igh-dose chemotherapy, followed by autologous transplant, appears to have some beneficial effects, especially when performed early in responding cancer patients. Post-transplant chemotherapy, added into the initial high-dose chemotherapy and autologous transplant, might improve the outcome. Early post-transplant treatment could possibly eliminate residual tumor burden and improve the cure rate. However, in this situation, chemotherapy regimens are often associated with severe toxicities. Transfer of drug resistance genes into bone marrow cells in order to protect patients from the bone marrow toxicity of chemotherapy is an attractive strategy that may allow safer administration of post-transplant chemotherapy. For our initial clinical study, we will enlist patients with diffuse large cell lymphoma and MCL who have a high risk of disease recurrence after high-dose chemotherapy treatment. After autologous transplant, patients will be administered methotrexate (MTX) and cytarabine (ARA-C), which are widely used in different combination regimens for patients with lymphoma. Those two drugs are known to be more effective when given at higher doses. However, their bone marrow toxicity also increases with their dose. A drug resistance gene that we have shown to protect bone marrow cells from MTX/ARA-C will be transferred into the patients’ peripheral blood stem cells, and those cells will be infused into the patients after high-dose chemotherapy administration.
We are developing a clinically applicable protocol for ex vivo transduction of hematopoietic progenitor/stem cells, as well as a retroviral vector containing drug resistance gene(s), which will protect patients against post-transplant chemotherapy related myelosuppression, and subsequently improve chemotherapy tolerance and provide better disease control. For the first pilot study we have elected to use a retroviral construct containing a mutant dihydrofolate reductase (DHFR) and cytidine deaminase (CD) fusion gene to protect patients from myelotoxicity, methotrexate (MTX) and cytarabine (Ara-C). If successful, these studies will provide the rationale to use retroviral gene transfer of other drug resistance genes to protect patients from a wide variety of drugs.
A high-dosage of myeloablative therapy with autologous hematopoietic stem cell transplant/rescue (auto-HSCT) will provide response in many cancer types, i.e., non-Hodgkin’s lymphomas (NHL). However, because of contamination of the stem cell product or persistence of residual tumor cells, disease relapse occurs in 40 to 70% of autografted patients. Early post-transplant treatment could possibly eliminate residual tumor burden and improve the cure rate. Nevertheless, in this situation, chemotherapy regimens are often associated with severe toxicities. MTX and Ara-C are cell cycle specific agents with a synergistic nature, and although MTX and Ara-C are not the first line treatment, they have been widely used in a combination for treatment of patients with NHL. Myelosuppression, a myeloprotection based gene therapy strategy, is the main dose-limiting toxicity for both MTX and Ara-C, and permits administration of higher doses, as well as more safe and effective treatment in the utilization of these drugs.
We have cloned several drug resistance genes and utilized these mutants for transduction into hematopoietic stem cells to generate resistance. The goal of transducing HSCs with a drug resistance gene is to increase the maximum tolerated chemotherapy dosage by protecting the bone marrow progenitors from the myelotoxicity of the relevant drug, and thus eliminating the residual tumor. We have generated a fusion gene construct with double mutant DHFR (Phe22-Ser31, F/S DHFR) and CD that conferred high levels of resistance to both MTX and Ara-C. This vector is considered to be of value in protecting hematopoietic progenitors from the toxicity of these anti-metabolites.
Different DHFR mutants have been widely used for conferring resistance to anti-folates. The DHFR mutants are used as selectable markers in vitro and in vivo in the context of gene therapy. We have shown both in a mouse mammary tumor (E0771) model and a human diffuse large cell lymphoma model (SKI-DLCL-1) that animals transplanted with mutant DHFR or FS/-DHFR-CD transferred bone marrow had better tolerance of post-transplant MTX, or MTX/Ara-C treatment tolerance, which led to higher tumor elimination rates in animals.
Retroviral vectors are designed to integrate the host genome and ectopic insertion of DNA. It is potentially a mutagenic event; as it can alter gene transcription, and regulatory and/or coding elements. The risk of insertional oncogenesis has been a concern and there have been a few reports documenting insertional oncogenesis. Prospective evaluation of retroviral insertion sites with linear amplification PCR (LAM-PCR) are considered an essential part of monitoring after gene therapy. We monitor the quantity of transfused CD34+ cells, the quantity of vector copies per cell, as well as transduction efficiency and have found increased vector copy numbers and transgene expression levels occur with higher transduction rates. Thus, our goal is to achieve an efficiency of gene transfer of approximately 30%, while balancing the need for significant transgene expression with safety concerns.
In vivo selective expansion of transgene carrying hematopoietic stem cells has been an important goal. However, forced clonal expansion of the transduced cells may also contribute to the selection of mutated malignant cells. In our NOD/SCID model post-transplant MTX/Ara-C, we had a transient increase in transgene expression. Nevertheless, the polyclonal nature of hematopoiesis was not altered either in primary or in secondary recipients. In this upcoming gene therapy clinical trial, adult patients will be prospectively followed by LAM-PCR and, in any case of persistent clonal dominance, the specific bands will be monitored with quantitative PCR as well as close follow-up of hematological parameters.
This gene therapy based approach requires cellular products to be generated under a highly regulated environment, in a facility that applies current Good Manufacturing Practice (GMP) guidelines. We are currently validating the procedures in the newly established Production Facility for Gene and Cellular Therapy (GMP facility) located in The Cancer Institute of New Jersey. We have generated a clinical grade producer cell line and optimized vector production and human CD34+ cell transduction conditions.
On the research and development side, we are currently working on developing new drug resistance gene constructs and safer vector backbones.
Tulin Budak-Alpdogan graduated from Hacettepe University Medical School in Turkey, and received further training there as an internist and hematologist. She earned her master’s degree in Basic and Tumor Immunology from the Oncology Institute at Hacettepe University. In 1999, Dr. Budak-Alpdogan first joined Dr. Joseph Bertino’s lab and then Dr. Sadelain’s laboratory at Memorial Sloan Kettering Cancer Center. In 2004, she joined UMDNJ-Robert Wood Johnson Medical School as a faculty member in the Department of Medicine. During the past two years, Dr. Tulin Budak-Alpdogan’s research has been supported by the Lymphoma Research Foundation and New Jersey Commission on Science and Technology.