Extra-adrenal expression of Cyp21a1 for gene therapy of congenital adrenal hyperplasia

用H-2aw18单倍型小鼠的杂合子培育出21-羟基酶缺陷型(Cyp21a1D)小鼠。构建了含小鼠Cyp21a1基因的逆转录病毒载体，并将其传递给培养的21OHD小鼠成纤维细胞。将Cyp21a1诱导的成纤维细胞注射到Cyp21a1D小鼠体内。用pAAV-CMV-SPORTH构建含Cyp21a基因的腺相关病毒载体(AAV-Cyp21a1)，注射入21OHD小鼠四肢肌肉。注射前后分别测定血清P4和DOC浓度。将转基因成纤维细胞移植到Cyp21a1D小鼠体内，血清P4/DOC比值变化不大。注射后4周，携带AAV-Cyp21a1的转基因21OHD小鼠DOC产量增加，P4/DOC比值下降(1223~39.2)。我们通过用腺相关病毒载体将Cyp21a1基因导入小鼠肌肉4周，成功地恢复了Cyp21a1D小鼠体内21OH活性的类固醇合成。

21-hydorxylase deficient (Cyp21a1D) mice were made by breeding with heterozygous of H-2aw18 haplotype mice. Retrovirus vector containing mouse Cyp21a1 cDNA was constructed and transmitted to fibroblasts cultured from 21OHD mice. Cyp21a1-induced fibroblasts were injected into Cyp21a1D mice. Adenoassociated virus vector containing Cyp21a cDNA (AAV-Cyp21a1) was constructed with pAAV-CMV-shuttle and injected into limbs muscles of 21OHD mice. Serum P4 and DOC concentrations were also measured before and after injection. Transplantation of the transgenic fibroblasts into Cyp21a1D mice showed slight change serum P4/DOC ratio. Transgenic 21OHD mice with AAV-Cyp21a1 showed increase DOC production and decreased P4/DOC ratio (1223 to 39.2) at 4weeks after injection. We succeeded recovering 21OH activity steroid production in in Cyp21a1D mice by introducing Cyp21a1 gene into muscle with adenoassociated virus vectors for 4 weeks.