Lentivirus-based gene transfer vectors
First Claim
1. A method of delivering a heterologous nucleic acid sequence to a target cell, comprising the step of infecting the target cell with a replication-defective lentivirus particle comprising a recombinant Equine Infectious Anemia Virus (EIAV) nucleic acid sequence comprising (i) cis-acting sequence elements required for reverse transcription of the recombinant EIAV nucleic acid sequence, (ii) a competent packaging signal, and (iii) the heterologous nucleic acid sequence.
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Abstract
A recombinant lentiviral vector expression system comprises a first vector that comprises a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome. The vector contains at least one defect in at least one gene encoding an EIAV structural protein, but is preferably a gag/pol expression vector. The expression system further comprises a second vector, also comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, and additionally containing a multiple cloning site wherein a heterologous gene may be inserted. The expression system also comprises a third vector which expresses a viral envelope protein. The first and third vectors are packaging signal-defective. When the expression system is transfected into a lentivirus-permissive cell, replication-defective EIAV particles may be produced, which particles are useful in delivering heterologous genes to a broad range of both dividing and non-dividing cells.
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Citations
38 Claims
- 1. A method of delivering a heterologous nucleic acid sequence to a target cell, comprising the step of infecting the target cell with a replication-defective lentivirus particle comprising a recombinant Equine Infectious Anemia Virus (EIAV) nucleic acid sequence comprising (i) cis-acting sequence elements required for reverse transcription of the recombinant EIAV nucleic acid sequence, (ii) a competent packaging signal, and (iii) the heterologous nucleic acid sequence.
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18. A method of delivering a heterologous nucleic acid sequence to a target cell, comprising the step of infecting the target cell with replication-defective lentivirus particles collected according to a method comprising:
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(a) transfecting a lentivirus-permissive cell with;
(i) a first vector comprising an Equine Infectious Anemia Virus (EIAV) nucleic acid sequence encoding EIAV gag and EIAV pol, wherein said vector (1) comprises at least one defect in at least one gene encoding an EIAV structural protein, and (2) comprises a defective packaging signal;
(ii) a second vector comprising a recombinant EIAV nucleic acid sequence comprising (1) cis-acting sequence elements required for reverse transcription of the EIAV nucleic acid sequence, (2) a competent packaging signal, and (3) a heterologous nucleic acid sequence; and
(iii) a third vector comprising a nucleic acid sequence of a virus, wherein said third vector (1) expresses a viral envelope protein, and (2) comprises a defective packaging signal;
(b) growing the cell under cell culture conditions sufficient to allow production of replication-defective lentivirus particles in the cell; and
(c) collecting said replication-defective lentivirus particles from the cell. - View Dependent Claims (19, 20, 21)
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- 22. A method of delivering a heterologous nucleic acid sequence to a target cell, comprising the step of infecting said target cell with a replication-defective lentivirus particle comprising a recombinant EIAV nucleic acid sequence encoding (i) a promoter, and (ii) the heterologous nucleic acid sequence, and wherein said recombinant EIAV nucleic acid sequence is defective in encoding at least one EIAV structural protein to result in a replication-defective lentivirus particle.
Specification