HIV-MSCV hybrid viral vector for gene transfer

A hybrid viral vector for transfer of selected genes to cells and mammals is provided. The vector is a hybrid of human immunodeficiency-based lentivirus and murine stem cell retrovirus.

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.

DETAILED DESCRIPTION OF THE INVENTION
 A hybrid viral vector has now been developed which exhibits high expression
 of an exogenous gene in non-dividing cells and which does not possess the
 potential hazards associated with use of the viral tat protein. The vector
 of the present invention is a HIV lentivirus/murine stem cell virus hybrid
 vector. More specifically, in the hybrid viral vector of the present
 invention an internal CMV enhancer/promoter of the HIV lentiviral vector
 is removed and a U3 region of a 3' HIV LTR of the HIV lentiviral vector is
 partially replaced by a U3 region of a 3' murine stem cell virus LTR (See
 FIG. 1). "Partially replaced" in the present invention is defined as
 replacement with a hybrid LTR that comprises approximately 50 bp of the 5'
 end of the HIV U3 region, the complete MSCV LTR, and approximately 50 bp
 of the 3' end of the HIV U3 region followed by the complete HIV RU5
 region.
 High gene expression has now been demonstrated using this hybrid viral
 vector. In these experiments, viral particles were generated via the
 vector in 293T cells using transient transfection with VSV-G pseudotyping.
 The viral particles were then used to transduce dividing and non-dividing
 human hematopoietic cells. For these experiments, the hybrid viral vector
 also comprised the exogenous gene encoding green fluorescent protein
 (GFP). FACS analysis of the transduced cells demonstrated a significant
 increase in the expression of the hybrid LTR resulting in a fluorescent
 signal 1.5 to 2 logs greater than the control cells transduced with an
 HIV-based vector containing the internal CMV enhancer/promoter. Thus, the
 hybrid viral vector of the present invention exhibits the high
 transduction efficiency of the HIV-based vector and the high expression of
 the MuLV-based vector even in non-dividing stem cells.
 The hybrid viral vector of the present invention was constructed using HIV
 vector pHR'CMVLacZ which was modified by replacing the original
 multicloning site (MCS) and LacZ with the MCS, internal ribosome entry
 site, and enhanced green fluorescent protein (EGFP) from the bicistronic
 MSCV based vector M1GR1. The resulting vector pHR'CMV-IRES-GFP was further
 modified by deleting the internal CMV enhancer/promoter and replacing the
 3'HIV LTR with a hybrid HIV/MSCV LTR. To generate the hybrid LTR, 50 bp of
 the 5' end of the HIV U3 region, the complete MSCV LTR, and the 50 bp of
 the 3' end of the HIV U3 region followed by the complete HIV RU5 region
 were individually amplified by PCR using primers listed in Table 1. The
 resulting 3 fragments were joined together by extension PCR and the
 resulting hybrid HIV/MSCV LTR was amplified by PCR using the primers
 HIV-U3 5'A (SEQ ID NO:1) and HIV-RU5 3'A (SEQ ID NO:6).
 TABLE 1
 Oligonucleotides Used to Generate the HIV/MSCV LTR
 Oligonucleotide
 Name Sequence
 HIV-U3 5'A ccgctcgagacctggaaaaacatg (SEQ ID NO:1)
 HIV-U3 3'A aggtggggtctttcaatccacagatcaaggatatc (SEQ ID NO:2)
 MSCV-LTR 3'C tgagggctcgccactacgggtacccgggcgacgca (SEQ ID NO:3)
 MSCV-LTR 5'C tgaaagaccccacctgtagg (SEQ ID NO:4)
 HIV-RU5 3'B agtggcgagccctcagatgc (SEQ ID NO:5)
 HIV-RU5 3'A ggactagtgaagcactcaaggcaagct (SEQ ID NO:6)
 The packaging plasmid CMVdeltaR8.2 and the VSV-G envelope plasmid PMD.G
 were used. Once produced, the viral vector was characterized in cells in
 culture using hematopoietic precursor cells and FACS analysis.
 The hybrid viral vector of the present invention is useful for transfer of
 selected genes into hematopoietic stem cells as well as other non-dividing
 cells including, but not limited to, cells of the skin, gastrointestinal
 tissue, cardiac tissue, and neuronal tissue. Techniques for transfer of
 selected genes into tissue or cells using viral vectors is
 well-established in the art. Further genes for selection and transfer via
 viral vectors are well known. One of skill can thus use these established
 techniques with the hybrid viral vector of the present invention to
 efficiently transfer selected genes to cells and mammals. The
 characteristics of high expression and safety make the hybrid viral vector
 of the present invention a desirable vector for gene transfer to both
 cells and mammals. In a preferred embodiment, the viral vector of the
 present invention is administered to a mammal, preferably a human. The
 vectors can be administered orally or parenterally, including
 intravenously, intramuscularly, intraperitoneally, intranasally,
 subcutaneously, or surgically. When administered parenterally, it is
 preferred that the vectors be given in a pharmaceutical vehicle suitable
 for injection such as a sterile aqueous solution or dispersion. Following
 administration, the mammal is monitored to detect changes in gene
 expression. Dose and duration of treatment is determined individually
 depending on the condition or disease to be treated. A wide variety of
 conditions or diseases can be treated based on the gene expression
 produced by administration of the gene of interest in the vector of the
 present invention.
 The following nonlimiting examples are provided to further illustrate the
 present invention.
 EXAMPLES
 Example 1
 Generation of Virus
 MSCV-Based Retrovirus: Retroviruses were generated using standard methods
 such as those described by Pear, W. S. et al. (1998. Blood 92:3780-3792).
 In this method M1GR1 and a vector that encodes the viral envelope protein
 VSV-G were transiently co-transfected into the packaging cell line GP that
 provides the retroviral gag-pol proteins. During the next three days, the
 transfected GP cells release retroviruses into the culture medium; each ml
 of media contains 10.sup.5 to 10.sup.6 infectious viral particles. The
 culture media were collected 48 and 72 hours post transfection and stored
 in small aliquots at -70.degree. C.
 HIV based lentivirus: Lentiviral particles were generated in 293T cells
 using transient transfection with plasmids encoding the RNA genome, viral
 protein, and VSV-G envelope protein following standard methods (Naldini,
 L. et al. 1996. Proc. Natl. Acad. Sci. 93:11382-11388). Viral particles
 were collected and stored as described for the MSCV-based retrovirus.
 The titer of the virus was determined by transducing the hematopoietic cell
 line K562. For transduction, 2-4 million cells, 1 ml of the harvested
 culture media, and 8 micrograms per milliliter of polybrene was
 centrifuged in a 24 well plate at 1500.times.g for 90 minutes. Afterwards,
 the cells were transferred to fresh media.
 Example 2
 Tissue Culture
 K562 cells were cultured in RPMI supplemented with 10% fetal bovine serum.
 293T cells and GP cells were cultured in DMEM supplemented with 10% fetal
 bovine serum. Primary human hematopoietic cells from bone marrow aspirates
 or umbilical cord blood were purified by Ficoll gradient followed by
 positive immuno-magnetic selection for CD34. CD34+ cells were cultured in
 IMDM supplemented with 12.5% horse serum and 12.5% fetal bovine serum. In
 some experiments the media were further supplemented with the growth
 factors stem cell factor, thrombopoietin, and flk-3.
 Primary CD34+ bone marrow or umbilical cord blood cells were transduced and
 cultured on a monolayer of primary bone marrow stromal cells for 5 weeks.
 Cells were trypsinized and plated into methylcellulose cultures with IL-3
 and GM-CSF. Colonies were evaluated 10-12 days later under phase and uv
 microscopy.