Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-07-06
2003-02-18
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C424S093100, C424S093210, C435S455000, C435S320100, C435S375000, C435S325000, C435S362000, C435S465000, C536S023100, C514S04400A
Reexamination Certificate
active
06521457
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to viruses as vectors useful in gene delivery, and more specifically to lentiviral vectors useful in gene delivery to non-dividing and dividing cells.
BACKGROUND OF THE INVENTION
The capacities to introduce a particular foreign or native gene sequence into a mammalian cell and to control the expression of that gene are of substantial value in the fields of medical and biological research. Such capacities provide a means for studying gene regulation, and for designing a therapeutic basis for the treatment of disease.
The introduction of a particular foreign or native gene into a mammalian host cell is facilitated by introducing a gene sequence into a suitable nucleic acid vector. A variety of methods have been developed which are capable of permitting the introduction of such a recombinant vector into a desired host cell. In contrast to methods which involve DNA transformation or transfection, the use of viral vectors can result in the rapid introduction of the recombinant molecule in a wide variety of host cells. In particular, viral vectors have been employed in order to increase the efficiency of introducing a recombinant nucleic acid vector into host cells. Viruses that have been employed as vectors for the transduction and expression of exogenous genes in mammalian cells include SV40 virus (see, e.g., H. Okayama et al.,
Molec. Cell. Biol
. 5, 1136-1142 (1985)); bovine papilloma virus (see, e.g., D. DiMaio et al.,
Proc. Natl. Acad. Sci. USA
79, 4030-4034 (1982)); adenovirus (see, e.g., J. E. Morin et al.,
Proc. Natl. Acad. Sci. USA
84, 4626 (1987)), adeno-associated virus (AAV; see, e.g., N. Muzyczka et al.,
J. Clin. Invest
. 94, 1351 (1994)); herpes simplex virus (see, e.g., A. I. Geller, et al.,
Science
241, 1667 (1988)), and others.
Retrovirus-based vectors are particularly favored as tools to achieve stable, integrated gene transfer of foreign genes into mammalian cells. Retroviruses that have been employed as vectors for the introduction and expression of exogenous genes in mammalian cells include the Moloney murine sarcoma virus (T. Curran et al.,
J. Virol
. 44, 674-682 (1982); A. Gazit et al,
J. Virol
. 60, 19-28 (1986)) and murine leukemia viruses (MULV; A. D. Miller,
Curr. Top. Microbiol. Immunol
. 158, 1-24 (1992).
Efforts to introduce recombinant molecules into mammalian cells have been hampered by the inability of many cells to be infected by the above-described viral or retroviral vectors. Limitations on retroviral vectors, for example, include a relatively restricted host range, based in part on the level of expression of the membrane protein that serves as the viral receptor. M. P. Kavanaugh et al.,
Proc. NatL. Acad. Sci USA
91, 7071-7075 (1994). Other limitations include the inability to integrate into non-dividing cells (e.g., neurons, hepatocytes, myofibers, hematopoietic stem cells), modest vector titers available with current packaging systems, and the fragility of vector particles that precludes purification and concentration.
Lentiviruses are a subgroup of retroviruses that are capable of infecting non-dividing cells. L. Naldini et al. report a lentiviral vector system based on the human immunodeficiency virus (HIV) that is capable of transducing heterologous gene sequences into non-proliferative HeLa cells and rat fibroblasts, as well as into human primary macrophages and terminally differentiated neurons. Science 272, 263-267 (1996). However, the use of such a system in humans raises serious safety concerns, due to the possibility of recombination by the vector into a virulent and disease-causing form.
Accordingly, a need remains for a safe and efficient lentiviral vector systems capable of mediating gene transfer into a broad range of dividing and non-dividing cells.
SUMMARY OF THE INVENTION
The present invention is directed to the transfer of heterologous gene sequences into cells using Equine Infectious Anemia Virus (EIAV)-derived vectors for gene delivery.
A first aspect of the present invention is a recombinant lentiviral vector expression system including a first vector comprising a nucleic acid sequence of at least part of the Equine Infectious Anemia Virus (EIAV) genome, wherein the vector (i) contains at least one defect in at least one gene encoding an EIAV structural protein, and (ii) contains a defective packaging signal. The expression system additionally includes a second vector comprising a nucleic acid sequence of at least part of the EIAV genome, wherein the vector (i) contains a competent packaging signal, and (ii) contains a multiple cloning site wherein a heterologous gene may be inserted. The vector expression system also includes a third vector comprising a nucleic acid sequence of a virus, wherein the third vector (i) expresses a viral envelope protein, and (ii) contains a defective packaging signal.
A second aspect of the present invention is a method of producing a replication-defective lentivirus particle, comprising transfecting a cell with a vector expression system of the invention as described above.
A third aspect of the present invention is a method of delivering a heterologous gene to a target cell, comprising transfecting said target cell with a vector expression system of the invention as described above.
A fourth aspect of the present invention is a method of producing a lentiviral stock comprising (a) transfecting a producer cell with a vector expression system of the invention as described above; (b) growing the producer cell under cell culture conditions sufficient to allow production of replication-defective lentivirus particles in the cell; and (c) collecting the replication-defective lentivirus particles from the producer cell.
The foregoing and other aspects of the invention are explained in detail in the drawings herein and the specification set forth below.
REFERENCES:
patent: 4650764 (1987-03-01), Temin et al.
patent: 5091309 (1992-02-01), Schlesinger et al.
patent: 5240846 (1993-08-01), Collins et al.
patent: 5501979 (1996-03-01), Geller et al.
patent: 5576206 (1996-11-01), Schlegel
patent: 5817491 (1998-10-01), Yee et al.
patent: 6013516 (2000-01-01), Verma et al.
patent: 0 453 242 (1991-04-01), None
patent: WO/95/14091 (1995-05-01), None
patent: WO/95/27044 (1995-10-01), None
patent: WO/95/31565 (1995-11-01), None
patent: WO/96/27672 (1996-09-01), None
patent: WO/96/35454 (1996-11-01), None
patent: WO/96/35777 (1996-11-01), None
patent: WO/96/35798 (1996-11-01), None
patent: WO/96/36364 (1996-11-01), None
patent: WO/97/12622 (1997-04-01), None
patent: WO 98/17815 (1998-04-01), None
patent: WO/98/17815 (1998-04-01), None
patent: WO/98/17816 (1998-04-01), None
Inder M. Verma et al, Gene therapy promises, problems and prospects, Nature vol. 389, Sep. 18, 1997.*
Stuart H. Orkin et al, Repoert And Recommendations Of The Panel To Assess The NIH Investment In Research On Gene Therapy, Dec. 7, 1995.*
Cohen;New Role for HIV: A Vehicle For Moving Genes Into Cells; Science, vol. 272:195 (1996).
Friedmann;Progress Toward Human Gene Therapy; Science, vol. 244:1275-81 (1989).
Hart, et al.;Reduced Retroviral Entry Into Polarized Airway Epithelia; 1996 Cystic Fibrosis Conference, Abstract No. 246.
Johnson, et al.;In Vivo Airway Gene Transfer and In Vitro Correction of CF Airway Cells Without Selection by a VSV-G Pseudotyped Retroviral Vector, 1996 Cystic Fibrosis Conference, Abstract No. 247.
Landau, et al.;Packaging System for Rapid Production of Murine Leukemia Virus Vectors with Variable Tropism; Virology, vol. 66, No. 8:5110-13 (1992).
Naldini, et al.;In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector; Science, vol. 272:263-67 (1996).
Pear, et al.;Production of high-titer helper-free retroviruses by transient transfection; Proc. Natl. Acad. Science, vol. 90:8392-96 (1993).
Perry, et al.;The Surface Envelope Protein Gene Region of Equine Infectious Anemia Virus Is Not an Important Determinant of Tropism In Vitro; Virology, vol. 66, No. 7:4085-97 (1992).
Tan, et al.;Inhibitory Activity of the Equine Infectious Anemia
Guzo David
Leffers, Jr. Gerald G.
Myers Bigel Sibley & Sajovec P.A.
University of North Carolina at Chapel Hill
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