Barquinero J, Kiem H P, von Kalle C, Darovsky B, Goehle S, Graham T, Seidel K, Storb R, Schuening F G
Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98104-2092.
Blood. 1995 Mar 1;85(5):1195-201.
We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.
我们研究了不同预处理方案对基因标记的造血重建细胞在犬体内植入的作用。用重组犬干细胞因子（rcSCF）或环磷酰胺处理后收集的外周血（PB）和/或骨髓细胞，在含载体的长期培养系统中进行转导。使用了三种病毒滴度相似的不同载体生产细胞系。其中两种，含新霉素的LN载体分别包装在具有嗜异性鼠逆转录病毒包膜的PA317细胞系或具有长臂猿白血病病毒（GALV）包膜的PG13细胞系中。在PA317细胞中产生的MFG/GC载体含有人类葡萄糖脑苷脂酶基因。19只犬在静脉内（A、C、D、E组）或髓内（B组）输注转导的自体造血细胞之前，分别接受了无预处理（A组，n = 5）、对双侧肱骨进行1000 cGy照射（B组，n = 5）、40 mg/kg的亚致死剂量环磷酰胺（C组，n = 4）、200或300 cGy全身照射（TBI）的亚致死剂量（D组，n = 3），或920 cGy TBI的致死剂量（E组，n = 3）。不同预处理组在将造血细胞输注到动物体内时的转导效率相似。对犬观察至少6个月。移植后至少每3周获取PB粒细胞，并通过聚合酶链反应分析转导基因的存在情况。移植后4周以上犬的阳性结果百分比，无预处理为0%，局部照射为5%，亚致死剂量环磷酰胺为18%，亚致死剂量TBI为33%，致死剂量TBI为17%。通过广义估计方程（GEE）技术分析预处理方案的影响，该技术将使用不同逆转录病毒载体和输注的单个核细胞数量视为潜在混杂变量，我们发现与仅接受局部照射或无预处理的犬相比，接受致死剂量TBI、亚致死剂量TBI或亚致死剂量环磷酰胺全身预处理的犬，基因标记的重建细胞植入显著改善（P <.001）。在实验设计的限制范围内，这些数据表明清髓性或骨髓抑制性预处理可改善基因标记的造血重建细胞的植入。
