Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1997-03-18
2002-10-29
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S455000, C435S373000, C435S366000, C435S372000, C435S320100, C536S023100, C536S023200, C536S023500, C536S024100
Reexamination Certificate
active
06472212
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns the field of gene therapy and more particularly relates to a method for genetically modifying primate bone marrow cells so that they have the capacity to regenerate in vivo, and to cells that produce recombinant retroviral vectors that can be used in such a method. The method is exemplified by the use of means which enhance the local concentration of retroviral particles derived from the murine leukemia virus in the vicinity of target primate stem cells.
BACKGROUND
Developments in the field of molecular biology have led to a better understanding of the genetic basis underlying the development of a large number of disorders. It is expected that the genes which are associated with the diseases that occur most frequently will have been identified, cloned and characterized before the end of this century.
So far, molecular genetics has contributed to medicine by the development of diagnostic tools and methods and the biotechnological production of pharmaceuticals. It may be expected, however, that it will also be possible to use the increasing knowledge of genetics for an essentially new therapeutic treatment, the so-called gene therapy. The purpose of gene therapy is to treat disorders by genetically modifying somatic cells of patients. The uses of gene therapy are not limited to hereditary disorders; the treatment of acquired diseases is also considered to be one of the possibilities. Although this field of study is still in a preliminary stage and must be developed, therapeutic possibilities are in the distance which can drastically improve medicine in the future (Anderson, (1984)
Science
226:41 0; Belmont and Caskey, (1986) in Gene Transfer, R. Kucherlapati, eds, Plenum press, New York and London, P. 411; and Williamson, (1982)
Nature
298:416.)
An important cell type for gene therapy purposes is the so-called hematopoietic stem cell which is the precursor cell of all circulating blood cells. This stem cell can multiply itself without losing its differentiating ability. In adult animals most stem cells are situated in the bone marrow. Very infrequently stem cells also start to circulate in the peripheral blood. This can be significantly augmented by treatment with stem cell mobilizing agents, including but not restricted to certain hematopoietic growth factors. In embryos, stem cells are by nature circulating much more frequently. Thus, bone marrow, peripheral blood after stem cell mobilization and embryonic blood, e.g. collected perinatally from the umbilical vein, are useful sources for stem cells. The underlying idea of a gene therapy directed to hematopoietic stem cells is that gene transfer to (a limited number of) stem cells may already be sufficient to replace the entire blood-forming tissue with genetically modified cells for a lifetime (Williamson, (1982)
Nature
298:416). This would enable treatment not only of diseases that are caused by a (hereditary) defect of blood cells, but also of diseases that are based on the inability to make a certain protein: the modified blood (forming) system could be a constant source of the protein, which could do its work at the places where necessary. It is also possible, with the introduction of genetic material into the blood system, to obtain resistance against infectious agents, to combat cancer, or even to overcome a predisposition to chronic diseases, such as rheumatism or diabetes. Finally, it is noted that in the treatment of some diseases it is to be preferred or necessary that the gene transfer to stem cells is performed on bone marrow cell populations from which certain cell types have been removed. One could for instance consider the use of gene therapy in the treatment of leukemia, in which case there should not occur any gene transfer to the leukemic cells.
One of the conditions for providing of such a bone marrow gene therapy protocol is a technique by which genes can be incorporated into the chromosomes of target cells, in such a manner that those genes are also passed on to the daughter cells and that the desired protein product is produced in those cells. In the invention described here, for this purpose use is made of recombinant retroviruses that carry with them the genes to be introduced and which are capable of delivering them to mammalian cells. They make use of the natural characteristic of retroviruses to integrate efficiently and stably into the genome of the infected cell, but not themselves to cause a productive infection because the retroviruses used are replication-defective and are not contaminated with wild-type viruses (Temin, (1986) in
Gene Transfer
, R. Kucherlapati, eds. Plenum Press, New York, p. 149 and Temin, (1990)
Hum. Gene Ther
. 1:1 1 1). The recombinant retroviruses which are used within the framework of the present invention are derived from viruses with a natural host-specificity that includes primates, or from viruses that can be pseudotyped with a host-specificity that includes primates. Said viruses include, but are not restricted to, murine leukemia viruses (MuLV; Weiss et al., (1984) RNA Tumor Viruses, New York) with a so-called amphotropic or xenotropic host-range, gibbon ape leukemia viruses (GaLV; Lieber et al.,
Proc. Natl. Acad. Sci. USA
72(1975) 2315-2319), and primate lentiviruses.
For the production of recombinant retroviruses, two elements are required: the so-called retroviral vector, which, in addition to the gene (or genes) to be introduced, contains all DNA elements of a retrovirus that are necessary for packaging the viral genome and the integration into the host genome; and the so-called packaging cell line which produces the viral proteins that are necessary for building up an infectious recombinant retrovirus (Miller, (1990),
Hum. Gene Ther
. 1:5).
As the presence of replication-competent viruses in a gene therapy protocol is considered highly undesirable, most modern packaging cell lines are constructed in a way such that the risk of recombination events whereby a replication-competent virus is generated, is minimized. This is effected by physically separating into two parts the parts of the virus genome that code for viral proteins and introducing them into the cell line separately (Danos and Mulligan, (1988)
Proc. Natl. Acad. Sci. USA
85:6460; Markowitz et al., (1988)
J. Virol
. 62:1120; and Markowitz et al., (1988)
Virology
167:400).
As the presence of both constructs is essential to the functioning of the packaging cell line and chromosomal instability occurs regularly, it is of great importance for the routine use of such cells in gene therapy procedures that, by means of a selection medium, selection for the presence of the constructs can be provided for. Therefore, these constructs are often introduced by means of a so-called cotransfection whereby both viral constructs are transfected together with a dominant selection marker. The possibility of selection thus provided is certainly not a trivial requirement, considering for instance the observation that we and various other research groups made, that virus-producing cells based on the packaging cell line &psgr;CRIP (Danos and Mulligan, (1988)
Proc. Natl. Acad. Sci. USA
85:6460) are not stable. That is to say that they are no longer resistant to the relevant selection media and during cultivation lose their capacity to produce retroviruses. One example, of importance for one of the embodiments of the present invention, is the so-called POC-1 cell line which was produced by us on the basis of &psgr;CRIP cells (Van Beusechem et al., (1990)
J. Exp. Med
. 172:729) which on account of its instability cannot be used for gene therapy on a routine basis. Therefore, in the invention described here, use is made of packaging cells which, by means of a dominant selection culture, do continue to produce stable virus.
Studies in mice have demonstrated that using amphotropic retroviral vectors, bone marrow stem cells can be provided with a new gene. After transplantation of these modified cells into lethally irradiated mice, the new gene could also be demonstrat
Valerio Domenico
Van Beusechem Victor Willem
Introgene B.V.
Nguyen Dave T.
Nguyen Quang
TraskBritt
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