Okee Moses, Bayiyana Alice, Musubika Carol, Joloba Moses L, Ashaba-Katabazi Fred, Bagaya Bernard, Wayengera Misaki
1 Department of Immunology and Molecular Biology, School of Biomedical Sciences, College of Health Sciences, Makerere University , Kampala, Uganda .
2 Unit of Genetics and Genomics, Department of Pathology, School of Biomedical Sciences, College of Health Sciences, Makerere University , Kampala, Uganda .
AIDS Res Hum Retroviruses. 2018 Jan;34(1):88-102. doi: 10.1089/AID.2017.0234. Epub 2018 Jan 5.
Efficiency of artificial restriction enzymes toward curing HIV has only been separately examined, using differing delivery vehicles. We compared the in vitro transduction and target-mutagenesis efficiency of consortium plasmid and adenoviral vector delivered HIV-1 pol gene targeting zinc finger nuclease (ZFN) with CRISPR/Cas, Custom-ZFN, CRISPR-Cas-9, and plasmids and vectors (murCTSD_pZFN, pGS-U-gRNA, pCMV-Cas-D01A, Ad5-RGD); cell lines (TZM-bl and ACH-2/J-Lat cells); and the latency reversing agents prostratin, suberoylanilide hydroxamic acid, and phorbol myristate acetate. Cell lines were grown in either Dulbecco's modified Eagle's medium or Roswell Park Memorial Institute with the antibiotics kanamycin, zeocin, and efavirenz. Efficiency was assayed by GFP/luciferase activity and/or validated by yeast MEL1 reporter assay, CEL1 restriction fragment assay, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Ad5-RGD vectors had better transduction efficiency than murCTSD and pGS-U-gRNA/pCMV-Cas-D01A plasmids. CRISPR/Cas9 exhibited better target-mutagenesis efficiency relative to ZFN (delivered by either plasmid or Ad5 vector) based on gel electrophoresis of pol gene amplicons within ACH-2 and J-Lat cells. Ad-5-RGD vectors enhanced target mutagenesis of ZFN, relative to murCTSD_pZFN plasmids, to levels of CRISPR/Cas9 plasmids. Similar reduction of luciferase activity among TZM-bl treated with Ad5-ZFN vectors relative to CRISPR/Cas-9 and murCTSD_pZFN plasmids was observed on challenge with HIV-1. qRT-PCR of HIV-1 pol gene transcripts affirmed that Ad5 (RGD) vectors enhanced target mutagenesis of ZFN. Whereas CRISPR/Cas-9 may possess inherent superior target-mutagenesis efficiency; the efficiency of ZFN (off-target toxicity withstanding) can be enhanced by altering delivery vehicle from plasmid to Ad5 (RGD) vectors.
人工限制酶治疗艾滋病病毒的效率仅使用不同的递送载体分别进行了检测。我们比较了通过联合质粒和腺病毒载体递送的靶向锌指核酸酶（ZFN）的HIV-1 pol基因与CRISPR/Cas、定制ZFN、CRISPR-Cas-9以及质粒和载体（murCTSD_pZFN、pGS-U-gRNA、pCMV-Cas-D01A、Ad5-RGD）的体外转导和靶向诱变效率；细胞系（TZM-bl和ACH-2/J-Lat细胞）；以及潜伏逆转剂苔藓抑素、辛二酰苯胺异羟肟酸和佛波酯。细胞系在添加抗生素卡那霉素、博来霉素和依法韦仑的杜氏改良伊格尔培养基或罗斯威尔公园纪念研究所培养基中培养。通过绿色荧光蛋白/荧光素酶活性测定效率，和/或通过酵母MEL1报告基因测定、CEL1限制性片段测定以及定量逆转录聚合酶链反应（qRT-PCR）进行验证。Ad5-RGD载体的转导效率优于murCTSD和pGS-U-gRNA/pCMV-Cas-D01A质粒。基于ACH-2和J-Lat细胞中pol基因扩增子的凝胶电泳，相对于ZFN（通过质粒或Ad5载体递送），CRISPR/Cas9表现出更好的靶向诱变效率。相对于murCTSD_pZFN质粒，Ad-5-RGD载体增强了ZFN的靶向诱变，使其达到CRISPR/Cas9质粒的水平。在用HIV-1攻击时，相对于CRISPR/Cas-9和murCTSD_pZFN质粒，用Ad5-ZFN载体处理的TZM-bl细胞中观察到类似的荧光素酶活性降低。HIV-1 pol基因转录本的qRT-PCR证实Ad5（RGD）载体增强了ZFN的靶向诱变。虽然CRISPR/Cas-9可能具有固有的更高靶向诱变效率；但通过将递送载体从质粒改为Ad5（RGD）载体，可以提高ZFN的效率（在不考虑脱靶毒性的情况下）。
