Abstract
BackgroundHuman papillomavirus (HPV) types 16 and 18 are the high-risk, sexually transmitted infectious causes of most cervical intraepithelial neoplasias (CIN) or cancers. While efficacious vaccines to reduce the sexual acquisition of these high-risk HPVs have recently been introduced, no virus-targeted therapies exist for those already exposed and infected. Considering the oncogenic role of the transforming (E6 and E7) genes of high-risk HPVs in the slow pathogenesis of cervical cancer, we hypothesize that timely disruption or abolition of HPV genome expression within pre-cancerous lesions identified at screening may reverse neoplasia. We aimed to derive model zinc finger nucleases (ZFNs) for mutagenesis of the genomes of two high-risk HPV (types 16 & 18).Methods and resultsUsing ZiFiT software and the complete genomes of HPV types16 and 18, we computationally generated the consensus amino acid sequences of the DNA-binding domains (F1, F2, & F3) of (i) 296 & 327 contextually unpaired (or single) three zinc-finger arrays (sZFAs) and (ii) 9 & 13 contextually paired (left and right) three- zinc-finger arrays (pZFAs) that bind genomic DNA of HPV-types 16 and 18 respectively, inclusive of the E7 gene (s/pZFAHpV/E7). In the absence of contextually paired three-zinc-finger arrays (pZFAs) that bind DNA corresponding to the genomic context of the E6 gene of either HPV type, we derived the DNA binding domains of another set of 9 & 14 contextually unpaired E6 gene-binding ZFAs (sZFAE6) to aid the future quest for paired ZFAs to target E6 gene sequences in both HPV types studied (pZFAE6). This paper presents models for (i) synthesis of hybrid ZFNs that cleave within the genomic DNA of either HPV type, by linking the gene sequences of the DNA-cleavage domain of the FokI endonuclease FN to the gene sequences of a member of the paired-HPV-binding ZFAs (pZFAHpV/E7 + FN), and (ii) delivery of the same into precancerous lesions using HPV-derived viral plasmids or vectors.ConclusionsWith further optimization, these model ZFNs offer the opportunity to induce target-mutagenesis and gene-therapeutic reversal of cervical neoplasia associated with HPV types 16 & 18.
Highlights
Human papillomavirus (HPV) types 16 and 18 are the high-risk, sexually transmitted infectious causes of most cervical intraepithelial neoplasias (CIN) or cancers
With further optimization, these model zinc finger nucleases (ZFNs) offer the opportunity to induce target-mutagenesis and gene-therapeutic reversal of cervical neoplasia associated with HPV types 16 & 18
The aim of this study was to identify and model deoxyribonucleic acids (DNA)-binding domains of zinc finger arrays (ZFAs) as precursors for (i) synthesizing ZFNs for target mutagenesis of two highrisk HPV –genomes, and development of a novel gene-therapeutic armament for reversing the primary oncogenic processes leading to cervical neoplasia
Summary
Human papillomavirus (HPV) types 16 and 18 are the high-risk, sexually transmitted infectious causes of most cervical intraepithelial neoplasias (CIN) or cancers. There are over 200 HPV types, and 30 to 40 of these are sexually transmitted, only types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 are denoted ‘highrisk’ HPVs [2,3] Infection with these HPV types may lead to the development of cervical, penile or anal intraepithelial neoplasia. When present in normal levels, p53 upregulates the p21 protein, which blocks the formation of the cyclin D/Cdk complex This prevents the phosphorylation of retinoblastoma protein (pRB) and in turn halts cell cycle progression by preventing the activation of the eukaryotic transcription factor E2F. Cervical cancer cells can be said to be addicted to and or to thrive on the expression of the E6 and E7 proteins of high-risk HPVs
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