Compositions and methods for delivering cargo to target cells

Abstract

This document provides compositions, systems, and methods for delivering cargo to target cells. The compositions, systems, and methods comprise one or more polynucleotides encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more capture portions for packaging cargo within the delivery vesicles. The one or more endogenous retroviral elements for forming delivery vesicles may comprise two or more of the following: a retroviral gag protein, a retroviral envelope protein, a retroviral reverse transcriptase, or a combination thereof. The retroviral gag protein alone, the retroviral envelope protein alone, or both the retroviral gag protein and the retroviral envelope protein may be endogenous.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 903,127, filed September 20, 2019, and U.S. Provisional Application No. 62 / 003,409, filed April 1, 2020. The entire contents of the above applications are hereby incorporated herein by reference.

[0003] Statement on Federally Funded Research

[0004] This invention was carried out with government support under approval number HL141201 granted by the National Institutes of Health (NIH). The government owns certain rights to this invention.

[0005] Reference to electronic sequence listing

[0006] The contents of the electronic sequence list (“BROD-4620WP_ST.25.txt”, 4,945 bytes in size, created on September 18, 2020) are incorporated herein by reference in their entirety. Technical Field

[0007] The topics disclosed herein generally relate to engineered delivery agents, compositions, systems, and their uses. Background Technology

[0008] Delivery systems are a crucial aspect of therapeutic efficacy. Delivering therapeutic agents into cells presents numerous challenges, including but not limited to limiting off-target effects, delivery efficiency, degradation, and more. Viruses and virus-like particles have been used to deliver various cargoes, such as gene therapy agents, to target cells. However, currently used vesicles and particles can be large in size and difficult to generate in a consistent manner. Therefore, simpler and improved delivery systems are needed. Summary of the Invention

[0009] In some exemplary embodiments, the present invention provides an engineered delivery system comprising one or more polynucleotides, wherein the one or more polynucleotides encode one or more endogenous retroviral elements for forming delivery vesicles and one or more capture portions for packaging cargo within delivery vesicles.

[0010] In some embodiments, one or more endogenous retroviral elements for forming delivery vesicles comprise two or more of the following: retroviral gag protein, retroviral envelope protein, retroviral reverse transcriptase, or combinations thereof.

[0011] 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.

[0012] In some implementations, the retroviral gag protein contains NC and MA domains.

[0013] In some embodiments, the retroviral gag protein is a gag homolog. In some embodiments, the gag homolog is Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In a specific embodiment, the gag homolog is PNMA4, PEG10, or RTL1.

[0014] In some embodiments, the envelope protein may be derived from a gamma retrovirus or a delta retrovirus. 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.

[0015] In some embodiments, the envelope protein includes 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.

[0016] In some implementations, the delivery system elicits a poor immune response.

[0017] In some embodiments, the cargo comprises nucleic acids, proteins, complexes thereof, or combinations thereof. In some embodiments, the cargo is linked to one or more envelope proteins via a adapter. In some embodiments, the adapter is a glycine-serine adapter. In some embodiments, the glycine-serine adapter is (GGS)3 (SEQ ID NO:1).

[0018] In some embodiments, the cargo comprises a ribonucleoprotein. In some embodiments, the cargo comprises a genetic regulator. In some embodiments, the genetic regulator comprises one or more components of a gene editing system and / or a polynucleotide encoding it. 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 includes CRISPR-Cas9. In some embodiments, a type V CRISPR-Cas system includes CRISPR-Cas12. In some embodiments, a type VI CRISPR-Cas system includes CRISPR-Cas13.

[0019] In some embodiments, the Cas protein of the CRISPR-Cas system can 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 includes 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.

[0020] In some implementations, the system may also include reverse transcriptase.

[0021] In some implementations, one or more capture portions include a DNA-binding portion, an RNA-binding portion, a protein-binding portion, or a combination thereof.

[0022] In some implementations, the delivery vesicles are virus-like particles.

[0023] In some embodiments, the system may further include a targeting portion capable of specifically binding to target cells. In some embodiments, the targeting portion comprises a membrane fusion protein. In some embodiments, the membrane fusion protein is the G envelope protein of vesicular stomatitis virus (VSV-G).

[0024] In some embodiments, the target cells are mammalian cells. In some embodiments, the mammalian cells are cancer cells. In some embodiments, the mammalian cells are infected by a pathogen. In some embodiments, the pathogen is a virus.

[0025] In another aspect, the present invention provides a delivery vesicle containing one or more components encoded in one or more polynucleotides in an engineered delivery system described herein.

[0026] In some embodiments, one or more components of the delivery vesicle comprise two or more of the following: retroviral gag protein, retroviral envelope protein, retroviral reverse transcriptase, or combinations thereof.

[0027] In some embodiments, the retroviral gag protein is an Agag homolog selected from the group consisting of: Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In a specific embodiment, the gag homolog is PNMA4, PEG10, or RTL1.

[0028] In some embodiments, the vesicle includes a cell-specific targeting portion. In some embodiments, the cell-specific targeting portion targets mammalian cells. In some embodiments, the cell-specific targeting portion includes a membrane fusion protein. In some embodiments, the membrane fusion protein is VSV-G.

[0029] In some embodiments, the mammalian cells are cancer cells. In some embodiments, the mammalian cells are infected by a pathogen. In some embodiments, the pathogen is a virus.

[0030] In another aspect, the present invention provides a system for delivering cargo to target cells, comprising delivery vesicles that encapsulate the cargo and endogenous reverse transcriptase.

[0031] In some embodiments, the delivery vesicles are virus-like particles. In some embodiments, the delivery vesicles consist of retroviral gag proteins and retroviral envelope proteins. In some embodiments, the retroviral gag proteins are derived from human endogenous retrovirus (HERV).

[0032] In some embodiments, the retroviral gag protein is Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In a specific embodiment, the retroviral gag protein is PNMA4, PEG10, or RTL1.

[0033] In some embodiments, the retroviral envelope protein is derived from HERV. In some embodiments, both the retroviral gag protein and the retroviral envelope protein are derived from HERV.

[0034] In some embodiments, the retroviral envelope protein includes 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.

[0035] In some embodiments, the cargo comprises nucleic acids, proteins, complexes thereof, or combinations thereof. In some embodiments, the cargo comprises ribonucleoproteins. In some embodiments, the cargo comprises a genetic regulator. In some embodiments, the genetic regulator comprises one or more components of a gene-editing system and / or a polynucleotide encoding it. 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 includes CRISPR-Cas9. In some embodiments, a type V CRISPR-Cas system includes CRISPR-Cas12. In some embodiments, a type VI CRISPR-Cas system includes CRISPR-Cas13.

[0036] In some implementations, the cargo is attached to one or more envelope proteins via a connector.

[0037] In some embodiments, the linker is a glycine-serine linker. In some embodiments, the glycine-serine linker is (GGS)3 (SEQ ID NO:1).

[0038] 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 includes incorporating a hairpin loop that binds to a hairpin-binding element on the envelope protein. In some embodiments, the hairpin loop is recognized by the MS2 aptamer.

[0039] In some embodiments, the system may also include a membrane fusion protein. In some embodiments, the membrane fusion protein is VSV-G.

[0040] In some embodiments, the target cells are mammalian cells. In some embodiments, the mammalian cells are cancer cells. In some embodiments, the mammalian cells are infected by a pathogen. In some embodiments, the pathogen is a virus.

[0041] In another aspect, the present invention provides a method for treating a disease, comprising administering any of the systems described herein to a subject in need, wherein delivery vesicles deliver cargo to one or more cells of the subject.

[0042] In some embodiments, the cargo may contain a therapeutic agent. In some embodiments, the therapeutic agent contains one or more components of the gene-editing system and / or a polynucleotide encoding it.

[0043] These and other aspects, objectives, 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 exemplary embodiments shown. Attached Figure Description

[0044] The features and advantages of the present invention can be understood by referring to the following detailed description and drawings of exemplary embodiments of the principles of the invention, and in the drawings:

[0045] Figure 1 - This shows the expression of various env proteins in HEK293T cells, with increased expression of Envw1, Envk1, and Envfrd.

[0046] Figure 2 - This shows the expression of various endogenous retroviral glycoproteins from particles pseudotyped with lentiviral proteins.

[0047] Figure 3 - This shows the expression of the Pnma3-RFP fusion construct (described at the top) compared to the lentivirus-RFP reporter gene in mouse neurons. Photomicrographs show organoid culture sections of the prefrontal cortex.

[0048] Figure 4 - This shows a map of various endogenous gag proteins that were tested for their ability to form capsids, secrete proteins, and transfer substances to new cells.

[0049] Figure 5 - Images showing transmission electron micrographs of the ability of various endogenous gag protein candidates to form a capsid.

[0050] Figure 6 - This demonstrates the ability of various endogenous gag proteins to be secreted from cells.

[0051] Figure 7A , Figure 7B - This shows gag constructs containing the Cas9 / gRNA complex in the absence (7A) and presence (7B) of the membrane fusion protein VSV-G.

[0052] Figure 8 - A schematic diagram illustrating the experimental outline.

[0053] Figure 9A , Figure 9B - This shows the sequence alignment of the number of introduced mutations, with the CRISPR complex transferred in vesicles containing RTL1 (9B) and control vesicles (9A).

[0054] Figure 10- A graph showing the number of insertions and deletions induced by editing complexes in vesicles containing various gag homologs.

[0055] Figures 11A to 11C - This demonstrates the ability of (11A)PNMA4, (11B)PEG10, and (11C)RTL1 to transfer the Cas9 / gRNA complex to new cells.

[0056] Figure 12 - Sequence alignment of knock-in mice expressing the HA tag on endogenous RTL-1.

[0057] Figure 13 - This shows nitrocellulose gels with HA tags for PEG10 and RTL1.

[0058] Figures 14A to 14D - Immunofluorescence images illustrating the ability of various gag homologs (14B to 14D) to form vesicles in the presence of VSV-G, compared to control particles (14A).

[0059] Figure 15A , Figure 15B - A graph showing the copy number of vesicles generated in the presence of various gag homologs is presented.

[0060] Figure 16 - A graph showing the fold change in viral infectivity with overexpression of various gag homologous proteins.

[0061] Figure 17 - This diagram illustrates various putative endogenous signaling systems on a scale of reduced immunogenicity.

[0062] Figure 18 - A schematic diagram illustrating the requirements for a coated VLP is shown.

[0063] Figure 19 - An electron micrograph showing the ability of various gag homologs to spontaneously form vesicles from cells.

[0064] Figure 20 - An electron micrograph showing the ability of various gag homologs to spontaneously form vesicles from cells.

[0065] Figure 21 - This shows the immunoprecipitation assay of various gag homologous proteins secreted from cells.

[0066] Figure 22 - A schematic diagram of a assay used to determine whether GAG is taken up by cells is shown.

[0067] Figures 23A to 23D- (23A, 23B) show the ability of various gag constructs to be taken up by cells and to introduce insertions and deletions into target sequences; (23A) SEQ ID NO: 9-18; (23B) SEQ ID NO: 19-26; (23C, 23D) show graphs of the ability of vesicles to be taken up in HEK293FT cells in the absence of VSV-G (23C) and in the presence of VSV-G (23D).

[0068] Figure 24 - This shows an immunoprecipitation assay demonstrating the ability of various constructs to be taken up by cells in the absence (left) and presence (right) of VSV-G.

[0069] Figure 25 - This shows a schematic diagram of two overlapping reading frames of PEG10.

[0070] Figure 26 - An immunoprecipitation gel showing the translation of ORF1 and ORF1 / 2 bands of PEG10.

[0071] Figure 27 - Immunoprecipitation reaction of whole-cell lysates of cells transfected with various PEG10 constructs.

[0072] Figure 28 Immunoprecipitation reaction of whole-cell lysates and VLP fractions of cells transfected with various PEG10 constructs.

[0073] Figure 29 - An immunoprecipitation assay to analyze the ability of VSV-G and SGCE to increase PEG10 secretion and uptake into target cells.

[0074] Figure 30 - An immunoprecipitation gel demonstrating the ability of various concentrations of sucrose buffer to improve the delivery efficiency of PEG10.

[0075] Figure 31 - This shows a chart of the percentage of missing insertions generated using various builds.

[0076] Figure 32 - It slowed down the localization of PEG10 in serum and cortical neurons in the brain via Western blotting and immunofluorescence staining.

[0077] Figure 33 - A graph showing early embryonic lethality in knockout mice lacking PEG10 is presented, demonstrating the importance of this gene in embryonic development.

[0078] Figure 34RNA sequencing gene ontology analysis of primary mouse neurons revealed three groups of differentially expressed genes: 1) genes involved in nuclear chromatin remodeling, 2) genes involved in the trans-golgi network (and exocytosis), and 3) SNARE and other genes encoding endosome proteins.

[0079] Figure 35 - A fluorescence micrograph showing the expression of the GFP / PEG10 reporter construct.

[0080] Figure 36 - This diagram illustrates the DNA methyltransferase identification mechanism (DamID) for mapping the binding sites of DNA-binding proteins and chromatin-binding proteins. DamID identifies binding sites by expressing the proposed DNA-binding protein along with the DNA methyltransferase as a fusion protein.

[0081] Figure 37 -DamID draws a schematic diagram.

[0082] Figure 38 - The ability of the PEG10-DAMID fusion construct to bind DNA and RNA was analyzed by cross-referencing DamID mapping data and ATAC sequencing data.

[0083] Figure 39 -Mass spectrometry analysis results of proteins enriched in VLP fractions from N2A cells.

[0084] Figure 40 - A schematic diagram of how PEG10 mediates secretion from cells.

[0085] Figure 41 - A schematic diagram of the construct for forming gag vesicles containing RNA is shown.

[0086] Figure 42 - A graph showing the ability of various gag homologs to generate RNA-containing vesicles in the absence of VSV-G.

[0087] Figure 43 - A graph showing the ability of various gag homologs to generate RNA-containing vesicles in the presence of VSV-G.

[0088] Figure 44 - A schematic diagram of a protocol for genome-wide screening of natural proteins across the blood-brain barrier is shown.

[0089] Figure 45 - Modification by transfecting cells passaged in step 1 with a second-generation packaging vector to reactivate the provirus. Figure 44 The scheme shown.

[0090] Figure 46 - This indicates the frequency at which the guide RNA is ultimately internalized in the target cell.

[0091] Figure 47 - This shows a nuclear type of CNS subgroup 14 days after the tail vein.

[0092] Figure 48 - A fluorescence micrograph showing the ability of different fusion agents (Arghap32 and Clmp) to further improve internalization efficiency.

[0093] Figure 49 - A schematic diagram illustrating the protocol used for transfecting constructs and evaluating the ability to generate insertions and deletions. Fusion and overexpression of Cas9 with PEG10 in cells allows for the generation of insertions and deletions in target cells.

[0094] Figure 50 - Analysis of the ability of various gag homologous proteins to act as natural fusion agents.

[0095] Figure 51 - Fluorescence micrographs showing the ability of different fusion agents (Arghap32 and CXADR) to further improve internalization efficiency.

[0096] Figure 52 - A graph showing the results of an analysis of the ability of various gags carrying Cas9 to be secreted from cells.

[0097] Figure 53 - Showing from Figure 52 A graph analyzing the ability of selected gags to be secreted from cells in the presence of VSV-G.

[0098] Figure 54 - This shows the difference between HIV (right) and... Figure 53 The chart generated by gag (left) shows the percentage of missing insertions.

[0099] Figure 55 - Analysis of the ability of various gag-IRES-Cas9 constructs to generate insertion and deletion in the presence of various fusion agents.

[0100] Figure 56 - This diagram illustrates the PEG10 cleavage pattern and protein imprinting of overexpressed, N-terminal and C-terminal tagged mouse PEG10 in HEK293FT cells.

[0101] Figures 57A to 57F- (57A) Protein imprinting of PEG10 cleavage pattern and a graph showing peptide abundance of intact PEG10; (57B) Protein imprinting of PEG10 cleavage pattern and a graph showing peptide abundance of the first reading frame of PEG10; (57C) Protein imprinting of PEG10 cleavage pattern and a graph showing peptide abundance of NC cleavage products; (57D) Protein imprinting of PEG10 cleavage pattern and a graph showing peptide abundance after cleavage at the protease domain of the second reading frame of PEG10; (57E) Protein imprinting of PEG10 cleavage pattern and a graph showing peptide abundance after cleavage at the RT domain of the second reading frame of PEG10; (57F) Protein imprinting of PEG10 cleavage pattern and a graph showing peptide abundance after cleavage at the C-terminus of the second reading frame of PEG10.

[0102] Figures 58A to 58B - Schematic diagram of protein blotting and PEG10 protease cleavage site and the resulting protein fragment (58A) with the putative cleavage prior to the Gag domain (58B).

[0103] Figure 59 A schematic diagram of the -PEG10 ORF1 / 2 gene and a protein blot showing the cleavage pattern of proteins isolated from VLP fractions and whole-cell lysates.

[0104] Figure 60 A schematic diagram of the -PEG10 protein shows that the absence of CCHC in the NC domain prevents it from binding to a specific sequence (SEQ ID NO: 2) that binds via a known myelin expression factor (MYEF).

[0105] Figure 61 - A protocol for a binding assay to determine whether PEG10 binds to DNA and a graph confirming the binding of PEG10 to DNA.

[0106] Figure 62 - This diagram illustrates the estimated location of the ORF1 cut site and the experiments conducted to confirm that location.

[0107] Figure 63 - This diagram illustrates the location of the ORF1 cleavage site and the assessment of effective load secretion.

[0108] Figure 64 – This shows fluorescence micrographs of the expression of GFP fusion constructs for various ORFs.

[0109] Figure 65 - A schematic diagram illustrating the hypothetical functions of various domains interacting with DNA.

[0110] Figure 66- A schematic diagram of PEG10 with mutations in various structural domains that define their functions.

[0111] Figure 67 - This diagram illustrates whether PEG10 is nuclear and can bind to DNA (such as MYEF), and then whether transcription is regulated by PEG10.

[0112] Figure 68 - This diagram illustrates how mutations in the nucleocapsid domain reduce the ability to bind the MYEF motif (SEQ ID NO:3).

[0113] Figure 69 - Footprint assay to determine the function of individual motifs in the PEG10 protein.

[0114] Figure 70 - This shows a quantitative protein blot of PEG10 in the blood of transgenic mice.

[0115] The images in this article are for illustrative purposes only and are not necessarily drawn to scale. Detailed Implementation

[0116] General definition

[0117] Unless otherwise defined, the 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 pertains. Definitions of common terms and techniques in molecular biology can be found in: *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); *Molecular Cloning: A Laboratory Manual*, 4th edition (2012) (Green and Sambrook); *Current Protocols in Molecular Biology* (1987) (edited by F.M. Usubel et al.); *The Series Methods in Enzymology* (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor); *Antibodies, A Laboratory Manual* (1988) (edited by Harlow and Lane); *Antibodies A Laboratory Manual*, 2nd edition 2013 (edited by E.A. Greenfield); *Animal Cell Culture* (1987) (edited by R.R. Freshney); 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 Edition, J. Wiley & Sons (New York, NY)(1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th edition, John Wiley & Sons (New York, NY 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0118] Unless the context clearly indicates otherwise, as used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents.

[0119] The terms “optional” or “optionally” mean that the event, situation, or alternative described below may or may not occur, and the description includes both cases in which said event or situation occurs and cases in which said event or situation does not occur.

[0120] The range of values ​​expressed by endpoints includes all numbers and fractions contained within the corresponding range, as well as the endpoints of the expression.

[0121] As used herein, the term “about” or “approximately” when referring to measurable values ​​such as parameters, quantities, durations, etc., is intended to encompass variations in and from the specified value, such as + / - 10% or less, + / - 5% or less, + / - 1% or less, and + / - 0.1% or less, provided that such variations are suitable for implementation in the disclosed invention. It should be understood that the values ​​referred to by the modifier “about” or “approximately” are themselves specifically and preferably disclosed.

[0122] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. A biological sample may contain (or be derived from) “body fluids.” This invention covers embodiments in which body fluids are selected from: amniotic fluid, aqueous humor, hyaline fluid, bile, serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculation, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, semen (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomitus, and one or more mixtures thereof. Biological samples include cell cultures, body fluids, and cell cultures derived from body fluids. Body fluids may be obtained from mammalian organisms, for example, by puncture or other collection or sampling procedures.

[0123] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rats, apes, humans, farm animals, loitering animals, and pets. This also includes tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro.

[0124] The terms “high,” “higher,” “increased,” “elevated,” or “elevation” refer to an increase to above baseline levels, for example, compared to a control. The terms “low,” “lower,” “reduced,” or “reduction” refer to a decrease to below baseline levels, for example, compared to a control.

[0125] The term "control" refers to any reference standard suitable for providing a comparison with the expression product in the test sample. In one embodiment, control includes obtaining a "control sample," detecting the level of the expression product in the control sample, and comparing it with the level of the expression product from the test sample. Such a control sample may contain any suitable sample, including but not limited to samples from control patients with known results (which may be stored samples or measurements from previous samples); normal tissue, fluid, or cells isolated from subjects (such as normal patients or patients with a disease of interest).

[0126] Various embodiments are described below. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation on the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other one or more embodiments. Throughout this specification, references to “one embodiment,” “an embodiment,” or “an example embodiment” mean that a specific feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment of the invention. Therefore, the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, as will be appreciated by those skilled in the art from this disclosure, specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. Moreover, while some embodiments described herein include features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0127] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference as if each individual publication, published patent document, or patent application were specifically and individually incorporated by reference.

[0128] Overview

[0129] The embodiments disclosed herein provide compositions, systems, and methods for delivering cargo to target cells. This disclosure includes polynucleotides encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more capture portions for packaging cargo within the delivery vesicles. Such vesicles may be virus-like particles. The vesicles can be used to deliver therapeutic agents to target cells. The polynucleotides may contain engineered genes that allow recruitment of cargo molecules or can be fused with cargo molecules that can be packaged in the generated vesicles. Customized polynucleotide compositions will allow for customized cargo and delivery, including cell-specific and cell-nonspecific delivery methods. In specific 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.

[0130] Engineered delivery system

[0131] In one aspect, the embodiments disclosed herein relate to engineered polynucleotides and vectors that encode vesicle-forming delivery systems derived from endogenous retroviral elements. In another aspect, the embodiments disclosed herein relate to the use of such engineered polynucleotides in methods of loading and / or packaging desired cargo molecules. In yet another aspect, the embodiments disclosed herein relate to such cargo-carrying delivery vesicles and methods of delivering cargo molecules to target cells using said delivery vesicles.

[0132] engineered polynucleotides

[0133] The embodiments disclosed herein include engineered polynucleotides encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more capture portions for packaging cargo within the delivery vesicles. The engineered polynucleotides may also include regulatory elements, such as promoters, enhancers, internal ribosome entry sites (IRES), repressors, inducers, etc., to control the expression of the vesicle-forming system. The engineered polynucleotides are designed for delivery to cells, cell-free systems, or any other suitable bioreactor to allow for the expression of delivery system components and the formation of the delivery vesicles, including packaging desired cargo molecules into the delivery vesicles.

[0134] In some embodiments, one or more endogenous retroviral elements for forming delivery vesicles are retroviral envelope proteins. In some embodiments, one or more endogenous retroviral elements for forming delivery vesicles are retroviral gag proteins. In some embodiments, both the retroviral gag protein and the retroviral envelope protein are endogenous. In some embodiments, the gag protein is endogenous while the envelope protein is virally derived. In some embodiments, the envelope protein is endogenous while the gag protein is virally derived. The system may also include cargo domain elements, such as peptide- or nucleotide-based elements that specifically bind to cargo of interest and are further detailed below.

[0135] The system may also include one or more targeting moieties capable of specifically binding to target cells. In some embodiments, the cargo may be linked to one or more envelope proteins via a connector. In some embodiments, the system may include regulatory molecules that control the expression of the vesicle formation system.

[0136] 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 regulatory sequences that direct constitutive expression of nucleotide sequences in many types of host cells, and regulatory sequences that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may direct expression primarily in the desired tissue of interest (e.g., muscle, neurons, bone, skin, blood, designated organs (e.g., liver, pancreas)) or specific cell types (e.g., lymphocytes)). Regulatory elements can also direct expression in a time-dependent manner (such as in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell type-specific. In some embodiments, the vector contains 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 combinations thereof. Examples of pol III promoters include, but are not limited to, the U6, 7SK, and H1 promoters. Examples of pol II promoters include, but are not limited to, the Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) (see, for example, Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter. The term “regulatory element” also encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I (Mol.Cell.Biol.,Vol.8(1),p.466-472,1988); SV40 enhancer; and intron sequence between exon 2 and exon 3 of rabbit β-globulin (Proc.Natl.Acad.Sci.USA.,Vol.78(3),p.1527-31,1981).The specific configurations of gRNA, reporter gene, pol II promoter, and pol III promoter in the context of this invention are described in more detail elsewhere herein.

[0137] In some embodiments, the regulatory sequence may be a regulatory element described in U.S. Patent No. 7,776,321, U.S. Patent Publication No. 2011 / 0027239, and International Patent Publication No. WO 2011 / 028929, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the vector may contain a minimal promoter. In some embodiments, the minimal promoter is a Mecp2 promoter, a tRNA promoter, or a U6 promoter. In another embodiment, 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.

[0138] Generally, a system may include vesicle-generating polynucleotides, vesicle-generating plasmids, vesicles generated by such plasmids, or both. The following sequences can be cloned into vectors. As used herein, a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. A vector is a replicon, such as a plasmid, bacteriophage, or granule, into which another DNA fragment can be inserted to induce replication of the inserted fragment. Generally, a vector is capable of replication 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 attached. 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, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which another DNA fragment can be inserted, such as by standard molecular cloning techniques. Another type of vector is the viral vector, in which a virus-derived DNA or RNA sequence is present for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by the virus for transfection into host cells. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors). Other vectors (e.g., non-episodic mammalian vectors) integrate into the host cell's genome upon introduction and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of the genes they are operatively linked to. Such vectors are referred to herein as "expression vectors." Common expression vectors used in recombinant DNA technologies are often in the form of plasmids.

[0139] Polynucleotides can be RNA or DNA molecules. They can be naturally occurring or recombinant. Polynucleotides can encode proteins or RNA molecules.

[0140] Polynucleotides may contain coding sequences for one or more components of the vesicles described herein. In some examples, polynucleotides contain sequences encoding barcode constructs. Polynucleotides may also contain sequences encoding another element, such as a perturbation element. As used herein, polynucleotides may be DNA, RNA, or hybrids thereof, including but not limited to cDNA, mRNA, genomic DNA, mitochondrial DNA, sgRNA, siRNA, shRNA, miRNA, tRNA, rRNA, snRNA, lncRNA, and synthetic (such as chemically synthesized) DNA or RNA or hybrids thereof. Polynucleotides may include natural nucleotides (such as A, T / U, C, and G), modified nucleotides, analogs of natural nucleotides (such as labeled nucleotides), or any combination thereof.

[0141] This invention also provides delivery vesicles for delivering polynucleotides encoding endogenous proteins. Such delivery vesicles or systems within the scope of this invention may be provided in any form, including but not limited to solids, semi-solids, emulsions, or particles. Similarly, any delivery system described herein, including but not limited to, lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene guns, may be provided as particulate delivery systems within the scope of this invention.

[0142] Generally speaking, "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In some preferred embodiments, the nanoparticles of the present invention have a maximum size (e.g., diameter) of 500 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum size in the range of 25 nm to 200 nm. In other preferred embodiments, the nanoparticles of the present invention have a maximum size of 100 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum size in the range of 35 nm to 60 nm. It should be understood that the particles or nanoparticles mentioned herein may be interchangeable where appropriate.

[0143] It should be understood that the size of the particles will vary depending on whether they are measured before or after loading. Therefore, in certain embodiments, the term "nanoparticles" may be used only for pre-loaded particles.

[0144] The nanoparticles covered by this invention can be provided in various forms, such as solid nanoparticles (e.g., metallic, nonmetallic, lipid-based solids, polymeric substances such as silver, gold, iron, and titanium), suspensions of nanoparticles, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles), can be prepared. Nanoparticles made of semiconductor materials can also be labeled as quantum dots, where electronic energy levels are quantized if they are small enough (typically below 10 nm). Such nanoscale particles are used as drug carriers or imaging agents in biomedical applications and are suitable for similar purposes as described in this invention.

[0145] Semi-solid and soft nanoparticles have been fabricated and are within the scope of this invention. Prototype nanoparticles with semi-solid properties are liposomes. Various types of liposome nanoparticles are currently used clinically as delivery systems for anticancer drugs and antibodies. Nanoparticles that are half hydrophilic and half hydrophobic are called Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at the water / oil interface and act as solid surfactants.

[0146] Self-assembling output compartments or nanoparticles containing RNA can be constructed using polyethyleneimine (PEI), which is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of polyethylene glycol (PEG). This system has been used, for example, as a tumor angiogenesis system targeting integrin expression and as a means of delivering siRNA that inhibits 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). Nanocomposites can be prepared by mixing equal volumes of cationic polymers and nucleic acids in an aqueous solution to produce a net molar excess of ionizable nitrogen (polymer) to phosphate (nucleic acid) in the range of 2 to 6. The electrostatic interaction between the cationic polymer and the nucleic acid results in the formation of a complex (polyplex) with an average particle size distribution of approximately 100 nm, thus referred to herein as a nanocomposite. It is envisioned that doses of approximately 100 to 200 mg of CRISPR Cas could be used for delivery in self-assembled nanoparticles by Schiffelers et al.

[0147] The nanocomposite of Bartlett et al. (PNAS, September 25, 2007, vol. 104, no. 39) can also be applied to this invention. The nanocomposite of Bartlett et al. was prepared by mixing equal volumes of an aqueous solution of a cationic polymer and nucleic acid to produce a net molar excess of ionizable nitrogen (polymer) to phosphate (nucleic acid) in the range of 2 to 6. The electrostatic interaction between the cationic polymer and the nucleic acid resulted in the formation of a complex with an average particle size distribution of approximately 100 nm, hence referred to herein as a nanocomposite. The DOTA-siRNA of Bartlett et al. 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). An amine-modified sense strand of the DOTA-NHS-ester with a 100-fold molar excess in carbonate buffer (pH 9) was added to a microcentrifuge tube. The contents were reacted by stirring at room temperature for 4 hours. DOTA-RNA sense conjugates were precipitated with ethanol, resuspended in water, and annealed with unmodified antisense strands to produce 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 were formed using a polycation containing cyclodextrin. Typically, nanoparticles were formed in water at a charge ratio of 3 (+ / -) and a siRNA concentration of 0.5 g / L. One percent of the adamantane-PEG molecules on the surface of the targeting nanoparticles were modified with Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5% (w / v) glucose carrier solution for injection.

[0148] The lipid particles developed by Qiaobing Xu's lab at Tufts University can be used / suited for this delivery system. See Wang et al., J. Control Release, 2017 Jan 31. pii:S0168-3659(17)30038-X. doi:10.1016 / j.jconrel.2017.01.037. [Electronic version published prior to publication]; Wang 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, Sep. 2014; and Wang et al., Agnew Chem Int Ed Engl., 53(11): 2893-8, Mar. 10, 2014.

[0149] U.S. Patent Publication No. 20110293703 also provides a library of amino alcohol lipid compounds prepared by the methods of the present invention. These amino alcohol lipid compounds can be prepared and / or screened using high-throughput techniques involving liquid handling procedures, robotics, microtiter plates, computers, etc. In some embodiments, the ability of screening amino alcohol lipid compounds to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into cells is considered.

[0150] U.S. Patent Publication No. 2013 / 0302401 relates to a class of poly(β-amino alcohols) (PBAAs) prepared using combinatorial polymerization. The PBAAs of this invention can be used as coatings (such as coatings for thin films or multilayer films for medical devices or implants), additives, materials, excipients, non-biofouling agents, micro-imagers, and cell encapsulation agents in biotechnological and biomedical applications. When used as surface coatings, these PBAAs induce varying levels of inflammation in vitro and in vivo depending on their chemical structure. The significant chemical diversity of such materials allows for the identification of polymer coatings that inhibit macrophage activation in vitro. Furthermore, these coatings reduce the recruitment of inflammatory cells and reduce fibrosis after subcutaneous implantation of carboxylate polystyrene microparticles. These polymers can be used to form polyelectrolyte complex capsules for cell encapsulation. This invention also has numerous other biological applications, such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of U.S. Patent Publication No. 20130302401 can be applied to the CRISPR Cas system of this invention or any other system.

[0151] In another embodiment, lipid nanoparticles (LNPs) were considered. Anti-transthyretin small interfering RNA has been encapsulated in lipid nanoparticles and delivered to humans (see, for example, Coelho et al., N EnglJ Med 2013; 369:819-29), and such systems are suitable for and applicable to the CRISPR Cas system of the present invention or any other system. Intravenous administration doses of approximately 0.01 to approximately 1 mg / kg body weight were considered. Drugs that reduce the risk of infusion-related reactions, such as dexamethasone, acetaminophen, diphenhydramine, or cetirizine and ranitidine, were considered. Multiple doses of approximately 0.3 mg / kg every 4 weeks for a total of 5 doses were also considered.

[0152] Zhu et al. (US20140348900) provided a process for preparing liposomes, lipid disks, and other lipid nanoparticles using a multi-port manifold, wherein a lipid solution stream containing an organic solvent is mixed with two or more aqueous solution streams (e.g., buffer solutions). In some aspects, at least some of the lipid and aqueous solution streams are not directly opposite to each other. Therefore, the process does not require diluting the organic solvent as an additional step. In some embodiments, one of the solutions may also contain an active pharmaceutical ingredient (API). This invention provides a robust process for manufacturing liposomes using different lipid formulations and different payloads. Particle size, morphology, and manufacturing scale can be controlled by varying the port size and the number of manifold ports, as well as by selecting the flow rates or velocities of the lipid and aqueous solutions.

[0153] LNP has been shown to be highly effective in delivering siRNA to the liver (see, for example, Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470), and is therefore being considered for delivering RNA encoding CRISPR Cas to the liver. A dose of approximately four 6 mg / kg LNP every two weeks is considered. Tabernero et al. demonstrated that tumor regression was observed after the first two cycles of LNP administration at 0.7 mg / kg, and by the end of six cycles, the patient had achieved a partial response, complete regression of lymph node metastases, and significant shrinkage of the liver tumor. This patient achieved a complete response after 40 doses, maintained remission, and completed treatment after more than 26 months of continuous dosing. Two patients with RCC and extrahepatic disease sites (including the kidneys, lungs, and lymph nodes) progressed after previous treatment with VEGF pathway inhibitors, with disease stabilizing for approximately 8 to 12 months in all sites. One patient with PNET and liver metastases continued with an 18-month (36-dose) extension study, with disease stabilizing.

[0154] In some implementations, the LNP contains nucleic acid, wherein the charge ratio of the nucleic acid backbone phosphate to the cationic lipid nitrogen atom is about 1:1.5-7 or about 1:4.

[0155] In some embodiments, the LNP further includes a shielding compound that is removable from the lipid composition under in vivo conditions. In some embodiments, the shielding compound is a bioinert compound. In some embodiments, the shielding compound does not carry any charge on its surface or on the molecule itself. In some embodiments, the shielding compound is polyethylene glycol (PEG), a hydroxyethyl glucose (HEG)-based polymer, polyhydroxyethyl starch (polyHES), and polypropylene. In some embodiments, the weight of PEG, HEG, polyHES, and polypropylene is about 500 to 10,000 Da or about 2,000 to 5,000 Da. In some embodiments, the shielding compound is PEG2000 or PEG5000.

[0156] In some implementations, sugar-based particles (e.g., GalNAc) may be used, as described herein and with reference to 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 those applicable to the delivery of all particles, unless otherwise explicitly stated. This may be considered as sugar-based particles and further details of other particle delivery systems and / or formulations are provided herein. Thus, GalNAc may be considered as particles in the other particle sense described herein, such that general uses and other considerations, such as the delivery of the particles, also apply to GalNAc particles. Solution-phase conjugation strategies may be used, for example, to attach tri-antennae GalNAc clusters (molecular weight ~2000) activated as PFP (pentafluorophenyl) esters to 5'-hexylamino-modified oligonucleotides (5′-HA ASO, molecular weight ~8000 Da; (Akinc et al., Bioconjugate Chem., 2015, 26(8), pp 1451-1455). Similarly, in vivo nucleic acid delivery using poly(acrylate) polymers has been described (see WO2013158141, which is incorporated herein by reference). In another alternative embodiment, premixing CRISPR nanoparticles (or protein complexes) with naturally occurring serum proteins can be used to improve delivery (Akinc A et al., 2010, Molecular Therapy vol.18no.7, 1357-1364).

[0157] References that may be used in conjunction with the teachings of this paper 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.2012134:16488-1691, 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.

[0158] As described by Patsuzyn et al. (Cell 172(1-2):275-288; 2018), measurements of intercellular transfer can be evaluated in multiple steps. In a specific implementation, an indirect assay for capsid formation in transfected HEK293 cells can be performed by chemical cross-linking followed by SDS-PAGE to detect the appearance of higher molecular weight bands corresponding to protein oligomers. Extracellular vesicle output can be performed by purifying extracellular vesicle fractions from culture medium, transfecting them, and using Western blotting to identify proteins other than the reported extracellular vesicle markers. Finally, the ability of capsid-containing extracellular vesicles to be taken up by recipient cells can be tested by placing culture medium or purified extracellular vesicle fractions from cells transfected with GFP-tagged Gag onto untransfected cells and using microscopy and / or FACS to look for fluorescent uptake. In addition to extracellular vesicle-mediated transfer, recombinant Arc can form capsids in vitro, which transfer blocked RNA to recipient cells in the absence of an inner membrane. Proteins can also be purified from bacteria or translated in vitro and the activity can be tested. The formation of the capsid structure in different assays can be confirmed using methods including, but not limited to, electron microscopy, dynamic light scattering, or Spectradyne particle analysis.

[0159] In a specific implementation, unassembled recombinant GAG-like proteins, nucleic acids, and / or proteins are assembled in a solution under low-salt conditions.

[0160] U.S. Patent No. 8,709,843, incorporated herein by reference, provides a drug delivery system for targeted delivery of particles containing a therapeutic agent to tissues, cells, and intracellular compartments. The present invention provides targeted particles comprising polymers conjugated with surfactants, hydrophilic polymers, or lipids. The teachings of U.S. Patent No. 8,709,843 can be applied to and / or adapted to the incorporation and / or delivery of one or more engineered delivery system molecules of the invention described herein.

[0161] U.S. Patent No. 5,543,158, incorporated herein by reference, provides biodegradable injectable particles having a biodegradable solid core containing bioactive material and a poly(alkylene glycol) moiety on the surface. The teachings of U.S. Patent No. 5,543,158 can be applied to and / or adapted to the incorporation and / or delivery of one or more engineered delivery system molecules of the invention described herein.

[0162] International Patent Publication No. WO2012135025 (also published as US20120251560), incorporated herein by reference, describes conjugated polyethyleneimine (PEI) polymers and conjugated aza-macrocyclic compounds (collectively, “conjugated lipomers” or “lipomers”). In some embodiments, such conjugated lipomers are envisioned for use in the context of the engineered delivery systems described herein to achieve in vitro, ex vivo, and in vivo expression of one or more components of the engineered delivery systems described herein, and some embodiments may result in the generation of engineered delivery particles from one or more engineered cells.

[0163] Furthermore, the engineered delivery system molecules described herein can be delivered using nanowires, for example, as described 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 to and / or are suitable for generating and / or delivering the CRISPR-Cas system molecules described herein.

[0164] One or more engineered delivery system molecules described herein may be contained in or otherwise incorporated into exosomes for delivery. Exosomes containing one or more engineered delivery molecules described herein may be used to deliver one or more engineered delivery system molecules to cells and / or subjects.

[0165] 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. Targeting the brain was achieved by engineering dendritic cells to express Lamp2b (an exosomal membrane protein fused with a neuron-specific RVG peptide). Purified exosomes were loaded with exogenous RNA via electroporation. Intravenously injected RVG-targeted exosomes specifically delivered GAPDH siRNA to neurons, microglia, and oligodendrocytes in the brain, resulting in specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and no non-specific uptake was observed in other tissues. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic agent for Alzheimer's disease. The teachings of Alvarez-Erviti et al. can be applied to and / or utilized in the generation and / or delivery of the CRISPR-Cas system molecules described herein.

[0166] In some implementations, the delivery system elicits a poor immune response or has low immunogenicity.

[0167] In some implementations, the delivery vesicles are virus-like particles (VLPs). As used herein, the term "virus-like particle" (VLP) refers to a structure that is similar to a virus in at least one property but has not yet been proven to be infectious. A VLP can be a non-replicating, non-infectious viral capsid containing a viral capsid but lacking all or part of the viral genome, particularly the replication component of the viral genome. VLPs are typically composed of one or more viral proteins, such as, but not limited to, those called capsids, coats, shells, surface and structural proteins (e.g., VP1, VP2). A VLP can also resemble a bacteriophage structure, being non-replicating and non-infectious, and lacking at least one or more genes encoding the bacteriophage replication mechanism, and also lacking one or more genes encoding one or more proteins responsible for viral attachment to or entry into the host.

[0168] Envelopes from various retroviral sources can be used for pseudogenotyping of vectors. The exact rules governing pseudogenotyping (i.e., which envelope proteins will interact with the nascent vector particles on the cytoplasmic side of the cell membrane to produce viable viral particles (Tato, Virology 88:71, 1978) and which will not (Vana, Nature 336:36, 1988)) are not well characterized. However, since a cell membrane buds to form the viral envelope, molecules normally present in the membrane are carried on the viral envelope. Therefore, many different potential ligands can be placed on the surface of viral vectors by manipulating cell lines to prepare gags and pols (in which vectors are generated) or by selecting various types of cell lines with specific surface markers. One surface marker that can be expressed in helper cells and can produce usable vector-cell interactions is another potential receptor for pathogenic viruses. Pathogenic viruses exhibit their usual interactions with cell surface markers or receptors on the surface of infected cells to produce virus-specific proteins (e.g., env) that cause viral infection. This reverses the vector's specificity for infection by using the same viral protein-receptor interaction, but with the interaction between the receptor on the vector and the viral protein on the cell.

[0169] One known virus involved in pseudotype formation is vesicular stomatitis virus (VSV), a prototypical member of the rhabdovirus family. It is an enveloped virus with a negative-sense RNA genome that causes self-limiting disease in livestock and is essentially non-pathogenic to humans. (Balachandran and Barber, 2000, IUBMB Life 50:135-8). Rhabdoviruses have a single-stranded negative-sense RNA genome of 11,000 to 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 viral particle contains a helical nucleocapsid core composed of genomic RNA and proteins. Generally, three proteins have been found to be associated with the nucleocapsid: N (nucleocapsid, tightly enclosing the genome), P (formerly known as NS, initially indicating non-structural), and L (large). The additional matrix (M) proteins are located within the membrane envelope and may interact with both the membrane and the nucleocapsid core. Individual glycoprotein (G) species cross the membrane and form spikes on the surface of the viral particle.

[0170] Endogenous retroviral elements

[0171] Human endogenous retroviruses (HERVs) comprise 8.29% of the draft human genome. Their prevalence stems from the accumulation of past retroviral infectious agents that have entered the germline, established a truce with host cells, and are expressed from the host genome. HERVs can be divided into approximately 100 distinct families based on sequence homology, each containing several to several hundred elements. Genes selected by the host from endogenous retroviruses are found to be active participants in several cellular processes, including viral defenses of Fv1 and Fv4 in mice and cell fusion in human placental development mediated by syncytiocytin. Although HERV transcripts have been detected in both normal and cancerous tissues (including T cells), their roles in normal cellular function and oncogenicity remain unclear. While the cellular conditions that promote HERV transcription are not fully understood, APOBEC has been shown to play a role in controlling endogenous retroviruses.

[0172] The strong similarity between HERV and retroviruses can be deduced from phylogenetic analyses of the reverse transcriptase domain of the pol gene or the transmembrane (TM) portion of the env gene, revealing an interleaving of the two elements and indicating a common history and shared ancestor (Tristem, M. (2000) J. Virol. 74, 3715-3730; Benit et al. (2001) J. Virol. 75 (11709-11719). Similarities have also been observed at the functional level.

[0173] Due to the close relationship between HERV and infectious retroviruses, and despite the fact that most HERVs have accumulated mutations, deletions and / or truncations, it is possible that some elements may still retain infectious retroviral functions, and the host may have converted these functions for its own benefit.

[0174] Genes encoding viral polypeptides capable of self-assembling into defective, non-reproducing viral particles can be obtained from the genomic DNA of DNA viruses or the genomic cDNA of RNA viruses, or from available subgenomic clones containing these genes. These genes will include those encoding viral capsid proteins (i.e., the proteins that make up the viral protein coat), and in the case of enveloped viruses (such as retroviruses), genes encoding viral envelope glycoproteins. Capsid protein maturation and particle self-assembly may also require additional viral genes. These can encode viral proteases responsible for processing capsid proteins or envelope glycoproteins. For example, the genomic structure of picornaviruses has been well characterized, and the protein synthesis patterns leading to viral particle assembly are well understood. Rueckert, R. in Virology (1985), BN Fields et al. (eds.), Raven Press, New York, pp. 705-738. In picornaviruses, the viral capsid protein is encoded by an RNA genome containing a single long reading frame and synthesized as part of a multiprotein, which is processed by a combination of cellular and viral proteases to produce the mature capsid protein. Therefore, the piconetic genes required for capsid self-assembly include capsid structural genes and viral proteases required for their maturation. Another type of virus from which genes encoding self-assembling capsid proteins can be isolated is lentivirus, with HIV being an example. Similar to piconetic capsid proteins, HIV gag protein synthesis occurs as a precursor polypeptide, which is subsequently processed by viral proteases into a mature capsid polypeptide. However, the gag precursor polypeptide can self-assemble into virus-like particles without protein processing. (Gheysen et al., Cell 59:103 (1989); Delchambre et al., The EMBO J.8:2653-2660 (1989)). Unlike piconetic capsids, the HIV capsid is surrounded by a loose membrane envelope containing viral glycoproteins. These are encoded by the viral env gene.

[0175] In an alternative embodiment, additional human proteins with Gag homology may be used to assemble virus-like capsids that mediate intercellular transfer of cargo. Such proteins include, but are not limited to, the extended PNMA gene family, including ZCC18, ZCH12, PNM8B, PNM6A, PMA6F, PMA6E, PNMA2, PNM8A, PNMA3, PNMA5, PNMA1, MOAP1, and CCDC8. In a specific embodiment, the GAG-like protein is Arc.

[0176] 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.

[0177] In some embodiments, one or more endogenous retroviral elements used to form delivery vesicles may comprise two or more of the following: retroviral gag protein, retroviral envelope protein, retroviral reverse transcriptase, or combinations thereof.

[0178] Retroviral Gag protein

[0179] Group-specific antigen (gag) proteins are core structural proteins or major components of the retroviral capsid. The HIV p17 matrix protein (MA) is a 17 kDa protein composed of 132 amino acids, containing the N-terminus of the Gag polyprotein. It is responsible for targeting the Gag polyprotein to the plasma membrane, but also contacts the HIV transmembrane glycoprotein gp41 in assembled viruses and plays a crucial role in recruiting the Env glycoprotein to the viral budding site.

[0180] Several studies have demonstrated that the expression of the gag gene alone in many systems leads to the efficient assembly and release of enveloped viral particles (Craven, RC et al. (1996). Dynamic interactions of Gag multiproteins. Current Topics in Microbiology and Immunology 214, pp. 65-94; Delchambre, M. et al. (1989). The Gag precursors of simian immunodeficiency virus assemble 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, HP et al. (1989), "Assembly and release of HIV-1 precursor." Pr55gagvirus-like particles from recombinant baculovirus-infected insect cells,” Cell59, 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 Virology5, pp.71-83; H.-G. et al. (1996), "Intracellular transport of retroviralcapsid components," Current Topics in Microbiology and Immunology 214, pp. 25-64; Madisen, L. et al. (1987), "Expression of the human immunodeficiency virus gaggene in insect cells," Virology 158, pp.248-250; Smith, AJ et al. (1990), "Humanimmunodeficiency virus type 1Pr55 gag and Pr 1 60gag-pol expressed from asimian 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). Therefore, the product of this gene possesses the structural information necessary for mediating intracellular transport, direct assembly into the capsid shell, and catalyzing a membrane extrusion process called budding.

[0181] Once Gag is translated, the Gag polyprotein is myristylated at its N-terminal glycine residue by N-myristyltransferase 1, a crucial modification for plasma membrane targeting. In its membrane-free form, the myristoyl fatty acid tail of the myristoyl amino acid (MA) is isolated in a hydrophobic pouch within the MA protein core. MA recognition of plasma membrane proteins activates a "myristoyl switch," in which the myristoyl group is extruded from the hydrophobic pouch in the MA and embedded into the plasma membrane.

[0182] The HIV nucleocapsid protein (NC) is a 7 kDa zinc finger protein in the Gag polyprotein and forms the viral nucleocapsid after viral maturation. The NC recruits the full-length viral genome RNA to the nascent viral particles.

[0183] The neuronal gene Arc shares homology with 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 in cells and when recombinantly expressed in bacteria. Arc capsids are capable of encapsulating their own mRNA, thereby mediating their intercellular transfer within extracellular vesicles. Purified Arc proteins can be used to reconstruct capsids with different DNAs or RNAs or proteins, or mixtures thereof, and can be packaged into capsids for delivery into cells. In some embodiments, lipids can be used to assemble the capsid to aid cellular uptake. Various embodiments may utilize different Arc orthologs.

[0184] In some embodiments, the polynucleotides described herein may comprise Gag homologs or their functional domains. The term "functional domain" refers to a polypeptide sequence having activity other than binding to a nucleic acid sequence recognized by a nucleic acid binding domain. By combining a nucleic acid binding domain with one or more effector domains, the polypeptides of the present invention can be used to target specific DNA sequences that specifically bind to the nucleic acid binding domain, mediated by the effector domain.

[0185] The molecular and genetic determinants of Gag-mediated intercellular communication can be determined by characterizing the mechanisms of capsid-mediated intercellular mRNA transfer, with particular attention to features that could allow such systems to be used for programmable cargo delivery. Different Gag proteins have evolved a variety of distinct RNA-binding domains for mediating specific encapsulation of their RNA genomes. The RNA-binding sequence specificity of human Gag homologs can be tested by protein pull-down and sequencing of associated RNA and / or by sequencing of extracellular vesicle fractions from HEK293 cells overexpressing each protein. Nucleic acid-binding domains can be exchanged between proteins, or additional RNA-binding domains with known specificity can be fused to test the extent to which binding specificity can be reprogrammed. Thus, Gag homologs or their functional domains may contain both an export compartment domain and a nucleic acid-binding domain.

[0186] Gag homologs can be selected from Arc, ASPRV1, Sushi-like proteins, Scan proteins, or PNMA proteins. In specific cases, the Gag homolog 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 some embodiments, the Gag homolog is an Arc protein, and in others, it is hARC or dARC1. Gag homologs may include ASPRV1. In other cases, the Gag homolog is PEG10, RTL3, RTL10, or RTL1. In some embodiments, the Gag homolog is a Scan protein, such as PGBD1. In some cases, the PEG10 Gag homolog is PEG10_i6 or PEG10_i2.

[0187] In some embodiments, the Gag homologous protein or its functional domain may include an export compartment domain and a nucleic acid binding domain. In specific embodiments, the nucleic acid binding domain may be modified relative to the native nucleic acid binding domain of the Gag homologous protein. In specific embodiments, the nucleic acid binding domain may be a non-native nucleic acid binding domain relative to the Gag homologous protein. In some embodiments, the Gag homologous protein may be an Arc or paraneoplastic Ma antigen (PNMA) protein.

[0188] In some implementations, recombinant GAG-like proteins can be expressed and purified from bacterial, yeast, insect, or mammalian cells. The recombinant GAG-like proteins can be purified under denaturing conditions and transferred to non-denaturing conditions via buffer exchange.

[0189] In some implementations, the retroviral gag protein is endogenous.

[0190] In some implementations, the retroviral gag protein may contain NC and MA domains.

[0191] In some implementations, as described herein, the retroviral gag protein may be a gag homolog.

[0192] In some embodiments, gag homologs may include, but are not limited to, Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12. In a specific embodiment, the gag homolog is Arc1, PNMA6a, or PNMA3. In a specific embodiment, the gag homolog is PEG10.

[0193] In some implementations, the gag homolog may contain a DNA-binding motif. As a specific example, and as discussed in Example 4, PEG10 contains a DNA-binding motif that allows packaging of a specified sequence of DNA.

[0194] As any person skilled in the art will understand, any system described herein can also be engineered into a minimal set of components and applied to any suitable endogenous element. See Examples 3 and 4, and... Figures 56 to 70 As described above, using PEG10 is just one example method, and any other endogenous element can be used after this method.

[0195] Reverse transcription of Env protein

[0196] Env is a retroviral gene encoding proteins that form the viral envelope. Expression of the env gene allows retroviruses to target and attach to specific cell types and penetrate the target cell membrane. The structures and sequences of several different env genes indicate that the Env protein is a type 1 fusion machinery. The type 1 fusion machinery initially binds to a receptor on the surface of the target cell, triggering a conformational change that allows the fusion protein to bind. The fusion peptide inserts itself into the host cell membrane, bringing the host cell membrane very close to the viral membrane, thus allowing membrane fusion. The sequence of the env gene can vary significantly among retroviruses; however, it is always located downstream of gag, pro, and pol. env mRNA must be spliced ​​to be expressed.

[0197] Env not only mediates viral entry into cells but is also a major target of cellular and antibody responses. It is synthesized as the precursor molecule gp160, which is subsequently processed by cellular proteases into the surface subunit (SU)gp120 and the transmembrane subunit (TM)gp41, existing as a trimer of the gp120-gp41 heterodimer on the viral or cell membrane. The SU protein domain determines viral tropism, as it is responsible for viral receptor binding. Therefore, the SU domain determines the specificity of the virus for a single receptor molecule. gp120 interacts with HIV's receptor and co-receptor molecules and mediates viral attachment to cells, while gp41 induces subsequent fusion between the virus and the cell membrane during the initial infection process for the release of the viral core components into the cell. The TM protein consists of three distinct domains: an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

[0198] In some implementations, the retroviral envelope protein is endogenous.

[0199] In some embodiments, the envelope protein may be derived from a gamma retrovirus. In some embodiments, the envelope protein may be derived from a delta retrovirus.

[0200] In some implementations, the envelope protein may be selected from, but is not 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.

[0201] In one aspect, the present invention provides the introduction of an RNA sequence into a transcript recruitment sequence, the transcript recruitment sequence forming a circular secondary structure and binding to an adaptor protein. In one aspect, the present invention provides a composition discussed herein, wherein the insertion of a different RNA sequence binding to one or more adaptor proteins is an aptamer sequence. In one aspect, the present invention provides a composition discussed herein, wherein the aptamer sequence comprises two or more aptamer sequences specific to the same adaptor protein. In one aspect, the present invention provides a composition discussed herein, wherein the aptamer sequence comprises two or more aptamer sequences specific to different adaptor proteins. In one aspect, the present invention provides a composition discussed 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, and PRR1. In one aspect, the present invention provides a composition discussed herein, wherein the cell is a eukaryotic cell. In another aspect, the present invention provides a composition discussed herein, wherein the eukaryotic cell is a mammalian cell, optionally a mouse cell. In another aspect, the present invention provides a composition discussed herein, wherein the mammalian cell is a human cell. Aspects of the present invention cover embodiments relating 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.

[0202] In some implementations, the adaptor protein domain is an RNA-binding protein domain. The RNA-binding protein domain recognizes a corresponding different RNA sequence, which can be an aptamer. For example, the MS2 RNA-binding protein recognizes and specifically binds to the MS2 aptamer (or vice versa).

[0203] Similarly, MS2 variant adaptor domains, such as the N55 mutant, and especially the N55K mutant, can also be used. This is the N55K mutant of the MS2 phage coat protein (demonstrated to have a higher binding affinity than wild-type MS2 in Lim, F., M. Spingola and DSPeabody. "Altering the RNA binding specificity of a translational repressor." Journal of Biological Chemistry 269.12(1994):9006-9010).

[0204] In some embodiments, the envelope protein may 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.

[0205] In some embodiments, both the retroviral gag protein and the retroviral envelope protein are endogenous. In some embodiments, the gag protein is endogenous while the envelope protein is derived from the virus. In some embodiments, the envelope protein is endogenous while the gag protein is derived from the virus.

[0206] Capture part

[0207] In some implementations, the vesicle includes one or more capture portions, for example, capture portions for packaging goods and / or recruiting designated goods into the vesicle.

[0208] As used herein, the term "nucleic acid capture moiety," or simply "capture moiety," refers to a portion that selectively binds to a target molecule. Optionally, this moiety may be immobilized on an insoluble support, such as in a microarray or on microparticles (such as beads). When used as a primer, the probe of the present invention may not be anchored to a solid support. The capture moiety "captures" the target molecule by hybridizing with and thus immobilizing the target. In the case where the moiety itself is immobilized, the target also becomes immobilized. This binding to the solid support can be achieved through a connecting portion that binds to either the capture moiety or the solid support.

[0209] The capture portion may contain one or more polynucleotide endogenous genes or plasmid endogenous genes, such as genes capable of recruiting plasmids into vesicles. The capture portion may contain exogenous genes or molecules capable of recruiting or capturing cargo molecules for vesicles. In some examples, the capture portion may interact with the cargo. The capture portion may be a nucleic acid-binding molecule, such as DNA, RNA, DNA-binding proteins, RNA-binding proteins, or combinations thereof. In some embodiments, the capture portion may be a protein-binding molecule, such as DNA, RNA, antibodies, nanobodies, antigens, receptors, ligands, fragments thereof, or combinations thereof. The capture portion may be fused with endogenous or exogenous genes.

[0210] In some implementations, one or more capture portions include a DNA-binding portion, an RNA-binding portion, a protein-binding portion, or a combination thereof.

[0211] In some embodiments, the capture portion may be, for example, a fluorescent portion, a radioactive isotope (e.g., 32 Labeling can be performed using antibodies, antigens, lectins, enzymes (e.g., alkaline phosphatase or horseradish peroxidase, which can be used for calorimetry), chemiluminescence, bioluminescence, or other labels well known in the art. In some embodiments, the 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 labeled as such, or alternatively, a labeled secondary probe may be used. A “secondary probe” comprises a nucleic acid sequence complementary to a region of the target nucleic acid sequence or a region of the capture moiety. The G region of the probe (which will typically not be complementary to the target) may be used to capture the secondary labeled nucleic acid probe.

[0212] In some embodiments, the capture portion is a nucleic acid hairpin. As used herein, the terms “nucleic acid hairpin,” “hairpin capture portion,” or simply “hairpin,” refer to a structure containing a single-molecule nucleic acid that comprises at least two complementary nucleic acid regions such that at least one intramolecular double strand can be formed. Hairpins are described, for example, in Cantor and Schimmel, “Biophysical Chemistry,” Part III, p. 1183 (1980). In some embodiments, the complementary nucleic acid regions are linked by a nucleic acid strand; in these embodiments, the hairpin comprises a single strand of nucleic acid. The region of the capture portion that connects the complementary regions is referred to herein as a “loop” or “connector.” In some embodiments, the loop comprises a nucleic acid strand or a modified nucleic acid. In some embodiments, the connector is not a hydrogen bond. In other embodiments, the loop comprises a connector region that is not based on nucleic acid; however, the capture portion in which the loop region is not a nucleic acid sequence is referred to herein as a hairpin. Examples of non-nucleic acid connectors suitable for loop regions are known in the art and include, for example, alkyl chains (see, for example, Doktycz et al. (1993) Biopolymers 33:1765). While it should be understood that the loop can be a single-stranded region of the hairpin, for the purposes of the following discussion, "single-stranded region" of the hairpin refers to the non-loop region of the hairpin. In embodiments where the loop is a nucleic acid strand, the loop preferably contains 2 to 20 nucleotides, more preferably 3 to 8 nucleotides. The size or configuration of the loop or linker is selected to allow complementary regions to form intramolecular duplexes. In a preferred embodiment, the hairpin used in the present invention will form at least one intramolecular duplex having at least 2 base pairs, more preferably at least 4 base pairs, and still more preferably at least 8 base pairs. The number of base pairs in the duplex region and its base composition can be selected to ensure any desired relative stability of duplex formation. For example, to prevent hybridization of non-target nucleic acids with the intramolecular duplex forming region of the hairpin, the number of base pairs in the intramolecular duplex region will typically be greater than about 4 base pairs. The intramolecular duplex will typically not have more than about 40 base pairs. In a preferred embodiment, the length of the intramolecular double strand is less than 30 base pairs, more preferably less than 20 base pairs.

[0213] Hairpins may be able to form more than one loop. For example, a hairpin capable of forming two intramolecular duplexes and two loops is referred to herein as a "double hairpin". In a preferred embodiment, the hairpin will have at least one single-stranded region substantially complementary to the target nucleic acid sequence. "Substantially complementary" means capable of hybridizing with the target nucleic acid sequence under the conditions employed. In a preferred embodiment, the "substantially complementary" single-stranded region is precisely complementary to the target nucleic acid sequence. In a preferred embodiment, the hairpin used in the present invention has a target complementary single-stranded region having at least 5 bases, more preferably at least 8 bases. In a preferred embodiment, the hairpin has a target complementary single-stranded region having less than 30 bases, more preferably less than 25 bases. The target complementary region will be selected to ensure that the target strand forms a stable duplex with the capture portion. In embodiments where the capture portion is used to detect the 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 located at the 3' or 5' end of the capture portion of the chain, or it can be located between two intramolecular double-stranded regions (e.g., between two double-stranded regions in a double hairpin).

[0214] cargo molecules

[0215] The delivery particles described herein can be used, and they may also contain a variety of different cargo molecules for delivery. Representative cargo molecules may include, but are not limited to, nucleic acids, polynucleotides, proteins, peptides, polynucleotide / peptide complexes, small molecules, sugars, or combinations thereof. Cargoes that can be delivered according to the systems and methods described herein include, but are not limited to, bioactive agents, including but not limited to therapeutic agents, imaging agents, and monitoring agents. Cargoes may be exogenous or endogenous materials.

[0216] Bioactive agents include any molecule that induces an effect in a cell. Bioactive agents can be proteins, nucleic acids, small molecules, carbohydrates, and lipids. When the cargo is or contains nucleic acids, the nucleic acid can be an isolated entity derived from a DNA-based vector. In these embodiments, the DNA-based vector itself is not the cargo. In other embodiments, the DNA-based vector itself may contain nucleic acid cargo. Therapeutic agents include chemotherapeutic agents, anticancer agents, antiangiogenic agents, tumor inhibitors, antimicrobial agents, enzyme substitutes, gene expression regulators, and expression constructs containing nucleic acids encoding therapeutic proteins or nucleic acids. Therapeutic agents can be peptides, proteins (including enzymes, antibodies, and peptide hormones), cytoskeleton ligands, nucleic acids, small molecules, non-peptide hormones, etc. To increase affinity for the nucleus, 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 nucleic acid materials and natural nucleic acid materials, including DNA, RNA, transposon DNA, antisense nucleic acids, dsRNA, siRNA, transcribed RNA, messenger RNA, ribosomal RNA, nucleolar small RNA, microRNA, ribonuclease, plasmids, expression constructs, etc.

[0217] Imaging agents include contrast agents, such as ferrofluid-based MRI contrast agents and gadolinium, fluorescein isothiocyanate, and 6-TAMARA for PET scans. Monitoring agents include reporter probes, biosensors, green fluorescent proteins, etc. Reporter probes include light-emitting compounds such as phosphors, radioactive and fluorescent moieties, such as rare earth chelates (e.g., europium chelates), Texas Red, rhodamine, fluorescein, FITC, fluo-3,5-hexadecanoyl fluorescein, Cy2, fluor X, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, dansyl, phycocytherin, phycocyanin, spectral orange, spectral green, and / or any one or more derivatives thereof. Biosensors are molecules that detect and transmit information about physiological changes or processes, for example, by detecting the presence of a chemical substance or changes in the presence of a chemical substance. Information obtained by a biosensor typically activates a signal detected by a transducer. Transducers typically convert biological reactions into electrical signals. Examples of biosensors include enzymes, antibodies, DNA, receptors, and regulatory proteins used as recognition elements, which can be used throughout the cell or isolated and used independently (D'Souza, 2001, Biosensors and Bioelectronics 16:337-353).

[0218] One or two or more different goods may be delivered by the delivery particles described herein.

[0219] In some embodiments, as described elsewhere herein, the cargo can be linked to one or more envelope proteins via a connector. Suitable connectors may include, but are not limited to, glycine-serine connectors. In some embodiments, the glycine-serine connector is (GGS)3 (SEQ ID NO:1).

[0220] In some embodiments, the cargo contains ribonucleoproteins. In specific embodiments, the cargo contains genetic regulators.

[0221] As used herein, the term "altered expression" may specifically refer to the alteration of the gene product produced by the cell. As used herein, the term "gene product" includes RNA (e.g., mRNA) transcribed from a gene or polypeptides encoded by a gene or translated from RNA.

[0222] Furthermore, as used herein, “altered expression” can encompass the regulation of the activity of one or more endogenous gene products. Therefore, the terms “altered expression,” “altering expression,” “modulating expression,” or “detecting expression,” or similar terms, can be used interchangeably with “altered expression or activity,” “altering expression or activity,” “modulating expression or activity,” or “detecting expression or activity,” or similar terms. As used herein, “modulating” or “to modulate” generally means reducing or inhibiting the activity of a target or antigen, or alternatively increasing the activity of a target or antigen, as measured using appropriate in vitro, cellular, or in vivo assays. Specifically, “modulating” or “to modulate” can mean: reducing or inhibiting the activity of a target or antigen (as determined by appropriate in vitro, cellular, or in vivo assays, typically depending on the target or antigen involved) by 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the activity of the target or antigen in the same assay under the same conditions but in the absence of the inhibitors / antagonists or activators / agonists described herein, or alternatively increasing the biological activity of the target or antigen (as determined by appropriate in vitro, cellular, or in vivo assays) 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.

[0223] As will be apparent to those skilled in the art, “modulation” can also refer to alterations (either increases or decreases) in the affinity, specificity, and / or selectivity of a target or antigen for one or more targets, compared to the same conditions but in the absence of a modulator. Again, this can be determined based on the target in any suitable manner and / or using any suitable assay known per se. In particular, the action as an inhibitor / antagonist or activator / agonist can result in an increase or decrease of 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 in the expected biological or physiological activity compared to the same assay under the same conditions but in the absence of an inhibitor / antagonist or activator / agonist. Modulation can also involve the activation of the target or antigen or the mechanism or pathway involved.

[0224] In some implementations, the genetic regulator may comprise one or more components of the gene editing system and / or a polynucleotide encoding it.

[0225] In some implementations, the gene editing system may be a CRISPR-Cas system.

[0226] CRISPR system

[0227] Generally, as used herein and in documents such as WO 2014 / 093622 (PCT / US2013 / 074667)), CRISPR-Cas or CRISPR system refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-related genes (“Cas”), including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active tracrRNA), tracr-mate sequences (in the context of endogenous CRISPR systems, encompassing “direct repeats” and partial direct repeats processed by tracrRNA), guide sequences (also referred to as “spacers” in the context of endogenous CRISPR systems), or the term “RNA” as used herein (e.g., RNA that guides Cas (such as Cas9), such as CRISPR RNA and trans-activating (tracr) RNA or single-stranded guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from CRISPR loci. Generally speaking, CRISPR systems are characterized by elements that facilitate the formation of CRISPR complexes at target sequence sites (also known as protospacers in the context of endogenous CRISPR systems). See, for example, 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.

[0228] Class 1 system

[0229] The methods, systems, and tools provided herein can be designed for use with class 1 CRISPR proteins. In some 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 (February 2020), and particularly as described on page 326. Figure 1The aforementioned literature is incorporated herein by reference in its entirety. Class 1 systems typically utilize multi-protein effector complexes, which in some embodiments may include ancillary proteins, such as one or more proteins in a cascade complex for antiviral defense called a CRISPR-associated complex, one or more adaptive proteins (e.g., Cas1, Cas2, RNA nucleases) and / or one or more accessory proteins (e.g., Cas4, DNA nucleases), proteins containing CRISPR-associated Rossman folds, and / or RNA transcriptase (CARF) domains. Although the sequence similarity of Class 1 systems is limited, Class 1 system proteins can be identified by their similar constructions, 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. Large subunits (e.g., Cas8 or Cas10) and small subunits (e.g., Cas11) are also typical features of Class 1 systems. See, for example... Figure 1 and Figure 2Koonin 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. Cascade, particularly class 1 proteins, may contain a dedicated complex of multiple Cas proteins that bind pre-crRNA and recruit additional Cas proteins (e.g., Cas6 or Cas5), which are nucleases directly responsible for processing the pre-crRNA. In another aspect, type I CRISPR proteins contain 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, types IV-A and IV-B, and types III-A, III-D, III-C, and III-B. Class 1 systems also include CRISPR-Cas variants, including type IA, type IB, type IE, type IF, and type IU variants. These variants can include variants carried by transposons and plasmids, including IF subtype versions encoded by a large family of Tn7-like transposons and a smaller group of Tn7-like transposons, and IB subtype systems encoded by Tn7-like transposons with similar degradation. Peters et al., PNAS 114(35)(2017); DOI: 10.1073 / pnas.1709035114; see also Makarova et al., the CRISPR Journal, v.1, n5. Figure 5 .

[0230] Type 2 systems

[0231] The compositions, systems, and methods described in more detail elsewhere in this document 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 differ from class 1 systems in that they possess a single, large, multi-domain effector protein. In some exemplary embodiments, a class 2 system can be a type II, V, or 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 (February 2020), which is incorporated herein by reference. Each type of class 2 system is further subdivided into subtypes. See Markova et al. 2020, in particular... Figure 2 Type 2, type II system can be divided into 4 subtypes: II-A, II-B, II-C1, and II-C2. Type 2, type V system 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. Type 2, type IV system can be divided into 5 subtypes: VI-A, VI-B1, VI-B2, VI-C, and VI-D.

[0232] The distinguishing feature of these types is that their effector complexes consist of a single, large, multi-domain protein. Type V systems differ from type II effectors (e.g., Cas9), which contain two nuclear domains, each responsible for cleaving one strand of the target DNA, with the HNH nuclease inserted into a Ruv-C-like nuclease domain sequence. Type V systems (e.g., Cas12) contain only a RuvC-like nuclease domain that cleaves both strands. Type VI (Cas13) is independent of the effectors of both type II and type V systems and contains two HEPN domains and the target RNA. The Cas13 protein also exhibits collateral activity triggered by target recognition. Some type V systems have also been found to possess this collateral activity in an in vitro context and have two single-stranded DNA molecules.

[0233] In some embodiments, the Type II system is a Type II system. In some embodiments, the Type II CRISPR-Cas system is a II-A CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-B CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-C1 CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-C2 CRISPR-Cas system. In some embodiments, the Type II system is a Cas9 system. In some embodiments, the Type II system includes Cas9.

[0234] In some implementations, the type 2 system is a V-type system. In some implementations, the V-type CRISPR-Cas system is a VA CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a V-B1 CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a V-B2 CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a VC CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a V-DC CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a VE CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a V-F1 CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a V-F1(V-U3) CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system is a V-F2 CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a V-F3 CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a VG CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a VH CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a VI CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a VK(V-U5) CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a V-U1 CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a V-U2 CRISPR-Cas system. In some embodiments, the V-type CRISPR-Cas system is a V-U4 CRISPR-Cas system. In some implementations, the V-type CRISPR-Cas system includes Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas14 and / or CasΦ.

[0235] In some embodiments, the Type 2 system is a Type VI system. In some embodiments, the Type VI CRISPR-Cas system is a VI-A CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a VI-B1 CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a VI-B2 CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a VI-C CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a VI-D CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system includes Cas13a (C2c2), Cas13b (group 29 / 30), Cas13c, and / or Cas13d.

[0236] CRISPR-Cas system cargo molecules

[0237] Generally, as used herein and in documents such as WO 2014 / 093622 (PCT / US2013 / 074667)), CRISPR-Cas or CRISPR system refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-related genes (“Cas”), including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active tracrRNA), tracr-mate sequences (in the context of endogenous CRISPR systems, encompassing “direct repeats” and partial direct repeats processed by tracrRNA), guide sequences (also referred to as “spacers” in the context of endogenous CRISPR systems), or the term “RNA” as used herein (e.g., RNA that guides Cas (such as Cas9), such as CRISPR RNA and trans-activating (tracr) RNA or single-stranded guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from CRISPR loci. Generally speaking, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at target sequence sites (also known as prespacer sequences in the context of endogenous CRISPR systems). See, for example, 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.

[0238] In some embodiments, a protospacer adjacent motif (PAM) or PAM-like motif guides the binding of the effector protein complex, as disclosed herein, to the 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 sequence). In other embodiments, the PAM may be a 3′ PAM (i.e., located downstream of the 5′ end of the protospacer sequence). The term “PAM” may be used interchangeably with the term “PFS” or “protospacer flanking site” or “protospacer flanking sequence”.

[0239] In a preferred embodiment, the CRISPR effector protein recognizes 3' PAM. In some embodiments, the CRISPR effector protein recognizes 3' PAM as 5'H, where H is A, C, or U.

[0240] In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which the guide sequence is designed to be complementary, where hybridization between the target sequence and the guide sequence facilitates CRISPR complex formation. The target sequence may comprise an RNA polynucleotide. The term "target RNA" refers to an RNA polynucleotide that is or contains the target sequence. In other words, the target RNA can be an RNA polynucleotide or a portion of an RNA polynucleotide, a portion of gRNA (i.e., the guide sequence) designed to be complementary to the target RNA, and the effector function mediated by the complex comprising the CRISPR effector protein and the gRNA will be directed to the target RNA. In some embodiments, the target sequence is located in the cell nucleus or cytoplasm.

[0241] In some exemplary embodiments, the CRISPR effector protein can be delivered using a nucleic acid molecule encoding a 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, in this case, is a sequence optimized for expression in a eukaryote (e.g., human) (i.e., optimized for expression in humans), or a sequence optimized for another eukaryote, animal, or mammal as discussed herein; see, for example, the SaCas9 human codon-optimized sequence in WO 2014 / 093622 (PCT / US2013 / 074667). While this is preferred, it should be understood that other examples are possible, and codon optimization for host species other than humans or for specific organs is known. In some embodiments, the enzyme-coding sequence encoding the CRISPR effector protein is a codon optimized for expression in a specific cell type (e.g., eukaryotic cell). Eukaryotic cells can be those derived from or originating from specific organisms (such as plants or mammals), including but not limited to human or non-human eukaryotic or animal or mammalian species as discussed herein, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. In some embodiments, processes that may be used to alter the genetic identity of the human germline and / or to alter the genetic identity of animals, which may cause suffering to humans and animals and offer essentially no medical benefit to humans or animals or animals affected by such processes, are excluded. Generally, codon optimization refers to the process of modifying nucleic acid sequences to enhance expression in host cells of interest, achieved by replacing at least one codon of the native sequence (e.g., about or more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) with codons more frequently or most frequently used in the genes of said host cell, while maintaining the native amino acid sequence. Various species exhibit specific preferences for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which in turn is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules, among other things. The dominance of a selected tRNA in a cell typically reflects the most frequently used codon in peptide synthesis. Therefore, genes can be tailored based on codon optimization to achieve 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 for codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, PA). In some implementations, one or more codons in the sequence encoding Cas (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more or all of these codons) correspond to the most frequently used codon for a specific amino acid.

[0242] In some embodiments, the methods described herein may include providing Cas transgenic cells in which one or more nucleic acids encoding one or more guide RNAs are provided or introduced, said nucleic acids being linked in the cell to a regulatory element containing a promoter of one or more genes of interest. As used herein, the term “Cas transgenic cell” refers to a cell in which a Cas gene has already been integrated into the genome, such as a eukaryotic cell. According to the invention, there are no particular limitations on the nature, type, or origin of the cells. Furthermore, the manner in which the Cas transgene is introduced into the cell may be varied and may be any method known in the art. In some embodiments, Cas transgenic cells are obtained by introducing the Cas transgene into isolated cells. In some other embodiments, Cas transgenic cells are obtained by isolating cells from a Cas transgenic organism. By way of example and not limitation, Cas transgenic cells as referred to herein may be derived from Cas transgenic eukaryotes, such as Cas knock-in eukaryotes. Reference is made to WO 2014 / 093622 (PCT / US13 / 74667), which is incorporated herein by reference. Methods involving targeting the Rosa locus, assigned to Sangamo BioSciences, Inc., may be modified to utilize the CRISPR Cas system of the present invention. Methods involving targeting the Rosa locus, assigned to Cellectis, may also be modified to utilize the CRISPR Cas system of the present invention. By way of further example, reference is made to Platt et al. (Cell; 159(2):440-455(2014)) describing Cas9 knock-in mice, which is incorporated herein by reference. Cas transgenes may also contain a Lox-Stop-polyA-Lox (LSL) cassette, thereby allowing Cas expression to be induced by Cre recombinase. Alternatively, Cas transgene cells can be obtained by introducing the Cas transgene into isolated cells. Delivery systems for transgenes are well known in the art. By way of example, Cas transgenes can be delivered in, for example, eukaryotic cells via vectors (e.g., AAV, adenovirus, lentivirus) and / or particles and / or nanoparticles, as also described elsewhere herein. Lentiviral and retroviral systems, as well as non-viral systems, for delivering CRISPR-Cas system components are well known in the art. AAV- and adenovirus-based systems for delivering CRISPR-Cas system components are well known in the art and are described herein (e.g., the engineered AAV of this invention).

[0243] Those skilled in the art will understand that when compounded with RNA capable of guiding Cas to target loci, cells such as those referred to herein (e.g., Cas transgenic cells) may contain genomic alterations in addition to having integrated Cas genes or mutations resulting from the sequence-specific effects of Cas.

[0244] In some embodiments, the present invention relates to vectors, for example, for delivering or introducing Cas and / or RNA (i.e., guide RNA) capable of guiding Cas to a target locus into cells and for propagating these components (e.g., in prokaryotic cells). This can be complementary to the delivery of one or more CRISPR-Cas components or the delivery of other genetically modified system components not yet delivered by engineered particles as described herein. As used herein, a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. A vector is a replicon, such as a plasmid, bacteriophage, or granule, into which another DNA fragment can be inserted to induce replication of the inserted fragment. Generally, a vector is capable of replication 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 already 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, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is the "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, such as through standard molecular cloning techniques. Another type of vector is the viral vector, in which a virus-derived DNA or RNA sequence is present for packaging into a virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by the virus for transfection into host cells. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attachable mammalian vectors) integrate into the host cell's genome upon introduction, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of the genes they are operatively linked to. Such vectors are referred to herein as "expression vectors." Common expression vectors used in recombinant DNA technologies are often in the form of plasmids.

[0245] Recombinant expression vectors may contain nucleic acids of the present invention in a form suitable for expression in host cells. This means that the recombinant expression vector includes one or more regulatory elements, which may be selected based on the host cell to be used for expression and are operatively linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell). Regarding recombination and cloning methods, reference is made to U.S. Patent Application 10 / 815,730, published September 2, 2004, as US2004-0171156 A1, the contents of which are incorporated herein by reference in their entirety. Therefore, embodiments disclosed herein may also include transgenic cells comprising a CRISPR effector system. In some exemplary embodiments, the transgenic cells may be used as separate discrete volumes. In other words, a sample containing a masking construct may be delivered to cells, for example, in suitable delivery vesicles, and if a target is present in the delivery vesicle, a CRISPR effector is activated and generates a detectable signal.

[0246] Vectors may include regulatory elements, such as promoters. Vectors may contain multiple Cas coding sequences and / or a single one, but may also contain at least 3 or 8 or 16 or 32 or 48 or 50 guide RNA (e.g., sgRNA) coding sequences, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 8, 3 to 16, 3 to 30, 3 to 32, 3 to 48, or 3 to 50 RNAs (e.g., sgRNA). In a single vector, each RNA (e.g., sgRNA) can have one promoter, which is advantageous when up to about 16 RNAs are present; and when a single vector provides more than 16 RNAs, one or more promoters can drive the expression of more than one RNA, for example, when 32 RNAs are present, each promoter can drive the expression of two RNAs, and when 48 RNAs are present, each promoter can drive the expression of three RNAs. With simple arithmetic and well-established cloning protocols, as well as the teachings in this disclosure, those skilled in the art can readily practice the invention with respect to RNAs for suitable exemplary vectors (such as AAV) and suitable promoters (such as the U6 promoter). For example, the packaging of AAV is limited to about 4.7 kb. The length of a single U6-gRNA (plus the cloning restriction site) is 361 bp. Therefore, those skilled in the art can readily assemble approximately 12 to 16 (e.g., 13) U6-gRNA cassettes into a single vector. This can be assembled by any suitable method, such as the Golden Gate strategy for TALE assembly (genome-engineering.org / taleffectors / ). Technicians can also use tandem guidance strategies to increase the number of U6-gRNAs by approximately 1.5-fold, for example, from 12 to 16 (e.g., 13) U6-gRNAs to approximately 18 to 24 (e.g., about 19) U6-gRNAs. Therefore, those skilled in the art can readily 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 way to increase the number of promoters and RNAs in a vector is to use a single promoter (e.g., U6) to express an array of RNAs separated by cleavable sequences.Another way to increase the number of promoter-RNAs in a vector is to express an array of promoter-RNAs separated by cleavable sequences within introns of a coding sequence or gene; and, in this case, it is advantageous to use a polymerase II promoter, which can increase expression and allow transcription of long RNAs in a tissue-specific manner (see, for example, nar.oxfordjournals.org / content / 34 / 7 / e53.shortandnature.com / mt / journal / v16 / n9 / abs / mt2008144a.html). In an advantageous embodiment, AAV can package U6 tandem gRNAs targeting up to about 50 genes. Thus, based on the knowledge in the art and the teachings of this disclosure, those skilled in the art can readily prepare and use vectors (e.g., single vectors) that express multiple RNAs or guide RNAs operatively or functionally linked to one or more promoters—especially regarding the number of RNAs or guide RNAs discussed herein—without requiring any excessive experimentation.

[0247] The guide RNA coding sequence and / or Cas coding sequence can be functionally or operatively linked to a regulatory element, thereby regulating the element to drive expression. The promoter can be a constitutive promoter and / or a conditional promoter and / or an inducible promoter and / or a tissue-specific promoter. Promoters can be selected from the following groups: 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, phosphoglycerate kinase (PGK) promoter, and EF1α promoter. The U6 promoter is a preferred promoter.

[0248] Other effectors used according to the invention can be identified by their proximity to the cas1 gene, for example, but not limited to, regions within 20 kb from the start and 20 kb from the end of the cas1 gene. In some embodiments, the effector protein comprises at least one HEPN domain and at least 500 amino acids, and wherein the C2c2 effector protein is naturally present in a prokaryotic genome within 20 kb upstream or downstream of the Cas gene or a 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, Cas... 13b, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologues, or modified versions thereof. In some example embodiments, the C2c2 effector protein is naturally present in the 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. In a further guided manner, 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 of its homologue. Homologous proteins may, but are not necessarily, structurally related, or only partially structurally related. An “orthologue” of a protein, as used herein, is a protein of a different species that performs the same or similar function as the protein of its orthologue. Orthologous proteins may, but are not necessarily, structurally related, or only partially structurally related.

[0249] In some embodiments, one or more elements of the nucleic acid targeting system are derived from a specific organism containing an endogenous CRISPR RNA targeting system. In some embodiments, the CRISPR RNA targeting system is present in *Eubacterium* and *Ruminococcus*. In some embodiments, the effector protein includes both targeting and associated ssRNA cleavage activity. In some embodiments, the effector protein includes a double HEPN domain. In some embodiments, the effector protein lacks a counterpart to the Helical-1 domain of Cas13a. In some embodiments, the effector protein is smaller than the two previously characterized CRISPR effector proteins, with a median size of 928 aa. This median size is 190 aa (17%) smaller than the median size of Cas13c, greater than 200 aa (18%) smaller than the median size of Cas13b, and greater than 300 aa (26%) smaller than the median size of Cas13a. In some embodiments, the effector protein does not require flanking sequences (e.g., PFS, PAM).

[0250] In some embodiments, the effector protein locus structure includes a WYL domain containing an accessory protein (as indicated by the three conserved amino acids following the initially identified sets of these domains; see, for example, WYL domain IPR026881). In some embodiments, the WYL domain accessory protein includes at least one helical-turn-helical (HTH) or ribbon-helical-helical (RHH) DNA-binding domain. In some embodiments, the accessory protein containing the WYL domain enhances the targeting and incidental ssRNA cleavage activity of the RNA-targeting effector protein. In some embodiments, the accessory protein containing the WYL domain includes an N-terminal RHH domain and a pattern of predominantly hydrophobic conserved residues, including an invariant tyrosine-leucine duplex corresponding to the original WYL motif. In some embodiments, the accessory protein containing the WYL domain is WYL1. WYL1 is a single WYL domain protein primarily associated with the genus *Ruminococcus*.

[0251] In other example embodiments, the type VI RNA-targeting Cas enzyme is Cas13d. In some embodiments, Cas13d is the indolent eubacterium DSM 15702 (Eubacterium siraeum DSM 15702) (EsCas13d) or the Ruminococcus species N15.MGS-57 (RspCas13d) (see, for example, 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 require flanking sequences (e.g., PFS, PAM).

[0252] The methods, systems, and tools provided herein can be designed for use with a class of CRISPR proteins, such as type I, III, or IV Cas proteins as described in Makarova et al., The CRISPR Journal, v.1, n., 5 (2018); DOI:10.1089 / crispr.2018.0033, and particularly as described on page 326. Figure 1 The aforementioned literature is incorporated herein by reference in its entirety. Class 1 systems typically utilize multi-protein effector complexes, which in some embodiments may include accessory proteins, such as one or more proteins in a cascade complex for antiviral defense called a CRISPR-associated complex, one or more adaptive proteins (e.g., Cas1, Cas2, RNA nucleases) and / or one or more accessory proteins (e.g., Cas4, DNA nucleases), proteins containing a CRISPR-associated Rossman fold (CARF) domain, and / or RNA transcriptases. Although the sequence similarity of Class 1 systems is limited, Class 1 system proteins can be identified by their similar constructions, including one or more repetition-associated mystery protein (RAMP) family subunits, such as Cas5, Cas6, and Cas7. RAMP proteins are characterized by having one or more RNA recognition motif domains. Large subunits (e.g., Cas8 or Cas10) and small subunits (e.g., Cas11) are also typical features of Class 1 systems. See, for example... Figure 1 and Figure 2Koonin EV, Makarova KS. 2019 Origins and evolution of CRISPR-Cassystems. Phil. Trans. R. Soc. B 374:20180087, DOI:10.1098 / rstb.2018.0087. In one embodiment, the class 1 system is characterized by the characteristic protein Cas3. Cascade, particularly class 1 proteins, may comprise a dedicated complex of multiple Cas proteins that bind pre-crRNA and recruit additional Cas proteins (e.g., Cas6 or Cas5), the complex being a nuclease directly responsible for processing the pre-crRNA. In one embodiment, the type I CRISPR protein comprises 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, types IV-A and IV-B, and types III-A, III-D, III-C, and III-B. Class 1 systems also include CRISPR-Cas variants, including type IA, type IB, type IE, type IF, and type IU variants. These variants can include variants carried by transposons and plasmids, including IF subtype versions encoded by a large family of Tn7-like transposons and a smaller group of Tn7-like transposons, and IB subtype systems encoded by Tn7-like transposons with similar degradation. Peters et al., PNAS 114(35)(2017); DOI:10.1073 / pnas.1709035114; see also Makarova et al., the CRISPR Journal, v.1, n5. Figure 5 .

[0253] Targeted portion

[0254] In some implementations, the engineered delivery system may also include a targeting portion capable of specifically binding to target cells. For the effective targeting of delivery vesicles to cells (such as cancer cells), it is useful for the targeting portion to have an affinity for cell surface receptors and to be bound to the targeting portion in an amount sufficient to have optimal affinity for cell surface receptors; and determining these aspects is within the skill of a person skilled in the art. In the field of active targeting, many cell-specific (e.g., tumor) targeting ligands exist.

[0255] Regarding active targeting, specifically targeting cell surface receptors (such as cancer cell surface receptors), targeting ligands on liposomes can enable liposomes to attach to cells (e.g., vascular cells) via non-internalized epitopes; this can increase the extracellular concentration of the delivered substance, thereby increasing the amount delivered to the target cells. Strategies for targeting cell surface receptors (such as those overexpressed on cancer cells) involve using receptor-specific ligands or antibodies. Many cancer cell types exhibit upregulation of tumor-specific receptors. For example, TfR and folate receptors (FRs) are heavily overexpressed by many tumor cell types in response to their increased metabolic demands. Folate can be used as a targeting ligand for specialized delivery due to its ease of conjugation to nanocarriers, its high affinity for FRs, and the relatively low frequency of FRs in normal tissues compared to their overexpression in activated macrophages and cancer cells (e.g., certain ovarian, breast, lung, colon, kidney, and brain tumors). Overexpression of FR on macrophages is an indicator of inflammatory diseases such as psoriasis, Crohn's disease, rheumatoid arthritis, and atherosclerosis; therefore, the folic acid-mediated targeting of the present invention can also be used to study, address, or treat inflammatory conditions and cancer. The folic acid-linked lipid particles or nanoparticles or liposomes or lipid bilayers of the present invention (“lipid entities of the present invention”) deliver their cargo intracellularly via receptor-mediated endocytosis. Intracellular transport can involve acidic compartments that facilitate cargo release, and importantly, cargo release can be altered or delayed until it reaches the cytoplasm or vicinity of the target organelle. Delivery of cargo using lipid entities of the present invention with a targeting portion (such as the folic acid-linked lipid entities of the present invention) can be superior to the non-targeting lipid entities of the present invention. Direct attachment of folic acid to the lipid head group may be detrimental to the intracellular delivery of the folic acid-conjugated lipid entities of the present invention, as their binding to cells may not be as efficient as folic acid attached to the surface of the lipid entities of the present invention via spacers, which allow for more efficient entry into cancer cells. The folic acid-conjugated lipid entities of the present invention can be used to deliver lipid complexes, such as liposomes, such as anionic liposomes, and viral or capsid or envelope or extraviral proteins, such as those discussed herein, such as adenoviruses or AAVs. Tf is a monomeric serum glycoprotein of approximately 80 kDa involved in the transport of iron throughout the body. Tf binds to TfR and translocates into cells via receptor-mediated endocytosis. TfR expression can be higher 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 lipid entities targeting TfR, such as those relating to hepatocytes, liver cancer cells, breast cells (such as breast cancer cells), colon cells (such as colon cancer cells), ovarian cells (such as ovarian cancer cells), head, neck and lung cells (such as head, neck and non-small cell lung cancer cells), and oral cells (such as oral tumor cells).

[0256] Regarding active targeting, the lipid entity of this invention can be multifunctional, i.e., employing more than one targeting moiety (such as CPP and Tf); a bifunctional system; for example, a combination of Tf and poly-L-arginine that can provide transvascular endothelial transport across the blood-brain barrier. EGFR is a tyrosine kinase receptor belonging to the ErbB receptor family, mediating cell growth, differentiation, and repair in cells (especially non-cancerous cells), but EGF is overexpressed in certain cells (such as many solid tumors, including colorectal cancer, non-small cell lung cancer, ovarian squamous cell carcinoma, renal cell carcinoma, head cancer, pancreatic cancer, cervical cancer, and prostate cancer, especially breast cancer). This invention includes a monoclonal antibody targeting EGFR linked to the lipid entity of this invention. HER-2 is commonly overexpressed in breast cancer patients and is also associated with lung cancer, bladder cancer, prostate cancer, brain cancer, and gastric cancer. HER-2 is encoded by the ERBB2 gene. This invention includes HER-2-targeting lipid entities of the present invention, such as anti-HER-2 antibody (or its binding fragment)-lipid entities of the present invention, HER-2-targeting polyethylene glycol-modified lipid entities of the present invention (e.g., having an anti-HER-2 antibody or its binding fragment), and HER-2-targeting maleimide PEG polymer lipid entities of the present invention (e.g., having an anti-HER-2 antibody or its binding fragment). During cell association, the receptor-antibody complex can be internalized by forming endosomes for delivery to the cytoplasm. Regarding receptor-mediated targeting, those skilled in the art consider ligand / target affinity and the number of receptors on the cell surface, as well as the fact that polyethylene glycol modification can act as a barrier against interaction with the receptor. Targeting using the antibody-lipid entities of the present invention can be advantageous. Multivalent presentation of the targeting portion can also increase antibody fragment uptake and signal transduction properties. In the practice of the present invention, those skilled in the art consider ligand density (e.g., a high ligand density on the lipid entities of the present invention may be advantageous for increased binding to target cells). Early prevention of macrophage attacks can be addressed using the spatially stable lipid entities of the present invention and by attaching ligands to the ends of molecules (such as PEG) anchored within the lipid entities of the present invention (e.g., lipid particles, nanoparticles, liposomes, or lipid bilayers). Targeting the microenvironment of cell clusters, such as the tumor microenvironment, can be advantageous; for example, targeting the vascular system of cell clusters (such as the tumor vascular system microenvironment). Therefore, the present invention includes targeting VEGF. VEGF and its receptor are well-known pro-angiogenic molecules and are well-characterized targets for anti-angiogenic therapies.Many small molecule inhibitors of receptor tyrosine kinases (such as VEGFR or basic FGFR) have been developed as anticancer agents, and this invention includes conjugating any one or more of these peptides to the lipid entities of this invention, such as phage IVO peptides (e.g., via or using a PEG terminus), tumor-homing peptide APRPG (SEQ ID NO:4) (such as APRPG-PEG modified). VCAMs and vascular endothelium play key roles in the pathogenesis of inflammation, thrombosis, and atherosclerosis. CAMs are involved in inflammatory conditions (including cancer) and are logical targets; E- and P-selectins, VCAM-1, and ICAM can be used to target the lipid entities of this invention, for example, using PEGylation. Matrix metalloproteinases (MMPs) belong to the zinc-dependent endopeptidase family. They are involved in tissue remodeling, tumor invasiveness, anti-apoptosis, and anti-metastasis. There are four MMP inhibitors called TIMP1 to TIMP4, which determine the balance between tumor growth inhibition and metastasis; the protein involved in tumor angiogenesis is MT1-MMP, expressed on newly formed blood vessels and tumor tissue. The proteolytic activity of MT1-MMP cleaves proteins (such as fibronectin, elastin, collagen, and laminin) on the plasma membrane and activates the degradation of soluble MMPs (such as MMP-2) in the matrix. Antibodies or fragments thereof (such as Fab' fragments) can be used in the practice of this invention, such as anti-human MT1-MMP monoclonal antibodies for attachment to lipid entities of this invention (e.g., via spacers, such as PEG spacers). αβ integrins, or integrins, are a group of transmembrane glycoprotein receptors that mediate cell attachment to surrounding tissues or the extracellular matrix. Integrins contain two distinct chains (heterodimers) called α subunits and β subunits. Tumor tissue-specific expression of integrin receptors can be used for targeted delivery in this invention; for example, the targeted portion may be an RGD peptide, such as a cyclic RGD. Aptamers are ssDNA or RNA oligonucleotides that confer high affinity and specific recognition of target molecules through electrostatic interactions, hydrogen bonds, and hydrophobic interactions rather than Watson-Crick base pairing, a typical oligonucleotide binding interaction. Aptamers as targeting moieties can offer advantages over antibodies: they can exhibit higher target antigen recognition compared to antibodies; they can be more stable and smaller in size; they can be readily synthesized and chemically modified for molecular conjugation; and they can be sequentially modified to improve selectivity and can be developed for recognizing poorly immunogenic targets. Such moieties, such as the sgc8 aptamer, can be used as targeting moieties (e.g., by covalent linking with the lipid entity of the present invention, for example, via spacers such as PEG spacers).The targeting portion can be stimuli-sensitive, for example, sensitive to externally applied stimuli (such as magnetic fields, ultrasound, or light); and pH triggering can also be used, for example, by using an unstable bond between a hydrophilic portion (such as PEG) and a hydrophobic portion (such as the lipid entity of the present invention), which is cleaved only upon exposure to relatively acidic conditions characteristic of a particular environment or microenvironment (such as intracellular vacuoles or acid-poisoned tumor masses). pH-sensitive copolymers can also be incorporated into embodiments of the present invention and can provide shielding; diortho esters, vinyl esters, cysteine-cleavable lipid polymers, diesters, and hydrazones are examples of some pH-sensitive bonds that are very stable at pH 7.5 but hydrolyze relatively quickly at pH 6 and lower, for example, a terminally alkylated copolymer of N-isopropylacrylamide and methacrylic acid, which facilitates the disruption of the stability of the lipid entity of the present invention and release in a pH-lowering compartment; or, the present invention includes ionic polymers (e.g., poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(acrylamide), and poly(acrylic acid)) for generating the pH-responsive lipid entity of the present invention. Temperature-triggered delivery is also within the scope of this invention. Many pathological areas (such as inflamed tissue and tumors) exhibit unique hyperthermia compared to normal tissue. Utilizing this hyperthermia is an attractive strategy in cancer therapy because hyperthermia is associated with increased tumor permeability and enhanced uptake. This technique involves localized heating of the site to increase microvascular pore size and blood flow, which can subsequently lead to increased extravasation in embodiments of the invention. The temperature-sensitive lipid entities of the invention can be prepared from thermosensitive lipids or polymers having a low critical solution temperature. Above this low critical solution temperature (e.g., at sites such as tumor sites or inflamed tissue sites), the polymer precipitates, thereby destroying the liposomes for release. Lipids having a specified gel-liquid phase transition temperature are used to prepare these lipid entities of the invention; and the lipid used for the thermosensitive embodiment can be dipalmitoylphosphatidylcholine. Thermosensitive polymers can also facilitate destabilization followed by release, and available thermosensitive polymers are poly(N-isopropylacrylamide). Another temperature-triggered system can employ hemolyzed lipid temperature-sensitive liposomes. This invention also includes redox-triggered delivery: delivery has been carried out by utilizing the redox potential difference between normal and inflamed or tumor tissues, and between the intracellular and extracellular environments; for example, GSH is an abundant reducing agent in cells, especially in the cytosol, mitochondria, and nucleus. The concentration of GSH in blood and extracellular matrix is ​​only one percent and one-thousandth of the intracellular concentration, respectively. This high redox potential difference caused by GSH, cysteine, and other reducing agents can break reducible bonds, disrupt the stability of the lipid entity of this invention, and lead to the release of the payload.Disulfide bonds can be used as cleavable / reversible linkers in the lipid entities of the present invention because they induce redox sensitivity due to disulfide-thiol reduction reactions. The lipid entities of the present invention can be made reductively sensitive by using two (e.g., two forms of disulfide-conjugated multifunctional lipids as disulfide bond cleavage (e.g., via tris(2-carboxyethyl)phosphine, dithiothreitol, L-cysteine, or GSH)) forms that can cause removal of the hydrophilic head group of the conjugate and alter membrane structure, thereby leading to the release of the payload. Calcein released from the reductively sensitive lipid entities of the present invention containing disulfide conjugates can be more useful than in reductively insensitive embodiments. Enzymes can also be used as triggers for payload release. Enzymes, including MMPs (e.g., MMP2), phospholipase A2, alkaline phosphatase, transglutaminase, or phosphatidylinositol-specific phospholipase C, have been found to be overexpressed in certain tissues (e.g., tumor tissues). In the presence of these enzymes, specifically, the engineered enzyme-sensitive lipid entities of the present invention can be disrupted and the payload released. MMP2-cleavable octapeptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) (SEQ ID NO:5) can be incorporated into the linker and can have antibody targeting, such as antibody 2C5. The invention also includes photo-triggered or energy-triggered delivery; for example, the lipid entities of the invention can be photosensitized such that light or energy can facilitate structural and conformational changes, leading to direct interaction between the lipid entities and target cells via membrane fusion, photoisomerization, photofragmentation, or photopolymerization; thus, such a moiety can be a benzoporphyrin photosensitizer. Ultrasound can be a form of energy for triggering delivery; lipid entities of the invention containing small amounts of specific gases (including air or perfluorocarbons) can be triggered for release by ultrasound (e.g., low-frequency ultrasound (LFUS)). Magnetic delivery: The lipid entities of the invention can be magnetized by incorporation with magnetite (such as Fe3O4 or γ-Fe2O3 (e.g., those with a size less than 10 nm)). Targeted delivery can then be performed by exposure to a magnetic field.

[0257] In addition to active targeting, this invention also includes intracellular delivery. Since liposomes follow the endocytic pathway, they are embedded in endosomes (pH 6.5-6) ​​and subsequently fuse with lysosomes (pH < 5), where they undergo degradation that reduces their therapeutic potential. Low endosome pH can be used to avoid degradation. Following conformational change / activation at lower pH, fused lipids or peptides disrupt the stability of the endosome membrane. Amines are protonated at acidic pH and cause endosome swelling and rupture via buffering. Unsaturated dioleoylphosphatidylethanolamine (DOPE) readily adopts an inverted hexagonal shape at low pH, which induces liposome fusion with the endosome membrane. This process disrupts the stability of DOPE containing lipid entities and releases the cargo into the cytoplasm; fused lipids GALA, cholesterol GALA, and PEG-GALA exhibit efficient endosome release; pore-forming protein listeriolysin O provides an endosome escape mechanism; and histidine-rich peptides have the ability to fuse with the endosome membrane, leading to pore formation and buffering proton pumps, causing membrane lysis.

[0258] Regarding active targeting, cell-penetrating peptides (CPPs) facilitate the uptake of macromolecules across the cell membrane, thereby enhancing the delivery of CPP-modified molecules within the cell. CPPs can be divided into two categories: amphiphilic helical peptides, such as transporters and MAPs, where lysine residues are the main contributors to positive charge; and Arg-rich peptides, such as TATp, Antennapedia, or penetratin. TATp is a transcription activator with 86 amino acids containing a highly basic (two Lys and six Arg residues out of nine) protein transduction domain that induces nuclear localization and RNA binding. Other CPPs used for liposome modification include: the minimal protein transduction domain of Antennapedia, a Drosophila homologous protein called penetratin, a 16-mer peptide (residues 43-58) located in the third helix of the homologous domain; a 27-amino acid-long chimeric CPP containing a peptide sequence from the N-terminus of the neuropeptide glycopeptide, which binds via the lysine residue mastoparan (a wasp venom peptide); VP22, a major structural component of HSV-1, which facilitates intracellular transport and transport of an amphiphilic model peptide (18-mer) via energy-dependent and non-energy-dependent mechanisms to the plasma membranes of mast cells and endothelial cells. This invention includes CPP-modified lipid entities of the invention for intracellular delivery via energy-dependent macropinocytosis followed by endosome escape. The invention also includes organelle-specific targeting. Lipid entities of the invention partially surface-functionalized with triphenylphosphonium (TPP) or lipid entities of the invention having the lipophilic cationic rhodamine 123 can efficiently deliver cargo to mitochondria. DOPE / sphingomyelin / stearyl-octa-arginine can deliver cargo into the mitochondria via membrane fusion. Lipid entities of the present invention, surface-modified with the lysosomal ligand octadecylrhodamine B, can deliver cargo to lysosomes. Ceramides can be used to induce lysosomal membrane permeation; the present invention includes intracellular delivery of lipid entities of the present invention having ceramides. The present invention also includes lipid entities of the present invention targeting the cell nucleus (e.g., via DNA embedding portions). The present invention also includes multifunctional liposomes for targeting, i.e., attaching more than one functional group to the surface of the lipid entities of the present invention, for example to enhance accumulation at desired sites and / or promote organelle-specific delivery and / or target specific cell types and / or respond to local stimuli (e.g., temperature (e.g., elevation), pH (e.g., decrease)), respond to externally applied stimuli (e.g., magnetic fields, light, energy, heat, or ultrasound), and / or promote intracellular delivery of cargo. All of these are considered active targeting portions.

[0259] Embodiments of the present invention include a delivery system comprising an actively targeted lipid particle or nanoparticle or liposome or lipid bilayer delivery system; or comprising a lipid particle or nanoparticle or liposome or lipid bilayer containing a targeting portion, thereby exhibiting active targeting or wherein the targeting portion is an actively targeted portion. The targeting portion may be one or more targeting portions, and the targeting portion may be used for any desired type of targeting, such as, for example, targeting cells (such as any cells mentioned herein); or targeting organelles (such as any organelles mentioned herein); or for targeting responses such as to physical conditions (such as heat, energy, ultrasound, light, pH), chemical substances (such as enzymes), or magnetic stimuli; or for targeting to achieve a specific result, such as delivering a payload to a specific location (such as through cell penetration).

[0260] It should be understood that for each possible targeting or active targeting portion discussed herein, there exists an aspect of the invention in which the delivery system includes such a targeting or active targeting portion. Similarly, the following table provides exemplary targeting portions that can be used in practice to implement the invention, and for each aspect of the invention, a delivery system including such a targeting portion is provided.

[0261] Table 1.

[0262]

[0263] Therefore, in embodiments of the delivery system, the targeting portion comprises a receptor ligand, such as, for example, hyaluronic acid for the CD44 receptor, galactose for hepatocytes, or an antibody or a fragment thereof (such as a binding antibody fragment targeting a desired surface receptor), and with respect to each targeting portion comprising a receptor ligand or antibody or a fragment thereof (such as a binding fragment thereof, such as targeting a desired surface receptor), there is an aspect of the invention wherein the delivery system comprises a targeting portion comprising a receptor ligand or antibody or a fragment thereof (such as a binding fragment thereof, such as targeting a desired surface receptor) or hyaluronic acid for the CD44 receptor, galactose for hepatocytes (see, for example, 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.Release 150(1), 2-22(2011); Sofou S “Antibody-targeted liposomes in cancer therapy and imaging,” Expert Opin.DrugDeliv.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 every document cited herein is hereby incorporated by reference.

[0264] Furthermore, referring to the teachings herein, those skilled in the art can readily select and apply desired targeting portions in the practice of the lipid entities of this invention. The invention includes an embodiment in which the delivery system comprises a lipid entity having a targeting portion.

[0265] In some implementations, the target cell may be a mammalian cell. In some implementations, the mammalian cell may be a cancer cell, as further described below.

[0266] In some embodiments, mammalian cells may be infected by pathogens. In some embodiments, the pathogen may be a virus, as further described below.

[0267] In some embodiments, the targeting portion comprises a membrane fusion protein. In some embodiments, the membrane fusion protein is the G envelope protein of vesicular stomatitis virus (VSV-G).

[0268] Membrane fusion is a common and important biological phenomenon that occurs when two separate lipid membranes merge into a single, continuous bilayer. Fusion reactions share common characteristics but are catalyzed by a variety of different proteins. These proteins mediate the initial recognition of the membrane destined for fusion and pull the membranes together to disrupt the stability of the lipid / water interface and initiate lipid mixing. A single fusion protein can do everything, or intracellular fusion reactions may require the assembly of protein complexes to ensure tight spatial and temporal regulation. Cellular fusion machinery is adapted to the needs of different reactions but operates on similar principles to achieve bilayer merging.

[0269] Membrane fusions range from cell fusion and organelle dynamics to vesicle transport and viral infection. Without exception, all these fusion events are driven by membrane fusion proteins (also known as fusion agents). Common fusion processes mediated by fusion proteins consist of a series of steps, including bringing two opposing lipid membranes close together, disrupting the lipid bilayer, and finally merging 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 protein called SNARE. SNARE proteins on vesicles (v-SNARE) and those on the target membrane (t-SNARE) not only provide recognition specificity but also the energy required for vesicle fusion.

[0270] Viral fusion is another important fusion event. Enveloped viruses, membrane-bound and derived from host cells, release their genome after the viral envelope fuses with the host cell membrane. Viral fusion proteins dominate the uncoating phase. Based on their structural characteristics, viral fusion proteins are classified into three types: type I, type II, and type III. Despite a long history of understanding viral fusion proteins, the underlying fusion mechanisms remain 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 VSV-G-triggered membrane fusion in acidic environments depends on a reversible conformational change, which reverts 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 viral particles. It mediates attachment and low-pH-induced fusion.

[0271] reverse transcriptase

[0272] In some implementations, the system also includes 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 retrotransposons to move genetic elements for proliferation within the host genome, by eukaryotic cells to extend telomeres at the ends of linear chromosomes in eukaryotic cells, and by some non-retroviral agents, such as hepatitis B virus (a member of the Hepadnaviridae family, which is a dsDNA-RT virus).

[0273] Retroviruses (RTs) possess three consecutive biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H activity, and DNA-dependent DNA polymerase activity. In general, these activities allow the enzymes to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then integrate into the host genome, and new RNA copies can be transcribed from the host genome by the host cell. The same reaction sequence is widely used in the laboratory to convert RNA into DNA for molecular cloning, RNA sequencing, polymerase chain reaction (PCR), or genomic analysis.

[0274] HIV reverse transcriptase also has ribonuclease activity that degrades viral RNA during cDNA synthesis, and DNA-dependent DNA polymerase activity that replicates sense cDNA strands into antisense DNA to form double-stranded viral DNA intermediates (vDNA).

[0275] Delivery vesicles

[0276] Within the scope of this invention, a delivery vesicle is also contemplated that contains one or more components encoded in one or more polynucleotides in the engineered delivery system described herein.

[0277] As described elsewhere in this document, such components include, but are not limited to, one or more polynucleotides encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more capture portions for packaging cargo within delivery vesicles. The one or more endogenous retroviral elements for forming delivery vesicles may comprise two or more of the following: retroviral gag protein, retroviral envelope protein, retroviral reverse transcriptase, or combinations thereof.

[0278] 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 contain NC and MA domains. In some embodiments, the retroviral gag protein may be a gag homolog. A gag homolog may be Arc1, Asprv1, PNMA1, PNMA3, PNMA4, PNMA5, PNMA6, PNMA7, PEG10, RTL1, MOAP1, or ZCCHC12.

[0279] In some embodiments, the envelope protein is derived from a gamma retrovirus or a delta retrovirus. 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.

[0280] In some implementations, as described elsewhere in this document, vesicle delivery elicits a poor immune response.

[0281] As described elsewhere herein, the cargo may comprise nucleic acids, proteins, complexes thereof, or combinations thereof. In a specific embodiment, the cargo comprises ribonucleoproteins. The cargo may comprise genetic regulators, which contain one or more components of a gene-editing system and / or polynucleotides encoding them.

[0282] The gene editing system can be a CRISPR-Cas system. As described elsewhere herein, the CRISPR-Cas system can be a type II, type V, or type VI CRISPR-Cas system. In a specific embodiment, 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; however, the present invention is not limited to these embodiments.

[0283] In some implementations, the vesicles also contain reverse transcriptase.

[0284] In some implementations, one or more capture portions include a DNA-binding portion, an RNA-binding portion, a protein-binding portion, or a combination thereof.

[0285] In some implementations, the delivery vesicles are virus-like particles.

[0286] In some implementations, the delivery vesicle may include a targeting portion, wherein the targeting portion is capable of specifically binding to target cells.

[0287] In some embodiments, the cell-specific targeting portion may comprise a membrane fusion protein. In some embodiments, as described elsewhere herein, the membrane fusion protein is VSV-G.

[0288] In some implementations, the cell-specific targeting portion targets mammalian cells. In some implementations, the mammalian cells may be cancer cells, as further described below.

[0289] In some implementations, mammalian cells are infected by a pathogen. In some implementations, the pathogen may be a virus, as further described below.

[0290] Methods for loading cargo molecules into delivery vesicle systems

[0291] Goods (e.g., nucleic acids and / or peptides) small enough to be encapsulated in delivery vesicles can be introduced into cells via transduction of viral or pseudoviral particles. The method of packaging the goods in viral particles can be accomplished using any suitable viral vector or vector system. Such viral vectors and vector systems are described in more detail elsewhere herein. As used in the context herein, “transduction” refers to the process of introducing foreign nucleic acids and / or proteins into cells (prokaryotic or eukaryotic) via viral or pseudoviral particles. After packaging in viral or pseudoviral particles, the viral particles can be exposed to cells (e.g., in vitro, ex vivo, or in vivo), whereby the viral or pseudoviral particles infect the cells and deliver the goods to the cells via transduction. The viral and pseudoviral particles can optionally be concentrated before exposure to target cells. In some embodiments, a viral titer of the composition containing viral and / or pseudoviral particles can be obtained, and a specified titer can be used to transduce cells.

[0292] In some embodiments, the viral vector is configured such that when the cargo is packaged, the cargo is outside the capsid or viral particle, i.e., the cargo is not inside the capsid (encapsulated or covered by the capsid) but exposed externally, allowing it to come into contact with the target genomic DNA. In some embodiments, the viral vector is configured such that all cargo is contained within the capsid after packaging.

[0293] A method for packaging cargo within vesicles involves using one or more "bioreactors" that generate and subsequently secrete one or more cargo-carrying vesicles. The bioreactors may contain cells, microorganisms, or cell-free systems. The bioreactors are generated by administering to cells one or more polynucleotides encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more capture portions for packaging cargo within the delivery vesicles. Targeting portions may also be administered to the cells, wherein the targeting portions are capable of specifically binding to target cells. Thus, the bioreactors may be able to generate cargo-carrying vesicles that deliver not only bioactive RNA molecules to the extracellular matrix but also to designated cells and tissues.

[0294] In some embodiments, the cargo molecule may be a polynucleotide or polypeptide, which may, alone or when delivered as part of a system, whether or not delivered with other components of the system, manipulate and modify the genome, epigenome, and / or transcriptome of the cell to which it is delivered. Such systems include, but are not limited to, the CRISPR-Cas system. Other gene modification systems, such as TALEN, zinc finger nucleases, Cre-Lox, morpholino, etc., are other non-limiting examples of gene modification systems, one or more of which may be delivered by engineered AAV particles as described herein.

[0295] This invention provides nucleic acid molecules, specifically polynucleotides, which in some embodiments encode one or more target peptides or polypeptides. The term "nucleic acid" in its broad sense includes any compound and / or substance comprising a nucleotide polymer. These polymers are often referred to as polynucleotides.

[0296] Exemplary nucleic acids or polynucleotides of the present invention include, but are not limited to: ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0297] In some embodiments, the polynucleotides of the present invention may be cyclic. As used herein, "cyclic polynucleotide" means a single-stranded cyclic polynucleotide that functions substantially similarly to and has RNA properties. The term "cyclic" is also intended to cover any secondary or tertiary configuration of the cyclic polynucleotide.

[0298] In some embodiments, the polynucleotide comprises 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 1,0 ... 500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50, 000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1, 500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000 and 2,000 to 100,000).

[0299] Vesicles formed by the bioreactor described herein can be separated by any suitable method known in the art. For example, vesicles may include tags that can bind antibodies or aptamers. Vesicles can also be separated and sorted by fluorescence-activated cell sorting (FACS) or by using size exclusion methods.

[0300] Method of delivering goods using delivery vesicles

[0301] Within the scope of this invention, a method for delivering cargo to one or more cells using the delivery vesicles described herein is also contemplated. As described, delivery vesicles can deliver cargo to one or more cells of a subject.

[0302] The system described herein may also 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 by binding to a target, including non-immunoglobulin scaffolds (including fibronectin, lipid transport proteins, protein A, ankyrin, thioredoxin, etc.). In some embodiments, membrane fusion proteins may include, but are not limited to, the G envelope protein of vesicular stomatitis virus (VSV-G), herpes simplex virus 1gB (HSV-1gB), Ebola virus glycoprotein, members of the SNARE protein family, and members of the syncytiocytin protein family.

[0303] In some embodiments, the goods may comprise a therapeutic agent. The terms "therapeutic agent," "therapeutic capable agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that, when administered to a subject, imparts certain beneficial effects. Beneficial effects include: enabling diagnostic identification; alleviating disease, symptoms, condition, or pathological symptoms; reducing or preventing the onset of disease, symptoms, condition, or pathological symptoms; and generally combating disease, symptoms, condition, or pathological symptoms.

[0304] Target cells may include, but are not limited to, mammalian cells, cancer cells, and cells infected by pathogens such as viruses, bacteria, fungi, or parasites. In some embodiments, the invention includes cargo delivery across the blood-brain barrier. As will be appreciated by those skilled in the art, vesicles can be engineered to exhibit tropism for any particular desired cell type.

[0305] Various delivery systems are known and can be used to administer pharmacological compositions, including but not limited to those encapsulated in liposomes, microparticles, microcapsules; microcells; polymers; capsules; tablets; etc. In one embodiment, the agent can be delivered in vesicles, particularly in liposomes. In liposomes, the agent is combined with an amphiphilic agent (such as a lipid) in aqueous solution, in the form of micelles, insoluble monolayers, liquid crystals, or lamellar aggregates. Lipids suitable for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, thioesters, lysophosphatidylcholine, phospholipids, saponins, bile acids, etc. The preparation of such liposomal formulations is within the art, as disclosed, for example, in U.S. Patent Nos. 4,837,028 and 4,737,323. In yet another embodiment, the pharmacological composition can be delivered in a controlled-release system (including, but not limited to, a delivery pump) (see, for example, Saudek et al., New Engl. J. Med. 321: 574 (1989)) and a semi-permeable polymeric material (see, for example, Howard et al., J. Neurosurg. 71: 105 (1989)). Additionally, the controlled-release system can be placed close to the therapeutic target (e.g., a tumor), thus requiring only a portion of the systemic dose. See, for example, Goodson, In: Medical Applications of Controlled Release, 1984. (CRC Press, Boca Raton, Fla.).

[0306] It should be understood that the therapeutic entities according to the invention can be administered in the presence of suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. A variety of suitable formulations can be found in all prescriptions known to pharmacists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), and particularly in Chapter 87 of Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) vesicles (such as Lipofectin). TM( ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be used in the treatments and therapies according to the invention, provided that the active ingredient in the formulation is not inactivated by the formulation and that 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 the citations therein are used to obtain additional information related to formulations, excipients and carriers familiar to medicinal chemists.

[0307] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rats, apes, humans, farm animals, loitering animals, and pets. This also includes tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro.

[0308] As used herein, the terms “requirement of treatment” or “need” refer to a judgment made by a caregiver (in the case of humans, a physician, nurse, nursing practitioner, or individual; in the case of animals (including non-human animals), a veterinarian) that the subject needs or will benefit from treatment. As a result of a disease treatable by the compounds of the present invention, this judgment is based on a number of factors within the caregiver’s experience, but these factors include knowledge that the subject is ill or will become ill.

[0309] As used in this context, “treatment” means to cure, alleviate, stabilize, prevent, or reduce the severity of at least one symptom or disease, pathological condition, or symptom. This term includes active treatment, which is treatment directly and specifically aimed at improving a disease, pathological condition, or symptom; and etiological treatment, which is treatment aimed at eliminating the cause of a related disease, pathological condition, or symptom. Additionally, this term includes paresthesia, which is treatment designed to alleviate symptoms rather than cure a disease, pathological condition, or symptom; preventative treatment, which is treatment aimed at minimizing or partially or completely inhibiting the development of a related disease, pathological condition, or symptom; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or symptom. It should be understood that while treatment aims to cure, alleviate, stabilize, or prevent a disease, pathological condition, or symptom, it does not need to actually result in a cure, alleviation, stabilization, or prevention. The effects of treatment can be measured or assessed in the manner described herein and known in the art as applicable to the disease, pathological condition, or symptom involved. Such measurement and assessment can be performed in a qualitative and / or quantitative manner. Therefore, for example, the characteristics or features of a disease, pathology, or symptom and / or the symptoms of a disease, pathology, or symptom can be reduced to any effect or any amount.

[0310] The compositions, agents, cells, or cell populations disclosed herein can be administered in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrathecally, intravenously, intralymphaticly, or intraperitoneally.

[0311] The medicament of the present invention can be administered by any suitable manner resulting in a concentration of compound that effectively treats or inhibits (e.g., by delaying) disease progression. The compound is mixed with a suitable carrier substance (e.g., a pharmaceutically acceptable excipient) that retains the therapeutic properties of the compound administered with it. An exemplary pharmaceutically acceptable excipient is physiological saline. A suitable carrier substance is typically present in an amount of 1 to 95% by weight of the total medicament. The medicament can be provided in a dosage form suitable for administration. Thus, the medicament can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, ointments, delivery devices, injections, implants, sprays, or aerosols.

[0312] Methods of administering pharmacological compositions (including 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 composition may be administered via any convenient route (e.g., by infusion or bolus injection, by absorption through the epithelium or mucosal layer of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.), the eye, etc.), and may be administered with other bioactive agents. Administration may be systemic or local. Furthermore, administration of the composition to the central nervous system via any suitable route (including intraventricular and intrathecal injection) may be advantageous. Lung administration may also be employed using inhalers or nebulizers and formulations with nebulizing agents. Local application of the agent to the area requiring treatment may also be desired; this can be achieved, for example, but not limited to, local infusion during surgery, local application, by injection, by catheter, by suppository, or by implantation.

[0313] The dosage of an agent that will effectively treat a specific condition or ailment will depend on the nature of the condition or ailment and can be determined by a person skilled in the art using standard clinical techniques. Additionally, in vitro assays may be optionally used to help determine the optimal dosage range. The precise dosage to be used in the formulation will also depend on the route of administration and the overall severity of the disease or ailment, and should be determined based on the judgment of the practitioner and the individual patient's situation. Ultimately, the attending physician will determine the dosage to treat each individual patient. In some embodiments, the attending physician will administer a low dose of the agent and observe the patient's response. A larger dose may be administered until the patient achieves optimal therapeutic effect, and no further dose increases are made thereafter. Generally, the daily dose range is from about 0.001 mg to about 100 mg per kilogram of mammalian body weight, preferably from 0.01 mg to about 50 mg per kilogram, and most preferably from 0.1 mg to 10 mg per kilogram, in a single dose or divided doses. On the other hand, in some cases, it may be necessary to use doses exceeding these limits. In some embodiments, the appropriate dose range for intravenous administration is typically about 5 to 500 micrograms (μg) of active compound per kilogram (kg) of body weight. The appropriate dose range for intranasal administration is typically about 0.01 pg / kg body weight to 1 mg / kg body weight. In some embodiments, the composition containing the agent of the invention is administered subcutaneously as a single dose to adult patients at a dose range of about 5 to 5000 μg / person, preferably about 5 to 500 μg / person. It is desirable to administer this dose once to three times daily. The effective dose can be extrapolated from dose-response curves obtained from in vitro or animal model testing systems. Suppositories typically contain 0.5% to 10% of the active ingredient by weight; oral formulations preferably contain 10% to 95% of the active ingredient. Ultimately, the attending physician will determine the appropriate duration of therapy using the composition of the invention. The dosage will also vary depending on the individual patient's age, weight, and response.

[0314] Preferably, the therapeutic agent can be administered in a therapeutically effective amount of the active ingredient. The term "therapeutically effective amount" means an amount that can elicit a biological or medical response sought by researchers, veterinarians, physicians or other clinicians in tissues, systems, animals or humans and, in particular, can prevent or alleviate one or more of the local or systemic symptoms or features of a disease or ailment being treated.

[0315] In some implementations, the therapeutic agent may contain one or more components of the gene editing system and / or a polynucleotide encoding it.

[0316] Example

[0317] Example 1 - Pseudogenization of lentiviruses using endogenous retroviral envelope proteins

[0318] The expression of various single env proteins was tested in HEK293T cells. Figure 1 Optimal expression was achieved using Envw1, Envkl, and Envfrd (Envw2). The vesicular stomatitis virus glycoprotein (VSV-G) mediates cell attachment and induces direct fusion between cell membranes. The applicant compared the pseudogenotyping efficiency of different env proteins with lentiviral DNA. Effective particle formation was observed using Envkl, Envw1, and Envfrd. Figure 2 ).

[0319] To examine whether the gag homolog Pnma3 was expressed in neuronal cells, the applicant fused Pnma3 with a red fluorescent reporter protein (RFP) and tested the expression of Pnma3 in mouse and rat neurons. The results showed that the expression of this fusion protein was comparable to that of the control RFP-lentiviral construct. Figure 3 ).

[0320] Example 2 - Screening endogenous gag protein candidates for capsid formation, protein secretion, and information transmission capabilities.

[0321] The ability of nine endogenous gag protein candidates to form vesicles in vitro was identified and screened. Figure 4 and Figure 5 Of the candidates tested, all except Asprvl were able to form vesicles. Figure 5 (and Table 2). However, only six can be secreted from cells (Table 3, ...). Figure 6 ).

[0322] Table 2. Vesicle-forming ability of gag protein candidates

[0323] Does it form a capsid outside the body? Asprv1 - Pnma1 + Pnma3 + Pnma4 + Pnma5 + Pnma6 + Pnma7 + Peg10 + Rtl1 +

[0324] Table 3. The ability of gag protein candidates to be secreted from cells

[0325] Secretory protein? Asprv1 - Pnma1 + Pnma3 - Pnma4 + Pnma5 + Pnma6 + Pnma7 - Peg10 + Rtl1 +

[0326] The applicant then tested the ability of various gag protein candidates to transfer the Cas9 / gRNA complex to another cell. In the absence of membrane fusion proteins ( Figure 7A None of the candidates successfully promoted this process. However, including VSV-G ( Figure 7B This is crucial for enabling the delivery of the complex to another cell (Table 4).

[0327] Table 4. The ability of gag protein candidates to deliver information to new cells

[0328] transfer? Asprv1 - Pnma1 - Pnma3 - Pnma4 + Pnma5 - Pnma6 - Pnma7 - Peg10 + Rtl1 +

[0329] Vesicles formed using PNMA4 and RTL1 exhibited the highest capacity to transfer gene editing complexes to new cells and induce insertion / deletion formation. Figure 10 ).

[0330] To evaluate whether the gag candidate would facilitate cellular secretion and subsequent information transfer from one cell to another, the applicant also generated knock-in mice expressing an HA tag on an endogenous gag protein. The DNA sequence encoding an exemplary HA-tagged RTL1 protein is shown in [the image / description]. Figure 12 As shown in the image.

[0331] Example 3 - Engineered Endogenous Vectors for Gene Therapy

[0332] The applicant aims to create a non-immunogenic vector capable of efficiently delivering gene therapy in vivo. While viral vectors are highly efficient, they can potentially be immunogenic, triggering an unwanted immune response against the vector itself in target cells, thus rendering the contained therapeutic agent ineffective. Lipid nanoparticles (LNPs) are easy to produce, but they have limited tropism and typically deliver only about 2% of their encoded payload. Exosomes are potentially non-immunogenic, but possess complex biological properties, and their efficacy is unclear. The applicant hopes to explore the potential of endogenous signaling systems to mediate intercellular gene transfer. For example, the human genome contains at least 40,000 genes encoding GAGs, each with varying immunogenic potential (…). Figure 17 Some highly expressed endogenous GAGs in Figure 4 As shown in the image.

[0333] The applicant analyzed the ability of several GAGs to spontaneously form vesicles. Figure 19 , Figure 20 To determine which GAGs could form vesicles, HA-tagged GAGs were overexpressed in HEK cells and the supernatant was collected. VLP fractionation was performed using PEG centrifugation. Figure 21 The applicant found that adding the VSV-G fusion agent improved the uptake of secreted GAG by target cells and promoted the generation of insertional deletions. Figures 23A to 23D , Figure 24 , Figure 52 and Figure 53 ).

[0334] Of all the GAGs tested, the applicant determined that PEG10 was the best candidate for mediating transfer and generating VLPs at levels equivalent to HIV lentivirus. Figure 24To optimize PEG10 delivery, the applicant seeks to understand the precise biological function of PEG10 and the extent to which it can be reprogrammed. The PEG10 gene comprises two overlapping reading frames encoding the same transcript in different isoforms. The shorter isoform possesses a CCHC-type zinc finger motif, which contains the sequence signature of the gag protein in most retroviruses and some retrotransposons, and functions in part through interaction with members of the TGF-β receptor family. The longer isoform possesses a DSG-shared sequence at the active site of the protease domain of the pol protein. The longer isoform is a result of a -1 translation frameshift also seen in some retroviruses. Figure 25 , Figure 26 ).

[0335] The applicant transfected cells with various PEG10 constructs and analyzed whole-cell lysates and VLP fractions by immunoprecipitation. Results showed that PEG10 VLPs were processed, but this processing did not require a protease domain. Figure 28 The applicant also found that the addition of VSV-G increased PEG10 secretion and allowed for uptake in target cells. Figure 29 ).

[0336] To improve delivery efficiency, the applicant cultured HEK293T cells in T225 flasks. The cells were transfected with various delivery components and then discharged at 45°C. μ Filtered using an m filter and ultracentrifuged with a 20% sucrose buffer. Resuspend the VLP at 250 °C. μ The suspension was placed in L of PBS, and 10 L of the suspension was added. μ L-sized aliquots were added to 20E3 cells. Insertion / deletion sequences were then detected by next-generation sequencing after 48 hours. Figure 31 These experiments revealed that PEG10 is a secreted capsid-forming protein, and that VSV-G enables PEG10 to deliver Cas9 to target cells and mediate the generation of insertions and deletions. The PEG10 VLP may be processed in its C-terminal domain. The applicant also found that the addition of SGCE increased PEG10 secretion but did not help increase entry (at least in HEK cells).

[0337] The applicant compiled and cloned a list of 165 additional genes that could serve as potential fusion agents (Tables 5 and 6). Each of these will be evaluated individually using HIV, PEG10, Arc, and Rtl1 GAG.

[0338] Table 5.

[0339]

[0340]

[0341] Table 6.

[0342] ADGRE5 FBLN2 MFAP2 SLC27A6 TMEM164 ANXA5 Frdm3 MTMR4 SLC32A1 TMEM18 ARHGAP20 GABRA1 NECTIN4 SLC38A2 tmem255a ARHGAP8 GNA11 OLFML2B SLC39A14 TMEM54 ATP1B1 GNAS OPCML SLC4A2 TRAF4 balap3 HTR7 OR5B17 SNTA1 ZCCHC14 cd63 IL27RA OSBPL6 SOBP Pnma6 Cldn1 IRS4 Pianp SPATA13 CLDN5 izumo2 PIGQ ST3GAL4 clmp JCAD PLA2G12A st8sia4 cpn2 KIR2DL3 PMEPA1 TCIRG1 CRISPLD2 KLHDC10 PTPRB TESC Egflam LY6K SLC13A5 TGFBR3 EML6 LYNX1 SLC14A1 THSD4 EXTL3 LYPD5 SLC22A3 TMED8

[0343] The applicant then determined that PEG10 could be found in both serum and cortical neurons in the brain. 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 nuclear chromatin remodeling, 2) genes involved in the trans-golgi network and exocytosis, and 3) SNARE and other genes encoding endosomes and transmembrane proteins.

[0344] To determine whether secreted GAGs are chromatin modifiers that bind DNA rather than RNA, the applicant performed DNA adenine methyltransferase identification (DamID), a protocol for mapping the binding sites of DNA-binding and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein along with a DNA methyltransferase as a fusion protein. The binding of the protein of interest to DNA localizes the methyltransferase to the region of the binding site. Adenosine methylation does not occur naturally in eukaryotes, therefore any adenine methylation in any region can be determined to have been induced by the fusion protein, meaning that the region is located near the binding site. Figure 36 To implement this protocol, the applicant digested the genome with DpnI, which only cut methylated GATC. A double-stranded adaptor with a known sequence was then ligated to the ends generated by DpnI. The ligation product was digested with DpnII, which cut unmethylated GATC, to ensure that only fragments flanked by contiguous methylated GATC were amplified in the subsequent PCR. PCR was then performed using primers matched to the adaptor, resulting in the specific amplification of the genomic fragments flanked by methylated GATC. Figure 37 The data obtained through DamID mapping was then cross-referenced with the ATAC sequencing data. Figure 38 The applicant overexpressed PEG10 and SGCE in N2A cells, fractionated the fractions by ultracentrifugation (VLP), and analyzed the proteins in the precipitates by mass spectrometry. The fractions were found to be rich in various proteins, including RNA turnover factors, transcription factors, and chromatin remodelers. Figure 39The applicant concludes that PEG10 is efficiently secreted from cells and can mediate the delivery of larger macromolecules. Because PEG10 is distributed throughout the body, it may bind to DNA and may itself be a substance delivered to, enters, and directly binds to DNA within cells. Figure 40 ).

[0345] Example 4 - Processing of PEG10 and Functional Properties of Processed Domains

[0346] The ability of PEG10 to form vesicles raises two central questions: 1) How is PEG10 processed, and 2) What is the role of each functional domain? To answer the first question, the applicant overexpressed mouse PEG10 with N-terminal and C-terminal HA tags in HEK293FT cells, immunoprecipitated PEG10 using HA magnetic beads, and analyzed the bands by Western blotting. The corresponding Coomassie staining bands were analyzed by mass spectrometry. The results showed that the protein was cleaved into all of the respective predicted domains ( Figure 56 , Figures 57A to 57F , Figure 58A and Figure 58B ).

[0347] To answer the second question, the applicant compared PEG10 to a previously identified protein called MYEF, a DNA-binding protein that binds to a highly specific 10-base-pair sequence in 3X repeats (such as...). Figure 59 (As shown on the right). The applicants determined that PEG10 binds to the exact same sequence, so they attempted to package particles expressing that DNA sequence. When PEG10 was overexpressed with plasmid DNA containing this sequence, the applicants noticed that PEG10 preferentially packaged and encapsulated the 10-base-pair DNA sequence and secreted plasmids carrying that sequence.

[0348] To quantify how much PEG10 circulates in the blood, the applicant engineered mice with a PEG10 antibody receptor tag and determined that PEG10 is expressed in mouse plasma at approximately 120 pg / μL. Figure 70 ).

[0349] ***

[0350] Various modifications and variations of the methods, pharmaceutical compositions, and kits described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in conjunction with specific preferred embodiments, it should be understood that further modifications are possible and the claimed invention should not be unduly limited to the specific embodiments described. Indeed, various modifications intended to the described modes of the invention will be apparent to those skilled in the art and are within the scope of the invention. This application is intended to cover any variations, uses, or modifications of the invention that generally follow the principles of the invention, and includes such deviations from this disclosure that are known in customary practice within the field to which the invention pertains and that may be applied to the essential features set forth above.

Claims

1. An engineered delivery system comprising one or more endogenous retroviral elements encoding one or more endogenous retroviral elements for forming delivery vesicles and one or more capture portions for packaging cargo within the delivery vesicles, wherein at least one of the one or more endogenous retroviral elements is a retroviral gag homolog selected from PEG10, RTL1, or PNMA4, wherein the cargo is linked, hybridized, fused, or selectively bound to the one or more capture portions within the delivery vesicles; and Membrane fusion proteins, including VSV-G.

2. The engineered delivery system of claim 1, wherein the one or more polynucleotides further encode one or more retroviral envelope proteins, retroviral reverse transcriptases, or combinations thereof.

3. The engineered delivery system of claim 2, wherein the retroviral reverse transcriptase is endogenous.

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

5. The engineered delivery system of claim 2, wherein the retroviral reverse transcriptase and the retroviral envelope protein are both endogenous.

6. The engineered delivery system of claim 2, wherein the retroviral gag homologous protein contains NC and MA domains.

7. The engineered delivery system of claim 1, wherein the retroviral gag homolog is PNMA4 or RTL1.

8. The engineered delivery system of claim 7, wherein the retroviral gag homolog is PEG10.

9. The engineered delivery system of claim 2, wherein the retroviral envelope protein is derived from γ retrovirus or δ retrovirus.

10. The engineered delivery system of claim 2, wherein the retroviral envelope protein is selected from envH1, envH2, envH3, envK1, envK2, envK3, envK4, envK5, envK6, envT, envW1, envfrd, envR(b), envR, envF(c)2, or envF(c)1.

11. The engineered delivery system of claim 2, wherein the retroviral gag homologous protein or the retroviral envelope protein comprises one or more of the capture portions.

12. The engineered delivery system of claim 11, wherein the capturing portion is a hairpin loop coupling element.

13. The engineered delivery system of claim 12, wherein the hairpin loop binding element is an MS2 adaptor protein.

14. The engineered delivery system of claim 1, wherein the delivery system has reduced immunogenicity.

15. The engineered delivery system of claim 2, wherein the cargo comprises one or more nucleic acids, proteins and / or complexes thereof.

16. The engineered delivery system of claim 2, wherein the cargo is connected to the one or more retroviral envelope proteins via a connector.

17. The engineered delivery system of claim 16, wherein the connector is a glycine-serine connector.

18. The engineered delivery system of claim 17, wherein the glycine-serine linker is (GGS)3.

19. The engineered delivery system of claim 15, wherein the cargo comprises ribonucleoprotein.

20. The engineered delivery system of claim 15, wherein the nucleic acid is DNA.

21. The engineered delivery system of claim 15, wherein the cargo contains a genetic regulator.

22. The engineered delivery system of claim 21, wherein the genetic regulator comprises one or more components of the gene editing system and / or a polynucleotide encoding thereas.

23. The engineered delivery system of claim 22, wherein the gene editing system is a CRISPR-Cas system.

24. The engineered delivery system of claim 23, wherein the CRISPR-Cas system is a type II, type V, or type VI CRISPR-Cas system.

25. The engineered delivery system of claim 24, wherein the type II CRISPR-Cas system includes CRISPR-Cas9.

26. The engineered delivery system of claim 24, wherein the V-type CRISPR-Cas system includes CRISPR-Cas12.

27. The engineered delivery system of claim 24, wherein the type VI CRISPR-Cas system includes CRISPR-Cas13.

28. The engineered delivery system of claim 23, wherein the engineered delivery system comprises one or more polynucleotides encoding a retroviral envelope protein, and wherein the Cas protein of the CRISPR-Cas system is modified to bind the one or more capture portions of the retroviral envelope protein.

29. The engineered delivery system of claim 23, wherein the engineered delivery system comprises one or more polynucleotides encoding a retroviral envelope protein, and wherein the guide molecule of the CRISPR-Cas system is modified to bind the one or more capture portions of the retroviral envelope protein.

30. The engineered delivery system of claim 28, wherein the modification comprises incorporating a hairpin loop that binds to a hairpin loop binding element on the retroviral envelope protein.

31. The engineered delivery system of claim 30, wherein the hairpin loop is recognized by the MS2 adaptor protein.

32. The engineered delivery system according to any one of claims 1 to 5, wherein the system further comprises reverse transcriptase.

33. The engineered delivery system of any one of claims 1 to 5, wherein the one or more capture portions comprise a DNA-binding portion, an RNA-binding portion, a protein-binding portion, or a combination thereof.

34. The engineered delivery system of any one of claims 1 to 5, wherein the delivery vesicle is a virus-like particle.

35. The engineered delivery system of any one of claims 1 to 5, the system further comprising a targeting portion, wherein the targeting portion is capable of specifically binding to target cells.

36. The engineered delivery system of claim 35, wherein the targeting portion comprises the membrane fusion protein.

37. The engineered delivery system of claim 35, wherein the target cell is a mammalian cell.

38. The engineered delivery system of claim 37, wherein the mammalian cell is a cancer cell.

39. The engineered delivery system of claim 37, wherein the mammalian cells are infected with a pathogen.

40. The engineered delivery system of claim 39, wherein the pathogen is a virus.

41. A delivery vesicle comprising one or more components encoded by one or more polynucleotides in an engineered delivery system according to any one of claims 1 to 40.

42. The delivery vesicle of claim 41, wherein one or more components further comprise a retroviral envelope protein, a retroviral reverse transcriptase, or both.

43. The delivery vesicle of claim 41, wherein the retroviral gag homolog is PNMA4 or RTL1.

44. The delivery vesicle of claim 43, wherein the retroviral gag homolog is PEG10.

45. The delivery vesicle of claim 41, wherein the delivery vesicle comprises a cell-specific targeting portion.

46. ​​The delivery vesicle of claim 45, wherein the cell-specific targeting portion targets mammalian cells.

47. The delivery vesicle of claim 46, wherein the mammalian cell is a cancer cell.

48. The delivery vesicle of claim 46, wherein the mammalian cell is infected with a pathogen.

49. The delivery vesicle of claim 48, wherein the pathogen is a virus.

50. A system for delivering cargo to target cells, comprising a delivery vesicle encapsulating the cargo and an endogenous reverse transcriptase, wherein the delivery vesicle comprises one or more endogenous retroviral elements for forming the delivery vesicle and one or more capture portions for packaging the cargo within the delivery vesicle, wherein at least one of the one or more endogenous retroviral elements is an endogenous retroviral gag homologous protein selected from PEG10, RTL1, or PNMA4, and wherein the cargo is linked, hybridized, fused, or selectively bound to the one or more capture portions within the delivery vesicle; and a membrane fusion protein comprising VSV-G.

51. The system of claim 50, wherein the delivery vesicle is a virus-like particle.

52. The system of claim 50 or 51, wherein the delivery vesicle further comprises a retroviral envelope protein.

53. The system of claim 50, wherein the endogenous retroviral gag homolog is PNMA4 or RTL1.

54. The system of claim 53, wherein the endogenous retroviral gag homolog is PEG10.

55. The system of claim 52, wherein the retroviral envelope protein is derived from HERV.

56. The system of claim 52, wherein the retroviral gag homologous protein or the retroviral envelope protein comprises one or more of the capture portions.

57. The system of claim 56, wherein the capturing portion is a hairpin loop coupling element.

58. The system of claim 57, wherein the hairpin loop binding element is an MS2 adaptor protein.

59. The system of claim 50, wherein the cargo comprises one or more nucleic acids, proteins and / or complexes thereof.

60. The system of claim 59, wherein the nucleic acid is DNA.

61. The system of claim 59, wherein the cargo comprises ribonucleoprotein.

62. The system of claim 57, wherein the cargo comprises a genetic regulator.

63. The system of claim 62, wherein the genetic regulator comprises one or more components of the gene editing system and / or a polynucleotide encoding thereas.

64. The system of claim 63, wherein the gene editing system is a CRISPR-Cas system.

65. The system of claim 64, wherein the CRISPR-Cas system is a type II, type V, or type VI CRISPR-Cas system.

66. The system of claim 65, wherein the type II CRISPR-Cas system includes CRISPR-Cas9.

67. The system of claim 65, wherein the V-type CRISPR-Cas system includes CRISPR-Cas12.

68. The system of claim 65, wherein the type VI CRISPR-Cas system includes CRISPR-Cas13.

69. The system of claim 52, wherein the cargo is connected to one or more retroviral envelope proteins via a connector.

70. The system of claim 69, wherein the connector is a glycine-serine connector.

71. The system of claim 70, wherein the glycine-serine linker is (GGS)3.

72. The system of claim 64, wherein the Cas protein of the CRISPR-Cas system is modified to bind to the capture portion of the retroviral envelope protein.

73. The system of claim 64, wherein the guide molecule of the CRISPR-Cas system is modified to bind to the capture portion of the retroviral envelope protein.

74. The system of claim 72, wherein the modification comprises incorporating a hairpin loop that binds to a hairpin loop binding element on the retroviral envelope protein.

75. The system of claim 74, wherein the hairpin loop is recognized by the MS2 adaptor protein.

76. The system of claim 50, wherein the target cell is a mammalian cell.

77. The system of claim 76, wherein the mammalian cell is a cancer cell.

78. The system of claim 76, wherein the mammalian cells are infected with a pathogen.

79. The system of claim 78, wherein the pathogen is a virus.

80. A method for loading cargo molecules into delivery vesicles, comprising incubating the cargo molecules and an engineered delivery system according to any one of claims 1 to 40 together with one or more bioreactors.

81. The method of claim 80, wherein the one or more bioreactors are cells, microorganisms, or cell-free systems.

82. Use of the delivery vesicles of any one of claims 41 to 49 in the preparation of a reagent for delivering cargo molecules, wherein the reagent is delivered to a target cell or cell population.

83. The use as described in claim 82, wherein delivery is in vivo.

84. The use as described in claim 82, wherein delivery is ex vivo.

85. The use as described in claim 82, wherein delivery is in vitro.

86. The use as claimed in any one of claims 82 to 85, wherein the cargo comprises one or more nucleic acids, proteins and / or complexes thereof.

87. The use as described in claim 86, wherein the nucleic acid is DNA.

88. The use as claimed in claim 86, wherein the cargo comprises ribonucleoprotein.

89. The use as claimed in claim 86, wherein the cargo comprises a genetic regulator.

90. The use as described in claim 82, wherein delivery across the blood-brain barrier occurs.

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