| Summary: | <p>Abstract</p> <p>Background</p> <p>Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that <it>ex-vivo </it>or even <it>in-vivo </it>abolition <it>or </it>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 <it>arrays </it>and <it>nucleases </it>(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.</p> <p>Methods and Results</p> <p><it>First, </it>we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) <it>arrays </it>(ZFAs or Zif<sub>HIV-pol</sub>) and (b) two zinc-finger <it>nucleases </it>(ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (Zif<sub>HIV-pol</sub>F<sub>N</sub>). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (Zif<sub>HIV-1</sub>F<sub>N</sub>). <it>Second, </it>a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either Zif<sub>HIV-pol</sub>F<sub>N </sub>or Zif<sub>HIV-1</sub>F<sub>N </sub>chimeric genes (termed <b>LV- 2xZif</b><sub><b>HIV-pol</b></sub><b>F</b><sub><b>N </b></sub>and <b>LV- 2xZif</b><sub><b>HIV-1</b></sub><b>F</b><sub><b>N, </b></sub>respectively) is proposed. <it>Third, </it>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.</p> <p>Conclusion</p> <p><b>LV-2xZif</b><sub><b>HIV-pol</b></sub><b>F</b><sub><b>N </b></sub>and <b>LV- 2xZif</b><sub><b>HIV-1</b></sub><b>F</b><sub><b>N </b></sub>may offer the <it>ex-vivo </it>or even <it>in-vivo </it>experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action <it>or </it>disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.</p>
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