Title:  Implantation of HSV-TK retrovirus producer cells to destroy glioma

United States Patent:  6,537,541

Issued:  March 25, 2003

Inventors:  Breakefield; Xandra O. (Newton, MA); Martuza; Robert L. (Chevy Chase, MD); Short; Marion Priscilla (Cambridge, MA)

Assignee:  The General Hospital Corporation (Boston, MA)

Appl. No.:  462500

Filed:  June 5, 1995

The present invention discloses compositions and methods for selectively killing neoplastic cells. Retroviral vectors are used to selectively express a gene in neoplastic cells. The gene or gene product targets the cells for selective killing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is drawn to the selective killing of neoplastic cells. Retrovirus vectors carrying a gene whose gene product is capable of targeting the neoplastic cells for selective cell death are utilized.

By neoplastic cells is intended dividing cells, usually rapidly dividing cells. For purposes of the invention, neoplastic cells include cells of tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas, and the like. Of particular interest are central nervous system tumors. These include astrocytomas, oligodendrogliomas, meningiomas, neurofibromas, ependymomas, Schwannomas, neurofibrosarcomas, glioblastomas, etc. The neoplastic cells of particular concern to the invention are those cells of brain tumors. Adult brain tumors are unique in that they constitute masses of dividing cells within a background of essentially non-dividing cells. Therefore, the present invention utilizes these metabolic differences to exploit the development of a targeted approach to selective killing of neoplastic cells. The invention can be utilized to selectively kill both benign and malignant neoplastic cells.

The retroviral vectors of the invention can integrate only into the genome of dividing cells. Thus, the vectors provide a useful vehicle for selective targeting of dividing cells. Retroviral vectors offer further advantages as there are no limitations in host range and these vectors have already been used successfully to infect many different cell types. For example, see Cepko, C., "Lineage analysis and immortalization of neural cells via retrovirus vectors," in Neuromethods, Vol. 16, pp. 177-218, Clifton, N.J., The Humana Press, Inc. (1989); Gilboa, E., BioEssays 5(6):252-257 (1987); Friedmann, T., Science 244:1275-1281 (1989).

In general, retroviral vectors are well known in the art. See, Breakefield et al., Molec. Neuro. Biol. 1:339 (1987); and, Shih et al., In: Vaccines 85, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1985) pages 177-180. Further, co-pending U.S. patent applications Ser. Nos. 07/304,619 and 07/508,731 are drawn to herpes simplex virus expression vectors. The disclosures of these applications are herein incorporated by reference. These applications provide further information on the construction and use of retrovirus vectors.

As indicated above, generally, the retrovirus vectors of the present invention are replication-defective and can be packaged into infectious retroviral particles by transfected cell lines which contain retroviral sequences coding for the proteins necessary for the packaging of retroviral RNA, but which cannot package their own RNA. See, Mann et al., Cell 33:153-159 (1983); Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988). Since retrovirus and vectors derived from them integrate into the host cell genome, their sequences are transmitted to all daughter cells. This feature of retroviruses has been successfully used for example, to trace cell lineages in the nervous system (Price et al., Proc. Natl. Acad. Sci. USA 84:156-160 (1987); Luskin et al., Neuron 1:635-647 (1988); Walsh and Cepko, Science 241:1342-1345 (1988)).

Genes for transfer into the neoplastic cells by the retroviral vectors are selected from those which target the host cell usually by the expression of a gene product in the host neoplastic cells. "Gene product" broadly refers to proteins encoded by the particular gene. However, for purposes of the invention, gene product also includes transcription products of the gene, particularly for use as anti-sense RNA. The host cells targeted by the present vectors are those cells into which the virus infects and expresses the desired gene product. The host cells thus constitute neoplastic cells infected by the retroviral vectors.

Genes are selected whose gene products serve to identify host cells, slow down or temporarily stimulate host cell growth in order to render the host cell more sensitive to chemotherapeutic agents and/or whose products target the host cell for cell death. Cell death can be accomplished by contacting the host cells, comprising the gene product, with a subsequent treatment, either physical or chemical treatment. Alternatively, the gene products themselves may serve to kill the host cells or slow down cell growth. Gene products which temporarily stimulate cell growth include for example, growth factors, including for example basic fibroblast growth factor (bFGF).

In this respect, one example of a useful gene product comprises imaging compounds which may be utilized for tumor location. The retrovirus is thus utilized as a means to diagnose the location and extent of the neoplastic growth. See, for example, Glatstein et al., Int. J. Radiat. Oncol. Biol. Phys. 11:299-314 (1985).

Genes are also selected whose products themselves are capable of selective cell killing. For example, the gene product may comprise anti-sense nucleic acid for essential cell proteins, such as replication proteins, which serve to render the host cells incapable of further cell growth and division. Anti-sense regulation has been described by Rosenberg et al., Nature 313:703-706(1985); Preiss et al., Nature 313:27-32 (1985); Melton, Proc. Natl. Acad. Sci. USA 82:144-148 (1985); Izart and Weintraub, Science 229:345-352 (1985); Kim and Wald, Cell 42:129-138 (1985); Pestka et al., Proc. Natl. Acad. Sci. USA 81:7525-7528 (1984); Coleman et al., Cell 37:683-691 (1984); and McGarry and Lindquist, Proc. Natl. Acad. Sci. USA 83:399-403 (1986).

Other genes which find use for slowing cell growth include tumor suppressor genes, genes which encode transcription factors which suppress cell growth, toxic proteins that are released by cells, and the like. For example, see Heinbrook et al., Proc. Natl. Acad. Sci. USA 87:4697 (1990), which describes a fusion protein with toxin coupled to the EGF ligand. Toxin genes have also been described, for example, Barker et al., Gene 86:285-290 (1990); Ito et al., Microb. Pathog. 8:47-60 (1990); Gannon et al., J. Gen. Microbiol. 136:1125-1136 (1990). Genes can also be inserted which alter cell growth characteristics or modulate cell growth, for example, a tumor suppressor gene such as the. Rb gene in retinoblastoma (Huang et al., Science 242:1563-1566 (1988)) or the p53 gene in colon cancer (Baker et al., Science 249:912-915 (1980)). Other suppressor or modulating genes may also be utilized.

Genes whose products serve to render the host cells more antigenic also find use in the invention. This antigenic effect may be accomplished by introducing new antigens on the surface of the host cells, thus augmenting the immune system in recognizing the tumor as a foreign body. The introduction of new antigens to the surface of the host cells is referred to as xenogenization of the cells (Austin et al., Ad. in Cancer Res. 30:301-345 (1979); Kobayashi et al., Ad. in Cancer Res. 30:279-299 (1979)). Any nonhuman surface antigen can be utilized including those described in Araki et al., Gene 89:195-202 (1990); Takle et al., Mol. Biochem. Parasitol. 37:57-64 (1989); Raney et al., J. Virol. 63:3919-3925 (1989); Tondravi, M. M., Curr. Genet. 14:617-626 (1988); and Miyanohara et al., Proc. Natl. Acad. Sci. USA 80:1-5 (1983).

The expression of nonhuman or unique surface antigens in neoplastic cells can also be utilized to locate such neoplastic cells by subsequent binding with labelled antibodies. See, for example, Le Doussal et al., Cancer Res. 50:3445-3452 (1990); Palabrica et al., Proc. Natl. Acad. Sci. USA 86:1036-1040 (1989); Berends et al., Cancer Immunol. Immunother. 26:243-249 (1988); and Welt et al., Proc. Natl. Acad. Sci. USA 84:4200-4204 (1987).

Alternatively, the gene or coding sequence may be selected whose products offer a conditional killing mechanism for dividing cells. In this manner, the expression of a particular protein followed by the subsequent treatment is effective in killing the neoplastic cells. The subsequent treatment comprises chemical and physical treatments. Agents for chemical treatments comprise the use of enzymes or other compounds which react with the gene product to kill the host cell. Physical treatments comprise subjection of the cells to radiation, UV light, and the like.

For example, the herpes simplex virus type I (HSV-1) thymidine kinase (TK) gene offers such a conditional killing mechanism for dividing cells. The selective advantage of using HSV-1-TK derived from the fact that the enzyme has a higher affinity for certain nucleoside analogues, such as acyclovir, ganciclovir and FIAU, than mammalian TK (McLaren et al., In: Herpes Virus and Virus Chemotherapy, R. Kono, ed., pp.57-61, Amsterdam, Elsevier (1985)). These drugs are converted to nucleotide-like precursors and incorporated into the DNA of replicating cells, thus disrupting the integrity of the genome, and ultimately leading to cell death. Several studies have successfully made use of the conditional toxicity of TK in development studies of transgenic mice (Borrelli et al., Nature 339:538-541 (1989); Heyman et al., Proc. Natl. Acad. Sci. USA 86:2698-2702 (1989)), as a selectable marker against non-homologous recombination events in cultured cells (Capecchi, M. R., Trends in Genetics 5(3):70-76 (1989)), for killing cells harboring wild type herpes viruses (Corey and Spear, N. Engl. J. Med. 314:686-691 (1986); Corey and Spear, N. Engl. J. Med. 314:749-756 (1986)), and in selecting for herpes virus mutants lacking TK activity (Coen et al., Science 234:53-59 (1986)).

The gene product may also encode a chemical or protein which renders the host cells radiosensitive and thus more susceptible to killing by radiation. Thus, upon subsequent subjection to radiation, the host cells are selectively killed. For example, the combination of the HSV-TK gene and ganciclovir, can be used. Cells bearing the HSV-TX gene show increased sensitivity to radiation in the presence of ganciclovir, as its metabolites interfere with DNA repair as well as DNA synthesis. See Snyderman et al., Arch. Otolaryngol. Head Neck Surg. 112:1147-1150 (1986); and Sealy et al., Cancer 54:1535-1540 (1984). Other strategies include selective transfer of cell surface antigenic markers, in conjunction with the development of tumor-specific immunoconjugates to improve targeting of chemotherapeutic agents. See, Reisfeld, R. A., in Molecular Probes Technology and Medical Applications, Albertini et al., Raven Press, New York (1989).

It is recognized that the gene of interest may be modified by any methods known in the art. For example, the gene may be placed under the control of heterologous regulatory regions, including the use of viral promoters, neoplastic cell or tumor specific promoters or control elements. In this manner, the gene product is further targeted to specific cell types. Methods for construction of such expression vectors are known in the art.

Generally, methods are known in the art for retroviral infection of the cells of interest. Typically, the virus is injected into the host at or near the site of neoplastic growth. For the most part, the virus is provided in a therapeutically effective amount to infect and kill target cells. Generally, the virus is provided for injection in a concentration in the range of about 10¹ to about 10¹⁰ plaque forming units (PFU), generally about 5.times.10⁴ to about 1.times.10⁶ PFU, more generally about 1.times.10⁵ to about 4.times.10⁵, although ranges may vary. Alternatively, the packaging cell line may be grafted near or into the tumor to provide a longer-lasting source of virus.

This selective killing of the retrovirus and delivery of the toxic gene can be enhanced by co-infection with a helper virus. That is, the helper virus augments gene delivery. In this manner, the packaging cell lines for making virus particles of the retrovirus vectors can be coinfected with a helper virus. Packaging cells or viral inoculum is then injected into the host at or near the site of infection. (See, Cepko, C. (1989), supra; Rosenberg et al., Science 242:1575-1578 (1988); and Mann et al., Cell 33:153-159 (1983)). Such helper viruses include ecotropic wild-type retroviruses, for example MoMLV (See, Danos et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988); Cepko, C., In Neuromethods, Vol. 16, Molecular Neurobiological Techniques, Boulton et al. (eds.), Clifton, N.J.: Humana (1989); and Mann et al., Cell 33:153-159 (1983)).

To utilize a helper virus, the packaging line or retroviral vectorinfected line can be subsequently infected with wild-type virus in culture and these cells can be grafted. (See, Rosenberg et al., Science 242:1575-1578 (1988) and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989)). The packaging cells are infected with the helper in the range of MOI of about 0.1 to about 20.

The sensitivity of the tumor cells to toxic agents is increased utilizing helper viruses. The helper viruses turn cells infected with retrovirus vectors into packaging cell lines. The results show that by co-infection with a helper virus, the retrovirus vectors of the invention are able to target more tumor cells, even those tumor cells away from the tumor mass. Furthermore, the tumor cells die faster and show more sensitivity to toxic agents when a helper virus is utilized.

The invention finds particular use in the treatment of glioblastomas.

Glioblastomas are the most common form of malignant brain tumor in man, and are almost always universally fatal. The glioblastoma represents approximately 30% or 50% of all primary brain tumors and, despite surgery, chemotherapy, and radiotherapy, are almost universally fatal. The mean survival is less than a year, and the five-year survival rate is only 3% or 5%. After treatment, reoccurrence of the disease often appears within two centimeters of the original site. Metastases are extremely rare; neurological disfunction and death are due to local growth and cerebral invasion. Therefore, the possible efficacy of local (non-systemic) treatments has been explored. A few of these include studies of local hypothermia, photodynamic is therapy, and interstitial radiation. Until the present invention, no therapeutic modality has made a substantial impact on the outcome of patients with malignant gliomas.

Claim 1 of 13 Claims

What is claimed is:

1. A method of transferring in vivo the HSV-tk gene into dividing glioma tumor cells in order to kill the tumor cells, the method comprising:

(a) introducing a retrovirus having (I) the HSV-tk gene, and (ii) at least one gene required for replication of the retrovirus into producer cells such that integration of the proviral DNA corresponding to the retrovirus into the genome of the producer cells results in the generation of a modified retrovirus wherein at least one of the genes required for replication of the retrovirus is replaced by the HSV-tk gene;

(b) selecting producer cells in which the modified retrovirus is incorporated as part of the genome of the producer cells;

(c) grafting the producer cells in the brain proximate to the dividing tumor cells, wherein the tumor cells are injected with the modified retrovirus being produced by the producer cells, thereby transferring the HSV-tk gene to the tumor cells; and

(d) killing the tumor cells by administering a substance that is metabolized by the expression product of the HSV-tk gene transferred to the tumor cells into a metabolite that kills the tumor cells.
 

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