Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy

Abstract

Background: Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype.

Methods and results: First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells) and latent HIV-infected cell lines.

Conclusion: LV-2xZifHIV-polFN and LV- 2xZifHIV-1FN may offer the ex-vivo or even in-vivo experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action or disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.

Figure 1

A graphic map of the distribution of the recognized target DNA sites by the 114 multi-Zif-arrays, along the entire length of the SIV/HIV-pol-gene. The figure offers a detailed graphics illustration of the distribution of target DNA sites along the full 3,182 bp lengths of the SIV/HIV-pol gene recognized by all 114 multi-Zif-arrays. For details, see Table 1 and Additional file 1.

Figure 2

A graphic map of the distribution of the target-DNA sites recognized by the two ZFNs obtained along the entire length of the SIV/HIV-pol-gene. This figure offers a graphic map of the distribution of target DNA sites recognized and cleaved by our two ZFNs, along the full 3,182 bp lengths of the SIV/HIV-pol gene. For details, see Table 2 and Additional file 2.

Figure 3

A graphic map of the distribution of the target-DNA sites recognized by 15 ZFNs obtained along the entire length of the HIV-1 genome. This figure offers a graphic map of distribution of target DNA sites recognized and cleaved by the 15 ZFNs along the full 9,182 bp lengths of the HIV-1 genome. For details, see additional file 3.