase the uptake and fusion of lipid bilayer particles with recipient cells. The present disclosure also identifies protein tags for conferring active cargo loading into lipid bilayer particles. The examples herein demonstrate the integration of these techniques by delivering a Cas9-sgRNA complex to edit primary human T cells, viral nucleocapsid derivatives (e.g., lentiviral nucleocapsids), or fluorescent proteins, and verifying the fusion of lipid vesicles with recipient cells. These approaches enable targeting of particles to various cells and efficient delivery of cargo.

[0051] To provide a substantial understanding of the technology, it should be appreciated that specific aspects, embodiments, implementations, variations, and features of the method are described below at various levels of detail.

[0052] In carrying out the method of the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al., (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al., (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.See (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al., eds (1996) Weir’s Handbook of Experimental Immunology.

[0053] Definitions Unless otherwise specified or indicated by context, the terms “a,” “an,” and “the” mean “one or more.” For example, “fusion protein,” “extracellular vesicle,” and “cell” should be interpreted to mean “one or more fusion proteins,” “one or more extracellular vesicles,” and “one or more cells,” respectively.

[0054] As used herein, the terms “about,” “approximately,” “substantially,” and “significantly” are understood by those skilled in the art and vary somewhat depending on the context in which they are used. When the use is not clear to those skilled in the art from the context in which these terms are used, “about” and “approximately” mean plus or minus no more than 10% of the particular term, and “substantially” and “significantly” mean plus or minus more than 10% of the particular term.

[0055] As used herein, "affinity" is a measure of the propensity of two or more binding partners to associate with each other. Those of skill in the art will recognize the various assays that can be used to assess affinity, and will further recognize appropriate controls for such assays. In some embodiments, affinity is evaluated in a quantitative assay. In some embodiments, affinity is evaluated over a plurality of concentrations (e.g., of one binding partner at a time). In some embodiments, affinity is evaluated in the presence of one or more potential competitors (e.g., that may be present in a relevant - e.g., physiological - setting). In some embodiments, affinity is referenced (e.g., has a known affinity above a certain threshold or has a known affinity below a certain threshold). In some embodiments, affinity can be evaluated relative to a contemporaneous reference; in some embodiments, affinity can be evaluated relative to a past reference. Typically, when affinity is evaluated relative to a reference, it is evaluated under comparable conditions.

[0056] As used herein, "binding moiety" refers to a moiety that binds to a target ligand described herein (e.g., a target ligand on the surface of a recipient cell or a population thereof). In many embodiments, the binding moiety of interest specifically binds to its target ligand in that it discriminates the target ligand from other potential binding partners in a particular interaction context. In some embodiments, the binding moiety exhibits specific binding to its target ligand relative to one or more other entities on the surface of the recipient cell(s). Alternatively or additionally, in some embodiments, the binding moiety exhibits preferential binding to its target ligand relative to one or more (or all) entities present on the surface of non-recipient cell(s) (e.g., non-recipient cell(s) that may be present in a system containing recipient cells). In some embodiments, the binding moiety binds to one or more target ligands and drives a specific biological activity that is linked only to a particular target ligand. In some embodiments, the binding moiety is a peptide binding moiety. In some embodiments, the binding moiety is a non-peptide binder. In some such embodiments, the production cell may be engineered to express a targeting chimeric polypeptide comprising a binding moiety that is subsequently modified (e.g., chemically modified) by attaching a non-polypeptide binding moiety such that the non-polypeptide binding moiety provides specific affinity for the target ligand. In some embodiments, the binder comprises (i) a targeting chimeric polypeptide and optionally a non-polypeptide moiety, and (ii) binds to a target cell when present on the surface of a lipid bilayer particle. Generally, the binding moiety can be or consist of a moiety of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.). In some embodiments, the binding moiety is a single chemical entity. In some embodiments, the binding moiety is a complex of two or more discrete chemical entities that associate with each other under relevant conditions by non-covalent interactions.For example, one of ordinary skill in the art will understand that in some embodiments, the binding moiety can include a "general" binding moiety (e.g., one of biotin / avidin / streptavidin and / or class - specific antibodies) and a "specific" binding moiety (e.g., an antibody or aptamer having a specific molecular target) that is linked to a partner of the general binding moiety. In some embodiments, such an approach can enable modular assembly of multiple affinity sites by linking different specific binding moieties to the same partner of the general binding moiety. In some embodiments, the binding moiety is a polypeptide (e.g., including an antibody or antibody fragment), or includes a polypeptide. In some embodiments, the binding moiety is a small molecule, or includes a small molecule. In some embodiments, the binding moiety is a nucleic acid, or includes a nucleic acid. In some embodiments, the binding moiety is an aptamer. In some embodiments, the binding moiety is a polymer; in some embodiments, the affinity moiety is not a polymer. In some embodiments, the binding moiety is non - polymeric in that it lacks a polymeric moiety. In some embodiments, the binding moiety is a carbohydrate, or includes a carbohydrate. In some embodiments, the binding moiety is a peptidomimetic, or includes a peptidomimetic. In some embodiments, the binding moiety is a scaffold protein, or includes a scaffold protein. In some embodiments, the binding moiety is a mimotope, or includes a mimotope. In some embodiments, the binding moiety is a stapled peptide, or includes a stapled peptide. In certain embodiments, the binding moiety is a nucleic acid such as DNA or RNA, or includes a nucleic acid.

[0057] As used herein, the phrase "characteristic array element" refers to an array element that represents a characteristic portion of a polymer (e.g., a polypeptide or nucleic acid) found within the polymer. One of ordinary skill in the art understands that the presence of a characteristic array element typically correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic array element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers. Characteristic array elements typically comprise at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic array element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., continuously linked monomers). In some embodiments, a characteristic array element comprises at least first and second stretches of contiguous monomers that are spaced apart by one or more spacer regions that may or may not differ in length between polymers that share the array element.

[0058] As used herein, "comparable" refers to two or more agents, entities, situations, sets of conditions, etc. that, while not necessarily identical to each other, are similar enough to allow a comparison between them such that a person of ordinary skill in the art can reasonably draw conclusions based on the observed differences or similarities. In some embodiments, comparable sets of conditions, situations, individuals, or groups are characterized by a plurality of substantially identical features and one or a few varying features. A person of ordinary skill in the art will understand, in context, how much identity is required in any given situation for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, a person of ordinary skill in the art will understand that sets of conditions, individuals, or groups are comparable to each other if the differences in the results or observed phenomena under different conditions, individuals, or groups, or observed under different conditions, individuals, or groups, are due to or indicative of variation in those varying features, and are characterized by a sufficient number and variety of substantially identical features to warrant a reasonable conclusion to that effect.

[0059] As used herein, "control" refers to an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation between the efficacy of cargo protein loading into EVs and the structure of the cargo protein, a positive control (a cargo protein known to exhibit the desired loading efficacy) and a negative control (a cargo protein that does not load into EVs) are typically employed.

[0060] As used herein, "corresponding to" refers to the relationship between two or more entities. For example, the term "corresponding to" can be used to specify the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., a suitable reference compound or composition). For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) can be identified as "corresponding to" a residue in a suitable reference polymer. For example, one of ordinary skill in the art, for purposes of simplification, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, and thus, for example, the amino acid "corresponding to" the residue at position 190 does not necessarily have to be the 190th amino acid in a particular amino acid chain, but rather corresponds to the residue found at position 190 in the reference polypeptide; one of ordinary skill in the art readily understands how to identify the "corresponding" amino acid. For example, one of ordinary skill in the art will understand that, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE can be utilized, for example, to identify "corresponding" residues in polypeptides and / or nucleic acids in accordance with the present disclosure. One of ordinary skill in the art will also understand that, in some cases, the term "corresponding to" can be used to describe an event or entity that shares a similarity with another event or entity (e.g., a suitable reference event or entity). By way of example, a gene or protein in one organism can, in some embodiments, be described as "corresponding to" a gene or protein from another organism in that it performs a similar role or function, and / or in that it exhibits a certain degree of sequence identity or homology, and / or in that it shares certain characteristic sequence elements.

[0061] As used herein, the term "engineered" refers to the aspect of being designed, produced, and / or manipulated by a human hand. For example, a polynucleotide is engineered when two or more sequences that are not linked in that order in nature are designed or otherwise caused by a human hand to be directly linked to each other in the engineered polynucleotide, and / or when certain residues in the polynucleotide are linked to entities or moieties that do not exist in nature and / or are not linked in nature by the action of a human hand. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide includes a regulatory sequence that is found in nature operably associated with a first coding sequence but not operably associated with a second coding sequence, and is linked by a human hand so as to be operably associated with the second coding sequence. Similarly, in some embodiments, a polypeptide can be considered "engineered" when it is encoded by or expressed from an engineered polynucleotide, and / or when it is produced other than by natural expression in a cell. Similarly, a cell or organism is considered "engineered" when its genetic, epigenetic, and / or phenotypic identity has been changed by an operation such that it is different compared to a suitable reference cell such as an identical cell that has not undergone such an operation. In some embodiments, the operation is a genetic operation or includes a genetic operation such that its genetic information is modified (e.g., by transformation, mating, somatic hybridization, transfection, introduction, or other mechanisms, a new genetic material that did not previously exist is introduced, or, for example, by substitution or deletion mutations, or mating protocols, a previously existing genetic material is modified or removed). In some embodiments, an engineered cell is a cell that has been engineered to contain and / or express a particular agent of interest (e.g., a protein, nucleic acid, and / or a particular form thereof) in an altered amount and / or according to an altered timing compared to such a suitable reference cell.It is a common practice and, as understood by those of ordinary skill in the art, the progeny of an engineered polynucleotide or cell are typically still referred to as "engineered" even if the actual engineering was performed on a precursor.

[0062] As used herein, an "engineered lipid bilayer particle" refers to a lipid bilayer particle engineered as described herein. For example, in some embodiments, a lipid bilayer particle can be considered "engineered" if it is synthetically produced, i.e., not produced by a cell. Alternatively or additionally, in some embodiments, a lipid bilayer particle can be considered "engineered" if it is produced by an engineered production cell. In some embodiments, an engineered lipid bilayer particle is produced by an engineered production cell that has been engineered to have a fusion factor entity polypeptide and / or a targeting chimeric polypeptide on its surface. In some such embodiments, the engineered production cell is different from a suitable reference cell in that it has been engineered to express a fusion factor entity polypeptide, a targeting chimeric polypeptide, or both, or to express one or both at different levels (e.g., elevated levels), such that lipid bilayer particles (e.g., CDMPC) produced (e.g., released) by such an engineered production cell bind to a recipient cell or cell population with significantly higher affinity and / or specificity than equivalent particles produced (e.g., released) by a reference cell.

[0063] As used herein, the term "extracellular vesicle" should be interpreted to include all nanoscale lipid vesicles secreted and / or budded by cells, such as exosomes and microvesicles, respectively. As used herein, the term "exosome" refers to extracellular vesicles derived from endocytic internal compartments or multivesicular bodies, and the term "microvesicle" refers to vesicles that bud directly from the cell surface. EVs, and their isolation and analysis are well known to those of skill in the art. See, for example, Doyle et al., Cells 8(7): 727 (2019), which is incorporated herein by reference in its entirety. Extracellular vesicles can be taken up by so-called extracellular vesicle (EV) recipient cells. As used herein, the term "recipient cell" can be interchangeable with the term "target cell".

[0064] As used herein, the term "cell-derived membrane particle" should be interpreted to include any membrane-derived vesicle or particle that can be generated by blebbing or budding, including hybrid vesicles generated by mixing vesicles produced from cells and synthetic vesicles, as well as vesicles or particles generated by mechanically treating cells. Thus, "cell-derived membrane particles" can include, but are not limited to, extracellular vesicles (as defined above), virus particles, virus-like particles (VLPs), apoptotic bodies, and platelet-like particles.

[0065] As used herein, the term "fusion factor entity polypeptide" refers to a polypeptide that mediates fusion between lipid bilayers. As documented by the present disclosure, in some embodiments, the presence of a fusion factor entity polypeptide in lipid bilayer particles or engineered lipid bilayer particles (particularly including on engineered lipid bilayer particles displaying a targeting chimeric polypeptide) increases the efficiency, specificity, and / or effectiveness of cargo delivery from such engineered lipid bilayer particles to a particular recipient cell of interest.

[0066] As used herein, the term "gene" means a segment of DNA that contains information for the regulated biosynthesis of an RNA product. One of ordinary skill in the art will understand that a gene typically includes an expressed sequence (e.g., an open reading frame that may include exons and introns). One of ordinary skill in the art will further understand that a gene typically includes one or more promoters and / or other untranslated regions (e.g., enhancer elements, repressor elements, chromatin binding sites, etc.) that can control, regulate, or otherwise affect expression.

[0067] As used herein, "homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid entities. One of ordinary skill in the art understands that homology can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. Entities are homologous at a position if the positions in the sequences being compared are occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that when aligned, comparing the two sequences, that percentage of bases (or amino acids) are the same. This alignment and percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation value = 10; matrix = BLOSUM62; description = 50 sequences; sort by HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs are published by the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percentage of homology and encode polypeptides having the same or similar biological activity. Two sequences are considered "non-homologous" or "non-identical" if they share less than 40% identity, or less than 25% identity, with each other.Furthermore, one of ordinary skill in the art will understand that "identical" polypeptides or nucleic acids often share one or more characteristic sequence elements that can confer shared structural and / or functional characteristics to, for example, the polypeptides that contain them.

[0068] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising", except that the latter terms are "open" transitional terms that do not limit the claim to the recited elements following these transitional terms only. The term "consisting of" is to be construed as a "closed" transitional term that limits the scope of the claim to only the recited elements following this transitional term, although it is included within the term "including". The term "consisting essentially of" is to be construed as a "partially closed" transitional term that is included within the term "including", but allows for additional elements following this transitional term, provided that such additional elements do not materially affect the basic and novel characteristics of the claim.

[0069] As used herein, the term "linker" refers to the portion of a multi-component agent that connects different elements to each other. For example, one of ordinary skill in the art will understand that polypeptides having a structure that includes two or more functional or organizational domains often include an extension of amino acids between such domains that connects them to each other. In some embodiments, a polypeptide that includes a linker element has an overall structure of the general form S1-L-S2, where S1 and S2 may be the same or different and represent two domains that are associated with each other by the linker. In some embodiments, the polypeptide linker has a length of at least 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized by not adopting a rigid three-dimensional structure and rather tending to impart flexibility to the polypeptide. In some embodiments, the linker is characterized in that it adopts a rigid three-dimensional structure and provides stability to the polypeptide. A variety of different linker elements that can be appropriately used when engineering polypeptides (e.g., fusion polypeptides) are known in the art (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1 121-1123).

[0070] As used herein, the terms "polynucleotide", "polynucleotide sequence", "nucleic acid" and "nucleic acid sequence" refer to nucleotides, oligonucleotides, polynucleotides (these terms may be used interchangeably), or any fragment thereof. As will be apparent from the context, these phrases may also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or antisense strand).

[0071] With respect to polynucleotide sequences, the terms "percent identity" and "identity %" refer to the percentage of residues that match between at least two polynucleotide sequences aligned using a standardized algorithm. Such algorithms can insert gaps into the sequences being compared in a standardized, reproducible manner in order to optimize the alignment between the two sequences, and thus achieve a more meaningful comparison of the two sequences. The percent identity of nucleic acid sequences can be determined as is understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A generally used and freely available set of sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI)'s Basic Local Alignment Search Tool (BLAST), which is available on its website from several sources, including NCBI (Bethesda, Md). The BLAST software suite includes various sequence analysis programs, including "blastn" which is used for aligning known polynucleotide sequences with other polynucleotide sequences from various databases. There is also a tool called "BLAST 2 Sequences" which is used to directly pairwise compare two nucleotide sequences. The "BLAST 2 Sequences" tool, which can be accessed interactively from the NCBI website, can be used with both blastn and blastp (described above).

[0072] With respect to a polynucleotide sequence, the percent identity may be measured over the length of the entire defined polynucleotide sequence as defined, for example, by a particular SEQ ID NO, or may be measured over a shorter length, for example, a fragment taken from a larger, defined sequence, such as a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 consecutive nucleotides. Such lengths are exemplary, and it is understood that the length of any fragment supported by the sequences shown in this specification, tables, figures, or sequence listings may be used to describe the length over which the percent identity may be measured.

[0073] With respect to a polynucleotide sequence, a “variant,” “mutant,” or “derivative” may be defined as a nucleic acid sequence having at least 50% sequence identity with a particular nucleic acid sequence over a certain length of the nucleic acid sequence, using blastn with the “BLAST 2 Sequences” tool available on the National Center for Biotechnology Information website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences - a new tool for comparing protein and nucleotide sequences,” FEMS Microbiol Lett. 174:247-250). Such a pair of nucleic acids may exhibit at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% or more sequence identity over a certain defined length.

[0074] Nucleic acid sequences that do not exhibit identity in height may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code where multiple codons can code for a single amino acid. This degeneracy can be utilized to vary the nucleic acid sequence and create multiple nucleic acid sequences that encode substantially the same protein. For example, a polynucleotide sequence as contemplated herein can encode a protein and can be codon-optimized for expression in a particular host. In the art, codon usage tables have been prepared for many host organisms including humans, mice, rats, pigs, Escherichia coli, plants, and other host cells.

[0075] With respect to a polynucleotide sequence, "recombinant nucleic acid" refers to a sequence having a sequence that is not naturally occurring or is made by an artificial combination of two or more distinct sequence segments. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificial manipulation of isolated nucleic acid segments, for example, by gene manipulation techniques known in the art. The term recombinant includes nucleic acids that have been changed only by addition, substitution, or deletion of a portion of the nucleic acid. Often, a recombinant nucleic acid can include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector used, for example, to transform cells.

[0076] The nucleic acids disclosed herein may be "substantially isolated or purified". The term "substantially isolated or purified" refers to a nucleic acid that has been removed from its natural environment and is free from at least 60%, preferably at least 75%, more preferably at least 90%, and even more preferably at least 95% of other components naturally associated therewith.

[0077] "Transformation" or "transfection" describes the process by which exogenous nucleic acid (e.g., DNA or RNA) is introduced into a recipient cell. Transformation or transfection can occur under natural or artificial conditions according to various methods well known in the art and can depend on any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method for transformation or transfection is selected based on the type of host cell to be transformed and includes, but is not limited to, bacteriophage or viral infection or non-viral delivery. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, electroporation, heat shock, particle bombardment, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patent Nos. 5,049,590, 5,049,386, 4,946,787; and 4,897,355), and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides are those of Felgner, WO91 / 17424; WO91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). The terms "transformed cell" or "transfected cell" include stably transformed or transfected cells in which the inserted DNA can replicate as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed or transfected cells that express the inserted DNA or RNA for a limited period. In another embodiment, the term also includes stably transfected cells.

[0078] The polynucleotide sequences contemplated herein may be present in an expression vector. For example, the vector may include: (a) a polynucleotide encoding an ORF of a cargo protein; and (b) a polynucleotide expressing an ABA binding domain, such as a pyrabactin resistance 1-like (PYL1) sequence or an abscisic acid-insensitive 1 (ABI1) sequence. The polynucleotide present in the vector may be operably linked to a prokaryotic or eukaryotic promoter. "Operably linked" refers to a situation where a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be adjacent or contiguous and may be in the same reading frame if necessary to ligate two protein-coding regions. Vectors contemplated herein may include a heterologous promoter (e.g., a eukaryotic promoter or a prokaryotic promoter) operably linked to a polynucleotide encoding a protein. A "heterologous promoter" refers to a promoter that is not the native or endogenous promoter of the protein or RNA to be expressed.

[0079] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as an mRNA or other RNA transcript), and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcription product and the encoded polypeptide may be collectively referred to as a "gene product". When the polynucleotide is derived from genomic DNA, expression includes mRNA splicing in eukaryotic cells.

[0080] The term "vector" refers to any means for introducing a nucleic acid (e.g., DNA) into a host organism or host tissue. There are various types of vectors, such as plasmid vectors, bacteriophage vectors, cosmid vectors, bacterial vectors, viral vectors, etc. As used herein, "vector" can refer to a recombinant nucleic acid engineered to express a heterologous polypeptide (e.g., a fusion protein disclosed herein). The recombinant nucleic acid typically contains cis-acting elements for expressing the heterologous polypeptide.

[0081] Any of the conventional vectors used for expression in eukaryotic cells can be used to directly introduce DNA into the target. For eukaryotic expression vectors, expression vectors containing control elements derived from eukaryotic viruses can be used (e.g., vectors containing a promoter or enhancer of SV40, CMV, or a retrovirus). Exemplary vectors include those that express a protein under the direction of a promoter such as the SV40 early promoter, SV40 late promoter, metallothionein promoter, human cytomegalovirus promoter, mouse mammary tumor virus promoter, and Rous sarcoma virus promoter. The expression vectors contemplated herein can contain eukaryotic or prokaryotic control sequences that regulate the expression of a heterologous protein (e.g., a fusion protein disclosed herein). Prokaryotic expression control sequences can include a constitutive or inducible promoter (e.g., T3, T7, Lac, trp, or phoA), a ribosome binding site, or a transcription terminator.

[0082] Vectors contemplated herein can be introduced and propagated in prokaryotes that can be used to amplify copies of the vector introduced into eukaryotic cells or as intermediate vectors in the production of vectors introduced into eukaryotic cells (e.g., amplifying a plasmid as part of a viral vector packaging system). Prokaryotes can be used to express one or more nucleic acids so as to amplify copies of the vector and provide a source of one or more proteins for delivery to a host cell or host organism. Protein expression in prokaryotes can be carried out using E. coli with a vector containing a constitutive or inducible promoter that directs the expression of a fusion protein containing the protein or a fragment thereof. The fusion vector adds amino acids to the encoded protein, such as at the amino terminus of the recombinant protein. Such fusion vectors can serve one or more of the following purposes: (i) increasing the expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; (iii) assisting in the purification of the recombinant protein by acting as a ligand in affinity purification (e.g., His tag); (iv) tagging the recombinant protein for identification (e.g., can be recognized by a labeled antibody such as green fluorescent protein (GFP) or an antigen (e.g., HA)); (v) facilitating the localization of the recombinant protein to a specific region within the cell (e.g., where the protein is fused to a nuclear localization signal (NLS) (e.g., at its N-terminus or C-terminus), which includes the NLS of SV40, nucleoplasmin, C-myc, the M9 domain of hnRNP A1, or a synthetic NLS). The importance of neutral and acidic amino acids in the NLS has been studied. (See Makkerh et al. (1996) Curr Biol 6(8):1025-1027). In many cases, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion protein portion and the recombinant protein so that the recombinant protein can be separated from the fusion protein after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase.

[0083] The presently disclosed methods can include delivering to a host cell one or more polynucleotides, or one or more vectors, one or more transcription products thereof, and / or one or more proteins transcribed therefrom, as described herein. Further contemplated are host cells produced by such methods, and organisms (such as animals, plants, or fungi) containing such cells or produced from such cells. The disclosed extracellular vesicles can be prepared by introducing a vector that expresses mRNA encoding the fusion proteins disclosed herein and cargo RNA. To introduce nucleic acids into mammalian cells or target tissues, conventional virus- and non-virus-based gene delivery methods can be used. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcription products of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have episomal or integrated genomes after delivery to cells.

[0084] In the methods contemplated herein, a host cell can be transiently or non-transiently transfected (i.e., stably transduced) with one or more vectors described herein. In some embodiments, the cells are transfected such that they occur naturally in a subject (i.e., in situ). In some embodiments, the cells to be transfected are harvested (i.e., excised) from a subject. In some embodiments, the cells are derived from cells harvested from a subject, such as a cell line. Suitable cells can include stem cells (e.g., embryonic stem cells and pluripotent stem cells). Cells transfected with one or more vectors described herein can be used to establish a new cell line containing sequences from the one or more vectors. In the methods contemplated herein, cells can be transiently transfected with components of the systems described herein (such as transient transfection with one or more vectors, or transfection with RNA), modified through the activity of the complex, and a new cell line can be established that contains cells with the modification but lacking other foreign sequences.

[0085] As used herein, the terms "protein", "polypeptide", or "peptide" may be used interchangeably to refer to a polymer of amino acids. Typically, a "polypeptide" or "protein" is defined as a long polymer of amino acids typically greater than 50, 60, 70, 80, 90, or 100 amino acids in length. A "peptide" is defined as a short polymer of amino acids, usually 50, 40, 30, 20 or less in length.

[0086] As used herein, a "protein" typically includes polymers of naturally or non-naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). Proteins contemplated herein may be further modified in vitro or in vivo to include non-amino acid moieties. Such modifications include acylation (e.g., O-acylation (ester), N-acylation (amide), S-acylation (thioester)), acetylation (e.g., acetylation (e.g., addition of an acetyl group to the N-terminus or lysine residue of a protein)), formylation lipoylation (e.g., addition of lipoic acid, a C8 functional group), myristoylation (e.g., addition of myristic acid, a C14 saturated acid), palmitoylation (e.g., palmitoylation (e.g., addition of palmitic acid, a C16 saturated acid)), alkylation (e.g., addition of an alkyl group such as methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., isoprenylation or prenylation (e.g., addition of an isoprenoid group such as farnesol or geranylgeraniol)), amidation at the C-terminus, glycosylation (e.g., addition of a glycosyl group to any of asparagine, hydroxylysine, serine or threonine to yield a glycoprotein). Glycation, considered a non-enzymatic attachment of sugar, polysialylation (e.g., addition of polysialic acid), glypiation (e.g., formation of a glycosylphosphatidylinositol (GPI) anchor), hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., usually addition of a phosphate group to serine, tyrosine, threonine or histidine) are different.

[0087] With respect to proteins, the term "amino acid residue" may also include amino acid residues selected from the group consisting of homocysteine, 2-aminoadipic acid, N-ethylasparagine, 3-aminoadipic acid, hydroxylysine, β-alanine, β-aminopropionic acid, allo-hydroxylysine, 2-aminobutyric acid, 3-hydroxyproline, 4-aminobutyric acid, 4-hydroxyproline, pipecolic acid, 6-aminocaproic acid, isodesmosine, 2-aminoheptanoic acid, alloisoleucine, 2-aminoisobutyric acid, N-methylglycine, sarcosine, 3-aminoisobutyric acid, N-methylisoleucine, 2-aminopimelic acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, desmosine, norvaline, 2,2'-diaminopimelic acid, norleucine, 2,3-diaminopropionic acid, ornithine, and N-ethylglycine.

[0088] The proteins disclosed herein may include "wild-type" proteins and their variants, mutants, and derivatives. As used herein, the term "wild-type" is a term of art understood by those of skill in the art and means the typical form of a living organism, strain, gene, or characteristic that occurs in nature, as distinguished from a mutant or variant. As used herein, "variant", "mutant", or "derivative" refers to a protein molecule having an amino acid sequence different from a reference protein or polypeptide molecule. A variant or mutant may have an insertion, deletion, or substitution of one or more amino acid residues relative to the reference molecule. A variant or mutant may also include a fragment of the reference molecule. For example, a mutant or variant may have one or more insertions, deletions, or substitutions of at least one amino acid residue relative to the reference polypeptide.

[0089] With respect to a protein, "deletion" refers to a change in the amino acid sequence in which one or more amino acid residues are deleted. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200 amino acid residues, or a range of amino acid residues bounded by any of these values (e.g., a deletion of 5-10 amino acids). A deletion can include an internal deletion or a terminal deletion (e.g., an N-terminal or C-terminal truncation of a reference polypeptide). A "variant", "mutant", or "derivative" of a reference polypeptide sequence can include a deletion with respect to the reference polypeptide sequence.

[0090] With respect to a protein, a "fragment" is a portion of an amino acid sequence that has the same sequence as the reference sequence but is shorter in length than the reference sequence. A fragment is composed of a sequence from which at least one amino acid residue has been removed from the full length of the reference sequence. For example, a fragment can be composed of 5-1000 consecutive amino acid residues of a reference polypeptide, respectively. In some embodiments, a fragment can be composed of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 consecutive amino acid residues of a reference polypeptide; in other embodiments, a fragment can include less than about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 consecutive amino acid residues of a reference polypeptide; or in yet other embodiments, a fragment has a length within a range bounded by any of these values (e.g., a range of 50-100 consecutive amino acids of a reference polypeptide). A fragment can be preferentially selected from a particular region of the molecule. The term "at least fragment" encompasses the full-length polypeptide. A fragment can include an N-terminal truncation, a C-terminal truncation, or both truncations with respect to the full-length protein. A "variant", "mutant", or "derivative" of a reference polypeptide sequence includes a fragment of the reference polypeptide sequence.

[0091] With respect to a protein, the terms "insertion" and "addition" refer to a change in the amino acid sequence that results in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more amino acid residues, or a range of amino acid residues bounded by any of these values (e.g., an insertion or addition of 5-10 amino acids). A "variant", "mutant", or "derivative" of a reference polypeptide sequence may include an insertion or addition with respect to the reference polypeptide sequence. A variant of a protein can have an N-terminal insertion, a C-terminal insertion, an internal insertion, or any combination of N-terminal insertion, C-terminal insertion, and internal insertion.

[0092] With respect to a protein, as used herein, "chimeric protein", "chimeric peptide", "fusion protein", or "fusion peptide" refers to a polypeptide produced by linking two or more functional domains from separate proteins or the same protein, either through an amino acid linker or directly linked, resulting in a single polypeptide having functional properties derived from each of the original proteins. In some embodiments, the linker is 10-50 amino acids in length, rich in glycine for flexibility, and rich in serine or threonine for solubility. In some embodiments, the linker adopts a rigid three-dimensional structure and is characterized by providing stability to the polypeptide. A "variant" of a reference polypeptide sequence may include a fusion polypeptide that includes the reference polypeptide.

[0093] For proteins, the terms "percent identity" and "identity %" refer to the percentage of residues that match between at least two amino acid sequences aligned using a standardized algorithm. Methods for amino acid sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions generally retain the charge and hydrophobicity of the substitution site and retain the structure (and thus function) of the polypeptide. The percent identity of amino acid sequences can be determined as is understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A commonly used and freely available set of sequence comparison algorithms is provided by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI), which is available on its website from several sources, including the NCBI (Bethesda, Md). The BLAST software suite includes various sequence analysis programs, including "blastp", which is used to align known amino acid sequences with other amino acid sequences from various databases. As described herein, variants, mutants, or fragments (e.g., variants, mutants, or fragments of a protein) may have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% amino acid sequence identity to a reference molecule.

[0094] For proteins, the percent identity can be measured over the length of the entire defined polypeptide sequence, such as defined by a particular SEQ ID NO., or over a shorter length, such as for example, a fragment taken from a larger, defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary, and it is understood that the length of any fragment supported by the sequences shown in the specification, tables, figures, or sequence listings can be used to describe the length over which the percent identity can be measured.

[0095] For proteins, the amino acid sequences of variants, mutants, or derivatives contemplated herein can include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative protein can include conservative amino acid substitutions relative to a reference molecule. "Conservative amino acid substitution" refers to a substitution in which an amino acid is replaced with a different amino acid and the substitution is predicted to least inhibit the properties of the reference polypeptide. In other words, a conservative amino acid substitution substantially conserves the structure and function of the reference polypeptide. The following table provides a list of exemplary conservative amino acid substitutions contemplated herein:

[0096] [Table 1]

[0097] Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone at the substitution site, e.g., beta-sheet or alpha-helix structure, (b) the charge or hydrophobicity of the molecule at the substitution site, and / or (c) the bulk of the side chain.

[0098] The disclosed protein, mutant, variant, or protein described herein may have one or more functional or biological activities (e.g., one or more functional or biological activities exhibited by the wild-type protein) as indicated by a reference polypeptide. For example, the disclosed protein, mutant, variant, or derivative thereof may have one or more biological activities including binding to a small molecule ABA and targeting EVs to recipient cells.

[0099] The disclosed proteins may be substantially isolated or purified. The term "substantially isolated or purified" refers to proteins that have been removed from their natural environment and are at least 60% free, preferably at least 75% free, more preferably at least 90% free, and even more preferably at least 95% free from other components with which they are naturally associated.

[0100] As used herein, the term "recombinant" when used in connection with, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the modification of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene not found in the native form (non-recombinant form) of the cell, or expresses a native gene that is abnormally expressed, under-expressed, or not expressed at all.

[0101] As used herein, the term "targeting domain" or "targeting peptide" refers to a peptide moiety that facilitates the specific binding of EVs to recipient cells. Exemplary "targeting domains" or "targeting peptides" include, but are not limited to, antibodies, any antibody fragment or antigen-binding fragment, such as, for example, Fab, Fab', and F(ab') 2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-bonded Fv (sdFv), de novo-designed binding molecules, affibodies, DARPINs, nanobodies, variable lymphocyte receptors (VLRs), and camelid antibodies. These antibody fragments are well known to those skilled in the art.

[0102] Recipient cell In particular, the present disclosure demonstrates the surprising effectiveness of the provided technology when applied to recipient cells, e.g., specific recipient cells of interest, or populations thereof. For example, the present disclosure documents the surprising specificity and / or efficiency of delivering cargo entities to recipient cells of interest, or populations thereof, using lipid bilayer particles such as cell-derived membrane particles (CDMPs) as described herein. Using the techniques of the present disclosure, lipid bilayer particles deliver cargo entities to recipient cells in an efficient and specific manner. In some embodiments, the provided technology delivers cargo entities when employed in vitro. In some embodiments, the provided technology achieves, for example, in vivo delivery of cargo entities to recipient cells.

[0103] In one embodiment, the recipient cells express a target ligand (e.g., a cell surface epitope). In some embodiments, the recipient cells express a specific target ligand or a specific combination of target ligands. In some embodiments, the recipient cells are CD5+, CD2+, or a combination thereof. In some embodiments, the recipient cells are immune cells. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are activated T cells. In some embodiments, the T cells are CD4+ and / or CD8+.

[0104] In some embodiments, the targeting chimeric polypeptides provided herein bind to a target ligand on the surface of recipient cells. In some embodiments, a particular targeting chimeric polypeptide (e.g., a targeting chimeric polypeptide on the surface of a lipid bilayer particle) binds to a target ligand on the surface of recipient cells if the recipient cells are present. In some embodiments, the target ligand (e.g., a target epitope) is expressed on all cells (universal features of the cell surface), or on a subset of cells, or on cells that occupy a subset of possible states (e.g., activated T cells versus resting T cells).

[0105] Without wishing to be bound by any particular theory, it is proposed that the unique combination of the fusion factor entity polypeptide and the targeting chimeric polypeptide provided herein achieves highly efficient targeted entry of lipid bilayer particles into specific recipient cells.

[0106] In some embodiments, the targeting chimeric polypeptides described herein bind to a specific target ligand on the recipient cell surface. Recipient cells can express a number of unique targeting ligands on their cell surface, and the targeting chimeric polypeptides can be designed to target one or more such target ligands. In some embodiments, the target ligand is present on the surface of the recipient cells. In some embodiments, the target ligand is an epitope, receptor, protein, carbohydrate, lipid, or a particular combination or conformational state thereof.

[0107] Engineered lipid bilayer particle In particular, the present disclosure provides engineered lipid bilayer particles, and preparations thereof, comprising (e.g., comprising on their surface) a targeting chimeric polypeptide, a fusion factor entity polypeptide, or a combination thereof, as described herein. The present invention provides, in some embodiments, engineered lipid bilayer particles and preparations thereof comprising a targeting chimeric polypeptide and a fusion factor entity polypeptide. The engineered lipid bilayer particles provided may be suitable for therapeutic use.

[0108] In some embodiments, the present disclosure (i) a targeting chimeric polypeptide, wherein (a) a targeting domain arranged such that the targeting domain is on the particle surface, the targeting domain comprising a binding moiety that specifically binds to a target ligand on the surface of a recipient cell of interest; and (b) a transmembrane domain directly or indirectly linked thereto comprising the targeting chimeric polypeptide; and (ii) a fusion factor entity polypeptide, wherein (a) a fusion factor moiety arranged such that the fusion factor moiety is on the particle surface; and (b) a transmembrane domain directly or indirectly linked thereto comprising a population of engineered lipid bilayer particles.

[0109] In some embodiments, the engineered lipid bilayer particles in the population display one or more targeting chimeric polypeptides and one or more fusion factor entity polypeptides. In some embodiments, at least a portion of the fusion factor entity polypeptide is present on the surface of the engineered lipid bilayer particle (e.g., the fusion factor entity moiety). In some embodiments, at least a portion of the targeting chimeric polypeptide is present on the surface of the engineered lipid bilayer particle (e.g., the targeting domain). Without wishing to be bound by any particular theory, it is proposed that a particular combination of a targeting chimeric polypeptide and a fusion factor entity polypeptide as described herein provides for highly efficient targeted entry of the engineered lipid bilayer particles into specific recipient cells. Indeed, without wishing to be bound by any particular theory, a particular targeting chimeric polypeptide as described herein (e.g., using a PDGFR transmembrane domain [or, in some embodiments, an equivalent transmembrane domain] and an affinity entity polypeptide, specifically including cases where such an affinity entity polypeptide is an antibody agent or includes an antibody agent) can provide certain display characteristics (e.g., orientation and / or other aspects of presentation, surface density, etc.), which are particularly compliant with a beneficial combination with a fusion factor entity polypeptide as described herein to achieve particle targeting as described herein.

[0110] In some embodiments, the engineered lipid bilayer particles are produced by one or more engineered producer cells as described herein, such that the engineered lipid bilayer particles display a targeting chimeric polypeptide and a fusion factor entity polypeptide.

[0111] In some embodiments, the population of engineered lipid bilayer particles is characterized in that the engineered lipid bilayer particles are smaller than eukaryotic cells. In some embodiments, the population of engineered lipid bilayer particles is characterized in that the average diameter of the engineered lipid bilayer particles is at most 1000 nm, such as at most 800 nm, such as at most 300 nm, such as at most 100 nm. In some embodiments, the population of engineered lipid bilayer particles is characterized in that the average diameter of the engineered lipid bilayer particles is at least 30 nm, such as at least 50 nm, such as at least 80 nm, such as at least 100 nm, such as at least 150 nm, such as at least 200 nm, such as at least 250 nm, such as at least 300 nm. In some embodiments, the population of engineered lipid bilayer particles is characterized in that the average diameter of the engineered lipid bilayer particles is from about 10 nm to about 1000 nm, such as from about 30 nm to about 800 nm, such as from about 50 nm to about 500 nm.

[0112] In some embodiments, the entry of engineered lipid bilayer particles into recipient cells can be affected by one or more attributes of the entry environment (e.g., pH, temperature, proximity-induced, mechanical tension or stress, rearrangement of lipid domains (e.g., rafts), radiation (e.g., nuclear, ultraviolet, visual, etc.), electrical signals, magnetic fields, etc., or combinations thereof).

[0113] In some embodiments, the engineered lipid bilayer particles display at least 5 copies of the fusion factor entity polypeptide, such as at least 10 copies, such as at least 50 copies, such as at least 100 copies, such as at least 200 copies, such as at least 300 copies, such as at least 400 copies, such as at least 500 copies of the fusion factor entity polypeptide.

[0114] In some embodiments, the engineered lipid bilayer particles display at least 10 copies of an affinity entity polypeptide, such as at least 50 copies, such as at least 100 copies, such as at least 200 copies, such as at least 300 copies, such as at least 400 copies, such as at least 500 copies of an affinity entity polypeptide.

[0115] In some embodiments, the engineered lipid bilayer particles are cell-derived membrane particles (CDMPs). In some embodiments, the CDMPs are selected from extracellular vesicles, virus particles, virus-like particles (VLPs), apoptotic bodies, platelet-like particles, and combinations thereof. In some embodiments, the extracellular vesicles are exosomes, microvesicles, and combinations thereof.

[0116] In some embodiments, the CDMP is an extracellular vesicle and can be selected from exosomes or microvesicles. In some embodiments, the CDMP can be virus particles, virus-like particles (VLPs), apoptotic bodies, and platelet-like particles. In some embodiments, the CDMP can be hybrid particles generated by mixing cell-derived particles or vesicles (e.g., particles or vesicles shed or budded from cells) with synthetic vesicles.

[0117] In some embodiments, the engineered lipid bilayer particles display a fusion factor entity polypeptide such as VSV-G or a functional variant thereof described herein. In some embodiments, the engineered lipid bilayer particles display a targeting chimeric polypeptide comprising a PDGFR transmembrane domain. In some embodiments, the engineered lipid bilayer particles display a targeting chimeric polypeptide that binds to VSV-G and CD2 or CD5 described herein.

[0118] Targeting chimeric polypeptide The present disclosure provides insights into particularly useful and / or effective targeting chimeric polypeptides for promoting specific binding of engineered lipid bilayer particles to target ligands (e.g., target ligands present on recipient cells). The present disclosure teaches that the targeting chimeric polypeptide and its binding to the target ligand can be useful for mediating targeted fusion of engineered lipid bilayer particles with recipient cells expressing the target ligand or a particular subset of recipient cells expressing the target ligand (e.g., providing specificity to the techniques described herein). Without wishing to be bound by a particular theory, it is proposed that the targeted fusion can be driven and / or influenced by one or more of proximity, affinity, and / or conformational changes.

[0119] In some embodiments, the targeting chimeric polypeptide mediates the binding of engineered lipid bilayer particles to the desired recipient cells. In some embodiments, the targeting chimeric polypeptide alone (e.g., in the absence of the fusion factor entity polypeptide) does not promote cell invasion and transduction. Further, in many embodiments, the combination of a targeting chimeric polypeptide as described herein and a fusion factor entity polypeptide as described herein achieves a significant improvement in the efficiency, specificity, and / or effectiveness of cargo delivery to a particular recipient cell of interest.

[0120] The targeting chimeric polypeptides provided herein are useful when designing binding to a particular type of recipient cell. In some embodiments, the targeting chimeric polypeptide binds to a specific target ligand, thereby directing binding to recipient cells expressing such specific target ligand. In some embodiments, when the targeting chimeric polypeptide is co-displayed in engineered lipid bilayer particles together with a fusion factor entity polypeptide, the binding of the targeting chimeric polypeptide promotes fusion of the fusion factor entity polypeptide with the recipient cell.

[0121] In some embodiments, the present disclosure provides a targeting chimeric polypeptide. In some embodiments, the present disclosure provides a nucleotide sequence encoding a targeting chimeric polypeptide.

[0122] In some embodiments, the techniques according to the present disclosure (e.g., systems, engineered lipid bilayer particles, and engineered production cells) include a targeting chimeric polypeptide. In some embodiments, the techniques according to the present disclosure include at least one targeting chimeric polypeptide. In some embodiments, the techniques according to the present disclosure include one or more targeting chimeric polypeptides.

[0123] In some embodiments, the targeting chimeric polypeptide includes a secretion signal. In some embodiments, the targeting chimeric polypeptide includes a FLAG tag. In some embodiments, the targeting chimeric polypeptide does not include a FLAG tag, for example, when the targeting chimeric polypeptide is a native polypeptide. In some embodiments, the targeting chimeric polypeptide includes an affinity moiety. In some embodiments, the targeting chimeric polypeptide includes a linker. In some embodiments, the targeting chimeric polypeptide does not include a linker, for example, when the targeting chimeric polypeptide is a native polypeptide. In some embodiments, the targeting chimeric polypeptide includes a membrane association moiety. In some embodiments, the targeting chimeric polypeptide includes an intra-particle moiety.

[0124] In some embodiments, the targeting chimeric polypeptide consists of or includes a transmembrane domain. In some embodiments, the transmembrane domain is a domain having high expression on the surface of the lipid bilayer particle.

[0125] In some embodiments, the targeting chimeric polypeptide is an engineered polypeptide. In some embodiments, the order from the N-terminus to the C-terminus of the targeting chimeric polypeptide is as follows: a secretion signal, a targeting domain, a linker, and / or a transmembrane domain. In some embodiments, the order from the N-terminus to the C-terminus of the targeting chimeric polypeptide is as follows: a secretion signal, a FLAG tag, a targeting domain, a linker, and / or a transmembrane domain.

[0126] In some embodiments, the targeting chimeric polypeptide is a wild-type polypeptide. In some embodiments, the targeting chimeric polypeptide is native to a particular production cell. In some embodiments, the targeting chimeric polypeptide is an engineered polypeptide. In some embodiments, the targeting chimeric polypeptide (e.g., an engineered targeting chimeric polypeptide) is a variant of a wild-type polypeptide and / or a native polypeptide.

[0127] In some embodiments, the order from the N-terminus to the C-terminus of the targeting chimeric polypeptide is as follows: a secretion signal, a targeting domain, a transmembrane domain, and / or an intra-particle portion.

[0128] In some embodiments, the affinity entity polypeptide comprises one or more modifications such as glycosylation, lipidation, phosphorylation, etc.

[0129] In some embodiments, a targeting chimeric polypeptide comprising a targeting domain. In some embodiments, the targeting chimeric polypeptide comprising a targeting domain comprises a binding moiety that specifically binds to a target ligand on the surface of a recipient cell of interest. In some embodiments, the binding domain is displayed on the surface of the lipid bilayer particle. It can be displayed in a manner that facilitates the binding of the lipid bilayer of the lipid bilayer particle to the target ligand on the surface of the recipient cell. In some embodiments, the targeting chimeric polypeptide further comprises a transmembrane domain. In some embodiments, the targeting domain is directly or indirectly linked to the transmembrane domain

[0130] In some embodiments, the targeting domain is an antibody agent or comprises an antibody agent. In some embodiments, the antibody agent is a single-chain antibody agent. In some embodiments, the antibody agent is an antibody, Fab, Fab’, F(ab’) 2 , Fd, scFv, single-chain antibody, disulfide-bonded Fvs (sdFv), affibody, DARPIN, nanobody, variable lymphocyte receptor (VLR), and camelid antibody, selected from the group consisting of.

[0131] The term "engineered high-affinity binding polypeptide" is equivalent to "de novo designed binding molecule" and is used interchangeably herein.

[0132] In some embodiments, the binding domain specifically binds to the surface of immune cells (e.g., lymphocytes such as CD4+ and / or CD8+ T cells). In some embodiments, the affinity moiety is characterized by binding to recipient cells expressing CD5, CD2, or a combination thereof.

[0133] In some embodiments, the present disclosure provides (a) a targeting domain that binds to human CD2, wherein the targeting domain is an antibody, Fab, Fab’, F(ab’) 2an antibody selected from the group consisting of Fd, scFv, single-chain antibody, disulfide-bonded Fvs (sdFv), de novo-designed binding molecules, affibody, DARPIN, nanobody, variable lymphocyte receptor (VLR), and camelid antibody, or comprising them): and (b) a chimeric targeting polypeptide comprising a transmembrane domain. In some embodiments, the targeting domain is scFv. The chimeric targeting polypeptide may optionally comprise a linker.

[0134] In some embodiments, the targeting domain binds to CD2. In some embodiments, the targeting domain comprises an anti-CD2 moiety or a fragment thereof. In some embodiments, the targeting domain has the amino acid sequence NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSGGGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSS (SEQ ID NO: 20), whichIt can be encoded by the nucleic acid sequence AACATCATGATGACGCAGAGCCCCAGCAGCCTGGCTGTTTCTGCTGGCGAGAAAGTGACCATGACCTGCAAGAGCAGCCAGAGCGTGCTGTACTCCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGTCTCCTAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACAGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATCAGTAGCGTGCAGCCCGAGGATCTGGCCGTGTACTACTGTCACCAGTACCTGAGCAGCCACACCTTTGGCGGCGGAACAAAGCTGGAAATCAAGAGAGGCGGAGGCGGATCAGGTGGCGGTGGATCTGGCGGTGGTGGATCTCAACTTCAGCAGCCAGGCGCAGAACTTGTGCGGCCTGGATCTAGCGTGAAGCTGAGCTGTAAAGCCAGCGGCTACACCTTCACCAGATACTGGATCCACTGGGTCAAGCAGCGGCCTATCCAGGGACTCGAGTGGATCGGCAATATCGACCCCAGCGACAGCGAGACACACTACAATCAGAAGTTCAAGGACAAGGCCACACTGACCGTGGACAAGTCTAGCGGCACAGCCTACATGCAGCTGTCCAGCCTGACAAGCGAGGACAGCGCCGTGTATTATTGCGCCACCGAGGACCTGTACTACGCCATGGAATATTGGGGCCAGGGCACCAGCGTGACCGTTAGCTCT (SEQ ID NO: 21). However, it should be noted that it is expected that the targeting domain capable of binding to CD2 functions. In some embodiments, the chimeric targeting polypeptide and lipid bilayer particle containing such a polypeptide are sufficient to deliver the content of the lipid bilayer particle (e.g., cargo entity) to recipient cells (e.g., lymphocytes) by CD2 binding only.,

[0135] In some embodiments, the present disclosure provides a chimeric targeting polypeptide comprising (a) a targeting domain that binds to human CD5, where the targeting domain is an antibody, Fab, Fab’, F(ab’) 2 , Fd, scFv, single-chain antibody, disulfide-bonded Fvs (sdFv), de novo designed binding molecule, affibody, DARPIN, nanobody, variable lymphocyte receptor (VLR), and camelid antibody, or comprises any of them), and (b) a transmembrane domain. In some embodiments, the targeting domain is a VLR. In some embodiments, the targeting domain is an scFv. The chimeric targeting polypeptide may optionally comprise a linker.

[0136] In some embodiments, the targeting domain binds to CD5. In some embodiments, the targeting domain comprises an anti-CD5 moiety or a fragment thereof. In some embodiments, the targeting domain comprises the amino acid sequence CPSQCSCSGTEVHCQRKSLASVPAGIPTTTRVLYLHVNEITKFEPGVFDRLVNLQQLYLGGNQLSALPDGVFDRLTQLTRLDLYNNQLTVLPAGVFDRLVNLQTLDLHNNQLKSIPRGAFDNLKSLTHIWLFGNPWDCACSDILYLSGWLGQHAGKEQGQAVCSGTNTPVRAVTEASTSPSKCP (SEQ ID NO: 24), which may be encoded by the nucleic acid sequence TGCCCCAGCCAGTGCAGCTGCTCCGGCACAGAAGTGCATTGCCAGAGAAAGTCCCTGGCCTCTGTGCCTGCCGGCATTCCTACCACAACCAGAGTGCTGTACCTGCACGTGAACGAGATCACCAAGTTCGAGCCCGGCGTGTTCGACAGACTGGTCAATCTCCAGCAGCTGTACCTCGGCGGCAATCAGCTTTCTGCTCTGCCCGATGGGGTGTTCGATAGGCTGACCCAGCTGACCAGACTGGACCTGTATAACAATCAGCTGACCGTGCTGCCAGCCGGCGTTTTCGATCGGCTCGTGAATCTCCAGACTCTGGACCTGCACAACAACCAGTTGAAGTCTATCCCCAGAGGGGCCTTCGACAACCTGAAGTCTCTGACCCACATCTGGCTGTTCGGCAACCCCTGGGATTGCGCCTGTAGCGACATCCTGTATCTGTCTGGCTGGCTGGGACAGCACGCCGGCAAAGAACAAGGACAGGCTGTGTGCAGCGGCACCAATACTCCAGTCAGAGCCGTGACCGAGGCCAGCACAAGCCCTTCTAAATGCCCT (SEQ ID NO: 25). However, it should be noted that a targeting domain capable of binding to CD5 is expected to function.In some embodiments, chimeric targeting polypeptides and lipid bilayer particles comprising such polypeptides are sufficient for delivering the contents of the lipid bilayer particles (e.g., cargo entities) to recipient cells (e.g., lymphocytes) by CD5 binding alone.

[0137] In some embodiments, the targeting chimeric polypeptide comprises a secretion signal. In some embodiments, the secretion signal has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the amino acid sequence METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 38). In some embodiments, the secretion signal is encoded by a polynucleotide having a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the nucleic acid sequence ATGGAAACGGACACCCTGCTGCTGTGGGTGCTGTTGTTGTGGGTGCCAGGATCTACAGGCGAC (SEQ ID NO: 39).

[0138] In some embodiments, the targeting chimeric polypeptide comprises a FLAG tag (such as a 3×FLAG tag). In some embodiments, the FLAG tag has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 40). In some embodiments, the secretion signal is encoded by a polynucleotide having a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the nucleic acid sequence GATTACAAGGACCACGATGGCGACTATAAGGATCACGACATCGACTACAAGGACGATGACGACAAG (SEQ ID NO: 41).

[0139] Fusion factor entity polypeptide Without wishing to be bound by any particular theory, it is proposed that a fusion factor entity polypeptide as described herein mediates the cellular entry of engineered lipid bilayer particles displaying such a fusion factor entity polypeptide. Certain fusion factor entity polypeptides may mediate cellular entry in the absence of a targeting chimeric polypeptide. However, the present disclosure documents that a combination of a fusion factor entity polypeptide and a targeting chimeric polypeptide as described herein can achieve significant specificity, efficiency, and / or effectiveness of cargo delivery to specific target cells of interest (e.g., immune cells such as human cells and / or T cells, such as human T cells) from engineered lipid bilayer particles containing them. In some embodiments, the fusion factor entity polypeptide is characterized by its ability to mediate fusion between lipid bilayers. In some embodiments, the fusion factor entity polypeptide mediates the transduction of recipient cells.

[0140] In some embodiments, the techniques according to the present disclosure include (e.g., utilize) a fusion factor entity polypeptide. In some embodiments, the techniques according to the present disclosure include (e.g., utilize) at least one fusion factor entity polypeptide (which is typically present in multiple copies on engineered lipid bilayer particles). In some embodiments, the techniques according to the present disclosure include one or more fusion factor entity polypeptides (e.g., each of which may be present in multiple copies on engineered lipid bilayer particles).

[0141] In some embodiments, the fusion factor entity polypeptide is a naturally occurring (e.g., wild-type) polypeptide. In some embodiments, the fusion factor entity polypeptide is native to a particular production cell. In some embodiments, the fusion factor entity polypeptide is an engineered polypeptide. In some embodiments, the fusion factor entity polypeptide (e.g., an engineered fusion factor entity polypeptide) is a variant of a wild-type polypeptide and / or a native polypeptide (e.g., including one or more amino acid substitutions).

[0142] In some embodiments, the fusion factor entity polypeptide includes a secretion signal. In some embodiments, the fusion factor entity polypeptide includes a fusion factor portion. In some embodiments, the fusion factor entity polypeptide includes a transmembrane domain. In some embodiments, the fusion factor entity polypeptide includes an intra-particle portion.

[0143] In some embodiments, the fusion factor entity polypeptide consists of or includes a fusion factor portion and a transmembrane portion.

[0144] In some embodiments, the order from the N-terminus to the C-terminus of the fusion entity polypeptide is as follows: secretion signal, fusion factor portion, transmembrane portion, fusion factor intra-particle portion, or combinations thereof.

[0145] In some embodiments, the fusion factor entity polypeptide, or a fusion factor portion thereof, has an amino acid sequence that includes characteristic sequence elements and / or an amino acid sequence that shares an overall degree of sequence identity with a reference fusion factor entity polypeptide (e.g., a wild-type fusion factor entity polypeptide and / or a fusion factor entity polypeptide). In some embodiments, the fusion factor entity polypeptide is a variant of such a reference fusion factor entity polypeptide. In some embodiments, the fusion factor entity polypeptide includes one or more modifications such as glycosylation, lipidation, phosphorylation, and the like.

[0146] In some embodiments, the fusion factor entity polypeptide is a constitutive fusion factor entity polypeptide in that its fusion factor entity activity is independent of a particular stimulus or condition.

[0147] In some embodiments, the fusion factor entity polypeptide is a conditional fusion factor entity polypeptide in that its fusion factor entity activity depends on or is induced by a particular stimulus or condition (e.g., pH, temperature, radiation (e.g., nuclear, ultraviolet, visual, etc.), electrical signal, magnetic field, etc., or a combination thereof).

[0148] In some embodiments, the fusion factor entity polypeptide binds to a specific target that drives fusion. In some embodiments, the fusion factor entity polypeptide binds to low density lipoprotein (LDL). In some embodiments, the fusion factor entity polypeptide binds to a receptor displayed on the recipient cell.

[0149] In some embodiments, the fusogenic entity polypeptide comprises a fusogenic moiety. A fusogenic moiety as provided herein mediates the entry of engineered lipid bilayer particles displaying a fusogenic entity polypeptide comprising a fusogenic moiety into recipient cells. In some embodiments, the fusogenic moiety mediates the transduction of lipid bilayer particles into recipient cells. The fusogenic moiety covers a moiety that promotes fusion between lipid bilayers or a functional moiety thereof.

[0150] In some embodiments, the fusogenic moiety is displayed on the surface of the engineered lipid bilayer particle (i.e., arranged such that the fusogenic moiety is on the surface of the particle). The fusogenic moiety may be displayed such that it can interact with a target ligand on the surface of the recipient cell. The fusogenic entity may be displayed in a manner that promotes fusion between the lipid bilayer of the engineered lipid bilayer particle and the lipid bilayer of the recipient cell.

[0151] In some embodiments, the fusogenic moiety targets a specific epitope on the recipient cell, and binding to this target epitope enables or enhances the uptake, fusion, and / or functional delivery of cargo contained within the engineered lipid bilayer particle. Such target epitopes may be expressed on all cells (universal features of the cell surface), or a subset of cells, or cells that occupy a subset of possible states (e.g., activated T cells versus resting T cells).

[0152] In some embodiments, the fusogenic moiety mediates fusion between the engineered lipid bilayer particle and the target cell in a manner that does not require binding of a fusogen to the target epitope.

[0153] In some embodiments, the fusogenic moiety enhances the fusion between the engineered lipid bilayer particle and the recipient cell to a level comparable to the fusion between the engineered lipid bilayer particle and the recipient cell in the absence of the engineered lipid bilayer particle showing a fusogenic entity polypeptide comprising the fusogenic moiety.

[0154] In some embodiments, the fusion factor entity polypeptide comprises a viral fusion factor moiety. In some embodiments, the fusion factor moiety is an enveloped virus fusion factor moiety. In some embodiments, the fusion factor moiety is a viral glycoprotein.

[0155] In some embodiments, the lipid bilayer particle comprises a viral glycoprotein for assisting in the fusion of the lipid bilayer particle and a recipient cell (e.g., lymphocyte). The viral glycoprotein is a lentiviral glycoprotein, or vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowlpox virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein. In particular, in some embodiments, the glycoprotein can be selected from glycoproteins selected from measles virus glycoprotein H, measles virus glycoprotein F, or a combination thereof.

[0156] In some embodiments, the fusion factor entity polypeptide is a polypeptide derived from vesicular stomatitis virus, measles virus, sindbis virus, tupaia paramyxovirus, nipah virus, chandipura virus, rabies virus, lymphocytic choriomeningitis virus, mokola virus, ross river virus, semliki forest virus, venezuelan equine encephalitis virus, ebola virus, marburg virus, lassa virus, avian leukosis virus, caprine arthritis-encephalitis retrovirus, moloney murine leukemia virus, gibbon ape leukemia virus, feline endogenous retrovirus (RD114), human T-lymphotropic virus type 1, human herpesvirus, maedi-visna virus, SARS-CoV, SARS-CoV-2, sendai virus, respiratory syncytial virus, human parainfluenza virus type 3, human parainfluenza virus type 4, hepatitis C virus, hepatitis C virus, influenza virus, fowl pest virus, Autographa californica multiple nuclear polyhedrosis virus, baboon endogenous retrovirus, coxsackievirus, Japanese encephalitis virus, dengue virus, Zika virus, West Nile virus, yellow fever virus, tick-borne encephalitis virus, herpes simplex virus 1, hendra virus, newcastle disease virus, Epstein-Barr virus, bourbon virus, varicella-zoster virus, severe fever with thrombocytopenia syndrome virus, hantavirus, vaccinia virus, simian immunodeficiency virus, human immunodeficiency virus, junin virus, machupo virus, bas Congo virus, la Crosse virus, human cytomegalovirus, human cytomegalovirus, togoto virus, or dourine virus.

[0157] In some embodiments, the fusion factor entity polypeptide is a glycoprotein selected from, or comprising, vesicular stomatitis glycoprotein (VSV-G).

[0158] In some embodiments, the fusion factor entity polypeptide is wild-type VSV-G or comprises wild-type VSV-G. In some embodiments, wild-type VSV-G has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% identical to the amino acid sequence MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 26). In some embodiments, the wild-type VSV-G fusion factor moiety has an amino acid sequence identical to SEQ ID NO: 26.

[0159]

[0160] In some embodiments, the fusion factor entity polypeptide comprises a fusion factor moiety. In some embodiments, the fusion factor moiety is a fragment of wild-type VSV-G or comprises a fragment of wild-type VSV-G. In some embodiments, the wild-type VSV-G fusion factor moiety has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSS (SEQ ID NO: 30). In some embodiments, the wild-type VSV-G fusion factor moiety has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 30.

[0161]

[0162] In some embodiments, the fusion factor entity polypeptide is a mutant VSV-G or comprises a mutant VSV-G. In some embodiments, the mutant VSV-G has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 28). In some embodiments, the mutant VSV-G fusion factor entity polypeptide has an amino acid sequence that is identical to SEQ ID NO: 28.

[0163]

[0164] In some embodiments, the fusion factor entity polypeptide comprises a fusion factor moiety. In some embodiments, the fusion factor moiety is or comprises a fragment of mutant VSV-G. In some embodiments, the mutant VSV-G fusion factor moiety has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSS (SEQ ID NO: 32). In some embodiments, the mutant VSV-G fusion factor moiety has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 32.

[0165]

[0166] In some embodiments, the fusion factor entity polypeptide comprises an intra-particle portion. In some embodiments, the intra-particle polypeptide portion has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence KLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 36). In some embodiments, the intra-particle polypeptide portion is encoded by a polynucleotide having a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence aaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaag (SEQ ID NO: 37).

[0167] In some embodiments, the fusion factor entity polypeptide is a measles virus polypeptide, such as the measles virus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is the measles virus glycoprotein H and / or F, or comprises them.

[0168] In some embodiments, the fusion factor entity polypeptide is a Sindbis virus polypeptide, such as the Sindbis virus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is the Sindbis virus glycoprotein E1 and / or E2, or comprises them.

[0169] In some embodiments, the fusion factor entity polypeptide is a Tupaia paramyxovirus polypeptide, such as the Tupaia paramyxovirus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is the Tupaia paramyxovirus glycoprotein H and / or F, or comprises them.

[0170] In some embodiments, the fusion factor entity polypeptide is or comprises a Nipah virus polypeptide such as a Nipah virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises Nipah virus glycoprotein G and / or F.

[0171] In some embodiments, the fusion factor entity polypeptide is or comprises a Chandipura virus polypeptide such as a Chandipura virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises Chandipura virus glycoprotein G.

[0172] In some embodiments, the fusion factor entity polypeptide is or comprises a rabies virus polypeptide such as a rabies virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises rabies virus glycoprotein G.

[0173] In some embodiments, the fusion factor entity polypeptide is or comprises a lymphocytic choriomeningitis virus polypeptide such as a lymphocytic choriomeningitis virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises lymphocytic choriomeningitis virus glycoproteins GP-1 and / or GP-2.

[0174] In some embodiments, the fusion factor entity polypeptide is or comprises a Mokola virus polypeptide such as a Mokola virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises Mokola virus glycoprotein G.

[0175] In some embodiments, the fusion factor entity polypeptide is a Ross River virus polypeptide, such as the Ross River virus glycoprotein, or includes them. In some embodiments, the fusion factor entity polypeptide is the Ross River virus glycoproteins E1 and / or E2, or includes them.

[0176] In some embodiments, the fusion factor entity polypeptide is a Semliki Forest virus polypeptide, such as the Semliki Forest virus glycoprotein, or includes them. In some embodiments, the fusion factor entity polypeptide is the Semliki Forest virus glycoproteins E1 and / or E2, or includes them.

[0177] In some embodiments, the fusion factor entity polypeptide is a Venezuelan equine encephalitis virus polypeptide, such as the Venezuelan equine encephalitis virus glycoprotein, or includes the Venezuelan equine encephalitis virus polypeptide. In some embodiments, the fusion factor entity polypeptide is the Venezuelan equine encephalitis virus glycoproteins E1 and / or E2, or includes them.

[0178] In some embodiments, the fusion factor entity polypeptide is an Ebola virus polypeptide, such as the Ebola virus glycoprotein, or includes them. In some embodiments, the fusion factor entity polypeptide is the Ebola virus glycoprotein GP, or includes it.

[0179] In some embodiments, the fusion factor entity polypeptide is a Marburg virus polypeptide, such as the Marburg virus glycoprotein, or includes them. In some embodiments, the fusion factor entity polypeptide is the Marburg virus glycoprotein GP, or includes it.

[0180] In some embodiments, the fusion factor entity polypeptide is a Lassa virus polypeptide such as the Lassa virus glycoprotein, or comprises a Lassa virus polypeptide. In some embodiments, the fusion factor entity polypeptide is the Lassa virus glycoprotein GPC or comprises it.

[0181] In some embodiments, the fusion factor entity polypeptide is an avian leukosis virus polypeptide such as the avian leukosis virus glycoprotein, or comprises an avian leukosis virus polypeptide. In some embodiments, the fusion factor entity polypeptide is the avian leukosis virus envelope glycoprotein or comprises it.

[0182] In some embodiments, the fusion factor entity polypeptide is a goat retrovirus polypeptide such as the goat retrovirus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is the goat retrovirus envelope glycoprotein or comprises it.

[0183] In some embodiments, the fusion factor entity polypeptide is a Moloney leukemia virus polypeptide, e.g., the Moloney leukemia virus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is the murine leukemia virus envelope glycoprotein or comprises them.

[0184] In some embodiments, the fusion factor entity polypeptide is a simian leukemia virus polypeptide, e.g., the simian leukemia virus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is the simian leukemia virus envelope glycoprotein or comprises them.

[0185] In some embodiments, the fusogenic entity polypeptide is a feline endogenous retrovirus polypeptide such as a feline endogenous retrovirus glycoprotein, or comprises them. In some embodiments, the fusogenic entity polypeptide is an RD114 glycoprotein, or comprises an RD114 glycoprotein.

[0186] In some embodiments, the fusogenic entity polypeptide is a human T-lymphotropic virus type 1 polypeptide such as a human T-lymphotropic virus type 1 glycoprotein, or comprises them. In some embodiments, the fusogenic entity polypeptide is a human T-lymphotropic virus type 1 glycoprotein SU and / or TM, or comprises them. In some embodiments, the fusogenic entity polypeptide is a human T-lymphotropic virus type 1 glycoprotein gp46, or comprises it.

[0187] In some embodiments, the fusogenic entity polypeptide is a human foamy virus polypeptide such as a human foamy virus glycoprotein, or comprises them. In some embodiments, the fusogenic entity polypeptide is a human foamy virus envelope glycoprotein, or comprises them.

[0188] In some embodiments, the fusogenic entity polypeptide is a maedi-visna virus polypeptide such as a maedi-visna virus glycoprotein, or comprises it. In some embodiments, the fusogenic entity polypeptide is a maedi-visna virus envelope glycoprotein, or comprises it.

[0189] In some embodiments, the fusogenic entity polypeptide is a SARS-CoV polypeptide (e.g., SARS-CoV 2) such as a SARS-CoV glycoprotein, or comprises it. In some embodiments, the pathogen entity polypeptide is a SARS-CoV spike glycoprotein (S), or comprises it.

[0190] In some embodiments, the fusion factor entity polypeptide is a Sendai virus polypeptide such as the Sendai virus glycoprotein, or comprises a Sendai virus polypeptide. In some embodiments, the fusion factor entity polypeptide is, or comprises, the Sendai virus glycoproteins HN and / or F.

[0191] In some embodiments, the fusion factor entity polypeptide is a respiratory syncytial virus polypeptide such as the respiratory syncytial virus glycoprotein, or comprises a respiratory syncytial virus polypeptide. In some embodiments, the fusion factor entity polypeptide is, or comprises, the respiratory syncytial virus glycoproteins G and / or F.

[0192] In some embodiments, the fusion factor entity polypeptide is, or consists of, human parainfluenza virus type 3 and / or type 4 polypeptides, for example, the respiratory human parainfluenza virus type 3 and / or type 4 glycoproteins. In some embodiments, the fusion factor entity polypeptide is, or comprises, the human parainfluenza virus type 3 and / or type 4 glycoproteins HN and / or F.

[0193] In some embodiments, the fusion factor entity polypeptide is a hepatitis C virus polypeptide such as the hepatitis C virus glycoprotein, or comprises such polypeptides. In some embodiments, the fusion factor entity polypeptide is, or comprises, the hepatitis C virus glycoproteins E1 and / or E2.

[0194] In some embodiments, the fusion factor entity polypeptide is an influenza virus polypeptide such as the influenza virus glycoprotein, or comprises an influenza virus polypeptide. In some embodiments, the fusion factor entity polypeptide is, or comprises, the influenza virus glycoproteins HA and / or NA.

[0195] In some embodiments, the pathogen entity polypeptide is or comprises a poultry pest virus polypeptide such as a poultry pest virus glycoprotein. In some embodiments, the pathogen entity polypeptide is or comprises the poultry pest virus glycoprotein HA.

[0196] In some embodiments, the fusion factor entity polypeptide is or comprises an Autographa californica multiple nucleopolyhedrovirus polypeptide, such as an Autographa californica multiple nucleopolyhedrovirus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises the Autographa californica multiple nucleopolyhedrovirus glycoprotein gp64.

[0197] In some embodiments, the fusion factor entity polypeptide is or comprises a baboon endogenous retrovirus polypeptide, or comprises a baboon endogenous retrovirus polypeptide such as a baboon endogenous retrovirus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises the baboon endogenous retrovirus glycoprotein envelope.

[0198] In some embodiments, the fusion factor entity polypeptide is or comprises a cocal virus polypeptide, such as the cocal virus glycoprotein (G).

[0199] In some embodiments, the fusion factor entity polypeptide is or comprises a Japanese encephalitis virus polypeptide, such as a Japanese encephalitis virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or comprises the Japanese encephalitis virus glycoprotein E.

[0200] In some embodiments, the fusion factor entity polypeptide is a dengue virus polypeptide, such as a dengue virus glycoprotein, or comprises a dengue virus polypeptide. In some embodiments, the fusion factor entity polypeptide is or comprises dengue virus glycoprotein E.

[0201] In some embodiments, the fusion factor entity polypeptide is a chikungunya virus polypeptide, such as a chikungunya virus glycoprotein, or comprises such polypeptides. In some embodiments, the fusion factor entity polypeptide is or comprises chikungunya virus glycoprotein E.

[0202] In some embodiments, the fusion factor entity polypeptide is a West Nile virus polypeptide, such as a West Nile virus glycoprotein, or comprises such polypeptides. In some embodiments, the fusion factor entity polypeptide is or comprises West Nile virus glycoprotein E.

[0203] In some embodiments, the fusion factor entity polypeptide is a yellow fever virus polypeptide, such as a yellow fever virus glycoprotein, or comprises such polypeptides. In some embodiments, the fusion factor entity polypeptide is or comprises yellow fever virus glycoprotein E.

[0204] In some embodiments, the fusion factor entity polypeptide is a tick-borne encephalitis virus polypeptide, such as a tick-borne encephalitis virus glycoprotein, or comprises such polypeptides. In some embodiments, the fusion factor entity polypeptide is or comprises tick-borne encephalitis virus glycoprotein E.

[0205] In some embodiments, the fusion factor entity polypeptide is or includes a herpes simplex virus polypeptide, such as a herpes simplex virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or includes herpes simplex virus glycoproteins HSV-1 gB, HSV-1 gH, HSV-1 gL, and / or HSV-1 gD.

[0206] In some embodiments, the fusion factor entity polypeptide is or includes a Hendra virus polypeptide, such as a Hendra virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or includes Hendra virus glycoproteins G and / or F.

[0207] In some embodiments, the fusion factor entity polypeptide is or includes a Newcastle disease virus polypeptide, such as a Newcastle disease virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or includes Newcastle disease virus glycoproteins F1 and / or F2.

[0208] In some embodiments, the fusion factor entity polypeptide is or includes an Epstein-Barr virus polypeptide, such as an Epstein-Barr virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or includes Epstein-Barr virus glycoproteins gB, gH, and / or gL. In some embodiments, the fusion factor entity polypeptide is or includes Epstein-Barr virus glycoprotein gp42.

[0209] In some embodiments, the fusion factor entity polypeptide is or includes a Bourbon virus polypeptide, such as a Bourbon virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is or includes Bourbon virus glycoprotein Gp.

[0210] In some embodiments, the fusion factor entity polypeptide is, or comprises, a varicella-zoster virus polypeptide, such as a varicella-zoster virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is, or comprises, varicella-zoster virus glycoproteins gB, gH, gE, and / or gL.

[0211] In some embodiments, the fusion factor entity polypeptide is, or comprises, a severe fever with thrombocytopenia syndrome virus polypeptide, such as a severe fever with thrombocytopenia syndrome virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is, or comprises, severe fever with thrombocytopenia syndrome virus glycoproteins gB, gH, and / or gL.

[0212] In some embodiments, the fusion factor entity polypeptide is, or comprises, a hantavirus polypeptide, such as a hantavirus glycoprotein. In some embodiments, the fusion factor entity polypeptide is, or comprises, hantavirus glycoproteins Gn and / or Gc.

[0213] In some embodiments, the fusion factor entity polypeptide is, or comprises, a vaccinia virus polypeptide, such as a vaccinia virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is, or comprises, vaccinia virus glycoproteins A28, A21, A16, F9, G9, G3, H2, J5, L5, L1, A33, A34, B5, and / or O3.

[0214] In some embodiments, the fusion factor entity polypeptide is, or comprises, a simian immunodeficiency virus polypeptide, such as a simian immunodeficiency virus glycoprotein. In some embodiments, the fusion factor entity polypeptide is, or comprises, the simian immunodeficiency virus Env glycoprotein.

[0215] In some embodiments, the fusogenic entity polypeptide is, or comprises, a human immunodeficiency virus (e.g., HIV-1) polypeptide, such as a human immunodeficiency virus glycoprotein. In some embodiments, the fusogenic entity polypeptide is, or comprises, a human immunodeficiency virus Env glycoprotein.

[0216] In some embodiments, the fusogenic entity polypeptide is, or comprises, a Junin virus polypeptide, such as a Junin virus glycoprotein. In some embodiments, the fusogenic entity polypeptide is, or comprises, the Junin virus glycoprotein complex GPC.

[0217] In some embodiments, the fusogenic entity polypeptide is, or comprises, a Machupo virus polypeptide, such as a Machupo virus glycoprotein. In some embodiments, the fusogenic entity polypeptide is, or comprises, the Machupo virus glycoprotein complex GPC.

[0218] In some embodiments, the fusogenic entity polypeptide is, or comprises, a Bass-Congo virus polypeptide, such as a Bass-Congo virus glycoprotein. In some embodiments, the fusogenic entity polypeptide is, or comprises, the Bass-Congo virus glycoprotein G.

[0219] In some embodiments, the fusogenic entity polypeptide is, or comprises, a La Crosse virus polypeptide, such as a La Crosse virus glycoprotein. In some embodiments, the fusogenic entity polypeptide is, or comprises, the La Crosse virus glycoproteins Gc and / or Gn.

[0220] In some embodiments, the fusion factor entity polypeptide is a human cytomegalovirus polypeptide such as a human cytomegalovirus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is, or comprises, human cytomegalovirus glycoproteins gH, gL, gO, UL128, UL130, and / or UL131A.

[0221] In some embodiments, the fusion factor entity polypeptide is a togavirus polypeptide, such as a togavirus glycoprotein, or comprises them. In some embodiments, the fusion factor entity polypeptide is, or comprises, the togavirus glycoprotein Gp.

[0222] In some embodiments, the fusion factor entity polypeptide is a dhori virus polypeptide, such as a dhori virus glycoprotein, or comprises a dhori virus polypeptide. In some embodiments, the fusion factor entity polypeptide is, or comprises, the dhori virus glycoprotein Gp.

[0223] In some embodiments, the fusion factor entity polypeptide comprises a non-viral fusion factor moiety.

[0224] In some embodiments, the fusion factor entity polypeptide comprises a human fusion factor moiety.

[0225] In some embodiments, the fusogenic entity polypeptide is a human fusogenic moiety selected from the group consisting of Syncytin-1, Syncytin-2, CD9, CD81, a biomarker, CD200 (OX-2G), DC-STAMP, OC-STAMP, E-cadherin (CADH1), cadherin 11 (CAD11), matrix metalloproteinase-9, zonula occludens-1 (ZO-1), myomaker, annexin A1, annexin A5, CD44, P2X purinoreceptor 7, IZUMO1, Juno, StartD7, receptor-like 1, related protein 4, CD63, connexin 43 (Cx43), CD36, MFR, tumor-related member 1, GLPR1-like protein 1, related protein 43, ERVV-1, ERVV-2, ERVH48-1, ERVMER34-1, ERV3-1, and ERVK13-1, or comprises them.

[0226] In some embodiments, the fusogenic entity polypeptide is a human fusogenic moiety selected from the group consisting of Syncytin-1, Syncytin-2, CD9, CD81, a biomarker, CD200 (OX-2G), DC-STAMP, OC-STAMP, E-cadherin (CADH1), cadherin 11 (CAD11), matrix metalloproteinase-9, zonula occludens-1 (ZO-1), myomaker, annexin A1, annexin A5, CD44, P2X purinoreceptor 7, IZUMO1, Juno, StartD7, receptor-like 1, related protein 4, CD63, connexin 43 (Cx43), CD36, MFR, tumor-related member 1, GLPR1-like protein 1, and related protein 43, or comprises them.

[0227] Transmembrane domain In some embodiments, the polypeptides described herein (e.g., targeting chimeric polypeptides, fusion polypeptides, cargo loading polypeptides, etc.) include a transmembrane domain (TMD). In some embodiments, the transmembrane domain enables the targeting domain to be displayed on the surface of the lipid bilayer particle. In some embodiments, the membrane association moiety positions the affinity moiety on the surface of the lipid bilayer particle such that it can bind to a recipient cell surface epitope. As used herein, the term "transmembrane domain" covers any "membrane association moiety" that can associate with a membrane (e.g., a lipid bilayer membrane). One of ordinary skill in the art will understand that in some embodiments, the "transmembrane domain" is a stretch of amino acids that together cause association with the membrane. In some embodiments, the transmembrane domain spans the membrane. In some embodiments, the transmembrane domain does not span the membrane. In some embodiments, the transmembrane domain associates with a membrane (e.g., a lipid bilayer membrane). In some embodiments, the transmembrane domain is located at the C-terminus of the polypeptide. In some embodiments, the transmembrane domain is characterized by a length of about 10 amino acids to about 300 amino acids. In some embodiments, the transmembrane domain is a heterologous transmembrane domain (e.g., relative to another portion of the polypeptide).

[0228] In some embodiments, the transmembrane domain is a viral transmembrane domain. In some embodiments, the transmembrane domain is a viral envelope transmembrane domain. In some embodiments, the transmembrane domain is a non-viral transmembrane domain. In some embodiments, the transmembrane domain is an engineered transmembrane domain.

[0229] Transmembrane domains are known in the art. Transmembrane domains (TMDs) mainly consist of non-polar amino acid residues and can cross a bilayer membrane once (single pass) or several times. TMDs usually consist of α-helices. Peptide bonds are polar and contain internal hydrogen bonds formed between the carbonyl oxygen atom and the amide nitrogen atom and may be hydrated. In the lipid bilayer where water is essentially excluded, peptides usually adopt an α-helical conformation to maximize internal hydrogen bonds. Typically, the length of the helix of 18 to 21 amino acid residues is sufficient to cover the normal width of the lipid bilayer. TMDs with an extracellular N-terminus and an intracellular C-terminus orientation are classified as type I TMDs, and TMDs with an extracellular C-terminus and an intracellular N-terminus orientation are classified as type II TMDs. In some of the disclosed extracellular embodiments, they are classified as type I or, if cytoplasmic, type II. In some embodiments, the transmembrane domain is a single-pass, type I transmembrane domain containing 18 to 21 amino acids, and at least about 90% of the amino acids are nonpolar. Suitable TMDs for the disclosed fusion proteins can include, but are not limited to, transmembrane domains of cell receptors such as the transmembrane domain of the platelet-derived growth factor receptor (PDGFR).In some embodiments, the transmembrane domain is the transmembrane domain of vesicular stomatitis virus, measles virus, sindbis virus, tupaia paramyxovirus, nipah virus, chandipura virus, rabies virus, lymphocytic choriomeningitis virus, mokola virus, ross river virus, semliki forest virus, venezuelan equine encephalitis virus, ebola virus, marburg virus, lassa virus, avian leukosis virus, jaagsiekte sheep retrovirus, moloney murine leukemia virus, gibbon ape leukemia virus, feline endogenous retrovirus (RD114), human T-lymphotropic virus type 1, human foamy virus, maedi visna virus, SARS-CoV, SARS-CoV-2, sendai virus, respiratory syncytial virus, human parainfluenza virus type 3, human parainfluenza virus type 4, hepatitis C virus, hepatitis C virus, influenza virus, fowl pest virus, Autographa californica multiple nucleopolyhedro virus, baboon endogenous retrovirus, kokobera virus, japanese encephalitis virus, dengue virus, zika virus, west nile virus, yellow fever virus, tick-borne encephalitis virus, herpes simplex virus 1, hendra virus, newcastle disease virus, epstein-barr virus, bourbon virus, varicella-zoster virus, severe fever with thrombocytopenia syndrome virus, hantavirus, vaccinia virus, simian immunodeficiency virus, human immunodeficiency virus, junin virus, machupo virus, bas Congo virus, la Crosse virus, human cytomegalovirus, human cytomegalovirus, togoto virus, or dhori virus.

[0230] In some embodiments, the PDGFR transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18 (AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR), which can be encoded by the nucleotide sequence GCCGTCGGCCAGGACACCCAAGAAGTGATCGTCGTCCCTCACAGCCTGCCTTTCAAGGTGGTGGTCATCAGCGCCATTCTGGCCCTGGTGGTGCTGACCATCATCAGCCTGATCATCCTGATTATGCTGTGGCAGAAGAAGCCCAGA (SEQ ID NO: 19).

[0231] The TMD may be directly linked to the targeting domain (e.g., scFv), or the TMD may be linked via a linker. In some embodiments, the linker that links the TMD and the targeting domain is (GGGGS) n and consists of the amino acid sequence, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more. In some embodiments, the linker that links the TMD and the targeting peptide comprises: (1) SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; or (2) any one of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 and an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0232] Thus, in some embodiments, the transmembrane domain may comprise the variant amino acid sequence AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 18), or a functional fragment thereof, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18.

[0233] In some embodiments, the fusion factor entity polypeptide comprises a transmembrane domain. In some embodiments, the transmembrane domain is or comprises the wild-type VSV-G transmembrane portion or a fragment thereof. In some embodiments, the fusion factor entity polypeptide transmembrane domain may comprise IASFFFIIGLIIGLFLVLRVGIHLCI (SEQ ID NO: 22) encoded by the nucleotide sequence attgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcatt (SEQ ID NO: 23). In some embodiments, the wild-type VSV-G fusion factor transmembrane portion has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the wild-type VSV-G fusogen transmembrane portion has an amino acid sequence identical to SEQ ID NO: 22.

[0234] In some embodiments, the transmembrane domain of a non-human transmembrane domain. In some embodiments, the transmembrane domain of a human transmembrane domain.

[0235] In some embodiments, the transmembrane domain is integrated into the membrane of the lipid bilayer particles at a high copy number.

[0236] Secretion signal In some embodiments, the polypeptides disclosed herein (e.g., targeting chimeric polypeptides and / or fusion factor entity polypeptides) include a secretion signal that is functional, for example, in mammalian cells. In some embodiments, the secretion signal utilized is a heterologous secretion signal. In some embodiments, the heterologous secretion signal comprises or consists of a non-human secretion signal. In some embodiments, the heterologous secretion signal comprises or consists of a viral secretion signal. In some embodiments, the secretion signal is characterized by a length of about 10 to about 40 amino acids, such as about 20 to about 30 amino acids. In some embodiments, the secretion signal is located at the N-terminus of the fusion factor entity polypeptide described herein. In some embodiments, the secretion signal preferably enables transport of the fusion factor entity polypeptide with which it is associated to a defined cellular compartment, preferably the cell surface, endoplasmic reticulum (ER), Golgi apparatus, or endosome-lysosome compartment.

[0237] In some embodiments, the secretion signal has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence MKCLLYLAFLFIGVNC (SEQ ID NO: 34). In some embodiments, the secretion signal is encoded by a polynucleotide having a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identical to the nucleic acid sequence atgaagtgccttttgtacttagcctttttattcattggggtgaattgc (SEQ ID NO: 35).

[0238] Affinity flag In some embodiments, the polypeptides disclosed herein (e.g., targeting chimeric polypeptides and / or fusion factor entity polypeptides) include a FLAG tag. In some embodiments, the FLAG tag utilized is a heterologous secretion tag. In some embodiments, the heterologous FLAG tag comprises or consists of a non-human FLAG tag. In some embodiments, the heterologous FLAG tag comprises or consists of a viral FLAG tag. In some embodiments, the FLAG tag signal is characterized by a length of about 15 to 30 amino acids. In some embodiments, the FLAG tag is positioned between the secretion signal sequence and the targeting domain sequence within the targeting chimeric polypeptides described herein. In some embodiments, the polypeptides described herein do not include a FLAG tag or other tag.

[0239] Cargo entity In some embodiments, the disclosed techniques include a cargo entity. In some embodiments, a lipid bilayer particle or a population of lipid bilayer particles includes a cargo entity. In some embodiments, the disclosed techniques include one or more cargo entities such as multiple cargo entities (e.g., a first cargo entity, a second cargo entity, etc., or combinations thereof). The cargo entities described herein can be of any chemical class, such as polypeptides, nucleic acids, saccharides, lipids, small entities, and combinations thereof.

[0240] In some embodiments, the cargo entity is part of a targeting chimeric polypeptide, a fusion factor entity polypeptide, or both. In some embodiments, the cargo entity binds to a targeting chimeric polypeptide, a fusion factor entity polypeptide, or both.

[0241] In some embodiments, the cargo entity is part of a polypeptide that is different from each of the targeting chimeric polypeptide and the fusion factor entity polypeptide. In some embodiments, the cargo entity is linked to a cargo loading domain. In some embodiments, the cargo loading domain comprises an abscisic acid-insensitive 1 (ABI1) sequence.

[0242] In some embodiments, the cargo loading domain is linked to the cargo entity and co-expressed with an ABA binding sequence (e.g., comprising a pyrabactin resistance 1-like (PYL1) sequence) and abscisic acid (ABA).

[0243] In some embodiments, the cargo loading domain is linked to the cargo entity.

[0244] In some embodiments, the cargo loading domain is linked to the cargo entity and co-expressed with an ABA binding sequence (e.g., comprising a pyrabactin resistance 1-like (PYL1) sequence).

[0245] In some embodiments, the cargo entity is a cytoplasmic cargo entity or a membrane-bound cargo entity.

[0246] In some embodiments, the cargo loading domain comprising the ABI1 sequence may be fused to a membrane polypeptide (i.e., a membrane-bound polypeptide). In other words, in some embodiments, the cargo entity may be a membrane polypeptide. Similarly, in some embodiments, the cargo loading domain comprising the abscisic acid-insensitive 1 (ABI1) sequence may be fused to a cytoplasmic polypeptide (i.e., a non-membrane-bound polypeptide). In other words, in some embodiments, the cargo entity may be a cytoplasmic polypeptide.

[0247] The loading system may be used to deliver a single cargo (or a single type of cargo) to a desired recipient cell, or may be used to deliver multiple cargos (e.g., different polypeptides) to a desired cell. For purposes of embodiments that include multiple different cargos, the disclosed loading system may be used to control the ratios of the various cargos that are loaded into lipid bilayer particles and then delivered to a desired recipient cell.

[0248] In some embodiments, the cargo entity is a polypeptide cargo entity. In some embodiments, the cargo entity is a cytoplasmic cargo molecule, where the cytoplasmic cargo entity can be a peptide, polypeptide, or protein of interest to be delivered to the recipient cell, such as an enzyme, a therapeutic agent (e.g., an antibody, an inhibitor, an agonist, an antagonist), or a fluorescent protein. In some embodiments, the cytoplasmic polypeptide cargo entity can be selected from any one or more of a base editor, a prime editor, a TALEN, a ZFN, a kinase, a kinase inhibitor, an activator or inhibitor of receptor signaling, an intrabody, a chromatin-modifying synthetic transcription factor, a native transcription factor, and mutants thereof. In some embodiments, the cytoplasmic polypeptide cargo entity is a CRISPR enzyme, such as a type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is a Cas9 protein (e.g., a naturally occurring bacterial Cas9, as well as any chimeric, mutant, homolog, or ortholog). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified variants thereof.

[0249] In some embodiments, the cargo entity is a nucleic acid cargo entity. In some embodiments, the nucleic acid cargo entity is a synthetic nucleic acid cargo entity. In some embodiments, the nucleic acid cargo entity comprises chemically modified nucleotides. Without wishing to be bound by any particular theory, synthetic nucleic acid cargo entities and / or nucleic acid cargo entities comprising chemically modified nucleotides provide stability to the nucleic acid cargo. This may be particularly useful for guide RNAs (e.g., for use in Cas gRNA complexes).

[0250] In some embodiments, the synthetic nucleic acid is an ASO or a chemically modified RNA.

[0251] In some embodiments, the cargo entity can be a nucleic acid (e.g., DNA or RNA) or other molecule. Nucleic acid cargo entities can include, but are not limited to, DNA encoding a polypeptide of interest, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, and combinations thereof. Other potential cargo entities include viruses and non-viral vectors (e.g., nucleocapsids) that are expressed intracellularly (or can be delivered to the cytoplasm of a cell), as well as ribonucleoprotein complexes such as CRISPR-type entities and endogenous complexes such as DICER or RISC bound to natural or synthetic RNAs such as miRNA, shRNA, etc. (but are not limited thereto). In some embodiments, a cargo that can be expressed intracellularly or physically delivered inside a cell can be (genetically or synthetically) fused to an ABI protein / peptide or an ABA binding sequence, which is explicitly contemplated herein. Genetic fusions can be achieved by expressing two or more components of a chimeric polypeptide or peptide in a lipid bilayer particle (e.g., a CDMP such as an EV) producing cell. Alternatively, a cargo entity fused to a loading domain is delivered to a lipid bilayer particle (e.g., a CDMP such as an EV) producing cell, leading to subsequent incorporation into the lipid bilayer particle. Alternatively, a cargo entity fused to a loading domain is inserted into a lipid bilayer membrane particle after secretion from the producing cell.

[0252] Additionally or alternatively, in some embodiments, the cargo entity can be a membrane-bound cargo molecule. In some embodiments, the membrane-bound cargo entity can include (i) a targeting peptide / protein and (ii) a transmembrane domain. Exemplary targeting peptides include any antibody fragment or antigen-binding fragment, such as Fab, Fab’, and F(ab’) 2 , Fd, scFv, single-chain antibody, disulfide-bonded Fv (sdFv), and nanobody, but are not limited thereto. The targeting peptide (e.g., scFv) can bind to a target of interest on a specific cell type such as a T cell. In some embodiments, the targeting peptide is, for example, Fab, Fab’, and F(ab’) that bind to CD2 or another target associated with T cells 2 , Fd, scFv, single-chain antibody, disulfide-bonded Fv (sdFv), de novo designed binding molecule, affibody, DARPIN, or nanobody. For example, other suitable targets include, but are not limited to, CD3, CD4, CD8, CD25, CD127, CD39, CD45RA, CTLA-4, PD-1

[0253] In some embodiments, the lipid bilayer particle targeting system according to any one of the above embodiments further includes abscisic acid (ABA), wherein the cargo loading domain of the chimeric polypeptide and / or the ABA binding sequence of the second chimeric polypeptide can bind to ABA, resulting in dimerization of the chimeric polypeptide and the second chimeric polypeptide

[0254] In some embodiments, the lipid bilayer particles comprise or consist of a viral nucleocapsid or derivative thereof, synthetic nucleic acid, transcription factor, recombinant enzyme, base editor, prime editor, nuclease (e.g., TALEN, ZFN, etc.), kinase, kinase inhibitor, activator or inhibitor of receptor signaling, intrabod, chromatin-modifying synthetic transcription factor, natural transcription factor, CRISPR-Cas family protein, DNA molecule, RNA molecule, ribonucleoprotein complex, or antisense oligonucleotide.

[0255] Also disclosed herein are nucleic acids encoding the chimeric polypeptides disclosed herein. For example, in some embodiments, the loading domain of the cargo chimeric loading polypeptide of the chimeric loading polypeptide isIt can be encoded by [[SEQ ID NO:5]] (SEQ ID NO:5), and has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83% identity with SEQ ID NO:5. ATGACCAGAGTGCCCCTGTACGGCTTCACCAGCATTTGTGGCAGACGGCCCGAAATGGAAGCCGCCGTGTCTACAATCCCCAGATTCCTCCAGAGCAGCAGCGGCTCCATGCTGGACGGCAGATTCGATCCTCAGAGCGCCGCTCACTTCTTCGGCGTGTACGATGGACATGGCGGAAGCCAGGTGGCCAACTACTGCCGCGAAAGAATGCATCTGGCCCTGGCCGAGGAAATCGCCAAAGAAAAGCCCATGCTGTGCGACGGCGACACCTGGCTGGAAAAGTGGAAGAAGGCCCTGTTCAACAGCTTCCTGAGAGTGGACAGCGAGATCGAGAGCGTGGCCCCTGAAACAGTGGGCAGCACATCTGTGGTGGCCGTGGTGTTTCCCAGCCACATCTTCGTGGCTAACTGCGGCGATAGCAGAGCCGTGCTGTGCAGAGGAAAAACAGCCCTGCCTCTGTCCGTGGACCACAAGCCTGATAGAGAGGATGAGGCCGCCAGAATTGAAGCCGCTGGCGGCAAAGTGATCCAGTGGAATGGCGCTAGAGTGTTCGGCGTGCTGGCCATGAGTAGATCCATCGGCGATAGATACCTGAAGCCTAGCATCATCCCCGATCCTGAAGTGACCGCCGTGAAGAGAGTGAAAGAGGACGACTGCCTGATCCTGGCCTCTGACGGTGTCTGGGACGTGATGACAGATGAAGAGGCCTGCGAGATGGCCCGGAAGAGAATCCTGCTGTGGCACAAGAAAAACGCCGTGGCCGGGGATGCTTCTCTGCTGGCTGACGAGAGAAGAAAAGAGGGCAAAGACCCCGCTGCCATGTCTGCCGCCGAGTACCTGTCTAAGCTGGCCATCCAGAGAGGCAGCAAGGACAACATCAGCGTGGTGGTCGTGGACCTGAAAA variant nucleic acid sequence having at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity, or a functional fragment thereof. Generally, a fragment is considered a functional fragment if it encodes a protein or peptide that increases the active loading of cargo entities into lipid bilayer particles, binds to an ABI1 binding protein, or enables a combination thereof.,

[0256] In some embodiments, the ABA binding sequence of the second chimeric peptide can be encoded by a variant nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO:1 or a functional fragment thereof. Generally, a fragment is considered a functional fragment if it encodes a protein or peptide that increases the active loading of cargo entities into lipid bilayer particles, binds to ABI1 or a variant or fragment thereof, or enables combinations thereof.

[0257] In some embodiments, any linker of the disclosed peptides can be encoded by any one of SEQ ID NO: 9 (ACTAGTGGCGGCGGAGGCAGCGGAGGCGGATCTGGCGGAGGATCT), SEQ ID NO: 11 (ACGCGTGGCGGCGGAGGCAGCGGAGGCGGATCTGGCGGAGGATCT), or SEQ ID NO: 13 (GGCGGCGGAGGAAGTGGCGGCGGATCTGGCGGAGGATCTACCGGT), or a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity with SEQ ID NO: 9, 11, or 13.

[0258] In some embodiments, the chimeric targeting polypeptide can optionally include a first cargo entity fused thereto. The first cargo entity is not particularly limited and can be any cargo entity disclosed herein. For example, in some embodiments, the first entity can be an ABA-binding sequence comprising a pyrabactin resistance 1-like (PYL1) sequence. In some embodiments, the PYL1 sequence comprises residues 33-209 of wild-type PYL1. In some embodiments, the PYL1 sequence is MGGGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 2), TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 3), or a variant amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to either SEQ ID NO: 2 or SEQ ID NO: 3, or a functional fragment of SEQ ID NO: 2, SEQ ID NO: 3, or a variant amino acid sequence thereof.For the purposes of the present disclosure, functional fragments of the ABA binding sequence can be about 5 amino acids in length, about 10 amino acids in length, about 15 amino acids in length, about 20 amino acids in length, about 25 amino acids in length, about 30 amino acids in length, about 35 amino acids in length, about 40 amino acids in length, about 45 amino acids in length, about 50 amino acids in length, about 55 amino acids in length, about 60 amino acids in length, about 65 amino acids in length, about 70 amino acids in length, about 75 amino acids in length, about 80 amino acids in length, about 85 amino acids in length, about 90 amino acids in length, about 95 amino acids in length, about 100 amino acids in length, about 105 amino acids in length, about 110 amino acids in length, about 115 amino acids in length, about 120 amino acids in length, about 125 amino acids in length, about 130 amino acids in length, about 135 amino acids in length, about 140 amino acids in length, about 145 amino acids in length, about 150 amino acids in length, about 155 amino acids in length, about 160 amino acids in length, about 165 amino acids in length, about 170 amino acids in length, about 175 amino acids in length, about 180 amino acids in length, about 185 amino acids in length, about 190 amino acids in length, about 195 amino acids in length, about 200 amino acids in length, about 205 amino acids in length, about 210 amino acids in length, about 215 amino acids in length, about 220 amino acids in length, about 225 amino acids in length, about 230 amino acids in length, about 235 amino acids in length, about 240 amino acids in length, about 245 amino acids in length, or about 250 amino acids in length. In other words, the functional fragment can be 5 - 50 amino acids, 5 - 40 amino acids, 5 - 30 amino acids, 5 - 20 amino acids, 5 - 15 amino acids, 10 - 50 amino acids, 10 - 40 amino acids, 10 - 30 amino acids, or 10 - 20 amino acids. Generally, a fragment is considered a functional fragment if it can increase the active loading of the cargo entity onto the lipid bilayer particle, if it can bind to ABI1 or its variant or fragment, or a combination thereof.

[0259] In another aspect, the present disclosure provides a nucleic acid encoding a chimeric peptide or lipid bilayer particle targeting system of any one of the above embodiments.

[0260] In another aspect, the present disclosure provides a production cell comprising a targeting chimeric polypeptide, a lipid bilayer particle targeting system, a lipid bilayer particle (e.g., a CDMP such as an extracellular vesicle), or a nucleic acid of any one of the above embodiments. In some embodiments, the production cell is a mammalian cell. Suitable mammalian cells include, but are not limited to, HEK293, HEK293FT, PER.C6, mesenchymal stem cells, megakaryocytes, iPSCs, T cells, red blood cells and erythrocyte precursors, and iPSC-derived versions of any of the foregoing cells.

[0261] In another aspect, the present disclosure provides a method for producing lipid bilayer particles that target recipient cells such as immune cells, the method comprising culturing a production cell of the foregoing aspect and harvesting the lipid bilayer particles produced by the production cell.

[0262] In some embodiments, the cargo entity is an intra-articular polymer aggregate (e.g., a lentiviral core, an AAV particle, other viral cores, a VLP core, and subunits thereof).

[0263] In some embodiments, the cargo entity is a nucleocapsid. In some embodiments, the nucleocapsid is a viral nucleocapsid. In some embodiments, the nucleocapsid is a recombinant viral nucleocapsid. In some embodiments, the nucleocapsid comprises a cargo nucleic acid and a cargo polypeptide.

[0264] In some embodiments, the cargo entity is an AAV nucleocapsid, an LVV nucleocapsid, or a fragment thereof, or encodes them.

[0265] Second chimeric polypeptide In addition to the chimeric targeting polypeptides described herein, in some embodiments, the provided lipid bilayer particles may further comprise a second chimeric polypeptide (i.e., a chimeric loading polypeptide) comprising a cargo loading domain comprising an abscisic acid-insensitive 1 (ABI1) sequence. In some embodiments, the second chimeric polypeptide may further comprise a linker that links a cargo entity (e.g., a cargo entity polypeptide) and the cargo loading domain. The linker is (1) SEQ ID NO: 10 (TSGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG), and SEQ ID NO: 17 (ESKYGPPAPPAP); or (2) an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 14, 15, 16, or 17.

[0266] The cargo loading domain of the second chimeric peptide may be a truncated variant of the wild-type protein that constitutes the extracellular vesicle targeting domain. For example, the cargo loading domain of the second chimeric peptide contains residues 126 to 423 of wild-type ABI1.In some embodiments, the cargo loading domain of the second chimeric peptide is MTRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO: 6), VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO: 7), a variant amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to either SEQ ID NO: 6 or SEQ ID NO: 7, or a functional fragment of SEQ ID NO: 6, SEQ ID NO: 7, or a variant amino acid sequence thereof.For the purposes of the present disclosure, functional fragments of ABI1 are about 5 amino acids in length, about 10 amino acids in length, about 15 amino acids in length, about 20 amino acids in length, about 25 amino acids in length, about 30 amino acids in length, about 35 amino acids in length, about 40 amino acids in length, about 45 amino acids in length, about 50 amino acids in length, about 55 amino acids in length, about 60 amino acids in length, about 65 amino acids in length, about 70 amino acids in length, about 75 amino acids in length, about 80 amino acids in length, about 85 amino acids in length, about 90 amino acids in length, about 95 amino acids in length, about 100 amino acids in length, about 105 amino acids in length, about 110 amino acids in length, about 115 amino acids in length, about 120 amino acids in length, about 125 amino acids in length, about 130 amino acids in length, about 135 amino acids in length, about 140 amino acids in length, about 145 amino acids in length, about 150 amino acids in length, about 155 amino acids in length, about 160 amino acids in length, about 165 amino acids in length, about 170 amino acids in length, about 175 amino acids in length, about 180 amino acids in length, about 185 amino acids in length, about 190 amino acids in length, about 195 amino acids in length, about 200 amino acids in length, about 205 amino acids in length, about 210 amino acids in length, about 215 amino acids in length, about 220 amino acids in length, about 225 amino acids in length, about 230 amino acids in length, about 235 amino acids in length, about 240 amino acids in length, about 245 amino acids in length, or about 250 amino acids in length. In other words, the functional fragment may be 5 - 50 amino acids, 5 - 40 amino acids, 5 - 30 amino acids, 5 - 20 amino acids, 5 - 15 amino acids, 10 - 50 amino acids, 10 - 40 amino acids, 10 - 30 amino acids, or 10 - 20 amino acids. Generally, a fragment is considered a functional fragment if it can increase the active loading of a cargo entity into lipid bilayer particles, if it can bind to an ABI1 binding protein, or a combination thereof.

[0267] Manufacturing and usage methods One skilled in the art, upon reading the present disclosure, will understand its significant scope of application. The engineered lipid bilayer particles provided may be suitable for therapeutic use. In some embodiments, the lipid bilayer particles or preparations provided herein can be contacted with recipient cells in vitro (e.g., CAR-T) or in vivo (e.g., HIV).

[0268] In particular, the ability to effectively deliver cargo entities (e.g., polypeptide cargo entities, nucleic acid cargo entities, saccharide cargo entities, lipid cargo entities, small cargo entities, composite cargo entities, etc.) to T cells is useful, inter alia, for providing engineered T cells for use, for example, in T cell therapy (e.g., CAR-T therapy). Alternatively or additionally, the ability to effectively deliver cargo entities is useful for the treatment of HIV (e.g., by delivering cargo entities that inhibit, interrupt or disrupt HIV or one or more aspects of its life cycle). In some embodiments, cargo entities such as siRNA, Cas9-sgRNA are useful for targeting HIV.

[0269] In another aspect, the present disclosure provides a method of targeting a cargo entity to a recipient cell (e.g., an immune cell such as a lymphocyte) using a lipid bilayer particle (CDMP) such as an EV. Generally, the lipid bilayer particle will comprise a targeting chimeric peptide of any one of the above embodiments (e.g., a targeting chimeric peptide comprising a targeting domain). Thus, the present disclosure provides a method of targeted delivery of a cargo entity to a recipient cell (e.g., an immune cell such as a lymphocyte), comprising administering to an individual a lipid bilayer particle as disclosed herein, wherein the lipid bilayer particle comprises the cargo entity.

[0270] In some embodiments, the cargo entity is a polypeptide cargo entity. In some embodiments, the cargo entity can be a peptide, polypeptide, or protein of interest to be delivered to recipient cells, such as an enzyme, a therapeutic agent (e.g., an antibody, an inhibitor, an agonist, and an antagonist), or a fluorescent protein. In some embodiments, the cytoplasmic polypeptide cargo entity can be selected from any one or more of a base editing factor, a prime editing factor, a TALEN, a ZFN, a kinase, a kinase inhibitor, an activator or inhibitor of receptor signaling, an intrabod, a chromatin-modifying synthetic transcription factor, a natural transcription factor, and mutants thereof. In some embodiments, the cytoplasmic polypeptide cargo entity is a CRISPR enzyme, e.g., a type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is a Cas9 protein (e.g., a naturally occurring bacterial Cas9, as well as any chimeric, mutant, homolog, or ortholog). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified variants thereof.

[0271] In some embodiments, the cargo entity is a nucleic acid cargo entity. In some embodiments, the first or second cargo entity can be a nucleic acid (e.g., DNA or RNA) or another molecule. Nucleic acid cargo entities can include, but are not limited to, DNA encoding a protein or peptide of interest, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, and combinations thereof. Other potential cargo entities include viral and non-viral vectors that are expressed intracellularly (or can be delivered to the cytoplasm of a cell), as well as ribonucleoprotein complexes such as CRISPR-type entities and endogenous complexes such as DICER or RISC bound to natural or synthetic RNAs such as miRNA and shRNA, among others. Indeed, one of ordinary skill in the art will recognize that any cargo that can be expressed intracellularly or physically delivered inside a cell can be fused to the ABI protein / peptide or ABA binding sequence and is hereby expressly contemplated. Genetic fusions can be achieved by expressing two or more components of a chimeric polypeptide or peptide in a lipid bilayer particle (e.g., a CDMP such as an EV) producing cell. Alternatively, a cargo entity fused to a loading domain is delivered to a lipid bilayer particle (e.g., a CDMP such as an EV) producing cell, leading to subsequent incorporation into the lipid bilayer particle. Alternatively, a cargo entity fused to a loading domain is inserted into a lipid bilayer particle after secretion from the producing cell.

[0272] Thus, in some embodiments, the cargo entity is or comprises a viral nucleocapsid, synthetic nucleic acid, transcription factor, recombinase, base editor, prime editor, nuclease (e.g., TALEN, ZFN, etc.), kinase, kinase inhibitor, activator or inhibitor of receptor signaling, intrabod, chromatin-modifying synthetic transcription factor, natural transcription factor, CRISPR-Cas family protein, DNA molecule, RNA molecule, or ribonucleoprotein complex.

[0273] In some embodiments, the cargo entity comprises a nucleic acid sequence encoding a chimeric antigen receptor.

[0274] The disclosed lipid bilayer particles can be formulated as part of a pharmaceutical composition for treating a disease or disorder, and the pharmaceutical composition can be administered to a patient in need thereof to deliver a cargo entity to recipient cells (e.g., lymphocytes) for treating the disease or disorder. Additionally or alternatively, the lipid bilayer particles can provide a nucleic acid sequence encoding a protein of interest, such as a chimeric antigen receptor (CAR), to recipient cells (e.g., lymphocytes) such that the CAR is expressed by the lymphocytes, as described in further detail below.

[0275] The present disclosure also provides an ex vivo method for targeting delivery of a cargo entity to lymphocytes, comprising obtaining a population of lymphocytes from an individual and contacting the population of lymphocytes with the lipid bilayer particles disclosed herein, wherein the lipid bilayer particles comprise the cargo entity. For the purposes of the ex vivo method, all of the above cargo entities are equally suitable and can be delivered to a population of recipient cells (e.g., immune cells such as lymphocytes). In some embodiments, the population of lymphocytes is obtained via apheresis. In some embodiments of the ex vivo method, the method can further comprise administering the population of recipient cells (e.g., immune cells such as lymphocytes) back to the individual after the recipient cells (e.g., immune cells such as lymphocytes) have contacted the lipid bilayer particles.

[0276] For example, a patient can undergo apheresis to isolate a population of recipient cells (e.g., immune cells such as lymphocytes), and then be contacted with lipid bilayer particles as disclosed herein that contain nucleic acids encoding ribonucleoprotein complexes and chimeric antigen receptors (CARs). The ribonucleoprotein complexes and nucleic acids encoding the CARs are delivered to the recipient cells (e.g., lymphocytes), and the nucleic acid sequences are integrated into the genome such that the CAR is expressed on the surface of the recipient cells (e.g., lymphocytes) and expressed by the recipient cells (e.g., lymphocytes). Expression of the CAR by the recipient cells (e.g., lymphocytes) can similarly be achieved in vivo by administering CD2-targeting lipid bilayer particles to the patient (e.g., by intravenous injection or infusion).

[0277] Manufacture In addition to targeting, the present disclosure also provides techniques (e.g., methods) for loading cargo into lipid bilayer particles and / or manufacturing otherwise engineered lipid bilayer particles (e.g., engineered lipid bilayer particles loaded with cargo) as described herein.

[0278] In some embodiments, techniques for loading cargo entities into lipid bilayer particles (e.g., CDMPs such as EVs) can include (a) expressing in a production cell a targeting chimeric polypeptide, a fusion factor entity polypeptide, and / or a nucleic acid encoding a cargo entity (e.g., mRNA), and (b) expressing in the production cell a targeting chimeric polypeptide and / or a fusion factor entity polypeptide. In some embodiments, the production cell is a eukaryotic cell, and optionally, the mRNA of the chimeric peptide comprising the cargo entity and / or the fusion factor entity polypeptide can be expressed from one or more vectors transfected into a suitable production cell for producing the disclosed extracellular vesicles. Note that the vector may also be stably transfected. A vector or vectors for expressing the mRNA of the chimeric peptide constituting the cargo entity may be packaged in a kit designed to prepare the disclosed extracellular vesicles. In some embodiments, the kit includes lipid bilayer particles having a synthetic nucleic acid (such as an antisense oligonucleotide) or a plasmid.

[0279] In some embodiments, the production cell is a mammalian cell. In some embodiments, the mammalian cell is optionally selected from HEK293, HEK293FT, mesenchymal stem cells, megakaryocytes, induced pluripotent stem cells (iPSCs), T cells, red blood cells, erythroid precursors, and iPSC-derived versions of any of the foregoing cells.

[0280] In some embodiments, the loading of the cargo entity of the chimeric loading peptide is enhanced as compared to passive cargo loading. For example, the methods provided herein (e.g., comprising lipid bilayer particles comprising a targeting chimeric polypeptide and / or a fusion factor entity polypeptide) can achieve cargo entities concentrated up to 23-fold in microvesicles and up to 49-fold in exosomes as compared to passive loading in these respective particles.

[0281] In another aspect, the present disclosure provides techniques for loading a plurality (e.g., two) of cargo entities into lipid bilayer particles such as CDMPs (e.g., extracellular vesicles (EVs)), including expressing in a product any one of the cell lipid bilayer particle loading systems (including a targeting chimeric polypeptide, a fusion entity polypeptide, or both) of the above embodiments.

[0282] In some embodiments, the co-localization of a cargo entity of a chimeric polypeptide or peptide and a second cargo entity of a second chimeric polypeptide is enhanced compared to passive cargo loading.

[0283] In some embodiments, the expression of a targeting chimeric polypeptide or a fragment thereof (e.g., VLR) alongside a viral cargo entity (e.g., a standard lentiviral packaging plasmid) results in a viral genome titer that does not result in a loss of 0% or more, or a loss of 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more. In some such embodiments, the standard lentiviral packaging plasmid can include a fusion factor (e.g., VSV-G) and a viral helper plasmid. This is comparable to the expression of a viral cargo entity (e.g., a standard lentiviral packaging plasmid) without the expression of a targeting chimeric polypeptide or a fragment thereof (e.g., VLR) in which a loss is observed.

[0284] Lymphocyte targeting Certain embodiments of the provided techniques are particularly useful or beneficial in achieving lymphocyte targeting (e.g., to human lymphocyte cells or a population thereof).

[0285] Those skilled in the art are aware that CRISPR-Cas9-mediated genomic manipulation of human T cells is an active area of research for the development of therapeutics for the treatment of cancer, autoimmune diseases, and infectious diseases. When the programmable nuclease Cas9 is delivered together with a single guide RNA (sgRNA) complementary to the target sequence, double-strand breaks are introduced, frameshift mutations are introduced into the coding gene, and protein expression is inhibited. Alternatively, by co-introducing a homology-directed repair template, specific mutations, insertions, or deletions can be inserted into the genome of recipient cells. Although this technology has various applications, its practical implementation remains challenging, at least due to the issues associated with in vivo delivery of Cas9. One approach that harnesses the advances underlying gene therapy is the adeno-associated virus (AAV) vehicle, but safety and efficacy are often limited by anti-vector immunity and restricted tissue tropism. Virus-like particles (VLPs) can also deliver Cas9 nuclease and base editors, but it is still unclear whether the immunogenicity of viral proteins limits these approaches. Synthetic nanoparticle-nucleic acid (e.g., mRNA) delivery is an alternative to viral vectors and has been successful in the in vivo delivery of mRNA for the sustained expression of chimeric antigen receptors in mouse T cells. However, it remains difficult to achieve efficient and specific T cell targeting in a way that results in transient expression of Cas9 necessary to avoid off-target effects. Such general difficulties are further compounded by the problem of what cargo to deliver to T cells with low endocytosis rates. Overall, there is a great opportunity to improve delivery systems that enable the delivery of biologics to T cells within patients.

[0286] A promising new strategy is the use of extracellular vesicles (EVs) for biomolecule transport. EVs are nanoscale, membrane-bound particles secreted by all cells that naturally encapsulate proteins and nucleic acids during biosynthesis. EVs mediate cell-to-cell communication, delivering their contents to recipient cells and influencing cellular function. Due to their inherent properties such as non-toxicity and non-immunogenicity, and the ability to manipulate surface and lumen cargo loading, EVs have become an attractive platform for delivering a wide range of therapeutic agents. To incorporate cargo into vesicles, cargo can be overexpressed in producer cells such that it is loaded during EV biosynthesis, or harvested vesicles can be physically or chemically modified. Genetically engineered cells that produce functionalized EVs may even be transplanted to continuously generate such particles in situ. Post-harvest modification of EVs may offer flexibility in cargo loading, but this approach requires more extensive purification and presents challenges from a manufacturing and regulatory perspective.

[0287] In several recent studies, the use of EVs to deliver Cas9 for the treatment of cancer, hepatitis B, and genetic diseases has been investigated, highlighting the promise of this approach for achieving intracellular Cas9 delivery. However, many exploratory studies have employed EV manipulation methods known to introduce artifacts into downstream experiments, obscuring the possibility that functional effects are due to the EVs. Of particular concern are methods of transfecting EV-producing cells with lipoplexes, loading EVs by electroporation, which is known to cause cargo aggregation, and isolating EVs using commercially available kits not intended for functional delivery applications, all of which have been shown to result in artifacts. 24-26 。

[0288] Here, we address this need by developing an integrated bioengineering strategy for genetically engineering cells to direct the self-organization and production of multifunctional EVs. As a motivating application, we systematically evaluate, compare, and generate technologies that enable EV targeting, active loading of protein cargos into EVs, and EV fusion to achieve functional cargo delivery to T cells. This investigation revealed the major limitations and facilitators of functional delivery via EVs, including potential mechanisms for enhanced delivery through receptor binding to T cells. We demonstrate these technologies by delivering a therapeutically relevant ability - Cas9 ribonucleoprotein complex (RNP) to disrupt the gene encoding the HIV co-receptor CXCR4 in primary human CD4 + T cells.

Example

[0289] The present technology is further illustrated by the following examples, which should not be construed as limiting in any sense. The examples herein are provided to explain the advantages of the present technology and to further assist those skilled in the art in preparing or using the compositions and systems of the present technology. The examples should in no way be construed as limiting the scope of the present technology as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. Also, each of the variations, aspects, or embodiments described above can further include or incorporate variations of any or all of the other variations, aspects, or embodiments of the present technology.

[0290] Example 1: Methods and Materials Plasmid construction. Plasmids were constructed using standard molecular biology techniques. Codon optimization was performed using the GeneArt gene synthesis tool (Thermo Fisher). PCR was carried out using Phusion DNA polymerase (New England Biolabs, NEB), and plasmid assembly was performed by restriction enzyme cloning. Plasmids were transformed into TOP10 competent E. coli (Thermo Fisher) and cultured at 37°C.

[0291] Plasmid backbone. A common expression vector was generated using modified pcDNA3.1 (Thermo Fisher V87020). Briefly, the hygromycin resistance gene and the SV40 promoter were removed, while the SV40 origin of replication and the poly(A) signal were left intact. The AmpR gene, the BsaI site of the 5'-UTR, and the BpiI site of the bGH poly(A) signal were mutated. The lentiviral vector pGIPZ (Open Biosystems) was obtained through the Northwestern High Throughput Analysis Laboratory. PlentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid No. 52961).

[0292] Plasmid source vectors. The fluorescent proteins enhanced blue fluorescent protein 2 (EBFP2), enhanced yellow fluorescent protein (EYFP), and dimer tomato (dTomato) were obtained from Addgene vectors (plasmid numbers 14893, 58855, and 18917, respectively) gifted by Robert Campbell, Joshua Leonard, and Scott Sternson. The monomeric teal fluorescent protein 1 (TFP1) was synthesized by Thermo Fisher. The scFv was synthesized from the published scFv sequence derived from the monoclonal antibody 9.6, and the PDGFR transmembrane domain was obtained from the pDisplay system vector (Addgene plasmid No. 61556, gifted by Robert Campbell). The C1C2 domain sequence was provided by Natalie Tigue and synthesized by Thermo Fisher. The constitutively active Cx43 and SLAM were synthesized by Thermo Fisher from the Uniprot sequences P17302 CXA1_HUMAN and Q13291-1 SLAF1_HUMAN isoform 1, respectively. The plasmid encoding the measles virus glycoprotein was gifted by Isabelle Clerc, Thomas Hope, and Richard D’Aquila. pX330 encoding Cas9 was gifted by Erik Sontheimer (UMass) and was originally gifted by Feng Zhang from Addgene plasmid No. The sequence of the CXCR4 sgRNA was provided by Judd Hultquist and is as follows: GAAGCGTGATGACAAAGAGG. The ABI domain and PYL were synthesized by Thermo Fisher and IDT, respectively.

[0293] Plasmid preparation. Bacteria were cultured overnight for 12 - 14 hours in 100 mL of LB + Amp culture medium. The culture was spun at 3,000 g for 10 minutes to pellet the bacteria. After resuspending the pellet, it was incubated for 30 minutes in 4 mL of 25 mM Tris® pH 8.0, 10 mM EDTA, 15% sucrose, 5 mg / mL lysozyme. The bacteria were lysed with 8 mL of 0.2 M NaOH and 1% SDS for 15 minutes, followed by neutralization with 5 mL of 3 M sodium acetate (pH 5.2) for 15 minutes. The precipitate was pelleted at 9,000 g for 20 minutes, the supernatant was filtered through cheesecloth, and incubated with 3 μL of 10 mg / mL RNAse A (Thermo Fisher) at 37°C for 1 - 3 hours. The sample was extracted with 5 mL of phenol chloroform, centrifuged at 7,500 g for 20 minutes, and the aqueous layer was recovered. The second phenol chloroform extraction was performed with 7 mL of the solvent. 0.7 volume of isopropanol was added to the recovered supernatant, the sample was inverted and incubated at room temperature for 10 minutes, and then centrifuged at 9,000 g for 20 minutes to pellet the DNA mixture. The pellet was dried briefly, resuspended in 1 mL of 6.5% PEG 20,000 and 0.4 M NaCl. The DNA was incubated overnight on ice and pelleted at 21,000 g for 20 minutes. The supernatant was removed, the pellet was washed with cold absolute ethanol, dried at 37°C, and then resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The DNA was diluted to 1 μg / μL using Nanodrop 2000 (Thermo Fisher).

[0294] Cell culture. HEK293FT cells (Thermo Fisher R70007) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco 31600-091) supplemented with 10% FBS (Gibco 16140-071), 1% penicillin-streptomycin (Gibco 15140-122), and 4 mM additional L-glutamine (Gibco 25030-081). Jurkat T cells (ATCC TIB-152) were cultured in Roswell Park Memorial Institute Medium (RPMI 1640, Gibco 31800-105) supplemented with 10% FBS, 1% pen-strep, and 4 mM L-glutamine. Sub-lines generated from these cell lines were cultured similarly. Adherent cells were removed from the plates using trypsin-EDTA (Gibco 25300-054), and the cells were re-cultured at a ratio of 1:5 or 1:10 every 2-3 days. Lenti-X cells (Takara) were cultured similarly with the addition of 1 mM sodium pyruvate (Thermo Fisher 11360-070). Primary human CD4 + T cells were cultured in RPMI supplemented with 10% FBS, 1% pen-strep, 5 mM HEPES, 5 mM sodium pyruvate, and 20 U / mL IL-2 (freshly added at the time of use). The cells were maintained at 37 °C, 5% CO 2 2. HEK293FT and Jurkat cells were determined to be mycoplasma negative by the MycoAlert Mycoplasma Detection Kit (Lonza LT07-318).

[0295] Transfection. For transfection of HEK293FT cells and derivative cell lines into 15 cm dishes for EV packaging, cells were plated at a density of 18x10 6 cells / dish (1×10 6 cells / mL) 6-12 hours prior to transfection. Cells were transfected by the calcium phosphate method with 30 μg of DNA and 1 μg of fluorescent transfection control. Plasmid DNA was diluted in 2 M CaCl 2(Final concentration 0.3 M) and mixed with 2×HEPES buffered saline (280 mM NaCl, 0.5 M HEPES, 1.5 mM Na 2 HPO 4 ) and added dropwise in a 1:1 ratio, and mixed 7 times by pipetting. The transfection solution was incubated for 3 minutes, mixed 8 times by pipetting, and gently added to the side of the plate. HEK293FT cells were transfected in a 10 cm dish. To package EV, the cells were plated at a density of 5x10 6 cells / dish (6.25x10 5 cells / mL), and transfected with 20 μg of DNA and 1 μg of transfection control as described above while dropping the transfection mixture into the dish. Lenti-X cells were also transfected in a 10 cm dish, but plated 24 hours before transfection. For transfection of HEK293FT cells in a 24-well plate, the cells were plated at a density of 1.7x10 5 cells / well (3.4x10 5 cells / mL), and transfected with 200 μg of DNA as described above while dropping the transfection mixture into the well. The medium was changed after 12 - 16 hours. Transfection of Jurkat lipofectamine was performed according to the manufacturer's protocol.

[0296] Generation of cell lines. To generate lentivirus, HEK293FT or Lenti-X cells were plated in a 10 cm dish at 5x10 6 cells / dish (6.25×10 5Plated at a density of cells / mL). For HEK293FT, after 6 - 12 hours, and for Lenti-X, after 24 hours, the cells were transfected with 10 μg of viral vector, 8 μg of psPAX2, and 3 μg of pMD2G via calcium phosphate transfection as described above. The medium was changed after 12 - 16 hours. Twenty-eight hours after medium change, lentivirus was harvested from the conditioned medium. The medium was centrifuged at 500 g for 2 minutes to remove cells, and the supernatant was filtered through a 0.45 μm pore filter (VWR). Lentivirus was concentrated from the filtered supernatant by ultracentrifugation at 100,420 g at 4°C using a SW41Ti rotor in an Ultra-Clear tube (Beckman Coulter 344059) in a Beckman Coulter Optima L-80 XP ultracentrifuge. The supernatant was aspirated, leaving a virus with a final volume of ~100 μL, and left on ice for at least 30 minutes before resuspending the concentrated lentivirus and used for transduction of ~1x10 5 cells plated at the time of transduction or the day before. If necessary, antibiotic selection was started 2 days after introduction using antibiotics at a concentration of 1 μg / mL puromycin (Invitrogen ant-pr) and 10 μg / mL blasticidin (Alfa Aesar J61883) for HEK293FT cells, and 0.2 μg / mL puromycin and 2 μg / mL blasticidin for Jurkat cells. The cells were stored in the antibiotic for at least 2 weeks while being subcultured every 1 - 2 days.

[0297] Sorting cells of the Cas9 reporter strain were resuspended in DMEM or RPMI supplemented with 10% FBS, 25 mM HEPES, and 100 μg / mL gentamicin (Amresco 0304) at a density of 1x10 7They were suspended at a concentration of cells / mL. Using a 488 nm laser (530 / 30 filter) and a 562 nm laser (582 / 15 filter), mTFP1-high-expressing cells lacking dTomato expression (top 10% or less) were sorted using a BD FacsAria Ilu. The cells were recovered in DMEM or RPMI supplemented with 20% FBS, 25 mM HEPES, and 100 μg / mL gentamicin. The cells were spin-downed and resuspended and recovered in normal growth medium supplemented with 100 μg / mL gentamicin.

[0298] Production, isolation, and characterization of EVs. EV-producing cell lines were plated in 10 cm or 15 cm dishes and transfected on the same day by the calcium phosphate method if necessary. The medium was changed to EV-depleted medium the next morning. The EV-depleted medium is DMEM supplemented with 10% exosome-depleted FBS (Gibco A27208-01), 1% pen-strep, and 4 mM L-glutamine. 41、42 . Briefly, the conditioning medium was debris-removed by centrifuging at 300 g for 10 minutes to remove cells, followed by centrifuging at 2,000 g for 20 minutes to remove dead cells and apoptotic bodies. The supernatant was centrifuged at 15,000 g for 30 minutes using a Beckman Coulter Avanti J-26XP centrifuge with a J-LITE JLA 16.25 rotor to pellet microvesicles. The supernatant was collected and exosomes were pelleted by ultracentrifugation at 120,416 g for 135 minutes using a Beckman Coulter Optima L-80 XP ultracentrifuge with an SW41 Ti rotor in polypropylene ultracentrifuge tubes (Beckman Coulter 331372). All centrifugation steps were performed at 4°C. The EV pellet was left in approximately 100 - 200 μL of conditioning medium, incubated on ice for at least 30 minutes after removing the supernatant, and then resuspended. The EV concentration was determined by NanoSight analysis. The sample was diluted with PBS, 10 8Analysis was performed on the order of particles / mL. NanoSight analysis was carried out using an NS300 (manufactured by Malvern) with software version 3.4. Videos of 30 seconds per sample were acquired three times using a 642 nm laser with a camera level of 14, an injection rate of 30, and a detection threshold of 7. The EV concentration was defined as the average value of the concentrations calculated from each video. The particle size distribution was created using the software. For TEM, 65 μL of 4% PFA was added to 200 μL of EV and fixed in an Eppendorf tube for 10 minutes. 15 μL of the fixed suspension was pipetted onto a plasma-cleaned (PELCO easiGlow), formvar / carbon-coated grid (EMS 300 mesh). After 10 minutes, the solution was removed by wicking with a filter paper wedge, and the grid was inverted and washed twice for 30 seconds on a drop of buffer, followed by washing once with diH 2 O. 2% uranyl acetate (Ted Pella) staining was performed twice and wiped off after 30 seconds. The grid was imaged with a JEOL JEM 1230 TEM (JEOL USA) at an acceleration voltage of 100 kV. Data was acquired with an Orius SC1000 CCD camera (Gatan). EVs were stored on ice and used within 10 days or stored at -80 °C for long-term storage.

[0299] Immunoblotting. For Western blot analysis, cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1% Triton® X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 tablet of protease inhibitor cocktail per 10 mL (Pierce PIA32953)) and incubated on ice for 30 minutes. The lysate was centrifuged at 12,000 g for 20 minutes at 4 °C, and the supernatant was collected. Protein concentration was determined by BCA assay (Pierce) according to the manufacturer's instructions. Samples were 1 - 2 μg of protein content (in the case of cell lysates), or 1×10 7 ~6×10 8Normalized by the number of vesicles (in the case of EVs). Samples were heated in Laemmli buffer (60 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 100 mM dithiothreitol, 0.01% bromophenol blue) at 70 °C (membrane-bound scFv and calnexin) or 95 °C (Cas9, CD9, CD81, Alix) for 10 minutes. Samples were loaded onto a 4-15% polyacrylamide gradient Mini-PROTEAN TGX precast protein gel (Bio-Rad) and run at 50 V for 10 minutes and then at 100 V for 1 hour. For anti-FLAG blots, the membrane was blocked with 3% milk in TBS (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, pH 8.0) for 30 minutes. After washing the membrane with TBS for 5 minutes, it was incubated overnight at 4 °C with a primary anti-FLAG antibody (Sigma F1804) diluted 1:1000 in 3% milk in TBS. The membrane was washed twice with TBS for 5 minutes and twice with TBST 1 (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, 0.05% Tween® 20, pH 8.0) for 5 minutes each. For all other blots, the membrane was blocked with 5% milk in TBST2 (50 mM Tris, 150 mM NaCl, 0.1% Tween® 20, pH 7.6) for 1 hour. Primary antibodies included anti-HA (Cell Signaling Technology 377245 C29F4, 1:1000), anti-CD9 (Santa Cruz Biotechnology sc-13118, 1:500), anti-CD81 (Santa Cruz Biotechnology sc-23962, 1:500, performed under non-reducing conditions), anti-Alix (Abcam Ab117600, 1:500), and anti-calnexin (Abcam Ab22595, 1:1000). The membrane was washed 3 times with TBST 2 for 5 minutes each before secondary antibody staining. HRP-conjugated anti-mouse (Cell Signaling Technology 7076) and anti-rabbit (Invitrogen 32460) secondary antibodies were diluted 1:3000 in 5% milk in TBST2. After incubating the membrane with the secondary antibody at room temperature for 1 hour, it was washed 3 times with TBST 2 (5-minute washes).The membrane was probed with Clarity Western ECL Substrate (Bio-Rad), exposed to film, developed, scanned, or imaged with an Azure c280 imager. Images were trimmed with Adobe Illustrator. No other image processing was performed.

[0300] Surface immunoblotting. Cells were transferred to FACS tubes (adherent cells were harvested using FACS buffer (PBS pH 7.4 containing 0.05% BSA and 2 mM EDTA) before staining), and centrifuged at 150 g for 5 minutes with 1 mL of FACS buffer. The supernatant was decanted, and the cells were resuspended in 50 μL of FACS buffer. 10 μL of human IgG (Thermo Fisher 027102) was added, and the cells were flicked and incubated at 4°C for 5 minutes. Then, the conjugated primary antibody was added at the manufacturer's recommended dilution, the cells were inverted and mixed, and incubated at 4°C for 30 minutes. Then, the cells were washed 3 times with 1 mL of FACS buffer, centrifuged at 150 g for 5 minutes, and the supernatant was decanted after each wash. Before flow cytometry, the cells were resuspended in 2 drops of FACS buffer. For Miltenyi Biotec antibodies, the cells were stained at 4°C for 15 minutes without blocking and washed once before flow cytometry according to the manufacturer's protocol. The antibodies used in this study were as follows: anti-FLAG-APC (Abcam ab72569), anti-CD2-APC (R&D Systems FAB18561A), anti-CD25-PE (Miltenyi REA945, 130-115-628), anti-SLAM-PE (Miltenyi REA151, 130-123-970), and anti-mouse IgG1-APC (R&DD Systems IC002A) or anti-human IgG1-PE (Miltenyi REA293, 130-113-438) were used as isotype controls as needed.

[0301] EV binding and uptake experiments Jurkat T cells or primary human CD4 + T cells, unless otherwise indicated, at an EV-to-cell ratio of 100,000:1 (usually 1×10 5per cell 1×10 10 EV) and incubated with EV. For Jurkat cells, the cells were plated in a 48-well plate with a total volume of 300 μL. Primary T cells were plated in a 96-well plate with a total volume of 200 μL. The cells were plated at the time of EV addition, and the wells were adjusted to the appropriate volume with RPMI. In the binding experiment, unless otherwise indicated, the cells were incubated at 37 °C for 2 hours, then washed three times with FACS buffer and centrifuged at 150 g for 5 minutes for Jurkat cells or 400 g for 3 minutes for primary T cells. Before flow cytometry, the cells were resuspended in 1 drop of FACS buffer. To adsorb EV onto aldehyde / sulfate latex beads (Thermo Fisher), EV was mixed with the beads at a ratio of 1x10 9 EV per 2 μL of beads diluted 1:10 in PBS. The sample volume was normalized with PBS, and the beads and EV were incubated at room temperature for 15 minutes. Then, the sample was adjusted to 200 μL with PBS and incubated with shaking at room temperature for 2 hours. The cells were blocked with an anti-CD2 antibody (Beckman Coulter A60794) that binds to the same epitope as the scFv or blank EV at 37 °C for 1 hour before EV incubation if indicated. In the EV uptake experiment of viral glycoprotein, the cells were incubated with EV at 37 °C for 16 hours. As preparation for analysis, the cells were washed twice with PBS, incubated with 2 drops of trypsin-EDTA at 37 °C for 5 minutes to remove vesicles bound to the surface. The cells were washed with RPMI, quenched the trypsin, and then washed two more times with FACS buffer before analysis.

[0302] Flow cytometry and analysis. Flow cytometry was performed using a BD LSR Fortessa Special Order Research Product. For dTomato, a 562 nm laser (582 / 15 filter) was used; for EYFP, a 488 nm laser (530 / 30 filter) was used; and for mTFP1, 488 nm and 405 nm lasers (530 / 30 filter and 525 / 50 filter, respectively) were used. For analysis, approximately 10,000 live cells were collected per sample. Data were analyzed using FlowJo v10 (FlowJo, LLC). Briefly, cells were identified using an FSC-A vs SSC-A plot, and singlet gating was performed using an FSC-A vs FSC-H plot (Figure 26). Fluorescence data were corrected for spectral bleed-through as needed. The mean fluorescence intensity (MFI) of single-cell samples was exported and averaged across three biological replicates. Autofluorescence from untreated cells was subtracted from other samples. The standard error of the mean was propagated through the calculations. Nine-peak Ultra Rainbow Calibration Particles (Spherotech URCP-100-2H) were used to create a calibration curve for converting fluorescence to absolute fluorescence units.

[0303] Confocal microscopy. Cells were transfected onto poly-L-lysine-coated glass coverslips by the calcium phosphate method and mounted on glass slides for imaging. Microscopic images were taken using a Leica SP5 II laser scanning confocal microscope with a 100× oil immersion objective lens. Brightfield images were acquired at a PMT setting of 443.0 V. For fluorescence excitation, a 514 nm laser was used at an intensity of 20% and a smart gain of 94%. The emission spectrum was captured at 520 - 540 nm using a HyD sensor. Images were captured at a resolution of 512×512 and a scanning speed of 400 Hz. Pseudo-color fluorescence images were contrast-adjusted in ImageJ such that 4% of the pixels were saturated.

[0304] Affinity chromatography. Affinity chromatography separations were performed as previously reported51 . Briefly, the anti-FLAG affinity column was prepared by loading anti-FLAG M2 affinity gel (Sigma A2220-1ML) into a 4 mL 1×5 cm glass column (Bio-Rad) and discharging it by gravity flow. The column was washed with 5 mL of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5), equilibrated by washing three times continuously with 1 mL of regeneration buffer (0.1 M glycine-HCl, pH 3.5), and then washed with 5 mL of TBS. The concentrated EVs were loaded onto the top of the column and chased with 1-2 mL of TBS. The column was incubated with EVs for 5 minutes. Then, the flow-through was re-loaded onto the column so that the medium containing EVs passed through the matrix five times. The column was washed with 10 mL of TBS before elution. EVs were eluted with 2.5 mL of elution buffer (100 μg / mL 3×FLAG peptide (Sigma F4799-4MG) in TBS), and after the void fraction (~1 mL) was discharged, it was incubated on the column for 5-10 minutes. EVs were fractionated five times into 0.5 mL fractions (about 8 drops were dripped from the column per fraction). The column was regenerated by washing three times continuously with 1 mL of regeneration buffer and stored at 4°C in storage buffer (50% glycerol, 0.02% sodium azide in TBS).

[0305] Cas9 in vitro cleavage assay. EV was prepared as described above using the components transiently transfected into a 10-cm dish at the following DNA ratios: 6 μg of anti-CD2 scFv, 9 μg of Cas9 vector, 5 μg of sgRNA vector, and 1 μg of mTFP1 transfection control. EV was lysed by incubating with mammalian protein extraction reagent (MPER, Thermo Fisher) for 10 minutes at room temperature (20 - 23°C) with gentle stirring. 200 ng of linearized target plasmid template was added to vesicles together with Cas9 reagent buffer (IDT, Alt-R CRISPR-Cas9 System), and the samples were incubated at 37°C for 1 hour. Proteinase K (Thermo Fisher) was added to the samples at 1 μL per 10 μL of reaction mixture and incubated at 55°C for 10 minutes. The samples were run on a 1% agarose gel stained with SYBR safe (Thermo Fisher) and imaged using a BioDoc-It imaging system (VWR).

[0306] Primary CD4 + Isolation, culture, and activation of T cells. The surprising finding is that the involvement of CD2 by either recombinant antibody or EV-displayed antibody enhances the functionality of exosome-mediated delivery in vitro, despite the fact that equivalent effects were not observed with delivery via microvesicles. The combination of this effect is not explainable by known features of CD2 / T cell biology but may be related to the finding that ligand involvement causes internalization of CD2. Both types of scFv-expressing vesicles specifically bound to CD2 and were to some extent internalized, but there may be differences in intracellular trafficking and fusion between the two vesicle populations. For example, transport via CD2 binding may prefer fusion over the original uptake pathway in a manner different from exosomes. Further research is needed to elucidate the underlying mechanism of this phenomenon.

[0307] EV function delivery experiment. EVs were produced as described above using components transiently transfected in 10 cm dishes at the following DNA ratios: 6 μg anti-CD2 scFv, 9 μg dual Cas9 and sgRNA vectors, 2.5 μg each of measles virus glycoproteins H / F or 3 μg VSV-G and 2 μg filler promoterless pcDNA, and 1 μg mTFP1 transfection control. To generate vesicles lacking scFv, a PDGFR-binding 3xFLAG tag construct was transfected at the same plasmid copy number in place of scFv into the same vector backbone. EVs were delivered to primary human CD4 + T cells as described above. Cells were cultured for 6 days in the presence of EVs, with fresh RPMI and IL-2 added every 2 - 3 days. For repeated dosing, 100 μL of medium was carefully removed from the top of each well and replaced with 100 μL of fresh EVs and medium. On day 6, cells were harvested, washed with PBS by centrifugation at 400 g for 3 minutes at 4 °C, and pelleted. Cells were resuspended in 100 μL of QuickExtract DNA Extract Solution (Lucigen QE9050), and genomic DNA was harvested according to the manufacturer's protocol. Briefly, samples were vortexed for 15 seconds, heated at 65 °C for 6 minutes, vortexed for 15 seconds, and heated at 98 °C for 2 minutes. DNA was stored at -80 °C.

[0308] High-throughput sequencing (NGS) library preparation. The first-round PCR amplification was performed using approximately 100 ng of genomic DNA as a template. The target CXCR4 region was amplified with the following primers: F1: 5’ACACTCTTTCCCTACACGCTCTTCCGATCTNNNNNGAGAAGCATGACGGACAAGTACAG3’ R1: 5’GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNTCCCAAAGTACCAGTTTGCCAC3’ The PCR protocol was as follows: 98°C for 3 minutes, (98°C for 15 seconds, 65°C for 30 seconds, 72°C for 3 seconds) × 15, 72°C for 5 minutes, 4°C for 5 minutes. The PCR product was purified using MagJET beads (Thermo Fisher K2821) and used as a template in the second-round PCR amplification with the following primers: F2: 5’AATGATACGGCGACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3’ R2: 5’CAAGCAGAAGACGGCATACGAGAT-Index-GTGACTGGAGTTCAGACGTGTGCTC3’ The PCR cycle was as follows: 98°C for 3 minutes, (98°C for 15 seconds, 69°C for 30 seconds, 72°C for 5 seconds) × 20, 72°C for 5 minutes, 4°C for 5 minutes. The PCR product was purified again using MagJET beads before HTS. The first-round and second-round PCRs were performed with a primer concentration of 200 nM and Phusion DNA polymerase.

[0309] HTS. The concentration of the genomic DNA sample was measured by Qubit using the HS dsDNA kit and pooled into an equimolar concentration library. The library was diluted to 4 nM by serial dilution. The library and PhiX were denatured with NaOH according to the Illumina MiSeq guide and diluted to 14 pM. The reaction mixture was composed of 8% PhiX and 92% library. The sample was run on an Illumina MiSeq using the MiSeq Reagent Kit v3, and paired-end reads were collected. The data was analyzed using custom code developed by 496code.

[0310] Statistical analysis. Statistical details are provided in the figure legends. Unless otherwise specified, three independent biological replicates (cells) or technical replicates (beads) were analyzed per condition, and the mean fluorescence intensity of approximately 10,000 live single cells or beads per sample was analyzed. Unless otherwise specified, error bars represent the standard error of the mean. Pairwise comparisons were performed using a two-sided Student's t-test in Excel under the null hypothesis that the two samples are equal. The significance threshold was set at 0.05. After the tests, the Benjamini-Hochberg procedure was applied within each panel of the figure to control the false discovery rate.

[0311] Example 2: Strategies for engineering multifunctional EVs to achieve delivery to T cells Our overall approach for the development of technologies aimed at enabling targeted delivery of biomolecules to T cells is to address each of the limiting steps of the process (Figure 1) - loading of cargo into EVs during biosynthesis, binding of EVs to specific recipient cells, uptake, and fusion of EVs with recipient cells to release the cargo into the cytoplasm of T cells. Our approach relies on fully genetically encodable functions, and we refer to this strategy as GEMINI - Genetically Encoded Multifunctional Integrated Nanovesicles.

[0312] Example 3: Engineered membrane scaffolds enable scFv expression in EVs We first investigated strategies for conferring targeting to EVs. A promising strategy to promote specific interactions between EVs and recipient cells and to enhance EV uptake is to display targeting moieties on the EV surface. This strategy has been explored using the presentation of small peptides, but we and others have demonstrated that these effects are modest and variable. 34. Recently, the display of high-affinity targeting domains, including nanobodies and single-chain variable fragments of antibodies (scFv), has enabled EV targeting to receptors such as EGFR and HER2. In these cases, the expression of scFv was achieved by fusion with the C1C2 lactadherin domain that binds to phosphatidylserine on the outer membrane leaflet of some EVs. We investigated whether this approach could mediate EV targeting to T cells using anti-CD2 scFv. Ligand binding induces CD2 internalization, and we hypothesized that such a mechanism would facilitate EV uptake upon docking with the receptor. This is thought to be particularly useful for delivery to T cells, which have a low endocytosis rate and are generally difficult to deliver with other vehicles. Also, we decided to avoid targets that might cause non-specific T cell activation such as CD3. We selected a well-defined display system based on the transmembrane domain of platelet-derived growth factor receptor (PDGFR). Using this general strategy, we hypothesized that targeting domains could be displayed on multiple EV populations. We considered three candidates for the design of the extracellular linker, as it could affect the transport, folding, and target binding of scFv. Namely, an α-helix that provides structure, a 40-residue glycine-serine sequence that provides flexibility, or the hinge region of IgG4 used in chimeric antigen receptors that display scFv on synthetic receptors. All three constructs were expressed at comparable levels in HEK293FT cells (Figures 7A-7B). To test for expression on EVs, two vesicle populations were isolated using a previously validated differential centrifugation method. Hereinafter, for convenience, the fraction separated at 15,000×g will be referred to as "microvesicles" (MV), and the fraction separated at 120,416×g will be referred to as "exosomes" (exo). The vesicles were enriched in canonical markers such as CD9, CD81, and Alix, and the endoplasmic reticulum protein calnexin was decreased (Figure 8A). Both populations consisted of vesicles with an average diameter of approximately 120-140 nm and showed the expected "cup-shaped" morphology (Figures 8B-8C). Importantly, all three scFv display constructs were substantially expressed in both vesicle populations (Figure 7C).

[0313] Example 4: Expression of anti-CD2 scFv enhances EV binding to Jurkat T cells To evaluate targeting, we harvested vesicles from HEK293FT producer cells stably expressing the scFv construct and cytoplasmic dTomato fluorescent protein. The EVs were incubated with Jurkat T cells that express high levels of CD2 for 2 hours (Figure 9), after which the cells were washed to remove unbound vesicles (Figure 10) and analyzed by flow cytometry. All three constructs enhanced the binding of microvesicles and exosomes to T cells (Figure 2A - 2C). Display of scFv on microvesicles enhanced the delivery of dTomato to T cells more than that on exosomes, although part (but not all) of this effect was due to greater dTomato uptake in microvesicles versus exosomes (Figure 11A - 11B). Since the coiled linker consistently yielded the greatest targeting effect, this design was carried forward in subsequent studies.

[0314] Example 5: CD2 - scFv binding mediates uptake by recipient cells

[0315] Next, we evaluated whether uptake of CD2 by EVs causes internalization (as mentioned above, ligand binding would of course cause internalization of CD2). To distinguish between EV binding and uptake, cells were trypsinized after incubation with EVs to remove non - internalized vesicles. Cells that received targeted vesicles had a slightly increased fluorescence compared to non - targeting controls (Figure 2D), indicating that CD2 targeting can mediate EV uptake.

[0316] Example 6: EV targeting via CD2 - scFv is specific To determine whether the observed interaction between vesicles and T cells is due to specific receptor binding, T11 on CD2 was used to block potential binding sites 1Antibodies that bind to the epitope were pre-incubated with recipient Jurkat cells. Pretreatment of the antibodies abolished the EV binding enhanced by the scFv (Figure 2E), demonstrating that our targeting is specific for CD2. In contrast, pre-incubation with non-target EVs (potential non-specific competitors) did not substantially reduce the background or the scFv-enhanced binding (Figure 12). These data indicate that the scFv mediates the specific binding of EVs to CD2.

[0317] Example 7: Optimization of scFv expression improves targeting Enhancing the activity of the interaction between a binding factor (e.g., a targeted therapeutic agent) and its receptor is a generally useful strategy for enhancing delivery and function in vivo. 43 To potentially utilize this mechanism, we sought to increase the expression of the scFv construct and thereby increase loading into vesicles by mass action. By optimizing the coding sequence of the scFv display construct for expression in human cells using a sliding window algorithm, we enhanced the cellular expression of the scFv (Figures 13A - 13B) and increased the loading of scFv into vesicles without affecting vesicle size or morphology (Figures 13C - 13E). EVs generated from cells stably expressing the optimized scFv construct showed enhanced specific binding to recipient cells (Figure 2F). At this final stage of limited optimization, the targeted EV binding to CD2 + Jurkat T cells exceeded 100-fold compared to non-targeted EVs. This optimized targeting system also resulted in enhanced EV binding and modest EV internalization in primary human CD4 + T cells that express CD2 at high levels (Figures 2G - 2H).

[0318] Example 8: The scaffold of CD2-scFv display affects loading and specificity In previous reports, scFv display on EVs has been achieved by fusing to the C1C2 lactadherin domain that binds to phosphatidylserine on the outer leaflet of some vesicles. To compare the PDGFR-based display strategy with other state-of-the-art EV scFv display systems, an optimized anti-CD2 scFv was fused to the C1C2 lactadherin domain scaffold (Figure 15A). Expression of both constructs in cells was similar, but the loading of the C1C2 scFv construct into vesicles was higher (compared to the PDGFR construct) (Figures 15B - 15C). Both systems gave similar microvesicle binding to Jurkat cells (Figure 15D). C1C2 display seemed to confer some enhancement in exosome binding to T cells (compared to PDGFR display), but C1C2 display targeting was uncertain, with only a subset of Jurkat recipient cells strongly binding to C1C2-display EVs, whereas PDGFR display targeting generally mediated delivery to the entire T cell population (Figure 15E). Since this pattern could be evidence of CD2-independent EV binding (which could be an artifact), it was investigated whether C1C2 display targeting was specific. Pre-incubating EVs with anti-CD2 antibody resulted in only partial reduction of C1C2-display-targeted EV binding (in contrast to PDGFR display targeting), suggesting that there is a substantial non-target-specific mechanism in C1C2 display targeting of EVs using this scFv (Figures 15F - 15G). From these observations, it was chosen to pursue effective and efficient PDGFR display of scFv to achieve EV targeting.

[0319] Example 9: Abscisic acid-inducible dimerization domain enables an active EV cargo loading system Next, we attempted to manipulate EVs containing scFv to load them with the therapeutic cargo of interest. Overexpression of cytoplasmic cargo in EV-producing cells results in passive loading of the cargo into vesicles during biosynthesis by mass action. Increasing the cargo content in EVs may result in a more potent delivery vehicle. To enhance the loading of cargo proteins and increase the likelihood that a given vesicle will take up both cytoplasmic cargo proteins and membrane-bound scFv, we designed a small molecule-controlled dimerization-based loading system (Figure 3A). Systems that use light or small molecules (e.g., rapamycin) as inducers have been reported to assist in the loading of EV cargo, but light is difficult to scale up to large volumes, and dimerization induced by rapamycin is so tight as to be functionally irreversible. Therefore, we explored a new strategy based on the abscisic acid (ABA)-induced interaction between a truncated version of abscisic acid-insensitive 1 (ABI) and a pyrabactin resistance-like (PYL) protein. This “ABA” system has several advantages. Namely, the association is rapid, dimerization is reversible, so cargo release in recipient cells may be possible, ABA is inexpensive and non-toxic, and loading by small molecule control is more amenable to biomanufacturing than light-based control. First, the ABI domain and the PYL domain were fused to the luminal side of the scFv construct and to the 5’ or 3’ terminus of the EYFP cargo protein localized in the cytoplasm or nucleus to examine the effect on protein expression and function. Fusion with the PYL domain decreased (or destabilized) the expression of EYFP (Figure 16A), but the scFv was resistant to fusion with either the ABI or PYL domain (Figures 16B-16C). Therefore, we proceeded with the scFv-PYL and EYFP-ABI (3’ fusion) constructs. ABA-induced dimerization of ABI and PYL in this setup was readily apparent by microscopy (Figures 3B and 17).

[0320] Example 10: Promoting Protein Uptake into EVs with ABI Domain Alone To examine the loading of cargo proteins, vesicles were adsorbed to latex beads and analyzed by flow cytometry. Surprisingly, no increase in EV loading due to ABA treatment was observed, and in all conditions, constructs containing the ABI domain showed a higher degree of loading than those lacking this domain (Figures 3C - 3D). This effect was not due to an ABI - dependent increase in protein expression in the producer cells (Figure 18A). Enhancement of loading by ABI was evident when paired with either scFv alone or the scFv - PYL construct, indicating that enhancement of loading by endogenous ABI is independent of the ABI - PYL interaction (Figure 3C). The presence of scFv, for unknown reasons, provided an additional advantage in protein loading compared to the control of EYFP - ABI alone (Figure 18B). To examine the role of intracellular localization in the EV loading process, a nuclear localization sequence (NLS) was introduced into EYFP - ABI and loading was compared with a pure cytoplasmic construct. ABA - induced dimerization had little effect on cargo loading, and addition of NLS to EYFP - ABI did not decrease loading into EVs (Figure 18C). Collectively, these data support the serendipitous finding that ABI constitutes a novel and potent EV cargo protein loading tag.

[0321] Example 11: The ABI domain mediates Cas9 loading into EVs Next, we investigated whether functional cargo can be loaded onto EVs using ABI. As a model, we selected the S. Pyogenes Cas9 ribonucleoprotein complex (RNP). RNPs can be synthesized in producer cells (thus consistent with the GEMINI strategy), and since RNPs must move to the nucleus of recipient cells to act on genomic targets, this system serves as a stringent test for functional delivery via EVs. ABI was fused to the N-terminus or C-terminus of Cas9, and generally the expression pattern was consistent with that observed with EYFP (Figure 19A). Thus, we advanced the Cas9-ABI (3’ fusion) construct. We also investigated whether the addition of the NLS or ABI domain affects Cas9 function. When expressed via transfection (along with cognate sgRNA) in reporter Jurkat T cells, the Cas9 fusion constructs showed similar nuclease activity (Figures 19B - 19C). When the Cas9 construct was expressed in producer cells, the NLS had a minimal effect on Cas9 loading into EVs, while the ABI domain significantly increased Cas9 loading (Figures 3E and 19D), although overall expression in producer cells was not increased (Figure 19E). These trends are consistent with those observed with EYFP, demonstrating the utility of the ABI loading tag across multiple cargo proteins.

[0322] Example 12: Co-loading of membrane scFv and ABI-fused Cas9 into EVs An important but mostly unexplored factor to consider when engineering EV-based therapeutics is the extent to which multiple types of cargo co-localize within the same vesicles in a population. ABI has successfully loaded proteins (alone) into EVs, but it was unclear whether dimerization of the cargo and display protein could facilitate co-loading into EVs (i.e., both scFv and Cas9 being co-loaded into individual vesicles). To evaluate this question, vesicles were generated from cells expressing scFv-PYL and Cas9-ABI treated with ABA or vehicle control, and anti-CD2 scFv-displaying vesicles were isolated by affinity chromatography via a 3×FLAG tag located at the N-terminus of the scFv (Figure 20A). High levels of Cas9 were found in scFv-enriched vesicles regardless of ABA treatment, indicating that ABI-tagging of the cargo is sufficient to achieve substantial co-localization of scFv and Cas9 in EVs (Figure 3F and Figure 20B).

[0323] Example 13: EV-loaded Cas9 exhibits nuclease function To evaluate whether Cas9 RNPs encapsulated in EVs are functional, a direct in vitro assay was developed. EVs from Cas9- and sgRNA-expressing cells were lysed and incubated with a plasmid encoding the sgRNA target sequence (Figure 3G). Plasmids treated with lysed RNP-containing EVs showed the expected specific cleavage products under all conditions tested. The presence or absence of the NLS did not affect cleavage efficiency in this assay, but Cas9 fused to the ABI domain showed a decrease in cleavage for both vesicle populations. This pattern is in contrast to what was observed in the transfection-based Cas9 assay (Figure 19C), and thus it is unclear whether this partial effect (e.g., a potential decrease in Cas9 turnover rate) has meaning in the context of cellular delivery. Therefore, both ABI+ and ABI- constructs were evaluated in subsequent experiments.

[0324] Example 14: Display of viral glycoproteins increases EV uptake by T cells To promote EV uptake and fusion, we considered displaying viral glycoproteins on EVs. First, we examined the vesicular stomatitis glycoprotein (VSV-G), which is commonly used for lentiviral pseudotyping and has been reported to effect fusion between EVs and recipient cells. VSV-G was transiently expressed in dTomato-expressing producer cells, and the resulting EVs were incubated with recipient T cells for 16 h, then treated with trypsin (to remove non-encapsulated vesicles) and analyzed by flow cytometry. VSV-G promoted EV uptake in both Jurkat T cells (Figs. 4A-4B) and primary human CD4 + T cells (Fig. 4C), demonstrating the utility of viral fusion proteins for delivering EVs to T cells.

[0325] To develop a T cell-specific EV fusion system (since VSV-G mediates fusion with most cell types), we considered using cleaved versions of the measles virus glycoproteins H and F, which have previously been used to assist lentiviral delivery to T cells. These proteins bind to signaling lymphocyte activation molecule F1 (SLAM) and complement regulatory factor CD46, both of which are expressed on diverse T cells. H and F are classically thought to mediate viral fusion at the cell surface, although viral endocytosis mediated by SLAM has also been reported. Using the same fluorescent EV uptake assay as above, we examined EV delivery to Jurkats (minimal SLAM expression), SLAM transgenic Jurkats, or primary human T cells expressing SLAM (Fig. 4D). The H and F proteins moderately promoted EV uptake into parental Jurkats (SLAM-), but these proteins strongly promoted EV uptake into SLAM transgenic Jurkats and primary human CD4 +The uptake of EVs by T cells was substantially promoted (Figs. 4E - 4F). Another non - viral protein - based strategy that has been reported to promote functional transfer by overexpressing constitutively active Cx43, a connexin protein involved in the formation of gap junctions on EV - producing cells, was also investigated. Since Cx43 did not increase the internalization of EVs by T cells, this approach was not further investigated (Fig. 22). Collectively, these results support the use of measles H / F glycoproteins as a means to promote EV uptake by SLAM + T cells.

[0326] Example 15: EVs mediate the functional delivery of Cas9 to primary T cells. To evaluate the functional delivery of Cas9 to recipient T cells, efficient cargo loading, T cell binding and fusion, followed by the release of active Cas9 RNPs are required. After verifying each of these steps individually, the evaluation of the combined technology was advanced. Specifically, the use of Cas9 targeting the CXCR4 locus in primary T cells was investigated using a previously validated sgRNA. Since viral glycoprotein expression is cytotoxic, at this point, the focus was shifted to the biomanufacture of EVs using the Lenti - X HEK293T cell line suitable for this task. EVs containing anti - CD2 scFv, NLS Cas9 - ABI with appropriate sgRNA, and VSV - G or measles virus glycoproteins H / F were added to primary human CD4 +After incubating with T cells for 6 days, genomic DNA was harvested for high-throughput sequencing (HTS), and targeted editing in a 64-nucleotide region centered on the predicted cleavage site was quantified and characterized. Interestingly, indels were identified at the predicted Cas9 cleavage sites in all vesicle treatments containing Cas9 RNP (Figures 5 and 23). VSV-G display on EVs resulted in higher editing efficiency than measles H and F proteins, and exosome treatment resulted in more editing than microvesicle treatment for the matched design. Most of the editing was classified as deletions, and the number of insertions and edits containing both insertions and deletions was small. This overall pattern is consistent with previous reports of Cas9 RNP editing at this locus, where the editing consists mostly of small deletions and insertions centered on the cleavage locus, and delivery of Cas9 via EVs using GEMINI results in an effect qualitatively comparable to electroporation of recombinant Cas9 RNP. To evaluate the role of ABI-mediated activity loading in functional delivery, EVs were generated with Cas9+ / -ABI and the editing efficiency in primary T cells was evaluated. The two Cas9 variants functioned relatively well in this context despite the previously noted trade-off in loading and specific cleavage activity (Figure 24).

[0327] By achieving functional delivery with the multi-functional EV, it became possible to next examine the specific contributions of the characteristics of each manipulated EV. In particular, since the anti-CD2 scFv results in a certain degree of binding and uptake in vitro, we sought to evaluate its unique contribution. To confirm the necessity of EV scFv-CD2 involvement in functional delivery, cells were pretreated with anti-CD2 antibody prior to EV addition to block the receptor on the recipient cells. Surprisingly, pretreatment with the anti-CD2 antibody was found to increase the editing rate regardless of the type of vesicle or viral glycoprotein system (Figures 6A-6B). To explain this observation, we hypothesized that the involvement of CD2 leads to a higher level of T cell activation, making the cells more susceptible to EV uptake and editing. To examine this possibility, T cells were incubated with EV scFvs or anti-CD2 antibody, and the surface expression of CD25 was analyzed 2 days after treatment. Since the expression of CD25 was hardly affected by any of the treatments, it was shown that the increased editing observed upon engagement with CD2 in T cell activation could not be explained (Figure 25). To examine how the editing efficiency changes depending on practical considerations such as the EV dose, and how CD2 involvement contributes to this process, EV delivery to T cells from two different donors was evaluated by either a single EV administration only, or by repeating daily EV administration for 6 days of incubation (Figures 6C-6D). As expected, repeated administration of EVs enhanced the editing efficiency in all cases, indicating that readministration is a useful handle for enhancing editing. In general, the involvement of scFv-CD2 enhanced editing via exosomes, but this effect was not apparent for editing via microvesicles. Finally, to evaluate what trends are maintained across experiments, a combined analysis was performed (normalizing to control for variables hypothesized to contribute to the variation in editing efficiency, such as donor T cell batch-specific sensitivity to Cas9 RNP-mediated editing) (Figures 6E-6F). Overall, these combined trends support the important conclusions described above.

[0328] Example 16: Consideration In this study, we developed a strategy that combines a genetically encoded general platform approach for targeting EVs to recipient cells with surface-displayed scFvs and tags them with vesicle-localization domains to actively load protein cargo into EVs and promote uptake and fusion into recipient cells by displaying viral glycoproteins. Applications that motivate the difficult goal of Cas9 delivery to T cells were useful for improving and validating the technologies that can be combined to achieve this goal.

[0329] An exciting aspect of EV-mediated delivery is the potential to target vesicles to the cell or receptor of interest by engineered interactions. Prior reports have demonstrated non-targeted EV-mediated transport to T cells with cargoes including AAV8 encapsulated in EVs and zinc finger fusion methyltransferases. EVs that bind to T cells have also been described as a way to bridge T cells and other cell targets by displaying anti-CD3 and anti-EGFR scFvs linked on the PDGFR transmembrane domain. To our knowledge, this study is the first to demonstrate the integration of EV targeting and uptake by T cells. We anticipate that the modularity of our targeting constructs will be useful for directing EVs to other cell types and receptors.

[0330] The key technology reported here was the serendipitous discovery that the ABI domain of the ABA dimerization system promotes the loading of EV cytoplasmic cargo proteins even in the absence of ABA. The mechanism of this effect is unknown. ABI is not predicted to localize to the cell membrane or endosomal pathway by WoLF PSORT (genscript.com / wolf-psort.html). The advantage of this system is that loading via ABI can be carried out more easily than tags that require overexpression of helper proteins to promote vesicular transport, such as multi-domain dimerization systems (using light, rapamycin, and Dmr domains) or WW domains and Ndfip1. Codiac BioSciences recently explored this particular Cas9 fusion strategy of deriving tags from membrane-bound proteins, which may be to reduce the turnover rate of Cas9 (Figure 19C). However, in the context of cell delivery where EVs have to overcome additional barriers of uptake, fusion, cytoplasmic release, and intracellular transport, higher cargo loading is likely to be beneficial. In such situations, the advantage of a high dose that can send more shots towards the goal may outweigh the slowness of the reaction rate. There is also a possibility of future improvement of the ABI fusion strategy to mitigate its impact on Cas9 activity.

[0331] The ultimate fate of EVs in recipient cells is often degradation via the endosome / lysosome pathway. Thus, developing ways to achieve vesicle fusion in recipient cells is desirable to achieve (or enhance) functional cargo delivery (i.e., into the cytoplasm). Here, we demonstrated that efficient internalization of EVs was achieved using VSV-G and measles virus glycoproteins H / F, with VSV-G in both Jurkat cells and primary T cells, and H / F in cells expressing the lymphocyte receptor SLAM. An important translational consideration is that mutant versions of the H / F protein have been developed to avoid neutralizing host antibodies such as those induced by the measles vaccine. However, in functional Cas9 delivery studies, higher Cas9 editing efficiencies were observed in primary T cells treated with VSV-G vesicles compared to H / F. Although modest at the doses used in this study, the conversion efficiencies we observed were equivalent to or greater than equivalent reports described in the literature. Perhaps the most rigorous and compelling comparator study achieved conversion efficiencies on the order of 0.1% in HEK293T reporter cells that constitutively express Cas9 (a cell type in which delivery of viral vectors and various biomolecules is considerably more efficient compared to T cells), using vesicles derived from MDA-MB-231 breast cancer cells loaded with sgRNA 12 times at high doses (approximately 1×10 10 about 1×10 compared to EVs 11It has been reported that it was necessary to administer [[ID=]],

[0332] A surprising finding is that equivalent effects were not observed with delivery via microvesicles, but functional delivery via exosomes was promoted in vitro when CD2 was involved by either recombinant antibodies or EV-displayed antibodies. The combination of this effect cannot be explained by known features of CD2 / T cell biology, but may be related to the finding that ligand engagement causes internalization of CD2. Both types of scFv-expressing vesicles specifically bound to CD2 and were internalized to some extent, but there may be differences in intracellular trafficking and fusion between the two vesicle populations. For example, transport via CD2 binding may prefer fusion over the original uptake pathway in a manner different from exosomes. Further research is needed to elucidate the underlying mechanism of this phenomenon.

[0333] A notable aspect of this study was the selection of a method to avoid artifacts seen in EV research. Particles from cells transfected with lipoplexes may mediate functional effects that are erroneously attributed to EVs. We minimized such risks by adopting a transfection method in which plasmids are less likely to translocate into T cells. Key comparative observations (e.g., differences in functional delivery by selection of viral glycoproteins) support the interpretation that true EV-mediated delivery has been quantified.

[0334] Example 17: Propositional functional introduction of Cas9 into T cells Further contemplated embodiments include using our active loading strategy to deliver the Cas9-sgRNA complex to recipient cells to mediate gene editing. An example of this strategy is to express Cas9-ABI (fused using our technology in one of the contemplated embodiments here) and anti-CXCR4-targeted sgRNA in HEK293FT cells; collecting EVs produced from these cells using standard methods; introducing these EVs into T cells (e.g., Jurkat T cells or primary human T cells), and then assessing whether the CXCR4 locus has been cleaved and repaired in these recipient T cells after a while (e.g., using high-throughput sequencing).

[0335] The technology adopted here is generalizable and applicable to large-scale production and biomanufacturing. Our strategy of genetically programming the self-assembly of multifunctional particles avoids the need for post-harvest chemical modification, requires further purification, reduces EV yield, and may cause regulatory problems. For some transgenes (e.g., viral glycoproteins that cannot be constitutively expressed due to toxicity), transient transfection was used, but such genes are regularly expressed from inducible promoters for the production of biopharmaceuticals. The integrated tools developed here for cell-derived membrane particle (CDMP) cargo loading and vesicle fusion are widely applicable to various applications and targets and are expected to provide a flexible platform for the manipulation of CDMPs therapeutics.

[0336] Example 18: Functional delivery into Jurkat T cells This example demonstrates the ability of extracellular vesicles containing a lentiviral core (EV-LV), a T cell targeting domain displayed on the PDGFR transmembrane body, and a fusogen to fuse with Jurkat T cells.

[0337] EV-LV was designed to present the following: 1) A non-targeting domain combined with no VSVG, VSVGwt, or VSVGmut; 2) An anti-CD2 targeting domain combined with no VSVG, VSVGwt, or VSVGmut; or 3) An anti-CD5 targeting domain combined with no VSVG, VSVGwt, or VSVGmut.

[0338] The production cell line LentiX used in this example to produce EV-LV is a subclone of the transformed embryonic kidney cell line HEK293. It is a lentivirus production cell and has been engineered to stably express the SV40 large T antigen. LentiX was plated and transfected with one or more plasmids (via calcium phosphate precipitation) according to the above research design. Figure 27 shows the plasmid constructs used in Example 18. The cell medium was appropriately changed. The produced lentivirus was harvested and its physical titer was determined by qPCR.

[0339] Next, Jurkat T cells or HEK293FT cells were transduced with 5 doses of non-concentrated (logarithmically spaced) virus with or without the addition of 8 μg / ml polybrene (a polymer that may reduce the electrostatic repulsion between the virus and the recipient cell membrane and improve transduction). Two days after transduction, flow-based fluorescence readings were taken and qPCR of the particle titer was performed in parallel.

[0340] Introduction of EV-LV and VSV-Gwt into Jurkat T cells All EV-LV displaying VSV-Gwt resulted in good transduction of Jurkat T cells. Doses of 30 - 100 μl of EV-LV displaying VSV-Gwt transduced 80 - 100% of Jurkat T cells (Figure 28A). The qPCR titer results and the virus genomes per cell obtained from plating 40,000 cells per well are shown in Figure 28B. Figure 29 shows the population-level histograms obtained by flow cytometry for representative samples of each condition reported in Figure 28.

[0341] Introduction of EV-LV and VSV-Gmut into Jurkat T cells EV-LV expressing VSV-Gmut alone minimally transduces Jurkat T cells (maximum 3.5%). Co-expression of either VSV-Gmut or a targeting domain (anti-CD2 or anti-CD5) restores the infectivity of VSV-Gmut alone (Figure 30A). Viral genomes per cell were obtained from qPCR titers and plating 40,000 cells per well (Figure 30B). Figure 31 shows population-level histograms obtained by flow cytometry for representative samples of each condition reported in Figure 30.

[0342] EV-LV was introduced into Jurkat T cells without any VSV-G Viruses that do not contain any VSV-G (e.g., VSV-Gwt or VSV-Gmut) generally do not infect Jurkat. This is due to the low magnitude of the fluorescence signal and may be due to protein translocation rather than transduction (Figure 32).

[0343] Introduction of EV-LV and various VSV-G into HEK293FT cells Viruses without VSV-Gwt or VSV-Gmut do not infect HEK (Figure 33A, D, G). Viruses with VSV-Gmut (Figure 33C, F) infect similarly to viruses with VSV-Gwt (Figure 33B, E, H). However, anti-CD5 VLR has lower infectivity compared to VSV-Gmut (Figure 33I).

[0344] EV-LV particles bearing either of two T cell targeting domains (anti-CD5 VLR or anti-CD2 scFv) and displaying non-compatible VSV-Gmut transfected Jurkat T cells in a dose-dependent manner.

[0345] Example 19: Functional delivery into primary human T cells This example demonstrates the ability of extracellular vesicles containing a lentiviral core (EV-LV), a T cell targeting domain displayed on the PDGFR transmembrane domain, and a fusogen to fuse with primary human T cells (CD4+ / CD8+).

[0346] The following combinations of T cell targeting domains and fusion factors were tested: - Without surface modification ver1 - Without surface modification ver7 - VSV-Gwt - VSV-Gmut - Anti-CD5 VLR - VSV-Gmut + Anti-CD5 VLR - VSV-Gwt + Anti-CD5 VLR ver1 - VSV-Gwt + Anti-CD5 VLR ver7

[0347] LentiX (lentivirus-producing cells) were plated and transfected with one or more plasmids according to the above study design (via calcium phosphate precipitation). Figure 34 shows the plasmid constructs used in Example 19. The cell medium was appropriately changed. The produced EV-LV was collected. The physical titer was determined by qPCR. Equal genomic copies of the virus were applied to T cells and HEK29FT cells. Three days after transduction, flow-based fluorescence readings were taken and co-staining was performed to confirm the identity of T cells as CD4+ and / or CD8+.

[0348] Introduction of EV-LV with VSV-Gmut or VSV-Gmut into primary human T cells All EV-LV expressing VSV-Gwt resulted in dose-dependent transduction of activated T cells (Figures 35A and B). No transduction was observed with VSV-Gmut alone. EV-LV expressing anti-CD5 VLR and VSV-Gmut showed transduction of activated T cells equivalent to or greater than that of VSV-Gwt (Figures 35A and B).

[0349] EV-LV introduced with VSV-Gmut or VSV-Gmut into HEK293TF cells 。 All EV-LVs expressing VSV-G (wt and mut) resulted in dose-dependent transduction of HEK293FT cells. The addition of anti-CD5 VLR to VSV-Gwt did not result in substantially higher transduction than VSV-Gwt alone. The addition of anti-CD5 VLR to VSV-Gmut did not result in higher transduction than VSV-Gmut alone. All of these observations appear to be due to the fact that HEK293FT does not express CD5.

[0350] EV-LV particles having anti-CD5 VLR and binding-incompatible VSV-Gmut transfect primary T cells in a dose-dependent manner. Inclusion of anti-CD5 VLR in VSV-Gmut restores the infectivity of the particles and results in comparable efficiency of transducing activated T cells as compared to VSV-Gwt alone. EV-LV particles containing anti-CD5 VLR and VSV-Gwt induce more than 5-fold more primary T cells as compared to the gold-standard EV-LV particles containing VSV-Gwt.

[0351] Equivalents The technology is not limited in terms of the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the technology. Many modifications and variations of the technology can be made, as will be apparent to those skilled in the art, without departing from its spirit and scope. In addition to those listed herein, functionally equivalent methods and apparatuses within the scope of the technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the technology. It is to be understood that the technology is not limited to a particular method, reagent, compound composition, or biological system. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0352] In addition, when a feature or aspect of the disclosure is described in terms of a Markush group, those skilled in the art will recognize that the disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush group.

[0353] As will be understood by those skilled in the art, for all purposes, and particularly from the perspective of providing a written description, all ranges disclosed herein include any and all possible sub-ranges and combinations thereof. It can be readily recognized that any recited range is to be considered as having been adequately described and enabled to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. of the same range. By way of non-limiting example, each range discussed herein can be readily decomposed into lower thirds, middle thirds, and upper thirds, etc. Also, as will be understood by those skilled in the art, all language such as "up to", "at least", "greater than", "less than", etc. includes the recited number and refers to ranges that can be subsequently decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, ranges include their individual members. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0354] All patents, patent applications, provisional applications, and publications mentioned or cited herein are hereby incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

1. (i) A targeting chimeric polypeptide, (a) A targeting domain arranged such that the targeting domain is on the surface of a particle, the targeting domain including a binding portion that specifically binds to a target ligand on the surface of a target recipient cell, (b) Transmembrane domains directly or indirectly linked thereto Targeting chimeric polypeptides including; and (ii) Fusion factor entity polypeptide, (a) A fusion factor portion arranged such that the fusion factor portion is on the particle surface, (b) Transmembrane domains directly or indirectly linked thereto Fusion factor entity polypeptide containing A collection of manipulated lipid bilayer particles, including [the specified element].

2. The population according to claim 1, wherein the targeting domain is an antibody agent or comprises one.

3. The population according to claim 2, wherein the antibody agent is a single-chain antibody agent.

4. Antibody agents are antibodies, Fab, Fab', F(ab'). ２ The group according to claim 2, selected from the group consisting of Fd, scFv, single-chain antibodies, disulfide-bonded Fvs (sdFv), affinibody, DARPIN, nanobody, variable lymphocyte receptor (VLR), and camelid antibodies.

5. The population according to claim 1, wherein the targeting domain is or comprises darpin or other manipulated high-affinity binding polypeptide.

6. The population according to claim 1, wherein the targeting domain is scFv.

7. The group according to claim 1, wherein the transmembrane domain of the targeting chimeric polypeptide includes an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

18.

8. The population according to claim 7, wherein the transmembrane domain of the targeting chimeric polypeptide comprises the amino acid sequence of SEQ ID NO:

18.

9. The population according to claim 1, wherein the targeting domain includes an amino acid sequence having at least 95% identity with the amino acid sequence of Sequence ID No.

20.

10. The population according to claim 9, wherein the targeting domain comprises the amino acid sequence of SEQ ID NO:

20.

11. The group according to claim 1, further comprising a first cargo entity linked to the transmembrane domain of a targeting chimeric polypeptide via a linker.

12. The population according to claim 11, wherein the linker comprises an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:

17.

13. The population according to claim 1, wherein the manipulated lipid bilayer particles are cell-derived membrane particles (CDMPs) selected from extracellular vesicles, viral particles, virus-like particles (VLPs), apoptotic bodies, platelet-like particles, and combinations thereof.

14. The population according to claim 13, wherein CDMP is an extracellular vesicle selected from the group consisting of exosomes, microvesicles, and combinations thereof.

15. The group according to claim 1, wherein the fusion factor entity polypeptide is a viral fusion factor.

16. The population according to claim 15, wherein the virus fusion factor is selected from lentiviral glycoprotein, or from glycoproteins selected from vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon leukemia virus glycoprotein (GaLV), amphiphilic mouse leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein.

17. The group according to claim 1, wherein the particles further comprise cargo entities.

18. The group according to claim 17, wherein the cargo entity is a polypeptide cargo entity.

19. The group according to claim 17, wherein the cargo entity is a nucleic acid cargo entity.

20. The group according to claim 18, wherein the polypeptide cargo entity is a polypeptide different from each of the targeting chimeric polypeptide and the fusion factor entity polypeptide, or a part thereof.

21. The group according to claim 20, wherein polypeptide cargo entities are linked to cargo loading domains.

22. The population according to claim 21, wherein the cargo loading domain comprises an abscisic acid-insensitive 1 (ABI1) sequence.

23. The group according to claim 1, wherein one or both of the targeting chimeric polypeptide and the fusion factor entity polypeptide further comprises an internal membrane cargo binding portion.

24. The group according to claim 23, wherein the cargo binding portion is bound to the polypeptide cargo.

25. The group according to claim 23, wherein the cargo binding portion binds to the nucleic acid cargo.

26. The group according to claim 1, wherein the particles further comprise a chimeric loading polypeptide comprising a cargo loading domain containing an abscisic acid-insensitive 1 (ABI1) sequence.

27. The population according to claim 26, wherein the cargo loading domain of the chimeric loading polypeptide is a cleaved variant of a wild-type protein containing an extracellular vesicle targeting domain.

28. The population according to claim 27, wherein the cargo loading domain of the chimeric loading polypeptide contains residues 126 to 423 of wild-type ABI1.

29. The population according to claim 26, wherein the cargo loading domain of the chimeric loading polypeptide comprises the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO:

7.

30. The group according to claim 13, wherein the CDMP comprises or contains a viral nucleocapsid, synthetic nucleic acid, transcription factor, recombinase, base editing factor, prime editing factor, nuclease (e.g., TALEN, ZFN, etc.), kinase, kinase inhibitor, receptor signaling activator or inhibitor, intrabody, chromatin modification synthetic transcription factor, native transcription factor, CRISPR-Cas family protein, DNA molecule, RNA molecule, or ribonucleoprotein complex.

31. Cells comprising the population described in any one of claims 1 to 30.

32. The cell according to claim 31, wherein the cell is a mammalian cell, and the mammalian cell is arbitrarily selected from HEK293, HEK293FT, mesenchymal stem cells, megakaryocytes, induced pluripotent stem cells (iPSCs), T cells, erythrocytes, erythrocyte precursors, and any iPSC-derived version of the said cell.

33. A method for producing lymphocyte-targeting lipid bilayer particles, comprising culturing the cells described in claim 31 and collecting the lipid bilayer particles produced by the cells.

34. A method for targeted delivery of cargo entities to lymphocytes, comprising administering the population described in claim 17 to an individual.

35. An ex vivo method for targeted delivery of cargo entities to lymphocytes, comprising obtaining a population of lymphocytes from an individual, and ex vivo contacting the population of lymphocytes with the population described in claim 17.

36. The method according to claim 34 or 35, wherein the cargo entity comprises a viral nucleocapsid, synthetic nucleic acid, transcription factor, recombinase, base editing factor, prime editing factor, nuclease (e.g., TALEN, ZFN, etc.), kinase, kinase inhibitor, receptor signaling activator or inhibitor, intrabody, chromatin modification synthetic transcription factor, native transcription factor, CRISPR-Cas family protein, DNA molecule, RNA molecule, or ribonucleoprotein complex.

37. The method according to claim 34 or 35, wherein the cargo entity comprises a nucleic acid sequence encoding a chimeric antigen receptor.

38. A method for producing a collection of lipid bilayer particles, The process includes isolating lipid bilayer particles produced by production cells engineered to express fusion factor entity polypeptides and targeting chimeric polypeptides. A method wherein lipid bilayer particles contain fusion factor entities and targeting chimeric polypeptides on their surface.

39. A method for delivering a cargo entity to a recipient cell, A step of contacting recipient cells with a population of lipid bilayer particles prepared from engineered production cells, wherein the engineered production cells are engineered to express a fusion factor entity polypeptide and a targeting chimeric polypeptide, A method wherein lipid bilayer particles contain fusion factor entities and targeting chimeric polypeptides on their surface.

40. A method for producing production cells, The process includes a step of manipulating production cells to express a fusion factor entity polypeptide and a targeting chimeric polypeptide, A method for producing lipid bilayer particles containing fusion factor entities and targeting chimeric polypeptides on the surface of production cells.

41. The population according to claim 1, wherein the targeting domain binds to CD5.

42. The population according to claim 41, wherein the targeting domain comprises an anti-CD5 entity or a fragment thereof.

43. The population according to claim 42, wherein the targeting domain comprises the amino acid sequence of SEQ ID NO:

24.

44. The population according to claim 43, wherein the targeting domain comprises the amino acid sequence encoded by Sequence ID No. 25.

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