Walsh C E, Mann M M, Emmons R V, Wang S, Liu J M
Clinical Pathology Department, NIH, Bethesda, MD, USA.
J Investig Med. 1995 Aug;43(4):379-85.
Fanconi anemia (FA) is an autosomal recessive inherited form of bone marrow failure. FA cells are characterized by their extreme sensitivity to DNA cross-linking agents that cause DNA instability and cell death. Four genetic complementation groups for FA have been identified and the gene for the complementation C group (FACC) has been cloned. Genetic transfer of the FACC gene should provide a growth advantage in transduced hematopoietic cells. We have previously demonstrated efficient retroviral-mediated gene transduction and correction of FA(C) cell lines and peripheral blood-derived CD34+ progenitors from patients carrying mutant FACC alleles. In this report we sought to define the optimal conditions for transduction of CD34+ progenitors from mobilized peripheral blood and umbilical cord blood.
Peripheral blood hematopoietic progenitors were obtained by G-CSF mobilization followed by apheresis. Human fetal cord blood cells were obtained from full-term gestation deliveries. Cells were immunoselected for CD34 antigen expression and then incubated with recombinant retroviruses containing a selectable marker gene (neomycin). Recombinant colony stimulating factors were added to facilitate viral transduction. Cells were plated in methylcellulose and resulting hematopoietic colonies were isolated and analyzed by PCR.
Transduction efficiency of peripheral blood progenitors (from normal individuals) using a retrovirus encoding the FACC cDNA was comparable to that of the retroviral producer G1Na.40 currently being used in clinical gene therapy marking studies. We extended our standard transduction protocol to analyze CD34+ and CD34+ CD38-subpopulations of progenitors derived from umbilical cord blood (from normal pregnancies). In addition, we tested whether FACC cDNA transduction could be improved by vector infection supported by autologous stroma. For FA(C) hematopoietic cell infection, vector supernatant transduction in the presence of recombinant human IL-3, IL-6, and SCF was found to be superior to transduction supported by autologous FA(C) patient stroma.
We documented efficient retroviral transduction of umbilical cord blood and peripheral blood enriched for hematopoietic progenitor cells. These results suggest the feasibility of a clinical gene therapy protocol utilizing progenitor cells from both peripheral blood and umbilical cord blood.
范可尼贫血（FA）是一种常染色体隐性遗传性骨髓衰竭疾病。FA细胞的特点是对导致DNA不稳定和细胞死亡的DNA交联剂极度敏感。已确定了FA的四个基因互补组，并且克隆了互补C组（FACC）的基因。FACC基因的基因转移应能为转导的造血细胞提供生长优势。我们之前已证明逆转录病毒介导的基因转导以及对携带突变FACC等位基因患者的FA（C）细胞系和外周血来源的CD34+祖细胞的校正。在本报告中，我们试图确定从动员的外周血和脐带血中转导CD34+祖细胞的最佳条件。
通过粒细胞集落刺激因子（G-CSF）动员后进行单采获得外周血造血祖细胞。从足月分娩中获取人胎儿脐带血细胞。对细胞进行免疫选择以检测CD34抗原表达，然后与含有选择标记基因（新霉素）的重组逆转录病毒一起孵育。添加重组集落刺激因子以促进病毒转导。将细胞接种于甲基纤维素中，分离得到的造血集落并通过聚合酶链反应（PCR）进行分析。
使用编码FACC cDNA的逆转录病毒对外周血祖细胞（来自正常个体）的转导效率与目前用于临床基因治疗标记研究的逆转录病毒生产株G1Na.40相当。我们扩展了标准转导方案以分析来自脐带血（来自正常妊娠）的祖细胞的CD34+和CD34+ CD38-亚群。此外，我们测试了自体基质支持的载体感染是否能改善FACC cDNA转导。对于FA（C）造血细胞感染，发现在重组人白细胞介素-3（IL-3）、白细胞介素-6（IL-6）和干细胞因子（SCF）存在下的载体上清转导优于自体FA（C）患者基质支持的转导。
我们记录了对富含造血祖细胞的脐带血和外周血进行高效逆转录病毒转导的情况。这些结果表明利用外周血和脐带血中的祖细胞进行临床基因治疗方案的可行性。
