Kajumbula Henry, Byarugaba Wilson, Wayengera Misaki
Dept of Medical Microbiology, School of Biomedical Science, College of Health Sciences, Makerere University, P O Box 7072, Kampala, Uganda.
Genet Vaccines Ther. 2012 Aug 31;10(1):8. doi: 10.1186/1479-0556-10-8.
Malaria causes immense human morbidity and mortality globally. The plasmodium species vivax and falciparum cause over 75 % clinical malaria cases. Until now, gene-based strategies against malaria have only been applied to plasmodium species and their mosquito-vector. Merozoites of these two respective plasmodium species target and invade red blood cells (RBCs) by using the duffy antigen receptor for chemokines (DARC), and Sialic Acid (SLC4A1) residues of the O-linked glycans of Glycophorin A. RBCs of naturally selected duffy-negative blacks are resistant to P.vivax tropism. We hypothesized that artificial aberration of the host-pathway by target mutagenesis of either RBC -receptors, may abolish or reduce susceptibility of the host to malaria. As a first step towards the experimental actualization of these concepts, we aimed to identify zinc finger arrays (ZFAs) for constructing ZFNs that target genes of either wild-type host-RBC- receptors.
In-Silico Gene & Genome Informatics
Using the genomic contextual nucleotide-sequences of homo-sapiens darc and glycophorin-a, and the ZFN-consortia software- CoDA-ZiFiT-ZFA and CoDA-ZiFiT-ZFN: we identified 163 and over 1,000 single zinc finger arrays (sZFAs) that bind sequences within the genes for the two respective RBC-receptors. Second, 2 and 18 paired zinc finger arrays (pZFAs) that are precursors for zinc finger nucleases (ZFNs) capable of cleaving the genes for darc and glycophorin-a were respectively assembled. Third, a mega-BLAST evaluation of the genome-wide cleavage specificity of this set of ZFNs was done, revealing alternate homologous nucleotide targets in the human genome other than darc or glycophorin A.
ZFNs engineered with these ZFA-precursors--with further optimization to enhance their specificity to only darc and glycophorin-a, could be used in constructing an experimental gene-based-malaria vaccine. Alternatively, meganucleases and transcription activator-like (TAL) nucleases that target conserved stretches of darc and glycophorin-a DNA may serve the purpose of abrogating invasion of RBCs by falciparam and vivax plasmodia species.
疟疾在全球范围内导致了巨大的人类发病和死亡。间日疟原虫和恶性疟原虫这两种疟原虫引发了超过75%的临床疟疾病例。到目前为止，针对疟疾的基于基因的策略仅应用于疟原虫物种及其蚊媒。这两种疟原虫的裂殖子分别通过趋化因子的达菲抗原受体（DARC）和血型糖蛋白A的O - 连接聚糖的唾液酸（SLC4A1）残基来靶向并侵入红细胞（RBC）。自然选择的达菲阴性黑人的红细胞对间日疟原虫的嗜性具有抗性。我们假设通过对红细胞受体进行靶向诱变来人为改变宿主途径，可能会消除或降低宿主对疟疾的易感性。作为实现这些概念的实验性第一步，我们旨在鉴定锌指阵列（ZFA），以构建靶向野生型宿主红细胞受体基因的锌指核酸酶（ZFN）。
电子基因与基因组信息学
利用智人darc和血型糖蛋白a的基因组上下文核苷酸序列，以及ZFN联盟软件CoDA - ZiFiT - ZFA和CoDA - ZiFiT - ZFN：我们鉴定出163个及1000多个单个锌指阵列（sZFA），它们分别与两种红细胞受体基因内的序列结合。其次，分别组装了2个和18个配对锌指阵列（pZFA），它们是能够切割darc和血型糖蛋白a基因的锌指核酸酶（ZFN）的前体。第三，对这组ZFN的全基因组切割特异性进行了mega - BLAST评估，揭示了人类基因组中除darc或血型糖蛋白A之外的其他交替同源核苷酸靶点。
用这些ZFA前体构建的ZFN，经过进一步优化以增强其对darc和血型糖蛋白a的特异性，可用于构建基于实验基因的疟疾疫苗。或者，靶向darc和血型糖蛋白a DNA保守区域的巨核酸酶和转录激活样效应物（TAL）核酸酶可用于消除恶性疟原虫和间日疟原虫对红细胞的侵袭。
