Kimura Shigemi, Ikezawa Makoto, Cao Baohong, Kanda Yasunari, Pruchnic Ryan, Cummins James, Huard Johnny, Miike Teruhisa, Suzuki Satoru
Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
J Gene Med. 2004 Feb;6(2):155-65. doi: 10.1002/jgm.480.
Adenoviral gene transfer to adult skeletal muscle is hindered by several major limitations, including host immune responses and maturation-dependent loss of myofiber infectivity. Ex vivo gene delivery is more efficient than direct viral injection in surmounting maturation-dependent adenoviral transduction. Here we investigated the use of helper cells to improve the efficiency of ex vivo gene transfer to adult mouse skeletal muscle.
New producer cells carrying the E1 gene of adenovirus type 5 (E32 cells) were developed using primary myoblasts from mdx mice. The E32 cells and 293 cells were infected with an E1-deleted first-generation adenovirus carrying the LacZ gene. These transduced helper cells were injected into the skeletal muscle of adult mdx and SCID mice.
LacZ-positive mature myofibers were detected in the skeletal muscle of adult mice sacrificed 5 days post-injection. The gene transfer efficiency using 293 cells and E32 cells was 6.2 and 3.6 times higher than myoblast-mediated gene transfer, respectively. Ex vivo gene transfer of these cell types led to a better outcome than did direct adenoviral injection.
We achieved more efficient adenoviral gene transduction by using 293 and E32 helper cells than by myoblast-mediated gene transfer and direct viral injection. These helper cells also enabled adenoviral gene transfer to mature myofibers. The mechanisms by which this method facilitated adenoviral gene transfer to mature myofibers remains unclear; however, we hypothesize that the in vivo occurrence of cytopathic effects (CPE) in the transduced 293 and E32 helper cell populations facilitated the improved adenoviral transduction of myofibers.
腺病毒基因转移至成年骨骼肌受到几个主要限制因素的阻碍，包括宿主免疫反应以及与成熟相关的肌纤维感染性丧失。在克服与成熟相关的腺病毒转导方面，体外基因递送比直接病毒注射更有效。在此，我们研究了使用辅助细胞来提高体外基因转移至成年小鼠骨骼肌的效率。
利用来自mdx小鼠的原代成肌细胞开发了携带5型腺病毒E1基因的新型生产细胞（E32细胞）。用携带LacZ基因的E1缺失第一代腺病毒感染E32细胞和293细胞。将这些转导的辅助细胞注射到成年mdx和SCID小鼠的骨骼肌中。
在注射后5天处死的成年小鼠骨骼肌中检测到LacZ阳性成熟肌纤维。使用293细胞和E32细胞的基因转移效率分别比成肌细胞介导的基因转移高6.2倍和3.6倍。这些细胞类型的体外基因转移比直接腺病毒注射产生了更好的结果。
与成肌细胞介导的基因转移和直接病毒注射相比，我们使用293和E32辅助细胞实现了更有效的腺病毒基因转导。这些辅助细胞还能使腺病毒基因转移至成熟肌纤维。该方法促进腺病毒基因转移至成熟肌纤维的机制尚不清楚；然而，我们推测转导的293和E32辅助细胞群体在体内出现的细胞病变效应（CPE）促进了肌纤维腺病毒转导的改善。
