Associate Professor, McGill University
Associate Physician, McGill University Health Centre
denis [dot] cournoyer [at] mcgill [dot] ca
Gene therapy of cancer by hematopoietic chemoprotection
One attractive strategy for cancer gene therapy is to transfer drug resistance genes into normal hematopoietic cells, thus protecting the patient from the main toxicity of most anti-cancer drugs.
Isoenzymes of the glutathione S-transferase (GST) family have been implicated in several models of resistance to an important class of drugs: the alkylating agents. Using retrovirus-mediated gene transfer of the rat Yc isoform of GST (GST-Yc), we were able to demonstrate an increase in alkylating drug resistance in unselected populations of cells that have been infected with the virus, including primary hematopoietic cells tested in vitro. Ongoing experiments are addressing the feasibility of in vivo chemoprotection with GST-Yc.
Another gene of interest for chemoprotection is the gene encoding the enzyme cytidine deaminase (CD) which metabolizes and inactivates nucleoside analogues such as cytosine arabinoside (ARA-C). In collaboration with Dr R.L. Momparler, who cloned the complementary DNA for the human CD gene, we have shown that murine fibroblasts transfected with a CD retrovirus vector become highly resistant to ARA-C (0% vs 73-90% of baseline colony formation at 10-6 M) (Momparler et al., Cancer Res. 3:331-338, 1996). The magnitude of the difference in survival between CD-transfected and control cells suggests that, in addition to chemoprotection, CD could be used as an in vivo selectable marker in vector constructions.
We have recently developed bicistronic retroviral vectors combining the expression of GST with CD. This combination broadens the spectrum of chemoprotection and should permit the in vivo selection of genetically modified hematopoietic cells, thus increasing the overall level of chemoprotection. This will shortly be tested in animals, and might subsequently lead to clinical trials of hematopoietic chemoprotection.
Gene therapy of inherited hematopoietic disorders by homologous recombination
Current efforts in the field of gene therapy are focused on the use of gene supplementation (i.e., randomly inserting a vector that supplements a defective function). However, the direct correction of the underlying mutation would be more desirable to correct dominant defects, to maintain complex gene regulation that is not achieved with the current gene transfer vectors, and to decrease the small but existing risks related to insertion mutagenesis. The two factors that limit the applicability of gene targeting to gene therapy are, first, the low frequency of homologous recombination events in human cells and, second, our inability to expand rare "targeted" primary human cells such as hematopoietic stem cells. We are conducting studies to improve the frequency of homologous recombination events between an incoming vector and a chromosomal locus in mammalian cells. The strategies under study are: (i) transient expression of a human (Nuc) or yeast (NUD1) recombination/repair endo-exonuclease (both cloned by T. K.-Y. Chow); (ii) inhibition of poly (ADP-ribosylation).
Gene therapy of HIV disease
Gene therapy has been proposed as a powerful approach to counteract HIV infection. The rationale for HIV gene therapy is to render the target cells for HIV infection resistant to the virus. Ideally, the genetic information interfering with the virus life cycle would be introduced into hematopoietic stem cells, resulting in the production of virus-resistant progeny cells (including the CD4+ lymphocytes and macrophages). Collaborators at Baylor College of Medicine, Dr Belmont and co-workers, have developed a unique retrovirus vector that expresses an artificial mutant virus protein (Trev) capable of inhibiting both Tat- and Rev-mediated HIV functions. This construct decreases HIV virus growth 10,000-fold in primary CD4+ T cells and makes such cells resistant to being directly killed by the virus. The Trev vector is being evaluated at Baylor College of Medicine in infants who develop maternally transmitted HIV infection in an FDA-approved Phase I clinical trial. However, the biological effects of the Trev vector in infants following transplantation of transduced cord blood cells might not predict its effects in adults receiving transduced peripheral blood stem cells (PBSC). We have recently been able to demonstrate that the Trev vector could be introduced efficiently into CD34+ human hematopoietic progenitor cells mobilized into the blood following G-CSF stimulation. We will next examine the ability of human monocytes produced from Trev-transduced progenitor cells in long-term cultures to resist viral challenge with laboratory and community isolates and monocytotropic HIV virus. We would next propose to conduct a small hypothesis-testing clinical trial to test the safety and in vivo biological activity of the Trev vector in adults with severe HIV disease.
Fonds de la Recherche en Santé du Québec
Medicine; Oncology; Human Genetics
Accepting students from
Experimental Medicine; Human Genetics