Methods for screening for next generation nucleic acid delivery vectors

Abstract

The technology described herein is directed to compositions and methods for delivery of nucleic acids to cells of interest, e.g., in vivo. Methods are described for the identification of viral envelope sequences that mediate infection or transduction of a target cell type using virus-like particles in a high-throughput manner. The methods and compositions described herein permit the delivery of, e.g., RNA encoding therapeutic polypeptides and / or gene editing machinery to cells of interest. Such delivery can be in a manner that limits, avoids or circumvents accumulation in or clearance of the delivery vehicles by the liver.

Description

METHODS FOR SCREENING FOR NEXT GENERATION NUCLEIC ACID DELIVERYVECTORSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This Application claims benefit under 35 U.S.C. § 119(e) ofthe U.S. Provisional Application No. 63 / 614, 196 filed December 22, 2023, the content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] Hie technology described herein relates to vims-like particles (VLPs) for nucleic acid delivery to a variety of organs, and methods of determining tropism of viral envelope proteins.BACKGROUND

[0003] The future of human medicine will require efficient, reliable, and safe methods to deliver macromolecular cargoes such as genes or gene editing tools to specific cells in vivo. Nucleic acid therapeutics encoding precise gene editing systems could provide cures for a wide variety of diseases as it has the potential to address both loss-of-function and gain-of-fiinction mutations. Hie inefficiency and lack of specificity of in vivo macromolecule deliver}’ presents the single largest barrier to the clinical success of genetic therapies.

[0004] At present, efficient in vivo gene transfer, and thus the potential for gene editing, is technically enabled only by lipid nanoparticles (LNPs) and adeno-associated vims (AAV). However, AAVs have several disadvantages for gene editing. First, AAV has a packaging limit of 4kb, smaller than what is required for many gene editing systems. Second, AAV expression lasts a long time (years), which increases the probability of off-target effects. Hiird, the immune system presents a significant challenge to the use of AAV, both in terms of pre-existing, neutralizing antibodies and innate immune response. Furthermore, systemically administered LNPs can only target the liver. Thus, delivering nucleic acids encoding therapeutic polypeptides or gene editing agents to specific cell types in the body is a grand challenge in biomedical research.SUMMARY

[0005] Vimses have naturally evolved to target various cells in the body through proteins on their surface that pemrit them to achieve tremendous specificity through interaction with proteins on the target cell’s surface. As different cell types have unique cell surface proteins, vimses can achieve extraordinary specificity. Vimses as vectors thus have a significant advantage in their likelihood to achieve cell-type specificity over lipid nanoparticles with different lipid compositions, which are unlikely to efficientlyevade the liver and have true specificity for different surface proteins or cell types. Identifying novel envelope proteins that mediate tropism to specific organs can serve as useful research tools as well as pcmrit modular delivery of, for example, gene editing therapeutics for potential treatment of a number of currently un-addressable diseases. Hie inventors have developed a pooled screening system using viruslike particles carrying different envelope proteins from different viruses. Through pseudotyping of a vesicle (e.g.. vesicle, or viral chassis, such as a lentiviral or other viral chassis, among others) with different envelope proteins and corresponding nucleic acid barcodes, each type of particle carries a nucleic acid barcode that can be read out with high-throughput sequencing. This system can be used to identify viral envelope proteins that mediate delivery to organs outside of the liver.

[0006] Accordingly, in one aspect, described herein is a plurality of virus-like particles (VLPs), wherein each vims-like particle comprises a membrane pseudotyped with or displaying a viral envelope protein, and a unique identifying barcode nucleic acid that is paired with the corresponding heterologous envelope protein.

[0007] In another aspect, described herein is a plurality of vims-like particles (VLPs), wherein each vims-like particle comprises a viral capsid, (e.g., lentiviral or other viral capsid); a membrane pseudotyped with a heterologous viral envelope protein; and a unique identifying barcode nucleic acid that is paired with the corresponding heterologous envelope protein.

[0008] In another aspect, described herein is a library comprising a plurality of vims-like particles (VLPs), wherein each vims-like particle comprises a lentiviral capsid; a membrane pseudotyped with a heterologous viral envelope protein; and a unique identifying barcode nucleic acid that is paired with the corresponding heterologous envelope protein.

[0009] In one embodiment of any of the aspects described herein, the barcode nucleic acid is linked to nucleic acid sequence encoding the heterologous envelope protein.

[0010] In another embodiment of this or other aspects described herein, the members of the plurality of VLPs each comprise distinct heterologous viral envelope proteins.

[0011] In another embodiment of this or other aspects described herein, the distinct heterologous viral envelope proteins are derived from naturally-occurring viruses.

[0012] In another embodiment of this or other aspects described herein, the distinct heterologous viral envelope proteins comprise envelope proteins derived from human endogenous retroviruses or human commensal viruses.

[0013] In another embodiment of this or other aspects described herein, the distinct heterologous viral envelope proteins comprise envelope proteins from a plurality of proteins listed in Table 1.

[0014] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 1 of the proteins listed in Table 1.

[0015] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 5 of the proteins listed in Table 1.

[0016] In another embodiment of this or other aspects described herein, tire distinct viral envelope proteins comprise envelope proteins from at least 50 of the proteins listed in Table 1.

[0017] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 90 of the proteins listed in Table 1.

[0018] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 96 of the proteins listed in Table 1.

[0019] In another embodiment of this or other aspects described herein, tire distinct viral envelope proteins comprise envelope proteins from at least 200 of tire proteins listed in Table 1.

[0020] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 300 of the proteins listed in Table 1.

[0021] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 400 of the proteins listed in Table 1.

[0022] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 500 of tire proteins listed in Table 1.

[0023] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 600 of the proteins listed in Table 1.

[0024] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 700 of tire proteins listed in Table 1.

[0025] In another embodiment of this or other aspects described herein, the distinct viral envelope proteins comprise envelope proteins from at least 766 of tire proteins listed in Table 1.

[0026] In another embodiment of this or other aspects described herein, the distinct heterologous viral envelope protein corresponds to a single unique identifying barcode nucleic acid.

[0027] In another embodiment of this or other aspects described herein, the heterologous envelope proteins each comprise a heterologous tag.

[0028] In another embodiment of this or other aspects described herein, the heterologous tag is an HA tag.

[0029] In another aspect, described herein is a population of cells comprising the plurality of any of the embodiments described herein, or the library of any of the embodiments described herein.

[0030] In another aspect, described herein is a method of determining individual tropism of a plurality of heterologous envelope proteins, the method comprising contacting a plurality of VLPs as described herein with a population of cells, and determining tire presence of the unique identifying barcode nucleic acids in one or more of the cells, wherein the location of each unique identifying barcode detected is an indication of the tropism of a corresponding heterologous envelope protein, hr one embodiment, the barcode nucleic acid(s) delivered by the VLP is expressed by the cells. In another embodiment, the barcode nucleic acid further comprises sequence encoding the heterologous viral envelope protein.

[0031] In another aspect, described herein is a method of determining tropism of a viral envelope protein, the method comprising: preparing a VLP comprising (a) a membrane pseudotyped with the viral envelope protein; and (b) a unique identifying barcode, wherein each unique barcode is paired with the corresponding heterologous envelope proteins; then contacting the prepared VLP with a population of cells; and determining the presence of the unique identifying barcodes nucleic acid in one or more of the cells, wherein the location of each unique identifying barcode detected is an indication of the tropism of the corresponding heterologous envelope protein. In one embodiment, the barcode nucleic acid(s) delivered by the VLP is expressed by the cells. In another embodiment, the barcode nucleic acid further comprises sequence encoding the heterologous viral envelope protein.

[0032] In another aspect, described herein is a method of determining tropism of a viral envelope protein, the method comprising: preparing a VLP comprising (a) a viral capsid (e.g., lentiviral); (b) a membrane pseudotyped with the viral envelope protein; and (c) a unique identifying barcode, wherein each unique barcode is paired with the corresponding heterologous envelope protein; then contacting the prepared VLP with a population of cells; and determining presence of the unique identifying barcodes in the cells, wherein the location of each unique identifying barcode detected is an indication of the tropism of the corresponding heterologous envelope protein. In one embodiment, the barcode nucleic acid(s) delivered by the VLP is expressed by the cells. In another embodiment, the barcode nucleic acid further comprises sequence encoding the heterologous viral envelope protein.

[0033] In one embodiment of this or other aspects described herein, the introducing or contacting of the plurality or library of VLPs or the introducing or contacting of the prepared VLP is in vitro or in vivo.

[0034] In another embodiment of this or other aspects described herein, the method of determining individual tropism of a plurality of heterologous envelope proteins further comprises isolating mRNA or other nucleic acid from cells prior to determining the expression or presence of the unique identifying barcodes present or expressed in tire cells.

[0035] In another aspect, described herein is a method of identifying viral envelope proteins effective for pseudotyping a virus-like particle membrane, the method comprising generating a library of constructs encoding viral envelope pscudotypc candidate proteins with a selectable tag peptide, producing a population of VLPs comprising the envelope pseudotype candidate proteins, and assessing pseudotype efficiency by detecting the selectable tag peptide.

[0036] In another aspect, described herein is a nucleic acid molecule comprising a 7SL RNA fused with mRNA encoding an RNA-guided endonuclease or base-editing enzyme.

[0037] In another aspect, described herein is a nucleic acid molecule comprising a 7SL RNA fused with a guide RNA for an RNA-guidcd endonuclease or base-editing enzyme.

[0038] In one embodiment of this or any other aspect described herein, the RNA-guided endonuclease is selected from Cas9. Cpfl. C2cl, C2c3, Casl2a. Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, and Casl3c. Cask CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, C2cl, C2c3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2c, Casl3a, Casl3b, and Casl3c.

[0039] In another embodiment of any one of the aspects described herein, the base-editing enzyme is selected from a nickase, a cytidine deaminase, an adenosine deaminase, ABE-SpRY, CBE-SpRY.ABE8e, ABE8e-SpRY, ABE8e-NRCH, NG-ABE8e, ABE-NRTH, ABEmax, BE1 , BE2, BE3, HF-BE3, BE4, BE4max, BE4-GAM, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR-BE3, VRER-BE3, Sa-BE3, SA-BE4, SaBE4-Gam, SaKKH-BE3, Casl2a-BE, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TadA*-dCas9, TadA-TadA*-Cas9, ABE7.9, ABE 6,3, and ABE7.10.

[0040] In another aspect, described herein is a virus-like particle comprising a nucleic acid molecule of any of the embodiments described herein.

[0041] In another aspect, described herein is a virus-like particle comprising a nucleic acid molecule of any of the embodiments described herein and an mRNA encoding an RNA-guided endonuclease that recognizes and functions with the guide RNA comprised by a nucleic acid molecule of any of the embodiments described herein.Definitions

[0042] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. Theterminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology, and molecular biology can be found in Hie Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter etal. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons. 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.). John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0043] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in tire art to which this invention belongs. If there is an apparent discrepancy between the usage of a tenu in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0044] As used herein, the term "virus-like particle" refers, at a minimum, to a cell membrane- derived membrane component displaying a transmembrane viral envelope protein or a solvent-exposed portion thereof, and a nucleic acid cargo within the membrane component. In some embodiments, the nucleic acid cargo comprises a barcode sequence that corresponds to the viral envelope protein. A vims- like particle's nucleic acid cargo component can encode tire envelope protein and the barcode. In someembodiments, a virus-like particle includes viral-derived structure that permits the packaging of the nucleic acid into tire particle, and / or viral-derived structure that permits the introduction of the nucleic acid to a target cell; thus, a virus-like particle can also include a capsid protein or a capsid that permits the packaging of the nucleic acid in a form permitting delivery into a cell. In some embodiments, a virus-like particle does not include viral-derived packaging structures.

[0045] As used herein, the term "tropism" or "viral tropism" refers to the relative ability or tendency for a given vims to infect a given cell or tissue type. A vims that has a tropism to a given cell or tissue type will infect that cell or tissue type more efficiently than it infects other cells or tissues in the same host. Tropism for enveloped viruses is generally determined by tire interactions of viral envelope proteins with proteins or other moieties expressed on the surface of a target cell. In general, the more specific or selective the interaction of the viral envelope protein is for a given cell-surface moiety, the greater tire tropism of a vims or vims-like particle bearing the envelope protein will be for a cell expressing or displaying that sell-surface moiety. In some embodiments, a vims with a tropism for a given cell or tissue type will infect that cell or tissue type at least 10X, 100X, 1000X, 104X, 105X or 106X more efficiently than it infects a non-target cell or tissue type in the same host. Methods for evaluating tropism are known to those skilled in the art; see, for example McCall L. I. (2021). Quo vadis? Central Rules of Pathogen and Disease Tropism. Frontiers in cellular and infection microbiology, 11, 640987.; Jang, M.J..Coughlin, G.M., Jackson, C.R. et al. Spatial transcriptomics for profiling the tropism of viral vectors in tissues. Nat Biotechnol 41, 1272-1286 (2023); Weinmann, J., Weis, S., Sippel, J. etal. Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nat Commun 11, 5432 (2020), which are incorporated herewith by reference. Tropism can also be evaluated as described herein below.

[0046] As used herein, the term “envelope protein” refers to a transmembrane protein on the outer surface of an enveloped vims that determines what cell, tissue or species types the vims can transduce.

[0047] As used herein, the terms “pseudotype” or “pseudotyping”, refer to a vims or vims-like particle, in which the viral envelope protein or proteins have been substituted w ith those of another vims. Pseudotyping permits not only the manipulation of viral tropism, but, as described herein, an approach for identifying viral envelope proteins that can permit such manipulation, e.g., to tailor delivery of nucleic acid cargo to preferred cell types.

[0048] As used herein, the terms “cargo” and “payload” are used interchangeably to describe the nucleic acid contents carried by a recombinant vims or by vims-like particles described herein.

[0049] Hie terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount, hr some embodiments, “reduce,” “reduction" or “decrease"or '‘inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level.

[0050] Hie tenus “increased”, “increase”, “enhance”, or “activate” arc all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0051] As used herein, the terms '‘protein" and "polypeptide" are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The tenns "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplar) polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0052] As used herein, the term “specific binding” refers to a chemical or physical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to tire second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times orgreater than the affinity for the third non-target entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.

[0053] Hie tcnu "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (e.g., mRNA) or antisense RNA derived from a nucleic acid fragment or fragments and / or to the translation of mRNA into a polypeptide.

[0054] "Expression products" include RNA transcribed from a gene or construct, and polypeptides obtained by translation of mRNA transcribed from a gene.

[0055] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. In this context, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplar}' detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods, hr some embodiments of any of the aspects, measuring can be a quantitative observation. Where detection of a nucleic acid barcode is concerned, “detecting” refers to the determination that a nucleic acid having that barcode sequence is present in a sample: such detecting can be performed, for example, via next generation sequencing.

[0056] As used herein, the term “heterologous” refers to that which is not endogenous to, or naturally occurring in, a referenced sequence, molecule (including e.g., a protein), virus, vims-like particle, cell, tissue, or organism. For example, a heterologous sequence of the present disclosure can be derived from a different species, or from the same species but substantially modified from an original form. Also for example, a nucleic acid sequence that is not normally expressed in a cell or a vims is a heterologous nucleic acid sequence with regard to that cell or vims. Hie term "heterologous" can refer to DNA, RNA, or protein that does not occur naturally as part of the organism in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature. It is DNA, RNA, or protein that is not endogenous to the vims or cell and has been artificially introduced into the vims or cell. A pseudotyped enveloped vims has a heterologous viral envelope protein. While VLPs as described herein are not naturally occurring, the viral envelope polypeptide comprised by a VLP as described herein can be considered to be heterologous to the VLP. Thus, a library of VLPs as described herein in which members have different viral envelope polypeptides includes heterologous viral envelope polypeptides.

[0057] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e g. a viral envelope polypeptide, a viral capsid polypeptide, etc.) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a barcode nucleic acid, a genomic component comprised by a VLP, a given viral polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0058] In some embodiments of any of the aspects, a vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of tire aspects, a vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0059] A vector can be an expression vector, e.g., for expressing genomic or polypeptide components of a VLP. As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. Hie sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0060] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes. The vector and / or particle may be utilized for tire purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Nonlimiting examples of a viral vector of this invention include an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric virus vector.

[0061] As used herein, '‘contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery' to cell culture medium, transfection, transduction, perfusion, injection, or other delivery method known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0062] The term “statistically significant" or '‘significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0063] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The tenn “about” when used in connection with percentages can mean ±1%.

[0064] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0065] Hie tenn "consisting of1refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0066] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0067] Hie singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0068] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0069] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0070] Fig. 1 demonstrates envelope proteins from one virus (such as VSV-G) can be pseudotyped or inserted into tire membrane of another type of virus.

[0071] Fig. 2 illustrates an overview of experimental design. A pooled library of virus-like particles carrying different envelope proteins and corresponding barcodes can be injected into a mouse. Sequencing of the barcodes for each envelope protein from different organs indicates where each viruslike particle localizes or delivers its cargo.

[0072] Fig. 3 illustrates that a non-replicating, pseudotyped virus-like particle (VLPs) can be constructed from four plasmids encoding an envelope protein (from a candidate virus of interest), Gag / Pol and Rev (proteins which package and administer the nucleic acid, e.g., RNA or DNA cargo), and the nucleic acid, e.g., RNA or DNA cargo (which can encode a barcode).

[0073] Figs. 4A-4B demonstrate viral purification using high-throughput “In-Sluny” Capto Core (CC) multimodal chromatography resin. Fig. 4A: Percentage of transduced cells measured by flow cytometry and Fig. 4B: Coomassic total protein stain to compare VSV-G viral isolation using cither traditional centrifugation or the In-Slurry CC method with different ratios of CC resin (CC1.25, CC2.5, or CC5)

[0074] Fig 5 demonstrates western blotting using anti-HA tag antibody of cell lysate (C) or purified supernatant (S) after production of viral particles pseudotyped with VSV-G envelope, MLV envelope, or MLV envelope modified to contain the VSV-G cytoplasmic tail. Although MLV envelope has much lower expression than VSV-G in producer cells, modifying the MLV envelope with tire VSV-G cytoplasmic tail allows for dramatic improvement in pseudotyping.

[0075] Figs 6A-6C demonstrate multiplexed library screen proof-of-principle validation. Fig. 6A: Schematic of experiment: Five different barcoded viral particles are tested for their transduction efficiency in three different cell lines (HEK293T, HepG2, or A549) Fig. 6B: individually by flow cytometry or Fig. 6C : pooled in a test library and their barcodes read out by NGS

[0076] Fig 7 demonstrates qPCR of purified VLPs made by transfecting cells with Gag and different guide RNA constructs, targeting either tire wild-type 7SL as a control, or the guide RNA. Construct 1 is able to package into the particle successfully.

[0077] Fig 8 shows experimental design of high-throughput pseudotyping evaluation.

[0078] Fig 9 demonstrates validation of a system using VLPs carrying luciferase, injected in an in vivo mouse model.

[0079] Figs 10A-10F show western blots of individually purified VLPs using MMR Slurry and then analyzed by Western blotting using an anti-HA tag antibody. The gel and corresponding expected size are shown.

[0080] Fig 11 shows mice injected with VLPs containing Nipah (NiV G / F), No envelope (control), or VSV-G envelope, each carrying a Luciferase reporter.

[0081] Fig. 12 shows sequencing readout of barcodes from three mice injected with a multiplexed library of VLPs containing VSV-G envelope, Nipah envelope, or no envelope (blank). Each VLP was barcoded and each ty pe of VLP had two different VLPs with different barcodes. Hie number of reads corresponding to each VLP is indicated by the color.

[0082] Fig. 13 shows validation of in vivo NGS-based sequencing platform. 4 preparations of viruslike particles (VLPs) were mixed together and injected into 4 mice with an exponentially decreasing dose from mouse 1 to mouse 4. Each VLP preparation carried a different nucleic acid barcode. Two of them displayed a functional fusogenic envelope (VSV-G), while two were made without any envelope protein. After injection, the mice w ere sacrificed and dissected to remove their liver and spleen. The DNA w as extracted, and the barcode sequences were enriched, amplified by PCR, and then sequenced using NGS. The counts of each barcode sequence in each organ represent how efficient a given VLP preparation was at functionally delivering to the cells in that organ.

[0083] Fig. 14 show's an in vivo screen of 96 different barcoded VLPs. 96 different VLP preparations displaying different envelope proteins, and carrying different barcodes, were mixed together and injected into 3 different mice. Using the same method as in Fig. 13, the barcode counts were measured for each organ and are displayed in the heatmap. Each row represents counts for one organ (e.g. Liver 3 represents the counts for barcodes in the liver of mouse 3). The negative control (NC) organs are organs from mice which received high doses of VLPs carrying different barcodes that were not present in any of the 96 VLPs used in this experiment. The Un-Enriched organs represent read counts from organs w here the barcode sequence was not enriched before PCR. Each column represents one different kind of VLP, carrying a unique kind of envelope protein.

[0084] Fig. 15 shows an arrayed in vitro screen of envelope proteins on HEK293T cells.

[0085] Fig. 16 shows an in vitro test screen comparing NGS and flow cytometry readouts on humanCD34+ HSCs.

[0086] Fig. 17A-17B shows comparison of envelope proteins. Fig. 17A: VLPs were made displaying two different envelope proteins, Yug Bogdanovac and VSV-G, indicated as B and V respectively. 100 uLof purified VLPs were assayed by anti-HA-tag western blot for the presence of the envelope proteins, which both carried an HA tag. V is far more abundant on VLPs than B. Fig. 17B: After modifying the cytoplasmic tail of B in two different ways (B-l and B-2), to cither truncate or replace with VSV-G, VLPs were made displaying V, B-l and B-2. Only 1 uL of VLPs were assay ed by anti-HA tag Western blot, showing that the two modifications lead to equivalent display and are able to rescue pseudotypingDETAILED DESCRIPTION

[0087] Hie technology described herein relates to the identification of viral envelope proteins that mediate viral tropism to given cell or tissue types, and to harnessing such viral envelope proteins for the delivery of nucleic acids to the given target cells or tissues. The technology described herein uses viruslike particles (VLPs), pseudotyped with viral envelope proteins for nucleic acid (e.g., RNA) delivery to preferred target cells or tissues. In some embodiments, the methods and compositions described herein thus permit the preparation of non-replicating virus-like particles that permit the targeted delivery of nucleic acid cargo to target cells in a manner that substantially avoids the liver accumulation or clearance of the VLPs that commonly occurs with other delivery approaches including but not limited to lipid nanoparticles.

[0088] Generally speaking, enveloped viruses are characterized by the presence of a lipid layer envelope surrounding a protein capsid that encapsulates the viral nucleic acid genome. Broadly, enveloped viruses have four components: the genome, the capsid / matrix, which is a shell of protein molecules that packages tire genome, a lipid membrane envelope surrounding the capsid, and the envelope proteins, which are embedded in tire lipid membrane and facilitate viral particle transduction of host cells. Tire envelope is typically derived from portions or components (phospholipid and protein) of the host cell membrane. Functionally, viral envelopes and associated envelope proteins help viruses bind to and to enter host cells and may help them to avoid the host immune system. The envelope can comprise a single layer of lipid or a lipid bilayer. Envelope proteins are inserted in, attached to, or anchored in the lipid layer. In some embodiments the envelope proteins are glycostructures such as glycoproteins and / or glycooligopeptides. Envelope proteins serve to bind to receptor sites, most often proteins, present or displayed on the host's cell membrane. Binding of a viral envelope protein to a host cell receptor promotes fusion of the viral lipid envelope with the host cell membrane. The viral envelope fusing with the host cell’s membrane allows the capsid and viral genome to enter and infect the host cell. Common features of enveloped viruses and envelope proteins are known to those skilled in the art, see, e.g., Rey et al, Cell 172: 1319-1334, 2018, which is incorporated by reference herein.

[0089] Enveloped viruses include but are not limited to: Argentine hemorrhagic fever virus, Australian bat virus, Autographa califomica multiple nucleopolyhedrovirus, Avian leukosis virus, baboon endogenous virus, Bolivian hemorrhagic fever virus, Boma disease virus, Breda virus, Bunyamwcra virus, Chandipura virus, Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Dengue fever virus, Duvenhage virus, Eastern equine encephalitis virus. Ebola hemorrhagic fever virus. Ebola Zaire virus, enteric adenovirus, Ephemerovirus. Epstein-Bar virus (EB V). European bat virus 1. European bat virus 2, Fug Synthetic gP Fusion, Gibbon ape leukemia vims, Hantavirus, Hendra vims, hepatitis A vims, hepatitis B vims, hepatitis C vims, hepatitis D vims, hepatitis E vims, hepatitis G Vims (GB vims C), herpes simplex vims type 1, herpes simplex vims type 2, human cytomegalovirus (HHV5), human foamy vims, human herpesvirus (HHV), human Herpesvirus 7, human herpesvirus type 6, human herpesvirus type 8, human immunodeficiency vims 1 (HIV-1), human metapneumovirus, human T-lymphotro pic vims 1, influenza A, influenza B, influenza C vims, Japanese encephalitis vims, Kaposi's sarcoma- associated herpesvirus (HHV8), Kaysanur Forest disease vims, La Crosse vims, Lagos bat vims, Lassa fever vims, lymphocytic choriomeningitis vims (LCMV), Machupo vims, Marburg hemorrhagic fever vims, measles vims, Middle eastern respiratory syndrome-related coronavirus, Mokola vims, Moloney murine leukemia vims, monkey pox, mouse mammary tumor vims, mumps vims, murine gammaherpesvirus, Newcastle disease vims, Nipah vims, Nipah vims, Norwalk vims, Omsk hemorrhagic fever vims, papilloma vims, parvovims, pseudorabies vims, Quaranfil vims, rabies vims, RD114 Endogenous Feline Retrovirus, respiratory7syncytial vims (RSV), Rift Valley fever vims, Ross River vims, Rotavirus, Rous sarcoma vims, rubella vims, Sabia-associated hemorrhagic fever vims, SARS- associated coronavirus (SARS-CoV), Sendai vims, Tacaribe vims, Thogotovims, tick-bome encephalitis causing vims, varicella zoster vims (HHV3), varicella zoster vims (HHV3), variola major vims, variola minor vims. Venezuelan equine encephalitis vims, Venezuelan hemorrhagic fever vims, vesicular stomatitis vims (VSV), Vesiculovims, West Nile vims, western equine encephalitis vims, and Zika Vims. Non-limiting examples of enveloped vims gly coprotein amino acid sequences can be found, for example in WO2022261148, which is incorporated by7reference herein.

[0090] In some examples, the envelope protein of a VLP as described herein recognizes a receptor present on a single type of cell. In another example, the envelope proteins of the VLP described herein recognizes a receptor present in multiple cell types (e.g., 2, 3, 4. 5, 6, or more different cell types), hi some examples, an envelope protein of a VLP described herein recognizes cell receptors present in a single type of tissue. In another example, the envelope proteins of the VLP described herein recognizes cell receptors present in multiple tissue types (e.g., 2, 3, 4, 5, 6, or more different tissue ty pes).

[0091] In some embodiments, the VLP comprises the exterior, solvent-exposed, or extracellular portion of a heterologous viral envelope protein. In some embodiments, the VLP comprises a fragment of a heterologous viral envelope protein which contains, at a minimum, the receptor binding domain of the envelope protein. Such a binding domain can be, for example, fused to a heterologous polypeptide chassis that anchors the binding domain in the envelope or lipid membrane component of a VLP as described herein. Alternatively, the receptor binding domain of a heterologous envelope protein can be displayed on a heterologous chassis or scaffold that displays the binding domain and links it to a barcoded nucleic acid chassis.

[0092] A VLP envelope polypeptide can further include a detectable marker polypeptide or tag sequence that permits the identification of cells infected or transduced by the VLP. Nonlimiting examples of detectable markers include the HA epitope tag derived from influenza A Hemagglutinin (Uniprot Q03909; residues 119-127; HA-tag); V5 tag; FLAG tag; C-myc tag; histidine tag (HIS tag); Avi tag; Strep tag; Strep tag II; S-peptide tag (S tag); ALFA tag; AU 1 tag; AU5 tag; Glu-Glu tag; HSV tag;KT3 tag; E tag; and C tag.

[0093] Hie host cell or tissue infected by a given enveloped virus or virus-like particle can be modified by switching all or part of the given virus' envelope protein with all or part of another virus' envelope protein. The process of switching envelope proteins is referred to as '’pscudotyping." and is well known in the art. Pseudotyping is described, for example, in Bischof et al. (Flexibility in cell targeting by pseudotyping lentiviral vectors. Methods Mol Biol. 2010; 614:53-68). Pseudotyping is most often used to modulate the cell type specificity of a viral vector by integration of foreign viral envelope proteins. Using this approach, host tropism can be altered and / or stability of tire virus can be decreased or increased.

[0094] Pseudotyping viral systems has also been widely employed, for example, to study highly infectious and pathogenic viruses, such as Ebola virus, Middle Eastern Respiratory Syndrome (MERS) virus, or SARS viruses (McWilliams et al.. Cell Rep. (2019) 26: 1718-26. e4. doi:10. 1016 / j.celrep.2019.01.069; Liu et al., Antiviral Res. (2018) 150:30-8. doi: 10.1016 / j. antiviral.2017. 12.007; and Fukushi et al., SARS- and Other Coronaviruses: Laboratory Protocols. Totowa, NJ: Humana Press (2008). p. 331-8). Hie two most commonly used pseudotyping systems are retro / lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVAG). The use of replication-restricted pseudoviruses bearing foreign viral coat proteins represents a safe and useful method that has been widely adopted by virologists to study viral entry, detection of neutralizing antibodies in serum samples, and therapeutic development under less stringent biosafety conditions (e.g., biosafety level-2 (BSL- 2)). Pseudotyped viruses have been used to produce vaccinecandidates against HIV (Racine et al., AIDS Research and Therapy. 14 (1): 55. doi : 10. 1 186 / sl2981 -017- 0179-2); Nipah henipavirus (Nie et al., Emerging Microbes & Infections. 8 (1): 272-281; doi: 10.1080 / 22221751.2019.1571871); Rabies lyssavirus (Mocschlcr ct al., Viruses. 8 (9): 254. doi: 10.3390 / v8090254), SARS-CoV (Kapadia et al., Virology. 376(1): 165-172. doi: 10.1016 / j. virol.2008.03.002); Zaire ebolavirus (Salata et al.. Viruses. 11 (3): 274. doi: 10.3390 / vl 1030274). and SARS-CoV-2 (Johnson et al.. Journal of Virology. 94 (21). doi: 10. 1128 / JVI.01062-20; and Condor Capcha et al., Front. Cardiovasc. Med., 15 January (2021)). Each of these references, among others, provide guidance on how to prepare pseudotyped viruses.

[0095] Envelope glycoproteins of any enveloped virus, including, but not limited to those viruses described herein above, or any enveloped virus known to infect human cells, can be used to prepare pseudotyped VLPs as described herein. Non-limiting examples of an envelope proteins include proteins listed in Table 1. In some embodiments, the envelope protein of a VLP is selected from a Hepatitis B virus (HBV) glycoprotein, a Hepatitis C vims (HCV) glycoprotein, a Marburg virus glycoprotein, an Ebola virus glycoprotein, a VSV-G glycoprotein; and the target cell is a liver cell, hr some cases, the envelope protein of a VLP is a pscudotyping viral glycoprotein, such as, but not limited to, a viral glycoprotein selected from an influenza virus hemagglutinin, a SARS-CoV glycoprotein, a respiratory syncytial virus glycoprotein, a human parainfluenza virus glycoprotein, and a VSV-G; and the target cell is a lung cell. In some cases, the envelope protein of a VLP is a pseudotyping viral glycoprotein, such as, but not limited to, a viral glycoprotein selected from a measles virus hemagglutinin and / or a measles virus fusion glycoprotein, and the target cell is a CD34+cell. In some cases, the envelope protein of a VLP is a pseudotyping viral glycoprotein, such as, but not limited to, a viral glycoprotein selected from a measles vims hemagglutinin and / or a measles vims fusion glycoprotein, an HTLV-1 glycoprotein, and a VSV- G glycoprotein; and the target cell is a CD8+T cell. In some cases, the envelope protein of a VLP is a pseudotyping viral glycoprotein, such as, but not limited to, a viral glycoprotein selected from a HIV-1 envelope, a HTLV-1 glycoprotein, a measles vims hemagglutinin, and a VSV-G glycoprotein; and the target cell is a CD4+ T cell. In some cases, the envelope protein of a VLP is a pseudotyping viral glycoprotein, such as, but not limited to, a Ross River vims glycoprotein or a VSV-G; and the target cell is a skeletal muscle cell. In some cases, the envelope protein of a VLP is a pseudotyping viral glycoprotein, such as, but not limited to. a viral glycoprotein selected from an Ebola vims glycoprotein, a Marburg vims glycoprotein, and a VSV-G; and the target cell is an ocular cell (e.g., in a retinal cell, a photoreceptor cell, etc.), hr some cases, the envelope protein of a VLP is a pseudotyping viral glycoprotein, such as, but not limited to, a viral glycoprotein selected from an Ebola vims glycoprotein, a Marburg vims glycoprotein, and a VSV-G; and tire target cell is an auditory cell (e.g., hair cells, cochlearcells, etc.). In some cases, the envelope protein of a VLP is a pseudotyping viral glycoprotein, such as, but not limited to, a viral glycoprotein selected from aa rabies glycoprotein, a Mokola virus glycoprotein, a Scmliki Forest vims glycoprotein, a Sindbis vims glycoprotein, a Venezuelan equine encephalitis vims glycoprotein, an influenza hemagglutinin glycoprotein, and a VSV-G; and wherein the target cell is a central nervous system cell (e.g., neurons (e.g., excitatory and inhibitory neurons); and glial cells (e.g.. oligodendrocytes, astrocytes and microglia)).

[0096] In some embodiments, the plurality or pool of viral envelope proteins comprise at least 1 envelope protein from Table 1. In some embodiments, the plurality or pool of viral envelope proteins comprise at least 5 envelope proteins from Tabic 1, at least 50 envelope proteins from Table 1, at least 90 envelope proteins from Table 1, at least 96 envelope proteins from Table 1, at least 100 envelope proteins from Table 1, at least 200 envelope proteins from Table 1. at least 300 envelope proteins from Table 1, at least 400 envelope proteins from Table 1, at least 500 envelope proteins from Table 1, at least 600 envelope proteins from Table 1, at least 700 envelope proteins from Table 1, or at least 766 envelope proteins from Table 1.

[0097] In some cases, an envelope protein includes a human endogenous retroviral (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule. Human endogenous retroviruses (HERVs) make up a significant portion (~8%) of the human genome. These viral elements integrated into the genome millions of years ago and were since then vertically transmitted through generations. A large majority of HERVs have lost functional activity through mutation or truncation, yet some endogenous retroviruses, such as the members of the HERV-K clade, still encode functional genes and have been shown to fomr retrovirus -like particles. Non-limiting examples of HERV envelope proteins can be found, for example in WO2023102550, which is incorporated by reference herein. In addition, HERVs databases are described in tire literature, see for example, L. Vargiu et al., Retrovirology, (2016). A HERVs database is also available and can be retrieved from the GenBank database. It is specifically contemplated that HERV glycoproteins can be included among the viral envelope proteins used to pseudotype VLPs as described herein.

[0098] Pseudotyping can be useful not only to change the tropism of one virus to mimic that of another virus or to direct it to another host cell or tissue type, but also to generate a library or pool of viruses or vims-like particles that can be screened as described herein to identify viral envelope proteins that mediate infection of selected target cell or tissue types.

[0099] The technology described herein includes assessment of pseudotype efficiency. In some embodiments, hemagglutinin (HA) tags are inserted into the envelope proteins prior to pseudotyping. As discussed further herein below', in some embodiments, an HA tag is inserted into the cytoplasmic domainof the envelope protein (other tags that are readily detected by, e.g., immunological methods, can also be used). In some embodiments, HA tag incorporation onto viral particles is assessed after viral particle purification by methods known to those skilled in the art, c.g., Western blotting using antibodies against the HA-tag. In some embodiments, high-throughput viral purification techniques are employed prior to HA tag detection. High-throughput viral purification techniques include but are not limited to In-Slurry binding of free proteins, e.g., in the media, into the interior of beads, e.g., Capto Core 700 resin.Virus-Like Particles

[0100] Hie technology described herein includes tire use of non-rcplicativc virus-like particles for tire delivery of a nucleic acid cargo to a cell, e.g., a mammalian cell, e.g., a murine cell, a rat cell, a bovine cell, an equine cell, a caprine cell, an ovine cell, a guinea pig cell or a human cell, among others. The delivery can be in vivo, ex vivo or in vitro to a mammal or mammalian cell or tissue. In one embodiment, the delivery is in vivo. In another embodiment, the mammal can be a humanized mammal, e g., a humanized mouse, which comprises a human or humanized immune system. In another embodiment, the delivery is in vivo to a rat cell, a bovine cell, an equine cell, a caprine cell, an ovine cell, a guinea pig cell or a human cell, hi another embodiment, the delivery is in vivo to a cell or tissue of a human.

[0101] VLPs are self-assembling, non-naturally occurring, multicomponent structures. A VLP comprises, at a minimum, an enveloped cell-derived lipid membrane component displaying a viral envelope polypeptide or a solvent-exposed portion thereof, and a nucleic acid comprising or encoding a barcode nucleic acid that corresponds to the viral envelope polypeptide. While it does not necessarily encode sequences necessary to generate an entire VLP, the nucleic acid component of a VLP can be referred to as the genome of the VLP. hi one embodiment, such genome can comprise or consist of a barcode nucleic acid. In another embodiment, such genome can comprise a barcoded nucleic acid and additional nucleic acid sequence encoding one or more polypeptide components of the VLP, including but not limited to the viral envelope polypeptide. In one embodiment, a virus-like particle’s nucleic acid component can encode the envelope polypeptide and the barcode. In one embodiment, a virus-like particle includes viral -derived structure necessary to introduce the nucleic acid to a target cell; thus, a virus-like particle can also include, in addition to the viral envelope polypeptide, one or more viral capsid polypeptides, or a capsid as needed to package tire nucleic acid in a form permitting delivery into a cell. In another embodiment, a virus-like particle does not include additional viral-derived structure such as viral capsid polypeptides.

[0102] In some embodiments, the VLP is derived from a retrovirus. In some embodiments, the VLP components can be selected from a Retroviridae virus, e.g. an Orthoretrovirinae virus or aSpumaretrovirinae virus. In some embodiments, the Orthoretrovirinae virus is selected from the group consisting of an Alpharctrovirus. Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lcntivirus. In some embodiments, the Spumaretrovirinae vims is selected from the group consisting of Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus, Simiispumavirus, or Spumavirus.

[0103] In some embodiments, a VLP is capable of self-assembling when one or more nucleic acids encoding the components of the VLP and the envelope glycoprotein are introduced into a eukaryotic host packaging cell and are expressed. A barcoded nucleic acid cargo can also be expressed in the host cell and integrated or encapsulated within the VLP upon self-assembly.

[0104] In preferred embodiments, the VLP is derived from a retroviral delivery vesicle generation system that can be composed of one or more polynucleotides that encode one or more effectors, optionally one or more polynucleotides that encode a barcoded cargo nucleic acid, and one or more polymucleotides that encode one or more packaging elements, one or more vesicle elements, or both. The cargo nucleic acid molecule can be modified with or include one or more packaging elements that complex or bind to a retroviral polypeptide and facilitate packaging of the nucleic acid payload molecule into the VLP. While the term ‘‘cargo nucleic acid" or simply “cargo” is referred to in the singular, it is contemplated that multiple copies of a cargo nucleic acid molecule, depending on type and other readily recognizable size constraints of the VLP, can be packaged within a single VLP.

[0105] In other embodiments, the VLP is derived from an extracellular vesicle generation system that generates extracellular vesicles displaying an envelope protein. In some embodiments, the cargo or nucleic acid is packaged into the VLP without tire use of additional viral capsid polypeptides. Methods for packaging cargo into an extracellular vesicle are known to those skilled in the art; see, for example, Zheng, Wenyi et al. “Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles.” Nature communications vol. 14,1 4734. 7 Aug. 2023, which is incorporated herein by reference and describes the preparation of extracellular vesicles that display functional entities on their surface, as well as containing cargo molecules. Fusion of a viral envelope polypeptide targeted to an extracellular vesicle with a nucleic acid-binding domain permits tire generation of extracellular vesicles that display a viral envelope polypeptide and comprise a barcoded nucleic acid corresponding to the envelope polypeptide. See also Osteikoetxea. Xabier et al. “Engineered Cas9 extracellular vesicles as a novel gene editing tool.” Journal of extracellular vesicles vol. 11,5 (2022): e 12225., which also describes the generation of extracellular vesicles with targeted cargo and is incorporated by reference herein. In some embodiments, the target cargo protein for an extracellular vesicle is fused to an RNA-binding domain and is targeted to the membrane by, for example, adding myristoylation or palmitoylation signalsto the protein. Moieties (e.g., polypeptides, viral envelope proteins, and / or such polypeptides or proteins associated with a nucleic acid cargo) targeted to the cell membrane in any of a number of different ways can become associated with a vesicle or VLP that buds off of that membrane, hr other embodiments, a transmembrane protein, e.g., a protein that gets packaged into vesicles, e.g., CD9, can be fused to an RNA binding domain. In some embodiments, the cargo is packaged using extracellular vesicle cargo loading methods, including, for example, targeting cargo to extracellular vesicles using proteins with vesicle sorting capabilities, e.g., TSAP2 or TSPAN3. In some embodiments, the cargo is targeted to extracellular vesicles with heterodimerization techniques that partner the cargo to one heterodimer of a pair and partner the other hctcrodimcr of the pair to a protein that accumulates in vesicles; non-limiting examples include, e.g., tetraspanins.

[0106] In some embodiments, the cargo is mRNA. In some embodiments, described herein, are methods for packaging cargo, e.g., mRNA, comprising a chimeric Gag-Pol-X protein, wherein X is an RNA binding domain, and the cargo mRNA has a cognate motif for X. In some embodiments, the RNA binding domain is attached by a flexible linker. In some embodiments, the linker is a flexible linker with an optimized HIV protease cleavage site.

[0107] In some embodiments, described herein are methods for packaging cargo, e.g., mRNA, comprising RT-deficient Gag-pol and cargo mRNA with Psi, LTR, and Gag fragment motifs.

[0108] In some embodiments, described herein are methods for packaging cargo, e.g., mRNA, comprising a chimeric EV-sorting transmembrane protein, with an RNA binding domain X, and an mRNA with tire cognate motif for X. In some embodiments, the EV-transmembrane protein includes but is not limited to CD63, TSPAN101, and CD9.

[0109] In some embodiments, the cargo is a gene editor and a donor DNA.

[0110] In some embodiments, described herein are methods for packaging cargo, e.g., a gene editor and a donor DNA, comprising a chimeric Gag-Pol-X protein, wherein X is a site-specific recombinase (SSR). In some embodiments, the HIV integrase has been deactivated. By way of non-limiting example, the VLP will enter a target cell, carrying the site-specific recombinase in protein form. The Pol will reverse-transcribe tire genome and tire SSR will integrate the gene using the att site, hr some embodiments, the SSR is attached by a linker. In some embodiments, the SSR is attached by a flexible linker with an optimized HIV protease cleavage site. In some embodiments, the other attachment site for the recombinase is present in the target cell genome of the desired site.

[0111] In some embodiments, methods for packing cargo comprise a chimeric Gag-Pol-X protein, wherein X is an SSR and the method further comprises another orthogonal att pair. In some embodiments, the att pair is with one att at each end of tire lentiviral genome. By way of non-limiting example, the VLPwill enter a target cell, carrying the site-specific recombinase in protein form. It will reverse-transcribe its genome and the SSR will first circularize the genome with one att pair and then integrate the gene into the genome using tire first att site.

[0112] In some embodiments, methods for packaging cargo, e.g.. a gene editor and a donor DNA, comprise a chimeric Gag-Pol-X protein, wherein X is a programmable nuclease, and a lentiviral genome encoding a therapeutic gene and guide RNAs. In some embodiments, the HIV integrase has been deactivated. In some embodiments, the programmable nuclease is attached by a linker. In preferred embodiments, the linker is a flexible linker with an optimized HIV protease cleavage site. By way of nonlimiting example, the VLP will enter a target cell, carry ing the editor in protein form. Uris editor will use tire guide RNAs to make a break or nick at a specific location in the genome, and the Pol will reverse transcribe the genome to provide a DNA donor for homologous recombination at the nicked / cut site. In some embodiments, methods for packaging cargo, e.g., a gene editor and a donor DNA, comprise a chimeric Gag-Pol-X protein, wherein X is an SSR, a chimeric Gag-Pol-Y protein, wherein Y is a programmable DNA editor or writer, and a lentiviral plasmid encoding the desired therapeutic gene and a guide RNA with one att site for the recombinase and another orthogonal att pair at each end. In some preferred embodiments, the HIV integrase has been deactivated, hi some embodiments, the recombinase and the programmable DNA editor or writer are attached by a linker. In preferred embodiments, the linker is a flexible linker with an optimized HIV protease cleave site. By way of non-limiting example, the VLP will enter a target cell, carrying the SSR and programmable editor in protein form. The programmable editor will use the guide RNA and create an att site in the genome at a desired location. Then Pol will reverse-transcribe its genome and the SSR will first circularize the genome with one att pair and then integrate the gene into the genome using the first att site.

[0113] In some embodiments, the retroviral system can be a lentiviral system, e.g.. a 3rdgeneration lentivirus system. Third-generation lentiviral vectors and vector systems thereof have increased safety over first- and second-generation lentiviral vectors and systems thereof because, for example, the various components of the viral genome are split between two or more different vectors but used together in vitro to make virus particles, they can lack a tat gene (when a constitutively active promoter is included upstream of the LTRs), and they can include one or more 3 ' LTR deletions to create self-inactivating (SIN) vectors having disrupted promoter / enhancer activity of the LTR. In some embodiments, a third- generation lentiviral vector system can include (i) a vector plasmid that contains the polynucleotide of interest and upstream promoter that are flanked by the 5 ’ and 3 ’ LTRs, which can optionally include one or more deletions present in one or both of the LTRs to render the vector self-inactivating; (ii) a “packaging vectors)” that can contain one or more genes involved in packaging a polynucleotide into avirus particle that is produced by the system (e.g. gag, pol, and rev) and upstream regulatory sequences (e.g. promoters)) to drive expression of the features present on the packaging vector, and (iii) an “envelope vector” that contains one or more envelope protein genes and upstream promoters. In certain embodiments, the third-generation lentiviral vector system can include at least two packaging vectors, with the gag-pol being present on a different vector than the rev gene.

[0114] In some embodiments, the system comprises four plasmids: one packaging plasmid which encodes the lentiviral capsid (gag) and packaging proteins (pro and pol), another packaging plasmid encoding the Rev regulatory protein, the transfer plasmid, which encodes the nucleic acid cargo or payload surrounded by lentiviral packaging signals or LTRs, and the envelope protein plasmid, which will vary for different members of a library and can be selected from any viral envelope polypeptide. Details of the genomic structure of lentiviruses and sequences required for self-assembly are known to those of ordinary skill in tire art.

[0115] Exemplary VLPs and how to prepare them are known in the art. As but one example, see WO2021113772A1, tire contents of which are incorporated by reference in their entirety herein. While VLPS including a ccll-dcrivcd membrane component arc considered herein, it is also contemplated that a vesicle comprising a synthetic membrane component could be used, incorporating and displaying a viral envelope polypeptide as for a VLP, and packaging a corresponding barcoded nucleic acid cargo.

[0116] The VLP system can include viral packaging elements, vesicle elements, or both. In some embodiments, the VLP system includes one or more polynucleotides that encode one or more packaging elements, one or more polynucleotides that encode one or more vesicle elements, or both. Packaging elements are genes / proteins that are involved in viral packaging of a cargo, such as a cargo polynucleotide. Vesicle elements are genes / proteins that are capable of generating a viral particle (or vesicle) and include envelope, capsid, and other structural gene / proteins that form the structural viral particle or vesicle that encapsulates a cargo, for example.

[0117] In some embodiments, the systems described herein comprise another polynucleotide comprising genes encoding packaging proteins. In some embodiments, packaging proteins may comprise lentiviral gag, pol, tat, and rev genes.

[0118] Each retroviral genome comprises genes designated gag, pol and env which encode for virion proteins and enzymes. These genes are flanked at both ends by regions termed long terminal repeats (LTRs). LTRs are responsible for integration and proviral transcription. These also serve as enhancerpromoter sequences. In other words, LTRs can control the expression of viral genes. Retroviral RNA encapsidation occurs as a consequence of a psi sequence located at the 5' end of the viral genome.

[0119] The LTRs themselves are identical sequences that can be divided into three elements, which are designated U3, R, and U5. U3 is derived from the single 3 'end sequence of RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the single 5 'end sequence of the RNA. Hie sizes of the three elements can vary considerably between different retroviruses.

[0120] In some embodiments, the gene of interest is under the control of an LTR sequence.

[0121] In some embodiments, the VLP is derived from a retrovirus, or comprises retroviral proteins.The major structural component of retrovimses is the polyprotein Gag, which also typically contain protease cleavage sites that, upon action by the viral protease, processes the Gag into subcomponents that in the case of the replication of the source virus, then self- assemble in the host cell to make the core inner shell of the virus. The expression of Gag alone is sufficient to mediate the assembly and release of viruslike particles (VLPs) from host cells. Gag proteins from all retroviruses contain an N-terminal membranebinding matrix (MA) domain, a capsid (CA) domain (with two subdomains), and a nucleocapsid (NC) domain that are structurally similar across retroviral genera but differ greatly in sequence. Outside these core domains, Gag proteins vary among retrovimses, and other linkers and domains may be present (Shur, F., ct al. The Structure of Immature Vims-Like Rous Sarcoma Vims Gag Particles Reveals a Structural Role for the plO Domain in Assembly. J Virol. 89(20): 10294 (2015)). The assembly pathway of Gag into immature particles in the host cell is mediated by interactions between MA (which is responsible for targeting Gag polyprotein to the plasma membrane), between NC and RNA, and between CA domains (which, in the context of the present disclosure, can assemble into a VLP capsid). In the context of the retrovimses, concomitant w ith, or shortly after, particle release, cleavage of Gag by the viral protease (PR) gives rise to separate MA, CA, and NC proteins, inducing a rearrangement of the internal viral structure, with CA fonning the shell of the mature viral core. Full proteolytic cleavage of Gag into its individual domains is necessary for vims infectivity for the native viruses.

[0122] Genes encoding viral polypeptides capable of self-assembly into defective, non-selfpropagating viral particles can be obtained from the genomic DNA of a DNA vims or the genomic cDNA of an RNA vims or from available subgenomic clones containing the genes. These genes will include those encoding viral capsid proteins (i.e., proteins that comprise the viral protein shell) and, in the case of enveloped vimses, such as retrovimses, the genes encoding viral envelope glycoproteins. Additional viral genes may also be required for capsid protein maturation and particle self-assembly. These may encode viral proteases responsible for processing of capsid protein or envelope glycoproteins. As an example, the genomic structure of picomavimses has been well characterized, and the patterns of protein synthesis leading to virion assembly are clear. Rueckert, R. in Virology (1985), B. N. Fields et al. (eds.) Raven Press, New York, pp 705-738. In picomavimses, the viral capsid proteins are encoded by an RNAgenome containing a single long reading frame and are synthesized as part of a polyprotein which is processed to yield the mature capsid proteins by a combination of cellular and viral proteases. Thus, tire picomavirus genes required for capsid self-assembly include both tire capsid structural genes and the viral proteases required for their maturation. Another virus class from which genes encoding selfassembling capsid proteins can be isolated is the lentiviruses, of which HIV is an example. Like the picomaviral capsid proteins, the HIV gag protein is synthesized as a precursor polypeptide that is subsequently processed, by a viral protease, into tire mature capsid polypeptides. However, the gag precursor polypeptide can self- assemble into virus-like particles in the absence of protein processing. Ghcyscn ct ah, Cell 59: 103 (1989); Dclchambrc ct ah, The EMBO J. 8:2653-2660 (1989). Unlike picomavirus capsids, HIV capsids are surrounded by a loose membranous envelope that contains the viral glycoproteins. These are encoded by the viral env gene.

[0123] The pol gene encodes the reverse transcriptase (RT), which contains the associated DNA polymerase, RNase H and integrase (IN), which mediate genome replication. The env gene encodes the surface glycoprotein (SU) and tire 11 (TM) transmembrane protein of the virion, which forms a complex that interacts specifically with cellular receptor proteins. This interaction ultimately leads to fusion infection of the viral membrane with the cell membrane. Retroviruses may also contain additional genes, which encode proteins in addition to gag, pol and env.

[0124] In some embodiments, the packaging proteins and / or vesicle proteins are encoded in one polynucleotide, in other embodiments, they are encoded in two separate polynucleotides.

[0125] In some embodiments, an engineered lentiviral vector system of the present invention can include one or more transfer plasmids. Transfer plasmids can be generated from various other vector backbones and can include one or more features that can work with other retroviral and / or lentiviral vectors in the system that can. for example, improve safety of the vector and / or vector system, increase virial titers, and / or increase or otherwise enhance expression of the desired insert to be expressed and / or packaged into the viral particle. Suitable features that can be included in a transfer plasmid can include, but are not limited to, 5’LTR, 3’LTR, SIN / LTR, origin of replication (Ori), selectable marker genes (e.g. antibiotic resistance genes), Psi (Y), RRE (rev response element), cPPT (central polypurinc tract), promoters, WPRE (woodchuck hepatitis post-transcriptional regulatory element), SV40 polyadenylation signal. pUC origin, SV40 origin, FI origin, and combinations thereof.

[0126] The VLPs described herein can use and further comprise a number of different cargo molecules for delivery-. Representative cargo molecules can include, but are not limited to, nucleic acids, polynucleotides, proteins, polypeptides, polynucleotide / polypeptide complexes, small molecules, sugars, or a combination thereof. Cargos that can be delivered in accordance with tire systems and methodsdescribed herein include, but are not necessarily limited to, biologically active agents, including, but not limited to, therapeutic agents, imaging agents, and monitoring agents. A cargo can be an exogenous material or an endogenous material, hr some embodiments, the cargo can be a “gene of interest”. In some embodiments, the cargo can be a plasmid encoding an envelope protein of interest. In some embodiments, the cargo can be a nucleic acid encoding a barcode. In some embodiments, the cargo can be a nucleic acid encoding an envelope protein of interest linked to a barcode sequence. VLPs can be produced in a packaging cell or cell line, and the VLP membrane component is derived from the packaging cell or cell line. The packaging cell or cell line is generally transduced or transfected with vectors, e.g., plasmid vectors, that encode the various components which, when combined, pemrit self-assembly of the VLP. The following describes methods for preparation of VLPs useful in tire methods and compositions described herein.

[0127] In some embodiments, the cargo polynucleotide is DNA. In some embodiments, the cargo polynucleotide is RNA. In some embodiments, the cargo polynucleotide is a polynucleotide (a DNA or an RNA) that encodes an RNA and / or a polypeptide. As used herein with reference to the relationship between DNA, cDNA, cRNA, RNA, protcin / pcptidcs, and the like ‘‘corresponding to” or “encoding” refers to the underlying biological relationship between these different molecules. As such, one of skill in the art would understand that operatively “corresponding to” can direct them to determine the possible underlying and / or resulting sequences of other molecules given the sequence of any other molecule which has a similar biological relationship with these molecules. For example, from a DNA sequence an RNA sequence can be detenuined and from an RNA sequence a cDNA sequence can be determined. In relation to a nucleic acid barcode, e.g., a barcode associated with a particular viral envelope protein, “corresponds to” or “corresponding to” refers to a relationship between the barcode sequence and the particular viral envelope protein - a different nucleic acid barcode sequence is assigned to each different viral envelope protein such that tire detection of the nucleic acid barcode in a cell indicates that the particular viral envelope protein assigned to or associated with that barcode mediated the introduction of the barcode to that cell.

[0128] In some embodiments, the VLPs described herein can comprise a reporter (e.g., a reporter protein). In some embodiments, the VLP comprises a nucleic acid encoding a reporter (e.g., a reporter protein). As used herein, a reporter is generally a protein or gene that can be detected when expressed in a target cell. In some embodiments, the presence or absence of a reporter in a target cell or a subset of a target cells in a population of cells provides the ability- to sort cells (e.g., using flow cytometry' and / or fluorescence-activated cell sorting).

[0129] In some embodiments, a reporter is a fluorescent protein. A fluorescent protein can be a green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP). Exemplary fluorescent proteins can be as described, c.g., in U.S. Pat. No. 7,060,869. The engineered particles displaying specific ligands deliver fluorescent protein into target cells upon cognate receptor-ligand interaction.VLP Cargos

[0130] VLPs as described herein deliver a nucleic acid cargo to a target cell, hr one embodiment, a library of VLPs, tire members of which comprise a nucleic acid barcode that corresponds to tire viral envelope polypeptide displayed by the given VLP, are introduced to an animal, and. after time sufficient to permit the VLPs to travel or translocate to and deliver their cargo to cells or tissues of the animal, cells and / or tissues of the animal are evaluated for the presence of nucleic acid barcodes, thereby identifying which VLP library' member(s), and therefore which viral envelope polypeptide(s) delivered the barcode to that cell or tissue. Detection of VLP nucleic acid cargo can be performed in situ, e.g., on sections of tissue prepared from tire animal, or in cells or tissues harvested or isolated from the animal. It is also contemplated that a library of VLPs can be contacted with cells or tissue explants in culture, followed by wash out to remove non-infecting VLPs and subsequent detection of barcode nucleic acids in the cells or tissue explants to identify the viral envelope proteins that mediate transduction of the given cells or tissues.

[0131] Hie VLP nucleic acid cargo can be simply a nucleic acid (RNA or DNA) comprising a barcode nucleic acid sequence, or can comprise a nucleic acid bearing sequence sufficient to direct the transcription of a sequence encoding a barcode nucleic acid. The barcode nucleic acid can comprise, for example, a single barcode sequence element, or a concatemeric barcode nucleic acid sequence including a plurality of copies of the same barcode sequence element. In some embodiments, the nucleic acid barcode element can be linked to nucleic acid sequence encoding the viral envelope polypeptide of the VLP. The barcoded nucleic acid component of a VLP will also comprise sequence sufficient for packaging of the barcoded nucleic acid component into the VLP. Genomic nucleic acid packaging signals for various viruses and VLP systems are known in the art. As one example, retroviral long terminal repeat (LTR) elements direct viral gene expression, facilitate genome replication and mediate packaging of the viral genome into the viral capsid. Sequence elements within, for example, lentiviral LTRs, that mediate viral genome packaging have been identified - the so-called psi sequences or psi elements mediate viral genome dimerization and packaging with the nucleocapsid polypeptide.Retroviral genome packaging and the signals required for it are described, for example, in Johnson et al.,PLOS Pathogen 6: 1001007 (2010) and Heng et ai., J. Mol. Biol. 417: 224-239 (2012), which are incorporated herein by reference. Where a VLP includes a capsid polypeptide or a capsid, inclusion of LTRs or LTR psi sequences appropriate for the given capsid in the viral cargo nucleic acid can permit efficient packaging of the cargo nucleic acid into the VLP.

[0132] In some embodiments of any of the aspects, the at least one RNA or DNA further comprises a barcode sequence. As used herein, a “nucleic acid barcode” or “barcode” are used interchangeably. Barcode refers to a short sequence of nucleotides which is unique to a particular nucleic acid cargo described herein, e.g., to a particular viral envelope polypeptide. In some embodiments of any of the aspects, the barcode sequence is unique or distinguishable from at least one other barcode sequence comprised by other nucleic acid cargo with a different viral envelope polypeptide as described herein. Nucleic acid barcodes, DNA barcodes, and barcoding strategies are known in the art, see, for example, Dahlman JE, et al. Proc Natl Acad Sci U S A. 2017 Feb 21;114(8)2060-2065; Wang Y, Wagner E. Pharmaceutics. 2020 Sep 18: 12(9): 888; Binan L, et al.. SLAS Technology. 2019 June: 24(3)298-307: Chen HC. Front Mol Biosci. 2023 Jul 11;10: 1141534; WO2021119402; WO2019213294: and 20230271187, which arc incorporated by reference herein.

[0133] Generally, barcode sequences can each have a length within a range of from about 5 nucleotides to about 40 nucleotides. For example, barcode sequences can each have a length of from about 5 nucleotides to about 35 nucleotides, from about 6 nucleotides to about 30 nucleotides, from about 7 nucleotides to about 25 nucleotides, from about 8 nucleotides to about 15 nucleotides. In some embodiments, barcode sequences can each have a length of 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. 32, 33, 34, 34 or 35 nucleotides.

[0134] In one embodiment, a barcode nucleic acid is the cargo nucleic acid. On other embodiments, the cargo nucleic acid comprises tire barcode nucleic acid or an element or construct that permits expression of a nucleic acid comprising a barcode sequence. In one embodiment, the barcode sequence can be present at the 5 ’-end of the cargo nucleic acid sequence. In another embodiment, the barcode sequence can be present at the 3 ’-end of the cargo nucleic acid sequence. The cargo nucleic acid sequence and the barcode sequence can be linked directly to each other, i.e., there are no nucleotides present between the cargo nucleic acid sequence and the barcode sequence. In some embodiments, at least one nucleotide can be present between the cargo nucleic acid sequence and the barcode sequence, as a linker. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides can be present between the cargo nucleic acid sequence and the barcode sequence.

[0135] It is noted that the barcodes can be interrogated using methods known to those of skill in the art including fluorescently labeled oligonucleotide / DNA / RNA hybridization, primer extension with labeled nucleotides, sequencing, e g., scqucncing-by-ligation, -synthesis or -hybridization.

[0136] In some embodiments, the nucleic acid cargo comprises an expression cassette that encodes for a fluorescent marker, e.g., GFP or MCherry. In some embodiments, nucleic acid cargo comprising barcodes can be quantitatively measured using next-generation sequencing. As used herein “nextgeneration sequencing” refers to oligonucleotide sequencing technologies that have the capacity to sequence oligonucleotides at speeds above those possible with conventional sequencing methods (e.g. Sanger sequencing), due to performing and reading out thousands to millions of sequencing reactions in parallel. Non-limiting examples of next-generation sequencing methods / platforms include Massively Parallel Signature Sequencing (Lynx Therapeutics): 454 pyro-sequencing (454 Life Sciences / Roche Diagnostics); solid-phase, reversible dye-terminator sequencing (Solexa / Illumina): SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNA nanoball sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Intelligen Bio-systems, Oxford Nanoporc Technologies, and Hclicos Bioscicnccs.

[0137] In some embodiments, a VLP bearing a viral envelope polypeptide determined to mediate infection or transduction of a given cell or tissue type can be used to deliver a nucleic acid cargo, e.g.. an RNA or DNA cargo, to a target cell or tissue of that type. The preparation or assembly of such VLPs can be performed substantially as described herein for members of a library of VLPs. The nucleic acid cargo can encode a therapeutic polypeptide, e.g., an enzyme or other polypeptide for which the subject is deficient. The nucleic acid cargo can encode a gene editing system, including but not limited to an RNA- guided nuclease system, e g., a CRISPR gene editing system or the like. Such gene editing systems and the components necessary for them (e.g., RNA-guided nuclease, guide RNA, etc.) are known to those of ordinary skill in the art. In one embodiment, a single VLP can comprise components necessary for a gene delivery or gene editing system. In other embodiments, the components of a gene editing system can be comprised by two or more VLPs, e.g., three VLPS, four VLPs or more. Where lipid nanoparticles used for therapeutic gene delivery (whether for gene editing or gene replacement) tend to accumulate in or be cleared by the liver, many naturally-occurring viruses have evolved the ability to substantially avoid such clearance by the liver. In one embodiment, a VLP as described herein substantially avoids clearance by and / or does not accumulate in the liver. In this context, “substantially avoids” means that a VLP bearing a given viral envelope protein either remains in circulation after being administered systemically, e.g., intravenously, for a greater period of time than a VLP that is substantially the same but carries a different envelope polypeptide, and / or accumulates in at least one tissue or cell type to a greater extent than itaccumulates in the liver. The greater period of time can be, for example, a circulating half-life at least 10% longer, at least 20% longer, at least 30% longer, at least 40% longer, at least 50% longer, at least 60% longer, at least 70% longer, at least 80% longer, at least 90% longer, at least 100% longer - i.c., at least 2X longer, at least 2.5X longer, at least 3X longer, at least 3.5X longer, at least 4X longer, at least 4.5X longer, at least 5X longer or more.

[0138] VLPs can be produced as a library in several different ways. One approach generates each VLP separately, e.g., using packaging cells or apparatus, e.g., in a multiwell culture format, followed by pooling of the different VLPs. In another approach, each heterologous envelope protein can be co- transduccd into cells, e.g., 293T cells, alongside cargo, e.g., a barcoded RNA or DNA cargo. In this approach, sequences encoding envelope proteins and barcoded cargo can be paired, for example, in 96 well-plates with the packaging cells, and the resulting cells are subsequently pooled to create a packaging cell library. In some embodiments, a library of VLPs can be produced using, e.g., methods as described by Dobson, C.S., et al., ‘'Antigen identification and high-throughput interaction mapping by reprogramming viral entry .” Nature methods vol. 19,4 (2022): 449-460, which is incorporated herein by reference Dobson ct al. describes not only a modular VSVG viral pscudotyping approach, but also a packaging system that maintains a protein / barcode linkage. This system, referred to as LeAPS, or the “lentivirus activated by promoter shuffling” approach. LeAPS permits generation of lentiviruses from previously transduced cells upon the re-introduction of helper plasmids by placing a strong promoter 5’ of the integrated viral genome - this is shown schematically in Figure 3 of the Dobson publication - and permits the production of a single library member per packaging cell. Uris approach can be adapted to pennit generation of a library of different VLPs while maintaining linkage of the nucleic acid barcode with the corresponding viral envelope protein.Capsid Polypeptides

[0139] In some embodiments, a VLP can include an enveloped virus capsid potypeptide or a capsid comprised of enveloped virus capsid polypeptide molecules. Capsid polypeptides can be selected from those comprised by any of the enveloped viruses known in the art or described herein, including but not limited to capsid polypeptides derived from lentiviruses or gammaretrovirus. The capsid or capsid polypeptide can comprise sequence sufficient to pennit self-assembly of tire capsid and nucleic acid cargo.

[0140] In certain embodiments, VLPs can be used to deliver RNA encoding gene editing machinery, e.g., RNA guided endonucleases, guide RNAs and base-editing enzymes. Non-limiting examples of such gene editing machinery include zinc-finger nucleases, TALENs, and CRISPR or other RNA-guidedendonucleases, optionally with guide RNAs or constructs encoding guide RNAs. Other examples of gene editing machinery, include base editing enzymes, e.g., cytosine base editors or adenine base editors. Examples of such base editing systems include those described in Cohn K.W. Lim ct al., Treatment of a Mouse Model of ALS by In Vivo Base Editing, Mol Ther. 2020 Jan 14. pii: S1525-0016(20)30011-3. doi: 10. 1016 / j.ymthe.2020.01.005: and Jonathan M. Levy et al., Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses, Nature Biomedical Engineering volume 4, pages97-l 10(2020), which are incorporated by reference herein in their entireties. Examples of base editing systems include those described in International Patent Publication Nos. WO 2019 / 071048 (c.g. paragraphs

[0933] -

[0938] ), WO 2019 / 084063 (c.g., paragraphs

[0173] -

[0186] ,

[0323] -

[0475] ,

[0893] -

[1094] ), WO 2019 / 126716 (e.g., paragraphs

[0290] -

[0425] ,

[1077] -

[1084] ), WO 2019 / 126709 (e.g., paragraphs

[0294] -

[0453] ), WO 2019 / 126762 (e.g., paragraphs

[0309] -

[0438] ), WO 2019 / 126774 (e.g., paragraphs

[0511] -

[0670] ), Cox DBT, et al., RNA editing with CRISPR-Casl3, Science. 2017 Nov 24:358(6366): 1019-1027; Abudayyeh 00, et al., A cytosine deaminase for programmable single-base RNA editing, Science 26 Jul 2019: Vol. 365, Issue 6451, pp. 382-386; Gaudclli NM ct al., Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage, Nature volume 551, pages 464-471 (23 November 2017); Komor AC, et al.. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016 May19;533(7603):420-4; Jordan L. Doman et al., Evaluation and minimization of Cas9- independent off- target DNA editing by cytosine base editors, Nat Biotechnol (2020). doi.org / 10.1038 / s41587-020-0414-6; and Richter MF et al., Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity, Nat Biotechnol (2020). doi.org / 10. 1038 / s41587-020-0453-z, which are incorporated by reference herein.

[0141] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A plurality of vims-like particles (VLPs), wherein each vims-like particle (VLP) comprises a) a membrane pseudotyped with a heterologous viral envelope protein; and b) a unique identifying barcode nucleic acid, wherein each unique barcode is paired with the corresponding heterologous envelope protein.2. The plurality of VLPs of paragraph 1, wherein the barcode nucleic acid is linked to nucleic acid sequence encoding the heterologous envelope protein.3. The plurality of paragraph 1, wherein members of the plurality of VLPs each comprise distinct heterologous viral envelope proteins.4. The plurality of paragraph 3, wherein the distinct heterologous viral envelope proteins are derived from naturally-occurring viruses.5. The plurality of paragraph 3, wherein the distinct heterologous viral envelope proteins comprise envelope proteins derived from human endogenous retroviruses or human commensal viruses.6. The plurality of any one of paragraphs 1-3. wherein the distinct heterologous viral envelope proteins comprise envelope proteins from a plurality of proteins listed in Table 1.7. The plurality of paragraph 6, wherein the distinct viral envelope proteins comprise envelope proteins from at least 1, at least 5, at least 96, at least 500 or at least 766 of the proteins listed in Table 1.8. The plurality of paragraph 1, wherein distinct heterologous viral envelope proteins correspond to a single unique identifying barcode nucleic acid.9. The plurality of any one of paragraphs 1-8. wherein the heterologous envelope proteins each comprise a heterologous tag.10. Tire plurality of paragraph 9, wherein the heterologous tag is an HA tag.11. Hie plurality of any one of paragraphs 1-10, wherein members of the plurality of VLPs each comprise a lentiviral capsid.12. A library comprising a plurality of VLPs, each VLP comprising a) a membrane pseudotyped with a heterologous viral envelope protein; and b) a unique identifying barcode nucleic acid, wherein each unique barcode is paired with the corresponding heterologous envelope proteins.13. Hie library of VLPs of paragraph 12, wherein the barcode nucleic acid is linked to nucleic acid sequence encoding the heterologous envelope protein.14. The library of VLPs of paragraph 13, wherein different members of the library comprise distinct heterologous viral envelope proteins.15. The library of VLPs of paragraph 12 or paragraph 13, wherein the distinct heterologous viral envelope proteins are derived from naturally-occurring viruses.16. Hie library of VLPs of any one of paragraphs 12-15, wherein the distinct heterologous envelope proteins comprise envelope proteins derived from human endogenous retroviruses or human commensal viruses.17. The library of VLPs of any one of paragraphs 12-16, wherein the distinct viral envelope proteins comprise envelope proteins from a plurality of proteins listed in Table 1.18. Hie library of VLPs of paragraph 17, wherein the distinct viral envelope proteins comprise envelope proteins from at least 1, at least 5, at least 96, at least 500 or at least 766 of the proteins listed inTable 1.19. The library of VLPs of any one of paragraphs 12-18, wherein the heterologous envelope proteins comprise a heterologous tag.20. Hie library’ of VLPs of paragraph 19, wherein the heterologous tag is an HA tag.21. The library of VLPs of any one of paragraphs 12-20, wherein the members of the library each comprise a lentiviral capsid.22. A population of cells comprising the plurality of any of paragraphs 1-11, or the library of any one of paragraphs 12-21.23. A method of determining individual tropism of a plurality of heterologous envelope proteins, the method comprising: introducing tire plurality of any of paragraph 1-11 or tire library of any one of claims 12-21 into a population of cells such that the VLPs are expressed; and determining expression of the unique identifying barcode nucleic acids expressed in the cells, wherein the location of each unique identifying barcode detected is an indication of the tropism of a corresponding heterologous envelope protein.24. Hie method of paragraph 23, wherein introducing is in vitro or in vivo.25. The method of paragraph 23, further comprising isolating mRNA from cells prior to determining the expression the unique identifying barcodes expressed in tire cells.26. A method of determining tropism of a viral envelope protein, the method comprising: preparing a VLP comprising a) a membrane pseudotyped with the viral envelope protein; and b) a unique identifying barcode, wherein each unique barcode is paired with the corresponding heterologous envelope proteins. introducing the prepared VLP in a population of cells such that the VLP is expressed: and determining expression of the unique identifying barcodes expressed in the cells, wherein the location of each unique identifying barcode detected is an indication of the tropism of the corresponding heterologous envelope protein.27. Tire method of paragraph 26, wherein tire VLP further comprises a lentiviral capsid.28. The method of paragraph 26, wherein introducing is in vitro or in vivo.29. The method of paragraph 26, further comprising isolating mRNA from cells prior to determining the expression the unique identifying barcodes expressed in the cells.30. A method of identifying viral envelope proteins effective for pseudotyping a viral particle membrane, the method comprising generating a library’ of constructs encoding viral envelope pseudotype candidate proteins with a selectable tag peptide, producing a population of VLPs comprising theenvelope pseudotype candidate proteins, and assessing pseudotype efficiency by detecting the selectable tag peptide.31. A nucleic acid molecule comprising a 7SL RNA fused with mRNA encoding an RNA-guidcd endonuclease or base-editing enzyme.32. A nucleic acid molecule comprising a 7SL RNA fused with a guide RNA for an RNA-guided endonuclease or base-editing enzyme.33. The nucleic acid molecule of paragraph 31 or 32, wherein the RNA-guided endonuclease is selected from Cas9, Cpfl, C2cl, C2c3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, and Casl3c. Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslOO, Csyl, Csy2, Csy3, Csel, Cse2. Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl. Csb2, Csb3, Csxl7, Csxl4. CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, C2cl, C2c3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, and Casl 3c.34. Hie nucleic acid molecule of paragraph 31 or 32, wherein the base-editing enzyme is selected from a nickase, a cytidine deaminase, an adenosine deaminase, ABE-SpRY, CBE-SpRY, ABE8c, ABE8e-SpRY, ABE8e-NRCH, NG-ABE8e, ABE-NRTH, AB Emax, BE1 , BE2, BE3, HF-BE3, BE4, BE4max, BE4-GAM, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR-BE3, VRER-BE3, Sa-BE3, SA-BE4, SaBE4-Gam, SaKKH-BE3, Casl2a-BE, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TadA*-dCas9, TadA-TadA*-Cas9, ABE7.9, ABE 6,3, and ABE7.10.35. A VLP comprising a nucleic acid molecule of any one of paragraphs 31-34.36. A VLP comprising a nucleic acid of paragraph 32 and an mRNA encoding an RNA-guided endonuclease that recognizes the guide RNA comprised by the nucleic acid of paragraph 3237. A VLP comprising a nucleic acid of claim 31 and a nucleic acid of paragraph 32, wherein the RNA- guided endonuclease or base-editing enzyme is guided by the guide RNA.

[0142] Hie technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1: Methods for Screening for Next Generation RNA Delivery Vectors

[0143] Delivering nucleic acids encoding therapeutic gene editing agents to specific cell types in the body is a grand challenge in biomedical research. AAV gene therapy has shown great success for some applications, but it has limitations for delivering Cas9 or other gene editing agents. For one. it would beadvantageous to express Cas9 transiently, and genes delivered by AAV are expressed for a long time. Expressing Cas9 for a long time increases the probability of off-target cuts. Additionally, the packaging limit of AAV imposes several constraints when it comes to delivering large proteins such as Cas9. It is possible to deliver mRNA encoding Cas9 in lipid nanoparticles (LNPs), but LNPs generally go to the liver. Even if LNPs can avoid the liver, they are ineffective at endosomal escape due to a lack of strong fusion mechanisms, unlike enveloped viruses, which have fusogenic envelope proteins. Hie inventors took advantage of viruses’ natural ability to efficiently infect a wide array of different organs in humans and extend the reach of gene editing beyond the liver.

[0144] Hie inventors developed a system to search viruses with envelope proteins that mediate tropism to specific organs. As this tropism will vary for different viruses, this natural capability of viruses to enter specific cell types was hijacked. Furthermore, since viruses have evolved for millions of years to infect humans and other animals, they have acquired intricate mechanisms for avoiding liver uptake that present a unique advantage for therapeutic delivery relative to synthetic nanoparticles. This system can find or assist in the development of new viral vectors that will efficiently deliver RNA to specific organs outside of the liver.

[0145] To facilitate this search, the inventors developed a pooled screening system of viral particles carrying different envelope proteins from different viruses. Using pseudotyping of lentivirus with different envelope proteins, each type of particle additionally carries a nucleic acid barcode that can be read out with high-throughput sequencing. This system can be used to perform multiplexed in vivo mouse screens to find viral envelopes that mediate delivery to organs outside of the liver. This system was used to find novel envelope proteins to develop non-replicating virus-like particles (VLPs) for targeted Cas9 mRNA delivery to a variety of organs. These targeted viral particles allow for the correction of genetic diseases currently inaccessible to gene editing, expanding the reach of nucleic acid therapeutics and genetic medicine.

[0146] Among the novel developments are: (1) methods for screening diverse envelope proteins on VLPs; (2) methods for high-throughput purification of VLPs and screening of pseudotyping; (3) methods for improvement of pseudotyping; (4) methods for multiplexed screening; (5) methods for packaging of guide RNAs into VLPs; and (6) improved machine learning models for envelope proteins.

[0147] The future of human medicine will require efficient, reliable, and safe methods to deliver macromolecular cargoes such as genes or gene editing tools to specific cells in vivo (1). With decades of basic biological discovery and clinical research, the understanding of the cell and molecular targets that should be modified for therapeutic treatments has increased. However, only a small fraction of targets are accessible to small molecules (which require deep binding pockets) or protein therapeutics (which canonly act extracellularly) (2). Nucleic acid therapeutics encoding precise gene editing systems could provide cures for a wide variety of diseases as it has tire potential to address both loss-of-function and gain-of-function mutations. Moreover, pennanent genetic alteration can result in therapeutic effects that could manifest indefinitely (1). Yet despite an increase in knowledge about interventions, the capacity to intervene in practice remains extremely limited. Hie inefficiency and lack of specificity of in vivo macromolecule delivery presents the single largest barrier to the clinical success of genetic therapies (3).

[0148] At present, efficient in vivo gene transfer, and thus the potential for gene editing, is technically effective only by lipid nanoparticles (LNPs) and adeno-associated virus (AAV). However, systemically administered LNPs can only target the liver (3, 4) and AAVs have several disadvantages for gene editing. First, AAV has a packaging limit of 4kb, smaller than what is required for many gene editing systems. Second, AAV expression lasts a long time (years), which increases the probability of off- target effects. Third, the immune system presents a significant challenge to the use of AAV, both in terms of pre-existing, neutralizing antibodies and innate immune response (3).

[0149] Hie inventors tackled the problem of targeted in vivo somatic gene therapy by mining biological diversity for novel viral particles with improved properties relative to AAV. Specifically, viruslike particles (VLPs) bearing envelope proteins that naturally evade the liver and target specific cell types were developed. Furthermore, a multiplexed screen to find envelope proteins with favorable tropism and other important characteristics for in vivo delivery was developed. These envelope proteins are used to develop RNA-carrying VLPs (5) for therapeutic delivery of gene editing cargo to specific cell types. The vectors can serve as useful research tools and can also enable modular delivery of gene editing therapeutics for potential treatment of several currently un-addressable diseases.

[0150] The invention is based on the notion that viruses have naturally evolved to target various cells in the body through proteins on their surface that enable them to achieve tremendous specificity through the interaction with proteins on the cell surface (6). As different cell types have unique cell surface proteins, viruses can achieve extraordinary specificity. Viruses as vectors thus have a significant advantage in their likelihood to achieve cell-type specificity over lipid nanoparticles with different lipid compositions, which are unlikely to efficiently evade the liver and have true specificity for different surface proteins or cell types. Most discovery efforts thus far have focused on one of the natural serotypes of AAV or engineering variants. Although AAV vectors have greatly pushed forw ard the field of gene therapy, they have several limitations, particularly for gene editing (1). Furthermore, given enormous diversity of the viruses that exist across the virome, there are many more unexplored viruses that would yield great vectors for in vivo delivery that have not been explored. A novel framework for screening thevirome to find such viruses and turn them into safe, cell type-specific therapeutic RNA shuttles was developed.Example 2: Method for screening diverse envelope proteins on VLPs

[0151] To find novel delivery vectors, the inventors developed a multiplexed screening platform to test different enveloped viruses in the same experiment. Broadly, enveloped viruses have four components - the genome, the capsid / matrix (a shell of proteins which packages the genome), a lipid membrane surrounding the capsid, and the envelope proteins (proteins which are embedded in the lipid membrane and help the viral particle transduce cells). Since different viruses have very’ different genomes and capsids, one challenge that needs to be overcome for this to work is a screening platform that can easily make different viruses using the same modular system. Such a system must produce different viral particles (with different envelope proteins) while maintaining genotype -phenotype linkage through DNA or RNA barcodes that correspond to the envelope protein. For this goal, the inventors took advantage of pseudotyping (7, 8), the process of putting envelope proteins from one virus onto membrane particles containing the capsid of another virus (such as HIV -derived lentivirus) (Figure 1). Uris allowed for tire creation of pseudotyped lentivirus particles where each envelope protein (derived from completely different viruses) is tied to a unique nucleic acid barcode packaged into the lentiviral capsid inside of the particles. A library' of these different viral particles (each carrying a different barcode indicating which envelope protein it carries) were then injected in pooled format into a mouse, followed by' organ dissection and high-throughput sequencing of the barcodes. Comparing barcode frequency in different organs / cell types enabled figuring out which viral particles can effectively escape the liver and get into other cell types (Figure 2). With tire individual candidate viral envelopes, virus like particles (VLPs) that contain the envelope proteins can be constructed, but instead of barcodes, reporter genes or other mRNAs of interest can be packaged (ex. Cas9) (5).

[0152] The inventors’ primary goal was to transform the discovery' and optimization of nextgeneration RNA shuttles through multiplexed in vivo screening of envelope proteins. Multiplexed screening enabled tire testing of the whole library’ that was constructed in a pooled format, where all library members (in this case, envelope proteins) were tested together inside one animal. To enable this, the experiment was designed or tested such that the readout of the property of interest was reducible to a relative frequency of nucleic acid barcodes where each barcode corresponded to a particular envelope protein in the pool.

[0153] Hie specific screening platform used was lentiviral virus-like particles (VLPs). Lentivirus derives from HIV-1 originally but has been modified to be non-replicating (9). In 3rdgeneration lentivirussystems, there are four plasmids: one packaging plasmid which encodes the lentiviral capsid (gag) and packaging proteins (pro and pol), another packaging plasmid encoding the Rev regulatory protein, the transfer plasmid, which contains the gene of interest surrounded by the lentiviral packaging signals or LTRs, and the envelope protein plasmid (often VSV-G). By ensuring the VLP only packages the gene of interest and no viral components, one can prevent vector replication and ensure safe experimentation (10). 3rdgeneration lentivirus system are considerably safer than 2ndgeneration lentivirus system due to the use of four plasmids instead of three (9).

[0154] For the screens described herein, the plasmid encoding the envelope protein was varied to test different envelope proteins (10). For each plasmid encoding tire envelope protein, a corresponding transfer vector containing a nucleic acid barcode was designed (Figure 3). An important prerequisite to discovering promising candidate envelope proteins in the screens as described was to select the correct ones to screen, especially given that screening has a finite throughput. The first test screen used a relatively small library of -100 members so that genes encoding library members could be ordered in a 96-well plate. Genes encoding these envelope proteins were synthesized in 96 well plate format and cloned them into the plasmid encoding the envelope protein, using a highly modular and efficient cloning strategy that the inventors designed.

[0155] This first library contained envelope proteins that had been previously used to pseudotype lentivirus (7, 8), including members such as VSV-G, three of its vesiculovirus envelope protein relatives (11), MLV (12), and Sindbis 2.2 1L1L (13). Additionally, all envelope proteins which have been previously used with lentivirus (7, 8, 10, 14), which is less than one hundred proteins, were synthesized. Ulis library includes Nipah virus envelope proteins, as well as other paramyxoviral envelope proteins, as there have been studies reporting that viral particles with these envelope proteins are capable of in vivo liver evasion in mice (15, 16). This library also includes HERV-K as it is the youngest endogenous retroviral protein - it is native to humans and therefore may have interesting immune-evasion properties (17, 18).

[0156] Hie first 100-member library included the following categories of envelope proteins:1. Envelope proteins previously used in pseudotyping studies2. Envelope proteins from human endogenous retroviruses which may have favorable immune evasion properties3. Envelope proteins from viruses that are known to infect mammals4. Envelope proteins from virus families such as VSV that have high titers and are known to very effective at transducing mammalian cells5. Envelope proteins from paramyxoviral families such as Nipah that have been reported to have interesting tropism and liver-evasion characteristics6. Envelope proteins from commensal human viruses

[0157] For categories #2 and #6, raw NGS RNA-Seq data were analyzed from human tissue atlas databases such as GTEx (19, 20) to find reads aligning to envelope proteins that may infect specific human tissues. Additionally, sequencing of viral particles isolated from human plasma was performed to find potential circulating human commensal viruses. By sequencing the RNA inside these vesicles and mapping it to existing envelope proteins, existing circulating commensal viruses whose envelope proteins may have favorable immune-evasion properties can be identified.

[0158] Computationally designed oligonucleotides can also be used to broadly enrich for envelope protein-like sequences and to increase the likelihood of finding rare circulating viral species. The inventors cast a wide net to identify specific classes or subfamilies of viral envelopes which have interesting properties for tropism and delivery. For any of these envelope proteins, variant libraries to mutagenize a given envelope protein can also be made. By generating and testing these large libraries that leverage recent improvements in next-generation DNA synthesis, the best envelope candidate can be significantly improved, as shown by other similar screens with AAV (21-24).Example 3: Method for high-throughput purification of VLPs and screening of pseudotyping

[0159] The inventors overcame several technical challenges to perform multiplexed experiments with a library of barcoded VLPs: production of a well-balanced library at the necessary scale, with minimal bias, diverse representation, and strong genotype -phenotype linkage. To have a well-controlled, balanced library, all the VLPs generated for screening had a lentiviral chassis, and tire only variable is the envelope protein. The added benefit is that the lentiviral production system has already been well- characterized and optimized (14). Generally, lend virus is produced by a transient transfection of tire four 3rdgeneration plasmids described in Figure 3. Transient transfection of plasmids at a low enough concentration to prevent barcode swapping results in too few VLPs to be meaningfully screened. Therefore, in order to produce barcoded VLPs in multiplex, a recently described two-step production process (25) to produce high-concentration barcoded VLP libraries was adopted. This approach prevents the expression of more than one library member per transfected packaging cell, which otherwise would disrupt genotype -phenotype linkage. By including an extra promoter in one of the plasmids, this system, termed Lentivirus Activated by Promoter Shuffling (LeAPS), allows high-titer viral particle production of pooled libraries in one batch of producer cells (25).

[0160] Another challenge to this approach is that not all envelope proteins pseudotype well onto lentiviral particles (26). As previously described, the process of producing viral particles with a capsid that derives from one type of vims but has envelope proteins from a different vims is called pseudotyping- the average number of a given type of envelope proteins on a VLP can be drought of as its pseudotyping efficiency. Efficient pseudotyping has been previously reported as a key obstacle for lentiviral engineering (10). This is because pseudotyping is not guaranteed to succeed, given that certain envelope proteins are not compatible with certain capsids. For example, many paramyxovirus glycoproteins cannot pseudotype onto lentivims due to their long cytoplasmic tails, which sterically interfere with the Gag capsid protein (27). Conversely, VSV-G and closely-related envelope proteins pseudoty pe extraordinarily well, likely partially due to tlieir short cy toplasmic tails (10). Additionally, HEK293T, which are commonly used, are not tire native host for the vims of origin of many of these envelope proteins, so some proteins may not be translated or processed correctly. Therefore, it is important both to identify which envelope proteins can pseudotype before screening the library, and to develop strategies for ameliorating the pseudoty ping efficiency of problematic envelope proteins.

[0161] To test which proteins pscudotypc efficiently, hemagglutinin (HA) tags were inserted into the cytoplasmic domain of all envelope proteins, and then HA tag incorporation onto viral particles was tested after viral particle purification from the cell supernatant. For this purpose, a novel high-throughput viral purification technique based on “In-Slurry” binding of free proteins in the media into the interior of beads (Capto Core 700) used for multimodal chromatography (28) that removed free proteins from viral supernatant was employed. Importantly, this technique can be performed in 96 well plate format to allow the parallel screening of hundreds of viral preparations in hours. This “In-Slurry” method was further optimized to tune the purity of tire viral preparations by changing the ratio of resin to supernatant. Preliminary results showed that purity of viral preparations was higher than that of centrifugation with a sucrose cushion (measured by total protein stain) and led to higher transduction efficiency (measured by flow cytometry) (Figure 4). These purified viruses were assessed for pseudotyping efficiency by Western blotting using antibodies against the HA -tag or other protein detection methods.

[0162] To assess pseudotyping, the following protocol was used: Mixed mode chromatography resin (MMR) slurry was prepared by taking Capto Core 700 resin (Cytiva) and centrifuging the resin at 800xg for 5 minutes, washing 3 times with PBS in a 50 mL falcon tube, and resuspending in a volume equal to the resin volume of PBS to produce a 50% slurry. For VLP isolation, a volume of MMR slurry corresponding to the volume of viral supernatant. The samples were mixed end over end for 45 minutes at room temperature, and then centrifuged at 800xg for 10 min. Finally, the supernatant is transferred and centrifuged in Coming CoStar X 0.45 pm filters at 2000xg for 10 minutes to separate tire CSF from theCapto Core beads. 4X LDS was added to the samples and then Western blotting was performed using an antibody against HA.Example 4: Methods for improvement of pseudotyping

[0163] After confirming that an envelope protein from a 96-well plate efficiently pseudotyped lentiviral particles, that envelope protein was added to the library. For a candidate envelope protein that does not pseudotype well, there are several strategies to improve pseudotyping. One such strategy, as also shown by previous reports, is truncation of the cytoplasmic tail, or replacement with that ofVSV-G, an envelope protein known to pscudotypc extremely well. Uris strategy was used to greatly improve pseudotyping of MLV envelope protein (Figure 5). The total envelope protein expression in cell lysate is highly different between VSV-G and some envelope proteins that do not pseudoty pe well, which indicates that steric hindrance or other forms of cytoplasmic tail incompatibility are only part of the story. Various strategies were investigated to improve overall expression of problematic envelope proteins by replacing and mutating the signal peptide as well as other domains of the envelope protein for a few candidate proteins that display ed poor pscudotyping.

[0164] After validating successful pseudotyping of candidate envelope proteins (whether in their original form or through the described modifications), those individual members were assembled into the library'. Using library members that were shown to pseudotype well, minimized the number of library members likely to be non-functional. These library members were assembled into the previously mentioned LeAPS system (25) to allow for the pooled production of the library' in a single batch of producer cells. Using a single batch of cells greatly improves the scale of the library production relative to isolating each different type of VLP individually.Example 5: Methods for Multiplexed Screening

[0165] The inventors screened the enveloped vims libraries to find variants that were capable of transducing specific populations of cells outside tire liver when administered intravenously. As stated in previous sections, tire liver is a major sink for most delivery vectors, which severely limits the viability of RNA therapeutics for most diseases. Thus, the main pooled screen involved different envelope proteins in vivo and detennining where each of these unique VLPs (carrying a different barcode) traveled in a mouse. By using NGS to read out the barcode, a quantitative read out of barcodes in different organs was obtained.

[0166] As a proof-of-principle, a small VLP library of five envelope proteins each carrying a unique barcode was constructed: VSV-G, Cocal G, Piry G, Maraba G, and Sindbis 2.2 1L1L. The VLPs were allproduced individually, carry ing a GFP expression cassette and computationally selected barcodes with universal flanking sequences in their RNA genome. If a VLP successfully transduces a cell, the Gag protein reverse transcribes its RNA genome and integrates it into the transduced cell genome, thereby enabling PCR amplification of the barcode from the genomic DNA. If a VLP does not transduce a cell, its barcode sequence remains as RNA and is not amplified. As such, isolation of the genomic DNA from cells transduced by a barcoded VLP pool, followed by PCR amplification of the barcode, yields a barcode pool where enriched barcodes correspond to VLPs that were particularly effective at transduction, and vice versa.

[0167] Three different immortalized cell types were transduced (human embryonic kidney HEK293T, liver carcinoma HepG2, and lung adenocarcinoma A549 cells) in vitro, with the different VLPs. either individually or all pooled together (Figure 6). Next, flow cytometry was performed to measure the infectivity of the VLPs individually and the results were compared to those of the NGS-based measurement of the pooled transduction. The relative infectivity of VSV-G and Sindbis were similar but the 3 relatives of VSV-G (Cocal G, Maraba G, and Pin G) had no measured infectivity in tire pooled fomiat, even though they perfonned comparably to VSV-G when used individually. It was also verified that a no-envelope control did not transduce cells, and its barcode was not observed in the pooled format. The disparity in the results is likely because VSV-G was highly abundant in the supernatant and may form an interference group with its relatives. Overall, from this experiment, the barcoding scheme was verified to work in vitro to measure pooled transduction efficiencies. Numerous studies have shown that in vitro vector tropism is not correlated with in vivo vector tropism - cells behave differently, and the biological barriers and fluidic conditions in vivo are difficult to mimic. Vectors which appear to function efficiently and specifically in vitro often do net work in vivo (3). Therefore, screening the libraries directly in vivo is the best way to discover workable vectors, and this multiplexed measurement technology enabled this to be done tractably at laboratory scale without using thousands of mice. The overall experimental scheme is the same as in vitro: a barcoded pool of VLPs was produced, this pool was injected into a mouse via tail vein, after~5-15 days, the mouse was euthanized and dissected, genomic DNA was extracted from each cell type / organ, and NGS was perfonned to measure barcode frequencies. Mice were surgically dissected into the following organs: liver, spleen, brain, heart, lung, gut, kidney, and muscle. Additionally, blood was drawn and separated by FACS into B cells, T cell, dendritic cells, macrophages, and hematopoietic stem cells (HSC). Once there is determination of hits in certain organs of interest, repeat iterations of the screen can be performed to look at which cell types within the organ were transduced.

[0168] One of the advantages of this pooled approach is that different VLPs are directly compared to each other in the same mouse. Additionally, built-in controls were also incorporated to compare tire VLPs to other vectors as well. Specifically, several AAV (such as AAV2, AAV8, and AAV9) and LNP formulations were able to be screened in the same in vivo experiment. By designing the same barcodes into the cargo of AAV (produced separately than the VLP library), the vectors were able to be directly compared to the gold standard for gene therapy and compared in the same mouse. Similarly, the DNA barcodes were encapsulated in some of the best reported LNPs. By using the same DNA barcodes as a readout, not only are there measurements comparing different envelope proteins against each other but there arc also measurements directly comparing different delivery modalities.

[0169] For the assembled library of different VLPs together with spiked-in AAV and LNP controls, there was a wide variety of systems that could be used to screen the library. Although the primary system used is in vivo mouse injection to study tropism, human systems for screening of the same library can also be used. In particular, the library' can be screened using human organ-on-chip systems (29, 30). As it has been shown that different organ on chip systems can be coupled together (31, 32), and some of these systems arc now commercially available, coupling of a liver organ-on-chip with a lung organ-on-chip can be used to see which VLPs enrich in human lung cells relative to liver cells. Using these strategies to screen large libraries of VLPs and other delivery vectors in a multiplexed format both in vivo and in vitro maximizes the chances of finding novel envelope proteins that can direct VLP tropism to organs of interest outside of the liver.

[0170] It is non-trivial to perform barcode measurements in vivo, due to the massive amount of background genomic DNA which make a simple DNA extraction and PCR intractable. The problem of having a very-large genomic DNA to barcode ratio can be overcome by designing restriction sites around the barcode. Smaller DNA barcodes could be separated using restriction enzymes away from the large genomic DNA with a size selection step prior to PCR. Nonetheless, this will determine the sensitivity in terms of how many barcodes can be detected per organ. An alternative approach would be to have the VLPs deliver a GFP reporter in addition to the barcode. Then, GFP-positive cells could be sorted out from the organs prior to barcode read-out. The trade-off with using this approach is that it becomes less scalable - organs must first be dissociated, and GFP-positive cells must be sorted.

[0171] In another experiment to demonstrate the platform, a multiplexed experiment with three VLPs: VSV-G, Nipah, and Blank (No envelope control) was performed. These VLPs (at equal ratios) were injected IV into mice (n=3), and after 7 days, sacrificed the mice, removed three organs (liver, spleen, and lung), extracted DNA, and sequenced the barcodes. Hie sequencing read out of thesebarcodes was compared to VLPs with VSV-G or Nipah envelopes carrying a Luciferase reporter that were injected individually into mice (Figs 11-12).Example 6: Methods of packaging of guide RNAs into VLPs

[0172] To create true next-generation RNA shuttles, the inventors also developed improved methods for packaging RNA into VLPs. Several groups have demonstrated mRNA packaging of cargo into lentivirus through modifications to the lentiviral system that remove tire integrase and reverse- transcriptasc functions of Gag, while fusing additional RNA-binding domains (38-43). However, these systems still contain Gag protein, which can elicit adverse effects when dosed in large quantities in vivo. Synthetic RNA binding domains can instead be fused to the internal domain of envelope proteins, including MS2, and thereby remove Gag from the system entirely. Even if one is using Gag, CRISPR systems require small, non-coding RNAs such as guide RNAs, and it is currently not possible to package both these RNAs and mRNA for the CRISPR protein into a VLP, even using Gag.

[0173] To increase the utility of the VLPs, the inventors developed new strategics to package several gRNAs per VLP. One strategy that was developed is to fuse the gRNA or pegRNA to the 7SL small noncoding RNA. This RNA has been shown to noncompetitively complex with the Gag protein and is found at high levels inside HIV particles. By exploring different methods of fusing this domain with guide RNAs and different domain architectures, transient guide RNA delivery via Gag into cells was optimized in order to achieve maximum in vivo editing efficiency with minimal off-target effects. Figure 7 shows qPCR data from these produced viral particles, where one of the modified constructs (Construct 1) results in the guide RNA being packaged as well as the wild-type 7SL RNA.

[0174] The inventors have implemented different methods for packaging cargo into VLPs. Delivery of mRNA into cells in vivo presents a challenge as wild-type VLPs deliver integrating DNA. Delivering mRNA can allow for temporally tunable expression of gene editors to minimize off-target effects. Below are descriptions of mRNA packaging methods that have been implemented.

[0175] 1. A chimeric Gag-Pol-X protein, where X is an RNA binding domain, and cargo mRNA with the cognate motif for X. X is attached by a flexible linker with an optimized HIV protease cleavage site. This results in packaging of capped mRNA into the VLP.

[0176] 2. RT-deficient Gag-pol and cargo mRNA with Psi, LTR, and Gag fragment motifs to achieve packaging of capped mRNA.

[0177] 3. A chimeric EV-sorting transmembrane protein such as CD63, TSPAN101, CD9, etc. with an RNA binding domain X, and an mRNA with tire cognate motif for X.

[0178] Any of the above methods can be additionally combined with the expression of mRNA stabilizing proteins in the producer cells to achieve greater efficiency.

[0179] Another challenge for gene editing is delivering a gene editor in a transient form while also delivering a donor DNA for the desired edit. Many large-scale gene edits benefit or require the presence of a donor DNA either encoding the edit or an entire gene to be inserted. While there are methods for delivering DNA, and methods for delivering gene editors in a transient form, there are no methods for delivering both in a single vector. Below are methods the inventors have developed to address this issue.

[0180] 1. A chimeric Gag-Pol-X protein where X is a site-specific recombinase (SSR) and HIV Int has been deactivated, and a lentiviral genome encoding the desired therapeutic gene, with one att site for tire SSR. X is attached by a flexible linker with an optimized HIV protease cleavage site. The other attachment site for the recombinase can be present in the target cell genome at the desired site. The VLP can enter a target cell, carrying the site-specific recombinase in protein form. Then the Pol reverse- transcribes the genome and the SSR integrates the gene using the att site.

[0181] 2. Same as 1, but the lentiviral genome carries another orthogonal att pair, with one att site at each end. Tire VLP enters a target cell, carrying the site-specific recombinase in protein form. Then it reverse-transcribes its genome and the SSR first circularizes the genome with one att pair and then integrates the gene into the genome using the first att site.

[0182] 3. A chimeric Gag-Pol-X protein where X is a programmable nuclease and the integrase has been deactivated, and a lentiviral genome encoding a therapeutic gene and guide RNAs. X is attached by a flexible linker with an optimized HIV protease cleavage site The VLP enters a target cell, earn ing the editor in protein fonu. This editor uses the guide RNAs to make a break or nick at a specific location in the genome, and the Pol reverse transcribes the genome to provide a DNA donor for homologous recombination at the nicked / cut site.

[0183] 4. A chimeric Gag-Pol-X protein where X is a site-specific recombinase and HIV Int has been deactivated, a chimeric Gag-Y protein where Y is a programmable DNA editorAvriter, and a lentiviral plasmid encoding the desired therapeutic gene and a guide RNA, with one att site for the recombinase and another orthogonal att pair at each end. X and Y are attached by a flexible linker with an optimized HIV protease cleavage site. Hie VLP enters a target cell, carrying the SSR and programmable editor in protein form. The programmable editor uses the guide RNA to create an att site in the genome at a desired location. Then Pol reverse-transcribes its genome and the SSR first circularizes the genome with one att pair and then integrates the gene into the genome using the first att site.Example 7: In vivo screen

[0184] Figure 13 shows the validation of in vivo NGS-based sequencing platform. 4 preparations of virus-likc particles (VLPs) were mixed together and injected into 4 mice with an exponentially decreasing dose from mouse 1 to mouse 4. Each VLP preparation carried a different nucleic acid barcode. Two of them displayed a functional fusogenic envelope (VSV-G). while two were made without any envelope protein. After injection, the mice were sacrificed and dissected to remove their liver and spleen. The DNA was extracted, and the barcode sequences were enriched, amplified by PCR, and then sequenced using NGS. Hie counts of each barcode sequence in each organ represent how efficient a given VLP preparation was at functionally delivering to the cells in that organ.

[0185] The heatmap displays the read counts of each barcode in two separate parts of each organ. Liver 1 represents the liver from the mouse with the highest dose, liver 2 is the liver from the mouse with the 2ndhighest dose, etc. The results show that the readout becomes noisy and reaches a limit of detection at the dose used in mouse 3 which corresponds to only 5e5 transducing units injected. This limit of detection is sufficiently low enough to support a highly multiplexed screen, indicating that the method has high sensitivity. In organs from mouse 1 and 2, the read counts for each of the barcodes are highly correlated, indicating the replicability of the NGS readout. Finally, no read counts are detected for the barcodes from the non-enveloped and non-fusogenic VLPs, indicating that the method has very low nonspecific background signal.

[0186] Figure 14 shows in vivo screen of 96 different barcoded VLPs. 96 different VLP preparations displaying different envelope proteins, and carrying different barcodes, were mixed together and injected into 3 different mice. Using tire same method as in Fig. 13, the barcode counts were measured for each organ and are displayed in the heatmap. Each row represents counts for one organ (e.g. Liver 3 represents the counts for barcodes in the liver of mouse 3). The negative control (NC) organs are organs from mice which received high doses of VLPs carrying different barcodes that were not present in any of tire 96 VLPs used in this experiment. The Un-Enriched organs represent read counts from organs where the barcode sequence was not enriched before PCR. Each column represents one different kind of VLP, carrying a unique kind of envelope protein.

[0187] Several results are demonstrated by this experiment. The first is that the method again has a low non-specific background, demonstrated by the lack of read counts in the NC organs and low read counts for H10-H12 in all organs, which represent non-enveloped VLPs. Secondly, Al -A3 and H7-H9 all represent VLPs displaying VSV-G and closely related envelopes, and their corresponding barcodes are measured primarily in tire liver and spleen with high replicability across mice. This is corroborated by both these experiments and in literature which show that these envelope proteins perfonn similarly toeach other and deliver to mostly the liver and spleen. Finally, there is one envelope protein, Yug Bogdanovac, (represented by G6 / G7) that demonstrates signal in non-liver / spleen organs, which shows the promise of this method for discovering envelope proteins effective for extra-hepatic delivery.Table 2: Virus used for envelope proteins in pooled library of VLPs in in vivo experiment of Figure 14.Example 8: In vitro screen with envelopes showing superior results relative to VSV-G

[0188] Figure 15 shows an arrayed in vitro screen of envelope proteins on HEK293T cells. A screen was performed of 80 different envelopes in vitro on HEK293T. VLPs were prepared displaying these envelope proteins and carrying a gene for GFP, and purified in high-throughput. 3 days after transduction with these envelope proteins, HEK293Ts were transduced with these envelopes. The envelopes listed have never been described for their use previously, and some (Alagoas, Carajas, and Middelburg) show improved transduction compared to VSV-G

[0189] Figure 16 shows in vitro test screen comparing NGS and flow cytometry readouts on human CD34+ HSCs. Five different VLPs displaying different envelope proteins and carrying GFP with different barcodes were prepared and either put onto human CD34+ cells (hematopoietic stem cells) individually, or pooled and put onto CD34+ cells. The cells which received individual VLP preparations were measured by flow cytometry to count the number of GFP+ cells as a measure of transduction efficiency of each envelope protein (x-axis). The cells which received the pooled VLPs w ere lysed and the barcodes w ere purified and sequenced to measure the relative transduction efficiencies in the pool (y- axis). This figure shows that the pooled sequencing measurement recapitulates the individual flow -based measurement as the two measurements are highly correlated, ft also shows that two envelope proteins, derived from RD114 and baboon endogenous virus, perform significantly better than VSV-G.

[0190] Figure 17 shows VLPs that display tw o different envelope proteins, Yug Bogdanovac and VSV-G, indicated as B and V respectively. 100 uL of purified VLPs assayed by anti-HA-tag western blot for the presence of the envelope proteins, which both carried an HA tag demonstrates that V is far more abundant on VLPs than B. Modification of the cytoplasmic tail of B in two different ways (B-l and B-2), to either truncate or replace with VSV-G, results in VLPs displaying V, B-l and B-2. Assaying only 1 uL of VLPs by anti-HA tag Western blot, shows that the two modifications lead to equivalent display and are able to rescue pseudotyping.References:1. Raguram A, Banskota S, Liu DR. Therapeutic in vivo delivery of gene editing agents. Cell. 2022 Jul 21;185( 15):2806-27. PubMed PMID: 35798006.2. Verdine GL, Walensky LD. The challenge of drugging undruggable targets in cancer: lessons learned from targeting BCL-2 family members. 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Drug delivery systems for RNA therapeutics. Nature reviews Genetics. 2022 May;23(5):265-80. PubMed PMID: 34983972. PMCID: PMC8724758.T able 1 : Viral envelope proteins for use in pooled library of VLPs . Sequences for the proteins are available publicly.

Claims

CLAIMSWhat is claimed herein is:

1. A plurality of virus-like particles (VLPs), wherein each virus-like particle (VLP) comprises a) a membrane pseudotyped with a heterologous viral envelope protein; and b) a unique identifying barcode nucleic acid, wherein each unique barcode is paired with the corresponding heterologous envelope protein.

2. The plurality of VLPs of claim 1, wherein the barcode nucleic acid is linked to nucleic acid sequence encoding the heterologous envelope protein.

3. The plurality of claim 1, wherein members of the plurality of VLPs each comprise distinct heterologous viral envelope proteins.

4. The plurality of claim 3, wherein the distinct heterologous viral envelope proteins are derived from naturally-occurring viruses.

5. The plurality of claim 3, wherein the distinct heterologous viral envelope proteins comprise envelope proteins derived from human endogenous retroviruses or human commensal viruses.

6. The plurality of any one of claims 1-3, wherein the distinct heterologous viral envelope proteins comprise envelope proteins from a plurality of proteins listed in Table 1.

7. The plurality of claim 6, wherein the distinct viral envelope proteins comprise envelope proteins from at least 1, at least 5, at least 96, at least 500 or at least 766 of the proteins listed in Table 1.

8. The plurality of claim 1, wherein distinct heterologous viral envelope proteins correspond to a single unique identifying barcode nucleic acid.

9. The plurality of any one of claims 1-8, wherein tire heterologous envelope proteins each comprise a heterologous tag.

10. Hie plurality of claim 9, wherein the heterologous tag is an HA tag.

11. Hie plurality of any one of claims 1-10, wherein members of the plurality of VLPs each comprise a lentiviral capsid.

12. A library comprising a plurality of VLPs, each VLP comprising a) a membrane pseudotyped with a heterologous viral envelope protein; and b) a unique identifying barcode nucleic acid, wherein each unique barcode is paired with the corresponding heterologous envelope proteins.

13. Hie library of VLPs of claim 12, wherein the barcode nucleic acid is linked to nucleic acid sequence encoding the heterologous envelope protein.

14. The library of VLPs of claim 13, wherein different members of the library comprise distinct heterologous viral envelope proteins.

15. The library of VLPs of claim 12 or claim 13, wherein the distinct heterologous viral envelope proteins are derived from naturally-occurring viruses.

16. Hie library’ of VLPs of any one of claims 12-15, wherein the distinct heterologous envelope proteins comprise envelope proteins derived from human endogenous retroviruses or human commensal viruses.

17. The library of VLPs of any one of claims 12-16, wherein the distinct viral envelope proteins comprise envelope proteins from a plurality of proteins listed in Table 1.

18. Hie library of VLPs of claim 17, wherein the distinct viral envelope proteins comprise envelope proteins from at least 1, at least 5, at least 96, at least 500 or at least 766 of the proteins listed in Table 1.

19. The library of VLPs of any one of claims 12-18, wherein the heterologous envelope proteins comprise a heterologous tag.

20. The library of VLPs of claim 19, wherein the heterologous tag is an HA tag.

21. Hie library of VLPs of any one of claims 12-20, wherein the members of the library each comprise a lentiviral capsid.

22. A population of cells comprising the plurality of any of claims 1-11, or the library of any one of claims 12-21.

23. A method of determining individual tropism of a plurality of heterologous envelope proteins, the method comprising: introducing the plurality of any of claims 1-11 or the library’ of any one of claims 12-21 into a population of cells such that the VLPs are expressed; and determining expression of the unique identifying barcode nucleic acids expressed in the cells, wherein the location of each unique identifying barcode detected is an indication of the tropism of a corresponding heterologous envelope protein.

24. Hie method of claim 23, wherein introducing is in vitro or in vivo.

25. Hie method of claim 23, further comprising isolating mRNA from cells prior to determining the expression the unique identifying barcodes expressed in the cells.

26. A method of determining tropism of a viral envelope protein, the method comprising: preparing a VLP comprising a) a membrane pseudotyped with the viral envelope protein; and b) a unique identifying barcode, wherein each unique barcode is paired with the corresponding heterologous envelope proteins, introducing the prepared VLP in a population of cells such that the VLP is expressed; anddetermining expression of the unique identifying barcodes expressed in the cells, wherein the location of each unique identifying barcode detected is an indication of the tropism of the corresponding heterologous envelope protein.

27. The method of claim 26, wherein the VLP further comprises a lentiviral capsid.

28. The method of claim 26, wherein introducing is in vitro or in vivo.

29. The method of claim 26, further comprising isolating mRNA from cells prior to determining the expression the unique identifying barcodes expressed in the cells.

30. A method of identifying viral envelope proteins effective for pseudotyping a viral particle membrane, the method comprising generating a library of constructs encoding viral envelope pscudotypc candidate proteins with a selectable tag peptide, producing a population of VLPs comprising the envelope pseudotype candidate proteins, and assessing pseudotype efficiency by detecting the selectable tag peptide.

31. A nucleic acid molecule comprising a 7ST RNA fused with mRNA encoding an RNA-guided endonuclease or base-editing enzyme.

32. A nucleic acid molecule comprising a 7SL RNA fused with a guide RNA for an RNA-guidcd endonuclease or base-editing enzyme.

33. The nucleic acid molecule of claim 31 or 32, wherein the RNA-guided endonuclease is selected fromCas9, Cpfl, C2cl, C2c3, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, and Casl3c. Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, CaslOO, Csyl, Csy2, Csy3, Csef, Cse2, Cscf, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbf, Csb2, Csb3, Csxl7, Csxl4, CsxfO, Csxf6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, C2cf, C2c3, Casl2a, Casf2b, Casf2c, Casf2d, Casf2e, Casl3a, Casf3b. and Cast 3c.

34. The nucleic acid molecule of claim 31 or 32, wherein the base-editing enzyme is selected from a nickase, a cytidine deaminase, an adenosine deaminase, ABE-SpRY, CBE-SpRY, ABE8e, ABE8e-SpRY, ABE8e-NRCH, NG-ABE8e, ABE-NRTH, ABEmax, BE1 , BE2, BE3, HF-BE3, BE4, BE4max, BE4-GAM, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR-BE3, VRER-BE3, Sa-BE3, SA-BE4, SaBE4-Gam, SaKKH-BE3, Casl2a-BE, Target-AfD, Target-AID-NG. xBE3, eA3A-BE3. A3A-BE3, BE-PLUS, TadA*-dCas9, TadA-TadA*-Cas9, ABE7.

9. ABE 6,3. and ABE7.10.

35. A VLP comprising a nucleic acid molecule of any one of claims 31-34.

36. A VLP comprising a nucleic acid of claim 32 and an mRNA encoding an RNA-guided endonuclease that recognizes tire guide RNA comprised by tire nucleic acid of claim 3237. A VLP comprising a nucleic acid of claim 31 and a nucleic acid of claim 32, wherein the RN A guided endonuclease or base-editing enzyme is guided by the guide RNA.

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