Compositions and methods for delivering cargo to target cells

Abstract

Compositions, systems, and methods for delivering a cargo to a target cell are provided.SOLUTION: Provided is a modified delivery system comprising one or more polynucleotides encoding one or more endogenous retroviral elements for forming a delivery vesicle and one or more capture moieties for packaging cargo within the delivery vesicle, wherein the one or more endogenous retroviral elements for forming the delivery vesicle comprise (i) an endogenous retroviral gag homologous protein, wherein the endogenous retroviral gag homologous protein is PNMA1, PNMA4, PNMA5, PNMA6, or RTL1, and the cargo is heterologous to the endogenous retroviral gag homologous protein.SELECTED DRAWING: Figure 18

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 903,127, filed September 20, 2019, and U.S. Provisional Application No. 63 / 003,409, filed April 1, 2020, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Statement on Federally Sponsored Research This invention was made with United States government support under Grant No. HL141201 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing ("BROD-4620WP_ST.25.txt", 4,945 bytes in size, created on September 18, 2020) are incorporated herein by reference in their entirety.

[0004] The subject matter disclosed herein relates generally to modified delivery agents, compositions, systems and uses thereof. [Background technology]

[0005] Delivery systems are an important aspect of therapeutic efficacy. Delivering therapeutic agents to the interior of cells presents many challenges, including but not limited to limiting off-target effects, delivery efficiency, degradation, etc. Viruses and virus-like particles have been used to deliver various cargoes (e.g., gene therapy drugs) to target cells. However, currently used vesicles and particles are large in size and can be difficult to produce in a consistent manner. Therefore, simpler and improved delivery systems are needed. Summary of the Invention

[0006] In certain exemplary embodiments, the present invention provides modified delivery systems comprising one or more polynucleotides that encode one or more endogenous retroviral elements for forming a delivery vesicle and one or more trapping moieties for packaging cargo within the delivery vesicle.

[0007] In some embodiments, the one or more endogenous retroviral elements for forming the delivery vesicle comprise two or more retroviral gag proteins, retroviral envelope proteins, retroviral reverse transcriptase, or a combination thereof.

[0008] In some embodiments, the retroviral gag protein may be endogenous. In some embodiments, the retroviral envelope protein may be endogenous. In some embodiments, both the retroviral gag protein and the retroviral envelope protein are endogenous.

[0009] In some embodiments, the retroviral gag protein comprises an NC domain and an MA domain.

[0010] In some embodiments, the retroviral gag protein is a gag homologous protein. In some embodiments, the gag homologous protein is Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In certain embodiments, the gag homologous protein is PNMA4, PEG10, or RTL1.

[0011] In some embodiments, the envelope protein may be derived from a gammaretrovirus or a deltaretrovirus. In some embodiments, the envelope protein is selected from envH1, envH2, envH3, envK1, envK2_1, envK2_2, envK3, envK4, envK5, envK6, envT, envW, envW1, envfrd, envR(b), envR, envF(c)2, or envF(c)1.

[0012] In some embodiments, the envelope protein comprises a cargo-binding domain. In some embodiments, the cargo-binding domain is a hairpin loop binding element. In some embodiments, the hairpin loop binding element is an MS2 aptamer.

[0013] In some embodiments, the delivery system elicits little or no immune response.

[0014] In some embodiments, the cargo comprises a nucleic acid, a protein, a complex thereof, or a combination thereof. In some embodiments, the cargo is linked to one or more envelope proteins by a linker. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the glycine-serine linker is (GGS)3 (SEQ ID NO: 1).

[0015] In some embodiments, the cargo comprises a ribonucleoprotein. In some embodiments, the cargo comprises a gene regulator. In some embodiments, the gene regulator comprises one or more components of a gene editing system and / or a polynucleotide encoding same. In some embodiments, the gene editing system is a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type II, Type V, or Type VI CRISPR-Cas system. In some embodiments, a Type II CRISPR-Cas system comprises CRISPR-Cas9. In some embodiments, a Type V CRISPR-Cas system comprises CRISPR-Cas12. In some embodiments, a Type VI CRISPR-Cas system comprises CRISPR-Cas13.

[0016] In some embodiments, the Cas protein of the CRISPR-Cas system may be modified to bind to the binding domain of the envelope protein. In some embodiments, the guide molecule of the CRISPR-Cas system is modified to bind to the binding domain of the envelope protein. In some embodiments, the modification comprises incorporating a hairpin loop that binds to a hairpin binding element on the envelope protein. In some embodiments, the hairpin loop can be recognized by the MS2 aptamer.

[0017] In some embodiments, the system may further comprise a reverse transcriptase.

[0018] In some embodiments, the one or more capture moieties comprise a DNA-binding moiety, an RNA-binding moiety, a protein-binding moiety, or a combination thereof.

[0019] In some embodiments, the delivery vesicle is a virus-like particle.

[0020] In some embodiments, the system may further comprise a targeting moiety, wherein the targeting moiety is capable of specifically binding to a target cell. In some embodiments, the targeting moiety comprises a membrane fusion protein. In some embodiments, the membrane fusion protein is the vesicular stomatitis virus G envelope protein (VSV-G).

[0021] In some embodiments, the target cell is a mammalian cell. In some embodiments, the mammalian cell is a cancer cell. In some embodiments, the mammalian cell is infected with a pathogen. In some embodiments, the pathogen is a virus.

[0022] In another aspect, the present invention provides delivery vesicles comprising one or more components encoded by one or more polynucleotides in the modified delivery systems described herein.

[0023] In some embodiments, one or more components of the delivery vesicle comprise two or more retroviral gag proteins, retroviral envelope proteins, retroviral reverse transcriptase, or combinations thereof.

[0024] In some embodiments, the retroviral gag protein is a gag homologous protein selected from the group consisting of Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In certain embodiments, the gag homologous protein is PNMA4, PEG10, or RTL1.

[0025] In some embodiments, the vesicle comprises a cell-specific targeting moiety. In some embodiments, the cell-specific targeting moiety targets a mammalian cell. In some embodiments, the cell-specific targeting moiety comprises a membrane fusion protein. In some embodiments, the membrane fusion protein is VSV-G.

[0026] In some embodiments, the mammalian cell is a cancer cell. In some embodiments, the mammalian cell is infected with a pathogen. In some embodiments, the pathogen is a virus.

[0027] In yet another aspect, the present invention provides a system for delivering cargo to a target cell, comprising a delivery vesicle encapsulating the cargo and an endogenous reverse transcriptase.

[0028] In some embodiments, the delivery vesicle is a virus-like particle. In some embodiments, the delivery vesicle is composed of a retroviral gag protein and a retroviral envelope protein. In some embodiments, the retroviral gag protein is derived from a human endogenous retrovirus (HERV).

[0029] In some embodiments, the retroviral gag protein is Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In specific embodiments, the retroviral gag protein is PNMA4, PEG10, or RTL1.

[0030] In some embodiments, the retroviral envelope protein is derived from a HERV. In some embodiments, both the retroviral gag protein and the retroviral envelope protein are derived from a HERV.

[0031] In some embodiments, the retroviral envelope protein comprises a cargo-binding domain. In some embodiments, the cargo-binding domain is a hairpin loop-binding element. In some embodiments, the hairpin loop-binding element is an MS aptamer.

[0032] In some embodiments, the cargo comprises a nucleic acid, a protein, a complex thereof, or a combination thereof. In some embodiments, the cargo comprises a ribonucleoprotein. In some embodiments, the cargo comprises a gene regulator. In some embodiments, the gene regulator comprises one or more components of a gene editing system and / or a polynucleotide encoding same. In some embodiments, the gene editing system is a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type II, Type V, or Type VI CRISPR-Cas system. In some embodiments, a Type II CRISPR-Cas system comprises CRISPR-Cas9. In some embodiments, a Type V CRISPR-Cas system comprises CRISPR-Cas12. In some embodiments, a Type VI CRISPR-Cas system comprises CRISPR-Cas13.

[0033] In some embodiments, the cargo is linked to one or more envelope proteins by a linker.

[0034] In some embodiments, the linker is a glycine-serine linker. In some embodiments, the glycine-serine linker is (GGS)3 (SEQ ID NO: 1).

[0035] In some embodiments, the Cas protein of the CRISPR-Cas system is modified to bind to the binding domain of the envelope protein. In some embodiments, the guide molecule of the CRISPR-Cas system is modified to bind to the binding domain of the envelope protein. In some embodiments, the modification comprises incorporating a hairpin loop that binds to the hairpin binding element on the envelope protein. In some embodiments, the hairpin loop is recognized by the MS2 aptamer.

[0036] In some embodiments, the system may further comprise a membrane fusion protein, hi some embodiments, the membrane fusion protein is VSV-G.

[0037] In some embodiments, the target cell is a mammalian cell. In some embodiments, the mammalian cell is a cancer cell. In some embodiments, the mammalian cell is infected with a pathogen. In some embodiments, the pathogen is a virus.

[0038] In yet another aspect, the present invention provides a method of treating a disease comprising administering any of the systems described herein to a subject in need of treatment, wherein the delivery vesicle delivers a cargo to one or more cells of the subject.

[0039] In some embodiments, the cargo may comprise a therapeutic agent, hi some embodiments, the therapeutic agent comprises one or more components of a gene editing system and / or a polynucleotide encoding same.

[0040] These and other aspects, objects, features, and advantages of the exemplary embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the illustrated exemplary embodiments.

[0041] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention may be utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0042] [Figure 1] Expression of various env proteins in HEK293T cells is shown, with increased expression of Envw1, Envk1, and Envfrd. [Figure 2] 1 shows the expression of various endogenous retroviral glycoproteins from particles pseudotyped with lentiviral proteins. [Figure 3] Expression of a Pnma3-RFP fusion construct (shown at the top) compared to a lentiviral-RFP reporter in mouse neurons is shown. Photomicrographs show slices of organotypic cultures from the prefrontal cortex. [Figure 4]Shown are maps of various endogenous gag proteins that were tested for their ability to form capsids, secrete proteins, and transfer material to new cells. [Figure 5] Transmission electron micrograph images showing the encapsidation ability of various candidate endogenous gag proteins. [Figure 6] 1 shows the ability of various endogenous gag proteins to be secreted from cells. [Figure 7] 7A and 7B show the gag construct containing the Cas9 / gRNA complex in the absence (7A) and presence (7B) of the membrane fusion protein VSV-G. [Figure 8] Schematic diagram showing the experimental setup. [Figure 9] Sequence alignment showing the number of mutations introduced by the CRISPR complex in vesicles containing RTL1 (9B) relative to control vesicles (9A). [Figure 10] Graph showing the number of indels induced by editing vesicle complexes containing various gag homology proteins. [Figure 11] (11A) PNMA4, (11B) PEG10, and (11C) RTL1 demonstrate the ability to transfer Cas9 / gRNA complexes to new cells. [Figure 12] Sequence alignment of knock-in mice expressing an HA tag on endogenous RTL-1. [Figure 13] Nitrocellulose gel showing HA-tagged PEG10 and RTL1. [Figure 14] Immunofluorescence images showing the ability of various gag homology proteins (14B-14D) to form vesicles in the presence of VSV-G compared to control particles (14A). [Figure 15] Graph showing the copy number of vesicles produced in the presence of various gag homologous proteins. [Figure 16] Graph showing the fold change in virus infectivity upon overexpression of various gag homologous proteins. [Figure 17] Schematic diagram showing various putative endogenous signaling systems on the scale of reduced immunogenicity. [Figure 18] Schematic showing requirements for enveloped VLPs. [Figure 19] Electron micrographs showing the ability of various gag homologous proteins to induce the spontaneous formation of vesicles from cells. [Figure 20] Electron micrographs showing the ability of various gag homologous proteins to induce the spontaneous formation of vesicles from cells. [Figure 21] Immunoprecipitation assay showing various gag homologous proteins secreted from cells. [Figure 22] Schematic diagram showing the assay used to determine whether GAGs are taken up by cells. [Figure 23] (23A, 23B) Graphs showing the ability of various gag constructs to be taken up by cells and introduce indels into target sequences; (23A) SEQ ID NOS: 9-18; (23B) SEQ ID NOS: 19-26; (23C, 23D) Graphs showing the ability of vesicles to be taken up by HEK293FT cells in the (23C) absence and (23D) presence of VSV-G. [Figure 24] Immunoprecipitation assay showing the ability of various constructs to be taken up by cells in the absence (left) and presence (right) of VSV-G. [Figure 25] Schematic diagram showing the two overlapping reading frames of PEG10. [Figure 26] Immunoprecipitation gel showing both the translated ORF1 and ORF1 / 2 bands of PEG10. [Figure 27] Immunoprecipitation reactions from whole cell lysates of cells transfected with various PEG10 constructs. [Figure 28] Immunoprecipitation reactions from whole cell lysates and VLP fractions of cells transfected with various PEG10 constructs. [Figure 29] Immunoprecipitation assays analyzing the ability of VSV-G and SGCE to promote PEG10 secretion and uptake into target cells. [Figure 30] Immunoprecipitation gels showing the ability of various concentrations of sucrose cushions to enhance the delivery efficiency of PEG10. [Figure 31]Graph showing the rate of indel generation using various constructs. [Figure 32] Western blot and immunofluorescence staining of delayed PEG10 location in both serum and cortical neurons in the brain. [Figure 33] 1 is a graph showing that knockout mice lacking PEG10 exhibit early embryonic lethality, demonstrating the importance of this gene in embryonic development. [Figure 34] RNA-seq gene ontology analysis of primary mouse neurons revealed three groups of differentially expressed genes: 1) genes involved in chromatin remodeling; 2) genes involved in the trans-Golgi network and exocytosis; and 3) SNARE and other genes encoding endosomal proteins. [Figure 35] Fluorescence micrograph showing expression of the GFP / PEG10 reporter construct. [Figure 36] Schematic diagram showing the DNA methyltransferase identification system (DamID) for mapping binding sites of DNA and chromatin-binding proteins. DamID identifies binding sites by expressing proposed DNA-binding proteins as fusion proteins with DNA methyltransferases. [Figure 37] Schematic diagram of DamID mapping. [Figure 38] PEG10-DAMID fusion constructs were analyzed for their ability to bind DNA and RNA by cross-referencing DamID mapping data with ATAC sequence data. [Figure 39] Mass spectrometry results of enriched proteins in the VLP fraction derived from N2A cells. [Figure 40] Schematic of how PEG10 mediates secretion from cells. [Figure 41] Schematic diagram showing the constructs that form RNA-containing gag vesicles. [Figure 42] Graph showing the ability of various gag homologous proteins to generate RNA-containing vesicles in the absence of VSV-G. [Figure 43]Graph showing the ability of various gag homologous proteins to generate RNA-containing vesicles in the presence of VSV-G. [Figure 44] Schematic showing the protocol for genome-wide screening of natural proteins that cross the blood-brain barrier. [Figure 45] A modification of the protocol shown in Figure 44 by transfecting cells passaged in step 1 with a second generation packaging vector to reactivate the provirus. [Figure 46] This shows the frequency with which guide RNAs are ultimately taken up by target cells. [Figure 47] The results of nuclear sorting of CNS subpopulations 14 days after tail vein injection are shown. [Figure 48] Fluorescence micrographs showing the ability of different fusion factors (Arghap32 and Clmp) to further enhance internalization efficiency. [Figure 49] Schematic diagram showing the protocol for transfection of constructs and evaluation of their indel generation ability. Cas9 is fused to PEG10 and overexpressed in cells, allowing for indel generation in target cells. [Figure 50] Analysis of various gag homologous proteins for their ability to act as natural fusogenic factors. [Figure 51] Fluorescence micrographs showing the ability of different fusion factors (Arghap32 and CXADR) to further enhance internalization efficiency. [Figure 52] Graph showing the analysis of various Cas9-carrying gags for their ability to be secreted from cells. [Figure 53] Graph showing analysis of the ability of selected gags from Figure 52 to be secreted from cells in the presence of VSV-G. [Figure 54] Graph showing the rate of indel generation from gag (left) in Figure 53 compared to HIV (right). [Figure 55] Analysis of the ability of different gag-IRES-Cas9 constructs to generate indels in the presence of various fusion factors. [Figure 56]Schematic diagram of PEG10 and Western blot showing the cleavage pattern of N-terminally and C-terminally tagged mouse PEG10 overexpressed in HEK293FT cells. [Figure 57A] Western blot of PEG10 cleavage pattern and graph showing peptide abundance of intact PEG10. [Figure 57B] Western blot of PEG10 cleavage pattern and graph showing peptide abundance of the first reading frame of PEG10. [Figure 57C] Western blot of PEG10 cleavage pattern and graph showing peptide abundance of NC cleavage products. [Figure 57D] Western blot of PEG10 cleavage pattern and graph showing peptide abundance after cleavage at the protease domain of the second reading frame of PEG10. [Figure 57E] Western blot of PEG10 cleavage pattern and graph showing peptide abundance after cleavage at the RT domain of the second reading frame of PEG10. [Figure 57F] Western blot of PEG10 cleavage pattern and graph showing peptide abundance after C-terminal cleavage of the second reading frame of PEG10. [Figure 58A] Western blot and schematic representation of the protease cleavage sites of PEG10 and the resulting protein fragments. [Figure 58B] Putative cleavage before the Gag domain. [Figure 59] Schematic diagram of the PEG10 ORF1 / 2 gene and Western blot showing the cleavage pattern of proteins isolated from VLP fractions and whole cell lysates. [Figure 60] 1 is a schematic diagram of the PEG10 protein, showing that the CCHC deletion in the NC domain results in the inability to bind to a specific sequence (SEQ ID NO: 2) to which the known myelin expression factor (MYEF) binds. [Figure 61] Binding experiment protocol to determine whether PEG10 binds to DNA, and graph confirming that PEG10 binds to DNA. [Figure 62] Schematic diagram showing the predicted location of the ORF1 cleavage site and experiments performed to confirm the location. [Figure 63] Schematic diagram showing the location of the ORF1 cleavage site and assessment of payload secretion. [Figure 64] Fluorescence micrographs showing the expression of GFP fusion constructs of various ORFs. [Figure 65] Schematic representation of hypothesized putative functions of various domains as they interact with DNA. [Figure 66] Schematic of PEG10 with mutations in various domains to determine function. [Figure 67] Schematic showing that if PEG10 is nuclear and can bind to DNA (like MYEF), then PEG10 regulates transcription. [Figure 68] Schematic diagram showing that mutations in the nucleocapsid domain resulted in a reduced ability to bind to the MYEF motif (SEQ ID NO: 3). [Figure 69] Footprinting assay to determine the function of individual motifs of the PEG10 protein. [Figure 70] Western blot showing quantification of PEG10 in the blood of transgenic mice.Figures herein are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE INVENTION

[0043] General definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2004. nd edition(1989)(Sambrook,Fritsch,and Maniatis);Molecular Cloning:A Laboratory Manual,4 thedition(2012)(Green and Sambrook);Current Protocols in Molecular Biology(1987)(F.M.Ausubel et al. eds.);the series Methods in Enzymology(Academic Press,Inc.):PCR 2:A Practical Approach(1995)(M.J.MacPherson,B.D.Hames,and G.R.Taylor eds.):Antibodies,A Laboratory Manual(1988)(Harlow and Lane,eds.):Antibodies A Laboratory Manual,2 ndedition 2013(EAGreenfield ed.);Animal Cell Culture(1987)(RIFreshney,ed.);Benjamin Lewin,Genes IX,published by Jones and Bartlet,2008(ISBN 0763752223);Kendrew et al.(eds.),The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd.,1994(ISBN 0632021829); Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons(New York, NY1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd edition (2011).

[0044] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly indicates otherwise.

[0045] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or substitute component may or may not occur, and the description includes cases where the event or circumstance occurs or does not occur.

[0046] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within each range and the recited endpoints.

[0047] As used herein, the term "about" or "approximately" when referring to a measurable value such as a parameter, amount, time duration, etc., is meant to encompass variations of and from the specified value, for example, variations of ±10% or less, ±5% or less, ±1% or less, and ±0.1% or less, of and from the specified value, insofar as such variations are appropriate for practice in the present invention. It should be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0048] As used herein, a "biological sample" may include whole cells and / or viable cells and / or cell debris. A biological sample may include (or be derived from) a "body fluid." The present invention encompasses embodiments in which the body fluid is selected from amniotic fluid, aqueous body fluid, vitreous humor, bile, serum, milk, cerebrospinal fluid, earwax (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph, mucus (including nasal discharge and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof. Biological samples include cell cultures, body fluids, and cell cultures derived from body fluids. Body fluids may be obtained from a mammalian organism, for example, by paracentesis or other collection or sampling procedure.

[0049] The terms "subject," "individual," or "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, livestock, game animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0050] The terms "high," "higher," "increased," "elevated," or "elevation" refer to an increase above basal levels, e.g., as compared to a control. The terms "low," "lower," "decreased," or "decreasement" refer to a decrease below basal levels, e.g., as compared to a control.

[0051] The term "control" refers to any reference standard suitable for providing a comparison with the expression product in a test sample. In one embodiment, control involves obtaining a "control sample" in which the expression product level is detected and compared to the expression product level from the test sample. Such control samples may include any suitable sample, including, but not limited to, a sample from a control patient with a known result (which may be a stored sample or a measurement of a previous sample); normal tissue, body fluid, or cells isolated from a subject, such as a normal patient or a patient with a condition of interest.

[0052] Various embodiments are described below. Note that specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects described herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and can be implemented in any other embodiment(s). Throughout this specification, references to "one embodiment," "an embodiment," or "an example embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," or "an example embodiment" in various places throughout this specification do not necessarily all refer to the same aspect, although they may. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments. Furthermore, some embodiments described herein include some features but not other features included in other embodiments, meaning that combinations of features from different embodiments are within the scope of the invention. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0053] All publications, published patent documents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0054] overview Embodiments disclosed herein provide compositions, systems, and methods for delivering cargo to target cells. The disclosure includes a polynucleotide encoding one or more endogenous retroviral elements for forming a delivery vesicle and one or more trapping moieties for packaging the cargo within the delivery vesicle. Such vesicles may be virus-like particles. The vesicles may be used to deliver therapeutic agents to target cells. The polynucleotide may contain a modified gene capable of recruiting or fusion to a cargo molecule and packaging it into the resulting vesicle. By adjusting the polynucleotide composition, it is possible to tailor the cargo and delivery, including both cell-specific and cell-nonspecific delivery methods. In certain embodiments, only one of the retroviral elements is an endogenous retroviral element. The endogenous retroviral element may be a retroviral gag protein or a retroviral envelope protein. The compositions, systems, and methods also include a retroviral reverse transcriptase. Preferably, the composition has reduced immunogenicity.

[0055] Modified Delivery Systems In one aspect, embodiments disclosed herein relate to modified polynucleotides and vectors encoding vesicles that form delivery systems derived from endogenous retroviral elements. In another aspect, embodiments disclosed herein relate to the use of such modified polynucleotides in methods for loading and / or packaging desired cargo molecules. In another aspect, embodiments disclosed herein relate to delivery vesicles carrying such cargo and methods of using said delivery vesicles to deliver cargo molecules to target cells.

[0056] Modified Polynucleotides Embodiments disclosed herein include modified polynucleotides encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more trapping moieties for packaging cargo within the delivery vesicles. The modified polynucleotides may further include regulatory elements, such as promoters, enhancers, internal ribosome entry sites (IRES), repressors, inducers, etc., for controlling expression of the vesicle-forming system. The modified polynucleotides are designed for delivery into cells, cell-free systems, or any other suitable bioreactor, allowing for the expression of delivery system components and the formation of delivery vesicles, including the packaging of desired cargo molecules into the delivery vesicles.

[0057] In some embodiments, one or more endogenous retroviral elements for forming the delivery vesicles comprise a retroviral envelope protein. In some embodiments, one or more endogenous retroviral elements for forming the delivery vesicles comprise a retroviral gag protein. In some embodiments, both the retroviral gag protein and the retroviral envelope protein are endogenous. In some embodiments, the gag protein is endogenous and the envelope protein is of viral origin. In some embodiments, the envelope protein is endogenous and the gag protein is of viral origin. The system may further comprise a cargo domain element, such as a peptide-based or nucleotide-based element, that specifically binds to a cargo of interest and is described in more detail below.

[0058] The system may further comprise one or more targeting moieties that can specifically bind to target cells. In some embodiments, the cargo may be linked to one or more envelope proteins by a linker. In some embodiments, the system may comprise a regulatory molecule that controls the expression of the vesicle-forming system.

[0059] The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences), and cellular localization signals (e.g., nuclear localization signals). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, e.g., cell cycle-dependent or developmental stage-dependent, which may or may not be tissue- or cell-type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, U6, 7SK, and H1 promoters.Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally containing the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally containing the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" also encompasses the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981). The specific configurations of gRNAs, reporter genes, and pol II and pol III promoters related to the present invention are described in more detail elsewhere herein.

[0060] In some embodiments, the regulatory sequences may be those described in U.S. Patent No. 7,776,321, U.S. Patent Publication No. 2011 / 0027239, and International Patent Publication No. WO2011 / 028929, the contents of which are incorporated by reference in their entireties. In some embodiments, the vector may include a minimal promoter. In some embodiments, the minimal promoter is a Mecp2 promoter, a tRNA promoter, or a U6 promoter. In further embodiments, the minimal promoter is tissue-specific. In some embodiments, the length of the vector polynucleotide, the minimal promoter, and the polynucleotide sequence is less than 4.4 Kb.

[0061] Generally, the system may include a vesicle-generating polynucleotide, a vesicle-generating plasmid, vesicles generated by such a plasmid, or both. The sequences described below can be cloned into a vector. As used herein, a "vector" is a tool that enables or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted, thereby replicating the inserted segment. Generally, a vector is replicable when associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, nucleic acid molecules without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which virally derived DNA or RNA sequences are present in a vector for packaging into a virus (e.g., retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are therefore replicated in conjunction with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

[0062] The polynucleotide may be an RNA molecule or a DNA molecule. The polynucleotide may be a natural polynucleotide or a recombinant polynucleotide. The polynucleotide may encode a protein molecule or an RNA molecule.

[0063] The polynucleotide may include a coding sequence for one or more components of the vesicles herein. In some examples, the polynucleotide includes a sequence encoding a barcode construct. The polynucleotide may further include a sequence encoding another element, such as a perturbation element. As used herein, a polynucleotide may be DNA, RNA, or a hybrid thereof, including, but not limited to, cDNA, mRNA, genomic DNA, mitochondrial DNA, sgRNA, siRNA, shRNA, miRNA, tRNA, rRNA, snRNA, lncRNA, and synthetic (e.g., chemically synthesized) DNA or RNA or a hybrid thereof. The polynucleotide may include natural nucleotides (e.g., A, T / U, C, and G), modified nucleotides, analogs of natural nucleotides such as labeled nucleotides, or any combination thereof.

[0064] The present invention also provides a delivery vesicle for delivering a polynucleotide encoding an endogenous protein.Such delivery vesicles or systems within the scope of the present invention can be provided in any form, including but not limited to, solid, semi-solid, emulsion, or colloidal particles.Thus, any of the delivery systems described herein, including but not limited to, lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene guns, can be provided as particulate delivery systems within the scope of the present invention.

[0065] Generally, "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In certain preferred embodiments, nanoparticles of the present invention have a maximum dimension (e.g., diameter) of 500 nm or less. In other preferred embodiments, nanoparticles of the present invention have a maximum dimension in the range of 25 nm to 200 nm. In other preferred embodiments, nanoparticles of the present invention have a maximum dimension of 100 nm or less. In other preferred embodiments, nanoparticles of the present invention have a maximum dimension in the range of 35 nm to 60 nm. It will be understood that references made herein to particle or nanoparticles can have the same meaning, where appropriate.

[0066] It will be understood that the size of the particles will vary depending on whether it is measured before or after loading, and therefore, in certain embodiments, the term "nanoparticle" may only apply to the particles before loading.

[0067] Nanoparticles encompassed by the present invention can be provided in different forms, such as solid nanoparticles (e.g., metals such as silver, gold, iron, titanium, etc.), non-metallic, lipid-based solids, polymers, nanoparticle suspensions, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles), can also be prepared. Nanoparticles made of semiconductor materials can be labeled with quantum dots if they are small enough (usually less than 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and can be adapted for similar purposes in the present invention.

[0068] Semi-solid and soft nanoparticles have been produced and are within the scope of the present invention. The prototypical semi-solid nanoparticle is the liposome. Various types of liposomal nanoparticles are currently used clinically as delivery systems for anti-cancer drugs and vaccines. Nanoparticles with half hydrophilic and half hydrophobic, called Janus particles, are particularly effective at stabilizing emulsions. They self-assemble at the water / oil interface and function as solid surfactants.

[0069] Self-assembled export compartments or nanoparticles containing RNA may be constructed from polyethyleneimine (PEI) PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of the polyethylene glycol (PEG). This system has been used, for example, to target integrin-expressing tumor neovasculature and deliver siRNA to inhibit vascular endothelial growth factor receptor-2 (VEGF-R2) expression, thereby achieving tumor angiogenesis (see, e.g., Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes can be prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid, resulting in a net molar excess of ionizable nitrogen (polymer) over phosphate (nucleic acid) ranging from 2 to 6. Electrostatic interactions between the cationic polymer and nucleic acid have led to the formation of polyplexes with an average particle size distribution of approximately 100 nm, hence the term nanoplex. Schiffelers et al.'s self-assembling nanoparticles are expected to deliver doses of CRISPR Cas of approximately 100–200 mg.

[0070] The nanoplexes of Bartlett et al. (PNAS, September 25, 2007, vol. 104, no. 39) may also be applied to the present invention. Bartlett et al.'s nanoplexes are prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid, resulting in a net molar excess of ionizable nitrogen (polymer) relative to phosphate (nucleic acid) ranging from 2 to 6. The electrostatic interaction between the cationic polymer and nucleic acid resulted in the formation of polyplexes with an average particle size distribution of approximately 100 nm, hence the term "nanoplex" herein. Bartlett et al.'s DOTA-siRNA was synthesized as follows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) (DOTA-NHS ester) was ordered from Macrocyclics (Dallas, TX). A 100-fold molar excess of amine-modified RNA sense strand containing DOTA-NHS ester in carbonate buffer (pH 9) was added to a microcentrifuge tube. The contents were allowed to react by stirring at room temperature for 4 hours. The DOTA-RNA sense complex was ethanol precipitated, resuspended in water, and annealed to the unmodified antisense strand to generate DOTA-siRNA. All liquids were pretreated with Chelex-100 (Bio-Rad, Hercules, CA) to remove trace metal contaminants. Tf-targeted and non-targeted siRNA nanoparticles can be formed using cyclodextrin-containing polycations. Typically, nanoparticles were formed in water with a charge ratio of 3 (+ / -) and an siRNA concentration of 0.5 g / L. 1% of the adamantane-PEG molecules on the surface of the targeted nanoparticles were modified with Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5% (wt / vol) glucose carrier solution for injection.

[0071] Lipid particles developed in the laboratory of Qiaobing Xu at Tufts University may be used / adapted for the delivery system of the present invention. Wang et al.,J.Control Release,2017 Jan 31.pii:S0168-3659(17)30038-X.doi:10.1016 / j.jconrel.2017.01.037. [Epub ahead of print];Altinoglu et al.,Biomater Sci.,4(12):1773-80,Nov.15,2016;Wang et al.,PNAS,113(11):2868-73 March 15,2016;Wang et al.,PloS One,10(11):e0141860.doi:10.1371 / journal.pone.0141860. eCollection 2015,Nov.3,2015;Takeda et al.,Neural Regen Res. 10(5):689-90, May 2015; Wang et al., Adv. Healthc Mater., 3(9):1398-403, September 2014; and Wang et al., Agnew Chem Int Ed Engl., 53(11):2893-8, March 10, 2014.

[0072] U.S. Patent Publication No. 20110293703 also provides libraries of amino alcohol lipidoid compounds prepared by the methods of the invention. These amino alcohol lipidoid compounds may be prepared and / or screened using high-throughput techniques, including liquid handlers, robots, microtiter plates, computers, etc. In certain embodiments, the amino alcohol lipidoid compounds are screened for their ability to transfect polypeptides or other agents (e.g., proteins, peptides, small molecules) into cells.

[0073] U.S. Patent Publication No. 2013 / 0302401 describes a class of poly(β-amino alcohols) (PBAAs) prepared using combinatorial polymerization. The PBAAs of this invention may be used in biotechnology and biomedical applications as coatings (such as film or multilayer film coatings for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cell encapsulation agents. When used as surface coatings, these PBAAs induced different levels of inflammation both in vitro and in vivo depending on their chemical structure. The great chemical diversity of this class of materials allowed for the identification of polymer coatings that inhibit macrophage activation in vitro. Furthermore, these coatings reduced inflammatory cell recruitment and fibrosis after subcutaneous implantation of carboxylated polystyrene microparticles. These polymers may also be used to form polyelectrolyte complex capsules for cell encapsulation. This invention may also have many other biological applications, such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of US Patent Publication No. 20130302401 may be applied to the CRISPR Cas system or any other system of the present invention.

[0074] In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anti-transthyretin small interfering RNA has been encapsulated in lipid nanoparticles and delivered to humans (see, e.g., Coelho et al., N Engl J Med 2013;369:819-29), and such systems may be adapted for use with the CRISPR-Cas system or any other system of the present invention. Doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are contemplated. Medication to reduce the risk of infusion-related reactions, such as dexamethasone, acetaminophen, diphenhydramine, or cetirizine, is contemplated, and ranitidine is also contemplated. Multiple doses of about 0.3 mg per kilogram every four weeks for five doses are also contemplated.

[0075] Zhu et al. (US20140348900) provide a process for preparing liposomes, lipid disks, and other lipid nanoparticles using a multiport manifold, in which a lipid solution stream containing an organic solvent is mixed with two or more streams of an aqueous solution (e.g., a buffer solution). In some aspects, at least some of the lipid and aqueous solution streams are not diametrically opposed to each other. Thus, this process does not require dilution of the organic solvent as an additional step. In some embodiments, one of the solutions may also contain an active pharmaceutical ingredient (API). The present invention provides a robust process for producing liposomes with different lipid formulations and different payloads. Particle size, morphology, and production scale can be controlled by varying the port size and number of manifold ports and selecting the flow rates or flow rates of the lipid and aqueous solutions.

[0076] LNP has been shown to be highly effective in delivering siRNA to the liver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470). Therefore, it is contemplated to deliver RNA encoding CRISPR Cas to the liver. Approximately four doses of 6 mg / kg LNP every two weeks may be contemplated. [Tabernero et al.] demonstrated that tumor regression was observed after the first two cycles of LNP administered at 0.7 mg / kg, and by the end of six cycles, the patient achieved a partial response with complete regression of lymph node metastases and substantial shrinkage of the liver tumor. This patient achieved a complete response after 40 doses, and the patient maintained remission and completed treatment after receiving more than 26 months of treatment. Two patients with RCC and extrahepatic disease sites (including kidney, lung, and lymph node) who had progressed after prior treatment with a VEGF pathway inhibitor remained stable at all sites for approximately 8 to 12 months, and a patient with PNET and liver metastases remained stable in an 18-month (36 doses) extension study.

[0077] In some embodiments, the LNP comprises a nucleic acid, where the charge ratio of nucleic acid backbone phosphate to cationic lipid nitrogen atoms is about 1:1.5-7 or about 1:4.

[0078] In some embodiments, the LNP also comprises a shielding compound, which is removable from the lipid composition under in vivo conditions. In some embodiments, the shielding compound is a biologically inert compound. In some embodiments, the shielding compound has no charge on its surface or on the molecule itself. In some embodiments, the shielding compound is polyethylene glycol (PEG), hydroxyethyl glucose (HEG)-based polymers, polyhydroxyethyl starch (polyHES), and polypropylene. In some embodiments, the PEG, HEG, polyHES, and polypropylene have a weight of about 500-10,000 Da, or about 2000-5000 Da. In some embodiments, the shielding compound is PEG2000 or PEG5000.

[0079] In some embodiments, sugar-based particles, such as GalNAc, may be used, as described herein and shown in WO2014118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136(49), 16958-16961, and the teachings herein, particularly with respect to delivery, apply to all particles unless otherwise expressly stated. This may be considered a sugar-based particle, and further details regarding other particle delivery systems and / or formulations are provided herein. Thus, GalNAc may be considered a particle in the sense of the other particles described herein, and as a result, general usage and other considerations, e.g., delivery of said particles, also apply to GalNAc particles. Solution-phase conjugation strategies can be used, for example, to attach triantennary GalNAc clusters (molecular weight approximately 2000) activated as PFP (pentafluorophenyl) esters to 5'-hexylamino-modified oligonucleotides (5'-HA ASO, molecular weight approximately 8000 Da; Ostergaard et al., Bioconjugate Chem., 2015, 26(8), pp1451-1455). Similarly, poly(acrylate) polymers have been described for in vivo nucleic acid delivery (see WO2013158141, incorporated herein by reference). In a further alternative embodiment, premixing of CRISPR nanoparticles (or protein complexes) with natural serum proteins can be used to improve delivery (Akinc A et al., 2010, Molecular Therapy vol.18 no.7, pp1357-1364).

[0080] References that may be used in conjunction with the teachings herein include Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al.,J.Am.Chem.Soc.2012 134:16488-1691,Weintraub,Nature 2013 495:S14-S16,Choi et al.,Proc.Natl.Acad.Sci.USA.2013 110(19):7625-7630,Jensen et al.,Sci.Transl. Med.5, 209ra152 (2013) and Mirkin, et al., Small, 10:186-192.

[0081] Measurement of cell-to-cell transfer may be assessed in multiple steps, as described by Patsuzyn et al. (Cell 172(1-2):275-288; 2018). In certain embodiments, indirect testing of encapsidation in transfected HEK293 cells may be performed by chemical crosslinking followed by SDS-PAGE to probe for the appearance of high-molecular-weight bands corresponding to protein oligomers. Export into extracellular vesicles may be performed by purifying the extracellular vesicle fraction from the culture medium after transfection and using Western blot to examine proteins in addition to reported extracellular vesicle markers. Finally, the ability of capsid-containing extracellular vesicles to be taken up by recipient cells may be tested by placing the culture medium or extracellular vesicle fractions purified from cells transfected with GFP-tagged Gag on untransfected cells and examining fluorescence uptake using microscopy and / or FACS. In addition to transport via extracellular vesicles, recombinant Arc can form encapsids in vitro and transport the encapsulated RNA into recipient cells in the absence of endosomal membranes. The protein can be purified from bacteria or translated in vitro and tested for activity. The formation of encapsidated structures in different assays can be confirmed using methods including, but not limited to, electron microscopy, dynamic light scattering, or Spectradyne particle analysis.

[0082] In certain embodiments, the unassembled recombinant GAG-like protein, nucleic acid and / or protein are mixed in a solution with low salt conditions.

[0083] U.S. Patent No. 8,709,843, incorporated herein by reference, provides a drug delivery system for targeted delivery of therapeutic agent-containing particles to tissues, cells, and intracellular compartments. The present invention provides targeted particles comprising a polymer, a hydrophilic polymer, or a lipid complexed to a surfactant. The teachings of U.S. Patent No. 8,709,843 may be applied and / or adapted to incorporate and / or deliver one or more of the modified delivery system molecules of the present invention described herein.

[0084] U.S. Patent No. 5,543,158 (incorporated herein by reference) provides biodegradable injectable particles having a biodegradable solid core comprising a biologically active agent and poly(alkylene glycol) moieties on the surface thereof. The teachings of U.S. Patent No. 5,543,158 may be adapted and / or used to incorporate and / or deliver one or more of the modified delivery system molecules of the present invention described herein.

[0085] International Patent Publication No. WO2012135025 (also published as US20120251560), incorporated herein by reference, describes conjugated polyethyleneimine (PEI) polymers and conjugated azamacrocycles (collectively referred to as "conjugated lipomers" or "lipomers"). In certain embodiments, it may be envisioned that such conjugated lipomers may be used in conjunction with the modified delivery systems described herein to achieve in vitro, ex vivo, and in vivo expression of one or more components of the modified delivery systems described herein, and some embodiments may produce modified delivery particles from the modified cell(s).

[0086] Additionally, the modified delivery system molecule(s) described herein may be delivered using a nanoclew, as described, for example, in Sun W et al, Cocoon-like self-degradable DNA nanoclew for anticancer drug delivery., J Am Chem Soc. 2014 Oct 22;136(42):14722-5. doi:10.1021 / ja5088024. Epub 2014 Oct 13.; or Sun W et al, Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing., Angew Chem Int Ed Engl. 2015 Oct 5;54(41):12029-33. doi:10.1002 / anie.201506030. Epub 2015 Aug 27. The teachings of Sun et al. can be applied and / or adapted to generate and / or deliver molecules of the CRISRP-Cas system described herein.

[0087] One or more of the modified delivery system molecules described herein can be contained in or incorporated into exosomes for delivery. Exosomes containing one or more modified delivery molecules described herein can be used to deliver the one or more modified delivery system molecule(s) to a cell and / or a subject.

[0088] Exosomes are endogenous nanovesicles that transport RNA and proteins and can deliver RNA to the brain and other target organs. To reduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used autologous dendritic cells to generate exosomes. Brain targeting was achieved by modifying dendritic cells to express the exosomal membrane protein Lamp2b fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous RNA by electroporation. Intravenously injected RVG-targeted exosomes specifically delivered GAPDH siRNA to neurons, microglia, and oligodendrocytes in the brain, resulting in specific gene knockdown. Preexposure to RVG exosomes did not attenuate knockdown, and nonspecific uptake into other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the potent mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target for Alzheimer's disease. The teachings of Alvarez-Erviti et al. can be applied and / or adapted to generate and / or deliver molecules of the CRISPR-Cas system described herein.

[0089] In some embodiments, the delivery system elicits little immune response or has low immunogenicity.

[0090] In some embodiments, the delivery vesicle is a virus-like particle (VLP). As used herein, the term "virus-like particle" (VLP) refers to a structure that resembles a virus in at least one attribute, but has not been shown to be infectious. A VLP can be a non-replicating, non-infectious viral shell that contains a viral capsid but lacks all or part of the viral genome, particularly the replication components of the viral genome. VLPs generally consist of one or more viral proteins, such as, but not limited to, proteins referred to as capsid, coat, shell, surface, and structural proteins (e.g., VP1, VP2). VLPs can resemble the structure of bacteriophages, are non-replicating, non-infectious, and lack at least the genes encoding the bacteriophage replication machinery and the genes encoding proteins involved in viral binding or entry into the host.

[0091] Envelopes from various retroviral sources can be used to determine the pseudotype of a vector. The exact rules for pseudotyping (i.e., which envelope proteins interact with the nascent vector particle on the cytoplasmic side of the cell membrane to generate viable virus particles (Tato, Virology 88:71, 1978) and which do not (Vana, Nature 336:36, 1988) has not been fully characterized. However, because a portion of the cell membrane is shed to form the viral envelope, molecules within the membrane are typically transported along the viral envelope. Therefore, by engineering the cell lines in which the vector is generated to produce gag and pol, or by selecting different types of cell lines with specific surface markers, multiple different potential ligands can be placed on the surface of the viral vector. One type of surface marker that can be expressed in helper cells and confer useful vector-cell interactions is the receptor for another potentially pathogenic virus. Pathogenic viruses display virus-specific proteins (e.g., env) on the surface of infected cells, which usually interact with cell surface markers or receptors to initiate viral infection. This uses the same viral protein-receptor interaction, but by having the receptor on the vector and the viral protein on the cell, it reverses the specificity of the vector's infection with respect to the potentially pathogenic virus.

[0092] One virus known to be involved in pseudotyping is vesicular stomatitis virus (VSV), the prototype member of the Rhabdoviridae family. It is an enveloped virus with a negative-strand RNA genome that causes self-limiting disease in livestock and is essentially nonpathogenic to humans. Balachandran and Barber (2000, IUBMB Life 50:135-8) report that rhabdoviruses have a single-stranded positive-strand RNA genome of 11,000-12,000 nucleotides (Rose and Schubert, 1987, Rhabdovirus genomes and their products, in *The Viruses: The Rhabdoviruses*, Plenum Publishing Corp., NY, pp. 129-166). The virus particle contains a helical nucleocapsid core composed of genomic RNA and protein. Three proteins, generally designated N (nucleocapsid, tightly encases the genome), P (formerly called NS, originally nonstructural), and L (large), are found associated with the nucleocapsid. An additional matrix (M) protein is located within the membrane envelope and likely interacts with both the membrane and the nucleocapsid core. A single glycoprotein (G) species spans the membrane, forming spikes on the surface of the virus particle.

[0093] Endogenous Retroviral Elements Human endogenous retroviral (HERV) sequences comprise 8.29% of the draft human genome. Their prevalence is attributed to the accumulation of past retroviral infectious agents that enter the germline, establish a truce with host cells, and are expressed from the host genome. HERVs can be grouped according to sequence homology into approximately 100 different families, each containing a few to several hundred elements. Genes derived from endogenous retroviruses adopted by the host have been shown to actively participate in several cellular processes, including viral defense via Fv1 and Fv4 in mice and syncytin-mediated cell fusion in human placental development. HERV transcripts have been detected in both normal and cancerous tissues, including T cells, but their role in normal cellular function and carcinogenesis remains unclear. While the cellular pathologies that drive HERV transcription are poorly understood, APOBECs have been shown to play a role in regulating endogenous retroviruses.

[0094] The strong similarity between modern HERVs and retroviruses can be inferred from phylogenetic analyses of the reverse transcriptase domain of the pol gene or the transmembrane (TM) portion of the env gene, which reveal interleaving of both types of elements, suggesting a common history and shared ancestry (Tristem, M. (2000) J. Virol. 74, 3715-3730; Benit et al. (2001) J. Virol. 75 (11709-11719)). Similarities are also observed at the functional level.

[0095] As a result of the close relationship between HERVs and infectious retroviruses, and despite the fact that most HERVs have accumulated mutations, deletions, and / or truncations, some elements may still possess infectious retroviral functions, which the host may repurpose to its own benefit.

[0096] Genes encoding viral polypeptides that are defective and capable of self-assembling into non-replicating viral particles can be obtained from the genomic DNA of DNA viruses or the genomic cDNA of RNA viruses, or from available partial genomic clones containing the genes. These genes include genes encoding viral capsid proteins (i.e., the proteins that comprise the viral protein shell) and, in the case of enveloped viruses such as retroviruses, 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 involved in processing capsid proteins or envelope glycoproteins. As an example, the genomic structure of picornaviruses has been well characterized, revealing the pattern of protein synthesis that leads to virion assembly. (Rueckert, R., in Virology (1985), BN Fields et al. (eds.), Raven Press, New York, pp. 705-738). In picornaviruses, viral capsid proteins are encoded by an RNA genome containing a single long reading frame and synthesized as part of a polyprotein, which is processed by a combination of cellular and viral proteases to generate mature capsid proteins. Therefore, picornavirus genes required for capsid self-assembly include both the capsid structural genes and the viral protease required for their maturation. Another class of viruses from which genes encoding self-assembling capsid proteins can be isolated are lentiviruses, one example of which is HIV. Similar to picornavirus capsid proteins, the HIV gag protein is synthesized as a precursor polypeptide, which is then processed by the viral protease to form the mature capsid polypeptide. However, the gag precursor polypeptide can self-assemble into virus-like particles without proteolytic processing.Gheysen et al., Cell 59:103 (1989); Delchambre et al., The EMBO J. 8:2653-2660 (1989). Unlike picornavirus capsids, the HIV capsid is surrounded by a loose membranous envelope containing viral glycoproteins, which are encoded by the viral env gene.

[0097] In alternative embodiments, additional human proteins with Gag homology may be used to assemble virus-like capsids that mediate cell-to-cell transport of cargo. Such proteins include, but are not necessarily limited to, the extended PNMA gene family, including ZCC18, ZCH12, PNM8B, PNM8B, PNM6A, PMA6F, PMA6E, PNMA2, PNM8A, PNMA3, PNMA5, PNMA1, MOAP1, and CCDC8. In a specific embodiment, the GAG-like protein is Arc.

[0098] In some embodiments, the endogenous retroviral element is an endogenous retroviral gag protein. In some embodiments, the endogenous retroviral element is an endogenous retroviral envelope protein. In some embodiments, the endogenous retroviral element is a retroviral reverse transcriptase. In some embodiments, one or more retroviral elements may be endogenous. In some embodiments, two or more retroviral elements may be endogenous.

[0099] In some embodiments, the one or more endogenous retroviral elements for forming the delivery vesicle may include two or more retroviral gag proteins, retroviral envelope proteins, retroviral reverse transcriptase, or combinations thereof.

[0100] Retroviral Gag protein The group-specific antigen (gag) protein is the core structural protein, or major component, of the retroviral capsid. The HIV p17 matrix protein (MA), a 17-kDa protein of 132 amino acids, comprises the N-terminus of the Gag polyprotein. It targets the Gag polyprotein to the plasma membrane but also contacts the HIV transmembrane glycoprotein gp41 of the assembled virus, which may play an important role in recruiting the Env glycoprotein to the viral budding site.

[0101] Several studies have shown that expression of the gag gene alone results in the efficient assembly and release of membrane-enveloped virions in multiple systems (Craven, R.C., et al. (1996). Dynamic interactions of the Gag polyprotein. Current Topics in Microbiology and Immunology 214, pp. 65-94; Delchambre, M., et al. (1989). The Gag precursor of simian immunodeficiency virus assembles into virus-like particles. EMBO 8, pp. 2653-60; Dickson, C., et al. (1984). "Protein biosynthesis and assembly," RNA tumor viruses (R. Weiss, N. Teich, H. Varmus, and J. Coffin, Eds.), Vol. 1, pp. 513-648, 2 vols. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Gheysen, H.P., et al. (1989), "Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells,” Cell 59,pp.103-12;Haffar,O.,et al.(1990),“Human immunodeficiency virus-like,non-replication,Gag-Env particles assemble in a recombinant vaccinia virus expression system,” J. Virol. 64, pp. 2653-59; Hunter, E. (1994), “Macromolecular interactions in the assembly of HIV and other retroviruses,” Sem. in Virology 5, pp. 71-83; Krausslich, H.-G., et al.(1996), “Intracellular transport of retroviral capsid components,” Current Topics in Microbiology and Immunology 214, pp. 25-64; Madisen, L., et al. (1987), “Expression of the human immunodeficiency virus gag gene in insect cells,” Virology 158, pp. 248-250; Smith, AJ, et al. al. (1990), “Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from a simian virus 40 late-replacement vector are efficiently processed and assembled into virus-like particles,” J. Virol. 64, pp. 2743-50; Sommerfelt, MA, et al. (1992), “Importance of the p12 protein in Mason-Pfizer monkey virus assembly and infectivity,” J. Virol. 66, pp. 7005-11; Wills, JW, et al. (1989), "Creation and expression of myristylated forms of Rous sarcoma virus Gag protein in mammalian cells," J. Virol. 63, pp. 4331-43). Thus, the product of this gene contains the structural information necessary to mediate intracellular transport, direct assembly into the capsid shell, and catalyze the membrane extrusion process known as budding.

[0102] Upon Gag translation, the Gag polyprotein is myristoylated at the N-terminal glycine residue by N-myristoyltransferase 1 (a modification important for plasma membrane targeting). In the membrane-unbound form, the MA myristoyl fatty acid tail is sequestered in a hydrophobic pocket within the core of the MA protein. Recognition of plasma membrane proteins by MA activates a "myristoyl switch," which extrudes the myristoyl group from the hydrophobic pocket within MA and embeds it in the plasma membrane.

[0103] The HIV nucleocapsid protein (NC), a 7-kDa zinc finger protein contained in the Gag polyprotein, forms the viral nucleocapsid after viral maturation and recruits the full-length viral genomic RNA to nascent virions.

[0104] The neuronal gene Arc is homologous to the Gag component of the Ty3 / gypsy retrotransposon and exhibits biochemical properties reminiscent of retroviral Gag proteins. Arc proteins assemble into virus-like capsids both intracellularly and when recombinantly expressed in bacteria. Arc capsids can mediate intercellular transport in extracellular vesicles and encapsulate their own mRNA. Purified Arc protein can be used to reconstitute capsids with different DNAs, RNAs, proteins, or mixtures thereof, which can then be packaged into capsids for delivery to cells. In some embodiments, capsids can be assembled using lipids to aid in cellular uptake. Various embodiments may utilize different Arc orthologs.

[0105] In some embodiments, the polynucleotides described herein may comprise a Gag homologous protein or a functional domain thereof. The term "functional domain" refers to a polypeptide sequence that has an activity other than binding to the nucleic acid sequence recognized by the nucleic acid binding domain. By combining a nucleic acid binding domain with one or more effector domains, the polypeptides of the present invention may be used to target one or more functions or activities mediated by the effector domain to a specific target DNA sequence to which the nucleic acid binding domain specifically binds.

[0106] The molecular and genetic determinants of Gag-mediated cell-cell communication can be determined by characterizing the mechanism of capsid-mediated intercellular mRNA transfer, focusing particularly on the properties that allow programmable cargo delivery using this system. Different Gag proteins have evolved diverse RNA-binding domains to mediate specific encapsidation of RNA genomes. The RNA-binding sequence specificity of human Gag homologous proteins can be tested by protein pull-down and sequencing of the associated RNA, and / or by sequencing extracellular vesicle fractions from HEK293 cells overexpressing each protein. Nucleic acid-binding domains can be swapped between proteins, or additional RNA-binding domains with known specificities can be fused to test the extent to which binding specificity can be reprogrammed. Thus, Gag homologous proteins, or their functional domains, can contain both an export compartment domain and a nucleic acid-binding domain.

[0107] The Gag homologous protein can be selected from Arc, ASPRV1, Sushi-Class protein, SCAN protein, or PNMA protein. In a specific example, the Gag homologous protein is a PNMA protein, such as ZCC18, ZCH12, PNM8B, PNM6A, PNMA6E_i2, PMA6F, PMAGE, PNMA1, PNMA2, PNM8A, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PNMA1, MOAP1, or CCD8. In an embodiment, the Gag homologous protein is an Arc protein, and in a specific embodiment, hARC or dARC1. The Gag homologous protein may include ASPRV1. In another example, the Gag homologous protein is PEG10, RTL3, RTL10, or RTL1. In a specific embodiment, the Gag homologous protein is a SCAN protein, such as PGBD1. In one example, the PEG10 Gag homologous protein is PEG10_i6 or PEG10_i2.

[0108] In some embodiments, a Gag homologous protein or functional domain thereof can include both an export compartment domain and a nucleic acid binding domain. In certain embodiments, the nucleic acid binding domain can be modified relative to the native nucleic acid binding domain of the Gag homologous protein. In certain embodiments, the nucleic acid binding domain can be a non-native nucleic acid binding domain relative to the Gag homologous protein. In some embodiments, the Gag homologous protein can be Arc or a paratumor-associated Ma antigen (PNMA) protein.

[0109] In some embodiments, recombinant GAG-like proteins may be expressed and purified from bacteria, yeast, insect cells, or mammalian cells. Recombinant GAG-like proteins may be purified under denaturing conditions and transferred to non-denaturing conditions by buffer exchange.

[0110] In some embodiments, the retroviral gag protein is endogenous.

[0111] In some embodiments, the retroviral gag protein may comprise an NC domain and an MA domain.

[0112] In some embodiments, the retroviral gag protein may be a gag homologous protein.

[0113] In some embodiments, gag homologous proteins may include, but are not necessarily limited to, Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In certain embodiments, the gag homologous protein is Arc1, PNMA6a, or PNMA3. In certain embodiments, the gag homologous protein is PEG10.

[0114] In some embodiments, gag homologous proteins may contain DNA binding motifs. As a specific example, and as described in Example 4, PEG10 contains a DNA binding motif that allows packaging of DNA of specific sequences.

[0115] As one of skill in the art will appreciate, any of the systems described herein can be further modified to a minimal set of components and applied to any suitable endogenous element. As described in Examples 3 and 4 and Figures 56-70, the use of PEG10 is merely exemplary of an approach that could be implemented with any other endogenous element.

[0116] Retroviral Env Protein Env is a retroviral gene that encodes a protein that forms the viral envelope. Expression of the env gene allows retroviruses to target and bind to specific cell types and invade the target cell membrane. The structure and sequence of several different env genes suggest that the Env protein is a type 1 fusion protein. Type 1 fusion proteins first bind to a receptor on the target cell surface, which triggers a conformational change that allows the fusion protein to bind. The fusion peptide inserts itself into the host cell membrane, bringing it into close proximity with the viral membrane and enabling membrane fusion. Although the sequence of the env gene can vary significantly between retroviruses, this gene is always located downstream of the gag, pro, and pol genes. The env mRNA must be spliced ​​for expression.

[0117] Env not only mediates viral entry into cells but is also a primary target for both cellular and antibody responses. It is synthesized as a precursor molecule, gp160, which is subsequently processed by cellular proteases into the surface subunit (SU) gp120 and the transmembrane subunit (TM) gp41, and exists on viral or cellular membranes as a trimer of gp120-gp41 heterodimers. The SU protein domain is involved in the viral receptor-binding function and thus determines the viral affinity. Thus, the SU domain determines the viral specificity for a single receptor molecule. gp120 interacts with HIV receptor and coreceptor molecules and mediates viral binding to cells, while gp41 triggers subsequent fusion between the viral and cellular membranes to release viral core components into cells during the initial infection process. The TM protein consists of three distinct domains: the extracellular domain, the transmembrane domain, and the cytoplasmic domain.

[0118] In some embodiments, the retroviral envelope protein is endogenous.

[0119] In some embodiments, the envelope protein may be derived from a gammaretrovirus. In some embodiments, the envelope protein may be derived from a deltaretrovirus.

[0120] In some embodiments, the envelope protein may be selected from, but is not necessarily limited to, envH1, envH2, envH3, envK1, envK2_1, envK2_2, envK3, envK4, envK5, envK6, envT, envW, envW1, envfrd, envR(b), envR, envF(c)2, or envF(c)1.

[0121] In one aspect, the present invention provides for the introduction of an RNA sequence into the transcript recruitment sequence that forms a loop secondary structure and binds to an adaptor protein. In one aspect, the present invention provides a composition as described herein, wherein the insertion of the separate RNA sequence(s) that bind to one or more adaptor proteins is an aptamer sequence. In one aspect, the present invention provides a composition as described herein, wherein the aptamer sequences are two or more aptamer sequences specific for the same adaptor protein. In one aspect, the present invention provides a composition as described herein, wherein the aptamer sequences are two or more aptamer sequences specific for different adaptor proteins.

[0013] In one aspect, the invention provides a composition described herein, wherein the adaptor protein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ΦCb5, ΦCb8r, ΦCb12r, ΦCb23r, 7s, or PRR1.

[0014] In one aspect, the invention provides a composition described herein, wherein the cell is a eukaryotic cell.

[0015] In one aspect, the invention provides a composition described herein, wherein the eukaryotic cell is a mammalian cell, optionally a mouse cell.

[0016] In one aspect, the invention provides a composition described herein, wherein the mammalian cell is a human cell. Aspects of the invention include embodiments related to the MS2 adaptor protein described in Konermann et al. "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex" Nature. 2014 Dec 10. doi:10.1038 / nature14136, the contents of which are incorporated herein by reference in their entirety.

[0122] In some embodiments, the adaptor protein domain is an RNA binding protein domain. The RNA binding protein domain recognizes a corresponding distinct RNA sequence, which can be an aptamer. For example, MS2 RNA binding protein recognizes and specifically binds to MS2 aptamer (or vice versa).

[0123] Similarly, MS2 variant adapter domains may also be used, such as N55 mutants, particularly the N55K mutant, which is the N55K mutant of the MS2 bacteriophage coat protein (shown to exhibit higher binding affinity than wild-type MS2 in Lim, F., M. Spingola, and D.S.Peabody. "Altering the RNA binding specificity of a translational repressor." Journal of Biological Chemistry 269.12 (1994):9006-9010).

[0124] In some embodiments, the envelope protein can include a cargo-binding domain. In some embodiments, the cargo-binding domain is a hairpin loop-binding element. In some embodiments, the hairpin loop-binding element is an MS2 aptamer.

[0125] In some embodiments, both the retroviral gag protein and the retroviral envelope protein are endogenous. In some embodiments, the gag protein is endogenous and the envelope protein is of viral origin. In some embodiments, the envelope protein is endogenous and the gag protein is of viral origin.

[0126] Supplementary part In some embodiments, the vesicles comprise one or more trapping moieties, e.g., for packaging cargo and / or recruiting specific cargo(s) to the vesicle.

[0127] As used herein, the term "nucleic acid capture moiety" or simply "capture moiety" refers to a moiety that selectively binds to a target molecule. Optionally, the moiety can be immobilized on an insoluble support, as in the case of a microarray, or on a microparticle such as a bead. When used as a primer, the probe of the present invention is unlikely to be immobilized on a solid support. The capture moiety can "capture" the target molecule by hybridizing to the target and thereby immobilizing the target. When the moiety itself is immobilized, the target is also immobilized. Such binding to the solid support may be via a linking moiety that binds to either the capture moiety or the solid support.

[0128] The capture moiety may comprise one or more genes endogenous to the polynucleotide or plasmid, such as a gene capable of recruiting the plasmid to the vesicle. The capture moiety may comprise an exogenous gene or a molecule capable of recruiting or capturing a cargo molecule from the vesicle. In some instances, the capture moiety may interact with the cargo. The capture moiety may be a nucleic acid-binding molecule, such as DNA, RNA, a DNA-binding protein, an RNA-binding protein, or a combination thereof. In some embodiments, the capture moiety may be a protein-binding molecule, such as DNA, RNA, an antibody, a nanobody, an antigen, a receptor, a ligand, a fragment thereof, or a combination thereof. The capture moiety may be fused to an endogenous gene or an exogenous gene.

[0129] In some embodiments, the one or more capture moieties comprise a DNA-binding moiety, an RNA-binding moiety, a protein-binding moiety, or a combination thereof.

[0130] In certain embodiments, the capture moiety can be, for example, a fluorescent moiety, a radioisotope (e.g., 32The capture moiety may be labeled with an antibody, antigen, lectin, enzyme (e.g., alkaline phosphatase or horseradish peroxidase (which can be used in calorimetry)), chemiluminescent, bioluminescent, or other labels known in the art. In certain embodiments, binding of the target strand to the capture moiety can be detected by chromatography or electrophoresis. In embodiments where the capture moiety does not contain a detectable label, the target nucleic acid sequence may be so labeled, or alternatively, a labeled secondary probe may be used. A "secondary probe" comprises a nucleic acid sequence that is complementary to either a region of the target nucleic acid sequence or a region of the capture moiety. Region G of the probe (which in most cases is not complementary to the target) may be useful for capturing a secondary labeled nucleic acid probe.

[0131] In some embodiments, the capture moiety is a nucleic acid hairpin. As used herein, the terms "nucleic acid hairpin," "hairpin capture moiety," or simply "hairpin" refer to a unimolecular nucleic acid-containing structure that contains at least two mutually complementary nucleic acid regions and can thereby form at least one intramolecular duplex. Hairpins are described, for example, in Cantor and Schimmel, "Biophysical Chemistry," Part III, p. 1183 (1980). In certain embodiments, the mutually complementary nucleic acid regions are connected via a nucleic acid strand; in these embodiments, the hairpin comprises a single-stranded nucleic acid. The region of the capture moiety that connects the mutually complementary regions is referred to herein as a "loop" or "linker." In some embodiments, the loop comprises a nucleic acid strand or a modified nucleic acid. In some embodiments, the linker is not hydrogen-bonded. In other embodiments, the loop comprises a linker region that is not nucleic acid-based; however, capture moieties in which the loop region is not a nucleic acid sequence are referred to herein as hairpins. Examples of non-nucleic acid linkers suitable for use in the loop region are known in the art and include, for example, alkyl chains (see, e.g., Doktycz et al. (1993) Biopolymers 33:1765). It is understood that the loop can be the single-stranded region of the hairpin; however, for purposes of the following discussion, the "single-stranded region" of the hairpin refers to the non-loop region of the hairpin. In embodiments in which the loop is a nucleic acid strand, the loop preferably comprises 2 to 20 nucleotides, more preferably 3 to 8 nucleotides. The size or configuration of the loop or linker is selected so that the mutually complementary regions can form an intramolecular duplex. In preferred embodiments, hairpins useful in the present invention form at least one intramolecular duplex having at least 2 base pairs, more preferably at least 4 base pairs, and even more preferably at least 8 base pairs. The number of base pairs in the double-stranded region, and its base composition, can be selected to ensure any desired relative stability of duplex formation. For example, to prevent non-target nucleic acids from hybridizing to the intramolecular duplex-forming region of the hairpin, the number of base pairs in the intramolecular duplex region will generally be greater than about 4 base pairs.The intramolecular duplex generally does not exceed about 40 base pairs, hi preferred embodiments, the intramolecular duplex is less than 30 base pairs, more preferably less than 20 base pairs in length.

[0132] A hairpin can form multiple loops. For example, a hairpin that can form two intramolecular duplexes and two loops is referred to herein as a "double hairpin." In preferred embodiments, the hairpin has at least one single-stranded region that is substantially complementary to the target nucleic acid sequence. "Substantially complementary" means capable of hybridizing to the target nucleic acid sequence under the conditions used. In preferred embodiments, a "substantially complementary" single-stranded region is exactly complementary to the target nucleic acid sequence. In preferred embodiments, hairpins useful in the present invention have a target-complementary single-stranded region having at least 5 bases, more preferably at least 8 bases. In preferred embodiments, the hairpin has a target-complementary single-stranded region having fewer than 30 bases, more preferably fewer than 25 bases. The target-complementary region is selected to ensure that the target strand forms a stable duplex with the capture moiety. In embodiments in which the capture moiety is used to detect a target strand from a large number of non-target sequences (e.g., when screening genomic DNA), the target-complementary region should be long enough to prevent binding of non-target sequences. The target-specific single-stranded region can be at either the 3' or 5' end of the capture moiety, or can be located between two intramolecular double-stranded regions (e.g., between the two double strands of a double hairpin).

[0133] cargo molecule The delivery particles described herein may be used and further comprise a plurality of different cargo molecules for delivery. Exemplary cargo molecules may include, but are not limited to, nucleic acids, polynucleotides, proteins, polypeptides, polynucleotide / polypeptide complexes, small molecules, sugars, or combinations thereof. Cargoes that can be delivered according to the systems and methods described herein include, but are not necessarily limited to, bioactive agents, including, but not limited to, therapeutic agents, imaging agents, and monitoring agents. Cargoes may be exogenous or endogenous substances.

[0134] Bioactive agents include any molecule that induces an effect within a cell. Bioactive agents can be proteins, nucleic acids, small molecules, carbohydrates, and lipids. When the cargo is or includes a nucleic acid, the nucleic acid can be a separate entity from the DNA-based carrier. In these embodiments, the DNA-based carrier is not itself the cargo. In other embodiments, the DNA-based carrier may itself include a nucleic acid cargo. Therapeutic agents include chemotherapeutic agents, anti-carcinogenic agents, anti-angiogenic agents, tumor suppressors, antibacterial agents, enzyme replacement agents, gene expression modulators, and expression constructs containing nucleic acids encoding therapeutic proteins or therapeutic nucleic acids. Therapeutic agents can be peptides, proteins (including enzymes, antibodies, and peptide hormones), cytoskeletal ligands, nucleic acids, small molecules, non-peptide hormones, etc. To enhance nuclear affinity, the agent can be conjugated to a nuclear localization sequence. Nucleic acids that can be delivered by the methods of the present invention include synthetic and natural nucleic acid materials, including DNA, RNA, transposon DNA, antisense nucleic acids, dsRNA, siRNA, transcribed RNA, messenger RNA, ribosomal RNA, small nuclear RNA, microRNA, ribozymes, plasmids, expression constructs, and the like.

[0135] Imaging agents include contrast agents such as ferrofluid-based MRI contrast agents and gadolinium agents for PET scans, fluorescein isothiocyanate, and 6-TAMARA. Monitoring agents include reporter probes, biosensors, green fluorescent protein, and the like. Reporter probes include luminescent compounds such as phosphors, radioactive moieties, and fluorescent moieties, such as rare earth chelators (e.g., europium chelators), Texas Red, rhodamine, fluorescein, FITC, fluo-3,5 hexadecanoyl fluorescein, Cy2, fluor X, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, dansyl, phycocriterin, phycocyanin, spectral orange, spectral green, and / or derivatives of any one or more of the above. Biosensors are molecules that detect and transmit information about physiological changes or processes, for example, by detecting the presence or change in the presence of a chemical substance. The information obtained by the biosensor typically activates a signal that is detected by a transducer. Transducers typically convert biological responses into electrical signals. Examples of biosensors include enzymes, antibodies, DNA, receptors, and regulatory proteins used as recognition elements, and can be used in whole cells or isolated and used independently (D'Souza, 2001, Biosensors and Bioelectronics 16:337-353).

[0136] One or more different cargoes may be delivered by the delivery particles described herein.

[0137] In some embodiments, the cargo may be linked to one or more envelope proteins by a linker, as described elsewhere herein. Suitable linkers may include, but are not necessarily limited to, a glycine-serine linker. In some embodiments, the glycine-serine linker is (GGS)3 (SEQ ID NO: 1).

[0138] In some embodiments, the cargo comprises a ribonucleoprotein. In certain embodiments, the cargo comprises a gene regulatory agent.

[0139] As used herein, the term "altered expression" can refer, inter alia, to altered production by a cell of a referenced gene product. As used herein, the term "gene product(s)" includes RNA transcribed from a gene (e.g., mRNA) or a polypeptide encoded by a gene or translated from RNA.

[0140] "Altered expression," as intended herein, can also encompass modulating the activity of one or more endogenous gene products. Thus, "altered expression," "altering expression," "modulating expression," or "detecting expression," or the like, can be used interchangeably with "altered expression or activity," "altering expression or activity," "modulating expression or activity," or "detecting expression or activity," respectively. As used herein, "modulating" or "to modulate" generally means decreasing or inhibiting the activity of a target or antigen, or increasing the activity of a target or antigen, as measured using a suitable in vitro, cellular, or in vivo assay. In particular, "modulating" or "to modulate" can mean reducing or inhibiting the (relevant or intended) activity or increasing the (relevant or intended) biological activity of a target or antigen by at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, as measured using a suitable in vitro, cellular, or in vivo assay (typically depending on the target or antigen involved), compared to the activity of the target or antigen in the same assay under the same conditions but in the absence of an inhibitor / antagonist or activator / agonist as described herein.

[0141] As will be apparent to those skilled in the art, "modulating" can also include causing a change (which can be either an increase or a decrease) in the affinity, avidity, specificity, and / or selectivity of a target or antigen for one or more targets under the same conditions but in the absence of a modulator. Again, the change can be measured in any suitable manner and / or using any suitable assay known per se, depending on the target. In particular, an inhibitor / antagonist or activator / agonist action can be an action that increases or decreases the intended biological or physiological activity by at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, respectively, compared to the biological or physiological activity in the same assay under the same conditions but in the absence of the inhibitor / antagonist or activator / agonist. Modulation can also include activating the target or antigen, or the mechanism or pathway in which it is involved.

[0142] In some embodiments, a gene regulatory agent may comprise one or more components of a gene editing system and / or polynucleotides encoding same.

[0143] In some embodiments, the gene editing system may be a CRISPR-Cas system.

[0144] CRISPR systems Generally, CRISPR-Cas or CRISPR system, as used herein and in documents such as WO2014 / 093622 (PCT / US2013 / 074667), refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (encompassing "direct repeats" and, for endogenous CRISPR systems, tracrRNA-processed partial direct repeats), guide sequences (also referred to as "spacers" for endogenous CRISPR systems), or "RNA(s)" (as that term is used herein (e.g., RNA(s) that guide a Cas, such as Cas9, e.g., a CRISPR RNA and a trans-activating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA))), or other sequences and transcripts from a CRISPR locus. Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence (also called protospacers for endogenous CRISPR systems). See, e.g., Shmakov et al. (2015) "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems," Molecular Cell, DOI: dx.doi.org / 10.1016 / j.molcel.2015.10.008.

[0145] Class 1 series The methods, systems, and tools provided herein can be designed for use with Class 1 CRISPR proteins. In certain exemplary embodiments, Class 1 systems can be Type I, Type III, or Type IV Cas proteins, as described in Makarova et al. "Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants," Nature Reviews Microbiology, 18:67-81 (Feb. 2020), incorporated herein by reference in its entirety, particularly as described in Figure 1, p. 326. Class 1 systems typically use multi-protein effector complexes, which in some embodiments can include auxiliary proteins, e.g., one or more proteins of a complex called the CRISPR-associated complex for antiviral defense (cascade), one or more adaptation proteins (e.g., Cas1, Cas2, RNA nuclease), and / or one or more accessory proteins (e.g., Cas4, DNA nuclease), a CRISPR-associated Rossmann fold (CARF) domain-containing protein, and / or an RNA transcriptase. Although class 1 systems share limited sequence similarity, class 1 system proteins can be distinguished by a similar architecture, including one or more repeat-associated mysterious protein (RAMP) family subunits, such as Cas5, Cas6, and Cas7. RAMP proteins are characterized by having one or more RNA recognition motif domains. A large subunit (e.g., Cas8 or Cas10) and a small subunit (e.g., Cas11) are also typical of class 1 systems. See, e.g., Figures 1 and 2. Koonin EV, Makarova KS. 2019 Origins and evolution of CRISPR-Cas systems. Phil. Trans. R.Soc.B 374:20180087, DOI:10.1098 / rstb.2018.0087. In one aspect, class 1 systems are characterized by the signature protein Cas3.Cascades, particularly Class 1 proteins, may comprise specialized complexes of multiple Cas proteins that bind to pre-crRNA and recruit additional Cas proteins, such as Cas6 or Cas5, which are nucleases directly involved in pre-crRNA processing. In one embodiment, Type I CRISPR proteins comprise an effector complex containing one or more Cas5 subunits and two or more Cas7 subunits. Class 1 subtypes include types IA, IB, IC, IU, ID, IE, and IF, IV-A and IV-B, and III-A, III-D, III-C, and III-B. Class 1 systems also include CRISPR-Cas variants, including type IA, IB, IE, IF, and IU variants, which can include transposon- and plasmid-borne variants, including subtype IF versions encoded by a large family of Tn7-like transposons, as well as a small group of Tn7-like transposons that encode similarly degraded subtype IB systems. See also Peters et al., PNAS 114(35)(2017); DOI:10.1073 / pnas.1709035114; Makarova et al., The CRISPR Journal, v.1, n5, Figure 5.

[0146] Class 2 series Compositions, systems, and methods described in more detail elsewhere herein can be designed and adapted for use with Class 2 CRISPR-Cas systems. Thus, in some embodiments, the CRISPR-Cas system is a Class 2 CRISPR-Cas system. Class 2 systems are distinguished from Class 1 systems by possessing a single, large, multidomain effector protein. In certain exemplary embodiments, the Class 2 system can be a Type II, Type V, or Type VI system, as described in Makarova et al., "Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants," Nature Reviews Microbiology, 18:67-81 (Feb. 2020), incorporated herein by reference. Each type of Class 2 system is further divided into subtypes. See Markova et al. 2020, particularly Figure 2. Class 2, Type II systems can be divided into four subtypes: II-A, II-B, II-C1, and II-C2. Class 2, type V systems can be divided into 17 subtypes: VA, V-B1, V-B2, VC, VD, VE, V-F1, V-F1(V-U3), V-F2, V-F3, VG, VH, VI, VK(V-U5), V-U1, V-U2, and V-U4. Class 2, type IV systems can be divided into 5 subtypes: VI-A, VI-B1, VI-B2, VI-C, and VI-D.

[0147] A distinctive feature of these types is that their effector complex consists of a single, large, multidomain protein. Unlike type II effectors (e.g., Cas9), which contain two core domains, each responsible for cleaving a single strand of target DNA, type V systems incorporate an HNH nuclease within the Ruv-C-like nuclease domain sequence. Type V systems (e.g., Cas12) contain only the RuvC-like nuclease domain, which cleaves both strands. Type VI (Cas13), which is unrelated to type II and type V effectors, contains two HEPN domains and a target RNA. The Cas13 protein also exhibits additional activity triggered by target recognition. Some type V systems have also been shown to possess this additional activity with two single-stranded DNA in vitro.

[0148] In some embodiments, the Class 2 system is a Type II system. In some embodiments, the Type II CRISPR-Cas system is a Type II-A CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a Type II-B CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a Type II-C1 CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a Type II-C2 CRISPR-Cas system. In some embodiments, the Type II system is a Cas9 system. In some embodiments, the Type II system comprises Cas9.

[0149] In some embodiments, the Class 2 system is a Type V system. In some embodiments, the Type V CRISPR-Cas system is a Type VA CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a Type V-B1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a Type V-B2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a Type VC CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a Type VD CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a Type VE CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a Type V-F1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a Type V-F1 (V-U3) CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type V-F2 CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type V-F3 CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type VG CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type VH CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type VI CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type VK (V-U5) CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type V-U1 CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type V-U2 CRISPR-Cas system. In some embodiments, the type V CRISPR-Cas system is a type V-U4 CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system comprises Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas14, and / or CasΦ.

[0150] In some embodiments, the Class 2 system is a Type VI system. In some embodiments, the Type VI CRISPR-Cas system is a Type VI-A CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a Type VI-B1 CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a Type VI-B2 CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a Type VI-C CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a Type VI-D CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system comprises Cas13a (C2c2), Cas13b (Group 29 / 30), Cas13c, and / or Cas13d.

[0151] CRISPR-Cas-based cargo molecules Generally, CRISPR-Cas or CRISPR system, as used herein and in documents such as WO2014 / 093622 (PCT / US2013 / 074667), refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (encompassing "direct repeats" and, for endogenous CRISPR systems, tracrRNA-processed partial direct repeats), guide sequences (also referred to as "spacers" for endogenous CRISPR systems), or "RNA(s)" (as that term is used herein (e.g., RNA(s) that guide a Cas, such as Cas9, e.g., a CRISPR RNA and a trans-activating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA))), or other sequences and transcripts from a CRISPR locus. Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence (also called protospacers for endogenous CRISPR systems). See, e.g., Shmakov et al. (2015) "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems," Molecular Cell, DOI: dx.doi.org / 10.1016 / j.molcel.2015.10.008.

[0152] In certain embodiments, a protospacer adjacent motif (PAM) or PAM-like motif directs binding of the effector protein complex disclosed herein to a target locus of interest. In some embodiments, the PAM may be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM may be a 3' PAM (i.e., located downstream of the 5' end of the protospacer). The term "PAM" may be used interchangeably with the terms "PFS" or "protospacer adjacent site" or "protospacer adjacent sequence."

[0153] In preferred embodiments, the CRISPR effector protein can recognize a 3' PAM. In certain embodiments, the CRISPR effector protein can recognize a 3' PAM that is 5' H, where H is A, C, or U.

[0154] In the context of CRISPR complex formation, the term "target sequence" refers to a sequence to which a guide sequence is designed to be complementary, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence may comprise an RNA polynucleotide. The term "target RNA" refers to an RNA polynucleotide that is or contains a target sequence. In other words, the target RNA may be an RNA polynucleotide or a portion of an RNA polynucleotide, and a portion of the gRNA, i.e., the guide sequence, is designed to be complementary to the target RNA, and the effector function mediated by the complex containing the CRISPR effector protein and the gRNA is directed to the target RNA. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0155] In certain exemplary embodiments, the CRISPR effector protein may be delivered using a nucleic acid molecule encoding the CRISPR effector protein. The nucleic acid molecule encoding the CRISPR effector protein may advantageously be a codon-optimized CRISPR effector protein. An example of a codon-optimized sequence is a sequence optimized for expression in a eukaryote, for example, a human (i.e., optimized for human expression), or a sequence optimized for another eukaryote, animal, or mammal as described herein; see, for example, the SaCas9 human codon-optimized sequence in WO2014 / 093622 (PCT / US2013 / 074667). Although the above is preferred, other examples are possible, and it is understood that codon optimization for host species other than humans, or codon optimization for specific organs, is known. In some embodiments, the enzyme-coding sequence encoding the CRISPR effector protein is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells may be cells of or derived from a particular organism, such as a plant or mammal, including, but not limited to, a human, or a non-human eukaryote or animal or mammal as described herein, e.g., a mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, processes for modifying the germline genetic identity of a human and / or processes for modifying the genetic identity of an animal likely to cause suffering to a human or animal without substantial medical benefit may be excluded, as well as animals resulting from such processes. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) with a codon more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit particular biases for particular codons for particular amino acids.Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Thus, with codon optimization, genes can be tailored for optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at kazusa.orjp / codon / , and these tables can be adapted in many ways. See Nakamura, Y., et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available, for example, GeneForge (Aptagen; Jacobus, PA), for optimizing codons for specific sequences for expression in specific host cells. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in the Cas-encoding sequence correspond to the most frequently used codon for a particular amino acid.

[0156] In certain embodiments, the methods described herein may include providing a Cas transgenic cell, into which one or more nucleic acids encoding one or more guide RNAs are provided or introduced operably linked to regulatory elements, including promoters, of one or more genes of interest. As used herein, the term "Cas transgenic cell" refers to a cell, such as a eukaryotic cell, in which a Cas gene has been integrated into its genome. The nature, type, or origin of the cell is not particularly limiting according to the present invention. Additionally, the method for introducing a Cas transgene into a cell may vary and may be any method known in the art. In certain embodiments, the Cas transgenic cell is obtained by introducing a Cas transgene into an isolated cell. In certain other embodiments, the Cas transgenic cell is obtained by isolating cells from a Cas transgenic organism. By way of example, and not limitation, the Cas transgenic cell referred to herein may be derived from a Cas transgenic eukaryotic organism, such as a Cas knock-in eukaryotic organism. See WO2014 / 09622 (PCT / US13 / 74667), which is incorporated herein by reference. Methods in U.S. Patent Publication Nos. 20120017290 and 20110265198, assigned to Sangamo BioSciences, Inc., which are directed to targeting the Rosa locus, may be modified to utilize the CRISPR-Cas system of the present invention. Methods in U.S. Patent Publication No. 20130236946, assigned to Cellectis, which are directed to targeting the Rosa locus, may also be modified to utilize the CRISPR-Cas system of the present invention. For further example, see Platt et al. (Cell; 159(2):440-455(2014)), which describes a Cas9 knock-in mouse (incorporated herein by reference). The Cas transgene may further comprise a Lox-Stop-polyA-Lox (LSL) cassette, allowing Cas expression to be induced by Cre recombinase. Alternatively, Cas transgenic cells may be obtained by introducing the Cas transgene into isolated cells. Transgene delivery systems are well known in the art.For example, Cas transgenes can be delivered to eukaryotic cells, for example, by vector (e.g., AAV, adenovirus, lentivirus) and / or particle and / or nanoparticle delivery, as described elsewhere herein. Lentivirus and retrovirus systems, as well as non-viral systems for delivering CRISPR-Cas system components, are generally known in the art. AAV and adenovirus-based systems for CRISPR-Cas system components are described herein as well as generally known in the art (e.g., the modified AAVs of the present invention).

[0157] Those skilled in the art will understand that cells such as the Cas transgenic cells referred to herein may contain additional genomic modifications in addition to having mutations resulting from the sequence-specific action of Cas when complexed with the integrated Cas gene or RNA that guides Cas to the target locus.

[0158] In certain embodiments, the present invention includes vectors, for example, for delivering or introducing Cas and / or RNA capable of guiding Cas to a target locus (i.e., guide RNA) into a cell, as well as for amplifying these components (e.g., in a prokaryotic cell). This can be in addition to the delivery of one or more CRISPR-Cas components or other gene modification system components not already delivered by the modified particles described herein. As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted, thereby replicating the inserted segment. Generally, a vector is replicable when associated with appropriate control elements. Generally, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that contain one or more free ends, nucleic acid molecules that do not contain free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in a vector for packaging into a virus (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAV)). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0159] A recombinant expression vector can contain the nucleic acid of the present invention in a form suitable for expression in a host cell, meaning that the recombinant expression vector contains one or more regulatory elements (which can be selected based on the host cell used for expression) operably linked to the nucleic acid sequence to be expressed. "Operably linked" in a recombinant expression vector is intended to mean that the nucleotide sequence of interest is linked to a regulatory element(s) in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or within a host cell when the vector is introduced into the host cell). For recombinant and cloning methods, reference is made to U.S. Patent Application No. 10 / 815,730, published September 2, 2004 as US2004-0171156A1, the contents of which are incorporated herein by reference in their entirety. Thus, the embodiments disclosed herein can also include transgenic cells containing a CRISPR effector system. In certain exemplary embodiments, the transgenic cells can function as individual, separate volumes. In other words, a sample containing the masking construct may be delivered to a cell, for example, in a suitable delivery vesicle, and if the target is present in the delivery vesicle, the CRISPR effector is activated and a detectable signal is generated.

[0160] The vector(s) may include regulatory element(s), e.g., promoter(s). The vector(s) may include a Cas coding sequence and / or may also include a sequence encoding a single, but possibly at least 3 or 8 or 16 or 32 or 48 or 50 guide RNA(s) (e.g., sgRNA), e.g., 1-2, 1-3, 1-4, 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s) (e.g., sgRNA). In a single vector, conveniently, if there are up to about 16 RNA(s), a promoter can be present for each RNA (e.g., sgRNA); and if a single vector provides more than 16 RNA(s), one or more promoter(s) can drive expression of multiple RNA(s). For example, if there are 32 RNA(s), each promoter can drive expression of two RNA(s), and if there are 48 RNA(s), each promoter can drive expression of three RNA(s). With simple arithmetic, well-established cloning protocols, and the teachings of this disclosure, one skilled in the art can easily implement the present invention for RNA(s) for a suitable exemplary vector, such as AAV, and a suitable promoter, such as the U6 promoter. For example, the packaging limit of AAV is approximately 4.7 kb. The length of a single U6-gRNA (and restriction site for cloning) is 361 bp. Thus, one skilled in the art can easily fit approximately 12 to 16, e.g., 13, U6-gRNA cassettes into a single vector. This can be assembled by any suitable means, such as the Golden Gate strategy used for TALE assembly (genome-engineering.org / taleffectors / ). One skilled in the art can also use a tandem guide strategy to increase the number of U6-gRNAs by approximately 1.5-fold, e.g., from 12-16, e.g., 13, to approximately 18-24, e.g., about 19, U6-gRNAs.Thus, one skilled in the art can easily achieve approximately 18 to 24, e.g., about 19, promoter RNAs, e.g., U6-gRNAs, in a single vector, e.g., an AAV vector. Another approach to increasing the number of promoters and RNAs in a vector is to use a single promoter (e.g., U6) to express an array of promoter RNAs separated by cleavable sequences. Yet another approach to increasing the number of promoter RNAs in a vector is to express an array of promoter RNAs separated by cleavable sequences within the coding sequence or intron of a gene; in this case, it is advantageous to use a polymerase II promoter, which increases expression and allows transcription of long RNAs in a tissue-specific manner (see, e.g., nar.oxfordjournals.org / content / 34 / 7 / e53.short and nature.com / mt / journal / v16 / n9 / abs / mt2008144a.html). In an advantageous embodiment, an AAV can package U6 tandem gRNAs targeting up to about 50 genes. Thus, from knowledge in the art and the teachings of this disclosure, one of skill in the art can readily make and use vector(s), e.g., a single vector, that expresses multiple RNAs or guides under the control of one or more promoters or operably or functionally linked together, without undue experimentation, particularly with respect to the number of RNAs or guides described herein.

[0161] The sequence encoding the guide RNA(s) and / or the sequence encoding the Cas can be functionally or operably linked to a regulatory element(s), which then drives expression. The promoter(s) can be constitutive promoter(s) and / or conditional promoter(s) and / or inducible promoter(s) and / or tissue-specific promoter(s). The promoter can be selected from the group consisting of RNA polymerase, pol I, pol II, pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. A preferred promoter is promoter U6.

[0162] Additional effectors for use in accordance with the present invention can be identified by their proximity to the Cas1 gene, for example, but not limited to, within a region 20 kb from the start of the Cas1 gene and 20 kb from the end of the Cas1 gene. In certain embodiments, the effector protein comprises at least one HEPN domain and at least 500 amino acids, and the C2c2 effector protein is naturally present in the prokaryotic genome within 20 kb upstream or downstream of the Cas gene or CRISPR array. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas12a, Cas13a, Cas13b, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csx1, Csx ... Examples of C2c2 effector proteins include sa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or variants thereof. In certain exemplary embodiments, the C2c2 effector protein is naturally present in a prokaryotic genome within 20 kb upstream or downstream of the Cas1 gene. The terms "orthologue" (also referred to herein as "ortholog") and "homologue" (also referred to herein as "homolog") are well known in the art. By way of further guidance, a "homologue" of a protein, as used herein, is a protein of the same species that performs the same or similar function as the protein to which it is homologous. Homologous proteins may, but are not necessarily, structurally related, or are only partially structurally related. As used herein, an "ortholog" of a protein is a protein of a different species that performs the same or similar function as the protein to which it is ortholog. Orthologous proteins may, but are not necessarily, structurally related, or are only partially structurally related.

[0163] In some embodiments, one or more elements of the nucleic acid targeting system are derived from a particular organism that contains an endogenous CRISPR RNA targeting system. In certain embodiments, the CRISPR RNA targeting system is found in Eubacterium and Ruminococcus. In certain embodiments, the effector protein contains targeted additional ssRNA cleavage activity. In certain embodiments, the effector protein contains a dual HEPN domain. In certain embodiments, the effector protein lacks the corresponding helical-1 domain of Cas13a. In certain embodiments, the effector protein is smaller than previously characterized Class 2 CRISPR effectors, with a median size of 928 aa. This median size is 190 aa (17%) smaller than Cas13c, more than 200 aa (18%) smaller than Cas13b, and more than 300 aa (26%) smaller than Cas13a. In certain embodiments, the effector protein does not require flanking sequences (e.g., PFS, PAM).

[0164] In certain embodiments, the structure of the effector protein locus includes a WYL domain containing accessory protein (denoted after the three amino acids conserved in the first identified group of these domains; see, e.g., WYL domain IPR026881). In certain embodiments, the WYL domain accessory protein includes at least one helix-turn-helix (HTH) or ribbon-helix-helix (RHH) DNA-binding domain. In certain embodiments, the WYL domain containing accessory protein increases both the targeting and concomitant ssRNA cleavage activity of the RNA-targeting effector protein. In certain embodiments, the WYL domain containing accessory protein includes an N-terminal RHH domain and a pattern of primarily hydrophobic conserved residues, including an invariant tyrosine-leucine doublet corresponding to the original WYL motif. In certain embodiments, the WYL domain containing accessory protein is WYL1. WYL1 is a single WYL domain protein primarily associated with Ruminococcus.

[0165] In another exemplary embodiment, the type VI RNA-targeting Cas enzyme is Cas13d. In certain embodiments, Cas13d is Eubacterium siraeum DSM 15702 (EsCas13d) or Ruminococcus species N15.MGS-57 (RspCas13d) (see, e.g., Yan et al., "Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein," Molecular Cell (2018), doi.org / 10.1016 / j.molcel.2018.02.028). RspCas13d and EsCas13d do not have flanking sequence requirements (e.g., PFS, PAM).

[0166] The methods, systems, and tools provided herein may be designed for use with Class 1 CRISPR proteins, which are Type I, Type III, or Type IV Cas proteins, as described in Makarova et al., The CRISPR Journal, v. 1, n., 5 (2018); DOI: 10.1089 / crispr.2018.0033 (incorporated herein by reference in its entirety), particularly as described in Figure 1, p. 326. Class 1 systems typically use multi-protein effector complexes, which in some embodiments may include auxiliary proteins, e.g., one or more proteins of a complex called the CRISPR-associated complex for antiviral defense (cascade), one or more adaptation proteins (e.g., Cas1, Cas2, RNA nuclease), and / or one or more accessory proteins (e.g., Cas4, DNA nuclease), a CRISPR-associated Rossmann fold (CARF) domain-containing protein, and / or an RNA transcriptase. Although class 1 systems share limited sequence similarity, class 1 system proteins can be distinguished by a similar architecture, including one or more repeat-associated mysterious protein (RAMP) family subunits, such as Cas5, Cas6, and Cas7. RAMP proteins are characterized by having one or more RNA recognition motif domains. A large subunit (e.g., Cas8 or Cas10) and a small subunit (e.g., Cas11) are also typical of class 1 systems. See, e.g., Figures 1 and 2. Koonin EV, Makarova KS. 2019 Origins and evolution of CRISPR-Cas systems. Phil. Trans. R. Soc. B 374:20180087, DOI:10.1098 / rstb.2018.0087. In one embodiment, class 1 systems are characterized by the signature protein Cas3. The Cascade, especially the Class 1 proteins, may comprise a dedicated complex of multiple Cas proteins that bind to the pre-crRNA and recruit additional Cas proteins such as Cas6 or Cas5, which are nucleases directly involved in pre-crRNA processing.In one embodiment, the type I CRISPR protein comprises an effector complex comprising one or more Cas5 subunits and two or more Cas7 subunits. Class 1 subtypes include types IA, IB, IC, IU, ID, IE, and IF, IV-A and IV-B, and III-A, III-D, III-C, and III-B. Class 1 systems also include CRISPR-Cas variants, including type IA, IB, IE, IF, and IU variants, which can include transposon- and plasmid-borne variants, including versions of subtype IF encoded by a large family of Tn7-like transposons, as well as a small group of Tn7-like transposons that encode degraded subtype IB systems. See also Peters et al., PNAS 114(35)(2017); DOI:10.1073 / pnas.1709035114; Makarova et al, the CRISPR Journal, v.1, n5, Figure 5.

[0167] targeting part In some embodiments, the modified delivery system can further comprise a targeting moiety that can specifically bind to target cells.In order to effectively target delivery vesicles to cells such as cancer cells, it is useful that the targeting moiety has affinity for cell surface receptors, and that the targeting moiety is linked in an amount sufficient to have optimal affinity for cell surface receptors; and it is within the scope of those skilled in the art to determine these aspects.In the field of active targeting, there are some cell-specific, for example, tumor-specific, targeting ligands.

[0168] Regarding active targeting, targeting cell surface receptors, such as cancer cell surface receptors, can involve the attachment of liposomes to cells, e.g., vascular cells, via non-internalizing epitopes; this can increase the extracellular concentration of the delivered substance, thereby increasing the amount delivered to the target cell. A strategy for targeting cell surface receptors, e.g., overexpressed cell surface receptors on cancer cells, is to use receptor-specific ligands or antibodies. Many cancer cell types exhibit upregulation of tumor-specific receptors. For example, TfR and folate receptors (FRs) are significantly overexpressed in many tumor cell types in response to increased metabolic demand. Folate can be easily conjugated to nanocarriers, has a high affinity for FRs, and is relatively rare in normal tissues compared to activated macrophages and cancer cells, e.g., certain ovarian, breast, lung, colon, kidney, and brain tumors. Therefore, it can be used as a targeting ligand for specialized delivery. Overexpression of FRs in macrophages is a sign of inflammatory diseases such as psoriasis, Crohn's disease, rheumatoid arthritis, and atherosclerosis; therefore, the folate-mediated targeting of the present invention can also be used to study, address, or treat inflammatory disorders and cancer. The folate-conjugated lipid particles or nanoparticles or liposomes or lipid bilayers of the present invention ("lipid entities of the present invention") deliver their cargo into cells via receptor-mediated endocytosis. Intracellular trafficking can be directed to acidic compartments, which promote cargo release, and, most importantly, cargo release can be altered or delayed until it reaches the cytoplasm or near the target organelle. Cargo delivery using lipid entities of the present invention with targeting moieties, such as folate-conjugated lipid entities of the present invention, may be superior to non-targeted lipid entities of the present invention. Direct conjugation of folate to the lipid headgroup may not be preferable for intracellular delivery of the folate-conjugated lipid entities of the present invention, as it may not bind to cells as efficiently as folate conjugated to the surface of the lipid entities of the present invention via a spacer (which may enter cancer cells more efficiently).The lipid entities of the present invention bound to folic acid can be used to deliver lipid complexes, e.g., liposomes, e.g., anionic liposomes, and viruses or capsids or envelopes or viral external proteins, e.g., adenovirus or AAV, as described herein. Tf is an approximately 80 kDa monomeric serum glycoprotein involved in iron transport throughout the body. Tf binds to TfR and enters cells by receptor-mediated endocytosis. TfR expression can be elevated in certain cells, such as tumor cells (compared to normal cells), and is associated with increased iron demand in rapidly proliferating cancer cells. Therefore, the present invention includes the use of TfR-targeted lipid entities of the present invention in, for example, hepatocytes, liver cancer cells, breast cells, e.g., breast cancer cells; colon cells, e.g., colon cancer cells; ovarian cells, e.g., ovarian cancer cells; head, neck, and lung cells, e.g., head, neck, and non-small cell lung cancer cells; and oral cells, e.g., oral tumor cells.

[0169] Furthermore, with regard to active targeting, the lipid entities of the present invention can be multifunctional, i.e., multiple targeting moieties, such as CPPs, can be used in conjunction with Tf; bifunctional systems can be used; for example, a combination of Tf and poly-L-arginine can be used, which can provide transport across the endothelium of the blood-brain barrier. EGFR is a tyrosine kinase receptor belonging to the ErbB family of receptors that mediates cell proliferation, differentiation, and repair in cells, particularly non-cancerous cells. However, EGF is overexpressed in certain cells, such as many solid tumors, including colorectal cancer, non-small cell lung cancer, ovarian squamous cell carcinoma, renal cancer, head cancer, pancreatic cancer, neck cancer, and prostate cancer, particularly breast cancer. The present invention encompasses EGFR-targeting monoclonal antibodies(ies) linked to the lipid entities of the present invention. HER-2 is often overexpressed in patients with breast cancer and is also associated with lung cancer, bladder cancer, prostate cancer, brain cancer, and gastric cancer. HER-2 is encoded by the ERBB2 gene. The present invention includes HER-2-targeting lipid entities of the present invention, such as anti-HER-2 antibody (or binding fragment thereof) lipid entities of the present invention, HER-2-targeting PEGylated lipid entities of the present invention (e.g., carrying an anti-HER-2 antibody or binding fragment thereof), and HER-2-targeting maleimide-PEG polymer lipid entities of the present invention (e.g., carrying an anti-HER-2 antibody or binding fragment thereof). Upon cell binding, the receptor-antibody complex can be internalized by endosome formation for delivery to the cytoplasm. With respect to receptor-mediated targeting, those skilled in the art will take into account ligand / target affinity and the amount of receptor on the cell surface, as well as the fact that PEGylation may function as a barrier to receptor interaction. The use of antibody-lipid entities for targeting of the present invention may be advantageous. Multivalent presentation of targeting moieties may also increase the uptake and signaling properties of antibody fragments. In practicing the present invention, those skilled in the art will also take into account ligand density (e.g., high ligand density on lipid entities of the present invention may be advantageous for increased binding to target cells). Early prevention by macrophages can be addressed with the sterically stabilized lipid entities of the present invention and by attaching ligands to the termini of molecules such as PEG that are immobilized on the lipid entities of the present invention (e.g. lipid particles or nanoparticles or liposomes or lipid bilayers).The cell mass microenvironment, such as the tumor microenvironment, can be targeted; for example, it may be advantageous to target the cell mass vasculature, such as the tumor vasculature microenvironment. Thus, the present invention encompasses targeting VEGF. VEGF and its receptors are well-known pro-angiogenic molecules and well-characterized targets for antiangiogenic therapy. Many small molecule inhibitors of receptor tyrosine kinases, such as VEGFR or basic FGFR, have been developed as anticancer drugs, and the present invention encompasses conjugating any one or more of these peptides to lipid entities of the present invention, e.g., phage IVO peptide(s) (e.g., via or by PEG terminus), tumor-homing peptide APRPG (SEQ ID NO: 4), such as APRPG-PEG-modified. The vascular endothelium, VCAM, plays an important role in the pathogenesis of inflammation, thrombosis, and atherosclerosis. CAMs are involved in inflammatory diseases, including cancer, and are logical targets; E-selectin and P-selectin, VCAM-1, and ICAM can be used to target the lipid entities of the present invention, e.g., by PEGylation. Matrix metalloproteinases (MMPs) belong to the zinc-dependent endopeptidase family. They are involved in tissue remodeling, tumor invasiveness, apoptosis, and resistance to metastasis. There are four MMP inhibitors, designated TIMPs 1-4, which determine the balance between tumor growth inhibition and metastasis; MT1-MMP is a protein involved in tumor vascular angiogenesis and is expressed in newly formed blood vessels and tumor tissue. The proteolytic activity of MT1-MMP cleaves proteins such as fibronectin, elastin, collagen, and laminin at the plasma membrane, activating soluble MMPs such as MMP-2, which degrade the matrix. Antibodies or fragments thereof, e.g., Fab' fragments, can be used in the practice of the present invention, such as anti-human MT1-MMP monoclonal antibodies linked to the lipid entities of the present invention via a spacer, e.g., a PEG spacer. αβ-integrins, or integrins, are a group of transmembrane glycoprotein receptors that mediate binding between cells and their surrounding tissue or extracellular matrix. Integrins contain two separate chains (heterodimers) called the α and β subunits.Tumor tissue-specific expression of integrin receptors can be exploited for targeted delivery in the present invention, whereby, for example, the targeting moiety can be an RGD peptide, such as cyclic RGD. Aptamers are ssDNA or RNA oligonucleotides that confer high affinity and specific recognition of target molecules through electrostatic, hydrogen, and hydrophobic interactions, in contrast to Watson-Crick base pairing, which is common in oligonucleotide binding interactions. Aptamers as targeting moieties may have advantages over antibodies: they can exhibit higher target antigen recognition than antibodies; they can be more stable and smaller in size than antibodies; they can be easily synthesized and chemically modified for molecular conjugation; and they can be modified to improve selectivity and developed to recognize less immunogenic targets. Such moieties, such as the sgc8 aptamer, can be used as targeting moieties (e.g., via covalent attachment to the lipid entities of the present invention, e.g., via a spacer, such as a PEG spacer). The targeting moiety may be stimulus-sensitive, e.g., sensitive to an externally applied stimulus such as a magnetic field, ultrasound, or light; or a pH trigger may be used, e.g., a labile bond between a hydrophilic moiety, such as PEG, and a hydrophobic moiety, such as a lipid entity of the invention, which bond is cleaved only upon exposure to the relatively acidic conditions characteristic of a particular environment or microenvironment, such as an intracellular vacuole or an acidic tumor mass.pH-sensitive copolymers can also be incorporated into embodiments of the present invention to provide shielding; diorthoesters, vinyl esters, cysteine-cleavable lipopolymers, double esters, and hydrazones are some examples of pH-sensitive linkages that are highly stable at pH 7.5 but relatively rapidly hydrolyze below pH 6, such as terminally alkylated copolymers of N-isopropylacrylamide and methacrylic acid, which promote destabilization of the lipid entities of the present invention and release in compartments with reduced pH; or, the present invention encompasses ionic polymers for generating pH-responsive lipid entities of the present invention (e.g., poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(acrylamide), and poly(acrylic acid)). Temperature-triggered delivery is also within the scope of the present invention. Many pathological regions, such as inflamed tissues and tumors, exhibit characteristic hyperthermia compared to normal tissue. Because hyperthermia is associated with increased tumor permeability and enhanced uptake, exploiting this hyperthermia is an attractive strategy for cancer therapy. This technique involves localized heating of the site to increase microvascular pore size and blood flow, which in turn can result in increased extravasation in embodiments of the present invention. The temperature-sensitive lipid entities of the present invention can be prepared from temperature-sensitive lipids or polymers with a low critical solution temperature. When the low critical solution temperature is exceeded (e.g., at a site such as a tumor or inflamed tissue), the polymer precipitates, rupturing and releasing the liposomes. Lipids with specific gel-to-liquid phase transition temperatures are used to prepare these lipid entities of the present invention; the lipid in temperature-sensitive embodiments can be dipalmitoylphosphatidylcholine. Temperature-sensitive polymers can also promote destabilization and subsequent release; a useful temperature-sensitive polymer is poly(N-isopropylacrylamide). Another temperature-triggered system can use lysolipid temperature-sensitive liposomes. The present invention also encompasses redox-triggered delivery: the difference in redox potential between normal tissue and inflamed or tumor tissue, and between the intracellular and extracellular environments, is utilized for delivery; for example, GSH is a reducing agent that is abundant in cells, particularly the cytosol, mitochondria, and nucleus.The concentrations of GSH in the blood and extracellular matrix are 100-1000 times lower than the intracellular concentration, respectively. This high redox potential caused by GSH, cysteine, and other reducing agents can disrupt reducible bonds, destabilize the lipid entities of the present invention, and result in the release of the payload. Disulfide bonds can be used as cleavable / reversible linkers in the lipid entities of the present invention because they are sensitive to redox via the reduction of disulfides to thiols; lipid entities of the present invention can be made reduction-sensitive by using two (e.g., two forms of disulfide-linked multifunctional lipids (e.g., via tris(2-carboxyethyl)phosphine, dithiothreitol, L-cysteine, or GSH) as disulfide bond cleavage can cause removal of the hydrophilic head group of the complex, altering membrane organization and resulting in release of the payload. Calcein release from reduction-sensitive lipid entities of the present invention containing disulfide complexes can be more useful than reduction-insensitive embodiments. Enzymes can also be used as triggers to release the payload, such as MMPs (e.g., MMP2), phospholipase A2, alkaline phosphatase, transglutaminase, etc. Enzymes such as phospholipase C or phosphatidylinositol-specific phospholipase C have been shown to be overexpressed in certain tissues, such as tumor tissue. In the presence of these enzymes, the specifically engineered enzyme-sensitive lipid entities of the present invention can be broken down, releasing the payload. An MMP2-cleavable octapeptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) (SEQ ID NO: 5) can be incorporated into the linker, allowing antibody targeting, e.g., antibody 2C5. The present invention also encompasses light- or energy-triggered delivery. For example, the lipid entities of the present invention can be photosensitive, and light or energy can promote structural and conformational changes, leading to direct interaction of the lipid entities of the present invention with target cells via membrane fusion, photoisomerization, photofragmentation, or photopolymerization; therefore, such moieties can be benzoporphyrin photosensitizers.Ultrasound can be one form of energy that induces delivery; lipid entities of the present invention that contain air or a small amount of certain gases, including perfluorinated hydrocarbons, can be induced to be released by ultrasound, for example, low-frequency ultrasound (LFUS).Magnetic delivery: lipid entities of the present invention can be magnetized by incorporating magnetite, such as Fe3O4 or γ-Fe2O3, for example, magnetite with a size of less than 10 nm.Targeted delivery can be by exposure to magnetic field.

[0170] Regarding active targeting, the present invention also encompasses intracellular delivery. As liposomes follow the endocytic pathway, they become trapped in endosomes (pH 6.5-6) ​​and subsequently fuse with lysosomes (pH <5), where they undergo degradation and become less therapeutic. A low endosomal pH can be utilized to avoid degradation. Fusogenic lipids or fusogenic peptides destabilize the endosomal membrane after conformational change / activation at low pH. Amines are protonated at acidic pH, causing swelling and rupture of endosomes through a buffering effect. Unsaturated dioleoylphosphatidylethanolamine (DOPE) readily adopts an inverted hexagonal shape at low pH, allowing liposomes to fuse with the endosomal membrane. This process destabilizes lipid entities, including DOPE, releasing the cargo into the cytoplasm; the fusogenic lipids GALA, cholesteryl-GALA, and PEG-GALA can exhibit highly efficient endosomal release; the pore-forming protein listeriolysin O can provide an endosomal escape mechanism; and histidine-rich peptides have the ability to fuse with endosomal membranes to form pores, buffering proton pumping and causing membrane lysis.

[0171] Regarding active targeting, cell-penetrating peptides (CPPs) facilitate the uptake of macromolecules through the cell membrane, thus enhancing the intracellular delivery of CPP-modified molecules. CPPs can be divided into two classes: amphipathic helical peptides, such as transportans and MAPs, in which lysine residues contribute primarily to the positive charge; and Arg-rich peptides, such as TATp, Antennapedia, or penetratin. TATp is an 86-amino acid transcriptional activator containing a highly basic (two Lys and six Arg of the nine residues) protein transduction domain that confers nuclear localization and RNA binding. Other CPPs that have been used to modify liposomes include: the minimal protein transduction domain of Antennapedia, a Drosophila homeoprotein called penetratin, a 16-mer peptide (residues 43-58) located in the third helix of the homeodomain; a 27-amino acid chimeric CPP containing a peptide sequence from the amino terminus of the neuropeptide galanin linked via a Lys residue, mastoparan, a bee venom peptide; VP22, a major structural component of HSV-1 that facilitates intracellular transport; and transportan (an 18-mer amphipathic model peptide) that translocates the plasma membrane of mast cells and endothelial cells by both energy-dependent and energy-independent mechanisms. The present invention encompasses lipid entities of the present invention modified with CPP(s) for intracellular delivery, which may proceed via energy-dependent macropinocytosis followed by endosomal escape. The present invention further encompasses organelle-specific targeting. Lipid entities of the present invention surface-functionalized with triphenylphosphonium (TPP) moieties or rhodamine 123, a lipid entity of the present invention having a lipophilic cation, can be effective in delivering cargo to mitochondria. DOPE / sphingomyelin / stearyl-octa-arginine can deliver cargo into mitochondria via membrane fusion. Octadecylrhodamine B, a lipid entity of the present invention surface-modified with a lysosomotropic ligand, can deliver cargo to lysosomes. Ceramide is useful for inducing permeabilization of lysosomal membranes; the present invention encompasses intracellular delivery of lipid entities of the present invention having ceramide.The present invention further encompasses lipid entities of the present invention that are targeted to the nucleus, for example, via a DNA-intercalating moiety. The present invention also encompasses multifunctional liposomes for targeting, i.e., for attaching multiple functional groups to the surface of the lipid entities of the present invention, for example, to enhance accumulation at a desired site, and / or promote organelle-specific delivery, and / or target specific cell types, and / or respond to local stimuli such as temperature (e.g., increased), pH (e.g., decreased), respond to externally applied stimuli such as magnetic fields, light, energy, heat, or ultrasound, and / or promote intracellular delivery of cargo. All of these are considered active targeting moieties.

[0172] The embodiments of the present invention include lipid particles or nanoparticles or liposomes or lipid bilayer delivery systems with active targeting; or lipid particles or nanoparticles or liposomes or lipid bilayers with targeting moieties, thereby providing active targeting, or delivery systems in which the targeting moiety is an active targeting moiety.The targeting moiety can be one or more targeting moieties, and the targeting moiety can be, for example, for targeting any cell mentioned herein; or for targeting organelles as described herein; or for targeting responses such as physical conditions, for example, heat, energy, ultrasound, light, pH, chemicals such as enzymes, or magnetic stimuli; or for achieving specific results, such as delivering a payload to a specific location by cell penetration, etc.

[0173] It should be understood that for each possible targeting moiety or active targeting moiety described herein, there is an embodiment of the invention in which a delivery system comprises such a targeting moiety or active targeting moiety. Similarly, the following table provides exemplary targeting moieties that can be used in the practice of the invention, and for each embodiment of the invention, provides a delivery system comprising such a targeting moiety. [Table 1]

[0174] Thus, in embodiments of the delivery system, the targeting moiety comprises a receptor ligand (e.g., hyaluronic acid for the CD44 receptor, galactose for hepatocytes, or an antibody or fragment thereof, e.g., a binding antibody fragment for a desired surface receptor), and for each targeting moiety comprising a receptor ligand, or an antibody or fragment thereof, e.g., a binding fragment thereof, for a desired surface receptor, there are aspects of the invention in which the delivery system comprises a targeting moiety comprising a receptor ligand, or an antibody or fragment thereof, e.g., a binding fragment thereof, for a desired surface receptor, or hyaluronic acid for the CD44 receptor, galactose for hepatocytes (see, e.g., Surace et al., “Lipoplexes targeting the CD44 hyaluronic acid receptor for efficient transfection of breast cancer cells,” J. Mol Pharm 6(4):1062-73; doi:10.1021 / mp800215d (2009); Sonoke et al., “Galactose-modified cationic liposomes as a liver-targeting delivery system for small interfering RNA,” Biol Pharm Bull.34(8):1338-42(2011);Torchilin,“Antibody-modified liposomes for cancer chemotherapy,” Expert Opin. Drug Deliv.5(9),1003-1025(2008);Manjappa et al,“Antibody derivatization and conjugation strategies:application in preparation of stealth immunoliposome to target chemotherapeutics to tumor,” J.Control.See Release 150(1), 2-22(2011); Sofou S, "Antibody-targeted liposomes in cancer therapy and imaging," Expert Opin. Drug Deliv. 5(2):189-204(2008); Gao J et al, "Antibody-targeted immunoliposomes for cancer treatment," Mini. Rev. Med. Chem. 13(14):2026-2035(2013); Molavi et al, "Anti-CD30 antibody conjugated liposomal doxorubicin with significantly improved therapeutic efficacy against anaplastic large cell lymphoma," Biomaterials 34(34):8718-25(2013) (each of which and the documents cited therein are incorporated herein by reference).

[0175] Furthermore, in view of the teachings herein, one of skill in the art will be readily able to select and apply a desired targeting moiety in the practice of the present invention with respect to the lipid entities of the present invention. The present invention includes an embodiment in which the delivery system comprises lipid entities having a targeting moiety.

[0176] In some embodiments, the target cell may be a mammalian cell, hi some embodiments, the mammalian cell may be a cancer cell, as further described below.

[0177] In some embodiments, the mammalian cell may be infected with a pathogen, hi some embodiments, the pathogen may be a virus, as further described below.

[0178] In some embodiments, the targeting moiety comprises a membrane fusion protein. In some embodiments, the membrane fusion protein is the G envelope protein of vesicular stomatitis virus (VSV-G).

[0179] Membrane fusion is a universal and important biological phenomenon that occurs when two separate lipid membranes fuse into a single, continuous bilayer. Fusion reactions share common features but are catalyzed by diverse proteins. These proteins mediate the initial recognition of membranes destined to fuse, bring them close together, destabilize the lipid / water interface, and initiate lipid mixing. While a single fusion protein may do it all, in other cases, the intracellular fusion reaction requires the assembly of protein complexes to ensure strict regulation in space and time. Although cell fusion machinery has been adapted to fit the needs of various reactions, it operates on similar principles to achieve bilayer merging.

[0180] Membrane fusion can range from cell fusion and organelle dynamics to vesicle trafficking and viral infection. Without exception, all of these fusion events are driven by membrane fusion proteins, also known as fusogenic factors. A typical fusion process mediated by fusion proteins consists of a series of steps, including the approach of two opposing lipid membranes, the disruption of the lipid bilayer, and finally the merging of the two lipid bilayers into one. Much of our understanding of membrane fusion comes from studies of vesicle fusion, which is driven by a special class of proteins called SNAREs. SNARE proteins on the vesicle (v-SNARE) and the SNARE proteins on the target membrane (t-SNARE) provide not only the recognition specificity but also the energy required for vesicle fusion.

[0181] Viral fusion is another important fusion event. Enveloped viruses, encapsulated by a host cell-derived membrane, release their genomes after fusion of the viral envelope with the host cell membrane. Viral fusion proteins govern the uncoating step. Viral fusion proteins are classified into three types: type I, type II, and type III, based on their structural features. Despite long-standing knowledge of viral fusion proteins, the underlying fusion mechanism remains a mystery. One such previously identified type III viral fusion protein is the vesicular stomatitis virus G protein (VSV-G). Previous studies have revealed that membrane fusion triggered by VSV-G in an acidic environment relies on a reversible conformational change that returns to its original state under neutral conditions. The fusion protein of VSV-G and related rhabdoviruses (e.g., rabies virus) is the only surface-expressed protein on bullet-shaped virions. It mediates both binding and low-pH-induced fusion.

[0182] reverse transcriptase In some embodiments, the system may further comprise a reverse transcriptase. Reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template (a process called reverse transcription). Reverse transcriptases are used by retroviruses to replicate their genomes. They are also used by mobile genetic elements in retrotransposons to propagate within host genomes, by eukaryotic cells to extend telomeres at the ends of their linear chromosomes, and by some non-retroviruses, such as hepatitis B virus, a member of the Hepadnaviridae family and a dsDNA-RT virus.

[0183] Retroviral RT possesses three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H, and DNA-dependent DNA polymerase activity. Collectively, these activities allow the enzyme to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can be integrated into the host genome, from which new RNA copies can be made via host cell transcription. This same series of reactions is widely used in laboratories to convert RNA to DNA for use in molecular cloning, RNA sequencing, polymerase chain reaction (PCR), or genome analysis.

[0184] HIV reverse transcriptase has ribonuclease activity, which degrades viral RNA during cDNA synthesis, and DNA-dependent DNA polymerase activity, which copies the sense cDNA strand into antisense DNA to form a double-stranded viral DNA intermediate (vDNA).

[0185] Delivery vesicles Contemplated within the scope of the present invention are delivery vesicles that contain one or more components encoded by one or more polynucleotides in the modified delivery systems described herein.

[0186] As described elsewhere herein, such components include, but are not necessarily limited to, one or more endogenous retroviral elements for forming the delivery vesicle and one or more polynucleotides encoding one or more trapping moieties for packaging cargo within the delivery vesicle. The one or more endogenous retroviral elements for forming the delivery vesicle may include two or more retroviral gag proteins, retroviral envelope proteins, retroviral reverse transcriptase, or a combination thereof.

[0187] In some embodiments, the retroviral gag protein may be endogenous. In some embodiments, the retroviral envelope protein may be endogenous. In some embodiments, both the retroviral gag protein and the retroviral envelope protein are endogenous. As described elsewhere herein, the retroviral gag protein may comprise an NC domain and an MA domain. In some embodiments, the retroviral gag protein may be a gag homology protein. The gag homology protein may be Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12.

[0188] In some embodiments, the envelope protein is derived from a gammaretrovirus or a deltaretrovirus, hi some embodiments, the envelope protein is selected from envH1, envH2, envH3, envK1, envK2_1, envK2_2, envK3, envK4, envK5, envK6, envT, envW, envW1, envfrd, envR(b), envR, envF(c)2, or envF(c)1.

[0189] As described elsewhere herein, in some embodiments, the delivery vesicles elicit little or no immune response.

[0190] As described elsewhere herein, the cargo may comprise a nucleic acid, a protein, a complex thereof, or a combination thereof. In certain embodiments, the cargo comprises a ribonucleoprotein. The cargo may comprise one or more components of a gene editing system and / or a gene regulatory agent comprising a polynucleotide encoding the same.

[0191] The gene editing system can be a CRISPR-Cas system.The CRISPR-Cas system can be type II, type V or type VI CRISPR-Cas system, as described elsewhere herein.In certain embodiments, the type II CRISPR-Cas system is CRISPR-Cas9, the type V CRISPR-Cas system is CRISPR-Cas12, and the type VI CRISPR-Cas system is CRISPR-Cas13, but the present invention should not be limited to these embodiments.

[0192] In some embodiments, the vesicles may further comprise a reverse transcriptase.

[0193] In some embodiments, the one or more capture moieties comprise a DNA-binding moiety, an RNA-binding moiety, a protein-binding moiety, or a combination thereof.

[0194] In some embodiments, the delivery vesicle is a virus-like particle.

[0195] In some embodiments, the delivery vesicle may further comprise a targeting moiety, which is capable of specifically binding to a target cell.

[0196] In some embodiments, the cell-specific targeting moiety may comprise a membrane fusion protein, hi some embodiments, the membrane fusion protein is VSV-G, as described elsewhere herein.

[0197] In some embodiments, the cell-specific targeting moiety targets a mammalian cell, which may be a cancer cell, as further described below.

[0198] In some embodiments, the mammalian cell is infected with a pathogen, which may be a virus, as further described below.

[0199] Methods for loading cargo molecules into delivery vesicle systems Cargo, e.g., nucleic acids and / or polypeptides, small enough to be packaged in delivery vesicles can be introduced into cells by transduction with viruses or pseudovirus particles. The method of packaging cargo into viral particles can be achieved using any suitable viral vector or vector system. Such viral vectors and vector systems are described in more detail elsewhere herein. As used herein in this context, "transduction" refers to the process by which foreign nucleic acids and / or proteins are introduced into cells (prokaryotic or eukaryotic) by viruses or pseudovirus particles. After packaging into viral particles or pseudovirus particles, the viral particles can be exposed to cells (e.g., in vitro, ex vivo, or in vivo), where they infect the cells and deliver the cargo to the cells via transduction. Viral particles and pseudovirus particles can optionally be concentrated before exposure to target cells. In some embodiments, the viral titer of a composition comprising a virus and / or pseudovirus particle can be obtained, and the specific titer can be used to transduce cells.

[0200] In some embodiments, the viral vector is configured so that when the cargo is packaged, the cargo(s) are outside the capsid or viral particle, meaning that the cargo is not inside (enveloped or surrounded by) the capsid, but is exposed to the outside so that it can contact the target genomic DNA. In some embodiments, the viral vector is configured so that all cargo(s) are contained within the capsid after packaging.

[0201] One approach to packaging cargo into vesicles involves the use of one or more "bioreactors" to generate and subsequently secrete one or more cargo-carrying vesicles. Bioreactors can include cells, microorganisms, or cell-free systems. Bioreactor cells are generated by administering one or more endogenous retroviral elements to form delivery vesicles and one or more polynucleotides encoding one or more trapping moieties to package the cargo into the delivery vesicles. A targeting moiety may be administered to the cells, and the targeting moiety can specifically bind to the target cell. Thus, the bioreactor may be capable of producing cargo-carrying vesicles that deliver bioactive RNA molecule(s) to specific cells and tissues as well as the extracellular matrix.

[0202] In some embodiments, the cargo molecule, whether delivered alone or as part of a system, can act to modify the genome, epigenome, and / or transcriptome of the cell to which it is delivered, regardless of whether it is delivered with other components of the system. Such systems include, but are not limited to, the CRISPR-Cas system. Other gene modification systems, such as TALEN, zinc finger nucleases, Cre-Lox, morpholinos, etc., are other non-limiting examples of gene modification systems in which one or more components can be delivered by the modified AAV particles described herein.

[0203] The present invention provides nucleic acid molecules, specifically polynucleotides that, in some embodiments, encode one or more peptides or polypeptides of interest. The term "nucleic acid" is used in its broadest sense to include any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides.

[0204] Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0205] In some embodiments, the polynucleotide of the present invention can be circular.As used herein, "circular polynucleotide" refers to a single-stranded circular polynucleotide that acts substantially like RNA and has the properties of RNA.The term "circular" also refers to any secondary or tertiary structure of circular polynucleotide.

[0206] In some embodiments, the polynucleotide is from about 30 to about 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000 , 100~1,500, 100~3,000, 100~5,000, 100~7,000, 100~10,000, 100~25,000, 100~50,000, 100~70,000, 100~100,000, 500~1,000, 500~1,500, 500~2,000, 500~3,000, 500~5,000, 500~7,000, 500~10,000, 500~25,000, 500~ 50,000, 500-70,000, 500-100,000, 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-5,000, 1,000-7,000, 1,000-10,000, 1,000-25,000, 1,000-50,000, 1,000-70,000, 1,000-100,000, 1,500-3,000, 1,500-5,000, 1,5 00-7,000, 1,500-10,000, 1,500-25,000, 1,500-50,000, 1,500-70,000, 1,500-100,000, 2,000-3,000, 2,000-5,000, 2,000-7,000, 2,000-10,000, 2,000-25,000, 2,000-50,000, 2,000-70,000, and 2,000-100,000).

[0207] The vesicles formed from the bioreactor described herein can be isolated by any suitable method known in the art. For example, the vesicles can contain tags that can bind to antibodies or aptamers. The vesicles can also be isolated and sorted by fluorescence-activated cell sorting (FACS) or size exclusion. Methods for delivering cargo using delivery vesicles

[0208] Also contemplated within the scope of the present invention are methods for delivering cargo to one or more cells using the delivery vesicles described herein. As described, the delivery vesicles can deliver cargo to one or more cells of a subject.

[0209] The systems described herein may include one or more targeting moieties capable of specifically binding to target cells. Such targeting moieties may include, but are not limited to, membrane fusion proteins, antibodies, peptides, cyclic peptides, small molecules, or related molecular structures that can be directed via binding to a target, including non-immunoglobulin scaffolds including fibronectin, lipocalin, protein A, ankyrin, thioredoxin, etc. In some embodiments, the membrane fusion protein is the G envelope protein of vesicular stomatitis virus (VSV-G), herpes simplex virus 1gB (HSV-1gB), Ebola virus glycoprotein, a member of the SNARE family of proteins, and a member of the syncytin family of proteins.

[0210] In some embodiments, the cargo may comprise a therapeutic agent. The terms "therapeutic agent," "therapeutic drug," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that has some beneficial effect upon administration to a subject. Beneficial effects include enabling a diagnostic determination; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or pathological condition; and generally combating a disease, symptom, disorder, or pathological condition.

[0211] Target cells may include, but are not limited to, mammalian cells, cancer cells, and cells infected with pathogens such as viruses, bacteria, fungi, or parasites. In some embodiments, the present invention involves delivering cargo across the blood-brain barrier. As will be appreciated by those skilled in the art, vesicles can be engineered to have tropism for any particular desired cell type.

[0212] Various delivery systems that can be used to administer pharmaceutical compositions are known, including, but not limited to, liposomes, microparticles, encapsulation in microcapsules; minicells; polymers; capsules; tablets, etc. In one embodiment, the drug may be delivered in vesicles, particularly liposomes. In liposomes, the drug is combined with other pharmaceutically acceptable carriers and amphiphilic agents, such as lipids that exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Lipids suitable for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, etc. The preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,837,028 and 4,737,323. In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system, including, but not limited to, a delivery pump (see, e.g., Saudek, et al., New Engl. J. Med. 321:574 (1989)) and a semipermeable polymeric material (see, e.g., Howard, et al., J. Neurosurg. 71:105 (1989)). Furthermore, controlled release systems can be placed near the therapeutic target (e.g., tumor), thus requiring only a fraction of the systemic dose. See, e.g., Goodson, In: Medical Applications of Controlled Release, 1984 (CRC Press, Boca Raton, Fla.).

[0213] It will be understood that administration of therapeutic entities according to the present invention may be in the presence of suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Numerous suitable formulations can be found in formularies known to all medicinal chemists, such as Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA complexes, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be suitable for treatment and therapy according to the present invention, so long as the active ingredient therein is not inactivated by the formulation, and the formulation is physiologically compatible and tolerable for the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998), and citations therein for additional information related to formulations, excipients, and carriers well known to medicinal chemists.

[0214] The terms "subject," "individual," or "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, livestock, game animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0215] As used herein, the term "in need of treatment" or "in need of" refers to a judgment made by a caregiver (e.g., a physician, nurse, practical nurse, or individual in the case of a human; a veterinarian in the case of an animal, including a non-human animal) that a subject needs or would benefit from treatment. This judgment is within the caregiver's realm of experience, but is made based on a variety of factors, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the compounds of the invention.

[0216] As used in this context, "treating" means curing, ameliorating, stabilizing, preventing, or reducing at least one symptom or the severity of a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically directed toward ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed toward eliminating the cause of the associated disease, pathological condition, or disorder. Furthermore, the term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than a cure of the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment employed to supplement another specific therapy directed toward ameliorating the associated disease, pathological condition, or disorder. It should be understood that treatment, while aimed at curing, ameliorating, stabilizing, or preventing a disease, pathological condition, or disorder, need not actually result in a cure, amelioration, stabilization, or prevention. The effectiveness of treatment can be measured or assessed as appropriate for the relevant disease, condition, or disorder, as described herein and known in the art. Such measurement and assessment can be made in qualitative and / or quantitative terms. Thus, for example, the characteristics or properties of a disease, condition, or disorder and / or the symptoms of a disease, condition, or disorder can be reduced to any effect or by any amount.

[0217] Administration of the compositions, agents, cells, or populations of cells disclosed herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions may also be administered to a patient by intravenous or intralymphatic injection, subcutaneously, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intrathecal, or intraperitoneal route.

[0218] Administration of the agents of the present invention may be by any suitable means that results in an effective compound concentration to treat or inhibit (e.g., by delaying) the onset of a disease. The compound is mixed with a suitable carrier substance, such as a pharmaceutically acceptable excipient that preserves the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable excipient is saline. Suitable carrier substances are generally present in an amount of 1 to 95% by weight of the total weight of the agent. The agent may be provided in a dosage form suitable for administration. Thus, the agent may be in the form of, for example, a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel, including hydrogel, paste, ointment, cream, plaster, drench, delivery device, injection, implant, spray, or aerosol.

[0219] Methods for administering pharmaceutical compositions containing agonists, antagonists, antibodies, or fragments thereof to an individual include, but are not limited to, intradermal, intrathecal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, inhalation, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), ocularly, etc., and can be administered together with other bioactive agents. Administration can be systemic or local. Furthermore, it may be advantageous to administer the compositions to the central nervous system by any suitable route, including intraventricular and intrathecal injection. Pulmonary administration may also be used by combining with an aerosolizing agent using an inhaler or nebulizer. It may also be desirable to administer the agent locally to the area requiring treatment; this may be achieved, for example, but not limited to, by local infusion during surgery, topical application, injection, catheter, suppository, or implant.

[0220] The amount of drug effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques by one of ordinary skill in the art. Additionally, in vitro assays may optionally be used to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration and the overall severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Ultimately, the attending physician will determine the amount of drug to treat each individual patient. In certain embodiments, the attending physician will administer a low dose of the drug and observe the patient's response. Higher doses of the drug may be administered until the optimal therapeutic effect for the patient is achieved, at which point the dose is not further increased. Generally, the daily dose range is about 0.001 mg to about 100 mg per kg of mammalian body weight, in single or divided doses, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg. However, it may be necessary to use doses outside these limits in some cases. In certain embodiments, a suitable dosage range for intravenous administration of a drug is generally about 5 to 500 micrograms (μg) of active compound per kilogram (Kg) of body weight. A suitable dosage range for intranasal administration is generally about 0.01 pg / kg to 1 mg / kg of body weight. In certain embodiments, a composition containing a drug of the present invention is injected subcutaneously into an adult patient as a single dose in the dose range of approximately 5 to 5,000 μg / human, preferably approximately 5 to 500 μg / human. This dose is preferably administered one to three times daily. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient. Ultimately, the attending physician will determine the appropriate duration of treatment using the compositions of the present invention. Dosage may also vary depending on the age, weight, and response of the individual patient.

[0221] Preferably, the therapeutic agent may be administered in a therapeutically effective amount of the active ingredient. The term "therapeutically effective amount" refers to an amount that can induce the biological or medical response in a tissue, system, animal or human that is sought by a researcher, veterinarian, physician or other clinician, and in particular, can prevent or alleviate one or more local or systemic symptoms or characteristics of the disease or condition being treated.

[0222] In some embodiments, the therapeutic agent may include one or more components of a gene editing system and / or polynucleotides encoding same. [Example]

[0223] Example 1 - Pseudotyping of lentiviruses using endogenous retroviral envelope proteins The expression of various individual env proteins was tested in HEK293T cells (Figure 1). The best expression was achieved with Envw1, Envk1, and Envfrd (Envw2). The glycoprotein of vesicular stomatitis virus (VSV-G) mediates cell adhesion and can induce direct fusion between membranes. Applicants compared the pseudotyping efficiency of different env proteins with lentiviral DNA. Efficient particle formation was observed with Envk1, Envw1, and Envfrd (Figure 2).

[0224] To confirm whether the gag homologous protein Pnma3 is expressed in neural cells, we fused it to a red fluorescent reporter protein (RFP) and tested its expression in mouse and rat neurons. The results showed that expression of this fusion protein was comparable to that of a control RFP-lentiviral construct (Figure 3).

[0225] Example 2 - Screening of endogenous gag protein candidates for their ability to form capsids, secrete proteins, and transmit signals Nine candidate endogenous gag proteins were identified and screened for their ability to form vesicles in vitro (Figs. 4 and 5). Of the candidates tested, all but Asprv1 were able to form vesicles (Fig. 5 and Table 2). However, only six were able to be secreted from cells (Table 3, Fig. 6). [Table 2] [Table 3]

[0226] Next, we tested the ability of various gag protein candidates to transport the Cas9 / gRNA complex to other cells. In the absence of a membrane fusion protein (Figure 7A), none of the candidates were able to successfully promote this process. However, the inclusion of VSV-G (Figure 7B) was important for delivering the complex to other cells (Table 4). [Table 4]

[0227] Vesicles formed using PNMA4 and RTL1 showed the best ability to transport gene editing complexes to new cells and induce indel formation (Figure 10).

[0228] To assess whether the gag candidates promoted secretion from cells and subsequent transfer of information from one cell to another, Applicants also generated knock-in mice expressing an HA tag on the endogenous gag protein. The DNA sequence encoding an exemplary HA-tagged RTL1 protein is shown in Figure 12.

[0229] Example 3 - Modification of endogenous vectors for gene therapy Applicant set out to create a non-immunogenic vector capable of efficiently delivering gene therapy in vivo. Viral vectors are highly efficient but immunogenic, eliciting unwanted immune responses in target cells against the vector itself, potentially rendering the therapeutic agent contained within ineffective. Lipid nanoparticles (LNPs) are easy to manufacture but have limited targeting and typically deliver only approximately 2% of their encoded payload. Exosomes are potentially non-immunogenic, but their biology is complex and their efficacy is unknown. Applicant sought to explore endogenous signaling systems for their potential to mediate intercellular gene transfer. For example, there are at least 40,000 encoded GAGs in the human genome, with varying immunogenic potential (Figure 17). Some highly expressed endogenous GAGs are shown in Figure 4.

[0230] Applicants analyzed several GAGs for their ability to spontaneously form vesicles (Figures 19 and 20). To determine which GAGs could form vesicles, HA-tagged GAGs were overexpressed in HEK cells and the supernatants were collected. The VLP fractions were centrifuged using PEG (Figure 21). Applicants found that the addition of VSV-G fusion factors both improved target cell uptake of secreted GAGs and promoted indel generation (Figures 23A-23D, 24, 52, and 53).

[0231] Of all the GAGs tested, Applicant determined that PEG10 was the best candidate for mediating transport comparable to HIV lentivirus and generating VLPs (Figure 24). To optimize PEG10 for delivery, Applicant sought to understand the precise biological function of PEG10 and the extent to which it could be reprogrammed. The PEG10 gene contains two overlapping reading frames of the same transcript encoding different isoforms. The shorter isoform contains a CCHC-type zinc finger motif, including sequences characteristic of gag proteins from most retroviruses and some retrotransposons, and functions in part by interacting with members of the TGF-β receptor family. The longer isoform contains the active site DSG consensus sequence of the protease domain of the pol protein. The longer isoform is the result of a -1 translational frameshift, also observed in some retroviruses (Figures 25 and 26).

[0232] Applicants transfected cells with various PEG10 constructs and analyzed whole-cell lysates and VLP fractions by immunoprecipitation. The results showed that PEG10 VLPs were processed, but that the protease domain was not required for this processing (Figure 28). Applicants also found that the addition of VSV-G promoted PEG10 secretion and enabled uptake into target cells (Figure 29).

[0233] To enhance delivery efficiency, we cultured HEK293T cells in T225 flasks. The cells were transfected with various delivery components, filtered through a 45 μm filter, and ultracentrifuged through a 20% sucrose cushion. The VLPs were resuspended in 250 μL of PBS, and a 10 μL aliquot of the suspension was added to 20E3 cells. Indels were then detected 48 hours later by next-generation sequencing (Figure 31). These experiments revealed that PEG10 is a secreted encapsidation protein and that VSV-G allows PEG10 to deliver Cas9 to target cells and mediate indel generation. PEG10 VLPs may be processed at the C-terminal domain. We also found that the addition of SGCE promoted PEG10 secretion but did not promote entry (at least in HEK cells).

[0234] Applicants have compiled and cloned a list of 165 additional genes that could act as potential fusogenic factors (Tables 5 and 6), each of which will be evaluated individually with HIV, PEG10, Arc, and Rtl1 GAG. [Table 5-1] [Table 5-2] [Table 6]

[0235] Applicants then determined that PEG10 could be found in both brain serum and cortical neurons (Figure 32). Consistent with previous reports, knockout mice lacking PEG10 exhibited early embryonic lethality, indicating the importance of this gene in embryonic development (Figure 33). Gene ontology analysis of primary mouse neurons revealed three groups of differentially expressed genes: 1) genes involved in chromatin remodeling, 2) genes involved in the trans-Golgi network and exocytosis, and 3) SNARE and other genes encoding endosomal and membrane proteins.

[0236] To investigate whether the secreted GAGs are chromatin modifiers that bind to DNA but not RNA, we performed DNA adenine methyltransferase identification (DamID), a protocol used to map the binding sites of eukaryotic DNA and chromatin-binding proteins. DamID identifies binding sites by expressing a proposed DNA-binding protein as a fusion protein with a DNA methyltransferase. When the protein of interest binds to DNA, the methyltransferase localizes to the region of the binding site. Because adenosine methylation does not occur naturally in eukaryotes, we can conclude that any region where adenine methylation is caused by the fusion protein is near the binding site (Figure 36). To perform this protocol, we digested the genome with DpnI, which cleaves only methylated GATCs. Then, double-stranded adapters with known sequences were ligated to the ends generated by DpnI. The ligation product was digested with DpnII, which cleaves unmethylated GATCs, ensuring that only fragments flanked by consecutive methylated GATCs were amplified in subsequent PCR. PCR was then performed using primers matching the adapter to specifically amplify the genomic fragment flanked by the methylated GATCs (Figure 37). The data obtained by DamID mapping was then cross-referenced with the ATAC sequence data (Figure 38). Applicant overexpressed PEG10 and SGCE in N2A cells, ultracentrifuged the VLP fraction, and analyzed the precipitated proteins by mass spectrometry. Various proteins, including RNA turnover factors, transcription factors, and chromatin remodelers, were found to be enriched in this fraction (Figure 39). Applicant concluded that PEG10 is efficiently secreted from cells and can mediate the delivery of macromolecules. Because PEG10 is found throughout the body, it is likely to bind to DNA and can be delivered to and enter cells, where it can directly bind to DNA (Figure 40).

[0237] Example 4 - PEG10 Processing and Functional Properties of the Processed Domain The ability of PEG10 to form vesicles raises two central questions: 1) how is PEG10 processed, and 2) what does each functional domain do? To answer the first question, Applicants overexpressed N- and C-terminal HA-tagged mouse PEG10 in HEK293FT cells, immunoprecipitated PEG10 using HA magnetic beads, and analyzed the bands by Western blotting. The corresponding Coomassie-stained bands were analyzed by mass spectrometry. The results showed that the protein was cleaved into all of the predicted domains (Figures 56, 57A-57F, 58A, and 58B).

[0238] To answer the second question, Applicants compared PEG10 to a previously identified protein known as MYEF, a DNA-binding protein that binds to a highly specific 10-base pair sequence in 3x repeats (shown on the right side of Figure 59). Applicants determined that PEG10 binds to the exact same sequence and therefore attempted to package particles expressing this DNA sequence. When PEG10 was overexpressed using plasmid DNA containing this sequence, Applicants noted that PEG10 preferentially packaged and encapsulated the 10-base pair DNA sequence and secreted the plasmid carrying that sequence.

[0239] To quantify the amount of PEG10 circulating in the blood, Applicants engineered mice with a PEG10 antibody receptor tag and determined that PEG10 was expressed at approximately 120 pg / μL in mouse plasma (Figure 70).

[0240] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the present invention has been described in connection with specific embodiments, it should be understood that further modifications are possible, and that the invention, as described in the claims, should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art are intended to be within the scope of the present invention. This application is intended to cover any variations, uses, or adaptations of the present invention that generally conform to the principles of the invention, and it will be understood that including such departures from the present disclosure is within known customary practice to which the invention pertains and may apply to the essential features described above. [Sequence table] SEQUENCE LISTING <110> The Broad Institute, Inc. Massachusetts Institute of Technology Zhang, Feng Segel, Michael <120> Compositions and Methods for Delivering Cargo to a Target Cell <130> BROD-4620WP <150> US 62 / 903,127 <151> 2019-09-20 <150> US 63 / 003,409 <151> 2020-04-01 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic <400> 1 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 2 <211> 30 <212> PRT <213> Artificial <220> <223> Synthetic <400> 2 Gly Cys Cys Thr Gly Thr Cys Thr Thr Thr Gly Cys Cys Thr Gly Thr 1 5 10 15 Cys Thr Thr Thr Gly Cys Cys Thr Gly Thr Cys Thr Thr Thr 20 25 30 <210> 3 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic <400> 3 Gly Thr Gly Thr Cys Cys Cys Cys 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic <400> 4 Ala Pro Arg Pro Gly 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic <400> 5 Gly Pro Leu Gly Ile Ala Gly Gln 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic <400> 6 Arg Gly Asx Tyr Cys 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial <220> <223> Synthetic <400> 7 Lys Cys Cys Tyr Ser Leu 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic <400> 8 Tyr Glu Val Gly His Arg Cys 1 5 <210> 9 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 9 ccccttcagt ctggaaagga ggacactgag g 31 <210> 10 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 10 ccccttcagt atggaaagga ggacactgag g 31 <210> 11 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 11 atcagtccag ccccttcagt ctggaaagga g 31 <210> 12 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 12 atcagtccag ccccttcagt atggaaagga g 31 <210> 13 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <400> 13 aatcagtcca gccccttcag tctggaaagg aggaca 36 <210> 14 <211> 37 <212> DNA <213> Artificial <220> <223> Synthetic <400> 14 aatcagtcca gccccttcaa gtctggaaag gaggaca 37 <210> 15 <211> 37 <212> DNA <213> Artificial <220> <223> Synthetic <400> 15 aatcagtcca gccccttcac gtctggaaag gaggaca 37 <210> 16 <211> 34 <212> DNA <213> Artificial <220> <223> Synthetic <400> 16 caatcagtcc agccccttca gtctggaaag gagg 34 <210> 17 <211> 37 <212> DNA <213> Artificial <220> <223> Synthetic <400> 17 caatcagtcc agccccttca cgggtctgga aaggagg 37 <210> 18 <211> 35 <212> DNA <213> Artificial <220> <223> Synthetic <400> 18 caatcagtcc agccccttca agtctggaaa ggagg 35 <210> 19 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic <400> 19 cagtccagcc ccttcagtct ggaaaggagg 30 <210> 20 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 20 tcagtccagc cccttcagtc tggaaaggag g 31 <210> 21 <211> 32 <212> DNA <213> Artificial <220> <223> Synthetic <400> 21 tcagtccagc cccttcaagt ctggaaagga gg 32 <210> 22 <211> 30 <212> DNA <213> Artificial <220> <223> Synthetic <400> 22 tcagtccagc cccttcagtc tggaaaggag 30 <210> 23 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 23 tcagtccagc cccttcaagt ctggaaagga g 31 <210> 24 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 24 tcagtccagc ccctttcagt ctggaaagga g 31 <210> 25 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 25 tcagtccagc cccttcaggt ctggaaagga g 31 <210> 26 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <400> 26 tcagtccagc cccttcatgt ctggaaagga g 31

Claims

1. A modified delivery system comprising one or more endogenous retroviral elements for forming a delivery vesicle and one or more polynucleotides encoding one or more trapping moieties for packaging cargo within the delivery vesicle.

2. 2. The system of claim 1, wherein the one or more endogenous retroviral elements for forming the delivery vesicle comprise two or more retroviral gag proteins, retroviral envelope proteins, retroviral reverse transcriptase, or a combination thereof.

3. The system of claim 2 , wherein the retroviral gag protein is endogenous.

4. The system of claim 2 , wherein the retroviral envelope protein is endogenous.

5. The system of claim 2 , wherein both the retroviral gag protein and the retroviral envelope protein are endogenous.

6. 4. The system of claim 2 or 3, wherein the retroviral gag protein comprises an NC domain and an MA domain.

7. The system according to any one of claims 2 to 6, wherein the retroviral gag protein is a gag homologous protein.

8. 8. The system of claim 7, wherein the gag homologous protein is Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12.

9. The system of claim 8, wherein the gag homologous protein is PNMA4, PEG10, or RTL1.

10. The system of claim 9, wherein the gag homologous protein is PEG10.

11. The system according to any one of claims 2 to 10, wherein the envelope protein is derived from a gammaretrovirus or a deltaretrovirus.

12. 12. The system of any of claims 2 to 11, wherein the envelope protein is selected from envH1, envH2, envH3, envK1, envK2_1, envK2_2, envK3, envK4, envK5, envK6, envT, envW, envW1, envfrd, envR(b), envR, envF(c)2, or envF(c)1.

13. The system according to any one of claims 2 to 12, wherein the envelope protein comprises a cargo-binding domain.

14. The system of claim 13 , wherein the cargo-binding domain is a hairpin loop-binding element.

15. The system of claim 13, wherein the hairpin loop binding element is an MS2 aptamer.

16. The system of any of claims 1 to 15, wherein the delivery system does not induce a significant immune response.

17. The system according to any one of claims 1 to 16, wherein the cargo comprises a nucleic acid, a protein, a complex thereof, or a combination thereof.

18. The system according to any one of claims 1 to 17, wherein the cargo is linked to one or more envelope proteins by a linker.

19. The system of claim 18, wherein the linker is a glycine-serine linker.

20. 20. The system of claim 19, wherein the glycine-serine linker is (GGS)3.

21. The system of claim 17 , wherein the cargo comprises a ribonucleoprotein.

22. The system of claim 17, wherein the nucleic acid is DNA.

23. The system of any preceding claim, wherein the cargo comprises a gene regulator.

24. 24. The system of Claim 23, wherein the gene regulatory agent comprises one or more components of a gene editing system and / or polynucleotides encoding same.

25. 25. The system of claim 24, wherein the gene editing system is a CRISPR-Cas system.

26. 26. The system of claim 25, wherein the CRISPR-Cas system is a Type II, Type V, or Type VI CRISPR-Cas system.

27. 27. The system of claim 26, wherein the type II CRISPR-Cas system comprises CRISPR-Cas9.

28. 28. The system of claim 27, wherein the Type V CRISPR-Cas system comprises CRISPR-Cas12.

29. 27. The system of claim 26, wherein the Type VI CRISPR-Cas system comprises CRISPR-Cas13.

30. 26. The system of claim 25, wherein a Cas protein of the CRISPR-Cas system is modified to bind to a binding domain of the envelope protein.

31. 26. The system of claim 25, wherein a guide molecule of the CRISPR-Cas system is engineered to bind to a binding domain of the envelope protein.

32. 31. The system of claim 30, wherein the modification comprises the incorporation of a hairpin loop that binds to a hairpin binding element on the envelope protein.

33. 33. The system of claim 32, wherein the hairpin loop is recognized by the MS2 aptamer.

34. The system of any one of claims 1 to 33, wherein the system further comprises a reverse transcriptase.

35. 35. The system of any of claims 1 to 34, wherein the one or more capture moieties comprise a DNA-binding moiety, an RNA-binding moiety, a protein-binding moiety, or a combination thereof.

36. The system of any of claims 1 to 35, wherein the delivery vesicle is a virus-like particle.

37. The system according to any of claims 1 to 36, further comprising a targeting moiety, said targeting moiety being capable of specifically binding to a target cell.

38. 38. The system of claim 37, wherein the targeting moiety comprises a membrane fusion protein.

39. 39. The system of claim 38, wherein the membrane fusion protein is the G envelope protein of vesicular stomatitis virus (VSV-G).

40. 39. The system of claim 38, wherein the membrane fusion protein is SGCE.

41. 38. The system of claim 37, wherein the target cell is a mammalian cell.

42. 42. The system of claim 41, wherein the mammalian cells are cancer cells.

43. 43. The system of claim 42, wherein the mammalian cell is infected with a pathogen.

44. 44. The system of claim 43, wherein the pathogen is a virus.

45. A delivery vesicle comprising one or more components encoded by said one or more polynucleotides in a modified delivery system according to any of the preceding claims.

46. 46. ​​The delivery vesicle of claim 45, wherein the one or more components comprise two or more retroviral gag proteins, retroviral envelope proteins, retroviral reverse transcriptase, or a combination thereof.

47. 47. The delivery vesicle of claim 46, wherein the retroviral gag protein is a gag homologous protein selected from the group consisting of Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, and ZCCHC12.

48. 48. The delivery vesicle of claim 47, wherein the gag homologous protein is PNMA4, PEG10, or RTL1.

49. 49. The delivery system of claim 48, wherein the gag homologous protein is PEG10.

50. 50. The delivery vesicle of any one of claims 45 to 49, wherein the vesicle comprises a cell-specific targeting moiety.

51. 51. The delivery vesicle of claim 50, wherein the cell-specific targeting moiety targets a mammalian cell.

52. 52. The delivery vesicle of claim 51, wherein the cell-specific targeting moiety comprises a membrane fusion protein.

53. 53. The delivery vesicle of claim 52, wherein the membrane fusion protein is VSV-G.

54. 53. The delivery vesicle of claim 52, wherein the membrane fusion protein is SGCE.

55. 52. The delivery vesicle of claim 51, wherein the mammalian cell is a cancer cell.

56. 52. The delivery vesicle of claim 51, wherein the mammalian cell is infected with a pathogen.

57. 57. The delivery vesicle of claim 56, wherein the pathogen is a virus.

58. A system for delivering cargo to a target cell comprising a delivery vesicle encapsulating the cargo and an endogenous reverse transcriptase.

59. 59. The system of claim 58, wherein the delivery vesicle is a virus-like particle.

60. 60. The system of claim 58 or 59, wherein the delivery vesicle is composed of retroviral gag protein and retroviral envelope protein.

61. 61. The system of claim 60, wherein the retroviral gag protein is derived from a human endogenous retrovirus (HERV).

62. 62. The system of claim 61, wherein the retroviral gag protein is Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12.

63. 63. The system of claim 62, wherein the retroviral gag protein is PNMA4, PEG10, or RTL1.

64. 64. The system of claim 63, wherein the retroviral gag protein is PEG10.

65. 59. The system of any of claims 58, wherein the retroviral envelope protein is derived from HERV.

66. 59. The system of claim 58, wherein both the retroviral gag protein and the retroviral envelope protein are derived from HERV.

67. 67. The system of any of claims 60 to 66, wherein the retroviral envelope protein comprises a cargo binding domain.

68. 68. The system of claim 67, wherein the cargo binding domain is a hairpin loop binding element.

69. 69. The system of claim 68, wherein the hairpin loop binding element is an MS2 aptamer.

70. 70. The system of any of claims 58 to 69, wherein the cargo comprises a nucleic acid, a protein, a complex thereof, or a combination thereof.

71. 71. The system of claim 70, wherein the nucleic acid is DNA.

72. 71. The system of claim 70, wherein the cargo comprises a ribonucleoprotein.

73. 73. The system of any of claims 58 to 72, wherein the cargo comprises a gene regulator.

74. 74. The system of Claim 73, wherein the gene regulatory agent comprises one or more components of a gene editing system and / or polynucleotides encoding same.

75. 75. The system of claim 74, wherein the gene editing system is a CRISPR-Cas system.

76. 76. The system of claim 75, wherein the CRISPR-Cas system is a Type II, Type V, or Type VI CRISPR-Cas system.

77. 77. The system of claim 76, wherein the type II CRISPR-Cas system comprises CRISPR-Cas9.

78. 77. The system of claim 76, wherein the Type V CRISPR-Cas system comprises CRISPR-Cas12.

79. 77. The system of claim 76, wherein the Type VI CRISPR-Cas system comprises CRISPR-Cas13.

80. 80. The system of any of claims 58 to 79, wherein the cargo is linked to one or more envelope proteins by a linker.

81. 81. The system of claim 80, wherein the linker is a glycine-serine linker.

82. 82. The system of claim 81, wherein the glycine-serine linker is (GGS)3.

83. 77. The system of claim 76, wherein a Cas protein of the CRISPR-Cas system is engineered to bind to a binding domain of the envelope protein.

84. 77. The system of claim 76, wherein a guide molecule of the CRISPR-Cas system is engineered to bind to a binding domain of the envelope protein.

85. 84. The system of claim 83, wherein the modification comprises the incorporation of a hairpin loop that binds to a hairpin binding element on the envelope protein.

86. 86. The system of claim 85, wherein the hairpin loop is recognized by the MS2 aptamer.

87. 87. The system of any one of claims 58 to 86, further comprising a membrane fusion protein.

88. 88. The system of claim 87, wherein the membrane fusion protein is VSV-G.

89. 88. The system of claim 87, wherein the membrane fusion protein is SGCE.

90. The system according to any one of claims 58 to 89, wherein the target cell is a mammalian cell.

91. 91. The system of claim 90, wherein the mammalian cell is a cancer cell.

92. 91. The system of claim 90, wherein the mammalian cell is infected with a pathogen.

93. 93. The system of claim 92, wherein the pathogen is a virus.

94. 44. A method of loading a cargo molecule into a delivery vesicle system, comprising incubating the cargo molecule and a modified delivery system according to any one of claims 1 to 43 with one or more bioreactors.

95. 95. The method of claim 94, wherein the one or more bioreactors are cells, microorganisms, or cell-free systems.

96. A method for delivering a cargo molecule, comprising delivering a delivery vesicle according to claims 45 to 57 to a target cell or cell population.

97. 97. The method of claim 96, wherein the delivery is in vivo.

98. 97. The method of claim 96, wherein the delivery is ex vivo.

99. 97. The method of claim 96, wherein the delivery is in vitro.

100. 100. The method of any of claims 96-99, wherein the cargo comprises a nucleic acid, a protein, a complex thereof, or a combination thereof.

101. 101. The method of claim 100, wherein the nucleic acid is DNA.

102. 101. The method of claim 100, wherein the cargo comprises a ribonucleoprotein.

103. 103. The method of any one of claims 100 to 102, wherein the cargo comprises a gene regulator.

104. 104. The method of any of claims 96-103, wherein delivery occurs across the blood-brain barrier.

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