Horn Peter A, Keyser Kirsten A, Peterson Laura J, Neff Tobias, Thomasson Bobbie M, Thompson Jesse, Kiem Hans-Peter
Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Blood. 2004 May 15;103(10):3710-6. doi: 10.1182/blood-2003-07-2414. Epub 2004 Jan 22.
The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.
由于慢病毒载体能够稳定整合到非分裂细胞的基因组中，因此其在造血干细胞转导中的应用引起了广泛关注。然而，已发表的大型动物研究报告显示，基因转导率差异很大，通常低于1%。在此，我们报告了使用慢病毒载体，通过非常短的18小时转导方案，对犬CD34(+)造血重建细胞进行转导。我们在3只犬的竞争性重建试验中，比较了慢病毒对来自干细胞因子(SCF)和粒细胞集落刺激因子(G-CSF)预处理骨髓或动员外周血的造血重建细胞的转导情况。所有犬均在9天内迅速植入。在所有谱系(包括B细胞、T细胞、粒细胞、红细胞以及血小板)中均检测到转基因表达，表明转导细胞实现了多谱系植入，总体长期标记水平高达12%。动员外周血细胞中的基因转导水平略高于预处理骨髓细胞。总之，我们展示了使用过夜转导方案对犬重建细胞进行高效慢病毒转导。这些结果对干细胞基因治疗方案的设计具有重要意义，尤其是对于那些在培养中维持干细胞是主要限制因素的疾病。
