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Fifty patients in the United States and the United Kingdom underwent HSCT with CD34+ cells transduced with a replication-defective lentivirus bearing ADA . At 24 months, all the patients were alive, and event-free survival was 96.7% among the U.S. patients and 95% among the U.K. patients. Only two patients did not have persistent engraftment. No viable virus or abnormal proliferations were noted.

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34+ stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34+ cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34+ cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

Access to cutting-edge technologies is essential for investigators to advance translational research. The Indiana Clinical and Translational Sciences Institute (CTSI) spans three major and preeminent universities, four large academic campuses across the state of Indiana, and is mandate to provide best practices to a whole state. To address the need to facilitate the availability of innovative technologies to its investigators, the Indiana CTSI implemented the Access Technology Program (ATP). The activities of the ATP, or any program of the Indiana CTSI, are challenged to connect technologies and investigators on the multiple Indiana CTSI campuses by the geographical distances between campuses (1-4 hr driving time). Herein, we describe the initiatives developed by the ATP to increase the availability of state-of-the-art technologies to its investigators on all Indiana CTSI campuses, and the methods developed by the ATP to bridge the distance between campuses, technologies, and investigators for the advancement of clinical translational research. The methods and practices described in this publication may inform other approaches to enhance translational research, dissemination, and usage of innovative technologies by translational investigators, especially when distance or multi-campus cultural differences are factors to efficient application.

In two earlier trials, patients with X-linked Severe Combined Immune Deficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). We have previously demonstrated that GMP-grade SIN gRV can be produced at high titers using transient transfection in bioreactors (van der Loo et al, Gene Ther 2011). Herein we report on the large-scale production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. The vector was certified and released for treatment of SCID-X1 in a multi-center international phase I/II trial which is currently recruiting patients in Paris, London, Los Angeles, Boston and Cincinnati.

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMTP140Kwc. Test mice had significant gene marking 8–12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMTP140K, thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMTP140K expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.

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 MGMT P140K 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-MGMT P140K wc.LDBM cells from 5-FU treated C57BL/6 donors were transduced with ecotropic MSCV-MGMTP140K wc 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 MGMT P140K expression. Low numbers of control animals may explain the failure to observe malignancies in this group; however, further studies are required to exclude MSCV-MGMTP140K wc 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.