In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement.
In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement.
复制标题
通过短片段同源替换体内和体外纠正 mdx 肌营养不良蛋白基因无义突变。
作者：
Robert Kapsa;Anita F. Quigley;Gordon S. Lynch;Kelly Steeper;Andrew J. Kornberg;P. Gregorevic;Lawrie Austin;Edward Byrne
细胞突变的靶向基因校正是治疗包括无义、错义和转录剪接突变在内的人类疾病的潜在策略。一种靶向基因修复方法，单链短片段同源置换（ssSFHR），已经成功修复了1%培养的气道上皮细胞中囊性纤维化跨膜传导调节因子（CFTR）位点上常见的deltaF508 3-bp微缺失。本研究研究了SFHR基因双链法变体在mdx小鼠杜氏肌营养不良（DMD）模型中的体外和体内应用修复。利用603 bp的野生型PCR产物修复mdx雄性小鼠培养成肌细胞和前胫肌（TA） Xp21.1天位点外显子23 c -t无义转移。多次转染和脂质转染试剂的变化都提高了体外SFHR效率，在15 - 20%的培养位点和0.0005 - 0.1%的TA中，mdx在dys位点成功转化为野生型核苷酸。mdx成肌细胞的遗传校正在培养中可持续28天，在TA中可持续至少3周。虽然观察到体外基因修复的频率很高，但这里使用的脂肪感染似乎对随后的细胞活力有不利影响，并且校正后的细胞不表达肌营养不良蛋白转录物。随着体外和体内基因修复效率的进一步提高，SFHR可能在人类DMD和其他遗传性神经肌肉疾病中找到一些应用。
Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common deltaF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was shown to persist for up to 28 days in culture and for at least 3 weeks in TA. While a high frequency of in vitro gene repair was observed, the lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans.
影响因子：
3.5
作者：
Im, WB;Phelps, SF;Chamberlain, JS
通讯作者：
Chamberlain, JS
人链特异性错配修复通过类似于细菌反应的双向机制发生。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Fang,WH;Modrich,P
通讯作者：
Modrich,P
影响因子：
4.4
作者：
SHARP, NJH;KORNEGAY, JN;BARTLETT, RJ
通讯作者：
BARTLETT, RJ