Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for <it>in-situ</it> reversal of associated cervical neoplasia

<p>Abstract</p> <p>Background</p> <p>Human 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).</p> <p>Methods and results</p> <p>Using 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/pZFA_HpV/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 (sZFA_E6) to aid the future quest for paired ZFAs to target E6 gene sequences in both HPV types studied (pZFA_E6). 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 <it>FokI</it> endonuclease F_N to the gene sequences of a member of the paired-HPV-binding ZFAs (pZFA_HpV/E7 + F_N), and (ii) delivery of the same into precancerous lesions using HPV-derived viral plasmids or vectors.</p> <p>Conclusions</p> <p>With 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.</p

Conservation significance of intact forest landscapes in the Scandinavian Mountains Green Belt

Context: As forest harvesting remains high, there is a crucial need to assess the remaining large, contiguous and intact forests, regionally, nationally and globally. Objectives: Our objective was to analyze the spatial patterns and structural connectivity of intact and primary forests in northern Sweden with focus on the Scandinavian Mountain region; one of the few remaining large European intact forest landscapes. Methods: Over 22 million ha with 14.5 million ha boreal and subalpine forest and with data consisting of a 60-70 year retrospective sequence, we analyzed distribution, density and connectivity of forests that have not been clear cut, using moving window and landscape analyzes derived from Circuitscape. Results: We revealed a contiguous, connected and semi-connected intact forest landscape forming a distinct Green Belt south to north along the mountain range. Almost 60% of the forestland remains intact, including contiguous clusters 10,000 ha and larger. The connectivity is particularly high in protected areas with primary forests outside contributing substantially to overall connectivity. We found gaps in connectivity in the southern parts, and furthermore low or absent connectivity across the whole inland and coastal areas of northern Sweden. Conclusions: Given its ecological values, the Scandinavian Mountains Green Belt is a key entity supporting ecological legacies, boreal biodiversity and ecosystem services, resilience and adaptive capacity, which needs to be safeguarded for the future. On the very large areas outside the mountain region, forestlands are severely fragmented, connectivity values are lost, and forest landscape restoration is needed for conservation and functional green infrastructure.