Glimm H, Flügge K, Möbest D, Hofmann V M, Postmus J, Henschler R, Lange W, Finke J, Kiem H P, Schulz G, Rosenthal F, Mertelsmann R, von Kalle C
Department of Internal Medicine I, Albert-Ludwigs-University, Freiburg, Germany.
Hum Gene Ther. 1998 Apr 10;9(6):771-8. doi: 10.1089/hum.1998.9.6-771.
Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34+ and CD34+/HLA-DRlo peripheral blood progenitor "stem" cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34+, CD34+/HLA-DRlo, and CD34+/CD38lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy.
明确的无血清条件对于临床基因导入造血干细胞的生物安全性和标准化具有很大的概念优势。在迄今为止报道的唯一一项研究中，塞卡尔等人通过对最初含有10%胎牛血清的逆转录病毒上清液进行复杂的浓缩程序实现了低血清条件。高成本、小体积、血清源性病原体可能的共富集、载体颗粒回收有限以及最终稀释培养基的低滴度限制了该程序的临床应用。未证明对原始造血祖细胞的转导。在本研究中，从PG13/LN生产细胞中产生了含有高滴度假型逆转录病毒载体PG13/LN的明确无血清培养基，无需物理富集程序。在含无血清载体的培养基中，定向造血祖细胞的转导效率很高，与含血清对照条件下的转导效率相似。在含该载体的培养基中，在白细胞介素-3、干细胞因子和fms样酪氨酸激酶-3配体存在的情况下，从CD34+和CD34+/HLA-DRlo外周血祖“干”细胞（PBSCs）产生的原始人类造血长期培养启动细胞衍生集落（LTC-IC衍生集落）的数量在7天的处理过程中增加了。所描述的程序允许对从CD34+、CD34+/HLA-DRlo和CD34+/CD38lo PBSCs产生的LTC-IC衍生集落进行高效转导。这是第一份证明体外原始外周血LTC-IC衍生集落增加以及它们在高滴度、含无血清载体的培养基中高效转导的报告，该培养基可仅由明确的药用级成分生产，使其非常适合临床基因治疗应用。
