Patent ID: 9260725
Filing Date: 2016-02-16
CPC Classification: A61K,C12N

Claim Text:
1. A method for transducing a target animal cell comprising contacting the target animal cell with a recombinant lentiviral vector under conditions to effect the transduction of the target animal cell by the recombinant lentiviral vector, wherein the recombinant lentiviral vector is produced by a method comprising: (I) (a) transfecting a host cell with: (i) a packaging plasmid comprising a stuffer sequence and a cPPT/cTS region that has reduced replication activity relative to wild-type cPPT/cTS replication activity, wherein the packaging plasmid comprises a pol gene and an RRE, and the stuffer sequence is positioned between the pol gene and the RRE of the packaging plasmid; (ii) an expression plasmid, which carries an env gene; and (iii) a lentiviral transfer vector comprising a therapeutic transgene; to yield a producer cell; (b) culturing the producer cell in a medium; and separating the producer cell from the medium to recover the recombinant lentiviral vector from the medium; (II) (a) transfecting a host cell with: (i) a packaging plasmid, wherein the packaging plasmid comprises a stuffer sequence and a cPPT/cTS region that has reduced replication activity relative to wild-type cPPT/cTS replication activity, wherein the stuffer sequence is of sufficient length to effectively provide a lentivirus having at least the size of a wild type lentiviral genome; (ii) an expression plasmid, which carries an env gene; and (iii) a lentiviral transfer vector comprising an expression cassette comprising a therapeutic transgene positioned under the control of a promoter that is active to promote detectable transcription of the transgene in a cell, a 3′ LTR and a 5′ LTR, wherein the 5′ LTR comprises a poly(A) hairpin sequence that inhibits viral replication to yield a producer cell; (b) culturing the producer cell in a medium; and (c) separating the producer cell from the medium to recover the recombinant lentiviral vector from the medium; or (III) (a) transfecting a host cell with: (i) a packaging plasmid, wherein the packaging plasmid comprises an RRE, a cPPT/cTS region that has reduced replication activity relative to wild-type cPPT/cTS replication activity and a stuffer sequence, wherein the stuffer sequence is of sufficient length to effectively provide a lentivirus having at least the size of a wild-type lentiviral genome; (ii) an expression plasmid, which carries an env gene; and (iii) a lentiviral transfer vector comprising an expression cassette comprising a transgene positioned under the control of a promoter that is active to promote detectable transcription of the transgene in a cell, a 3′ LTR and a 5′ LTR, wherein the 5′ LTR comprises a poly(A) hairpin sequence that inhibits viral replication; to yield a producer cell; (b) culturing the producer cell in a medium; and (c) separating the producer cell from the medium to recover the recombinant lentiviral vector from the medium.