459. Murine Study To Evaluate Long-Term Transgene Expression of the MSCV-MGMTP140K wc Retroviral Vector in Support of a Phase I Gene Transfer Trial-Limitations of the Murine Model as a Pre-Clinical Tool
Authors: Elke Will, Jeff Bailey, Todd Schuesler, Brenden Balcik, Ben Burzynski, David Witte, Gerlinde Layh-Schmitt, Christof vonKalle, Christopher Baum, Brian P. Sorrentino, Lars M. Wagner, Lilith Reeves, David A. Williams
Journal: Molecular Therapy (2006)

Retroviral gene therapy vectors expressing the MGMTP140K transgene have been shown to protect hematopoietic cells from toxicity associated with a combined cancer treatment using 6-benzylguanine (6-BG) and an alkylating agent such as Temozolomide (TEM). In a Phase I gene transfer trial, high grade astrocytoma patients having poor prognoses using standard therapies, will undergo escalating dose treatments with 6-BG and TEM following MGMTP140K gene transfer into autologous hematopoietic stem cells. Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted preclinical model defined to assess their safety, and, in particular their risk related to insertional mutagenesis. This study was designed as a murine pre-clinical study to assess the long term effects of the transduction of hematopoietic cells with the retroviral vector to be used in the clinical trial, MSCV-MGMTP140Kwc.LDBM cells from 5-FU treated C57BL/6 donors were transduced with ecotropic MSCV-MGMTP140Kwc vector on recombinant fibronectin CH296 and transplanted into 40 lethally irradiated (11.75 Gy) C57BL/6 recipient test mice (10 controls received mock-transduced cells). Titer measured on non-hematopoietic cells led to an unintended high MOI on the transplanted cells of 4. The animals were observed for up to 12 months. Gene marking was determined by quantitative PCR and by intracellular staining of the human MGMT transgene product.All mice were found to have significant gene marking in the peripheral blood with 0.1-2 vector copies per cell. The majority of the animals (80%) demonstrated more than 40% peripheral blood cells expressing human MGMT protein 8-12 months post transplantation, thus confirming persistent vector expression. Unexpectedly, 5 test mice have been diagnosed with malignant lymphoma. None of the control mice have been found to have developed malignancies, although the full pathologic evaluation is pending for 5 of the 8 remaining control animals. Laser capture microdissection (LCM) of tumor cells with subsequent quantitative PCR detected no vector in the tumor cells of any of the 5 animals with malignancies, whereas vector was consistently detected in non-malignant hematopoietic tissue.These results indicate that the malignancies were not caused by insertional mutagenesis or MGMTP140K expression. Low numbers of control animals may explain the failure to observe malignancies in this group; however, further studies are required to exclude MSCV-MGMTP140Kwc gene transfer as a causative factor for development of malignancies. A new murine study is initiated to distinguish host vs. donor cells, use a lower irradiation dose, include equal numbers of control animals, avoid 5-FU and utilize a transduction protocol with a MOI similar to the clinical protocol. Retroviral gene therapy vectors expressing the MGMTP140K transgene have been shown to protect hematopoietic cells from toxicity associated with a combined cancer treatment using 6-benzylguanine (6-BG) and an alkylating agent such as Temozolomide (TEM). In a Phase I gene transfer trial, high grade astrocytoma patients having poor prognoses using standard therapies, will undergo escalating dose treatments with 6-BG and TEM following MGMTP140K gene transfer into autologous hematopoietic stem cells. Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted preclinical model defined to assess their safety, and, in particular their risk related to insertional mutagenesis. This study was designed as a murine pre-clinical study to assess the long term effects of the transduction of hematopoietic cells with the retroviral vector to be used in the clinical trial, MSCV-MGMTP140Kwc. LDBM cells from 5-FU treated C57BL/6 donors were transduced with ecotropic MSCV-MGMTP140Kwc vector on recombinant fibronectin CH296 and transplanted into 40 lethally irradiated (11.75 Gy) C57BL/6 recipient test mice (10 controls received mock-transduced cells). Titer measured on non-hematopoietic cells led to an unintended high MOI on the transplanted cells of 4. The animals were observed for up to 12 months. Gene marking was determined by quantitative PCR and by intracellular staining of the human MGMT transgene product. All mice were found to have significant gene marking in the peripheral blood with 0.1-2 vector copies per cell. The majority of the animals (80%) demonstrated more than 40% peripheral blood cells expressing human MGMT protein 8-12 months post transplantation, thus confirming persistent vector expression. Unexpectedly, 5 test mice have been diagnosed with malignant lymphoma. None of the control mice have been found to have developed malignancies, although the full pathologic evaluation is pending for 5 of the 8 remaining control animals. Laser capture microdissection (LCM) of tumor cells with subsequent quantitative PCR detected no vector in the tumor cells of any of the 5 animals with malignancies, whereas vector was consistently detected in non-malignant hematopoietic tissue. These results indicate that the malignancies were not caused by insertional mutagenesis or MGMTP140K expression. Low numbers of control animals may explain the failure to observe malignancies in this group; however, further studies are required to exclude MSCV-MGMTP140Kwc gene transfer as a causative factor for development of malignancies. A new murine study is initiated to distinguish host vs. donor cells, use a lower irradiation dose, include equal numbers of control animals, avoid 5-FU and utilize a transduction protocol with a MOI similar to the clinical protocol.