Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-03-28
2002-08-13
Wortman, Donna (Department: 1645)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S091400, C435S235100, C435S354000, C435S456000, C435S457000, C435S476000, C536S023100, C536S023400, C536S023720, C424S199100, C424S204100, C424S218100
Reexamination Certificate
active
06432699
ABSTRACT:
1. FIELD OF THE INVENTION
The invention involves viral vectors that can be used to transduce a target cell, i.e., to introduce genetic material into the cell. The targets of interest are eukaryotic cells and particularly human cells. The transduction can be done in vivo or in vitro. More particularly the invention concerns viral vectors that have chimeric envelope proteins and contain the IgG-binding domain of protein A. These vectors when used in conjunction with antibodies targeting a particular cell are particularly useful for gene therapy.
2. BACKGROUND OF THE INVENTION
A variety of viral based vectors have been employed to transfer and to express a gene of interest into a eukaryotic target cell. Recombinant DNA techniques are used to replace one or more of the genes of the virus with the gene of interest operably linked to a promoter that is functional in the target cell. The construct, termed a viral vector, infects the target cell, using the physiological infective “machinery” of the virus, and expresses the gene of interest instead of the viral genes. Because not all the genes of the virus are present in the vector, infection of the target by the vector does not produce viral particles. Viruses that have been used to infect human or mammalian target cells include herpes virus, adenovirus, adeno-associated virus and derivatives of leukemia-type retroviruses. Among the retroviruses of particular interest in the transduction of cells of human origin are constructs based on amphotropic retroviruses.
2.1. Use of Amphotropic and Ecotropic Retrovirus Vectors
Retroviruses are particularly well suited for transduction of eukaryotic cells. The advantages of a vector based this type of virus include its integration into the genome of the target cell so that the progeny of the transduced cell express the gene of interest. Secondly, there are well developed techniques to produce a stock of infectious vector particles that do not cause the production of viral particles in the transduced target cell. Lastly, the production and purification of stocks vector particles having titers of 10
6
TCIU/ml can be accomplished.
One disadvantage of the use of retroviral vectors is that there is presently no practical general, method whereby a particular tissue or cell type of interest can be specifically transduced. Previous efforts to this end have included surgical procedures to limit to specific organs the physical distribution of the viral vector particles (Ferry, N. et al., 1991
, Proc. Natl. Acad. Sci
. 88:8377). Alternatively, practitioners have taken advantage of the fact that type C retroviruses only infect dividing cells. Thus, a population of cells, e.g., bone marrow cells, was removed from a subject and cultured ex vivo in the presence of growth factors specific for the specific target cell which, thus, comprises most of dividing cells in the culture. See, e.g., Wilson, J. M. et al., 1990
, Proc. Natl. Acad. Sci
. 87:439-47; Ohashi, T. et al., 1992
, Proc. Natl. Acad. Sci
. 69:11332-36. After transduction the dividing cells must be harvested and, 30 for many purposes, reimplanted into the subject. The technical difficulties of the ex vivo culture technique combined with the unavailability of growth factors of specific for some types of cells have limited the application of this approach.
A second difficulty presented by the use retroviral based vectors is that a recombination may occur between sequences of vector and an endogenous retrovirus. Such recombination can give rise to a replication competent virus that can cause the production of infectious particles by the target cell. In contrast to herpes virus or adenovirus infection, retroviral infections are not necessarily self-limiting.
Notwithstanding these difficulties, retrovirus vectors based on amphotropic murine leukemia retroviruses that infect human cells, have been approved for use in human gene therapy of certain diseases, for example adenosine deaminase and low density lipoprotein receptor deficiencies and Gaucher's Disease. See, e.g., Miller A. D., 1992
, Nature
357:455; Anderson, W. F., 1992
, Science
256:808; and Crystal, R. G., 1995
, Science
270:404-410.
One approach to overcoming the limitations of using amphotropic retrovirus vectors in human cells has been to mutate the gene encoding the protein on the viral surface that determines the specificity of infection of the virus, the gp70 protein. Using recombinant DNA technology a “mutant” virus is constructed that has had small regions of the gp70 sequence replaced by predetermined sequences. The limits of this approach are set by the requirement for knowledge of the sequence that will enable infection of the target of interest. However, when this knowledge was available, the anticipated alteration in viral specificity has been observed (Valsesia-Wittmann, S., 1994
, J. Virol
. 68:4609-19).
Retrovirus vectors are the most efficient tools available today to stably transduce genes into the genomes of vertebrate cells. Murine leukemia retrovirus (MLV)-based vectors commonly used for gene transfer are classified on the basis of their host range as either ecotropic or amphotropic. Murine ecotropic virions can only infect mouse or rat cells, but murine amphotropic viruses can infect cells of most species, including human cells. Because of their ability to infect such a broad spectrum of cells, a major drawback to the use of amphotropic virus vectors is the fact that these vectors lack target-cell specificity.
Several attempts to alter the host range of retroviruses have been reported to date. Recently, direct modifications of the envelope protein of murine leukemia virus (MLV) have been shown to redirect the viral tropism. A recombinant virus containing a fragment encoding a single Fv antibody chain at the N terminal region of the MLV env gene has been shown to recognize the corresponding epitopes and infect human cells (Russell, S. J. et al., 1993
, Nucleic Acids Res
. 21:1081-1085; Somia, N. V. et al., 1995
, Proc. Natl. Acad. Sci. USA
92:7570-7574; Marin, M. et al., 1996
, J. Virol
. 70:2957-2962). Kasahara et al. have made a chimeric ecotropic virus containing an erythropoietin-envelope fusion protein (Kasahara, N. et al., 1994
, Science
266:1373-1376). This chimeric virus has been shown to infect human cells bearing the erythropoietin receptor. However, this type of approach suffers from at least two limitations. First, each targetable vector must be constructed de novo. It is unlikely:that the incorporation of different targeting elements in the envelope of the virus can always be achieved with equal success and without reducing the virus titers than can be obtained. Second, virions constructed to directly bind to specific targets in human cells are intrinsically unsafe, as wild-type recombinants could produce potentially harmful effects patients treated with such vectors. By contrast, virions constructed as outlined in this manuscript are uninfectious to human cells in the absence of an accompanying targeting reagent, such as a mAb, which is produced separately and only provided in conjunction with the virus at a convenient time.
2.2. Known Viral Vector Complexes to Transduce Target Cells
An alternative to altering the specificity of binding of the gp70 protein itself is to employ a second, novel structure that binds or is bonded to both the viral particle and to the target cell. In one example of this approach, lactose molecules were covalently coupled, by a non-specific reaction, to the envelope proteins of an ecotropic retrovirus, which does not normally infect human cells. A human hepatocellular carcinoma that was known to have receptors for lactose-containing proteins was found to be susceptible to transduction by this vector complex, although the integration of the transduced gene of interest in the target cell chromosome was not directly demonstrated (Neda, H. et al., 1991
, J. Biol. Chem
. 266:14143). No evidence of expression was observed in a hepatocellular carcinoma that lacked the lactose specific receptor. The method of Neda results in a varia
Meruelo Daniel
Ohno Kouichi
Darby & Darby
New York University
Wortman Donna
Zeman Robert A.
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