Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-11-12
2001-04-17
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S455000, C435S091330, C536S023100, C536S024100, C536S024200
Reexamination Certificate
active
06218186
ABSTRACT:
BACKGROUND OF THE INVENTION
The hematopoietic stem cell (HSC) is an ideal target cell for gene therapy because a single modified cell can potentially regenerate the entire hematopoietic system in which every cell contains the modification. While murine HSC can be transduced using retroviral vectors based on murine leukemic viruses (MuLV), human HSC resist such manipulation (Dinauer, M. C. et al. 1999
. Blood
94:914-922; Persons, D. A. et al. 1997
. Blood
90:1777-1786; Hawley, R. G. et al. 1996
. Proc. Natl. Acad. Sci. USA
93:10297-10302).
One possible explanation is that viral integration of these vectors requires cell division (Miller, D. G. et al. 1990
. Molec. Cell Biol
. 10:4239-4242). In human hematopoietic stem cells that divide slowly, the virus may be neutralized within the cytoplasm before division occurs. This issue is typically addressed by optimizing culture conditions for maximal cell replication. While this strategy increases the transduction of hematopoietic progenitor cells, transduction of the human HSC remains poor (Kohn, D. B. et al. 1998
. Nature Med
. 4:775-780; Kohn, D. B. et al. 1995
. Nature med
. 1:1017-1023; Knaan-Shanzer, S. et al. 1996
. Gene Therapy
3:323-333). Furthermore, ex vivo initiation of replication of the stem cells is associated with commitment, differentiation, and loss of the re-population potential (Gothot, A. et al. 1998
. Blood
92:2641-2649).
The murine stem cell virus (MSCV) based retroviral vectors is another effective vehicle for delivery and expression of exogenous genes in human hematopoietic progenitor cells. Again, a major limitation of this MULV-based vector, however, is that viral integration requires cell division.
These findings suggest that a vector that is less dependent on replication would be better at transducing human HSC.
Unlike the MuLV, the human immunodeficiency virus (HIV) can integrate into the genome of non-dividing cells. The HIV virus encodes a gag core protein with a nuclear localization signal that allows entry into the nucleus by active transport through the nuclear pore (Bukrinsky, M. I. et al. 1993
. Nature
365:666-669). Furthermore, the HIV vpr and vif accessory proteins contribute to transduction of some non-dividing cells (Connor, R. I. et al. 1995
. Virology
206:935-944; Zufferey, R. D. et al. 1997
. Nature Biotech
. 15:871-875; Kafri, T. et al. 1997
. Nature genetics
17:314-317). This distinctive property has been exploited to engineer vectors based on the HIV. These vectors can efficiently transduce non-dividing human hematopoietic stem cells (Evans, J. T. et al. 1999
. Human Gene Therapy
10:1479-1489; Case, S. S. et al. 1999
. Proc. Natl. Acad. Sci. USA
96:2988-2993; Uchida, N. et al. 1998
. Proc. Natl. Acad. Sci. USA
95:11939-11944; Miyoshi, H. et al. 1999
. Science
283:682-686; Sutton, R. E. et al. 1998
. J. Virol
. 72:5781-5788), and other cells such as neurons (Naldini, L. et al. 1996
. Proc. Natl. Acad. Sci. USA
93:11382-11388), skeletal muscle (Kafri, T. et al. 1997
. Nature Genetics
17:314-317), and hepatocytes (Kafri, T. et al. 1997
. Nature Genetics
17:314-317).
One disadvantage of the HIV-based vectors has been the weak expression by the HIV LTR enhancer/promoter in hematopoietic cells. To compensate for this weakness, some HIV-based vectors express the HIV accessory protein tat that enhances transcription off the HIV LTR (Evans, J. T. et al. 1999
. Human Gene Therapy
10:1479-1489; Case, S. S. et al. 1999
. Proc. Natl. Acad. Sci. USA
96:2988-2993; Uchida, N. et al. 1998
. Proc. Natl. Acad. Sci. USA
95:11939-11944). However, tat also enhances the expression of some cellular genes (Chirivi, R. G. et al. 1999
. Blood
94:1747-1754; Weiss, J. M. et al. 1999
. J. Immunol
. 163:2953-2959; Barillari, G. et al. 1999
. J. Immunol
. 164:1929-1935; Demarchi, F. et al. 1999
. J. Virol
. 73:7080-7086; Maggirwar, S. B. et al. 1999
. J. Neurochem
. 73:578-586; Lefevre, E. A. et al. 1999
. J. Immunol
. 163:1119-1122) potentially disrupting the normal function of the transduced cell, although hematopoiesis in mice appears not to be grossly affected by over-expression of tat (Frazier, A. L. and J. V. Garcia. 1994. AIDS Res.
Human Retrovir
. 10:1517-1519). Furthermore, tat protein itself can enter cells (Schwarze, S. R. et al. 1999
. Science
185:1569-1572) and thus may lead to tat expression in bystander non-target cells. Other HIV-based vectors use an internal CMV enhancer/promoter to drive the expression (Miyoshi, H. et al. 1999
. Science
283:682-686). However, the expression in hematopoietic cells is low (Miyoshi, H. et al. 1999
. Science
283:682-686), approximately 2 logs less than expression by the MuLV-based vector.
The recently described HIV-based lentiviral vector has been shown to be efficient in integrating into non-cycling cells (Verma, I. M. and N. Somia. 1997
. Source Nature
389:239-30 242). Studies to determine the usefulness of this vector have been performed by Choi, J. K. and A. Gewirtz. (1998
. Blood
92:468a). Using the enhanced green fluorescent protein (EGFP) as the reporter protein, it was found that the cellular expression of the lentivirus vector either from the HIV LTR promoter/enhancer or from an internal CMV promoter/enhancer was poor. FACS analysis of transduced hematopoietic cells demonstrated that EGFP fluorescent intensity in the lentiviral-transduced cells was only one-half log greater than control cells. This value negatively contrasted with the 2 to 3 log fluorescence signal augmentation observed in cells transduced with the MSCV-based system. To obtain better expression, Uchida et al. (1998
. Proc. Natl. Acad. Sci. USA
95:11939-11944) successfully utilized a HIV-based vector system that also expressed the viral transcription co-factor tat that is critical for high expression off the HIV LTR. However, the uncharacterized physiological effect and immunological response of expression the viral tat protein is a problem for clinical safety.
Thus, the HIV-based vectors need further improvements.
A hybrid HIV/murine stem cell virus(MSCV) vector has now been developed wherein the original internal CMV enhancer/promoter is removed and the U3 region of the HIV LTR is partially replaced by the U3 region of the MSCV LTR. As demonstrated herein, this hybrid provides a safe vector with a high transduction efficiency. Thus, this new hybrid viral vector has advantages over previously available vectors.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a hybrid viral vector which comprises a human immunodeficiency virus-based lentivirus and a murine stem cell retrovirus. In a preferred embodiment, this vector further comprises a selected gene for transduction via the viral vector into mammalian cells.
Another object of the present invention is to provide a method for transducing a selected gene into a cell which comprises administering to the cell a hybrid viral vector containing a human immunodeficiency virus-based lentivirus, a murine stem cell retrovirus and the selected gene.
Yet another object of the present invention is a method for delivering a selected gene to cells in a mammal which comprises administering to the mammal a hybrid viral vector containing a human immunodeficiency virus-based lentivirus, a murine stem cell retrovirus and the selected gene.
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Barillari et al.,
Choi John Kim
Gewirtz Alan M.
Crouch Deborah
Licata & Tyrrell P.C.
Nguyen Quang
Trustees of the University of Pennsylvania
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