# US 11,078,495 B2 · Methods and compositions for integration defective lentiviral vectors > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,495 B2 - Document ID: US11078495B2 - Status: Granted - Kind: B2 - Application number: 15/768,438 - Filed: 2016-10-14 - Published: 2018-11-08 - Granted: 2021-08-03 - Earliest claimed: 2015-10-15 - Assignees: The University of North Carolina at Chapel Hill - Applicants: The University of North Carolina at Chapel Hill - Inventors: Tal Kafri - Web page: https://patentgrep.com/patent/US11078495B2 ## CPC - C12N 15/86: Viral vectors - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 2310/122: Hairpin - C12N 2310/14: interfering nucleic acids [NA] - C12N 2310/531: Stem-loop; Hairpin - C12N 2330/51: Specially adapted vectors - C12N 2710/22022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2740/16043: viral genome or elements thereof as genetic vector - C12N 2830/30: being an enhancer not forming part of the promoter region - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention provides an integration-defective lentiviral vector based on a parental lentivirus and related methods, the integration-defective lentiviral vector including one or more of the following: (a) a mutation, deletion or other modification of one or more binding sites for a host factor involved in gene silencing; (b) an addition of one or more binding sites for a transcription activator, which can be natural (such as but not limited to ubiquitous and/or tissue/cell type specific) including but not limited to SP1 NFkB, or synthetic including but not limited to binding sites for tetracycline regulated trans activators tTA, rtTA, tT65, and/or rtT65; (c) one or more nucleic acid sequences from a SV40 genome, wherein the one or more sequences are obtained from a region of the SV40 genome upstream to the SV40 poly-adenylation signal; (d) a shRNA expression cassette, which encodes a shRNA directed to a host gene involved in epigenetic silencing and/or in DNA repair pathways; or (e) any combination of (a), (b), (c) and (d), wherein as compared to the parental lentivirus, the integration-defective lentiviral vector resists gene silencing. ## Claims ### Claim 1 An integration-defective lentiviral vector (IDLV) based on a parental lentivirus, the integration-defective lentiviral vector comprising one or more of the following: a) a mutation, deletion or other modification selected from the group consisting of i) deletion of an AP-4 binding site; ii) mutation of contiguous nucleotides CNRG to DNRG or CNYG or CNRH, wherein N=A or C or G or T, R=A or G, Y=C or T, H=A or C or T, and D=A or G or T; iii) deletion of a negative regulatory element (NRE); iv) insertion of an NEκB binding site; v) insertion of an SP1 binding site; and vi) any combination thereof; b) insertion of one or more binding sites for a transcription activator, which can be natural or synthetic, and incorporated into the 3′ U3 region in either a self-inactivating (SIN) or a non-SIN form of the lentiviral vector and wherein the transcription activator is selected from the group consisting of SP1; NFkB; a binding site for tetracycline regulated trans activators tTA, rtTA, tT65 and/or rtT65; and any combination thereof; c) insertion of one or more nucleic acid sequences from a SV40 genome, wherein the one or more nucleic acid sequences are a 3f46 region, s2f2R region and/or a USE region of the SV40 genome upstream to the SV40 poly-adenylation signal, and inserted in the lentiviral vector in the 3′ LTR either in the opposite or same orientation to the long terminal repeats (LTRs); and d) any combination of (a), (b) and (c), wherein as compared to the parental lentivirus, the integration-defective lentiviral vector resists gene silencing. ### Claim 2 (depends on 1) The lentiviral vector of claim 1, further comprising a short hairpin RNA (shRNA) expression cassette, which encodes a shRNA directed to a host gene involved in epigenetic silencing and/or ubiquitination or proteasome activity. ### Claim 3 (depends on 2) The lentiviral vector of claim 2, wherein the shRNA expression cassette is directed to a host gene involved in epigenetic silencing selected from the group consisting of AP4, p300, YY1, histone deacetylases (HDACs), histone methylases, histone ubiquitinating enzymes, histone sumoylating proteins, histone phosphorylating proteins, BMI, and Ring1B. ### Claim 4 (depends on 2) The lentiviral vector of claim 2, wherein the shRNA expression cassette is controlled by a PolIII promoter, a U6 promoter, a H1 promoter, or a PolII promoter. ### Claim 5 (depends on 1) The lentiviral vector of claim 1, further comprising a heterologous sequence. ### Claim 6 (depends on 5) The lentiviral vector of claim 5, wherein the heterologous sequence is selected from the group consisting of one or more marker genes, therapeutic genes, antiviral genes, antitumor genes, cytokine genes, genes encoding antigens, sequences that can associate with the host chromatin, sequences that encode a protein that can associate with the host DNA and with the host chromatin and with the nucleic acid of the vector; sequences that encode a protein having DNA methylation activity; sequences that encode genome-editing proteins CAS9, Zinc-finger-nucleases, or TALENs and de-differentiation factors to generate iPS; sequences that encode guide RNAs for CAS9 editing activity; sequences for homologous recombination and NHEJ-based gene editing using gene editing proteins CAS9, Zinc-finger-nucleases, or TALENs, or for gene editing following chemical and/or physical and/or biological induction of DNA damage; and any combination thereof. ### Claim 7 An integration-defective lentiviral vector (IDLV) based on a parental lentivirus, the integration-defective lentiviral vector comprising one or more of the following: a) a mutation, deletion or other modification selected from the group consisting of i) deletion of an AP-4 binding site; ii) mutation of contiguous nucleotides CNRG to DNRG, CNYG or CNRH, wherein N=A or C or G or T, R=A or G, Y=C or T, H=A or C or T, and D=A or G or T; iii) deletion of a TATA box; iv) deletion of a negative regulatory element (NRE); v) insertion of an NEκB binding site; vi) insertion of an SP1 binding site; and vii) any combination thereof; (b) insertion of one or more binding sites for a transcription activator, which can be natural or synthetic, and incorporated into the 3′ U3 region in either a self-inactivating (SIN) or a non-SIN form of the lentiviral vector and wherein the transcription activator is selected from the group consisting of SP1; NFkB; a binding site for tetracycline regulated trans activators tTA, rtTA, tT65 and/or rtT65; and any combination thereof; (c) insertion of one or more nucleic acid sequences from a SV40 genome, wherein the one or more nucleic acid sequences are a 3f46 region, s2f2R region and/or a USE region of the SV40 genome upstream to the SV40 poly-adenylation signal, and inserted in the lentiviral vector either in the opposite or same orientation to the long terminal repeats (LTRs); (d) a short hairpin RNA (shRNA) expression cassette, which encodes a shRNA directed to a host gene involved in epigenetic silencing and/or ubiquitination or proteasome activity; and (e) any combination of (a), (b), (c) and (d), wherein as compared to the parental lentivirus, the integration-defective lentiviral vector resists gene silencing, and further wherein the integration-defective lentiviral vector comprises a site-directed recombination site. ### Claim 8 (depends on 1) A recombinant retroviral particle comprising the vector of claim 1. ### Claim 9 (depends on 1) An inducible retroviral vector packaging cell line comprising the integration-defective lentiviral vector of claim 1 and at least one construct encoding one or more proteins required for the integration-defective lentiviral vector to be packaged and including an integration defective integrase or integration competent integrase. ### Claim 10 (depends on 1) A retroviral vector kit comprising: (a) the integration-defective lentiviral vector according to claim 1 in a transfer cassette; and (b) a packaging cell line comprising at least one construct encoding one or more proteins required for the viral vector to be packaged. ### Claim 11 (depends on 8) A method for expressing a nucleotide sequence of interest in a cell of an animal without integration of the nucleotide sequence into the genome of the cell of the animal, the method comprising infecting the cell of the animal with the recombinant retroviral particle of claim 8. ### Claim 12 (depends on 11) The method of claim 11, wherein the cell of the animal is contacted with a proteasome inhibitor and/or an HDAC inhibitor before, concurrent with and/or after contact with the recombinant retroviral particle. ### Claim 13 (depends on 7) The lentiviral vector of claim 7, wherein the shRNA expression cassette is directed to a host gene involved in epigenetic silencing selected from the group consisting of AP4, p300, YY1, histone deacetylases (HDACs), histone methylases, histone ubiquitinating enzymes, histone sumoylating proteins, histone phosphorylating proteins, BMI, and Ring1B. ### Claim 14 (depends on 7) The lentiviral vector of claim 7, wherein the shRNA expression cassette is controlled by a PolIII promoter, a U6 promoter, a H1 promoter, or a PolII promoter. ### Claim 15 (depends on 7) The lentiviral vector of claim 7, further comprising a heterologous sequence. ### Claim 16 (depends on 7) The lentiviral vector of claim 7, wherein the heterologous sequence is selected from the group consisting of one or more marker genes, therapeutic genes, antiviral genes, antitumor genes, cytokine genes, genes encoding antigens, sequences that can associate with the host chromatin, sequences that encode a protein that can associate with the host DNA and with the host chromatin and with the nucleic acid of the vector; sequences that encode a protein having DNA methylation activity; sequences that encode genome-editing proteins CAS9, Zinc-finger-nucleases, or TALENs and de-differentiation factors to generate iPS; sequences that encode guide RNAs for CAS9 editing activity; sequences for homologous recombination and NHEJ-based gene editing using gene editing proteins CAS9, Zinc-finger-nucleases, or TALENs, or for gene editing following chemical and/or physical and/or biological induction of DNA damage; and any combination thereof. ### Claim 17 (depends on 7) A recombinant retroviral particle comprising the vector of claim 7. ### Claim 18 (depends on 7) An inducible retroviral vector packaging cell line comprising the integration-defective lentiviral vector of claim 7 and at least one construct encoding one or more proteins required for the integration-defective lentiviral vector to be packaged and including an integration defective integrase or integration competent integrase. ### Claim 19 (depends on 7) A retroviral vector kit comprising: (a) the integration-defective lentiviral vector according to claim 7 in a transfer cassette; and (b) a packaging cell line comprising at least one construct encoding one or more proteins required for the viral vector to be packaged. ### Claim 20 (depends on 17) A method for expressing a nucleotide sequence of interest in a cell of an animal without integration of the nucleotide sequence into the genome of the cell of the animal, the method comprising infecting the cell of the animal with the recombinant retroviral particle of claim 17. ### Claim 21 (depends on 20) The method of claim 20, wherein the cell of the animal is contacted with a proteasome inhibitor and/or an HDAC inhibitor before, concurrent with and/or after contact with the recombinant retroviral particle.