Development Of Methods For Ex Vivo Cultured And Immunologically And/or Geneticall

与NIH研究所的一些研究人员合作，开展了体外培养扩增的淋巴造血细胞的复杂加工系统的临床前开发，并随后进行了免疫和/或遗传操作，如下所示： 使用抗CD25免疫毒素选择性去除同种异体供者淋巴细胞的同种异体反应性T细胞的制备(与NHLBI，干细胞移植计划合作)：2001财年，我们完成了这一复杂过程的开发和放大，并于2001年9月在异基因造血移植环境中启动了选择性去除供者特异性同种异体反应性的DLI临床试验。在2002财年，3名患者参加了临床试验，并接受了他们的移植加SD细胞。今年，我们还计划评估两种细胞操纵策略作为免疫毒素的替代品：一种是免疫磁分离法，另一种是用光敏剂孵育后的紫外光激活杀灭细胞。在2006财年，作为免疫毒素替代品的紫外线激活杀灭细胞被提交给NHLBI赞助的IND临床试验。 为临床试验准备供体Th2 T细胞(与NCI合作)：这一过程的开发结合了CD8/CD20去除和CD3/CD28珠状刺激，产生了95%的CD4+和1%的CD8+的淋巴细胞产物。这项临床试验于2001年3月启动，到2002财年为止，已有27名患者接受治疗。在2004财年，在小鼠模型中，在西罗莫司存在下培养的Th2细胞促进了异基因HPC的植入，并降低了GVHD的发生率。临床试验开始使用CD8/20耗竭过程，后来在2005财年期间通过免疫磁性选择用CD4+浓缩来取代CD8/20。 在2006财年，使用这些培养产品的试验继续登记。 纤维连接蛋白转导：一种使用纤维连接蛋白涂层袋子改进基因转导的方法以前被开发出来，并被纳入慢性肉芽肿疾病的基因治疗的临床试验中；该研究于2001年完成。2001财年末，这种方法被用于利用新载体进行ADA缺乏症基因治疗的新临床试验。到目前为止，已经观察到非常高的转导效率(&gt；80%)，已经治疗了2名患者。在2002财年，对X连锁SCID患者的自体PBSC和骨髓的基因转导方法进行了改进(使用了新的细胞因子)并得到了验证，这项临床试验将开始，等待科学和FDA对明显由媒介诱导的肿瘤发生的儿童的调查结果的解决。在2006财年，ADA缺乏症和X连锁SCID的治疗方案都重新启动。对于X连锁的SCID方案，评估了一种使用超速离心机的载体浓缩方法。初步实验表明，转导效率没有差异。

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.