Bauer G, Sauter S, Ibanez C, Rice C R, Valdez P, Jolly D, Kohn D B
Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, CA 90027, USA.
Biol Blood Marrow Transplant. 1998;4(3):119-27. doi: 10.1053/bbmt.1998.v4.pm9923409.
Using retroviral supernatants derived from the amphotropic murine packaging cell line PA317 and the amphotropic canine packaging cell line (DA), cord blood and mobilized peripheral blood CD34+ cells were transduced with the vector LN (neomycin resistance) and the vector L-TR/TAT neo (neomycin resistance in conjunction with a double-hammerhead ribozyme conferring anti-HIV activity). Different multiplicities of infection (MOI) were applied in the setup according to vector titrations on NIH-3T3 cells. PA317-based supernatants were tested at MOI of 10 and 30. Purified concentrated DA-derived vector preparations were tested at MOI of 10, 30, 100, and 300. Immediately after transduction, CD34+ cells were plated into colony assays in the presence and absence of G418 to evaluate the amount of gene transfer and potential toxic effects of the vectors on colony growth. The remaining cells were subjected to G418 selection in liquid culture for 12 days and subsequently challenged with HIV-1JR-FL to test for efficacy of the anti-HIV gene in macrophages derived from transduced CD34+ cells. Transduction by the PA317-packaged vectors was maximal at the lowest MOI used and did not increase with increasing MOI. In contrast, transduction by the DA-packaged vectors could be progressively increased using increased MOI. The net transduction efficiency per unit of reverse transcriptase activity in the DA vector preparations was 8.7-fold higher than in the PA317 vector supernatants. HIV-1 challenge of the cells transduced by the ribozyme vector derived from the PA317 packaging cells resulted in a 1.5 log inhibition of p24 output compared with the control cells containing neomycin resistance only. A 2.5 log inhibition of p24 output could be observed in the cell population transduced with DA-packaged vector supernatants. Compared with retroviral supernatants from PA317 packaging cell lines, DA packaging line-derived vector preparations demonstrated higher transduction efficiency into CD34+ cells, particularly at higher MOI, and increased efficacy of the transferred anti-HIV gene when challenged with HIV-1JR-FL. The increase in transduction efficiency may be due to a higher ratio of intact vs. defective vector particles in the DA-derived vector preparations.
利用源自嗜性鼠包装细胞系PA317和嗜性犬包装细胞系（DA）的逆转录病毒上清液，用载体LN（新霉素抗性）和载体L-TR/TAT neo（新霉素抗性与赋予抗HIV活性的双锤头核酶结合）转导脐血和动员的外周血CD34+细胞。根据在NIH-3T3细胞上的载体滴定，在实验设置中应用了不同的感染复数（MOI）。基于PA317的上清液在MOI为10和30时进行测试。纯化浓缩的源自DA的载体制剂在MOI为10、30、100和300时进行测试。转导后立即将CD34+细胞接种到有和没有G418的集落测定中，以评估基因转移量以及载体对集落生长的潜在毒性作用。其余细胞在液体培养中进行12天的G418选择，随后用HIV-1JR-FL攻击，以测试转导的CD34+细胞来源的巨噬细胞中抗HIV基因的功效。PA317包装的载体在使用的最低MOI时转导效率最高，并且不会随着MOI的增加而增加。相比之下，使用增加的MOI可以逐渐提高DA包装的载体的转导效率。DA载体制剂中每单位逆转录酶活性的净转导效率比PA317载体上清液高8.7倍。与仅含有新霉素抗性的对照细胞相比，源自PA317包装细胞的核酶载体转导的细胞受到HIV-1攻击后，p24产量受到1.5个对数的抑制。在用DA包装的载体上清液转导的细胞群体中，可以观察到p24产量受到2.5个对数的抑制。与PA317包装细胞系的逆转录病毒上清液相比，源自DA包装细胞系的载体制剂对CD34+细胞表现出更高的转导效率，特别是在较高的MOI时，并且在用HIV-1JR-FL攻击时，转移的抗HIV基因的功效增强。转导效率的提高可能是由于源自DA的载体制剂中完整与缺陷载体颗粒的比例更高。
