Xu L, Stahl S K, Dave H P, Schiffmann R, Correll P H, Kessler S, Karlsson S
Molecular and Medical Genetics Section, NINDS, Bethesda, MD 20892.
Exp Hematol. 1994 Feb;22(2):223-30.
Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme glucocerebrosidase (GC), and is an excellent candidate for gene replacement therapy. To develop a clinically acceptable protocol for this purpose, we created two amplified (A) high-titer retroviral vector-producer cell lines to efficiently transduce hematopoietic stem and progenitor cells. GP+envAm12/A-LGSN (A-LGSN), contained the GC cDNA driven by the retroviral long terminal repeat (LTR) and the neomycin phosphotransferase gene expressed from the simian virus 40 early promoter. GP+envAm12/A-LG4 (A-LG4) contained only the GC gene driven by the LTR. Both A-LGSN and A-LG4 contained multiple proviral copies and gave approximately 10-fold higher titers on 3T3 cells compared to their unamplified counterparts. These vectors were packaged in GP+envAm12 cells because vectors produced in this cell line transduced hematopoietic cells more efficiently than other packaging cells tested. Bone marrow mononuclear cells and purified CD34+ cells were infected with virus supernatants four times in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) over 96 hours in culture. Cells were then plated in semisolid cultures and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies were scored for vector presence by polymerase chain reaction (PCR). Transduction efficiency of CFU-GM colonies derived from CD34+ cells was improved considerably using the amplified vectors in the GP+envAm12 packaging line. For A-LGSN, A-LG4, and unamplified LGSN, transduction efficiencies were 41, 42, and 25%, respectively. Therefore, multiple proviral copies resulting in higher titer improves retroviral transduction of human hematopoietic progenitor cells. Hematopoietic cells from Gaucher patients were transduced and placed into long-term bone marrow culture (LTBMC). Viral supernatant from the amplified producer lines transduced long-term culture initiating cells (LTCIC) efficiently (30 to 50%) using this clinically acceptable protocol. Both sustained mRNA expression and GC enzyme production are achieved in the long-term culture of LTCIC and lead to correction of the GC deficiency in their progeny cells.
戈谢病是一种由葡糖脑苷脂酶（GC）缺乏引起的溶酶体贮积症，是基因替代疗法的理想候选对象。为了为此制定一个临床可接受的方案，我们创建了两种扩增（A）的高滴度逆转录病毒载体生产细胞系，以有效转导造血干细胞和祖细胞。GP+envAm12/A-LGSN（A-LGSN）包含由逆转录病毒长末端重复序列（LTR）驱动的GC cDNA和由猿猴病毒40早期启动子表达的新霉素磷酸转移酶基因。GP+envAm12/A-LG4（A-LG4）仅包含由LTR驱动的GC基因。A-LGSN和A-LG4都包含多个前病毒拷贝，与未扩增的对应物相比，在3T3细胞上的滴度高出约10倍。这些载体在GP+envAm12细胞中包装，因为在该细胞系中产生的载体比测试的其他包装细胞更有效地转导造血细胞。在白细胞介素-3（IL-3）、白细胞介素-6和干细胞因子（SCF）存在的情况下，骨髓单个核细胞和纯化的CD34+细胞在96小时的培养过程中用病毒上清液感染4次。然后将细胞接种到半固体培养物中，并通过聚合酶链反应（PCR）对集落形成单位-粒细胞/巨噬细胞（CFU-GM）集落进行载体存在情况评分。在GP+envAm12包装细胞系中使用扩增载体可显著提高源自CD34+细胞的CFU-GM集落的转导效率。对于A-LGSN、A-LG4和未扩增的LGSN，转导效率分别为41%、42%和25%。因此，多个前病毒拷贝导致更高滴度可提高人类造血祖细胞的逆转录病毒转导效率。对戈谢病患者的造血细胞进行转导，并置于长期骨髓培养（LTBMC）中。使用这种临床可接受的方案，来自扩增生产细胞系的病毒上清液有效地转导了长期培养起始细胞（LTCIC）（30%至50%）。在LTCIC的长期培养中实现了持续的mRNA表达和GC酶的产生，并导致其后代细胞中GC缺乏的纠正。
