Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Physiol Genomics. 2011 Aug 16;43(15):911-6. doi: 10.1152/physiolgenomics.00006.2011. Epub 2011 May 31.
In the course of developing a low-density lipoprotein receptor (LDLR) gene therapy treatment for homozygous familial hypercholesterolemia (HoFH), we planned to examine the efficacy in a nonhuman primate model, the rhesus macaque heterozygous for an LDL receptor mutation fed a high-fat diet. Unexpectedly, our initial cDNA sequencing studies led to the identification of a heretofore unidentified splicing isoform of the rhesus LDLR gene. Compared with the publicly available GenBank reference sequence of rhesus LDLR, the novel isoform contains a 21 bp in frame insertion. This sequence coincides with part of exon 5 and creates a site for the restriction enzyme MscI. Using this site as a marker for the 21 bp in-frame insertion, we conducted a restriction enzyme screen to examine for the prevalence of the novel isoform in rhesus liver tissue cDNA and its homolog in human liver tissue cDNA. We found that the novel isoform is the predominant LDLR cDNA found in rhesus liver and the sole LDLR cDNA found in human liver. Finally, we compared the in vivo functionality of the novel and previously identified rhesus LDLR splicing isoforms in a mouse model of HoFH.
在开发用于治疗纯合家族性高胆固醇血症（HoFH）的低密度脂蛋白受体（LDLR）基因治疗的过程中，我们计划在食高脂肪饮食的携带 LDLR 基因突变的恒河猴非人类灵长类动物模型中检验其疗效。出乎意料的是，我们最初的 cDNA 测序研究导致鉴定出了一种以前未被识别的恒河猴 LDLR 基因的剪接异构体。与公开的恒河猴 LDLR 的 GenBank 参考序列相比，新的异构体包含 21 个碱基的框内插入。该序列与外显子 5 的一部分重合，并创建了限制酶 MscI 的作用位点。使用该位点作为 21 个碱基框内插入的标记，我们进行了限制酶筛选，以检查新异构体在恒河猴肝组织 cDNA 及其在人肝组织 cDNA 中的同源物中的普遍性。我们发现，新异构体是恒河猴肝脏中发现的主要 LDLR cDNA，也是人肝脏中唯一发现的 LDLR cDNA。最后，我们在 HoFH 的小鼠模型中比较了新鉴定的和先前鉴定的恒河猴 LDLR 剪接异构体的体内功能。
