Division of Hematology and Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Blood. 2012 Mar 29;119(13):3038-41. doi: 10.1182/blood-2011-09-382317. Epub 2012 Jan 23.
In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.
在之前的工作中，我们将一种携带人凝血因子 IX 基因的腺相关病毒（AAV）载体注入到患有严重血友病 B 的男性的骨骼肌中。在载体注射后长达 1 年的时间里，对注射肌肉进行活检，通过 Southern blot 显示出基因转移的证据，通过免疫组化和免疫荧光染色显示出蛋白表达的证据。尽管该过程看起来是安全的，但循环 F.IX 水平仍然低于治疗水平（<1%）。最近，我们从一位 10 年前接受过注射的受试者的肌肉组织中获得了样本，该受试者因与基因转移无关的原因死亡。通过 Western blot、免疫组化和免疫荧光染色，我们在注射的肌肉组织中显示出持续的因子 IX 表达。使用 RT-PCR 在注射的骨骼肌中检测到 F.IX 转录本，并且分离的全基因组 DNA 检测到存在转移的 AAV 载体序列呈阳性。这是迄今为止从肠外给予的 AAV 载体中报告的最长的转基因表达，对肌肉定向基因转移的未来具有广泛的影响。
