Preclinical development of complex processing systems for ex vivo culture-expanded lymphohematopoietic cells, with subsequent immunologic and/or genetic manipulation, have been carried out in collaboration with a number of NIH institute investigators, as follows:? ? Preparation of allogeneic donor lymphocytes selectively depleted for alloreactive T-cells using an anti-CD25 immunotoxin (collaboration with NHLBI, Stem Cell Transplant Program): In FY2001, we completed development and scale up of this complex process, and in September 2001, initiated a phase I clinical trial of DLI selectively depleted of donor-specific alloreactivity in the setting of allogeneic hematopoietic transplantation. In FY2002, 3 patients were entered on the clinical trial and received their transplants plus SD cells. This year, we also made plans to evaluate 2 cell manipulation strategies as alternatives to the immunotoxin: one, an immunomagnetic separation method and the other, UV light-activated killing of cells after incubation with a photosensitizing agent. During FY2006, the UV light-activating killing of cells as an alternative to immunotoxin was submitted in an NHLBI sponsored IND clinical trial. ? ? Preparation of donor Th2 T cells for clinical trials (collaboration with NCI): Development of this process incorporated CD8/CD20 depletion and CD3/CD28 bead stimulation, which produces a lymphocyte product that is 95% CD4+ and < 1% CD8+. The clinical trial was initiated in March 2001, and 27 patients have been treated to date through FY2002. In FY2004, Th2 cells cultured in the presence of sirolimus, in murine models, promoted engraftment of allogeneic HPC with lower occurrence of GVHD. The clinical trial was initiated using CD8/20 depletion process which was later replaced by CD4+ enrichment by immunomagnetic selection during FY2005. ? During FY2006, the trials using these cultured products continued enrollment. ? ? ? Fibronectin transduction: A method for improved gene transduction using fibronectin-coated bags was previously developed and incorporated into the clinical trial of gene therapy for chronic granulomatous disease; that study was completed in 2001. At the end of FY2001, this method was adapted to a new clinical trial of gene therapy in ADA deficiency that takes advantage of new vectors. To date, very high transduction efficiencies (>80%) have been observed, and 2 patients have been treated. In FY2002, methods were improved (with new cytokines) and validated for gene transduction of autologous PBSC and bone marrow of patients with X-linked SCID, and this clinical trial will begin pending resolution of scientific and FDA investigation of the child with apparent vector-induced oncogenesis. During FY2006, both protocols for ADA deficiency and X-linked SCID were re-initiated. For the X-linked SCID protocol, a method of vector concentration using an ultra-centrifuge was evaluated. Initial experiments showed no difference in transduction efficiency.
