Abstract
We report clinical and molecular data for the first subject enrolled in our gene therapy clinical trial. This patient had follicular transformed large cell lymphoma that relapsed with a large mediastinal mass >2 years after CHOP, and despite 3 cycles of ICE, had persistent residual disease. After mobilization with rituximab, cyclophosphamide (Cy), and G-CSF, 3X106 CD34+ cells/kg were cryopreserved directly. CD34+ cells from a 2nd apheresis product were transduced with retroviral vector, SF1m (SFFV LTRs, MESV leader and MDR-1 cDNA), in fibronectin CH-296 peptide (Retronectin) coated bags, serum-free, in cytokines. The final product was 95% CD34+, 96% viable, and represented 5.5X106 CD34+ cells/kg. Post transduction, 11% of the cells effluxed rhodamine (a function of the MDR-1 encoded p-glycoprotein), corresponding to 20–30% transduction efficiency due to production of alternatively spliced mRNAs, as compared to 1% of the non-transduced cells. The patient received low dose (100 cGy) total body irradiation (TBI) followed by infusion of the transduced (Tr) cells, then 4 cycles of vincristine, dose escalating VP-16, and prednisone. The platelet count nadir improved with each cycle (18, 65, 98, and 131 K after cycles 1, 2, 3, 4) as did the ANC nadir, 100 for cycle 1 v. 640 for cycle 4, likely due to selection and expansion of MDR-1 expressing cells. The patient was in CR prior to a 2nd myeloablative transplant of the original unmanipulated cells after high dose Cy/TBI (1200cGy). For 21 months (m) after infusion of Tr cells, the patient remained well, until he suffered a fatal myocardial infarction. The post mortem showed no evidence of lymphoma or other malignancy. PB and bone marrow (BM) samples collected every 1–3m were examined by RTQ-PCR and nested PCR for presence of the transgene. Tr cells were detected by RTQ-PCR as early as 2 weeks post-transplant. CFU assays of BM samples collected 1, 3, and 6m after transplant demonstrated increasing resistance to doxorubicin with increased time after Tr cell infusion. The transgene was not detected by nested PCR in colonies 1m after initial transplant, however, after 3m, the MDR-1 transgene was present in 3.1% of the colonies and in 10.2% of colonies after 6m. The more sensitive RTQ-PCR showed 16.3%, 25.5%, and 23.9% colonies positive at 1,3, and 6m after transplant. Thus far, insertion site analysis by inverse PCR revealed 13 products representing insertions in non-coding sequence of chromosomes 4, 12, 16, and 20; more sensitive analysis is in progress. Nested and RTQ-PCR of PB and BM samples from 6 weeks and on CFU from BM 14m after the 2nd transplant were negative. RCR safety testing was negative by RTQ-PCR for retroviral 4070A and VSVG envelopes. Thus, autologous engraftment of genetically modified cells with MDR-1 was achieved with 100 cGy TBI in a patient with poor risk NHL who subsequently achieved ‘myeloprotection’ with debulking chemotherapy. Definitive treatment with Cy/TBI and autologous infusion of unmodified cells provided a unique safety feature to eliminate in vivo residual transduced cells, protecting against the risk of insertional mutagenesis.
Identification of the insertion site of transgenic DNA based on cyclization of the target gene with the flanking sequence and nested inverse PCR
