Lentiviral vectors

Details Lentiviral vectors Lentiviral vectors

1999-05-25

2003-03-11

Guzo, David (Department: 1636)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C435S235100, C435S320100, C435S325000, C435S366000, C435S455000, C435S456000, C435S457000, C435S005000, C435S006120, C536S023100, C536S023720, C536S024100, C424S093100, C424S093200, C424S093600

Reexamination Certificate

active

06531123

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to improved lentivirus-derived packaging and transducing vectors useful for the expression of genes at high levels in eukaryotic cells. The improved vectors are safer, yet permit increased efficiency of packaging the recombinant viral genome and increased long-term gene expression.
BACKGROUND OF THE INVENTION
1. Gene Transfer; Gene Therapy
Viral vectors transduce genes into target cells with high efficiencies owing to specific virus envelope-host cell receptor interaction and viral mechanisms for gene expression. Consequently, viral vectors have been used as vehicles for the transfer of genes into many different cell types including whole embryos, fertilized eggs, isolated tissue samples, and cultured cell lines. The ability to introduce and express a foreign gene in a cell is useful for the study of gene expression and the elucidation of cell lineages (J. D. Watson et al.,
Recombinant DNA
, 2d Ed., W. H Freeman and Co., NY [1992], pp. 256-263). Retroviral vectors, capable of integration into the cellular chromosome, have also been used for the identification of developmentally important genes via insertional mutagenesis (J. D. Watson et al., supra, p. 261). Viral vectors, and retroviral vectors in particular, are also used in therapeutic applications (e.g., gene therapy), in which a gene (or genes) is added to a cell to replace a missing or defective gene or to inactivate a pathogen such as a virus.
In view of the wide variety of potential genes available for therapy, it is clear that an efficient means of delivering these genes is sorely needed in order to fulfill the promise of gene therapy as a means of treating infectious, as well as non-infectious diseases. Several viral systems including murine retrovirus, adenovirus, parvovirus (adeno-associated virus), vaccinia virus, and herpes virus have been developed as therapeutic gene transfer vectors (For review see, A. W. Nienhuis et al.,
Hematology
, Vol. 16
:Viruses and Bone Marrow
, N. S. Young (ed.), pp. 353-414 [1993]).
Factors affecting viral vector usage include tissue tropism, stability of virus preparations, genome packaging capacity, and construct-dependent vector stability. In addition, in vivo application of viral vectors is often limited by host immune responses against viral structural proteins and/or transduced gene products.
One of the key issues in human gene therapy is the toxicity and safety to the treatment subjects. Gene therapy applications in humans have met with problems associated with the host immune responses against the gene delivery vehicles or the therapeutic gene products. Viral vectors (e.g., adenovirus) which co-transduce several viral genes together with the therapeutic gene(s) are particularly problematic. For example, readministration is necessary for adenovirus vectors because of the transient nature of viral gene expression. As such, a host immune response to the vector or the therapeutic gene product may be detrimental (B. C. Trapnell and M. Gorziglia, Curr. Op. Biotechnol., 5:617-625 [1994]; and S. K. Tripathy et al., Nature Med., 2:545-550 [1996]).
Although MLV vectors have not been reported to induce cytotoxicity and do not elicit strong host immune responses, lentiviral vectors such as HIV-1 which carry several immunostimulatory gene products have the potential to cause cytotoxicity and induce strong immune responses in vivo. The latter are known to induce strong cell-mediated immune responses upon transient exposure (M. Clerici et al., J. Inf. Dis., 165:1012-1019 [1992]; M. Clerici et al., J. Amer. Med. Assoc., 271:42-46 [1994]; L. A. Pinto et al., J. Clin. Invest., 96:867-876 [1995]; and S. Rowland-Jones et al., Nature Med., 1:59-64 [1995]). However, this may not be a concern for lentiviral derived transducing vectors, as the latter need not encode any viral genes in the transducing vector.
Of course, in some instances, the purpose of the vector is to provoke a clinically useful immune response against an encoded protein.
Another important issue related to the lentiviral vector usage is that of possible cytopathogenicity upon exposure to some cytotoxic viral proteins. Exposure to HIV-1 proteins may induce cell death or functional unresponsiveness in T cells (N. Chirmule et al., J. Virol., 69:492-498 [1995]; C. J. Li et al., Science 268:429-431 [1995]; J. D. Lifson et al., Science 232:1123-1127 [1986]; I. G. Macreadie et al., Mol. Microbiol., 19:1185-1192 [1996]; and T. Nosaka et al., Exp. Cell. Res., 209:89-102 [1993]). During the development of the present invention, it was observed that direct gene transfer into tissue culture cells by the calcium-phosphate DNA co-precipitation method could induce more than 80% cell death which is caused mainly by necrosis and a residual percentage, approximately 2-4%, by programmed cell death
A final concern is the possibility of generating replication-competent, virulent virus by recombination.
Safety concerns have prompted much effort towards the development of non-viral vector systems, such as liposome-mediated gene transfer, naked DNA injections and gene gun technology. However, all of these non-viral gene transfer methods lack the ability to allow permanent integration of foreign genes into the host cell chromosomes, and are relatively inefficient. For long term expression of therapeutic genes in target cells, efficient means of transduction and genome integration are essential.
2. Retroviruses; Retroviral Vectors
The term “retrovirus” is used in reference to RNA viruses that utilize reverse transcriptase during their replication cycle. The retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles. At each end of the provirus are structures called “long terminal repeats” or “LTRs.” The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. There are several genera included within the family Retroviridae, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, and Spumavirus. Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. They are integrated into the host DNA, and are capable of transmitting sequences of host DNA from cell to cell. This has led to the development of retroviruses as vectors for various purposes including gene therapy.
Retroviral vectors derived from the amphotropic Moloney murine leukemia virus (MLV-A), use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The amphotropic MLV vector system has been well established and is a popular tool for gene delivery (See e.g., E. M. Gordon and W. F. Anderson, Curr. Op. Biotechnol., 5:611-616 [1994]; and A. D. Miller et al., Meth. Enzymol., 217:581-599 [1993]).
Other retroviruses, including human foamy virus (HFV) and human immunodeficiency virus (HIV) have gained much recent attention, as their target cells are not limited to dividing cells and their restricted host cell tropism can be readily expanded via pseudotyping with vesicular stomatitis virus G (VSV-G) envelope glycoproteins (See e.g., J. C. Burns et al., Proc. Natl. Acad. Sci. USA 90:8033-8037 [1993]; A. M. L. Lever, Gene Therapy. 3:470-471 [1996]; and D. Russell

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