# US 10,870,865 B2 · Particle for the encapsidation of a genome engineering system > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,870,865 B2 - Document ID: US10870865B2 - Status: Granted - Kind: B2 - Application number: 16/301,317 - Filed: 2017-05-12 - Published: 2020-03-05 - Granted: 2020-12-22 - Earliest claimed: 2016-05-13 - Assignees: Flash Therapeutics - Applicants: Flash Therapeutics - Inventors: Pascale Bouille; Régis Gayon; Lucille Lamouroux; Alexandra Iche - Web page: https://patentgrep.com/patent/US10870865B2 ## CPC - C12N 15/86: Viral vectors - C07K 14/005: from viruses - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12N 15/907: {in mammalian cells} - C07K 2319/735: containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) - C07K 2319/85: containing an RNA binding domain - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2740/16023: Virus like particles [VLP] - C12N 2740/16043: viral genome or elements thereof as genetic vector - C12N 2740/16052: relating to complementing cells and packaging systems for producing virus or viral particles - C12N 2795/18122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2800/24: Vectors characterised by the absence of particular element, e.g. selectable marker, viral origin of replication - C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT - C12N 2800/50: Vectors for producing vectors - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to a retroviral particle comprising a protein derived from the Gag polyprotein, an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest bound to an encapsidation sequence, each encapsidation sequence being recognized by a binding domain introduced into the protein derived from the Gag polyprotein and/or into the integrase, and at least one of said sequences of interest of the encapsidated non-viral RNAs comprises a part coding a nuclease. ## Claims ### Claim 1 A retroviral particle, comprising a protein derived from the Gag polyprotein, an envelope protein, optionally an integrase, and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest bound to an encapsidation sequence, each encapsidation sequence being recognized by a heterologous binding domain introduced into the protein derived from the Gag polyprotein and/or into the integrase, in which the at least two encapsidated non-viral RNAs differ by their sequence of interest, wherein: at least one of said sequences of interest of the encapsidated non-viral RNAs comprises a part coding for a nuclease, said nuclease being chosen from the group constituted by a nuclease associated with CRISPR system; and the sequence of interest of the other encapsidated non-viral RNA corresponds to at least one recognition element of a guide RNA. ### Claim 2 (depends on 1) The retroviral particle according to claim 1, in which the nuclease is Cas9. ### Claim 3 (depends on 1) The retroviral particle according to claim 1, which further comprises at least a third encapsidated non-viral RNA having a sequence of interest corresponding to a second recognition element of a guide RNA or to an additional guide RNA. ### Claim 4 (depends on 1) The retroviral particle according to claim 1, comprising a nucleocapsid protein, an envelope protein, optionally an integrase, and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest bound to an encapsidation sequence, each encapsidation sequence being recognized by a heterologous binding domain introduced into the nucleocapsid protein and/or into the integrase. ### Claim 5 (depends on 4) The retroviral particle according to claim 4, in which the heterologous binding domain is introduced into the nucleocapsid protein, and a second heterologous binding domain may be introduced into the nucleocapsid and/or into the integrase. ### Claim 6 (depends on 1) The retroviral particle according to claim 1, comprising a nucleocapsid protein, an envelope protein, optionally an integrase, and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest bound to an encapsidation sequence, at least one encapsidation sequence being the stem-loop motif of the MS2 bacteriophage repeated 12 times, said stem-loop motif being recognized by the Coat protein of the MS2 bacteriophage introduced into the nucleocapsid protein. ### Claim 7 (depends on 6) The retroviral particle according to claim 6, wherein the encapsidated non-viral RNAs comprises, as encapsidation sequence, the stem-loop motif of the MS2 bacteriophage repeated 12 times is an RNA coding Cas9, and a second encapsidated non-viral RNA is an RNA corresponding to at least one recognition element of a guide RNA or coding a guide, said encapsidated non-viral RNAs comprising as encapsidation sequence the stem-loop motif of the MS2 bacteriophage repeated 2 times. ### Claim 8 (depends on 1) The retroviral particle according to claim 1, comprising a nucleocapsid protein, an envelope protein, optionally an integrase, and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest bound to an encapsidation sequence, at least one encapsidation sequence being the stem-loop motif of the PP7 bacteriophage repeated 2 times, said stem-loop motif being recognized by the Coat protein of the PP7 bacteriophage introduced into the nucleocapsid protein. ### Claim 9 (depends on 1) The retroviral particle according to claim 1, in which the heterologous binding domain is introduced into the integrase, and a second heterologous binding domain may be introduced into the nucleocapsid and/or into the integrase. ### Claim 10 (depends on 1) The retroviral particle according to claim 1, which is a lentiviral particle. ### Claim 11 (depends on 1) A composition comprising the particle according to claim 1. ### Claim 12 (depends on 1) A kit for producing particles according to claim 1, comprising: (i) an expression plasmid comprising at least two different non-viral RNA sequences, each RNA sequence comprising a sequence of interest for which an encapsidation sequence is inserted upstream of, downstream of or within this sequence, or, alternatively, a first and a second expression plasmid each comprising a sequence of interest upstream or downstream of which an encapsidation sequence is inserted, at least one of these sequences of interest coding for a nuclease chosen from a group constituted by nucleases associated with CRISPR system, the other sequence of interest corresponding to at least one recognition element of a guide RNA, (ii) an encapsidation plasmid coding for a protein derived from the Gag polyprotein and/or a chimeric integrase, comprising a binding domain allowing recognition of an encapsidation sequence, and, (iii) an envelope plasmid coding for an envelope protein. ### Claim 13 (depends on 12) The kit according to claim 12, further comprising a second encapsidation plasmid coding for: a protein derived from the wild-type Gag polyprotein, when the first encapsidation plasmid codes for a protein derived from the chimeric Gag polyprotein, and/or a wild-type integrase, when the first encapsidation plasmid codes for a chimeric integrase. ### Claim 14 (depends on 1) A manufacturing method for producing the particle according to claim 1, comprising a step of co-transfection of cells with: (i) an expression plasmid comprising at least two different non-viral RNA sequences, each RNA sequence comprising a sequence of interest for which an encapsidation sequence is inserted upstream of, downstream of or within this sequence, or, alternatively, a first and a second expression plasmid each comprising a sequence of interest upstream or downstream of which an encapsidation sequence is inserted, at least one of these sequences of interest coding for a nuclease chosen from a group constituted by nucleases associated with CRISPR system, the other sequence of interest corresponding to at least one recognition element of a guide RNA, (ii) an encapsidation plasmid coding for a protein derived from the Gag polyprotein and/or a chimeric integrase comprising a binding domain allowing recognition of an encapsidation sequence, and, (iii) an envelope plasmid coding for an envelope protein; and recovery of the supernatant from the transfected cells comprising a plurality of the particle. ### Claim 15 (depends on 14) The method according to claim 14, in which the step of co-transfection is further carried out with a second encapsidation plasmid coding for: a protein derived from the wild-type Gag polyprotein, when the first encapsidation plasmid codes for a protein derived from the chimeric Gag polyprotein, and/or a wild-type integrase, when the first encapsidation plasmid codes for a chimeric integrase. ### Claim 16 (depends on 14) A composition obtained by the method according to claim 14. ### Claim 17 (depends on 1) A method for generating in cells a DNA double-strand break, a point mutation, a sequence deletion including gene knock-out, a sequence insertion or gene replacement, comprising transducing the cells with the particle according to claim 1.