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Fanconi anemia (FA) is a rare recessive syndrome, characterized by congenital anomalies, bone marrow failure, and predisposition to cancer. Two earlier clinical trials utilizing gamma-retroviral vectors for the transduction of autologous FA hematopoietic stem cells (HSCs) required extensive in vitro manipulation and failed to achieve detectable long-term engraftment of transduced HSCs. As a strategy for minimizing ex vivo manipulation, we investigated the use of a "rapid" lentiviral transduction protocol in a murine Fanca(-/-) model. Importantly, while this and most murine models of FA fail to completely mimic the human hematopoietic phenotype, we observed a high incidence of HSC transplant engraftment failure and low donor chimerism after conventional transduction (CT) of Fanca(-/-) donor cells. In contrast, rapid transduction (RT) of Fanca(-/-) HSCs preserved engraftment to the level achieved in wild-type cells, resulting in long-term multilineage engraftment of gene-modified cells. We also demonstrate the correction of the characteristic hypersensitivity of FA cells against the cross-linking agent mitomycin C (MMC), and provide evidence for the advantage of using pharmacoselection as a means of further increasing gene-modified cells after RT. Collectively, these data support the use of rapid lentiviral transduction for gene therapy in FA.

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-MGMT(P140K)wc. 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 MGMT(P140K), 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 MGMT(P140K) 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.

Fanconi anemia (FA) is a genetic syndrome characterized by the uniform development of aplastic anemia. Current therapy for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors are not optimal. We have opened two phase I clinical trials to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivo over un-corrected cells (gene transfer study). To date, 7 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.9|[times]|106 CD34+ cells/kg (range 0.3|[ndash]|2.9|[times]|106 CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells is possible but challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1|[times]|105 viable CD34+ cells/kg for ex vivo culture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected. Despite collection before significant pancytopenia, an average of only 5|[times]|105 CD34+ cells/kg (range 1.5|[ndash]|10|[times]|105 CD34+ cells/kg) was purified from these 3 cases representing |[sim]|10% of the expected yield from normal individuals. These cells underwent ex vivo gene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final product determined by real-time PCR analysis of progenitors averaged 48% (range 40|[ndash]|62%). Equivalent correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivo manipulation was 82|[ndash]|110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5|[ndash]|3.5|[times]|105 nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. FAA vector sequences were detected in the PB of one patient early post-infusion (+4 weeks) but none was detected after 8 weeks. Despite no long term detection of the corrective gene in the PB and BM, both patients exhibited a transient but marked improvement (10|[ndash]|20% increase) in their peripheral blood hemoglobin (> 6 months) and platelets counts (2|[ndash]|3 months). The data demonstrates that gene transfer efficiency in the clinical setting has been significantly improved, however the collection of adequate numbers of HSC may be the critical barrier to the success of genetic correction attempts in FA.

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.