Hurwitz D R, Kirchgesser M, Merrill W, Galanopoulos T, McGrath C A, Emami S, Hansen M, Cherington V, Appel J M, Bizinkauskas C B, Brackmann H H, Levine P H, Greenberger J S
ALG Company, Marlboro, MA 01752, USA.
Hum Gene Ther. 1997 Jan 20;8(2):137-56. doi: 10.1089/hum.1997.8.2-137.
Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfected to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adherent bone marrow stromal cell cultures, established and expanded from iliac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3.84 micrograms/10(6) cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases, the stromal cells were cryopreserved following transfection, and subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 micrograms/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following reinfusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Vector-modified stromal cells were detectable, by polymerase chain reaction (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 x 10(8)), with a total hFIX expression capacity of 281 micrograms/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.
犬骨髓基质细胞从最初10 - 20毫升骨髓抽吸物中扩增至超过10⁹个细胞，并在体外转染以高水平表达人生长激素（hGH）。经体外修饰的骨髓基质细胞用于基因治疗模型系统，以在犬体内全身递送转基因产物。从8只犬的髂嵴骨髓抽吸物建立并扩增的贴壁骨髓基质细胞培养物，用hGH基因质粒表达载体转染，结果显示在体外每24小时每10⁶个细胞表达0.54 - 3.84微克hGH。转染的质粒载体不具有真核生物复制起点，也不具有有效整合到宿主细胞基因组所需的序列。因此，预期表达是短暂的。转染细胞通过注入前肢静脉或直接注入髂嵴骨髓自体重新引入每只犬体内。在2个案例中，基质细胞在转染后冷冻保存，随后解冻并注入。在1个案例中，扩增的基质细胞先冷冻保存，然后解冻、再培养、转染并注入。重新引入的细胞数量范围为2.2×10⁷至2.6×10⁹，hGH总表达能力范围为62至1400微克/24小时。每只犬的血浆在细胞回输后2至5天内平均3.1天（标准差±0.8天）可检测到hGH。血浆峰值水平范围为0.10至1.76纳克/毫升。基于注入细胞的总表达能力和犬体重，所有犬获得了相似的hGH表达值。通过聚合酶链反应（PCR）分析，在所有4只分析的犬回输后外周循环中可检测到载体修饰的基质细胞。在3只犬中，修饰的基质细胞在8.5 - 15周内被检测到。此外，回输后在2只犬的髂嵴骨髓中分别在9周和13周检测到修饰的基质细胞。在另一个实验中，培养的骨髓基质细胞用人类凝血因子IX（hFIX）质粒载体转染。将总hFIX表达能力为281微克/24小时的修饰细胞（5.57×10⁸）回输，导致血浆中hFIX连续9天可检测到，第1天峰值水平为8纳克/毫升。这些结果表明，体外骨髓基质细胞系统是一种潜在的强大方法，可将分泌的转基因产物递送至大型动物的全身循环。
