Technologies
Suicide genes
Suicide gene therapy, also referred as gene-directed enzyme prodrug therapy (GDEPT), is a novel approach to render cancer cells more sensitive to chemotherapeutics by maximizing the therapeutic index and minimizing the unfavorable side effects of these agents. Suicide gene therapy is a two step treatment. In the first step, a "suicide" gene encoding an enzyme able to activate a non- or mildy toxic prodrug to a cytotoxic metabolite is delivered to the tumor. In the second step, a prodrug is administrated systemically and converted by the "suicide" enzyme to a toxic drug leading to cell death. Because current gene delivery system allow transfection of only a very small percentage of tumor cells, the efficacy of suicide gene therapy depends critically on the "bystander effects", the ability of the transfected cells to transfer death signals to neighboring tumor cells.
InvivoGen Therapeutics has developed two chimeric bifunctional suicide genes to increase the sensitivity of tumor cells to the chemotherapeutic agents, 5-fluorocytosine (5-FC) and gemcitabine (dFdC).
Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC), an antifungal agent, into 5-fluorouracil (5-FU), a widely applied anticancer agent. CD is found in bacteria, yeast and fungi but is absent in mammalian cells. The cytotoxic effect of 5-FU is largely exerted following its conversion to 5-FUMP and 5-FdUMP. 5-FdUMP binds irreversibly to thymidylate synthase hence blocking DNA synthesis by deprivation of dTTP. As 5-FU is able to diffuse passively through cell membranes, it induces a bystander killing effect. The potential of the CD gene / 5-FC prodrug system to induce tumor regression has been demonstrated in various animal models. However, the efficacy of the system is limited, many tumor cells remain relatively resistant to 5-FU as a consequence of possible defects in downstream cellular metabolism of 5-FU.
In mammalian cells, the conversion of 5-FU to 5-FUMP which involves two enzymes is a rate-limiting step. In contrast, in bacteria and yeast, this conversion is catalyzed by a single enzyme, uracil phosphoribosyltransferase (UPRT). Our strategy to optimize the CD / 5-FC system is to associate the UPRT activity to the CD activity. Both enzymes should work cooperatively to enhance the sensitivity of tumor cells to 5-FC and 5-FU. The fcy::fur gene was constructed by fusing in frame the yeast genes, fcy and fur, encoding CD and UPRT respectively. Since the fcy and fur genes are of non-human origin and thus prone to silencing by methylation when applied in vivo, we generated a CpG-free allele of the fusion gene.
The increased potency of the fcy::fur gene was demonstrated in vitro and in vivo. Tumor cells transduced with the bifunctional chimeric gene were a 100-fold more sensitive to 5-FC than cells transduced with the CD gene alone. Furthermore, the fcy::fur / 5-FC suicide system generates a stronger bystander effect.
The fcy::fur gene is available commercially from InvivoGen . The use of the fcy::fur gene for cancer gene therapy covered by US Patent: 5, 856, 153.
The cytidine analogue gemcitabine (dFdC) is a promising new chemotherapeutic agent for the treatment of colorectal, breast, pancreatic, renal, and lung cancer. Gemcitabine is phosphorylated to its mononucleotide (dFdCMP) by deoxycytidine kinase (DCK) and subsequently by nucleotide kinases to its active metabolites, dFdCDP and dFdCTP. dFdCTP is incorporated into DNA and thereby blocks DNA replication. dFdCDP inhibits ribonucleotide reductase leading to depletion of the DNA precursor pool.The first two phosphorylation steps, which are catalyzed by DCK and uridylate monophosphate kinase (UMK), are rate limiting in tumors. The substrates of these two enzymes, dFdC and dFdCMP respectively, are labile due to increased deaminase activities. Furthermore, many solid tumors are resistant to gemcitabine due to a deficiency in DCK activity caused by mutational inactivation of DCK.
To overcome these limitations, we have engineered a bifunctional chimeric gene encoding both DCK and UMK activies. The dck::umk gene was generated by fusing in frame the human dck and umk genes. The ability of the dck::umk gene to increase the sensitivity of tumor cells to gemcitabine was demonstrated in vitro and in vivo in a model of pancreatic cancer. Our very encouraging preclinical data have prompted us to initiate a phase I/II clinical trial in patients with pancreatic adenocarcinoma.
InvivoGen Therapeutics has developed two chimeric bifunctional suicide genes to increase the sensitivity of tumor cells to the chemotherapeutic agents, 5-fluorocytosine (5-FC) and gemcitabine (dFdC).
Fcy::fur combines the activity of the yeast cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) and sensitize the cells to 5-FC
Dck::umk is a fusion of the human deoxycytidine kinase (DCK) and uridylate monophosphate kinase (UMK) genes that metabolizes gemcitabine into toxic metabolites.
Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC), an antifungal agent, into 5-fluorouracil (5-FU), a widely applied anticancer agent. CD is found in bacteria, yeast and fungi but is absent in mammalian cells. The cytotoxic effect of 5-FU is largely exerted following its conversion to 5-FUMP and 5-FdUMP. 5-FdUMP binds irreversibly to thymidylate synthase hence blocking DNA synthesis by deprivation of dTTP. As 5-FU is able to diffuse passively through cell membranes, it induces a bystander killing effect. The potential of the CD gene / 5-FC prodrug system to induce tumor regression has been demonstrated in various animal models. However, the efficacy of the system is limited, many tumor cells remain relatively resistant to 5-FU as a consequence of possible defects in downstream cellular metabolism of 5-FU.
In mammalian cells, the conversion of 5-FU to 5-FUMP which involves two enzymes is a rate-limiting step. In contrast, in bacteria and yeast, this conversion is catalyzed by a single enzyme, uracil phosphoribosyltransferase (UPRT). Our strategy to optimize the CD / 5-FC system is to associate the UPRT activity to the CD activity. Both enzymes should work cooperatively to enhance the sensitivity of tumor cells to 5-FC and 5-FU. The fcy::fur gene was constructed by fusing in frame the yeast genes, fcy and fur, encoding CD and UPRT respectively. Since the fcy and fur genes are of non-human origin and thus prone to silencing by methylation when applied in vivo, we generated a CpG-free allele of the fusion gene.
The increased potency of the fcy::fur gene was demonstrated in vitro and in vivo. Tumor cells transduced with the bifunctional chimeric gene were a 100-fold more sensitive to 5-FC than cells transduced with the CD gene alone. Furthermore, the fcy::fur / 5-FC suicide system generates a stronger bystander effect.
The fcy::fur gene is available commercially from InvivoGen . The use of the fcy::fur gene for cancer gene therapy covered by US Patent: 5, 856, 153.
The cytidine analogue gemcitabine (dFdC) is a promising new chemotherapeutic agent for the treatment of colorectal, breast, pancreatic, renal, and lung cancer. Gemcitabine is phosphorylated to its mononucleotide (dFdCMP) by deoxycytidine kinase (DCK) and subsequently by nucleotide kinases to its active metabolites, dFdCDP and dFdCTP. dFdCTP is incorporated into DNA and thereby blocks DNA replication. dFdCDP inhibits ribonucleotide reductase leading to depletion of the DNA precursor pool.The first two phosphorylation steps, which are catalyzed by DCK and uridylate monophosphate kinase (UMK), are rate limiting in tumors. The substrates of these two enzymes, dFdC and dFdCMP respectively, are labile due to increased deaminase activities. Furthermore, many solid tumors are resistant to gemcitabine due to a deficiency in DCK activity caused by mutational inactivation of DCK.
To overcome these limitations, we have engineered a bifunctional chimeric gene encoding both DCK and UMK activies. The dck::umk gene was generated by fusing in frame the human dck and umk genes. The ability of the dck::umk gene to increase the sensitivity of tumor cells to gemcitabine was demonstrated in vitro and in vivo in a model of pancreatic cancer. Our very encouraging preclinical data have prompted us to initiate a phase I/II clinical trial in patients with pancreatic adenocarcinoma.