Enhancement of receptor engagement mediated biological product delivery

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

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 341,914, filed on May 13, 2022, which is incorporated herein by reference in its entirety. Government Support Statement

[0002] This invention was made with government support under Grant No. P30 AI117943 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

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

[0004] The emerging field of nucleic acid therapeutics has attracted a lot of attention, especially because it has the potential to treat a variety of diseases, disorders, or conditions that may not be easily addressed by other means. However, as a recent review pointed out, "While nucleic acid therapeutics can expand the range of treatable diseases, their broader application is limited by multiple delivery challenges" (Gupta et al., "Nucleic acid delivery for therapeutic applications" in Adv. Drug Delivery Reviews 178: 113834, 2021).

[0005] The following discussion is intended only to assist the reader in understanding the present disclosure and is not admitted to describe or constitute prior art.

[0006] Secreted extracellular vesicles (EVs), such as exosomes and microvesicles, are nanoscale lipid vesicles that are produced by a variety of cell types and transfer proteins, nucleic acids, and other molecules between cells in the human body and between cells in other animals. Many viruses are known to use EVs to deliver their genomes to other cells (e.g., enveloped viruses). EVs have a wide range of potential therapeutic uses and are attractive platforms for the delivery of a variety of therapeutic drugs. For example, targeted exosomes have been shown to be effective for delivering RNA to neural cells and tumor cells in mice. Membrane particles from other cell sources may also be used for similar purposes.

[0007] Controlling the composition of EVs in the lumen and / or membrane is challenging. While various passive approaches exist for both, better engineering of EV composition is needed to realize their therapeutic potential. The disclosed technology aims to address these limitations of current technologies. Summary of this technology

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

[0009] Among other things, the present disclosure provides insight that combinations of certain affinity agent polypeptides and fusogen agent polypeptides on the surface of lipid bilayer particles can impart surprisingly improved delivery properties to these particles, such as enabling more efficient delivery and / or delivery to specific cell types.

[0010] The present disclosure identifies the source of one or more problems associated with many conventional strategies for delivering a payload (e.g., nucleic acid payload delivery) to a recipient cell of interest, particularly including techniques intended to achieve in vivo delivery. Among other things, the present disclosure identifies the source of specific problems associated with conventional strategies for achieving payload delivery using viral fusogens (e.g., VSV-G) or variants thereof; the present disclosure demonstrates that combining a fusogen entity polypeptide (e.g., a viral fusogen entity polypeptide) with a targeting chimeric polypeptide, as described herein, can address these identified problems and / or otherwise achieve particularly beneficial results (e.g., particularly precise and / or efficient payload delivery).

[0011] The present disclosure is particularly concerned with the challenges associated with effectively delivering a payload (e.g., a nucleic acid payload) to a specific immune cell (e.g., a T cell). The present disclosure records the particular effects of the provided technology in delivering a payload (e.g., a nucleic acid payload) to a T cell (e.g., an activated T cell).

[0012] In addition, the present disclosure provides engineered lipid bilayer particles and preparations thereof, the surfaces of which contain fusion proto-entity polypeptides and targeted chimeric polypeptides as described herein. In some embodiments, the particles and / or preparations provided are characterized by specific load delivery characteristics. In some embodiments, the particles and / or preparations provided achieve load delivery (e.g., specific load delivery and / or enhanced load delivery) to specific cells or cell populations of interest; in some embodiments, such delivery is performed in vivo. In addition, the present disclosure provides specific combinations of fusion proto-entity polypeptides and targeted chimeric polypeptides as described herein, which can drive specific functions.

[0013] In some embodiments, the provided technology can be used to deliver viral vectors (e.g., lentiviral cores, adeno-associated virus particles), and / or virus-like particles within lipid bilayer particles. Alternatively or additionally, in some embodiments, the provided technology can also be used to deliver non-viral vectors (e.g., nucleic acid payloads that are not packaged in protein core or capsid structures).

[0014] In addition, the present disclosure establishes the challenges faced by in vivo gene delivery to cells, especially delivery to certain immune system cells (such as T cells), especially the in vivo delivery of cargo (e.g., payload) specifically and efficiently targets specific receptor cells of interest (e.g., T cells). Although some methods partially meet the delivery of cargo (e.g., payload) to target cells, especially T cells, in vitro in a specific and efficient manner, there is still an unmet need for specific in vivo delivery and non-toxic in vivo and in vitro delivery, and there is also an unmet need for more efficient in vitro delivery to cells.

[0015] In addition, in some embodiments, the present disclosure provides techniques (eg, systems, engineered lipid bilayers, production cells, manufacturing and delivery methods) for mediating fusion of engineered lipid bilayer particles with recipient cells (eg, delivery of cargo).

[0016] Certain particularly useful applications of the provided technology include, for example, CAR T cell therapy, such as in the manufacture of CAR T cells for tumor treatment, immune system disorders, and other applications.

[0017] In addition, in some embodiments, the present disclosure provides technologies for enhancing the delivery of specific cargoes (e.g., payloads) and / or delivery to specific recipient cells or cell populations, including specific delivery to specific immune cells or cell populations, in particular delivery to T cells or T cell populations.

[0018] In some embodiments, the present disclosure achieves the specificity and / or efficiency of payload delivery through the combined activity of fusion original entity polypeptide and targeting chimeric polypeptide. In some embodiments, the provided technology achieves delivery showing higher specificity and / or efficiency compared to a specific reference; in some embodiments, such references may be a sufficiently comparable system including fusion original entity polypeptide and targeting chimeric polypeptide, but not both. In many embodiments, a suitable reference may be a sufficiently comparable system including fusion original entity polypeptide but not including targeting chimeric polypeptide. Alternatively or additionally, in some embodiments, a suitable reference may be a sufficiently comparable system including a specific viral fusion original entity polypeptide (e.g., VSV-G or its variants) and, for example, lacking a targeting chimeric polypeptide described herein. In some embodiments, a suitable reference does not use the same affinity agent polypeptide, even if it includes at least one surface agent having a certain degree of affinity for the surface of a receptor cell or its population.

[0019] The present disclosure provides targeted chimeric polypeptides, fusion protomer entity polypeptides, and systems and methods using the same for targeting 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 production cells, methods for manufacturing lipid bilayer particle preparations, methods for delivering cargo entities to recipient cells, and recipient cells containing one or more cargo entities, which may further comprise targeted chimeric polypeptides and fusion protomer entity polypeptides (e.g., received by fusion of the receptor cell membrane with the lipid bilayer particles as described herein), and these receptor cells also have a nucleus.

[0020] In one aspect, the present disclosure provides a targeting chimeric polypeptide comprising: (a) a targeting domain that binds to a target ligand (e.g., a target ligand present on the surface of a receptor cell of interest; specifically including human cells and / or immune cells, such as T cells, further particularly including CD2 and / or CD5, e.g., human CD2 and / or human CD5), wherein the targeting domain comprises an antibody reagent, such as Fab, Fab', F(ab')2, Fd, scFv, single-chain antibodies, disulfide-linked Fvs (sdFv), de novo designed binding molecules, affibodies, DARPIN, nanobodies, variable lymphocyte receptors (VLR), camelid antibodies, etc.; and optionally (b) a transmembrane domain. In certain embodiments, the targeting domain is an scFv.

[0021] In certain embodiments, the transmembrane domain comprises AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (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 targeted chimeric polypeptide can further comprise a first cargo entity connected to the transmembrane domain via a linker. In some embodiments, the linker comprises: (1) Selected from SEQ ID NO: 10 (TSGGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG), and SEQ ID NO: The amino acid sequence of NO: 17 (ESKYGPPAPPAP); or (2) having an amino acid sequence that is 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% identical to any one of SEQ ID NO: 10, 12, 14, 15, 16 or 17.

[0025] In another aspect, the present disclosure provides lipid bilayer particles (eg, cell-derived membrane particles (CDMPs)) comprising a targeting chimeric polypeptide disclosed herein and / or a fusogen entity polypeptide disclosed herein.

[0026] In some embodiments, the lipid bilayer particle is a CDMP. In some embodiments, the CDMP is selected from an extracellular vesicle, a viral particle, a virus-like particle (VLP), an apoptotic body, a platelet-like particle, and a combination 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 fusion original entity polypeptide. In some embodiments, the fusion original entity polypeptide is a viral polypeptide (e.g., glycoprotein). In some embodiments, the viral glycoprotein is selected from a lentiviral glycoprotein or a glycoprotein selected from vesicular stomatitis (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic mouse leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowl plague 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 lipid bilayer particles 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 mouse leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, lymphocytic choriomeningitis virus (LCMV) glycoprotein, and any combination thereof. Expression of such glycoproteins or glycoprotein combinations (e.g., measles virus glycoprotein H and measles virus glycoprotein F) can be in embodiments independent of (i.e., not including) the targeting chimeric polypeptides disclosed herein, because these glycoproteins independently provide novel uses for binding and fusion of lipid bilayer particles to receptor cells.

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

[0029] In some embodiments, the lipid bilayer particle comprises a cargo entity as described herein.

[0030] In some embodiments, the disclosed lipid bilayer particles (e.g., CDMPs) may further comprise a chimeric loading polypeptide comprising a cargo loading domain comprising an abscisic acid insensitive 1 (ABI1) sequence and optionally a cargo entity. In some embodiments, the chimeric loading polypeptide comprises a cargo loading domain and a cargo entity, the cargo loading domain comprising an abscisic acid insensitive 1 (ABI1) sequence. In some embodiments, the chimeric loading polypeptide further comprises a linker connecting the cargo entity and the cargo loading domain. In some embodiments, the linker of the chimeric loading polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 (TSGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGGSGGSGGGSGGGSGGGSGGGSGGGS), and SEQ ID NO: 17 (ESKYGPPAPPAP); or has an amino acid sequence similar to SEQ ID NO: 18 (ESKYGPPAPPAP); NO: 10, 12, 14, 15, 16 or 17 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%, 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 of NO: 10, 12, 14, 15, 16 or 17.

[0031] In some embodiments, the cargo loading domain of the chimeric cargo polypeptide is a truncated variant of the wild-type protein that comprises an extracellular vesicle targeting domain. In some embodiments, the cargo loading domain of the chimeric cargo polypeptide comprises residues 126-423 of wild-type ABI1. In some embodiments, the cargo loading domain of the chimeric cargo polypeptide comprises: MTRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALP LSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK(SEQ ID NO:6), VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLS VDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK(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 any one of SEQ ID NO: 6 or 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 chimeric cargo polypeptide cargo entity is a cytoplasmic cargo molecule. In some embodiments, the chimeric cargo polypeptide cargo entity is a membrane-bound cargo entity.

[0033] In some embodiments, the first cargo entity is or comprises an ABA binding sequence. In some embodiments, the first cargo entity is or comprises an ABA binding sequence, and the ABA binding sequence comprises a pyrabactinresistance 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 comprises MGGGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 2), TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 3), with SEQ ID NO: 3 NO:2 or 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 any one of NO:2 or 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 further comprise abscisic acid (ABA).

[0036] In some embodiments, the lipid bilayer particle comprises or contains 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, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin-modified 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, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin-modified 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 present disclosure provides nucleic acids encoding the chimeric targeting polypeptides and / or fusion proentity polypeptides disclosed herein.

[0038] On the other hand, the present disclosure provides a production cell comprising a targeting chimeric polypeptide disclosed herein and / or a fusion original 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 (iPSC), T cells, erythrocytes, erythrocyte precursors, and iPSC-derived versions of any of the foregoing cells. On the other hand, the present disclosure provides a method for producing lipid bilayer particles, comprising culturing a production cell comprising a targeting chimeric polypeptide disclosed herein and / or a fusion original entity polypeptide, a lipid bilayer particle disclosed herein (e.g., CDMP) or a nucleic acid disclosed herein, and harvesting the lipid bilayer particles (e.g., CDMP) produced by the cell.

[0039] In another aspect, the present disclosure provides a method for targeted delivery of a cargo entity to a recipient cell (eg, an immune cell such as a lymphocyte), 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 recombinase, a base editor, a prime editor, a nuclease (e.g., TALEN, ZFN, etc.), a kinase, a kinase inhibitor, a receptor signaling activator or inhibitor, an intracellular antibody, 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 a cargo entity to a recipient cell (e.g., an immune cell, such as a lymphocyte), comprising obtaining a population of recipient cells (e.g., lymphocytes) from an individual, and contacting the population of recipient cells (e.g., lymphocytes) ex vivo with a lipid bilayer particle disclosed herein, wherein the lipid bilayer particle comprises a cargo entity.

[0043] In some embodiments, the lymphocyte population is obtained by apheresis.

[0044] In some embodiments, the ex vivo method can further include administering the population of recipient cells (e.g., lymphocytes) back to the individual after the recipient cells (e.g., lymphocytes) have been contacted with the lipid bilayer particles (e.g., such that 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 claimed. Other objects, advantages and novel features will be apparent to those skilled in the art from the following brief description of the drawings and the detailed description of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings include the following figures:

[0047] Figure 1 Overview of the GEMINI strategy showing genetically engineered multifunctional EVs. EV cargo proteins and nucleic acids are expressed in producer cells to facilitate incorporation into a variety of vesicle populations: microvesicles budding from the cell surface, or exosomes generated by endosomal internalization into multivesicular bodies. Surface-displayed targeting and fusion proteins facilitate binding to and uptake by recipient cells, followed by cargo release via cell surface fusion or endosomal escape. In the proof-of-principle application performed in this study, the goal was to deliver Cas9-sgRNA complexes to T cells for gene knockout, as described in subsequent sections.

[0048] Figure 2 Display of scFvs on EVs was shown to mediate specific, targeted binding and uptake by T cells. Figure 2 A. Strategy for targeting EVs to T cells (left) and illustration of EV binding assay (right). Figure 2 B. Targeted EV binding to Jurkat (2 h incubation). To assess potential differences in dTomato loading, mean EV fluorescence was analyzed separately ( Fig.11 ). Figure 2 C. Depicted in Figure 2 Representative histograms of the distribution of helical linker EV-mediated fluorescence in recipient cells analyzed in B. Figure 2 D. Differentiation of binding and internalization of EVs targeted to Jurkat. After 6 h of incubation, bound and non-internalized EVs were removed by trypsin digestion. Figure 2 E. Specificity of EV targeting to CD2. Preincubation with anti-CD2 antibody abolished EV targeting to Jurkat. Figure 2 F. Enhanced targeting by codon-optimized expression of scFv constructs. Fold increase relative to non-targeting control is indicated in blue. Figure 2 G. Targeting EVs with primary human CD4 + Binding of T cells (2 h incubation). Figure 2 H. Differentiation of binding and internalization of EVs targeted to primary human CD4+ T cells. All experiments were performed in biological triplicates and error bars represent standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001). EV dTomato loading was assessed as Fig.11 shown.

[0049] Figure 3 We show that cargo proteins are actively loaded into EVs by tagging them with the ABI domain of the abscisic acid dimerization system. Figure 3 A. Schematic representation of abscisic acid-based dimerization of EV cargo proteins and subsequent loading into vesicles. Figure 3 B. ABA-induced dimerization between PYL and ABI domains. Exemplary microscopy shows binding of anti-CD2 scFv-PYL (membrane bound) and EYFP-ABI (cytoplasmic) in the presence of ABA. Full image as Fig.17 shown. Figure 3C. ABI induces cargo loading into EVs. EVs produced under the indicated conditions were adsorbed to aldehyde / sulfate latex beads and analyzed by flow cytometry to determine overall mean fluorescence. Experiments were performed in biological triplicate and error bars represent standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001). Figure 3 D. Figure 3 Representative histograms of EYFP + / - ABI conditions in C. Figure 3 E. Active loading of Cas9-ABI with and without NLS into EV. 6.0×10 8 EV. The expected band size (~160 or 195 kDa, arrow) corresponds to the Cas9- / +ABI domain. The complete blot is shown in Fig.19 Provided by D. Figure 3 F. Analysis of ABA-dependent loading of Cas9-ABI into EVs enriched with anti-CD2 scFv-PYL by affinity chromatography. 1.3 × 10 7 Microvesicles (MV) or 2.0×10 7 Exosomes. Expected band size: 195 kDa (arrow) The complete blot is as follows Fig. 20 Provided by B. Figure 3 G. Biological activity of EV-associated Cas9. Vesicles were lysed and incubated with linearized target plasmid in Cas9 nuclease reaction buffer at 37°C for 1 hour. Expected cleavage band sizes: 7.6 and 4.6 kb (arrows).

[0050] Figure 4 Viral glycoproteins displayed on EVs were shown to mediate uptake by receptor T cells. Figure 4 A. Schematic representation of viral glycoproteins facilitating EV uptake and fusion at the cytoplasmic membrane or in endosomes. Figure 4 B. Uptake of dTomato-labeled VSV-G EVs by Jurkat T cells. Figure 4 C. Primary human CD4 + Uptake of dTomato-labeled VSV-G EVs by T cells. Figure 4 D. Surface expression of SLAM on T cells. Unmodified Jurkat, transgenic SLAM-expressing Jurkat, or primary human CD4 + Surface expression of SLAM on T cells. Figure 4E. Jurkat (+ / -SLAM) uptake of dTomato-labeled measles virus glycoprotein H / F EV. Figure 4 F. Primary human CD4 + T cell uptake of EVs labeled with measles virus glycoprotein H / F. In all cases, EVs were incubated with cells for 16 h and surface-bound vesicles were removed with trypsin. Experiments were performed in biological triplicate and error bars represent standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001). EV dTomato loading was assessed as Fig.21 shown.

[0051] Figure 5 We show that EVs mediate functional delivery of Cas9-gRNA in primary human T cells. Figure 5 A. Graphical representation of the functional delivery assessment. 2.0×10 10 EV and 5.0×10 4 CD4 + T cells were incubated for 6 days before genomic DNA extraction and HTS analysis. Figure 5 B. Indel frequencies detected at the CXCR4 locus targeted by Cas9. The sgRNA recognition site (green), PAM sequence (underlined, red), and predicted cleavage site (amplicon position 26, scissors) are shown. The total percentage of HTS reads classified as "edited" is represented by the area below the histogram track shown for each sample. Figure 5 C. Distribution of EV-Cas9-mediated edits, by type. DNA amplicon positions are plotted on the abscissa and observed edit lengths are plotted on the ordinate, with the size of each dot scales with the number of edits that meet that description. Each read is uniquely classified as a deletion, insertion, or substitution, such that no single read produces more than one dot in this plot. In the case of substitutions, the positive ordinate reports the inserted portion of the edit and the negative ordinate reports the deleted portion of the edit, so that each edit corresponds to two dots in this case. In this plot, deletions are reported by placing a dot at the midpoint of the deleted segment. To help interpret the apparent “V” pattern, the dots are colored blue to represent cases where one end of the deleted segment corresponds to a predicted cut region, likely corresponding to a subset of the observed DNA repair outcomes. Sample dots are colored as shown in the figure. Figure 5As shown in B.

[0052] Figure 6 We show that CD2 engagement and repeated dosing enhance EV-mediated functional cargo delivery and vary with vesicle subpopulations. Figure 6 A. Schematic representation of the strategy to explore the requirement for scFv-CD2 engagement by blocking CD2. Figure 6 B. Blocking CD2 on recipient cells prior to adding EVs increases total editing of all vesicle types. 4 CD4 + T cells were incubated at 8.0 × 10 9 EVs were grown for 6 days before genomic DNA extraction and HTS analysis. Heatmap coloring ranges from 0-6% of total Cas9-mediated editing. Figure 6 C-6D. Schematic representation of experiments exploring Cas9-mediated editing with repeated EV administration and various CD2 engagement modes ( Figure 6 C) and evaluation ( Figure 6 D) Shows two independent experiments using different donor cells and EV preparations. EV doses were: donor 1—5 × 10 4 Cells 1.25×10 10 Microvesicles (MV) or 5.50×10 9 Exosomes (exo); Donor 2—5×10 4 Cells 1.50×10 10 Microvesicles (MV) or 7.50×10 9 Exosomes (exo). Heatmap coloring as Figure 6 As shown in B. Figure 6 E. EV-mediated delivery of Cas9 function showed consistent trends across 3 donors and EV batches. For each of three independent experiments, editing efficiency was normalized to samples that received VSV-G exosomes (open bars). Figure 6 F. Figure 6 Combined analysis of the experiments presented in D. Within each vesicle population, editing efficiency was normalized to samples that received multiple doses of VSV-GEV (open bars); this normalization strategy was designed to control for the largest expected source of variability (i.e., intrinsic donor / T cell batch-specific susceptibility to EVs and editing). Error bars represent one standard deviation.

[0053] Figure 7 EVs harvested from cell lines expressing anti-CD2 scFv were shown to contain the full-length scFv. Figure 7 A. Surface staining of stable cell lines expressing anti-CD2 scFvs with different linkers between the binding and transmembrane domains (via 3x FLAG tag). Figure 7 B-7C. scFv in the stable HEK293FT production cell line ( Figure 7 B) or EVs harvested from these cell lines ( Figure 7 C) expression. Expected band size: ~38-40 kDa (arrow). Each lane was loaded with 0.5 μg of cell lysate or 1.0×10 8 EV.

[0054] Figure 8 EVs harvested by differential ultracentrifugation were shown to display characteristic surface markers, size distribution, and morphology. Figure 8 A. Detection of CD9 (25 kDa), CD81 (26 kDa), and Alix (96 kDa) in microvesicle (MV) and exosome (Exo) EV fractions. The EV fraction contains the least calnexin (~90 kDa). The expected band position is indicated by the arrow. Each lane was loaded with 3 μg of cell lysate or 4.5×10 8 Vesicle. Figure 8 B. Representative NTA size distribution of EV subpopulations. Numbers above the histograms indicate the mode size. Error bars (black) indicate the standard error of the mean calculated for each bin. Figure 8 C. Representative TEM of EV subpopulations.

[0055] Fig. 9 Jurkat T cells were shown to express CD2 on the cell surface. Cells were surface stained for CD2 expression and analyzed by flow cytometry.

[0056] Fig.10 Repeated washing is shown to remove non-specifically bound EVs from Jurkat. EVs loaded with dTomato were incubated with Jurkat T cells for 2 h at 37 °C and washed for varying numbers of times to remove excess vesicles before analysis by flow cytometry. Cells were treated with trypsin for 5 min after EV incubation and used as a reference (representative of complete removal of EVs from the cell surface). Three washes were performed in all subsequent binding assays.

[0057] Fig.11 It was shown that EVs harvested from fluorescent cells had similar mean fluorescence in subsets. Fig.11 A. Figure 2B. Assessment of EV dTomato loading. EVs were adsorbed to acetaldehyde / sulfate latex beads and analyzed by flow cytometry to determine overall population fluorescence. Experiments were performed in biological triplicate and error bars represent standard error of the mean. Fig.11 B. Fig.11 Representative histograms of the fluorescence distribution of EV-loaded beads shown in A. Fig.11 C-11H. Figure 2 D and Fig.12 ( Fig.11 C) Figure 2 E( Fig.11 D) Figure 2 F( Fig.11 E) Figure 2 G-2H( Fig.11 F), Fig.15 D( Fig.11 G) and 15F( Fig.11 H) Assessment of EV dTomato loading.

[0058] Fig.12 Showing that blocking EV recipient cells with blank EVs did not affect targeted or background binding. Recipient Jurkat T cells were incubated for 1 h in the presence or absence of non-fluorescent, non-targeted EVs (red) to block scavenger receptors and then incubated with fluorescent, targeted vesicles (purple) for 2 h. Experiments were performed in biological triplicate and error bars represent standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001). EV dTomato loading was assessed as Fig.11 As shown in C.

[0059] Fig.13 Codon optimization was shown to increase scFv display on EVs without altering EV morphology. Fig.13 A. Expression of scFv constructs in EV production cell lysates with different levels of codon optimization. Low-expressing (unoptimized) constructs were used in previous experiments, medium-expressing constructs were generated by artificial codon optimization, and high-expressing constructs were optimized by FisherGeneArt synthesis. Low-expressing and high-expressing constructs were further evaluated. 0.2 μg of cell lysate was loaded per lane. Expected band size: ~40 kDa (arrow). Fig.13 B. Expression of original and optimized scFv constructs in stable EV production cell lines. 0.2 μg of cell lysate was loaded per lane. Fig.13C.scFv in Fig.13 EVs harvested from cells are shown in B. 4.5 × 10 8 EV. Fig.13 D. Representative NTA size distribution of EV subpopulations displaying optimized scFvs. Numbers above the histograms indicate the mode size. Error bars (black) indicate the standard error of the mean calculated for each bin. Fig.13 E. Representative TEM of EVs displaying optimized scFv.

[0060] Fig.14 Primary T cells were shown to express CD2 on the cell surface. Cells were surface stained for CD2 expression and analyzed by flow cytometry.

[0061] Fig.15 showed that different scFv display technologies resulted in different EV targeting properties. Fig.15 A. Cartoon highlighting the structure of the PDGFR transmembrane domain scFv display and the lactadherin C1C2 domain anchored to phosphatidylserine. Fig.15 B. Expression of scFv constructs in EV production cell lysate. 1 μg of cell lysate was loaded per lane. Expected band sizes: ~40 kDa and ~75 kDa (black arrows). Fig.15 C. Loading the scFv construct into Fig.15 EVs produced by the cell lines in B. 5.0 × 10 8 EV. Fig.15 D. Targeted EVs bind to Jurkat T cells after 2 h of incubation. Fig.15 E. corresponds to Fig.15 Representative histograms of the summary data reported in D. Red boxes indicate subpopulations of cells showing skewed, high exosome binding. Fig.15 F. Recipient Jurkat T cells were incubated in the presence or absence of anti-CD2 antibody for 1 h and then incubated with EVs for 2 h. Fig.15 G. corresponds to Fig.15 Representative histograms of the summary data reported in F. Flow cytometry experiments were performed in biological triplicate, and error bars ( Fig.15 D-15F) Means with standard errors are indicated. EV dTomato loading was assessed as Fig.11 Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001).

[0062] Fig.16 showed that ABA-binding domains can be incorporated into EV cargo proteins. Fig.16 A. Expression of EYFP fused to ABI and PYL ABA binding domains with and without NLS in transiently transfected HEK293FT cells was analyzed by flow cytometry. Experiments were performed in biological triplicate and error bars represent standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001). Fig.16 B. Surface staining of HEK293FT transfected with anti-CD2 scFv constructs fused to ABI or PYL at the C-terminus (via 3x FLAG tag). Fig.16 C.From Fig.16 Expression of anti-CD2 scFv constructs of B. 2 μg of cell lysate loaded per lane. Expected band sizes: -40, 62 and 75 kDa (arrows).

[0063] Fig.17 ABA was shown to induce dimerization between the ABT and PYL domains. Fig.17 A-17B. Using ethanol (EtOH) Fig.17 A) or ABA( Fig.17 B) HEK293FT cells transfected with anti-CD2 scFv-PYL and EYFP-ABI were treated and imaged by confocal microscopy. Bright field, fluorescence, contrast-adjusted and pseudo-colored fluorescence and overlays are shown.

[0064] Fig.18 We show that the ABI domain increases EV cargo loading independent of total protein expression. Fig.18 A. Expression of EYFP and EYFP-ABI in transiently transfected HEK293FT cells in the presence of anti-CD2 targeting constructs was analyzed by flow cytometry. The key observation is that addition of the ABI domain does not increase overall cargo protein expression in producer cells. Fig.18 B. Figure 3 Replicate of EYFP-ABI EV loading trends in the presence of scFv shown in C. Fig.18C. Comparison of EYFP loading into EVs with ABA-binding constructs under ABA-induced dimerization conditions with and without NLS. The addition of NLS did not significantly affect EYFP loading, nor did ABA-induced dimerization significantly affect loading of nuclear-localized cargoes. Experiments were performed in biological triplicate and error bars represent standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001).

[0065] Fig.19 We show that the ABI domain increases the loading of Cas9 into EVs and that Cas9-ABI retains functionality. Fig.19 A. Expression of Cas9 fused to ABI or PYL domains in transiently transfected HEK293FT cells. 2 μg of cell lysate was loaded per lane. Expected band sizes: -160, 183 and 195 kDa (arrows). Fig.19 B. Cartoon showing the Cas9 reporter construct. Successful editing by Cas9 results in the loss of the stop codon and (in the case of some random fractions) repair-mediated frameshift inducing expression of dTomato. Fig.19 C. The absence of NLS or the presence of ABI domain does not substantially reduce the editing efficiency of Cas9 in transiently transfected Jurkat T cells. Cells were analyzed by flow cytometry 3 days after transfection. The experiment was repeated three times biologically, and the error bars represent the standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001). Samples with high cellular autofluorescence were excluded from the analysis. Fig.19 D. Complete blot of Cas9 EV active loading data Figure 3 As shown in E. Fig.19 E. Cellular expression of Cas9 with and without ABI domain or NLS. 2 μg of cell lysate was loaded per lane.

[0066] Fig. 20 showed that EV populations can be separated by affinity chromatography to analyze cargo loading patterns. Fig. 20A. Validation of affinity chromatography technique. Vesicles containing 3×FLAG-tagged scFv were flowed through an anti-FLAG affinity matrix and analyzed for FLAG tag to demonstrate enrichment in the eluted population. 1.5×10 7 EVs. Expected band size: ~62 kDa (arrow). Fig. 20 B. Figure 3 Full blot of affinity-isolated EV Cas9 contents with and without ABA-induced dimerization presented in F.

[0067] Fig.21 Evaluation of EV dTomato loading for vesicle uptake and fusion experiments are shown. Fig.21 A-21E. Figure 4 B( Fig.21 A) Figure 4 C( Fig.21 B) Figure 4 E( Fig.21 C) Figure 4 F( Fig.21 D) and Fig. 22 ( Fig.21 E) EV fluorescence control. EVs were adsorbed onto aldehyde / sulfate latex beads and analyzed by flow cytometry to determine overall population fluorescence. Experiments were performed in biological triplicate and error bars represent standard error of the mean.

[0068] Fig. 22 Display of Cx43 on EVs does not result in increased EV uptake by Jurkat T cells. dTomato EVs were incubated with Jurkat for 16 h. Cells were trypsinized to remove surface-bound vesicles and then analyzed by flow cytometry. Experiments were performed in biological triplicate and error bars represent standard error of the mean. Statistical tests included two-tailed Student's t-tests using the Benjamini-Hochberg method to reduce the false discovery rate (*p<0.05, **p<0.01, ***p<0.001). EV dTomato loading was assessed in Fig.21 E.

[0069] Fig.23 We show that EV-mediated Cas9-gRNA editing results in the generation of indels around the predicted CXCR4 cleavage site. Fig.23 A-23D. In primary human CD4 + Point mutations observed in T cells ( Fig.23 A) missing ( Fig.23 B) Insert Fig.23C) and substitutions (defined as edits containing both insertions and deletions) ( Fig.23 D) as a function of the percentage of sequenced reads classified as edited. Each edit observed was uniquely classified into one of these four categories. The predicted Cas9 cleavage site is Figure 5 Position 26 of the amplicon shown.

[0070] Fig.24 The presence of the ABI active loading domain generally does not affect Cas9 editing efficiency. 4 CD4 + T cells were incubated at 8.0 × 10 9 EVs were maintained for 6 days followed by genomic DNA extraction and NGS analysis. Heatmap coloring ranges from 0-0.3% of total Cas9 editing.

[0071] Fig.25 CD2 engagement does not affect the activation state of primary T cells. Primary human CD4 + T cell staining. Unstimulated cells were used as a control for background anti-CD25 staining and isotype controls were used to determine the effect of treatment on general cellular staining. Fluorescence was normalized using calibration beads to allow comparison of signals across days. MEPE: Entity Equivalent PE.

[0072] Fig.26 A representative flow cytometry gating strategy for EV delivery experiments is shown. Fig.26 A. Identify live cells based on their FSC-A and SSC-A profiles, and identify singlets from live cells based on their FSC-A and FSC-H profiles. Quantify the mean fluorescence intensity from singlets. Fig.26 B. Identification of aldehyde / sulfate latex beads based on their FSC-A and SSC-A profiles. Quantification of mean fluorescence intensity from the beads.

[0073] Fig. 27An overview of the plasmids used in Example 18 is shown. The first plasmid encodes the lentiviral auxiliary genes: gag, pol, rev and tat, where expression is driven by the cytomegalovirus promoter (pCMV). The second plasmid encodes the lentiviral backbone, including the 5' long terminal repeat (LTR) and the 3' LTR, in which the U3 promoter sequence has been destroyed by deletion, thereby generating a self-inactivating lentiviral vector. The lentiviral payload is driven by the human elongation factor la promoter (phEFla) and contains the mNeonGreen fluorescent protein. The 3' portion of the lentiviral genome includes a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to stabilize the lentiviral genomic RNA and the payload encoding the mRNA driven from the vector. The third plasmid encodes a fusogen (driven by the pCMV promoter); the fusogen gene contained in the vector includes a wild-type vesicular stomatitis virus glycoprotein, VSV-G (VSVGwt), or a mutant version of VSV-G that cannot bind to its cognate receptor (VSVGmut), or a non-coding sequence (blank). The fourth plasmid encodes a membrane displayed targeting chimeric polypeptide (referred to herein as "binder"); the binder gene contained in this vector includes a single chain variable fragment of an antibody that binds to CD2 (anti-CD2 scFv), or a variable lymphocyte receptor binding domain that binds to CD5 (anti-CD5 VLR).

[0074] Fig.28 A shows transduction of Jurkat T cells with extracellular vesicles containing lentiviral cores (EV-LVs) displaying wild-type (wt) VSV-G as a fusion proent entity and in combination with a targeting domain selected from anti-CD2 and anti-CD5. Four EV-LV doses were tested, 1 ul, 2.5 ul, 10 ul, 25 ul and 100 ul, with or without polybrene. B shows transduction of Jurkat T cells evaluated under similar conditions to A, but in this case, the dose is reported as viral genomes added per recipient Jurkat T cell (quantified by qPCR).

[0075] Fig.29 AC shows transduction of Jurkat T cells with EV-LV displaying wild-type (wt) VSV-G and A no targeting domain; B anti-CD2; or C anti-CD5 VLR; according to flow-based fluorescence readout. These histograms correspond to Fig.28 Virus doses and samples reported in B.

[0076] Fig.30AB A shows transduction of Jurkat T cells with EV-LV displaying mutated (mut) VSV-G as the fusion progeny entity and in combination with a targeting domain selected from anti-CD2 and anti-CD5. Four EV-LV doses were tested, 1 ul, 2.5 ul, 10 ul, 25 ul and 100 ul, with or without polybrene. B shows that Jurkat T cell transduction was evaluated under similar conditions to A, but in this case, the dose is reported as viral genomes added per recipient Jurkat T cell (quantified by qPCR).

[0077] Fig.31 AC shows transduction of Jurkat T cells with EV-LV displaying mutated (mut) VSV-G and A no targeting domain; B anti-CD2; or C anti-CD5 VLR; according to flow-based fluorescence readout. These histograms correspond to Fig.30 Virus doses and samples reported in B.

[0078] Fig.32 AB A shows transduction of Jurkat T cells with EV-LV displaying mutant (mut) VSV-G and the indicated affinity reagents; this figure is redrawn Fig.31 The highest EV-LV dose condition in is shown for comparison. B shows transduction of Jurkat T cells with bilayer lipid particles displaying only the targeting domain. The bilayer lipid particles do not display the fusogen. This figure is redrawn Fig.31 for comparison.

[0079] Fig.33 AI shows transduction of HEK293FT cells with EV-LV. In each case, EV-LV were formulated such that their surface contained neither fusogen nor targeting chimeric polypeptide (A), only targeting chimeric polypeptide (D, G), only fusogen (B, C), or a combination of fusogen and targeting chimeric polypeptide (E, F, H, I).

[0080] Fig.34An overview of the plasmids used in Example 19 is shown. The first plasmid encodes the lentiviral auxiliary genes: gag, pol, rev and tat, where expression is driven by the cytomegalovirus promoter (pCMV). The second plasmid encodes the lentiviral backbone, including the 5' long terminal repeat (LTR) and the 3' LTR, in which the U3 promoter sequence has been destroyed by deletion, thereby generating a self-inactivating lentiviral vector. The lentiviral payload is driven by the human elongation factor 1a promoter (phEF1a) and contains the miRFP720 fluorescent protein. The 3' portion of the lentiviral genome includes a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to stabilize the lentiviral genomic RNA and the payload encoding the mRNA driven from the vector. The third plasmid encodes a fusion progenitor (driven by the pCMV promoter); the fusion progenitor gene contained in the vector includes the wild-type vesicular stomatitis virus glycoprotein VSV-G (VSVGwt) or a mutant version of VSV-G that cannot bind to its cognate receptor (VSVGmut). The fourth plasmid encodes a targeting chimeric polypeptide (referred to herein as "binder"); the binder gene contained in this vector includes a variable lymphocyte receptor binding domain that binds to CD5 (anti-CD5 VLR).

[0081] Fig.35 AB A shows transduction of activated human CD4+T cells with EV-LV displaying wild-type (wt) VSV-G or mutated (mut) VSV-G as fusion progenitor entities and combined with anti-CD5 targeting chimeric polypeptides, B shows transduction of activated human CD8+T cells with EV-LV displaying wild-type (wt) VSV-G or mutated (mut) VSV-G as fusion progenitor entities and combined with anti-CD5 targeting chimeric polypeptides. The use of two different backbones that differ only in backbone sequence (backbone versions 1 or 2 as shown) shows reproducibility between various lentiviral backbones.

[0082] Fig.36 Shown are activated human CD4+ T cells transduced with EV-LV displaying wild-type (wt) VSV-G or mutated (mut) VSV-G as fusogen entities and in combination with anti-CD5 target chimeric polypeptides. These histograms correspond to Fig.35 Virus doses and samples reported in A. The reproducibility between various lentiviral backbones was shown using two different backbones that differed only in backbone sequence (backbone versions 1 or 2 as indicated).

[0083] Fig.37 Shown are activated human CD8+ T cells transduced with EV-LV displaying wild-type (wt) VSV-G or mutated (mut) VSV-G as fusogen entities combined with an anti-CD5 targeting domain. These histograms correspond to Fig.35 Virus doses and samples reported in B. Two different backbones (version 1 and version indicated with suffixes) were used to show reproducibility between various lentiviral backbones.

[0084] Fig.38 HEK293FT cells were transduced with EV-LV displaying wild-type (wt) VSV-G or mutated (mut) VSV-G as fusion proents in combination with an anti-CD5 targeting domain. Two different backbones that differed only in backbone sequence (backbone versions 1 or 2 as shown) were used to show reproducibility between the various lentiviral backbones. Detailed description

[0085] The present disclosure provides surprising insights and useful techniques for delivering cargo entities to interested receptor cells. In addition, the present disclosure recognizes that the extracellular vesicles displaying the fusion protein comprising a targeting domain and a transmembrane domain (e.g., a PDGFR transmembrane domain) show particularly efficient and / or effective vesicle targeting to specific receptor cells. Exemplary useful such fusion proteins are described in PCT Publication WO2019 / 199941 (titled "Extracellular vesicles of membrane proteins comprising targeting affinity domains", and disclosed on October 17, 2019, the content of which is incorporated herein by reference in its entirety), which proves that when displayed on various vesicle types (e.g., exosomes, microvesicles), this fusion protein can mediate vesicle uptake by receptor cells. The present disclosure provides surprising further progress related to such fusion proteins, demonstrating that, for example, their combination with the fusion pro-entity polypeptides described herein can achieve significantly efficient and / or effective targeted delivery of cargo to specific receptor cells of interest, including specific human and / or immune cells, particularly T cells (e.g., human T cells), on various lipid bilayer particles, thereby providing unique and important value in this field.

[0086] Those skilled in the art will understand after reading this specification that when a cargo entity is delivered to a recipient cell according to the teachings herein, in some embodiments such cargo entity can modify (e.g., genetically modify) the recipient cell, such as for a variety of applications, including various therapeutic applications. The cargo entity can be delivered to a specific recipient cell via lipid bilayer particles, including cell-derived membrane particles (CDMPs) (e.g., extracellular vesicles (EVs)).

[0087] As noted herein, particularly useful embodiments of the technology provided achieve genetic modification of T cells. Genetically modified T cells can achieve applications from cancer immunotherapy to HIV treatment, but the delivery of T cell targeted therapy remains challenging. Extracellular vesicles (EVs) and other cell-derived membrane particles (CDMPs) are nanoscale particles secreted by cells that naturally encapsulate and transfer proteins, nucleic acids, and nucleic acid-protein complexes, such as viral nucleocapsids (e.g., enveloped viruses), making them attractive and clinically relevant biocompatible delivery tool engineering platforms. The present disclosure provides a set of technologies for genetically engineering production cells to produce multifunctional lipid bilayer particles (e.g., CDMPs such as EVs)—without the need to employ chemical modifications that complicate biomanufacturing. The present disclosure further provides high-affinity targeting domains (e.g., targeting chimeric polypeptides) and / or fusion protoentities displayed on the surface of lipid bilayer particles to achieve specific, effective binding to receptor cells (e.g., immune cells, such as T cells). Fusion protoentity polypeptides (e.g., glycoproteins) can increase the uptake of lipid bilayer particles and fusion with receptor cells. The present disclosure also identifies protein tags to enable active cargo loading into lipid bilayer particles. The examples herein demonstrate the integration of these technologies by delivering Cas9-sgRNA complexes to edit primary human T cells, delivering viral nucleocapsid derivatives (e.g., lentiviral nucleocapsids), or delivering fluorescent proteins to verify the fusion of lipid vesicles with recipient cells. These methods can target particles to a range of cells, thereby effectively delivering cargo.

[0088] It should be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in varying levels of detail in order to provide a substantial understanding of the present technology.

[0089] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, 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., NY); MacPherson et al., (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al., (1995) PCR 2: APractical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hamesand 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. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed.(2003) GeneTransfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., eds (1996) Weir's Handbook of Experimental Immunology. definition

[0090] Unless otherwise stated or indicated by the context, the terms "a", "an" and "the" mean "one or more". For example, "a fusion protein", "an extracellular vesicle" and "a cell" should be interpreted as meaning "one or more fusion proteins", "one or more extracellular vesicles" and "one or more cells", respectively.

[0091] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If the use of these terms is unclear to one of ordinary skill in the art given the field in which they are used, "about" and "approximately" will mean plus or minus ≤10% of the particular term, and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0092] "Affinity" as used herein is a measure of the tightness with which two or more binding partners bind to each other. Those skilled in the art know that a variety of assays can be used to assess affinity, and also know suitable controls for such assays. In some embodiments, affinity is assessed in quantitative assays. In some embodiments, affinity is assessed at multiple concentrations (e.g., one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competing entities (e.g., may be present in a relevant--e.g., physiological--environment). In some embodiments, affinity is assessed relative to a reference (e.g., with a known affinity above a specific threshold or with a known affinity below a specific threshold). In some embodiments, affinity can be assessed relative to a reference performed simultaneously; in some embodiments, affinity can be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.

[0093] As used herein, "binding moiety" is used herein to refer to a portion that binds to a target ligand of interest as described herein (e.g., a target ligand on a receptor cell surface, or a population thereof). In many embodiments, the binding moiety of interest is a binding moiety that specifically binds to its target ligand, which distinguishes its target ligand from other potential binding partners in a specific interaction context. In some embodiments, relative to one or more other entities on the receptor cell surface, the binding moiety displays specific binding to its target ligand. Alternatively or additionally, in some embodiments, relative to one or more (or all) entities present on the surface of a non-receptor cell (e.g., a non-receptor cell that may be present in a system including a receptor cell), the binding moiety displays preferential binding to its target ligand. In some embodiments, the binding moiety binds to one or more target ligands and drives a specific biological activity associated only with a specific target ligand. In some embodiments, the binding moiety is a peptide binding moiety. In some embodiments, the binding moiety is a non-peptide binding agent. In some such embodiments, the production cell can be engineered to express a targeting chimeric polypeptide comprising a binding portion, which is subsequently modified (e.g., chemically modified) by attaching a non-polypeptide binding portion so that the non-polypeptide binding portion provides specific affinity for the target ligand. In some embodiments, the binding agent comprises (i) a targeting chimeric polypeptide, and optionally a non-polypeptide portion, and (ii) binds to a target cell when present on the surface of a lipid bilayer particle. In general, the binding portion can be or comprise a portion of any chemical class (e.g., polymers, non-polymers, small molecules, polypeptides, carbohydrates, lipids, nucleic acids, etc.). In some embodiments, the binding portion is a single chemical entity. In some embodiments, the binding portion is a complex of two or more discrete chemical entities that bind to each other through non-covalent interactions under relevant conditions. For example, one skilled in the art will appreciate that in some embodiments, the binding portion may comprise a "universal" binding portion (e.g., one of biotin / avidin / streptavidin and / or a class-specific antibody) and a "specific" binding portion (e.g., an antibody or aptamer with a specific molecular target) connected to a partner of the universal binding portion. In some embodiments, such methods can allow modular assembly of multiple affinity moieties by connecting different specific binding moieties to the same universal binding moiety partner. In some embodiments, the binding moiety is or comprises a polypeptide (including, for example, an antibody or antibody fragment). In some embodiments, the binding moiety is or comprises a small molecule. In some embodiments, the binding moiety is or comprises 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 moieties are non-polymeric because they lack a polymeric moiety.In some embodiments, the binding moiety is or comprises a carbohydrate. In some embodiments, the binding moiety is or comprises a peptide mimetic. In some embodiments, the binding moiety is or comprises a scaffold protein. In some embodiments, the binding moiety is or comprises a mimeotope. In some embodiments, the binding moiety is or comprises a stapled peptide. In certain embodiments, the binding moiety is or comprises a nucleic acid, such as DNA or RNA.

[0094] As used herein, the phrase "characteristic sequence element" refers to a sequence element representing the characteristic part of the polymer found in a polymer (for example, in a polypeptide or nucleic acid). It is understood by those skilled in the art that the presence of a characteristic sequence element is generally related to the presence or level of a specific activity or property of a polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a specific polymer as a member (or non-member) of a specific family or group of such polymers. The characteristic sequence element generally comprises at least two monomers (for example, amino acids or nucleotides). In some embodiments, the characteristic sequence 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 (for example, monomers connected in succession). In some embodiments, the characteristic sequence element at least comprises the first and second sections of continuous monomers separated by one or more spacers, and the length of the spacer may vary or not vary between the various polymers of the shared sequence element.

[0095] As used herein, "comparable" refers to two or more reagents, entities, situations, condition groups (sets of conditions) etc., which may be different from each other, but are sufficiently similar to allow comparisons between them, so that those skilled in the art will appreciate that it is possible to reasonably draw conclusions based on observed differences or similarities. In some embodiments, comparable condition groups, environment, individuality or colony are characterized by a plurality of substantially identical features and one or a small amount of different features. It will be appreciated by those of ordinary skill in the art that in context, in any given case, two or more such reagents, entities, situations, condition groups etc. are considered to be comparable, requiring which degree of identicalness. For example, it will be appreciated by those of ordinary skill in the art that when characterized with substantially identical features of sufficient quantity and type, thereby drawing reasonable conclusions, that is, the difference in the result or observed phenomenon obtained under or with the environment, individuality or colony of different groups is caused by the variation of those different features or shows the variation of these features, so the environment, individuality or colony of these groups are comparable to each other.

[0096] As used herein, a "control" is an alternative sample used for comparison purposes in an experiment. A control can be "positive" or "negative". For example, when the purpose of the experiment is to determine the correlation between the efficacy of cargo protein loading to EV 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 not loaded onto EV) are typically used.

[0097] As used herein, "corresponding to" refers to a 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., relative to an appropriate 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 determined as "corresponding to" a residue in an appropriate reference polymer. For example, a person of ordinary skill will understand that, for the purpose of simplicity, residues in a polypeptide are typically designated using a canonical numbering system based on a reference to a related polypeptide, so that, for example, an amino acid "corresponding to" a residue at position 190 does not actually have to be the 190th amino acid in a particular amino acid chain, but rather corresponds to a residue at position 190 in a reference polypeptide; a person of ordinary skill in the art will readily understand how to determine a "corresponding" amino acid. For example, those skilled in the art are aware of various sequence alignment strategies, including software programs such as 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 that can be used, for example, to determine "corresponding" residues in polypeptides and / or nucleic acids according to the present disclosure. Those skilled in the art will also understand that in some cases, the term "corresponding to" can be used to describe an event or entity that has a relevant similarity to another event or entity (e.g., an appropriate reference event or entity). As just one example, a gene or protein in one organism can be described as "corresponding to" a gene or protein from another organism to indicate that, in some embodiments, it plays a similar role or performs a similar function and / or that it shows a certain degree of sequence identity or homology, or shares certain characteristic sequence elements.

[0098] As used herein, the term "engineered" refers to aspects designed, produced and / or manipulated by human hands. For example, when two or more sequences that are not connected together in this order in nature are designed by human hands or otherwise caused to be directly connected to each other in an engineered polynucleotide, and / or when specific residues in a polynucleotide are non-naturally present and / or are caused to be connected to an entity or part (an entity or moiety) that is not connected to it in nature by the action of human hands, the polynucleotide is considered to be "engineered". For example, in some embodiments described and / or utilized herein, the engineered polynucleotide contains a regulatory sequence found in nature that is operably associated with a first coding sequence but not operably associated with a second coding sequence, and is connected by human hands so that it is operably associated with the second coding sequence. Similarly, in some embodiments, if a polypeptide is encoded or expressed by an engineered polynucleotide, and / or if it is produced in a cell in a non-naturally expressed manner, the polypeptide can be considered to be "engineered". Similarly, a cell or organism is considered "engineered" if it has been manipulated so that its genetic, epigenetic and / or phenotypic characteristics are changed relative to an appropriate reference cell, such as an otherwise identical cell that has not been manipulated. In some embodiments, the manipulation is or includes genetic manipulation so that its genetic information is changed (e.g., new genetic material that did not previously exist is introduced, such as by transformation, mating, somatic cell hybridization, transfection, transduction or other mechanisms, or previously existing genetic material is changed or removed, such as by substitution or deletion mutations, or by mating methods). In some embodiments, an engineered cell is a cell that has been manipulated so that it contains and / or expresses a specific agent of interest (e.g., a protein, nucleic acid and / or a specific form thereof) with an altered amount and / or altered timing relative to an appropriate reference cell. As is common practice and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell are still generally referred to as "engineered", even if the actual manipulation was performed on a previous entity.

[0099] As used herein, "engineered lipid bilayer particles" refer to lipid bilayer particles engineered as described herein. For example, in some embodiments, if the lipid bilayer particles are synthetically produced, i.e., not produced by cells, it can be considered to be "engineered". As an alternative or supplement, in some embodiments, if the lipid bilayer particles are produced by engineered production cells, it can be considered to be "engineered". In some embodiments, engineered lipid bilayer particles are produced by production cells engineered to have fusion progen (fusogen) entity polypeptides and / or targeted chimeric polypeptides on their surfaces. In some such embodiments, the difference between the engineered production cell and the appropriate reference cell is that it has been engineered to express fusion progen (fusogen) entity polypeptides, targeted chimeric polypeptides, or both, or to express one or both of them at different levels (e.g., elevated levels), so that the lipid bilayer particles (e.g., CDMP) produced (e.g., released) by such engineered production cells are compared to similar particles produced (e.g., released) by reference cells, with significantly higher affinity and / or specific binding to receptor cells or cell populations.

[0100] As used herein, the term "extracellular vesicle" is understood to include all nanoscale lipid vesicles, such as exosomes and microvesicles, secreted and / or sprouted by cells, respectively. As used herein, the term "exosome" refers to extracellular vesicles derived from internal endocytic compartments and multivesicular bodies, and the term "microvesicle" refers to vesicles that sprout directly from the cell surface. EV and its separation and analysis are well known to those skilled in the art. For example, see Doyle et al., Cells 8(7): 727(2019), the entire contents of which are incorporated herein by reference. Extracellular vesicles can be taken up by so-called extracellular vesicle (EV) receptor cells. As used herein, the term "receptor cell" can be interchanged with the term "target cell".

[0101] As used herein, the term "cell-derived membrane particles" should be understood to include any membrane-derived vesicles or particles that can be produced by blebbing or budding, and can include hybrid vesicles produced by mixing vesicles produced by cells and synthetic vesicles, as well as vesicles or particles produced by mechanical treatment of cells. Therefore, "cell-derived membrane particles" can include, but are not limited to, extracellular vesicles (as defined above), viral particles, virus-like particles (VLPs), apoptotic bodies, and platelet-like particles.

[0102] As used herein, the term "fusogen entity polypeptide" refers to a polypeptide that mediates fusion between lipid bilayers. As described in the present disclosure, in some embodiments, the presence of a fusogen entity polypeptide in an engineered lipid bilayer particle (particularly included on an engineered lipid bilayer particle displaying a targeting chimeric polypeptide) increases the efficiency, specificity and / or effectiveness of cargo delivery from such engineered lipid bilayer particles to specific receptor cells of interest.

[0103] As used herein, the term "gene" refers to a DNA segment containing information for regulating biosynthesis of an RNA product. It will be understood by those skilled in the art that a gene typically includes an expressed sequence (e.g., an open reading frame, which may, for example, include exons and introns. It will be further understood by the skilled artisan that a gene typically includes one or more promoters and / or other untranslated regions (e.g., enhancer elements, repressor elements, chromatin binding sites, etc.), which may control, regulate, or otherwise affect expression.

[0104] As used herein, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid entities. It is understood by those skilled in the art that homology can be determined by comparing the positions in each sequence compared for comparison purposes. When a certain position in the compared sequence is occupied by the same base or amino acid, the entity is homologous at that position. The degree of homology between sequences depends on the number of matches or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having 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 compared, the percentage of bases (or amino acids) is the same when comparing two sequences. This comparison and percentage homology or sequence identity can be determined using software programs known in the art. In some embodiments, comparison is performed using default parameters. A comparison 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; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at 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. If two sequences are less than 40% identical to each other or less than 25% identical to each other, they are considered "unrelated" or "non-homologous." In addition, those skilled in the art will understand that "homologous" polypeptides or nucleic acids can generally share one or more characteristic sequence elements, for example, the characteristic sequence elements may confer common structural and / or functional properties to the polypeptides containing it.

[0105] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising" in that the latter terms are "open" transition terms that do not limit a claim to only the listed elements following these transition terms. The term "consisting of, while encompassed by the term "comprising," should be interpreted as a "closed" transition term that limits a claim to only the listed elements following the transition term. The term "consisting essentially of, while encompassed by the term "comprising," should be interpreted as a "partially closed" transition term that allows for the addition of additional elements after the transition term, provided that these additional elements do not materially affect the basic and novel features of the claim.

[0106] As used herein, the term "joint" refers to the part of a multi-element reagent, which connects different elements to each other. For example, it is understood by those of ordinary skill in the art that the polypeptide whose structure includes two or more functional or organizational domains generally includes a section of amino acids connecting them to each other between these domains. In some embodiments, the polypeptide comprising the joint element has the overall structure of the general form S1-L-S2, wherein S1 and S2 can be identical or different, representing two domains connected to each other by a joint. In some embodiments, the length of the polypeptide joint is 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 in that it does not tend to adopt a rigid three-dimensional structure, but provides flexibility for the polypeptide. In some embodiments, the linker is characterized in that it adopts a rigid three-dimensional structure and provides stability for the polypeptide. When engineering polypeptides known in the art (e.g., fusion polypeptides), a variety of different linker elements can be appropriately used (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123).

[0107] As used herein, the terms "polynucleotide", "polynucleotide sequence", "nucleic acid" and "nucleic acid sequence" refer to nucleotides, oligonucleotides, polynucleotides (these terms are used interchangeably), or any fragments thereof. As will be clear from the context, these phrases may also refer to DNA or RNA (which may be single-stranded or double-stranded and may represent the sense strand or the antisense strand) of genomic, natural or synthetic origin.

[0108] With respect to polynucleotide sequences, the terms "percent identity" and "% identity" refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm can insert spaces in the compared sequences in a standardized and repeatable manner so as to optimize the alignment between the two sequences and thus achieve a more meaningful comparison of the two sequences. The percent identity of a nucleic acid sequence can be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, the entire contents of which are incorporated herein by reference). The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) provides a set of commonly used and freely available sequence comparison algorithms, and BLAST is available from multiple sources, including NCBI, Bethesda, Md., on its website. The BLAST software suite includes various sequence analysis programs, including "blastn", which is used to align known polynucleotide sequences with other polynucleotide sequences from multiple databases. There is also a tool called "BLAST 2Sequences" for directly comparing two nucleotide sequences in pairs. "BLAST 2 Sequences" can be accessed and used interactively on the NCBI website. The "BLAST 2 Sequences" tool can be used with blastn and blastp (described above).

[0109] With respect to polynucleotide sequences, percent identity can be determined over the length of an entire defined polynucleotide sequence, e.g., as defined by a particular SEQ ID number, or can be determined over a shorter length, e.g., over the length of a fragment taken from a larger, defined sequence, e.g., 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 only, and it is understood that any fragment length supported by the sequences shown herein (in the tables, figures, or sequence listing) can be used to describe a length over which percent identity can be determined.

[0110] With respect to polynucleotide sequences, a "variant," "mutant," or "derivative" can be defined as a nucleic acid sequence that has at least 50% sequence identity with a particular nucleic acid sequence over a certain length of one of the nucleic acid sequences, as determined using blastn and using the "BLAST 2 Sequences" tool available on the website of the National Center for Biotechnology Information. (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 can exhibit, for example, 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 greater sequence identity over a certain defined length.

[0111] Due to the degeneracy of the genetic code, a nucleotide sequence that does not show a high degree of identity may still encode a similar amino acid sequence, wherein a plurality of codons can encode a single amino acid. It should be understood that this degeneracy can be utilized to change the nucleotide sequence to produce a plurality of nucleotide sequences that all encode substantially identical proteins. For example, the polynucleotide sequence considered herein can encode proteins and can be codon optimized for expression in a particular host. In the art, codon usage frequency tables have been compiled for many host organisms, including humans, mice, rats, pigs, Escherichia coli, plants and other host cells.

[0112] With respect to polynucleotide sequences, "recombinant nucleic acid" is a non-naturally occurring sequence or a sequence having an artificial combination of two or more originally separated sequence fragments. This artificial combination is often achieved by chemical synthesis or more commonly by artificial manipulation of separated nucleic acid segments, such as by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that are altered only by adding, replacing or deleting a portion of a nucleic acid. Typically, a recombinant nucleic acid can include a nucleic acid sequence that is operably linked to a promoter sequence. Such a recombinant nucleic acid can be part of a vector used, for example, to transform a cell.

[0113] The nucleic acids disclosed herein can be "substantially isolated or purified." The term "substantially isolated or purified" refers to a nucleic acid that is removed from its natural environment and is 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 it is naturally associated.

[0114] "Transformation" or "transfection" describes the process of introducing exogenous nucleic acid (e.g., DNA or RNA) 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 rely on any known method for inserting exogenous nucleic acid sequences into prokaryotic or eukaryotic host cells. The method used for transformation or transfection is selected according to the type of host cell to be transformed, and can include, but is not limited to, phage or viral infection or non-viral delivery. Non-viral delivery methods of nucleic acids include lipofection, nuclear transfection, microinjection, electroporation, heat shock, particle bombardment, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virus particles (artificial virions) and agents to enhance DNA uptake. Lipofection is described in, for example, U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM Lipofectin TM ). Cationic and neutral lipids suitable for effective receptor recognition liposome transfection of polynucleotides include those described in WO 91 / 17424 of Felgner; WO 91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). The term "transformed cells" or "transfected cells" includes stably transformed or transfected cells in which the inserted DNA is capable of replicating 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 time. In another embodiment, the term also includes stably transfected cells.

[0115] The polynucleotide sequences considered herein may be present in an expression vector. For example, a vector may comprise: (a) a polynucleotide encoding an ORF of a cargo protein; (b) a polynucleotide expressing an ABA binding domain, such as a pyranoside resistance 1-like (PYL1) sequence or an abscisic acid insensitive 1 (ABI1) sequence. The polynucleotides present in the vector may be operably linked to a prokaryotic or eukaryotic promoter. "Operably linked" refers to a situation in which a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Operably linked DNA sequences may be very close or continuous, and when two protein coding regions need to be linked, they may be in the same reading frame. The vector considered herein may comprise a heterologous promoter (e.g., a eukaryotic or prokaryotic promoter) operably linked to a polynucleotide encoding a protein. "Heterologous promoter" refers to a promoter that is not a native or endogenous promoter of the expressed protein or RNA.

[0116] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0117] The term "vector" refers to a means by which a nucleic acid (e.g., DNA) can be introduced into a host organism or host tissue. There are many types of vectors, including plasmid vectors, phage vectors, cosmid vectors, bacterial vectors, and viral vectors. As used herein, a "vector" may refer to a recombinant nucleic acid that has been engineered for expression of a heterologous polypeptide (e.g., a fusion protein disclosed herein). The recombinant nucleic acid typically comprises a cis-acting element for expression of a heterologous polypeptide.

[0118] Any conventional vector for expression in eukaryotic cells can be used to introduce DNA directly into a subject. Expression vectors containing regulatory elements from eukaryotic viruses can be used for eukaryotic expression vectors (e.g., vectors containing SV40, CMV or retroviral promoters or enhancers). Exemplary vectors include vectors that express proteins under the guidance of promoters such as 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 may include eukaryotic or prokaryotic control sequences that regulate the expression of heterologous proteins (e.g., fusion proteins disclosed herein). Prokaryotic expression control sequences may include constitutive or inducible promoters (e.g., T3, T7, Lac, trp or phoA), ribosome binding sites or transcription terminators.

[0119] The vectors considered herein can be introduced and propagated in prokaryotes, which can be used to amplify the copy number of the vector introduced into eukaryotic cells or as an intermediate vector (e.g., amplify a plasmid as a part of a viral vector packaging system) in the production of vectors introduced into eukaryotic cells. Prokaryotes can be used to amplify the copy number of the vector and express one or more nucleic acids, such as providing a source for one or more proteins delivered to a host cell or host organism. The expression of proteins in prokaryotes can be carried out using Escherichia coli, which has a vector comprising a constitutive or inducible promoter for the expression of a fusion protein comprising a protein or a fragment thereof. The fusion vector adds many amino acids to the encoded protein, such as to the amino terminus of a recombinant protein. Such fusion vectors can be used for one or more purposes, such as: (i) increasing the expression of recombinant protein; (ii) increasing the solubility of recombinant protein; (iii) aiding in the purification of recombinant protein by acting as a ligand in affinity purification (e.g., histidine tag); (iv) tagging recombinant protein for identification (e.g., green fluorescent protein (GFP) or an antigen that can be recognized by a labeled antibody (e.g., HA); (v) promoting the localization of recombinant protein to a specific area of ​​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 may include 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 NLS has been studied. (See Makkerh et al. (1996) Curr Biol 6(8):1025-1027). Usually, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusogen part and the recombinant protein to enable the recombinant protein to be separated from the fusogen part after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin and enterokinase.

[0120] The currently disclosed method may include delivering one or more polynucleotides, such as one or more vectors described herein, one or more transcripts thereof, and / or one or more proteins transcribed therefrom to a host cell. Further contemplate host cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising such cells or produced by such cells. The disclosed extracellular vesicles can be prepared by introducing vectors expressing mRNA encoding the fusion proteins disclosed herein and cargo RNA. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Non-viral vector delivery systems include DNA plasmids, RNA (transcripts of vectors such as 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 free or integrated genomes after delivery to cells.

[0121] In the methods contemplated herein, host cells can be transiently or non-transiently transfected (i.e., stably transduced) with one or more vectors described herein. In some embodiments, cells are transfected when they exist naturally in the subject (i.e., in situ). In some embodiments, the transfected cells are taken from the subject (i.e., explanted). In some embodiments, the cells are derived from cells taken from the subject, such as cell lines. Suitable cells may 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 new cell lines comprising one or more vector-derived sequences. In the methods contemplated herein, cells can be transiently transfected with components of the system described herein (e.g., by transient transfection of one or more vectors, or by transfection with RNA), and modified by the activity of the complex to establish new cell lines comprising cells containing modifications but lacking any other exogenous sequences.

[0122] As used herein, the terms "protein" or "polypeptide" or "peptide" are used interchangeably to refer to polymers of amino acids. Generally, "polypeptide" or "protein" is defined as a relatively long amino acid polymer, which is generally greater than 50, 60, 70, 80, 90 or 100 amino acids in length. "Peptide" is defined as a relatively short amino acid polymer, which is generally 50, 40, 30, 20 or fewer amino acids in length.

[0123] "Proteins" contemplated herein generally comprise 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. These modifications can include, but are not limited to, acylation (e.g., O-acylation (ester), N-acylation (amide), S-acylation (thioester)), acetylation (e.g., addition of an acetyl group at the N-terminus of a protein or at a lysine residue), formylation, fatty acylation (e.g., attachment of lipoic acid, a C8 functional group), myristoylation (e.g., myristate, attachment of a C14 saturated acid), palmitoylation (e.g., palmitate, attachment of a C16 saturated acid), alkylation (e.g., addition of an alkyl group, such as a methyl group, at a lysine or arginine residue), isoprenylation or prenylation (e.g., addition of an isoprenoid group, such as farnesol or geranylgeraniol), C-terminal amidation, glycosylation (e.g., addition of a sugar group on asparagine, hydroxylysine, serine, or threonine to produce a glycoprotein). In contrast to glycation, which is considered the non-enzymatic attachment of sugars, polysialylation (e.g., addition of polysialic acid), glycoformulation (e.g., glycosylphosphatidylinositol (GPI) anchor formation), hydroxylation, iodination (e.g., thyroid hormones), and phosphorylation (e.g., addition of a phosphate group, typically on serine, tyrosine, threonine, or histidine).

[0124] With respect to proteins, the term "amino acid residue" may also include residues contained in the sequence consisting of homocysteine, 2-aminoadipic acid, N-ethylasparagine, 3-aminoadipic acid, hydroxylysine, β-alanine, β-alanine, allo-hydroxylysine, The amino acid residue is selected from the group consisting of amino acid residues selected from the group consisting of acid, 2-aminobutyric acid, 3-hydroxyproline, 4-aminobutyric acid, 4-hydroxyproline, pipecolic acid, 6-aminohexanoic acid, isodesmosine, 2-aminoheptanoic acid, allo-isoleucine, 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.

[0125] Proteins disclosed herein may include "wild-type" proteins and variants, mutants, and derivatives thereof. The term "wild-type" as used herein is a term understood by those skilled in the art, and refers to a typical form of an organism, strain, gene, or feature present in nature, as distinguished from a mutant or variant form. 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 one or more amino acid residue insertions, deletions, or substitutions relative to a reference molecule. A variant or mutant may include a fragment of a reference molecule. For example, relative to a reference polypeptide, a mutant or variant molecule may have one or more insertions, deletions, or substitutions of at least one amino acid residue.

[0126] With respect to proteins, "deletion" refers to an alteration of the amino acid sequence resulting in the lack of one or more amino acid residues. The deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100 or 200 amino acid residues or a range of amino acid residues defined by any of these values ​​(e.g., 5-10 amino acid deletions). Deletions may include internal deletions or terminal deletions (e.g., N-terminal truncations or C-terminal truncations of a reference polypeptide). A "variant", "mutant" or "derivative" of a reference polypeptide sequence may include a deletion relative to a reference polypeptide sequence.

[0127] With respect to proteins, a "fragment" is a portion of an amino acid sequence that is identical in sequence to a reference sequence but shorter in length than the reference sequence. The sequence contained in the fragment may be up to the entire length of the reference sequence, minus at least one amino acid residue. For example, the fragment may contain 5 to 1000 consecutive amino acid residues of the reference polypeptide, respectively. In some embodiments, the fragment may contain at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 consecutive amino acid residues of the reference polypeptide; in other embodiments, the fragment may contain 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 the reference polypeptide; or in other embodiments, the length of the fragment is within the range defined by any of these values ​​(e.g., a range of 50-100 consecutive amino acids of the reference polypeptide). Fragments may be preferentially selected from certain regions of the molecule. The term "at least a fragment" encompasses the full-length polypeptide. Fragments may include N-terminal truncations, C-terminal truncations, or both truncations relative to the full-length protein. A "variant," "mutant," or "derivative" of a reference polypeptide sequence may include a fragment of the reference polypeptide sequence.

[0128] With respect to proteins, the terms "insertion" and "addition" refer to changes in an amino acid sequence that result 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 defined by any of these values ​​(e.g., 5-10 amino acids are inserted or added). A "variant," "mutant," or "derivative" of a reference polypeptide sequence may include an insertion or addition relative to a reference polypeptide sequence. Variants of a protein may have an N-terminal insertion, a C-terminal insertion, an internal insertion, or any combination of an N-terminal insertion, a C-terminal insertion, and an internal insertion.

[0129] With respect to proteins, as used herein, "chimeric protein", "chimeric peptide", "fusion protein" or "fusion peptide" refers to a polypeptide produced by connecting or directly linking two or more functional domains from separate or the same protein via an amino acid linker, thereby producing a single polypeptide with functional properties derived from each of the original proteins. In some embodiments, the linker is 10-50 amino acids in length and is rich in glycine for flexibility and serine or threonine for solubility. In some embodiments, the linker is characterized in that it adopts a rigid three-dimensional structure and provides stability to the polypeptide. A "variant" of a reference polypeptide sequence may include a fusion polypeptide comprising a reference polypeptide.

[0130] With respect to proteins, the phrases "percent identity" and "% identity" refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods for amino acid sequence alignment are well known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally retain the charge and hydrophobicity of the substitution site, thereby retaining the structure (and therefore the function) of the polypeptide. The percent identity of an amino acid sequence can be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, the entire contents of which are incorporated herein by reference). The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) provides a set of commonly used and freely available sequence comparison algorithms, and BLAST is available from multiple sources, including NCBI, Bethesda, Md., on its website. The BLAST software suite includes different sequence analysis programs including "blastp", which is used to align a known amino acid sequence with other amino acid sequences from multiple databases. As described herein, a variant, mutant or fragment (e.g., a protein variant, mutant or fragment thereof) can have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% amino acid sequence identity relative to a reference molecule.

[0131] With respect to proteins, percent identity can be determined over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or can be determined over a shorter length, for example, a fragment taken from a larger, defined polypeptide sequence, for example, 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 consecutive residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein (in the tables, figures or sequence listing) can be used to describe the length over which percent identity can be determined.

[0132] With respect to proteins, the amino acid sequence of variants, mutants or derivatives contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant or derivative protein may include conservative amino acid substitutions relative to a reference molecule. A "conservative amino acid substitution" is a substitution in which one amino acid is substituted for another different amino acid, where the substitution is expected to have minimal interference with the properties of a reference polypeptide. In other words, a conservative amino acid substitution substantially retains the structure and function of a reference polypeptide. The following table provides a list of exemplary conservative amino acid substitutions contemplated herein:

[0133] Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of ​​the substitution, for example, maintaining a beta-sheet or alpha-helical conformation, (b) the charge or hydrophobicity of the molecule at the site of substitution, and / or (c) most side chains.

[0134] The proteins, mutants, and variants disclosed herein may have one or more functions or biological activities exhibited by a reference polypeptide (e.g., one or more functions or biological activities exhibited by a wild-type protein). For example, the disclosed proteins, their mutants, variants, or derivatives may have one or more biological activities, including binding to small molecule ABA and targeting EV to receptor cells.

[0135] The disclosed proteins can be substantially isolated or purified. The term "substantially isolated or purified" refers to a protein that is separated from its natural environment and is 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 it is naturally associated.

[0136] As used herein, the term "recombinant" when used to refer to, for example, a cell or a 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 by the alteration 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 genes not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.

[0137] As used herein, the term "targeting domain" or "targeting peptide" refers to a peptide portion that promotes specific binding of EV to a receptor cell. Sample "targeting domain" or "targeting peptide" includes, but is not limited to, antibodies and any antibody fragment or antigen binding fragment, such as Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked 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. Receptor cells

[0138] In addition, the present disclosure demonstrates the surprising effects of the provided technology when applied to receptor cells, e.g., specific receptor cells of interest or their populations. For example, the present disclosure describes the surprising specificity and / or efficiency of delivering cargo entities to receptor cells of interest or their populations using lipid bilayer particles, such as cell-derived membrane particles (CDMPs) described herein. Using the technology of the present disclosure, lipid bilayer particles deliver cargo entities to receptor cells in an effective and specific manner. In some embodiments, the provided technology delivers cargo entities when used ex vivo. In some embodiments, the provided technology achieves, for example, in vivo delivery of cargo entities to receptor cells.

[0139] In some embodiments, the receptor cells express target ligands (e.g., cell surface epitopes). In some embodiments, the receptor cells express specific target ligands or specific combinations of target ligands. In some embodiments, the receptor cells are CD5+, CD2+, or a combination thereof. In some embodiments, the receptor 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+.

[0140] In some embodiments, the targeting chimeric polypeptides provided herein bind to a target ligand on the surface of a receptor cell. In some embodiments, a specific 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 a receptor cell. In some embodiments, the target ligand (e.g., a target epitope) is expressed on all cells (a universal feature of the cell surface), or expressed on a cell subset, or expressed on a subpopulation (e.g., activated T cells relative to resting T cells) cell in a possible state.

[0141] Without wishing to be bound by any particular theory, it is proposed that the unique combination of fusogen entity polypeptides and targeting chimeric polypeptides provided herein enables efficient targeting of lipid bilayer particles into specific receptor cells.

[0142] In some embodiments, the targeting chimeric polypeptides described herein bind to a specific target ligand on the surface of a receptor cell. Receptor cells can express many unique target ligands on the cell surface, and the targeting chimeric polypeptide can be designed to target one or more such target ligands. In some embodiments, the target ligand is located on the surface of the receptor cell. In some embodiments, the target ligand is an epitope, a receptor, a protein, a carbohydrate, a lipid, or a specific combination or conformational state thereof. Engineered lipid bilayer particles

[0143] In addition, the present disclosure provides engineered lipid bilayer particles and preparations thereof, which comprise (e.g., comprising on its surface) targeting chimeric polypeptides as described herein, fusogen entity polypeptides or combinations thereof. In some embodiments, the present invention provides engineered lipid bilayer particles and preparations thereof comprising targeting chimeric polypeptides and fusogen entity polypeptides. The engineered lipid bilayer particles provided may be suitable for therapeutic applications.

[0144] In some embodiments, the present disclosure provides a population of engineered lipid bilayer particles comprising: (i) a targeting chimeric polypeptide comprising (a) a targeting domain, which is configured such that the targeting domain is located on the surface of the particle, wherein the targeting domain comprises a binding moiety that specifically binds to a target ligand on the surface of a receptor cell of interest; and is directly or indirectly linked to (b) a transmembrane domain, and (ii) a fusogen entity polypeptide comprising (a) a fusogen portion, which is arranged so that the fusogen portion is located on the surface of the particle; and is directly or indirectly attached to (b) Transmembrane domain.

[0145] In some embodiments, the engineered lipid bilayer particles in the population display one or more targeting chimeric polypeptides and one or more fusion original (fusogen) entity polypeptides. In some embodiments, at least a portion of the fusion original (fusogen) entity polypeptide is present on the surface of the engineered lipid bilayer particles (e.g., fusion original (fusogen) part). In some embodiments, at least a portion of the targeting chimeric polypeptide is present on the surface of the engineered lipid bilayer particles (e.g., targeting domain). Without wishing to be bound by any particular theory, it is proposed that the specific combination of the targeting chimeric polypeptide described herein and the fusion original (fusogen) entity polypeptide provides efficient targeting entry of the engineered lipid bilayer particles into specific receptor cells. Indeed, without wishing to be bound by any particular theory, it is proposed that certain targeting chimeric polypeptides described herein (e.g., utilizing a PDGFR transmembrane domain [or, in some embodiments, a similar transmembrane domain] and an affinity entity polypeptide, particularly including instances where such affinity entity polypeptide is or comprises an antibody agent) may provide particular display properties (e.g., orientation and / or other aspects of presentation, surface density, etc.) that make them particularly suitable for beneficial combination with the fusogen entity polypeptides described herein to achieve particle targeting as described herein.

[0146] In some embodiments, the engineered lipid bilayer particle is produced by one or more engineered production cells as described herein such that the engineered lipid bilayer particle displays a targeting chimeric polypeptide and a fusogen entity polypeptide.

[0147] In some embodiments, the engineered lipid bilayer particle population is characterized in that the engineered lipid bilayer particles are smaller than eukaryotic cells. In some embodiments, the engineered lipid bilayer particle population is characterized in that the average diameter of the engineered lipid bilayer particles is at most 1000nm, for example at most 800nm, for example at most 300nm, for example at most 100nm. In some embodiments, the engineered lipid bilayer particle population is characterized in that the average diameter of the engineered lipid bilayer particles is at least 30nm, for example at least 50nm, for example at least 80nm, for example at least 100nm, for example at least 150nm, for example at least 200nm, for example at least 250nm, for example at least 300nm. In some embodiments, the engineered lipid bilayer particle population is characterized in that the average diameter of the engineered lipid bilayer particles is about 10nm to about 1000nm, for example, about 30nm to about 800nm, for example, about 50nm and about 500nm.

[0148] In some embodiments, entry of engineered lipid bilayer particles into recipient cells may be affected by one or more properties of the entry environment (e.g., pH, temperature, proximity-induced, mechanical tension or stress, rearrangement of lipid domains (e.g., lipid rafts), radiation (e.g., nuclear, ultraviolet, visible, etc.), electrical signals, magnetic fields, etc., or combinations thereof).

[0149] In some embodiments, the engineered lipid bilayer particle displays at least 5 copies of a fusogen 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 a fusogen entity polypeptide.

[0150] In some embodiments, the engineered lipid bilayer particle displays at least 10 copies of the 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 the affinity entity polypeptide.

[0151] In some embodiments, the engineered lipid bilayer particles are cell-derived membrane particles (CDMPs). In some embodiments, the CDMPs are selected from extracellular vesicles, viral 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.

[0152] In some embodiments, the CDMP is an extracellular vesicle, which may be selected from exosomes or microvesicles. In some embodiments, the CDMP may be a viral particle, a virus-like particle (VLP), an apoptotic body, and a platelet-like particle. In some embodiments, the CDMP may be a hybrid particle produced by mixing cell-derived particles or vesicles (e.g., particles or vesicles blebbed or budded from cells) and synthetic vesicles.

[0153] In some embodiments, the engineered lipid bilayer particles display a fusogen entity polypeptide, such as VSV-G or a functional variant thereof described herein. In some embodiments, the engineered lipid bilayer particles display a targeted chimeric polypeptide comprising a PDGFR transmembrane domain. In some embodiments, the engineered lipid bilayer particles display a VSV-G as described herein and a targeted chimeric polypeptide that binds CD2 or CD5. Targeting chimeric peptides

[0154] The present disclosure provides insights into particularly useful and / or effective targeted chimeric polypeptides to promote specific binding of engineered lipid bilayer particles to target ligands (e.g., target ligands present on receptor cells). The present disclosure teaches that targeted chimeric polypeptides and their binding to target ligands can be used to mediate targeted fusion of engineered lipid bilayer particles to receptor cells expressing target ligands or specific subsets of receptor cells expressing target ligands (e.g., to provide specificity for the techniques described herein). Without wishing to be bound by any particular theory, it is proposed that targeted fusion can be driven and / or influenced by one or more of proximity, affinity, and / or conformational changes.

[0155] In some embodiments, the targeting chimeric polypeptide mediates the binding of engineered lipid bilayer particles to the receptor cells of interest. In some embodiments, the targeting chimeric polypeptide alone (e.g., in the absence of the fusogen entity polypeptide) does not promote cell entry and transduction. Moreover, in many embodiments, the combination of the targeting chimeric polypeptide described herein and the fusogen polypeptide described herein achieves a significant improvement in the efficiency, specificity and / or effectiveness of the delivery of goods to specific receptor cells of interest.

[0156] When designing the combination with a specific type of receptor cell, the targeting chimeric polypeptide provided herein is useful. In some embodiments, the targeting chimeric polypeptide binds to a specific target ligand, thereby guiding the binding to a receptor cell expressing such a specific target ligand. In some embodiments, when the targeting chimeric polypeptide is co-displayed with a fusogen entity polypeptide in an engineered lipid bilayer particle, the binding of the targeting chimeric polypeptide promotes the fusion of the fusogen entity polypeptide with a receptor cell.

[0157] In some embodiments, the present disclosure provides a targeted chimeric polypeptide. In some embodiments, the present disclosure provides a nucleotide sequence encoding a targeted chimeric polypeptide.

[0158] In some embodiments, the technology according to the present disclosure (e.g., systems, engineered lipid bilayer particles, and engineered production cells) comprises a targeting chimeric polypeptide. In some embodiments, the technology according to the present disclosure comprises at least one targeting chimeric polypeptide. In some embodiments, the technology according to the present disclosure comprises one or more targeting chimeric polypeptides.

[0159] In some embodiments, the targeting chimeric polypeptide comprises a secretion signal. In some embodiments, the targeting chimeric polypeptide comprises a FLAG tag. In some embodiments, the targeting chimeric polypeptide does not comprise a FLAG tag, for example, when the targeting chimeric polypeptide is a native polypeptide. In some embodiments, the targeting chimeric polypeptide comprises an affinity portion. In some embodiments, the targeting chimeric polypeptide comprises a linker. In some embodiments, for example, when the targeting chimeric polypeptide is a native polypeptide, the targeting chimeric polypeptide does not comprise a linker. In some embodiments, the targeting chimeric polypeptide comprises a membrane-associated portion. In some embodiments, the targeting chimeric polypeptide comprises an intragranular portion.

[0160] In some embodiments, the targeting chimeric polypeptide consists of or comprises a transmembrane domain. In some embodiments, the transmembrane domain is a domain that has high expression on the surface of lipid bilayer particles.

[0161] In some embodiments, the targeting chimeric polypeptide is an engineered polypeptide. In some embodiments, the order of the targeting chimeric polypeptide from N-terminus to C-terminus is as follows: secretion signal, targeting domain, linker and / or transmembrane domain. In some embodiments, the order of the targeting chimeric polypeptide from N-terminus to C-terminus is as follows: secretion signal, FLAG tag, targeting domain, linker and / or transmembrane domain.

[0162] 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.

[0163] In some embodiments, the order of the targeting chimeric polypeptide from N-terminus to C-terminus is as follows: secretion signal, targeting domain, transmembrane domain, and / or intraparticle portion.

[0164] In some embodiments, the affinity entity polypeptide comprises one or more modifications, such as glycosylation, lipidation, phosphorylation, etc.

[0165] In some embodiments, the targeting chimeric polypeptide comprises a targeting domain. In some embodiments, the targeting chimeric polypeptide comprising a targeting domain comprises a binding portion that specifically binds to a target ligand on the surface of a receptor cell of interest. In some embodiments, the binding domain is displayed on the surface of a lipid bilayer particle. It can be displayed in a manner that promotes the binding of the lipid bilayer of the lipid bilayer particle to the target ligand on the surface of the receptor cell. In some embodiments, the targeting chimeric polypeptide further comprises a transmembrane domain. In some embodiments, the targeting domain is directly or indirectly connected to the transmembrane domain.

[0166] In some embodiments, the targeting domain is or comprises an antibody reagent. In some embodiments, the antibody reagent is a single-chain antibody reagent. In some embodiments, the antibody reagent is selected from the group consisting of antibodies, Fab, Fab', F(ab')2, Fd, scFv, single-chain antibodies, disulfide-linked Fv (sdFv), affibodies, DARPINs, nanobodies, variable lymphocyte receptors (VLRs), and camelid antibodies.

[0167] The term "engineered high affinity binding polypeptide" is equivalent to "de novo designed binding molecule" and is used interchangeably herein.

[0168] In some embodiments, the binding domain specifically binds to the surface of an immune cell (e.g., a lymphocyte, such as a CD4+ and / or CD8+ T cell). In some embodiments, the affinity portion is characterized in that it binds to a receptor cell expressing CD5, CD2, or a combination thereof.

[0169] In some embodiments, the disclosure provides a chimeric targeting polypeptide comprising: (a) a targeting domain that binds to human CD2, wherein the targeting domain is or comprises an antibody reagent selected from the group consisting of an antibody, Fab, Fab', F(ab')2, Fd, scFv, single-chain antibody, disulfide-linked Fv (sdFv), a de novo designed binding molecule, an affibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody: and (b) a transmembrane domain. In some embodiments, the targeting domain is a scFv. The chimeric targeting polypeptide may optionally comprise a linker.

[0170] In some embodiments, the targeting domain binds CD2. In some embodiments, the targeting domain comprises an anti-CD2 portion or fragment thereof. In some embodiments, the targeting domain comprises the amino acid sequence NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSGGGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSS(SEQ ID NO:20),It can be encoded by a nucleic acid sequence (SEQ ID NO: 21). However, it should be noted that targeting domains capable of binding to CD2 are expected to work. In some embodiments, the chimeric targeting polypeptide and lipid bilayer particles comprising such polypeptides, binding to CD2 alone is sufficient to deliver the contents of the lipid bilayer particles (e.g., cargo entities) to recipient cells (e.g., lymphocytes).

[0171] In some embodiments, the present disclosure provides a chimeric targeting polypeptide comprising: (a) a targeting domain that binds to human CD5, wherein the targeting domain is or comprises an antibody reagent selected from the group consisting of an antibody, Fab, Fab', F(ab')2, Fd, scFv, single-chain antibody, disulfide-linked Fv (sdFv), a de novo designed binding molecule, an affibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody: and (b) a transmembrane domain. In some embodiments, the targeting domain is a VLR. In some embodiments, the targeting domain is a scFv. The chimeric targeting polypeptide may optionally comprise a linker.

[0172] In some embodiments, the targeting domain binds CD5. In some embodiments, the targeting domain comprises an anti-CD5 portion or fragment thereof. In some embodiments, the targeting domain comprises the amino acid sequence CPSQCSCSGTEVHCQRKSLASVPAGIPTTTRVLYLHVNEITKFEPGVFDRLVNLQQLYLGGNQLSALPDGVFDRLTQLTRLDLYNNQLTVLPAGVFDRLVNLQTLDLHNNQLKSIPRGAFDNLKSLTHIWLFGNPWDCACSDILYLSGWLGQHAGKEQGQAVCSGTNTPVRAVTEASTSPSKCP(SEQ ID NO:24), which can be determined by the nucleic acid sequence (SEQ ID NO:25) encoding. However, it should be noted that targeting domains capable of binding to CD5 are expected to work. In some embodiments, chimeric targeting polypeptides and lipid bilayer particles comprising such polypeptides, binding to CD5 alone is sufficient to deliver the contents of the lipid bilayer particles (e.g., cargo entities) to recipient cells (e.g., lymphocytes).

[0173] 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 amino acid sequence ATGGAAACGGACACCCTGCTGCTGTGGGTGCTGTTGTTGTGGGTGCCAGGATCTACAGGCGAC (SEQ ID NO: 39).

[0174] In some embodiments, the targeting chimeric polypeptide comprises a FLAG tag (e.g., a 3x 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 amino acid sequence GATTACAAGGACCACGATGGCGACTATAAGGATCACGACATCGACTACAAGGACGATGACGACAAG (SEQ ID NO: 41). Fusion entity polypeptide

[0175] Without wishing to be bound by any particular theory, it is proposed that the fusion original (fusogen) entity polypeptide described herein mediates the engineered lipid bilayer particles displaying this fusion original (fusogen) entity polypeptide to enter cells. Some fusion original (fusogen) entity polypeptides may mediate entry into cells in the absence of a targeting chimeric polypeptide. However, the disclosure records that the combination of fusion original (fusogen) entity polypeptides described herein and targeting chimeric polypeptides can achieve the outstanding specificity, efficiency and / or effectiveness of delivering goods from the engineered lipid bilayer particles comprising them to a specific target cell of interest (for example, human cells and / or immune cells, such as T cells, such as human T cells). In some embodiments, the fusion original (fusogen) entity polypeptide is characterized by its ability to mediate fusion between lipid bilayers. In some embodiments, the fusion original (fusogen) entity polypeptide mediates the transduction of receptor cells.

[0176] In some embodiments, the technology according to the present disclosure includes (e.g., utilizes) a fusogen entity polypeptide. In some embodiments, the technology according to the present disclosure includes (e.g., utilizes) at least one fusogen entity polypeptide (which is typically present in multiple copies on the engineered lipid bilayer particle). In some embodiments, the technology according to the present disclosure includes one or more fusogen entity polypeptides (e.g., each polypeptide can be present in multiple copies on the engineered lipid bilayer particle.

[0177] In some embodiments, the fusion original (fusogen) entity polypeptide is a naturally occurring (e.g., wild-type) polypeptide. In some embodiments, the fusion original (fusogen) entity polypeptide is native to a specific production cell. In some embodiments, the fusion original (fusogen) entity polypeptide is an engineered polypeptide. In some embodiments, the fusion original (fusogen) entity polypeptide (e.g., an engineered fusion original (fusogen) entity polypeptide) is a variant (e.g., comprising one or more amino acid substitutions) of a wild-type polypeptide and / or a native (native) polypeptide.

[0178] In some embodiments, the fusogen entity polypeptide comprises a secretion signal. In some embodiments, the fusogen entity polypeptide comprises a fusogen portion. In some embodiments, the fusogen entity polypeptide comprises a transmembrane domain. In some embodiments, the fusogen entity polypeptide comprises an intragranular portion.

[0179] In some embodiments, the fusogen entity polypeptide consists of or comprises a fusogen portion and a transmembrane portion.

[0180] In some embodiments, the order of the fusogen entity polypeptide from N-terminus to C-terminus is as follows: secretion signal, fusogen portion, transmembrane portion, fusogen intraparticle portion, or a combination thereof.

[0181] In some embodiments, the fusogen entity polypeptide or its fusogen portion has an amino acid sequence that includes characteristic sequence elements and / or has overall sequence identity with a reference fusogen entity polypeptide (e.g., a wild-type fusogen entity polypeptide and / or a fusogen entity peptide). In some embodiments, the fusogen entity polypeptide is a variant of such a reference fusogen entity polypeptide. In some embodiments, the fusogen entity polypeptide includes one or more modifications, such as glycosylation, lipidation, phosphorylation, etc.

[0182] In some embodiments, the fusogen entity polypeptide is a constitutive fusogen entity polypeptide whose fusogenic activity is not dependent on specific stimuli or conditions.

[0183] In some embodiments, the fusogen entity polypeptide is a conditional fusogen entity polypeptide, whose fusogen activity depends on or is triggered by a specific stimulus or condition (e.g., pH, temperature, radiation (e.g., nuclear, ultraviolet, visible light, etc.), electrical signal, magnetic field, etc., or a combination thereof).

[0184] In some embodiments, the fusogen entity polypeptide binds to a specific target to drive fusion. In some embodiments, the fusogen entity polypeptide binds to low-density lipoprotein (LDL). In some embodiments, the fusogen entity polypeptide binds to a receptor displayed on a receptor cell.

[0185] In some embodiments, the fusogen entity polypeptide comprises a fusogen portion. The fusogen portion provided herein mediates the entry of engineered lipid bilayer particles displaying a fusogen entity polypeptide comprising a fusogen portion into a recipient cell. In some embodiments, the fusogen portion mediates transduction of lipid bilayer particles to a recipient cell. The fusogen portion encompasses a portion characterized in that it promotes fusion between lipid bilayers or its functional portion.

[0186] In some embodiments, the fusogen moiety is displayed on the surface of the engineered lipid bilayer particle (i.e., arranged so that the fusogen moiety is located on the surface of the particle). The fusogen moiety can be displayed in a manner that enables it to interact with a target ligand on the surface of a receptor cell. The fusogen entity can be displayed in a manner that promotes fusion of the lipid bilayer of the engineered lipid bilayer particle with the lipid bilayer of a receptor cell.

[0187] In some embodiments, the fusogen moiety targets a specific epitope on a receptor cell such that binding of the target epitope enables or enhances uptake, fusion, and / or functional delivery of cargo contained within the engineered lipid bilayer particle. Such a target epitope may be expressed on all cells (a universal feature 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).

[0188] In some embodiments, the fusogen moiety mediates fusion between the engineered lipid bilayer particle and the target cell in a manner that does not require the fusogen to bind to the target epitope.

[0189] In some embodiments, the fusogen portion enhances fusion between the engineered lipid bilayer particle and the recipient cell comparable to fusion between the engineered lipid bilayer particle and the recipient cell where the engineered lipid bilayer particle does not display the fusogen entity polypeptide comprising the fusogen portion.

[0190] In some embodiments, the fusogen entity polypeptide comprises a viral fusogen portion. In some embodiments, the fusogen portion is an enveloped viral fusogen portion. In some embodiments, the fusogen portion is a viral glycoprotein.

[0191] In some embodiments, the lipid bilayer particles include viral glycoproteins to help the lipid bilayer particles fuse with receptor cells (e.g., lymphocytes). The viral glycoproteins may be selected from lentiviral glycoproteins or glycoproteins selected from vesicular stomatitis (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic mouse leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein. Specifically, in some embodiments, the glycoprotein may be measles virus glycoprotein H, measles virus glycoprotein F, or a combination thereof.

[0192] In some embodiments, the fusogen entity polypeptide is from vesicular stomatitis virus, measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, rabies virus, lymphocytic choriomeningitis virus, Mokola virus, Ross River 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-Venes virus 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, avian plague virus, Autographa californica multiplenucleopolyhedro virus, baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, dengue virus, Zika virus, West Nile virus, yellow fever virus, tick-borne encephalitis virus, herpes simplex virus type 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, varicella zoster virus, severe fever with thrombocytopenia virus, Hanta virus, vaccinia virus, simian immunodeficiency virus, human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus virus), La Crosse virus, human cytomegalovirus, human cytomegalovirus, Thogoto virus, or Dhori virus.

[0193] In some embodiments, the fusogen entity polypeptide is or comprises a glycoprotein selected from vesicular stomatitis glycoprotein (VSV-G).

[0194] In some embodiments, the fusogen entity polypeptide is or comprises wild-type VSV-G. In some embodiments, the wild-type VSV-G has the amino acid sequence MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGV In some embodiments, the wild-type VSV-G fusogen portion has an amino acid sequence that is identical to SEQ ID NO: 26.

[0195] In some embodiments, wild-type VSV-G is encoded by

[0196] In some embodiments, the fusogen entity polypeptide comprises a fusogen portion. In some embodiments, the fusogen portion is or comprises a fragment of wild-type VSV-G. In some embodiments, the wild-type VSV-G fusogen portion has an amino acid sequence similar to KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQS CGYATVTDAEAAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKH WGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSS (SEQ ID NO:30) 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 an amino acid sequence. In some embodiments, the wild-type VSV-G fusogen portion has an amino acid sequence identical to the amino acid sequence of SEQ ID NO:30.

[0197] In some embodiments, the wild-type VSV-G fusion progenitor is encoded in part by

[0198] In some embodiments, the fusogen entity polypeptide is or comprises a mutated VSV-G. In some embodiments, the mutated VSV-G has an amino acid sequence similar to MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQ SCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMAD In some embodiments, the mutant VSV-G fusion entity polypeptide 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 KDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO:28). In some embodiments, the mutant VSV-G fusion entity polypeptide has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO:28.

[0199] In some embodiments, the mutant VSV-G is encoded by

[0200] In some embodiments, the fusogen entity polypeptide comprises a fusogen portion. In some embodiments, the fusogen portion is or comprises a fragment of a mutated VSV-G. In some embodiments, the mutated VSV-G fusogen portion has an amino acid sequence similar to KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQS CGYATVTDAEAAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKH WGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSS (SEQ ID NO:32) 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 an amino acid sequence. In some embodiments, the mutated VSV-G fusogen portion has an amino acid sequence identical to the amino acid sequence of SEQ ID NO:32.

[0201] In some embodiments, the mutant VSV-G fusogen portion is encoded by

[0202] In some embodiments, the fusogen entity polypeptide comprises an intragranular portion. In some embodiments, the intragranular 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 intragranular 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 amino acid sequence aaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaag (SEQ ID NO: 37).

[0203] In some embodiments, the fusogen entity polypeptide is or comprises a measles virus polypeptide, such as a measles virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a measles virus glycoprotein H and / or F.

[0204] In some embodiments, the fusogen entity polypeptide is or comprises a Sindbis virus polypeptide, such as a Sindbis virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises Sindbis virus glycoprotein E1 and / or E2.

[0205] In some embodiments, the fusogen entity polypeptide is or comprises a Tupaia paramyxovirus polypeptide, such as a Tupaia paramyxovirus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Tupaia paramyxovirus glycoprotein H and / or F.

[0206] In some embodiments, the fusogen entity polypeptide is or comprises a Nipah virus polypeptide, such as a Nipah virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Nipah virus glycoprotein G and / or F.

[0207] In some embodiments, the fusogen entity polypeptide is or comprises a Chandipura virus polypeptide, such as a Chandipura virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Chandipura virus glycoprotein G.

[0208] In some embodiments, the fusogen entity polypeptide is or comprises a rabies virus polypeptide, such as a rabies virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a rabies virus glycoprotein G.

[0209] In some embodiments, the fusogen entity polypeptide is or comprises a lymphocytic choriomeningitis virus polypeptide, such as a lymphocytic choriomeningitis virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a lymphocytic choriomeningitis virus glycoprotein GP-1 and / or GP-2.

[0210] In some embodiments, the fusogen entity polypeptide is or comprises a Mokola virus polypeptide, such as a Mokola virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Mokola virus glycoprotein G.

[0211] In some embodiments, the fusogen entity polypeptide is or comprises a Ross River virus polypeptide, such as a Ross River virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Ross River virus glycoprotein El and / or E2.

[0212] In some embodiments, the fusogen entity polypeptide is or comprises a Semliki Forest virus polypeptide, such as a Semliki Forest virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Semliki Forest virus glycoprotein El and / or E2.

[0213] In some embodiments, the fusogen entity polypeptide is or comprises a Venezuelan equine encephalitis virus polypeptide, such as a Venezuelan equine encephalitis virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Venezuelan equine encephalitis virus glycoprotein El and / or E2.

[0214] In some embodiments, the fusogen entity polypeptide is or comprises an Ebola virus polypeptide, such as an Ebola virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises an Ebola virus glycoprotein GP.

[0215] In some embodiments, the fusogen entity polypeptide is or comprises a Marburg virus polypeptide, such as a Marburg virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Marburg virus glycoprotein GP.

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

[0217] In some embodiments, the fusogen entity polypeptide is or comprises an avian leukosis virus polypeptide, such as an avian leukosis virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises an avian leukosis virus envelope glycoprotein.

[0218] In some embodiments, the fusogen entity polypeptide is or comprises a Jaagsiekte sheep retrovirus polypeptide, such as a Jaagsiekte sheep retrovirus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Jaagsiekte sheep retrovirus envelope glycoprotein.

[0219] In some embodiments, the fusogen entity polypeptide is or comprises a Moloney murine leukemia virus polypeptide, such as a Moloney murine leukemia virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Moloney murine leukemia virus envelope glycoprotein.

[0220] In some embodiments, the fusogen entity polypeptide is or comprises a Gibbon Ape Leukemia Virus polypeptide, such as a Gibbon Ape Leukemia Virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Gibbon Ape Leukemia Virus envelope glycoprotein.

[0221] In some embodiments, the fusogen entity polypeptide is or comprises a feline endogenous retrovirus polypeptide, such as a feline endogenous retrovirus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a RD114 glycoprotein.

[0222] In some embodiments, the fusogen entity polypeptide is or comprises a human T-lymphotropic virus type 1 polypeptide, such as a human T-lymphotropic virus type 1 glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises human T-lymphotropic virus type 1 glycoprotein SU and / or TM. In some embodiments, the fusogen entity polypeptide is or comprises human T-lymphotropic virus type 1 glycoprotein gp46.

[0223] In some embodiments, the fusogen entity polypeptide is or comprises a human foamy virus polypeptide, such as a human foamy virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a human foamy virus envelope glycoprotein.

[0224] In some embodiments, the fusogen entity polypeptide is or comprises a Maedi-visna virus polypeptide, such as a Maedi-visna virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Maedi-visna virus envelope glycoprotein.

[0225] In some embodiments, the fusogen entity polypeptide is or comprises a SARS-CoV polypeptide (e.g., SARS-CoV 2), such as a SARS-CoV glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a SARS-CoV spike glycoprotein (S).

[0226] In some embodiments, the fusogen entity polypeptide is or comprises a Sendai virus polypeptide, such as a Sendai virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Sendai virus glycoprotein HN and / or F.

[0227] In some embodiments, the fusogen entity polypeptide is or comprises a respiratory syncytial virus polypeptide, such as a respiratory syncytial virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a respiratory syncytial virus glycoprotein G and / or F.

[0228] In some embodiments, the fusogen entity polypeptide is or comprises a human parainfluenza virus type 3 and / or type 4 polypeptide, such as a respiratory human parainfluenza virus type 3 and / or type 4 glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a human parainfluenza virus type 3 and / or type 4 glycoprotein HN and / or F.

[0229] In some embodiments, the fusogen entity polypeptide is or comprises a hepatitis C virus polypeptide, such as a hepatitis C virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises hepatitis C virus glycoprotein E1 and / or E2.

[0230] In some embodiments, the fusogen entity polypeptide is or comprises an influenza virus polypeptide, such as an influenza virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises an influenza virus glycoprotein HA and / or NA.

[0231] In some embodiments, the fusogen entity polypeptide is or comprises an avian plague virus polypeptide, such as an avian plague virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises an avian plague virus glycoprotein HA.

[0232] In some embodiments, the fusogen entity polypeptide is or comprises an Autographa californica multiple nucleopolyhedro virus polypeptide, such as an Autographa californica multiple nucleopolyhedro virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises an Autographa californica multiple nucleopolyhedro virus glycoprotein gp64.

[0233] In some embodiments, the fusogen entity polypeptide is or comprises a baboon endogenous retroviral polypeptide, such as a baboon endogenous retroviral glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a baboon endogenous retroviral glycoprotein envelope.

[0234] In some embodiments, the fusogen entity polypeptide is or comprises a Cocal virus polypeptide, such as Cocal virus glycoprotein (G).

[0235] In some embodiments, the fusogen entity polypeptide is or comprises a Japanese encephalitis virus polypeptide, such as a Japanese encephalitis virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Japanese encephalitis virus glycoprotein E.

[0236] In some embodiments, the fusogen entity polypeptide is or comprises a dengue virus polypeptide, such as a dengue virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a dengue virus glycoprotein E.

[0237] In some embodiments, the fusogen entity polypeptide is or comprises a Zika virus polypeptide, such as a Zika virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Zika virus glycoprotein E.

[0238] In some embodiments, the fusogen entity polypeptide is or comprises a West Nile virus polypeptide, such as a West Nile virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises West Nile virus glycoprotein E.

[0239] In some embodiments, the fusogen entity polypeptide is or comprises a yellow fever virus polypeptide, such as a yellow fever virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a yellow fever virus glycoprotein E.

[0240] In some embodiments, the fusogen entity polypeptide is or comprises a tick-borne encephalitis virus polypeptide, such as a tick-borne encephalitis virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a tick-borne encephalitis virus glycoprotein E.

[0241] In some embodiments, the fusogen entity polypeptide is or comprises a herpes simplex virus polypeptide, such as a herpes simplex virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a herpes simplex virus glycoprotein HSV-1gB, HSV-1gH, HSV-1gL and / or HSV-1gD.

[0242] In some embodiments, the fusogen entity polypeptide is or comprises a Hendra virus polypeptide, such as a Hendra virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Hendra virus glycoprotein G and / or F.

[0243] In some embodiments, the fusogen entity polypeptide is or comprises a Newcastle Disease Virus polypeptide, such as a Newcastle Disease Virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises Newcastle Disease Virus glycoprotein F1 and / or F2.

[0244] In some embodiments, the fusogen entity polypeptide is or comprises an Epstein-Barr virus polypeptide, such as an Epstein-Barr virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises an Epstein-Barr virus glycoprotein gB, gH and / or gL. In some embodiments, the fusogen entity polypeptide is or comprises an Epstein-Barr virus glycoprotein gp42.

[0245] In some embodiments, the fusogen entity polypeptide is or comprises a Bourbon virus polypeptide, such as a Bourbon virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Bourbon virus glycoprotein Gp.

[0246] In some embodiments, the fusogen entity polypeptide is or comprises a varicella zoster virus polypeptide, such as a varicella zoster virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a varicella zoster virus glycoprotein gB, gH, gE and / or gL.

[0247] In some embodiments, the fusogen entity polypeptide is or comprises a severe fever with thrombocytopenia virus polypeptide, such as a severe fever with thrombocytopenia virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises severe fever with severe fever with thrombocytopenia virus glycoprotein gB, gH and / or gL.

[0248] In some embodiments, the fusogen entity polypeptide is or comprises a hantavirus polypeptide, such as a hantavirus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a hantavirus glycoprotein Gn and / or Gc.

[0249] In some embodiments, the fusogen entity polypeptide is or comprises a vaccinia virus polypeptide, such as a vaccinia virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a vaccinia virus glycoprotein A28, A21, A16, F9, G9, G3, H2, J5, L5, L1, A33, A34, B5 and / or O3.

[0250] In some embodiments, the fusogen entity polypeptide is or comprises a simian immunodeficiency virus polypeptide, such as a simian immunodeficiency virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a simian immunodeficiency virus Env glycoprotein.

[0251] In some embodiments, the fusogen entity polypeptide is or comprises a human immunodeficiency virus (eg, HIV-1) polypeptide, such as a human immunodeficiency virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a human immunodeficiency virus Env glycoprotein.

[0252] In some embodiments, the fusogen entity polypeptide is or comprises a Junin virus polypeptide, such as a Junin virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Junin virus glycoprotein complex GPC.

[0253] In some embodiments, the fusogen entity polypeptide is or comprises a Machupo virus polypeptide, such as a Machupo virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Machupo virus glycoprotein complex GPC.

[0254] In some embodiments, the fusogen entity polypeptide is or comprises a Bas-Congo virus polypeptide, such as a Bas-Congo virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Bas-Congo virus glycoprotein G.

[0255] In some embodiments, the fusogen entity polypeptide is or comprises a La Crosse virus polypeptide, such as a La Crosse virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a La Crosse virus glycoprotein Gc and / or Gn.

[0256] In some embodiments, the fusogen entity polypeptide is or comprises a human cytomegalovirus polypeptide, such as a human cytomegalovirus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a human cytomegalovirus glycoprotein gH, gL, gO, UL128, UL130 and / or UL131A.

[0257] In some embodiments, the fusogen entity polypeptide is or comprises a Thogoto virus polypeptide, such as a Thogoto virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Thogoto virus glycoprotein Gp.

[0258] In some embodiments, the fusogen entity polypeptide is or comprises a Dhori virus polypeptide, such as a Dhori virus glycoprotein. In some embodiments, the fusogen entity polypeptide is or comprises a Dhori virus glycoprotein Gp.

[0259] In some embodiments, the fusogen entity polypeptide comprises a non-viral fusogen portion.

[0260] In some embodiments, the fusogen entity polypeptide comprises a human fusogen portion.

[0261] In some embodiments, the fusogen entity polypeptide is or comprises a polypeptide selected from syncytin-1, syncytin-2, CD9, CD81, muscle marker, CD200 (OX-2G), DC-STAMP, OC-STAMP, E-cadherin (CADH1), cadherin 11 (CAD11), matrix metalloproteinase-9, tight junction protein-1 (ZO-1), myomerger, annexin A1, annexin A5 (Annexin A2), annexin B1 (Annexin B3), annexin B4 (Annexin B5), annexin B6 (Annexin B7), annexin B8 (Annexin B9), annexin B10 (Annexin B11), annexin B12 (Annexin B13), annexin B14 (Annexin B15), annexin B15 (Annexin B16), annexin B17 (Annexin B18), annexin B20 (Annexin B21), annexin B22 (Annexin B23), annexin B24 (Annexin B25), annexin B26 (Annexin B27), annexin B28 (Annexin B29), annexin B30 (Annexin B31), annexin B31 (Annexin B32), annexin B32 (Annexin B33), annexin B34 (Annexin B35), annexin B36 (Annexin B37), annexin B37 (Annexin B38), annexin B39 (Annexin B40), annexin B41 (Annexin B42), annexin B43 (Annexin B44), annexin B45 (Annexin B46), annexin B47 (Annexin B48), annexin B49 (Annexin B5 A5), CD44, P2X purinergic receptor 7, IZUMO1, Juno, StartD7, receptor-like protein 1, associated protein 4, CD63, connexin 43 (Cx43), CD36, macrophage fusion receptor (MFR), tumor-associated member 1, GLPR1-like protein 1, associated protein 43, human fusogen part of the group consisting of ERVV-1, ERVV-2, ERVH48-1, ERVMER34-1, ERV3-1 and ERVK13-1.

[0262] In some embodiments, the fusogen entity polypeptide is or comprises a human fusogen portion selected from the group consisting of Syncytin-1, Syncytin-2, CD9, CD81, muscle marker, CD200 (OX-2G), DC-STAMP, OC-STAMP, E-Cadherin (CADH1), Cadherin 11 (CAD11), matrix metalloproteinase-9, tight junction protein-1 (ZO-1), myomerger, Annexin A1, Annexin A5, CD44, P2X purinergic receptor 7, IZUMO1, Juno, StartD7, receptor-like protein 1, related protein 4, CD63, connexin 43 (Cx43), CD36, MFR, tumor-associated member 1, GLPR1-like protein 1 and related protein 43. Transmembrane domain

[0263] In some embodiments, polypeptides described herein (e.g., targeting chimeric polypeptides, fusion original (fusogen) entity polypeptides, cargo loading polypeptides, etc.) include transmembrane domains (TMDs). In some embodiments, transmembrane domains allow targeting domains to be displayed on the surface of lipid bilayer particles. In some embodiments, membrane-associated parts position affinity parts on the surface of lipid bilayer particles so that they can bind to receptor cell surface epitopes. As used herein, the term "transmembrane domain" encompasses any "membrane-associated part" that can be associated with a membrane (e.g., a lipid bilayer membrane). It will be understood by those skilled in the art that, in some embodiments, "transmembrane domains" are amino acids that cause association with a membrane. In some embodiments, transmembrane domains span membranes. In some embodiments, transmembrane domains do not span membranes. In some embodiments, transmembrane domains are combined with membranes (e.g., lipid bilayer membranes). In some embodiments, transmembrane domains are located at the C-terminus of a polypeptide. In some embodiments, transmembrane domains are 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 (eg, to another portion of the polypeptide).

[0264] 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.

[0265] Transmembrane domain is known in the art. Transmembrane domain (TMD) is mainly composed of non-polar amino acid residues, can be single (single transmembrane) or multiple through bilayer. TMD is usually composed of α helix. Peptide bond is polar and can include internal hydrogen bond formed between carbonyl oxygen atom and amide nitrogen atom, and these hydrogen bonds can be hydrated. In lipid bilayer, water is basically excluded, and peptide usually adopts α-helical configuration, so that its internal hydrogen bonding is maximized. The helical length of 18-21 amino acid residues is usually enough to cross the usual width of lipid bilayer. TMD oriented to the outer N-terminal of cytoplasm and the C-terminal of cytoplasm is classified as type I TMD, and TMD oriented to the outer C-terminal of cytoplasm and the N-terminal of cytoplasm is classified as type II TMD. In some embodiments outside the disclosed cytoplasm, they are classified as type I, or if it is cytoplasm, they are classified as type II. In some embodiments, the transmembrane domain is a single transmembrane, type I transmembrane domain comprising 18-21 amino acids, wherein at least about 90% of the amino acids are non-polar. Suitable TMDs for the disclosed fusion proteins may include, but are not limited to, transmembrane domains of cell receptors, such as platelet-derived growth factor receptor (PDGFR) transmembrane domains.In some embodiments, the transmembrane is vesicular stomatitis virus, measles virus, Sindbis virus, Tupaiaparamyxovirus, Nipah virus, Chandipura virus, rabies virus, lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, avian leukosis virus, Jaagsiektesheep retrovirus, Moloney murine leukemia virus, Gibbon ape leukemia virus, feline endogenous retrovirus (RD114), human T-lymphotropic virus type 1, human foamy virus, Maedi-Venes virus 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, avian plague virus, Autographa californica multiple nucleopolyhedro virus, baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, dengue virus, Zika virus, West Nile virus, yellow fever virus, tick-borne encephalitis virus, herpes simplex virus type 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, varicella zoster virus, severe fever with thrombocytopenia virus, Hanta virus, vaccinia virus, simian immunodeficiency virus, human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus virus), La Crosse virus, human cytomegalovirus, human cytomegalovirus, Thogoto virus, or Dhori virus transmembrane domain.

[0266] 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).

[0267] The TMD can be directly connected to a targeting domain (e.g., scFv) or the TMD can be connected via a joint. In some embodiments, the joint connecting the TMD and the targeting domain comprises an amino acid sequence of (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. In some embodiments, the linker connecting the TMD and the targeting peptide comprises: (1) 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; 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% sequence identity to any one of SEQ ID NO: 10, 12, 14, 15, 16, or 17.

[0268] Thus, in some embodiments, the transmembrane domain can comprise AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (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% sequence identity to SEQ ID NO: 18, or a functional fragment thereof.

[0269] In some embodiments, the fusogen entity polypeptide comprises a transmembrane domain. In some embodiments, the transmembrane domain is or comprises a wild-type VSV-G transmembrane portion or a fragment thereof. In some embodiments, the fusogen entity polypeptide transmembrane domain may comprise IASFFFIIGLIIGLFLVLRVGIHLCI (SEQ ID NO: 22), which may be represented by the nucleotide sequence attgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcatt (SEQ ID NO: 23). In some embodiments, the wild-type VSV-G fusogen 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 the amino acid SEQ ID NO: 22. In some embodiments, the wild-type VSV-G fusogen transmembrane portion has an amino acid sequence that is identical to the amino acid SEQ ID NO: 22.

[0270] In some embodiments, the transmembrane domain is a non-human transmembrane domain. In some embodiments, the transmembrane domain is a human transmembrane domain.

[0271] In some embodiments, the transmembrane domain is integrated into the membrane of the lipid bilayer particle at a high copy number. secretion signal

[0272] In some embodiments, the polypeptide disclosed herein (e.g., a targeted chimeric polypeptide and / or a fusogen entity polypeptide) comprises a secretion signal, for example, a secretion signal that is functional in a mammalian cell. In some embodiments, the secretion signal utilized is a heterologous secretion signal. In some embodiments, the heterologous secretion signal comprises a non-human secretion signal or is composed of a non-human secretion signal. In some embodiments, the heterologous secretion signal comprises a viral secretion signal or is composed of a viral secretion signal. In some embodiments, the secretion signal is characterized by a length of about 10 to about 40 amino acids, for example, about 20 to about 30 amino acids. In some embodiments, the secretion signal is located at the N-terminus of the fusogen entity polypeptide described herein. In some embodiments, the secretion signal preferably allows the fusogen entity polypeptide associated therewith to be transported to a defined cell compartment, preferably a cell surface, endoplasmic reticulum (ER), Golgi apparatus or endosomal-lysosomal compartment.

[0273] 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 amino acid sequence atgaagtgccttttgtacttagcctttttattcattggggtgaattgc (SEQ ID NO: 35). Affinity Tags

[0274] In some embodiments, the polypeptide disclosed herein (e.g., a targeting chimeric polypeptide and / or a fusogen entity polypeptide) comprises a FLAG tag. In some embodiments, the FLAG tag used is a heterologous secretion tag. In some embodiments, the heterologous FLAG tag comprises a non-human FLAG tag or consists of a non-human FLAG tag. In some embodiments, the heterologous FLAG tag comprises a viral FLAG tag 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 located between the secretion signal sequence and the targeting domain sequence within the targeting chimeric polypeptide described herein. In some embodiments, the polypeptide described herein does not comprise a FLAG tag or any other tag. Goods Entity

[0275] In some embodiments, the disclosed technology includes a cargo entity. In some embodiments, a lipid bilayer particle or a population of lipid bilayer particles comprises a cargo entity. In some embodiments, the disclosed technology includes one or more cargo entities, such as multiple cargo entities (e.g., a first cargo entity, a second cargo entity, etc. or a combination thereof). The cargo entities described herein can be of any chemical class, such as polypeptides, nucleic acids, sugars, lipids, small entities, and combinations thereof.

[0276] In some embodiments, the cargo entity is part of a targeting chimeric polypeptide, a fusogen entity polypeptide, or both. In some embodiments, the cargo entity is associated with a targeting chimeric polypeptide, a fusogen entity polypeptide, or both.

[0277] In some embodiments, the cargo entity is a portion of a polypeptide that is different from each of the targeting chimeric polypeptide and the fusogen entity polypeptide. In some embodiments, the cargo entity is connected to a cargo loading domain. In some embodiments, the cargo loading domain comprises an abscisic acid insensitive 1 (ABI1) sequence.

[0278] In some embodiments, a cargo loading domain is linked to a cargo entity and co-expressed with an ABA binding sequence (e.g., including a pyranosin resistance 1-like (PYL1) sequence) and abscisic acid (ABA).

[0279] In some embodiments, the cargo loading domain is physically connected to the cargo.

[0280] In some embodiments, a cargo loading domain is linked to a cargo entity and co-expressed with an ABA binding sequence (e.g., including a pyranoside resistance 1-like (PYL1) sequence).

[0281] In some embodiments, the cargo entity is a cytoplasmic cargo entity or a membrane-bound cargo entity.

[0282] In some embodiments, a cargo loading domain comprising an ABI1 sequence can be fused to a membrane polypeptide (i.e., a membrane-bound polypeptide). In other words, in some embodiments, the cargo entity can be a membrane polypeptide. Similarly, in some embodiments, a cargo loading domain comprising an abscisic acid-insensitive 1 (ABI1) sequence can be fused to a cytoplasmic polypeptide (i.e., a non-membrane-bound polypeptide). In other words, in some embodiments, the cargo entity can be a cytoplasmic polypeptide.

[0283] The loading system can be used to deliver a single cargo (or a single type of cargo) to a desired recipient cell, or it can be used to deliver multiple cargoes (e.g., different polypeptides) to a desired cell. For purposes of embodiments including multiple different cargoes, the disclosed loading system can be used to control the ratio at which the various cargoes are loaded into the lipid bilayer particles and subsequently delivered to the desired recipient cells.

[0284] In some embodiments, the cargo entity is a polypeptide cargo entity. In some embodiments, the cargo entity is a cytoplasmic cargo molecule, wherein the cytoplasmic cargo entity can be a peptide, polypeptide or protein of interest to be delivered to a recipient cell, 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 editor, a guide editor (prime editor), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a kinase, a kinase inhibitor, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin modification synthetic transcription factor, a natural transcription factor and a mutant form 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 cutting. In some embodiments, the CRISPR enzyme catalyzes RNA cutting. In some embodiments, the CRISPR enzyme is a Cas9 protein (e.g., a naturally occurring bacterial Cas9 and any chimera, 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, and homologs or modified variants thereof.

[0285] 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 a particular theory, synthetic nucleic acid cargo entities and / or nucleic acid cargo entities comprising chemically modified nucleotides provide stability for nucleic acid cargoes. This may be particularly useful for guiding RNA (e.g., for Cas gRNA complexes).

[0286] In some embodiments, the synthetic nucleic acid is an ASO or a chemically modified RNA.

[0287] In some embodiments, the cargo entity may be a nucleic acid (e.g., DNA or RNA) or another molecule. Nucleic acid cargo entities may include, but are not limited to, DNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, and combinations thereof encoding polypeptides of interest. Other potential cargo entities include, but are not limited to, viral and non-viral vectors (e.g., nucleocapsids) and ribonucleoprotein complexes expressed intracellularly (or delivered to the cytosol of the cell), such as CRISPR-type entities and endogenous complexes, such as DICER or RISC bound to natural or synthetic RNAs such as miRNA, shRNA, etc. In some embodiments, cargo that can be expressed in cells or physically delivered to the interior of cells can be fused (genetically or synthetically) with ABI proteins / peptides or ABA binding sequences and are explicitly contemplated herein. Genetic fusion can be achieved by expressing two or more components of a chimeric polypeptide or peptide in a lipid bilayer particle (e.g., CDMP, such as EV) production cell. Alternatively, a cargo entity fused to a loading domain can be delivered to a lipid bilayer particle (e.g., CDMP, such as EV) production cell for subsequent incorporation into a lipid bilayer particle. Alternatively, the cargo entity fused to the loading domain can be inserted into the lipid bilayer particle after secretion of the paper bilayer particle from the producer cells.

[0288] 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) targeting peptide / protein and (ii) transmembrane domain. Exemplary targeting peptides include, but are not limited to, any antibody fragment or antigen binding fragment, such as Fab, Fab'1 and F(ab')2, Fd, scFv, single-chain antibody, disulfide-linked Fv (sdFv) and nano antibodies. Targeting peptides (e.g., scFv) can be combined with specific cell types, such as targets of interest on T cells. In some embodiments, the targeting peptide can be Fab, Fab' and F(ab')2, Fd, scFv, single-chain antibody, disulfide-linked Fv (sdFv), de novo designed binding molecules, affibodies, DARPIN or nano antibodies combined with, for example, CD2 or other targets associated with T cells. For example, other suitable targets include, but are not limited to, CD3, CD4, CD8, CD25, CD127, CD39, CD45RA, CTLA-4 and PD-1.

[0289] In some embodiments, the lipid bilayer particle targeting system of any of the above embodiments further comprises 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, so that the chimeric polypeptide and the second chimeric polypeptide dimerize.

[0290] In some embodiments, the lipid bilayer particle comprises or contains therein a viral nucleocapsid or a derivative thereof, 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, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin-modified synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, a ribonucleoprotein complex, or an antisense oligonucleotide.

[0291] Also disclosed herein are nucleic acids encoding the chimeric polypeptides disclosed herein. For example, in some embodiments, the cargo loading domain of the chimeric loading polypeptide can be composed of ATGACCAGAGTGCCCCTGTACGGCTTCACCAGCATTTGTGGCAGACGGCCCGAAATGGAAGCCGCCGTGTCTACAATCCCCAGATTCCTCCAGAGCAGCAGCGGCTCCATGCTGGACGGCAGATTCGATCCTCAGAGCGCCGCTCACTTCTTCGGCGTGTACGATGGACATGGCGGAAGCCAGGTGGCCAACTACTGCCGCGAAAGAATGCATCTGGCCCTGGCCGAGGAAATCGCCAAAGAAAAGCCCATGCTGTGCGACGGCGACACCTGGCTGGAAAAGTGGAAGAAGGCCCTGTTCAACAGCTTCCTGAGAGTGGACAGCGAGATCGAGAGCGTGGCCCCTGAAACAGTGGGCAGCACATCTGTGGTGGCCGTGGTGTTTCCCAGCCACATCTTCGTGGCTAACTGCGGCGATAGCAGAGCCGTGCTGTGCAGAGGAAAAACAGCCCTGCCTCTGTCCGTGGACCACAAGCCTGATAGAGAGGATGAGGCCGCCAGAATTGAAGCCGCTGGCGGCAAAGTGATCCAGTGGAATGGCGCTAGAGTGTTCGGCGTGCTGGCCATGAGTAGATCCATCGGCGATAGATACCTGAAGCCTAGCATCATCCCCGATCCTGAAGTGACCGCCGTGAAGAGAGTGAAAGAGGACGACTGCCTGATCCTGGCCTCTGACGGTGTCTGGGACGTGATGACAGATGAAGAGGCCTGCGAGATGGCCCGGAAGAGAATCCTGCTGTGGCACAAGAAAAACGCCGTGGCCGGGGATGCTTCTCTGCTGGCTGACGAGAGAAGAAAAGAGGGCAAAGACCCCGCTGCCATGTCTGCCGCCGAGTACCTGTCTAAGCTGGCCATCCAGAGAGGCAGCAAGGACAACATCAGCGTGGTGGTCGTGGACCTGAAA(SEQ ID NO:5),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:5, or a functional fragment thereof. In general, a fragment is considered to be a functional fragment if the protein or peptide encoded by the fragment is capable of increasing active loading of a cargo entity into a lipid bilayer particle, binding to an ABI1 binding protein, or a combination thereof. ,

[0292] In some embodiments, the ABA binding sequence of the second chimeric peptide can be composed of (SEQ ID NO: 1), 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. In general, a fragment is considered to be a functional fragment if the protein or peptide encoded by the fragment is capable of increasing active loading of a cargo entity into a lipid bilayer particle, binding to ABI1 or a variant or fragment thereof, or a combination thereof.

[0293] In some embodiments, the linker of any disclosed peptide can be composed of one of SEQ ID NO: 9 (ACTAGTGGCGGCGGAGGCAGCGGAGGCGGATCTGGCGGAGGATCT), 11 (ACGCGTGGCGGCGGAGGCAGCGGAGGCGGATCTGGCGGAGGATCT), or 13 (GGCGGCGGAGGAAGTGGCGGCGGATCTGGCGGAGGATCTACCGGT), or a peptide having the same sequence as SEQ ID NO: or 100% sequence identity to one of NO:9, 11 or 13.

[0294] In some embodiments, the chimeric targeting polypeptide may optionally include a first cargo entity fused thereto. The first cargo entity is not particularly limited and may be any cargo entity disclosed herein. For example, in some embodiments, the first entity may be an ABA binding sequence comprising a pyranostatin 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 comprises MGGGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 2), TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN(SEQ ID NO:3), or with SEQ ID NO:3 ID NO:2 or 3, 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, or a functional fragment of SEQ ID NO:2, SEQ ID NO:3, or a variant amino acid sequence thereof. For purposes of the present disclosure, a functional fragment of an ABA binding sequence can be about 5 amino acids long, about 10 amino acids long, about 15 amino acids long, about 20 amino acids long, about 25 amino acids long, about 30 amino acids long, about 35 amino acids long, about 40 amino acids long, about 45 amino acids long, about 50 amino acids long, about 55 amino acids long, about 60 amino acids long, about 65 amino acids long, about 70 amino acids long, about 75 amino acids long, about 80 amino acids long, about 85 amino acids long, about 90 amino acids long, about 95 amino acids long, about 100 amino acids long, about 105 amino acids long, about 110 amino acids long, about 115 amino acids long, about 120 amino acids long, about 125 amino acids long. In some embodiments, the present invention relates to a 5-amino acid sequence of the present invention. The 5-amino acid sequence of the present invention is about 140 amino acids long, about 145 amino acids long, about 150 amino acids long, about 155 amino acids long, about 160 amino acids long, about 165 amino acids long, about 170 amino acids long, about 175 amino acids long, about 180 amino acids long, about 185 amino acids long, about 190 amino acids long, about 195 amino acids long, about 200 amino acids long, about 205 amino acids long, about 210 amino acids long, about 215 amino acids long, about 220 amino acids long, about 225 amino acids long, about 230 amino acids long, about 235 amino acids long, about 240 amino acids long, about 245 amino acids long, or about 250 amino acids long.In other words, a 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. In general, a fragment is considered to be a functional fragment if it can increase the active loading of the cargo entity into the lipid bilayer particle, the binding to ABI1 or its variants or fragments thereof, or a combination thereof.

[0295] In another aspect, the present disclosure provides a nucleic acid encoding the chimeric peptide or lipid bilayer particle targeting system of any one of the above embodiments.

[0296] On the other hand, the present disclosure provides a production cell comprising a targeted chimeric polypeptide, a lipid bilayer particle targeting system, a lipid bilayer particle (e.g., a CDMP, e.g., an extracellular vesicle), or a nucleic acid of any 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, erythrocytes, and erythrocyte precursor cells, and iPSC-derived versions of any of the foregoing cells.

[0297] In another aspect, the present disclosure provides a method for producing lipid bilayer particles targeted to receptor cells, such as immune cells, comprising culturing the production cells of the aforementioned aspects, and harvesting the lipid bilayer particles produced by the production cells.

[0298] In some embodiments, the cargo entity is an intraparticle macromolecular assembly (e.g., lentiviral core, AAV particle, other viral cores, VLP cores and subunits thereof).

[0299] 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.

[0300] In some embodiments, the cargo entity is or encodes an AAV nucleocapsid, or an LVV nucleocapsid, or a fragment thereof. Second chimeric polypeptide

[0301] 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 peptide may further comprise a linker connecting a cargo entity (e.g., a cargo entity polypeptide) and a cargo loading domain. The linker may comprise (1) an amino acid sequence selected from SEQ ID NO: 10 (TSGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGGSGGSGGGSGGSGGGSGGGSGGGSGG) and SEQ ID NO: 17 (ESKYGPPAPPAP); or (2) an amino acid sequence having the same amino acid sequence as SEQ ID NO: 18 (ESKYGPPAPPAP); NO: 10, 12, 14, 15, 16 or 17 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%, 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 NO: 10, 12, 14, 15, 16 or 17.

[0302] The cargo loading domain of the second chimeric peptide can be a truncated variant of a wild-type protein comprising an extracellular vesicle targeting domain. For example, the cargo loading domain of the second chimeric peptide comprises residues 126-423 of wild-type ABI1. In some embodiments, the cargo loading domain of the second chimeric peptide comprises: MTRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALP LSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK(SEQ ID NO:6), VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLS VDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK(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 any one of SEQ ID NO: 6 or 7, or SEQ ID NO:6, a functional fragment of SEQ ID NO:7, or a variant amino acid sequence thereof. For the purposes of the present disclosure, a functional fragment of ABI1 can be about 5 amino acids long, about 10 amino acids long, about 15 amino acids long, about 20 amino acids long, about 25 amino acids long, about 30 amino acids long, about 35 amino acids long, about 40 amino acids long, about 45 amino acids long, about 50 amino acids long, about 55 amino acids long, about 60 amino acids long, about 65 amino acids long, about 70 amino acids long, about 75 amino acids long, about 80 amino acids long, about 85 amino acids long, about 90 amino acids long, about 95 amino acids long, about 100 amino acids long, about 105 amino acids long, about 110 amino acids long, about 115 amino acids long, about 120 amino acids long, about 125 amino acids long. , about 130 amino acids long, about 135 amino acids long, about 140 amino acids long, about 145 amino acids long, about 150 amino acids long, about 155 amino acids long, about 160 amino acids long, about 165 amino acids long, about 170 amino acids long, about 175 amino acids long, about 180 amino acids long, about 185 amino acids long, about 190 amino acids long, about 195 amino acids long, about 200 amino acids long, about 205 amino acids long, about 210 amino acids long, about 215 amino acids long, about 220 amino acids long, about 225 amino acids long, about 230 amino acids long, about 235 amino acids long, about 240 amino acids long, about 245 amino acids long or 250 amino acids long. In other words, a 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. In general, a fragment is considered to be a functional fragment if it can increase active loading of a cargo entity into a lipid bilayer particle, binding to an ABI1 binding protein, or a combination thereof. How to make and use

[0303] Those skilled in the art will understand its amazing scope of application after reading this disclosure. The engineered lipid bilayer particles provided can be suitable for therapeutic applications. In some embodiments, the lipid bilayer particles or preparations provided herein can be contacted with ex vivo (such as CAR-T) or in vivo (such as HIV) receptor cells.

[0304] In addition, the ability to effectively deliver cargo entities (e.g., polypeptide cargo entities, nucleic acid cargo entities, sugar cargo entities, lipid cargo entities, small cargo entities, complex cargo entities, etc.) to T cells is useful for providing engineered T cells, for example, for use as T cell therapy (e.g., CAR-T therapy). As an alternative or supplement, the ability to effectively deliver cargo entities is useful in treating HIV (e.g., by delivering cargo entities that inhibit, interrupt, or disrupt one or more aspects of HIV or its life cycle). In some embodiments, cargo entities such as siRNA, Cas9-sgRNA, etc. can be used to target HIV.

[0305] In another aspect, the present invention provides a method for 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). Typically, the lipid bilayer particle will comprise a targeting chimeric peptide (e.g., a targeting chimeric peptide comprising a targeting domain) of any of the above embodiments. Thus, 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), comprising administering to an individual a lipid bilayer particle disclosed herein, wherein the lipid bilayer particle comprises a cargo entity.

[0306] 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 delivered to a recipient cell, 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 editor, a guide editor (prime editor), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a kinase, a kinase inhibitor, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin modification synthetic transcription factor, a natural transcription factor and a mutant form 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 cutting. In some embodiments, the CRISPR enzyme catalyzes RNA cutting. In some embodiments, the CRISPR enzyme is a Cas9 protein (e.g., a naturally occurring bacterial Cas9 and any chimera, 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, homologs thereof, or modified variants thereof.

[0307] In some embodiments, the cargo entity is a nucleic acid cargo entity. In some embodiments, the first or second cargo entity may be a nucleic acid (e.g., DNA or RNA) or another molecule. Nucleic acid cargo entities may include, but are not limited to, DNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, and combinations thereof encoding polypeptides of interest. Other potential cargo entities include, but are not limited to, viral and non-viral vectors (e.g., nucleocapsids) expressed intracellularly (or delivered to the cytosol of cells), and 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. In fact, those skilled in the art will recognize that any cargo that can be expressed in cells or physically delivered to the interior of cells can be fused (genetically or synthetically) to ABI proteins / peptides or ABA binding sequences and is explicitly contemplated herein. Genetic fusion can be achieved by expressing two or more components of a chimeric polypeptide or peptide in a lipid bilayer particle (e.g., CDMP, such as EV) production cell. Alternatively, the cargo entity fused to the loading domain can be delivered to a lipid bilayer particle (e.g., CDMP, such as EV) production cell for subsequent incorporation into the lipid bilayer particle. Alternatively, the cargo entity fused to the loading domain can be inserted into the lipid bilayer particle after the lipid bilayer particle is secreted by the production cell.

[0308] Thus, in some embodiments, the cargo entity is or includes 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, a receptor signaling activator or inhibitor, an intracellular antibody, 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.

[0309] In some embodiments, the cargo entity comprises a nucleic acid sequence encoding a chimeric antigen receptor.

[0310] The disclosed lipid bilayer particles can be formulated as part of a pharmaceutical composition for treating a disease or condition, and the pharmaceutical composition can be administered to a patient in need to deliver the cargo entity to a receptor cell (e.g., a lymphocyte) to treat the disease or condition. 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 a receptor cell (e.g., a lymphocyte) so that the CAR is expressed by the lymphocyte, as described in more detail below.

[0311] The present disclosure also provides an in vitro method for targeted delivery of cargo entities to lymphocytes, comprising obtaining a lymphocyte population from an individual and contacting the lymphocyte population with the lipid bilayer particles disclosed herein in vitro, wherein the lipid bilayer particles contain cargo entities. For the in vitro method, all of the cargo entities described above are similarly applicable and can be delivered to a recipient cell (e.g., immune cell, such as lymphocyte) population. In some embodiments, a lymphocyte population is obtained by a single collection. In some embodiments of the in vitro method, the method may further include administering the recipient cell (e.g., immune cell, such as lymphocyte, etc.) population back to the individual after the recipient cell (e.g., immune cell, such as lymphocyte) is contacted with the lipid bilayer particles.

[0312] For example, the patient can be separated by a single sampling to separate the receptor cell (e.g., immune cell, such as lymphocyte) population, and then contacted with the lipid bilayer particles disclosed herein, which contain ribonucleoprotein complexes and nucleic acids encoding chimeric antigen receptors (CAR). The ribonucleoprotein complex and the nucleic acid encoding CAR will be delivered to the receptor cells (such as lymphocytes), and the nucleic acid sequence will be integrated into the genome and expressed by the receptor cells (e.g., lymphocytes), so that CAR is expressed on the surface of the receptor cells (i.e., lymphocytes). The expression of receptor cell (e.g., lymphocyte) CAR can also be achieved in vivo by administering CD2-targeted lipid bilayer particles (such as intravenous injection or infusion) to the patient. Production

[0313] In addition to targeting, the present disclosure also provides techniques (eg, methods) for loading cargo into lipid bilayer particles and / or otherwise producing engineered lipid bilayer particles (eg, cargo-loaded engineered lipid bilayer particles), as described herein.

[0314] In some embodiments, the technology for loading cargo entities into lipid bilayer particles (CDMPs, such as EVs) may include expressing (a) nucleic acids (e.g., mRNAs) encoding targeted chimeric polypeptides, fusogen entity polypeptides, and / or cargo entities in production cells, and (b) expressing targeted chimeric peptides and / or fusogen entity polypeptides in production cells. In some embodiments, production cells are eukaryotic cells. Optionally, mRNAs of chimeric peptides containing cargo entities and / or fusogen entity polypeptides may be expressed from one or more vectors transfected into suitable production cells to produce the disclosed extracellular vesicles. Note that vectors may also be stably transfected. One or more vectors (vectors or vectors) for expressing mRNAs of chimeric peptides containing cargo entities may be packaged in a kit designed for preparing the disclosed extracellular vesicles. In some embodiments, the kit includes lipid bilayer particles with synthetic nucleic acids (e.g., antisense oligonucleotides) or plasmids.

[0315] 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 (iPSC), T cells, erythrocytes, erythropoetic precursors, and iPSC-derived versions of any of the foregoing cells.

[0316] In some embodiments, the cargo entity loading of the chimeric loading peptide is enhanced compared to passive cargo loading. For example, the methods provided herein (e.g., lipid bilayer particles comprising targeting chimeric polypeptides and / or fusion proent polypeptides) can achieve up to 23-fold enrichment of cargo entities in microvesicles and up to 49-fold enrichment in exosomes compared to passive loading in corresponding particles.

[0317] In another aspect, the present disclosure provides a technique (e.g., a method) for loading multiple (e.g., two) cargo entities into lipid bilayer particles, such as CDMPs (e.g., extracellular vesicles (EVs)), comprising expressing a lipid bilayer particle loading system (comprising a targeting chimeric polypeptide, a fusion entity polypeptide, or both) of any of the above embodiments in a production cell.

[0318] In some embodiments, 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.

[0319] In some embodiments, the expression of a targeting chimeric polypeptide or fragment thereof (e.g., a VLR) together with a viral cargo entity (e.g., a standard lentiviral packaging plasmid) results in a loss of viral genome titer of no more than 0%, no more than 10%, no more than 20%, no more than 30%, no more than 40%, or no more than 50%. In some such embodiments, a standard lentiviral packaging plasmid may include a fusogen (e.g., VSV-G) and a viral helper plasmid. This is comparable to the loss observed when expressing a viral cargo entity (e.g., a standard lentiviral packaging plasmid) without expressing a targeting chimeric polypeptide or fragment thereof (e.g., a VLR). Lymphocyte targeting

[0320] Certain embodiments of the provided technology are particularly useful or advantageous in achieving lymphocyte targeting (eg, targeting human lymphocytes or populations thereof).

[0321] Those skilled in the art are aware that CRISPR-Cas9-mediated engineering of the human T cell genome is an active area of ​​research for the development of drugs to treat cancer, autoimmune diseases, and infectious diseases. Delivery of the programmable nuclease Cas9 and a single-stranded guide RNA (sgRNA) complementary to the targeting sequence results in the generation of double-strand breaks that can introduce frameshift mutations in the coding gene and ablate protein expression. In addition, co-delivery of homology-directed repair templates can insert specific mutations, insertions, or deletions into the genome of the recipient cell. Despite the multiple applications of this technology, translation of this strategy remains difficult, at least in part due to challenges associated with the in vivo delivery of Cas9. One approach to exploiting fundamental advances in gene therapy is to use adeno-associated virus (AAV) vectors, but their safety and efficacy are often limited by anti-vector immunity and limited tissue tropism. Virus-like particles (VLPs) can also deliver Cas9 nucleases or base editors, but it is unclear whether the immunogenicity of viral proteins will similarly limit these approaches. Synthetic nanoparticle-nucleic acid (e.g., mRNA) delivery is an alternative to viral vectors and has been successfully used to deliver mRNA in vivo to enable sustained expression of chimeric antigen receptors in mouse T cells. However, achieving efficient and specific T cell targeting, transient expression of Cas9, and avoiding off-target effects remain challenging. These prevalent difficulties are compounded by the fact that T cells exhibit low endocytosis rates, making delivery of any cargo to T cells a challenge. Overall, there is a great opportunity to improve delivery systems to deliver biologics to T cells in patients.

[0322] One promising emerging strategy is to utilize extracellular vesicles (EVs) to deliver biomolecular cargo. Extracellular vesicles are nanometer-sized, membrane-enclosed particles secreted by all cells that naturally encapsulate proteins and nucleic acids during biogenesis. EVs mediate intercellular communication and deliver their contents to recipient cells to affect cellular function. Their inherent properties, such as lack of toxicity and immunogenicity, as well as the ability to engineer surface and lumen cargo loading, make EVs an attractive platform for the delivery of a wide range of therapeutics. Cargo can be incorporated into vesicles during EV biogenesis by overexpressing the cargo in producer cells or by physically or chemically modifying the vesicles after harvesting. Cells that have been genetically engineered to produce functionalized EVs can even be implanted to continuously generate such particles in situ. While modification of EVs after harvesting can confer flexibility in cargo loading, this approach requires more extensive purification and poses manufacturing and regulatory challenges.

[0323] Several recent studies have investigated the use of EVs to deliver Cas9 for the treatment of cancer, hepatitis B, and genetic diseases, highlighting the promise of this approach for achieving intracellular Cas9 delivery. However, many of these exploratory studies have employed EV engineering approaches known to introduce artifacts in downstream experiments, obscuring possible functional effects of the EVs. Of particular concern are approaches that rely on transfection of EV producer cells with lipid complexes, loading EVs using electroporation methods known to result in cargo aggregation, or isolating EVs using commercial kits not designed for functional delivery applications, all of which have been shown to introduce artifacts. 24-26

[0324] Here, we address this need by developing an integrated bioengineering strategy for genetically engineered cells that directs the self-assembly and production of multifunctional EVs. As an instructive application, we systematically evaluated, compared, and generated technologies that enable EV targeting, active loading of protein cargo into EVs, and EV fusion to achieve functional cargo delivery to T cells. This exploration identified key limiting factors and drivers of EV-mediated functional delivery, including potential mechanisms for receptor binding-mediated enhanced delivery to T cells. We validated these technologies by demonstrating therapeutically relevant capabilities—delivery of Cas9 ribonucleoprotein complexes (RNPs) to primary human CD4 + In T cells, the gene encoding the HIV co-receptor CXCR4 was eliminated. Example

[0326] The technology of the present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate the advantages of the present technology and to further assist those of ordinary skill in the art in preparing or using the compositions and systems of the present technology. These examples should never be construed as limiting the scope of the present technology as defined by the appended claims. These examples may include or incorporate any variation, aspect, or embodiment of the above-mentioned present technology. The above-mentioned variation, aspect, or embodiment may further each include or incorporate a variation of any or all other variations, aspects, or embodiments of the present technology. Example 1: Methods and Materials

[0327] Plasmid construction. Plasmids were constructed using standard molecular biology techniques. Codon optimization was performed using the GeneArt gene synthesis tool (Thermo Fisher). PCR was performed 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 (E. Coli) (Thermo Fisher) and grown at 37°C.

[0328] Plasmid backbone. Modified pcDNA3.1 (Thermo Fisher V87020) was used to generate a universal expression vector. In brief, the hygromycin resistance gene and SV40 promoter were removed, leaving the SV40 replication origin and poly(A) signal intact. The Bsal site in the AmpR gene and 5'-UTR and the Bpil site in 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 number 52961).

[0329] Plasmid source vectors. Fluorescent proteins enhanced blue fluorescent protein 2 (EBFP2), enhanced yellow fluorescent protein (EYFP), and dimeric tomato (dTomato) were derived from Addgene vectors donated by Robert Campbell, Joshua Leonard, and Scott Sternson (plasmid numbers 14893, 58855, and 18917, respectively). Monomeric cyan fluorescent protein 1 (TFP1) was synthesized by Thermo Fisher. scFv was synthesized from a previously published scFv sequence derived from monoclonal antibody 9.6, and the PDGFR transmembrane domain was derived from the pDisplay system vector (Addgene plasmid number 61556, donated by Robert Campbell). The C1C2 domain sequence was provided by Natalie Tigue and synthesized by Thermo Fisher. Constitutively active Cx43 and SLAM were derived from Uniprot sequences P17302 CXA1_HUMAN and Q13291-1SLAF1_HUMAN isoform 1 was synthesized by Thermo Fisher. The plasmid encoding measles virus glycoprotein was donated by Isabelle Clerc, Thomas Hope, and Richard D'Aquila. pX330 encoding Cas9 was donated by Erik Sontheimer (University of Massachusetts) and was originally derived from Addgene plasmid number 42230 donated by Feng Zhang. The CXCR4 sgRNA sequence was provided by Judd Hultquist and is shown below: GAAGCGTGATGACAAAGAGG. ABI and PYL domains were synthesized by Thermo Fisher and IDT, respectively.

[0330] Plasmid preparation. Bacteria were grown overnight in 100 mL LB + Amp culture for 12-14 hours. The culture was spun at 3,000 g for 10 minutes to precipitate the bacteria, and the precipitate was resuspended in 4 mL of 25 mM Tris pH 8.0, 10 mM EDTA, 15% sucrose and 5 mg / mL lysozyme and incubated for 30 minutes. The bacteria were lysed in 8 mL of 0.2 M NaOH and 1% SDS for 15 minutes, and then neutralized in 5 mL of 3 M sodium acetate (pH 5.2) for 15 minutes. The precipitate was precipitated at 9,000 g for 20 minutes, the supernatant was filtered through cheesecloth, and incubated at 37 ° C with 3 μL of 10 mg / mL RNAse A (Thermo Fisher) for 1-3 hours. The sample was extracted with 5 mL of phenol chloroform, and the water layer was recovered after centrifugation at 7,500 g for 20 minutes. A second phenol chloroform extraction was performed with 7 mL of solvent. 0.7 volumes of isopropanol were added to the recovered supernatant, the sample was inverted and incubated at room temperature for 10 minutes, and then centrifuged at 9,000g for 20 minutes to precipitate the DNA mixture. The precipitate was briefly dried and resuspended in 1 mL of 6.5% PEG 20000 and 0.4M NaCl. The DNA was incubated on ice overnight and precipitated at 21,000g for 20 minutes. The supernatant was removed, and the precipitate was washed with cold anhydrous ethanol and dried at 37°C, then resuspended in TE buffer (10mM Tris, 1mM EDTA, pH 8.0). The DNA was diluted to 1μg / μL using Nanodrop2000 (Thermo Fisher).

[0331] 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% penicillin-streptomycin and 4 mM L-glutamine. Sublines generated from these cell lines were cultured in the same manner. Cells were subcultured every 2-3 days at a ratio of 1:5 or 1:10, and adherent cells were removed from the plates using trypsin-EDTA (Gibco 25300-054). Lenti-X cells (Takara) were cultured in the same manner, 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 when used). Cells were maintained at 37°C, 5% CO2. HEK293FT and Jurkat cells were negative for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza LT07-318).

[0332] Transfection. To transfect HEK293FT cells and derived cell lines for EV packaging in 15 cm dishes, culture 18 × 10 6 cells / dish (1×10 6 Cells were plated at a density of 10 cells / mL. Cells were transfected by the calcium phosphate method with 30 μg DNA plus 1 μg fluorescent transfection control. Plasmid DNA was mixed with 2M CaCl2 (final concentration 0.3M) and added dropwise in a 1:1 ratio to 2× HEPES buffered saline solution (280mM NaCl, 0.5M HEPES, 1.5mM Na2HPO4) and mixed seven times by pipetting. The transfection solution was incubated for 3 minutes, mixed eight times by pipetting, and gently added to the side of the plate. To transfect HEK293FT cells for EV packaging in a 10 cm dish, cells were plated at 5×10 6 cells / dish (6.25×105 Cells were plated at a density of 1.7 × 10 cells / mL and transfected as described above with 20 μg DNA plus 1 μg transfection control, and the transfection mixture was added dropwise to the culture dish. Lenti-X cells were transfected in the same manner in 10 cm culture dishes, but were plated 24 hours prior to transfection as recommended by the manufacturer (Takara). For transfection of HEK293FT cells in 24-well plates, cells were plated at 1.7 × 10 5 cells / well (3.4×10 5 Cells were plated at a density of 10 cells / mL and transfected with 200 μg DNA as described above, and the transfection mixture was added dropwise to the wells. The medium was changed after 12-16 hours. Jurkat lipofectamine transfection was performed according to the manufacturer's protocol.

[0333] Cell line generation. To generate lentivirus, HEK293FT or Lenti-X cells were plated at 5 × 10 6 cells / dish (6.25×10 5 Cells were plated at a density of 10 cells / mL in a 10 cm dish. After 6-12 hours for HEK293FT or 24 hours for Lenti-X, cells were transfected with 10 μg viral vector, 8 μg psPAX2 and 3 μg pMD2G by calcium phosphate transfection as described above. The medium was changed after 12-16 hours. After 28 hours of changing the medium, the lentivirus was harvested from the conditioned medium. The medium was centrifuged at 500g for 2 minutes to clear the cells, and the supernatant was filtered through a 0.45 pm pore size filter (VWR). The lentivirus was concentrated from the filtered supernatant by ultracentrifugation at 100,420g in an Ultra Clear tube (Beckman Coulter 344059) at 4°C in a Beckman Coulter Optima L-80XP ultracentrifuge equipped with a SW41Ti rotor. The supernatant was aspirated, leaving a final volume of ∼100 μL of virus, and the concentrated lentivirus was placed on ice for at least 30 min before being resuspended and used to transduce ∼1 × 10 5 Cells were plated at the time of transduction or the day before. Where appropriate, drug selection was initiated 2 days after transduction, using antibiotic concentrations of 1 μg / mL puromycin (Invitrogen ant-pr) and 10 μg / mL blasticidin (Alfa Aesar J61883) for HEK293FT cells, or 0.2 μg / mL puromycin and 2 μg / mL blasticidin for Jurkat cells. Cells were maintained in antibiotics for at least two weeks, with subcultures performed every one to two days.

[0334] Cas9 reporter cell line sorting. Cells were sorted at 1×10 7 Cells were prepared for fluorescence activated cell sorting (FACS) by resuspending in DMEM or RPMI (supplemented with 10% FBS, 25mM HEPES and 100μg / mL gentamicin (Amresco0304) as appropriate) at a concentration of 10 cells / mL. The highest mTFP1 expressers (top 10% or less) lacking any dTomato expression were sorted on a BD FacsAria Ilu using a 488nm laser (530 / 30 filter) and a 562nm laser (582 / 15 filter). Cells were collected in DMEM or RPMI (supplemented with 20% FBS, 25mM HEPES and 100μg / mL gentamicin as appropriate). The cells were centrifuged and resuspended in normal growth medium containing 100μg / mL gentamicin for recovery.

[0335] EV production, isolation and characterization. EV-producing cell lines were plated in 10 or 15 cm culture dishes and transfected by the calcium phosphate method on the same day where appropriate. The medium was replaced with EV-depleted medium the next morning. EV-depleted medium was prepared by adding 10% exosome-depleted FBS (Gibco A27208-01), 1% pen-strep and 4mM L-glutamine to DMEM. EVs were harvested from conditioned medium by differential centrifugation 24-36 hours after medium replacement as described previously. 41,42 Briefly, conditioned media was cleared of debris by centrifugation at 300 g for 10 min to remove cells, followed by centrifugation at 2,000 g for 20 min to remove dead cells and apoptotic bodies. The supernatant was centrifuged at 15,000 g for 30 min in a Beckman Coulter Avanti J-26XP centrifuge equipped with a J-LITE JLA 16.25 rotor to pellet microvesicles. The supernatant was collected and ultracentrifuged at 120,416 g for 135 min in a Beckman Coulter Optima L-80XP ultracentrifuge equipped with a SW41 Ti rotor using polypropylene ultracentrifuge tubes (BeckmanCoulter 331372) to pellet exosomes. All centrifugation steps were performed at 4°C. After removal of the supernatant, the EV pellet was placed in ~100-200 μL of conditioned media and incubated on ice for at least 30 min before resuspending. EV concentration was determined by NanoSight analysis. Samples were diluted with PBS to a concentration of approximately 10 8Particles / mL for analysis. NanoSight analysis was performed on a NS300 (Malvern) with software version 3.4. Three 30-second videos were acquired for each sample using a 642 nm laser, with a camera level of 14, an infusion rate of 30, and a detection threshold of 7. Default settings were used for blur, minimum track length, and minimum expected particle size. EV concentration was defined as the average of the concentrations calculated from each video. Size distributions were generated by the software. For TEM, 65 μL of 4% PFA was added to 200 uL EVs and samples were fixed in Eppendorf tubes 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 with a wedge of filter paper and the grid was washed twice by inverting it on a drop of buffer for 30 seconds and then once with diH2O. 2% uranyl acetate (Ted Pella) stain was applied twice and blotted after 30 seconds. The grids were allowed to dry and then stored in a grid box until use. The grids were imaged in a JEOL JEM 1230TEM (JEOL USA) with an accelerating voltage of 100 KV. Data were collected using an Orius SC1000CCD camera (Gatan). EVs were stored on ice and used within 10 days, or stored at -80°C for long-term storage.

[0336] 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 and one protease inhibitor cocktail tablet (Pierce PIA32953) per 10 mL) and incubated on ice for 30 minutes. Lysates were cleared by centrifugation at 12,000 g for 20 minutes at 4°C and the supernatant was harvested. Protein concentration was determined using the BCA assay (Pierce) according to the manufacturer's instructions. Samples were counted at 1 to 2 μg of protein (for cell lysates) or 1 × 10 7 Up to 6×10 8Each (for EV) was standardized. The samples were heated at 70 ° C (for membrane-bound scFv and calnexin) or 95 ° C (for Cas9, CD9, CD81 and Alix) in Laemmli buffer (60mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 100mM dithiothreitol, 0.01% bromophenol blue) for 10 minutes. The samples were loaded onto a 4-15% polyacrylamide gradient Mini-PROTEAN TGX precast protein gel (Bio-Rad), run at 50V for 10 minutes, and then run at 100V for 1 hour. The protein was transferred to a PVDF membrane (Bio-Rad) at 100V for 45 minutes. For anti-FLAG blotting, the membrane was blocked in 3% milk in TBS (50mM Tris, 138mM NaCl, 2.7mMKC1, pH 8.0) for 30 minutes. The membrane was washed once in TBS for 5 minutes and then incubated overnight at 4°C in an anti-FLAG primary antibody (Sigma F1804) diluted 1:1000 with 3% milk in TBS. Prior to secondary antibody staining, the membrane was washed once in TBS for 5 minutes and twice in TBST 1 (50mM Tris, 138mM NaCl, 2.7mM KCl, 0.05% Tween 20, pH 8.0) for 5 minutes each. For all other blots, the membrane was blocked in 5% milk in TBST 2 (50mM Tris, 150mM NaCl, 0.1% Tween 20, pH 7.6) for 1 hour. The membrane was incubated overnight at 4°C in the primary antibody diluted in 5% milk in TBST 2. The primary antibodies included anti-HA (Cell Signaling Technology 377245C29F4, 1: 1000), anti-CD9 (Santa Cruz Biotechnology sc-13118, 1: 500), anti-CD81 (Santa Cruz Biotechnology sc-23962, 1: 500, run under non-reducing conditions), anti-Alix (Abeam Abl 17600, 1: 500) and anti-calnexin (Abam Ab22595, 1: 1000). Before secondary antibody staining, the membrane was washed 3 times in TBST 2 for 5 minutes each. HRP-conjugated anti-mouse (Cell Signaling Technology 7076) and anti-rabbit (Invitrogen 32460) secondary antibodies were diluted 1: 3000 with 5% milk in TBST 2. The membrane was incubated with the secondary antibody for 1 hour at room temperature and then washed 3 times in TBST 2 (washing for 5 minutes).Membranes were probed with Clarity Western ECL Substrate (Bio-Rad) and exposed to film, developed and scanned or imaged using an Azure c280 imager. Images were cropped using Adobe Illustrator. No other image processing was performed.

[0337] Surface immunoblotting. The cells were transferred to a FACS tube containing 1 mL of FACS buffer (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. 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, the cells were flicked to mix, and incubated at 4 ° C for 5 minutes. The conjugated primary antibody was then added according to the dilution recommended by the manufacturer, the cells were flicked to mix and incubated at 4 ° C for 30 minutes. The cells were then 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 performing flow cytometry, the cells were resuspended in two drops of FACS buffer. For MiltenyiBiotec antibodies, 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 are as follows: anti-FLAG-APC (Abam 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&D Systems IC002A) or anti-human IgG1-PE (Miltenyi REA293, 130-113-438) were used as isotype controls where appropriate.

[0338] EV binding and uptake experiments. Jurkat T cells or primary human CD4 + T cells and EVs were cultured at a 100,000:1 EV:cell ratio (usually 1 × 10 5 Cells 1×10 10EVs) were incubated at 37 °C for 2 h and then centrifuged at 150 g for 5 min for Jurkat cells and at 400 g for 3 min for primary T cells, unless otherwise stated. For Jurkat, cells were plated in 48-well plates in a total volume of 300 μL. For primary T cells, cells were plated in 96-well plates in a total volume of 200 μL. Cells were plated when EVs were added and the wells were adjusted to the appropriate volume with RPMI. For binding experiments, unless otherwise stated, cells were incubated at 37 °C for 2 h and then washed 3 times in FACS buffer and centrifuged at 150 g for 5 min for Jurkat cells and at 400 g for 3 min for primary T cells. Cells were resuspended in a drop of FACS buffer before flow cytometry. For adsorption of EVs to aldehyde / sulfate latex beads (Thermo Fisher), EVs were incubated with beads at a rate of 1 × 10 per 2 μL of beads diluted 1:10 with PBS. 9 EVs were mixed in a ratio of 1:1. The volume of each sample was normalized with PBS, and the beads and EVs were incubated at room temperature for 15 minutes. The samples were then made up to 200 μL with PBS and incubated with shaking at room temperature for 2 hours. Prior to the specified EV incubations, cells were blocked for 1 hour at 37°C using an anti-CD2 antibody that binds to the same epitope as the scFv (Beckman Coulter A60794) or blank EVs. For EV uptake experiments of viral glycoproteins, cells were incubated with EVs at 37°C for 16 hours. In preparation for analysis, cells were washed twice in PBS and incubated with two drops of trypsin-EDTA at 37°C for 5 minutes to remove surface-bound vesicles. The cells were washed with RPMT to quench trypsin and then washed twice more with FACS buffer before analysis.

[0339] Analysis Flow cytometry and analysis. Flow cytometry was performed on a BD LSR Fortessa Special Order Research Product using a 562 nm laser (582 / 15 filter) for dTomato, a 488 nm laser (530 / 30 filter) for EYFP, and 488 nm and 405 nm lasers (530 / 30 and 525 / 50 filters, respectively) for mTFP1. Approximately 10,000 viable cells were collected for each sample for analysis. Data were analyzed using FlowJo v10 (FlowJo, LLC). Briefly, cells were identified using a plot of FSC-A versus SSC-A, and singlets were gated using a plot of FSC-A versus FSC-H ( Fig.26). Fluorescence data were spectrally compensated for bleed-through where appropriate. Mean fluorescence intensity (MFI) was derived for single-cell samples and averaged across three biological replicates. Autofluorescence from untreated cells was subtracted from the other samples. Standard errors of the mean were calculated. Where indicated, calibration curves were generated using 9-peak UltraRainbowCalibration Particles (Spherotech URCP-100-2H) to convert fluorescence to absolute fluorescence units.

[0340] Confocal microscopy. Cells were transfected onto poly-L-lysine-coated glass coverslips by the calcium phosphate method and mounted on slides for imaging. Microscopic images were acquired on a Leica SP5 IT laser scanning confocal microscope using a 100x oil immersion objective. Bright-field images were acquired at a PMT setting of 443.0 V. Fluorescence excitation was performed using a 514 nm laser with an intensity of 20% and an intelligent gain of 94%. Emission spectra were acquired in the range of 520–540 nm using a HyD sensor. Images were acquired at a resolution of 512 × 512 and a scan speed of 400 Hz. Pseudo-colored fluorescence images were contrast adjusted in ImageJ software to achieve 4% pixel saturation.

[0341] Affinity chromatography. Affinity chromatography separation was performed as previously reported. 51In brief, an anti-FLAG affinity column was prepared by loading anti-FLAG M2 affinity gel (Sigma A2220-1ML) into a 4mL 1×5cm glass column (Bio-Rad) and draining by gravity flow. The column was washed with 5mL TBS (50mM Tris-HCl, 150mM NaCl, pH 7.5) and balanced with 3 consecutive 1mL washes of regeneration buffer (0.1M glycine-HCl, pH 3.5), followed by another 5ml TBS wash. The concentrated EVs were loaded onto the top of the column and subsequently 1-2mL TBS was added (chased with 1-2mL of TBS). The column was incubated with EVs for 5 minutes before continuing. The effluent was reloaded onto the column so that the medium containing EVs passed through the matrix five times. The column was washed with 10mL TBS before elution. EVs were eluted with 2.5 mL of elution buffer (100 μg / mL 3x FLAG peptide (Sigma F4799-4MG) in TBS) and incubated on the column for 5-10 min after the void fraction was drained (approximately 1 mL). Five EV fractions were collected in 0.5 mL portions (approximately 8 drops from the column each). The column was regenerated by three consecutive washes with 1 mL of regeneration buffer and then stored in storage buffer (50% glycerol, 0.02% sodium azide in TBS) at 4°C.

[0342] Cas9 in vitro cleavage assay. EVs were prepared as described above by transiently transfecting the components in a 10 cm dish with the following DNA ratios: 6 μg anti-CD2 scFv, 9 μg Cas9 vector, 5 μg sgRNA vector, and 1 μg mTFP1 transfection control. EVs were lysed by incubating with mammalian protein extraction reagent (MPER, Thermo Fisher) for 10 min at room temperature (20-23°C) with gentle agitation. 200 ng of linearized target plasmid template and Cas9 reagent buffer (IDT, Alt-R CRISPR-Cas9 System) were added to the vesicles, and the samples were incubated at 37 for 1 hour. Proteinase K (Thermo Fisher) was added to the samples at 1 μL per 10 μL reaction mixture and incubated at 55°C for 10 min. Samples were run on 1% agarose gels stained with SYBR safe (Thermo Fisher) and imaged using the BioDoc-It imaging system (VWR).

[0343] Primary CD4 +T cell isolation, culture, and activation. A surprising finding was that engagement of CD2 enhanced exosome-mediated functional delivery in vitro for both recombinant and EV-displayed antibodies, although similar benefits were not observed for microvesicle-mediated delivery. This combination of effects cannot be explained by known features of CD2 / T cell biology, although it may be related to the finding that ligand engagement triggers CD2 internalization. Although both types of scFv-displaying vesicles specifically bound CD2 and were internalized to some extent, there may be differences in intracellular trafficking and fusion between the two vesicle populations. For example, CD2-binding-mediated trafficking may favor fusion in a different way that favors exosomes compared to the native uptake pathway. This phenomenon warrants further investigation to elucidate the underlying mechanism.

[0344] EV functional delivery experiments. EVs were prepared as described above by transiently transfecting the following DNA ratios in 10 cm dishes: 6 μg anti-CD2 scFv, 9 μg dual Cas9 and sgRNA vectors, 2.5 μg or 3 μg VSV-G of each measles virus glycoprotein H / F, 2 μg filler promoter-less pcDNA, and 1 μg mTFP1 transfection control. To generate vesicles lacking scFv, a PDGFR-binding 3x FLAG tag construct in the same vector backbone was transfected in place of the scFv at the same plasmid copy number. EVs were delivered to primary human CD4 + T cells. Cells were cultured in the presence of EVs for 6 days, with fresh supplemented RPMI and IL-2 added every 2-3 days. For repeat dosing, 100 μL of medium was carefully removed from the top of each well and replaced with 100 μL of fresh EVs and medium. Cells were harvested on day 6, washed with PBS, and pelleted by centrifugation at 400 g for 3 minutes at 4°C. Cells were resuspended in 100 μL QuickExtract DNA extraction 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.

[0345] High-throughput sequencing (NGS) library generation. Approximately 100 ng of genomic DNA was used as a template in the first round of PCR amplification. The CXCR4 region of interest was amplified using the following primers: F1: 5'ACA CTC TTT CCC TAC ACG CTC TTC CGATCT NNN NNG AGA AGC ATG ACG GAC AAG TAC AG 3'R1: 5'GTG ACT GGA GTT CAG ACG TGTGCT CTT CCG ATC TNN NNN TCC CAA AGT ACC AGT TTG CCA C 3'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 of PCR amplification using the following primers: F2: 5'AAT GAT ACG GCG ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T 3'R2: 5'CAA GCA GAA GAC GGC ATA CGA GAT-Index-GTG ACT GGA GTT CAG ACG TGT GCT C3'PCR cycles were as follows: 98°C 3 min, (98°C 15 sec, 69°C 30 sec, 72°C 5 sec) × 20, 72°C 5 min, 4°C 5 min. Prior to HTS, the PCR product was purified again using MagJET beads. Both the first and second rounds of PCR were performed using a primer concentration of 200 nM and Phusion DNA polymerase.

[0346] HTS. Genomic DNA sample concentrations were measured on Qubit using the HS dsDNA kit and pooled into libraries at equimolar concentrations. The libraries were serially diluted to 4 nM. The libraries and PhiX were denatured with NaOH and diluted to 14 pM according to the Illumina MiSeq guide. The reaction mixture consisted of 8% PhiX and 92% library. Samples were run on Illumina MiSeq using MiSeq ReagentKit v3 and paired-end reads were collected. Data were analyzed using custom code developed by 496code.

[0347] Statistical analysis. Statistical details are described in the figure descriptions. Unless otherwise stated, three independent biological replicates (cells) or technical replicates (beads) were analyzed for each condition, and the average fluorescence intensity of approximately 10,000 live single cells or beads was analyzed for each sample. Unless otherwise stated, error bars represent the standard error of the mean. Paired comparisons were performed using two-tailed Student's t-tests in Excel, with the null hypothesis being that the two samples were equal. The significance threshold was set to 0.05. After testing, the Benjamini-Hochberg procedure was applied in each panel of a given figure to reduce the false discovery rate. Example 2: Strategies for engineering multifunctional EVs for delivery to T cells.

[0348] Our overall approach to developing technology to achieve the goal of targeted delivery of biomolecules to T cells is to address each limiting step in the process ( Figure 1 ) - cargo loading into EVs during biogenesis, EV binding to specific receptor cells, uptake, and fusion of EVs with receptor cells to release cargo into the cytoplasm of T cells. Our approach relies entirely on genetically encodeable functions, and we call this strategy GEMINI - genetically encoded multifunctional integrated nanovesicles. Example 3: Engineered membrane scaffolds displaying scFv on EVs

[0349] We first investigated strategies to confer targeting properties on EVs. To promote specific interactions between EVs and recipient cells and facilitate EV uptake, a promising strategy is to display targeting moieties on the surface of EVs. This strategy was pioneered by displaying small peptides, although we and others have shown that these effects are modest and variable. 34Recently, the display of high-affinity targeting domains, including nanobodies and antibody single-chain variable fragments (scFvs), has enabled EV targeting of receptors such as EGFR and HERZ. In these cases, scFv display was achieved by fusion to the C1C2 lactadherin domain, which binds to phosphatidylserine on the outer membrane leaflet of certain EVs. We investigated whether this approach could mediate EV targeting to T cells using an anti-CD2 scFv. We chose CD2 as a target for several reasons; ligand engagement triggers internalization of CD2, and we hypothesized that this mechanism could enhance EV uptake upon receptor docking. This could be particularly useful for conferring delivery to T cells, which exhibit low rates of endocytosis and are often challenging to deliver via other vectors. We also chose to avoid targets such as CD3 that could induce nonspecific T cell activation. We chose a unique display system based on the transmembrane domain of the platelet-derived growth factor receptor (PDGFR); we hypothesized that targeting domains could be displayed on multiple EV populations using this general strategy. Since the design of the extravesicle linker may affect scFv trafficking, folding, and target binding, we investigated three candidates: an alpha helix to provide structure, a 40-residue glycine-serine sequence to provide flexibility, or an IgG4 hinge region for chimeric antigen receptors to display the scFv on the synthetic receptor. All three constructs were expressed at comparable levels in HEK293FT cells ( Figure 7 A-7B). To test the display on EVs, two vesicle populations were isolated using a previously validated differential centrifugation method. EVs are best defined by the isolation method used for their isolation; for convenience, the fraction isolated at 15,000 × g is referred to below as “microvesicles” (MV) and the fraction isolated at 120,416 × g is referred to as “exosomes” (exo). Vesicles were enriched in typical markers such as CD9, CD81, and Alix, while the endoplasmic reticulum protein calnexin was low ( Figure 8 A). Both populations contained vesicles with an average diameter of ∼120-140 nm and exhibited the expected “cup-shaped” morphology ( Figure 8 Importantly, all three scFv display constructs were expressed substantially in both vesicle populations ( Figure 7 C). Example 4: Display of anti-CD2 scFv enhances EV binding to Jurkat T cells

[0350] To assess targeting, we harvested vesicles from HEK293FT producer cells stably expressing our scFv constructs and the cytoplasmic dTomato fluorescent protein. EVs were incubated with Jurkat T cells expressing high levels of CD2 ( Fig. 9 ) for 2 h, and then the cells were washed to remove unbound vesicles ( Fig.10) and analyzed by flow cytometry. All three constructs enhanced the binding of microvesicles and exosomes to T cells ( Figure 2 A-2C). Displaying scFv on microvesicles enhanced dTomato delivery to T cells more than displaying scFv on exosomes, although some—but not all—of this effect can be attributed to the greater incorporation of dTomato in microvesicles compared with exosomes ( Fig.11 A-11B). Because the helical linker consistently provided the greatest targeting effect, this design was continued in subsequent work. Example 5: CD2-scFv binding mediates receptor cell uptake

[0351] We next assessed whether EV engagement with CD2 triggers internalization (as described, ligand binding naturally triggers CD2 internalization). To distinguish EV binding from uptake, cells were treated with trypsin after incubation with EVs to remove non-internalized vesicles. Cells that received targeted vesicles showed a modest increase in fluorescence relative to non-targeted controls ( Figure 2 D), indicating that CD2 targeting can mediate EV uptake. Example 6: CD2-scFv-mediated EV targeting is specific

[0352] To determine whether the observed vesicle-T cell interactions were caused by specific receptor binding, we pre-incubated recipient Jurkat cells with an antibody that binds to the same T111 epitope on CD2 as our scFv to block potential binding sites. Antibody pretreatment abolished the scFv-enhanced EV binding ( Figure 2 E), demonstrating that our targeting is specific for CD2. In contrast, preincubation with non-targeting EVs (potential nonspecific competitors) did not significantly reduce background or scFv-enhanced binding ( Fig.12 ). These data together indicate that scFv mediates specific binding of EV to CD2. Example 7: Optimization of scFv expression increases targeting

[0353] Increasing the affinity of the interaction between binders (e.g., targeted therapeutic drugs) and their receptors is a generally useful strategy to enhance delivery and function in vivo. 43 To potentially exploit this mechanism, we sought to increase the expression of our scFv constructs and thus their loading into vesicles by mass action. By optimizing the coding sequence of our scFv display constructs for expression in human cells using a sliding window algorithm, we enhanced the cellular expression of our scFv ( Fig.13 A-13B) and increased scFv loading on vesicles without affecting vesicle size or morphology ( Fig.13C-13E). EVs produced by cells stably expressing the optimized scFv constructs exhibited enhanced specific binding to recipient cells ( Figure 2 F) At the end of this limited optimization, targeting EVs and CD2 + The binding of Jurkat T cells was increased 100-fold compared to non-targeted EVs. This optimized targeting system also enhanced the binding of primary human CD4 + EV binding and modest EV internalization in T cells ( Figure 2 G-2H), these cells express high levels of CD2 ( Fig.14 ). Example 8: CD2-scFv display scaffold affects loading and specificity

[0354] Previous reports have achieved scFv display on EVs via fusion to the C1C2 lactadherin domain, which binds to phosphatidylserine on the outer membrane leaflet of some vesicles. To compare our PDGFR-based display strategy with other state-of-the-art EV scFv display systems, we fused our optimized anti-CD2 scFv to the C1C2 lactadherin domain scaffold ( Fig.15 A). We observed similar expression of both constructs in cells, but a higher loading of the C1C2 scFv construct into vesicles (compared to the PDGFR construct) was observed. Fig.15 B-15C). Both systems enabled the microvesicles to bind to Jurkat cells ( Fig.15 D). C1C2 display appears to enhance exosome binding to T cells somewhat (compared to PDGFR display), but targeting of C1C2 display is unequal, with only a subset of Jurkat recipient cells strongly binding to C1C2-displayed EVs, whereas PDGFR display targeting generally mediates delivery to the entire T cell population ( Fig.15 E). As this pattern could provide evidence for CD2-independent EV binding (which would comprise an artifact), we investigated whether C1C2 display targeting was specific. Preincubation of EVs with anti-CD2 antibodies mediated only a partial reduction in C1C2 display-targeted EV binding (in contrast to PDGFR display targeting), suggesting that there are a number of non-target-specific mechanisms involved in C1C2 display targeting of EVs using this scFv. Fig.15 Given these observations, we chose to proceed with EV targeting using the well-validated and highly efficient scFv display of PDGFR. Example 9: Abscisic acid-induced dimerization domain enables active EV cargo loading system

[0355] We next sought to engineer our scFv-containing EVs to load therapeutic cargo of interest. Overexpression of cytoplasmic cargo in EV-producing cells results in passive loading into vesicles by mass action during biogenesis. Increasing the cargo capacity of EVs may result in more efficient delivery vehicles. To improve cargo loading and increase the probability that a given vesicle will incorporate both a cytoplasmic cargo protein and our membrane-bound scFv, we designed a small entity-regulated dimerization-based loading system ( Figure 3 A). Systems using light or small entities (e.g., rapamycin) as inducers have been reported to aid EV cargo loading, but light is difficult to scale up to large volumes, and rapamycin-induced dimerization is very tight and functionally irreversible. We therefore explored a new strategy based on the interaction between abscisic acid-insensitive 1 (ABI) induced by the plant hormone abscisic acid (ABA) and truncated versions of pyraclostrobin resistance-like (PYL) proteins. This “ABA” system has several advantages: binding is rapid; dimerization is reversible, potentially allowing cargo release in recipient cells; ABA is inexpensive and nontoxic; and small-molecule-regulated loading is more readily applicable to biomanufacturing than light control. We first investigated fusion of the ABI and PYL domains to the luminal side of our scFv constructs and to the 5’ or 3’ end of cytoplasmic or nuclear-localized EYFP cargo proteins to determine the effects on protein expression and function. Fusion to the PYL domain reduced expression of (or destabilized) EYFP ( Fig.16 A), while scFv is resistant to fusion with ABI or PYL domains ( Fig.16 B-16C). Therefore, we continued to study the scFv-PYL and EYFP-ABI (3' fusion) constructs. In this setup, ABA-induced dimerization of ABI and PYL was easily visualized by microscopy ( Figure 3 B and Fig.17 ). Example 10: The ABI domain alone drives protein incorporation into EVs

[0356] To investigate cargo protein loading, vesicles were adsorbed to latex beads and analyzed by flow cytometry. Surprisingly, no increase in EV loading was observed with ABA treatment, and constructs containing the ABI domain showed a higher degree of loading than constructs lacking this domain under all conditions ( Figure 3 C-3D). This effect was not due to an ABI-dependent increase in protein expression in producer cells ( Fig.18A). ABI-enhanced loading was evident when paired with either scFv alone or scFv-PYL constructs, indicating that intrinsic ABI-enhanced loading is independent of ABI-PYL interactions ( Figure 3 C) For unknown reasons, the presence of scFv has an increased benefit for protein loading compared to the EYFP-ABI only control ( Fig.18 B). To investigate the role of subcellular localization on the EV loading process, we introduced a nuclear localization sequence (NLS) into EYFP-ABI and compared its loading with that of a purely cytoplasmic construct. ABA-induced dimerization again had a negligible effect on cargo loading, and the addition of an NLS to EYFP-ABI did not reduce loading into EVs ( Fig.18 C) Altogether, these data support the serendipitous finding that ABI constitutes a novel, efficient protein loading tag for EV cargo. Example 11: ABI domain mediates Cas9 loading into EVs

[0357] We next investigated whether ABIs could be used to load EVs with functional cargo. As a model, we chose the Streptococcus pyogenes (S. Pyogenes) Cas9 ribonucleoprotein complex (RNP)—RNPs can be synthesized in producer cells (thus consistent with the GEMINI strategy), and because RNPs must reach the nucleus of recipient cells to act on genomic targets, this system constitutes a rigorous test of functional EV-mediated delivery. ABIs were fused to either the N-terminus or the C-terminus of Cas9, and overall, the expression pattern matched that observed with EYFP ( Fig.19 A). Therefore, we continued to use the Cas9-ABI (3' fusion) construct. We also investigated whether the addition of the NLS or ABI domain would affect Cas9 function. When expressed in reporter Jurkat T cells by transfection (together with the cognate sgRNA), the Cas9 fusion constructs exhibited similar nuclease activity ( Fig.19 B-19C). When the Cas9 constructs were expressed in producer cells, the NLS had minimal effect on Cas9 loading into EVs, whereas the ABI domain significantly increased Cas9 loading ( Figure 3 E and Fig.19 D), but did not increase overall expression in producer cells ( Fig.19 E) These trends are consistent with those observed for EYFP and demonstrate the utility of the ABI loading tag for a variety of cargo proteins. Example 12: Co-loading of membrane scFv and ABI-fused Cas9 into EVs

[0358] An important but unexplored factor to consider when constructing EV-based therapeutics is the extent to which multiple cargo types localize to the same vesicles within a population. Although ABIs (alone) successfully load proteins into EVs, it remains unknown whether dimerization of cargo and display protein can enhance co-loading into EVs (i.e., co-loading of scFv and Cas9 into a single vesicle). To evaluate this question, we generated vesicles from cells expressing scFv-PYL and Cas9-ABI and treated with ABA or expressing vector control, and isolated vesicles displaying anti-CD2 scFv using affinity chromatography via a 3x FLAG tag at the N-terminus of the scFv ( Fig. 20 A). High levels of Cas9 were found in scFv-enriched vesicles, independent of ABA treatment, indicating that ABI labeling of the cargo is sufficient to achieve substantial colocalization of scFv and Cas9 in EVs ( Figure 3 F and Fig. 20 B). Example 13: EV-loaded Cas9 exhibits nuclease function

[0359] To assess whether EV-encapsulated Cas9 RNPs are functional, we developed a direct in vitro assay. EVs from cells expressing Cas9 and sgRNA were lysed and incubated with a plasmid encoding the sgRNA target sequence ( Figure 3 G). Plasmids treated with lysed RNP-containing EVs showed the expected specific cleavage products in all conditions tested. The presence or absence of NLS did not affect cleavage efficiency in this assay, but Cas9 fused to the ABI domain showed reduced cleavage of both vesicle populations. This pattern contrasts with that observed in transfection-based Cas9 assays ( Fig.19 C), it was therefore unclear whether this partial effect (e.g., potential reduction in Cas9 turnover) would be meaningful in the context of cellular delivery. Therefore, in subsequent experiments, both ABI+ and ABI- constructs were evaluated. Example 14: Viral glycoprotein display increases EV uptake by T cells

[0360] To promote EV uptake and fusion, we investigated displaying viral glycoproteins on EVs. We first investigated vesicular stomatitis glycoprotein (VSV-G), which is commonly used in pseudotyped lentivirus production and reported to enable EV fusion with recipient cells. VSV-G was transiently expressed in producer cells expressing dTomato, and the resulting EVs were incubated with recipient T cells for 16 h, followed by trypsinization (to remove non-internalized vesicles) and analysis by flow cytometry. VSV-G enhanced the uptake and fusion of Jurkat T cells ( Figure 4 A-4B) and primary human CD4 + T cells ( Figure 4C) EV uptake, establishing the utility of viral fusion proteins for EV delivery to T cells.

[0361] To develop an EV fusion system that would be more specific for T cells (since VSV-G mediates fusion with most cell types), we investigated the use of truncated versions of the measles virus glycoproteins H and F, which have been previously used to aid lentiviral delivery to T cells. These proteins bind the signaling lymphocyte activation entity F1 (SLAM) and / or the complement regulator CD46, both of which are expressed on different T cells. H and F are traditionally thought to mediate viral fusion at the cell surface, although there are also reports that viral endocytosis can be mediated by SLAM. In the same fluorescent EV uptake assay described above, we investigated the effects of EV delivery to Jurkat (which minimally express SLAM), SLAM transgenic Jurkat, or primary human T cells expressing SLAM ( Figure 4 D) H and F proteins confer modest EV uptake to parental Jurkat (SLAM-), but these proteins significantly enhance EV uptake in SLAM transgenic Jurkat and primary human CD4 + EV uptake by T cells Figure 4 E-4F). We also explored another reported nonviral protein-based strategy that has been reported to promote functional transfer by overexpressing constitutively active Cx43, a connexin involved in gap junction formation, on EV-producing cells. Cx43 did not confer increased EV internalization to T cells in our hands, so this approach was not pursued further ( Fig. 22 Altogether, these results support the use of the Measles H / F glycoprotein as a method to enhance EV uptake by SLAM+ T cells. Example 15: EV-mediated functional delivery of Cas9 to primary T cells.

[0362] Evaluation of functional delivery of Cas9 to recipient T cells requires efficient cargo loading, T cell binding and fusion, and subsequent release of active Cas9 RNP, and after validating each of these steps individually, we began evaluating combinatorial techniques—the first combinatorial test of the GEMINI strategy. Specifically, we investigated the application of Cas9 targeting the CXCR4 locus in primary T cells using previously validated sgRNAs. Due to the cytotoxicity of viral glycoprotein expression, we turned to EV biomanufacturing using the Lenti-X HEK293T cell line, which is well suited to this challenge; this line was chosen for its ability to produce high titers of lentivirus. EVs containing anti-CD2 scFv, NLS Cas9-ABI, and appropriate sgRNAs, and either VSV-G or measles virus glycoprotein H / F were co-cultured with primary human CD4 +T cells were incubated for 6 days and then genomic DNA was harvested for high-throughput sequencing (HTS) to quantify and characterize targeted editing in a 64-nucleotide region centered on the expected cleavage site. Excitingly, indels were identified at the predicted Cas9 cleavage site for all vesicles treated with Cas9 RNP ( Figure 5 and Fig.23 ). For matched designs, VSV-G display on EVs produced higher editing efficiencies than measles H and F protein display, and exosome treatment provided more edits than microvesicle treatment. Most edits were classified as deletions with a small number of insertion events or edits consisting of both insertions and deletions. This overall pattern is consistent with previous reports of Cas9 RNP editing at this site, i.e., edits primarily consisted of small deletions and insertions centered on the cleavage site, indicating that EV-mediated Cas9 delivery using GEMINIs produces effects that are qualitatively comparable to electroporation of recombinant Cas9 RNP. To evaluate the role of ABI-mediated active loading in functional delivery, we generated EVs with Cas9+ / -ABI and assessed editing efficiencies in primary T cells. Although a trade-off between loading and specific cleavage activity has been noted previously, in this case both Cas9 variants performed equally well ( Fig.24 ).

[0363] The functional delivery achieved by our multifunctional EVs allowed us to further explore the specific contribution of each engineered EV feature. In particular, we sought to assess the unique contribution of the anti-CD2 scFv, as it can confer a degree of in vitro binding and uptake. To determine the requirement for EV scFv-CD2 engagement for functional delivery, we pretreated cells with anti-CD2 antibodies prior to adding EVs to block the receptor on recipient cells. Surprisingly, we found that pretreatment with anti-CD2 antibodies increased editing efficiency across a variety of vesicle types and viral glycoprotein systems ( Figure 6 A-6B). To explain this observation, we hypothesized that engagement of CD2 might result in higher levels of T cell activation, rendering the cells more susceptible to EV uptake and editing; if confirmed, this would be a novel consequence of CD2 engagement. To investigate this possibility, we incubated T cells with EV scFv or anti-CD2 antibody and analyzed surface expression of CD25 2 days after treatment. The effects of any treatment on CD25 expression were minimal, indicating that T cell activation could not explain the increased editing observed upon CD2 engagement ( Fig.25To investigate how editing efficiency varies with practical considerations such as EV dose and to explore how CD2 engagement contributes to this process, we evaluated EV delivery to T cells from two different donors using either a single EV dose or repeated daily EV administration for 6 days of incubation ( Figure 6 C-6D). As expected, repeated EV administration increased editing efficiency in all cases, suggesting that repeated dosing is a useful approach to promote editing. Overall, scFv-CD2 engagement enhanced exosome-mediated editing, although this effect was not as pronounced for microvesicle-mediated editing. Finally, to assess which trends held true across experiments, we performed comprehensive analyses (normalization to control for variables hypothesized to cause variation in editing efficiency, such as batch-specific sensitivity of donor T cells to Cas9RNP-mediated editing) ( Figure 6 E-6F). Overall, these combined trends support the key conclusions above. Example 16: Discussion

[0364] 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, actively loading protein cargo into EVs by tagging with vesicle localization domains, and promoting uptake and fusion with recipient cells by displaying viral glycoproteins. The incentivizing application of achieving Cas9 delivery to T cells—a challenging goal—proved useful for refining and validating technologies that can be combined with each other to achieve this goal.

[0365] An exciting aspect of EV-mediated delivery is the potential to target vesicles to cells and receptors of interest through engineered interactions. Previous reports have demonstrated non-targeted EV-mediated delivery to T cells with cargoes including EV-encapsulated AAV8 or zinc-finger-fused methyltransferases. EVs that bind to T cells have also been described as a method to cross-link T cells and other cellular targets by displaying linked anti-CD3 and anti-EGFR scFvs on the PDGFR transmembrane domain. To our knowledge, this study is the first to demonstrate integration of EV targeting and T cell uptake. We anticipate that the modularity of our targeting constructs will facilitate directing EVs to other cell types and receptors.

[0366] The key technology reported here involves the serendipitous discovery that the ABI domain (from the ABA dimerization system) promotes loading of EV cytoplasmic cargo proteins even in the absence of ABA. The mechanism of this effect is unclear. ABI is not predicted by WoLFPSORT (genscript.com / wolf-psort.html) to localize to the cell membrane or endosomal pathways. The advantage of this system is that ABI-mediated loading is easier to achieve compared to multidomain dimerization systems (using light, rapamycin, or Dmr domains) or tags that require overexpression of auxiliary proteins to promote transport to vesicles, such as WW domains and Ndfip1. Other active loading tags have recently been explored by Codiak Biosciences, in which the tags are derived from membrane-associated proteins, although the reversibility of this interaction has not yet been determined. Although increasing Cas9 loading did not result in additional DNA cleavage in our in vitro assays, perhaps because this particular Cas9 fusion strategy reduces Cas9 turnover (FTG.19C), higher cargo loadings may be beneficial in a cellular delivery setting where EVs must overcome the additional hurdles of uptake, fusion, cytoplasmic release, and intracellular trafficking. In this setting, the advantage of more “hits” on the target from a higher dose outweighs the slower reaction rate. Future work may also allow for optimization of the ABI fusion strategy to mitigate any effects on Cas9 activity.

[0367] The final fate of EVs in recipient cells is typically degradation in the endosomal / lysosomal pathway, so developing methods to achieve vesicle fusion in recipient cells is desirable to achieve (or enhance) functional cargo delivery (i.e., to the cytoplasm). Here, we demonstrate efficient internalization of EVs using VSV-G and measles virus glycoproteins H / F, by Jurkat and primary T cells for VSV-G and by cells expressing the lymphocyte receptor SLAM for H / F. An important translational consideration is that mutant versions of the H / F proteins have been developed to evade neutralizing host antibodies, such as those induced by the measles vaccine. However, in functional Cas9 delivery studies, we observed higher Cas9 editing efficiencies in primary T cells treated with VSV-G vesicles compared to H / F, likely due to increased fusion of VSV-G in the acidic endosomal environment. The transformation efficiencies we observed, despite the modest doses used in this study, meet or exceed similar reports in the literature. Perhaps the most rigorous and compelling comparative study reported that 12 repeated high doses (~1 × 10 11 EV, compared with our ~1×10 10Vesicles from MDA-MB-231 breast cancer cells loaded with sgRNA were administered in a controlled manner (compared to EVs) to achieve transformation efficiencies on the order of 0.1% in HEK293T reporter cells constitutively expressing Cas9 (a cell type that is quite efficient for delivery of viral vectors and various biomolecules compared to T cells). We observed significantly higher transformation efficiencies in our system, and the transformation efficiency increased with repeated administration of both EV types. In the specific HIV application under consideration, even transformation of a limited pool of T cells to resist HIV infection could have therapeutic benefits. The potential use of EVs in HIV treatment has been investigated through approaches such as Cas9-mediated proviral excision in microglia, inhibition of viral replication with zinc finger-fused methyltransferases, or killing of infected cells using HIV Env-targeted vesicles, but these initial demonstrations have not yet been developed into methods for T cell-specific delivery and treatment through clinically translatable approaches. Another important finding is that while the exact editing efficiency varied depending on the donor and EV dose (a pattern observed with other methods for Cas9 RNP delivery), the overall trend was highly conserved when controlling for these effects, demonstrating reproducibility. These results are particularly exciting when one notes that the quantified efficiency is limited by the rates of Cas9-mediated cleavage and DNA repair, meaning that we are certainly underestimating the number of functional delivery events and that other cargo types and mechanisms may result in higher rates of functional delivery.

[0368] A surprising finding was that CD2 engagement enhanced exosome-mediated functional delivery in vitro, either by recombinant antibodies or EV-displayed antibodies, although similar benefits were not observed for microvesicle-mediated delivery. This combination of effects cannot be explained by known features of CD2 / T cell biology, although it may be related to the finding that ligand engagement triggers CD2 internalization. Although both scFv-displaying vesicles were able to specifically bind CD2 and be internalized to some extent, there may be differences in the intracellular trafficking and fusion of the two vesicle populations. For example, CD2-binding-mediated trafficking may favor fusion in a different way that favors exosomes than the native uptake pathway. This phenomenon warrants further investigation to elucidate the underlying mechanisms.

[0369] A notable feature of this study is the choice of methodology, which avoids artifacts found in EV studies. A common and often overlooked artifact in EV functional delivery experiments is the risk of transfer of residual producer cell transfection reagents; particles from cells transfected with lipoplexes can mediate functional effects that are falsely attributed to EVs. We minimized this risk by using a transfection method that is unlikely to transfer plasmids to T cells. Key comparative observations (e.g., differences in functional delivery due to viral glycoprotein selection) demonstrate that true EV-mediated delivery was quantified. Example 17: Predictive Functional Delivery of Cas9 to T Cells

[0370] Further contemplated embodiments include using our active loading strategy to deliver Cas9-sgRNA complexes to recipient cells to mediate gene editing. An example of this strategy is expressing Cas9-ABI (fused using our technology in one of the embodiments considered here) and anti-CXCR4-targeting sgRNA in EIEK293FT cells; harvesting EVs produced by these cells using standard methods; delivering these EVs to T cells (e.g., Jurkat T cells or primary human T cells); after a period of time, assessing whether the CXCR4 site in these recipient T cells has been cleaved and repaired (e.g., using high-throughput sequencing).

[0371] The techniques employed here are general and amenable to large-scale production and biomanufacturing. Our strategy for genetically programming the self-assembly of multifunctional particles avoids the need for post-harvest chemical modifications, which require further purification, reduce EV yields, and may pose regulatory challenges. Although we used transient transfection for some transgenes (e.g., viral glycoproteins that cannot be constitutively expressed due to toxicity), such genes are typically expressed from inducible promoters for the production of biologics. We anticipate that the integrated tools developed here for cargo loading and vesicle fusion of cell-derived membrane particles (CDMPs) will be broadly applicable to a range of applications and targets, providing a flexible platform for engineering CDMP therapeutics. Example 18: Functional delivery to Jurkat T cells

[0372] This example demonstrates the ability of extracellular vesicles containing a lentiviral core (EV-LV), a T cell targeting domain displayed on the PDGFR transmembrane, and a fusogen to fuse with Jurkat T cells.

[0373] EV-LV is designed to display 1) Combination of no targeting domain with no VSVG, VSVGwt, or VSVGmut; 2) a combination of an anti-CD2 targeting domain with no VSVG, VSVGwt, or VSVGmut; or 3) Combination of anti-CD5 targeting domain with no VSVG, VSVGwt or VSVGmut.

[0374] The production cell line LentiX used in this example to produce EV-LV is a subclone of the transformed embryonic kidney cell line HEK293. LentiX is a lentiviral production cell that has been engineered to stably express the SV40 large T antigen. According to the above study design, LentiX is plated and transfected with one or more plasmids (by calcium phosphate precipitation). Fig. 27The plasmid constructs used in Example 18 are shown. The cell culture medium was changed appropriately. The produced lentivirus was harvested and the physical titer was determined by qPCR.

[0375] Jurkat T cells or HEK293FT cells were then transduced with 5 doses of unconcentrated (logarithmically spaced) virus with or without 8ug / ml polybrene (a polymer that reduces electrostatic repulsion between the virus and recipient cell membranes and can improve transduction). Flow-based fluorescence readings were performed two days after transduction, and qPCR was performed in parallel to measure particle titers. EV-LV with VSV-Gwt was delivered to Jurkat T cells.

[0376] All EV-LVs displaying VSV-Gwt provided good transduction of Jurkat T cells. 30-100ul doses of EV-LVs displaying VSV-Gwt transduced 80-100% of Jurkat T cells ( Fig.28 A) Viral genomes per cell obtained from qPCR titer results and 40,000 cells plated per well are shown in Fig.28 B. Fig.29 Depicts the results obtained by flow cytometry Fig.28 Population-level histograms of representative samples for each condition reported in . EV-LV with VSV-Gmut was delivered to Jurkat T cells.

[0377] EV-LV displaying VSV-Gmut (alone) transduced Jurkat T cells minimally (by 3.5%). Co-expression of VSV-Gmut with one of the targeting domains (anti-CD2 or anti-CD5) complemented the infectivity of VSV-Gmut alone. Fig.30 A). Viral genomes per cell obtained from qPCR titer results and 40,000 cells plated per well ( Fig.30 B). Fig.31 Depicts the results obtained by flow cytometry Fig.30 Population-level histograms of representative samples for each condition reported in . EV-LV without any VSV-G was delivered to Jurkat T cells.

[0378] Viruses without any VSV-G (e.g., VSV-Gwt or VSV-Gmut) generally did not infect Jurkats. Based on the low intensity of the fluorescence signal, this may be due to protein transfer rather than transduction ( Fig.32 ). EV-LV with various VSV-G were delivered to HEK293FT cells.

[0379] Viruses without VSV-Gwt or VSV-Gmut did not infect HEK ( Fig.33A, D, G). Viruses with VSV-Gmut ( Fig.33 C, F) and virus with VSV-Gwt ( Fig.33 B, E, and H) were similar in infection. The exception was the anti-CD5 VLR, which had VSV-Gmut but less transduction ( Fig.33 I).

[0380] EV-LV particles displaying one of the two T cell targeting domains (anti-CD5 VLR or anti-CD2 scFv) and displaying VSV-Gmut without binding ability transduced Jurkat T cells in a dose-dependent manner. Example 19: Functional delivery to primary human T cells

[0381] This example demonstrates the ability of extracellular vesicles comprising 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+).

[0382] The following T cell targeting domain and fusogen combinations were tested: - No surface modification version 1 - No surface modification version 7 -VSV-Gwt -VSV-Gmut - Anti-CD5 VLR -VSV-Gmut+anti-CD5 VLR -VSV-Gwt + anti-CD5 VLR version 1 -VSV-Gwt + anti-CD5 VLR version 7

[0383] LentiX (lentiviral producer cells) were plated and transfected (by calcium phosphate precipitation) with one or more plasmids according to the study design described above. Fig.34 Plasmid constructs used in Example 19 are shown. Cell culture medium was changed as appropriate. The resulting EV-LV was harvested. Physical titers were determined by qPCR. Equal genome copy numbers of virus were used for T cells and HEK29FT cells, respectively. Flow-based fluorescence readout was performed three days after transduction, and staining was performed to confirm the identity of the T cells as CD4 + and / or CD8 + . EV-LV delivery with VSV-Gmut or VSV-Gmut to primary human T cells

[0384] All EV-LVs displaying VSV-Gwt produced a dose-dependent transduction of activated T cells ( Fig.35A and B). Although no transduction was observed with VSV-Gmut alone, EV-LV displaying anti-CD5 VLR and VSV-Gmut demonstrated transduction of activated T cells as good as or better than VSV-Gwt ( Fig.35 A and B). EV-LV with VSV-Gmut or VSV-Gmut was delivered to HEK293TF cells.

[0385] Both EV-LVs of G (wt and mut) produced dose-dependent transduction of HEK293FT cells. Addition of anti-CD5 VLR with VSV-Gwt did not result in significantly higher transduction compared to VSV-Gwt alone. Addition of anti-CD5 VLR with VSV-Gmut did not result in significantly higher transduction compared to VSV-Gmut alone. Both observations may be attributed to the fact that HEK293FT does not express CD5.

[0386] EV-LV particles displaying anti-CD5 VLR and non-binding VSV-Gmut transduced primary T cells in a dose-dependent manner. Inclusion of anti-CD5 VLR and VSV-Gmut restored the infectivity of the particles, making them as effective as VSV-Gwt alone in transducing activated T cells. EV-LV particles displaying anti-CD5 VLR and VSV-Gwt transduced primary T cells ~5 times better (~5x) than the gold standard EV-LV particles displaying only VSV-Gwt. Equivalent method

[0387] The present technology is not limited to the specific embodiments described in the present application, and these embodiments are intended to be used as separate descriptions of the individual aspects of the present technology. Without departing from the spirit and scope of the present technology, many modifications and changes can be made to the present technology, which will be apparent to those skilled in the art. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art according to the foregoing description. Such modifications and changes are intended to fall within the scope of the present technology. It should be understood that the technology of the present invention is not limited to specific methods, reagents, compound compositions or biological systems, which can certainly vary. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments, and are not intended to be limited.

[0388] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0389] As will be understood by those skilled in the art, for any and all purposes, particularly in providing written description, all scopes disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof.Any listed scope can be easily identified as fully describing and enabling the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each scope discussed herein can be easily decomposed into lower third, middle third, and upper third, etc. All languages ​​can also be understood by those skilled in the art, such as "up to", "at least", "greater than", "less than", etc. including the enumerated numbers and referring to the scope that can be subsequently divided into sub-ranges as described above. Finally, as will be understood by those skilled in the art, scope includes each individual member.Therefore, for example, a group with 1-3 units refers to a group with 1, 2 or 3 units. Similarly, a group with 1-5 units refers to a group with 1, 2, 3, 4 or 5 units, etc.

[0390] All patents, patent applications, provisional applications, and publications mentioned or cited herein are 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. Engineered lipid bilayer particle population, including: (i) a targeting chimeric polypeptide comprising: (a) a targeting domain, which is configured so that the targeting domain is located on the surface of the particle, wherein the targeting domain comprises a binding moiety that specifically binds to a target ligand on the surface of a receptor cell of interest; and is directly or indirectly linked to (b) a transmembrane domain; and (ii) a fusogen entity polypeptide comprising (a) a fusogen portion, which is arranged so that the fusogen portion is located on the surface of the particle; and is directly or indirectly attached to (b) Transmembrane domain.

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

3. A population according to claim 2, wherein the antibody reagent is a single chain antibody reagent.

4. A population according to claim 2, wherein the antibody agent is selected from the group consisting of antibodies, Fab, Fab', F(ab')2, Fd, scFv, single-chain antibodies, disulfide-linked Fv (sdFv), affibodies, DARPINs, nanobodies, variable lymphocyte receptors (VLRs) and camelid antibodies.

5. A population according to claim 1, wherein the targeting domain is or comprises a DARPIN or other engineered high affinity binding polypeptide.

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

7. A population according to claim 1, wherein the transmembrane domain of the targeting chimeric polypeptide comprises AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (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.

8. A population according to claims 1-7, wherein the targeting domain comprises the amino acid sequence NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSGGGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSS (SEQ ID NO: 20).

9. A population according to claims 1-8, further comprising a first cargo entity connected to the transmembrane domain of the targeting chimeric polypeptide via a linker.

10. A population according to claim 9, wherein the linker comprises: (1) Selected from SEQ ID NO: 10 (TSGGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG) and SEQ ID The amino acid sequence of NO:17 (ESKYGPPAPPAP); or (2) having an amino acid sequence that is 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% identical to any one of SEQ ID NO: 10, 12, 14, 15, 16 or 17.

11. A population according to claims 1-10, wherein the engineered lipid bilayer particles are cell-derived membrane particles (CDMPs).

12. A population according to claim 11, wherein the CDMP is selected from the group consisting of extracellular vesicles, viral particles, virus-like particles (VLPs), apoptotic bodies, platelet-like particles, and combinations thereof.

13. The population according to claim 12, wherein the CDMP is an extracellular vesicle selected from the group consisting of exosomes, microvesicles, and combinations thereof.

14. A population according to claims 1-13, wherein the fusogen entity polypeptide is a viral fusogen.

15. A population according to claims 1-13, wherein the fusogen entity polypeptide is a non-viral fusogen.

16. The colony according to claim 14, wherein the viral fusogen is selected from the group consisting of vesicular stomatitis virus, measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, rabies virus, lymphocytic choriomeningitis virus, Mokola virus, Ross River 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-Venes virus 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, avian plague virus, Autographa californica multiplenucleopolyhedro virus, baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, dengue virus, Zika virus, West Nile virus, yellow fever virus, tick-borne encephalitis virus, herpes simplex virus type 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, varicella zoster virus, severe fever with thrombocytopenia virus, Hanta virus, vaccinia virus, simian immunodeficiency virus, human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus virus), La Crosse virus, human cytomegalovirus, human cytomegalovirus, Thogoto virus, or Dhori virus.

17. A population according to claim 14, wherein the viral fusogen is selected from lentiviral glycoproteins or is a glycoprotein selected from the group consisting of vesicular stomatitis virus (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic mouse leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein and lymphocytic choriomeningitis virus (LCMV) glycoprotein.

18. A population according to any one of claims 1 to 17, wherein the particles further comprise a cargo entity.

19. A population according to claim 18, wherein the cargo entity is a polypeptide cargo entity.

20. The population according to claim 18, wherein the cargo entity is a nucleic acid cargo entity.

21. The population according to claim 18, wherein the cargo entity is a part of the targeting chimeric polypeptide, such that the targeting chimeric polypeptide comprises the targeting domain, the transmembrane domain and the polypeptide cargo entity.

22. The population according to claim 19, wherein the cargo entity is a portion of the fusogen entity polypeptide, such that the fusogen entity polypeptide comprises a fusogen portion and the polypeptide cargo entity.

23. The population according to claim 19, wherein the targeted chimeric polypeptide further comprises a first polypeptide cargo entity, and the fusogen entity polypeptide comprises a fusogen portion and a second polypeptide cargo entity.

24. The population according to claim 18, wherein the polypeptide cargo entity is a polypeptide or a portion thereof that is different from each of the targeting chimeric polypeptide and the fusogen entity polypeptide.

25. A population according to claim 24, wherein the polypeptide cargo entity is linked to a cargo loading domain.

26. A population according to claim 25, wherein the cargo loading domain comprises an abscisic acid insensitive 1 (ABI1) sequence.

27. A population according to claim 24, wherein the polypeptide cargo entity further comprises a transmembrane domain.

28. The population according to claim 24, wherein the polypeptide cargo entity is bound to the intramembrane portion of the targeting chimeric polypeptide or the fusogen entity polypeptide.

29. The population according to claim 20, wherein the nucleic acid cargo entity is bound to a membrane-associated polypeptide associated with a membrane of the particle.

30. A population according to claim 29, wherein said membrane-associated polypeptide is or comprises said targeting chimeric polypeptide or said fusogen entity polypeptide.

31. A population according to claims 1-30, wherein one or both of said targeting chimeric polypeptide and said fusogen entity polypeptide further comprises an intramembrane cargo binding moiety.

32. A population according to claim 31, wherein the cargo binding moiety binds a polypeptide cargo.

33. The population according to claim 31, wherein the cargo binding moiety binds a nucleic acid cargo.

34. A population according to claims 1-33, wherein the particle further comprises a chimeric loading polypeptide, the chimeric loading polypeptide comprises the cargo loading domain, the cargo loading domain comprises an abscisic acid insensitive 1 (ABI1) sequence.

35. A population according to claim 34, wherein the chimeric cargo polypeptide further comprises a linker connecting the cargo entity and the cargo loading domain.

36. A population according to claim 35, wherein said linker of said chimeric cargo polypeptide comprises: (1) Selected from SEQ ID NO: 10 (TSGGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG) and SEQ ID The amino acid sequence of NO:17 (ESKYGPPAPPAP); or (2) having an amino acid sequence that is 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% identical to any one of SEQ ID NO: 10, 12, 14, 15, 16 or 17.

37. A population according to any one of claims 34-36, wherein the cargo-loading domain of the chimeric loading polypeptide is a truncated variant of the wild-type protein comprising an extracellular vesicle targeting domain.

38. A population according to any one of claims 34-37, wherein the cargo loading domain of the chimeric loading polypeptide comprises residues 126-423 of wild-type ABI1.

39. A population according to any one of claims 34 to 38, wherein the cargo loading domain of the chimeric loading polypeptide comprises: MTRVPLYGFTSICGRRPEMEAAVSTIPRFLQSSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALP LSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK(SEQ ID NO:6), VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLS VDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK(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 any one of SEQ ID NO: 6 or 7, or A functional fragment of SEQ ID NO:6, SEQ ID NO:7, or a variant amino acid sequence thereof.

40. A population according to any one of claims 34 to 39, wherein the chimeric cargo polypeptide-carrying cargo entity is a cytoplasmic cargo molecule.

41. A population according to any one of claims 39-40, wherein the first cargo entity is an ABA binding sequence comprising a pyranostatin resistance 1-like (PYL1) sequence.

42. A population according to claim 41, wherein the PYL1 sequence comprises residues 33-209 of wild-type PYL1.

43. A population according to claim 41 or 42, wherein the PYL1 sequence comprises MGGGAPTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO: 2), 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 any one of SEQ ID NO: 2 or 3, or A functional fragment of SEQ ID NO:2, SEQ ID NO:3, or a variant amino acid sequence thereof.

44. A population according to any one of claims 39-43, wherein the CDMP further comprises abscisic acid (ABA).

45. A population according to any one of claims 11-44, wherein the CDMP comprises or contains 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, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin-modified synthetic transcription factor, a natural transcription factor, a CRISPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.

46. ​​A population according to claims 1-45, wherein the population is frozen.

47. A population according to claim 46, wherein the population is frozen in glycerol, or is dried, or is in culture.

48. A targeted chimeric polypeptide comprising: (a) a targeting domain, which comprises a binding moiety that specifically binds to a target ligand on the surface of a receptor cell of interest; and is directly or indirectly linked to (b) a transmembrane domain.

49. The targeted chimeric polypeptide according to claim 46, wherein the targeting domain is or comprises an antibody agent.

50. The targeted chimeric polypeptide according to claim 47, wherein the antibody agent is a single chain antibody agent.

51. The targeted chimeric polypeptide according to claim 47, wherein the antibody agent is selected from the group consisting of antibodies, Fab, Fab', F(ab')2, Fd, scFv, single chain antibodies, disulfide-linked Fv (sdFv), affibodies, DARPINs, nanobodies, variable lymphocyte receptors (VLRs) and camelid antibodies.

52. The targeted chimeric polypeptide according to claim 46, wherein the targeting domain is or comprises a DARPIN or other engineered high affinity binding polypeptide.

53. The targeted chimeric polypeptide according to claim 49, wherein the targeting domain is a scFv.

54. The targeted chimeric polypeptide of claims 46 to 51, wherein the transmembrane domain comprises AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (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%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, or a functional fragment thereof.

55. The targeted chimeric polypeptide according to any one of claims 46 to 52, wherein the targeting domain comprises the amino acid sequence NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSG GGGSQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSS(SEQ ID NO:20).

56. The targeted chimeric polypeptide according to any one of claims 46-53, further comprising a first cargo entity connected to the transmembrane domain via a linker.

57. The targeted chimeric polypeptide according to claims 46-54, wherein the linker comprises: (1) Selected from SEQ ID NO: 10 (TSGGGGGSGGGSGGGS), SEQ ID NO: 12 (TRGGGGSGGGSGGGS), SEQ ID NO: 14 (GGGGSGGGSGGGSTG), SEQ ID NO: 15 (DQSNSEEAKKEEAKKEEAKKSNS), SEQ ID NO: 16 (SGGGSGGGSGGGSGGSGGSGGGSGGSGGSGGGSGGGSGGG) and SEQ ID The amino acid sequence of NO:17 (ESKYGPPAPPAP); or (2) having an amino acid sequence that is 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% identical to any one of SEQ ID NO: 10, 12, 14, 15, 16 or 17.

58. A system comprising a first polynucleotide encoding a fusogen entity polypeptide; and A second polynucleotide encodes a targeting chimeric polypeptide.

59. A system according to claim 58, wherein the first and second polynucleotides are part of a single polynucleotide molecule.

60. The system according to claims 58-59, wherein the first and second polynucleotides are separate polynucleotide molecules.

61. The system of claims 58-60, wherein at least one of the first and second polynucleotides is circular.

62. The system according to claims 58-61, wherein both the first and second polynucleotides are circular.

63. The system according to claims 58-62, wherein at least one of the first and second polynucleotides is linear.

64. The system according to claims 58-63, wherein the first and second polynucleotides are both linear.

65. The system according to claims 58-64, wherein the first and second polynucleotides are placed in separate containers.

66. The system according to claims 58-64, wherein the first and second polynucleotides are placed in the same container.

67. The system of claims 58-66, wherein the first and second polynucleotides comprise expression elements sufficient to direct production of the encoded polypeptides in an engineered production cell.

68. The system according to claim 57, wherein the cells are mammalian cells.

69. The system according to claims 67-68, wherein the expression element is a promoter.

70. The system of claim 69, wherein the promoter is a small molecule inducible promoter.

71. The system according to claims 58-70, wherein the fusion pro-entity polypeptide comprises: a fusogen portion; and Transmembrane domain.

72. The system according to claim 71, wherein the fusogen portion and the membrane association portion are directly linked or indirectly linked via a linker.

73. The system according to claims 71-72, wherein the fusogen moiety is characterized by being able to promote fusion between lipid bilayers.

74. The system according to claims 71-73, wherein the fusogen portion is a viral fusogen portion.

75. The system according to claims 71-73, wherein the fusogen moiety is a non-viral fusogen moiety.

76. The system according to claims 71-75, wherein the transmembrane domain is a viral transmembrane domain or a derivative thereof.

77. The system according to claims 71-75, wherein the transmembrane domain is a non-viral transmembrane domain.

78. The system of claims 58-77, wherein the targeting chimeric polypeptide comprises: (a) a targeting domain, which comprises a binding moiety that specifically binds to a target ligand on the surface of a receptor cell of interest; and (b) a transmembrane domain.

79. The system according to claim 78, wherein the affinity portion and the membrane association portion are directly linked or indirectly linked via a linker.

80. Nucleic acid encoding a chimeric targeting polypeptide and / or a fusogen entity polypeptide according to any one of claims 1 to 45.

81. A cell comprising the population of any one of claims 1-45 or the nucleic acid of claim 80.

82. A cell according to claim 81, wherein the cell is a mammalian cell, wherein the mammalian cell is optionally selected from HEK293, HEK293FT, mesenchymal stem cells, megakaryocytes, induced pluripotent stem cells (iPSC), T cells, erythrocytes and erythrocyte precursor cells, and iPSC-derived versions of any of the foregoing cells.

83. A method for producing lipid bilayer particles targeting lymphocytes, comprising culturing the cells of claim 23 or 24, and harvesting the lipid bilayer particles produced by the cells.

84. A method for targeted delivery of a cargo entity to lymphocytes, comprising administering to an individual a population according to any one of claims 7-21, wherein the lipid bilayer particles comprise a cargo molecule.

85. A method according to claim 84, wherein the cargo entity comprises 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, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRTSPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.

86. The method according to claim 84 or 85, wherein the cargo entity comprises a nucleic acid sequence encoding a chimeric antigen receptor.

87. An ex vivo method for targeted delivery of a cargo entity to lymphocytes, comprising obtaining a lymphocyte population from an individual and contacting the lymphocyte population ex vivo with a population according to any one of claims 7 to 21, wherein the lipid bilayer particles comprise a cargo molecule.

88. A method according to claim 87, wherein the cargo entity comprises 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, a receptor signaling activator or inhibitor, an intracellular antibody, a chromatin-modifying synthetic transcription factor, a natural transcription factor, a CRTSPR-Cas family protein, a DNA molecule, an RNA molecule, or a ribonucleoprotein complex.

89. The method according to claim 87 or 88, wherein the cargo entity comprises a nucleic acid sequence encoding a chimeric antigen receptor.

90. The method according to any one of claims 87-89, wherein the lymphocyte population is obtained by apheresis.

91. The method according to any one of claims 87-90, further comprising administering the lymphocyte population back to the individual after contacting the lymphocytes with the lipid bilayer particles.

92. A method for producing a lipid bilayer particle population, the method comprising the steps of: Isolation of lipid bilayer particles produced by engineered production cells engineered to express expressing a fusogen entity polypeptide; and Targeting entity peptides, The lipid bilayer particles are made to contain a fusogen entity and a targeting chimeric polypeptide on their surface.

93. A method for delivering a cargo entity to a recipient cell, the method comprising the steps of: The recipient cell is contacted with a population of lipid bilayer particles produced by an engineered production cell, wherein the engineered production cell is engineered to express Fusogen: Entity polypeptide; as well as Targeting chimeric peptides, The lipid bilayer particle is such that it contains a fusogen entity and a targeting chimeric polypeptide on its surface.

94. A method for producing a production cell, the method comprising the following steps pf: Engineered production units to express Fusogen entity polypeptide; and Targeting chimeric peptides, The producer cells are caused to produce lipid bilayer particles containing a fusogen entity and a targeting chimeric polypeptide on their surface.

95. A recipient cell comprising a cargo entity, and wherein the recipient cell membrane comprises a targeting chimeric polypeptide and a fusogen entity polypeptide, and a cell nucleus.

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