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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 Zif HIV-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 (Zif HIV-pol F N ). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (Zif HIV-1 F N ).  Second,  a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either Zif HIV-pol F N  or Zif HIV-1 F N  chimeric genes (termed  LV- 2xZif   HIV-pol   F   N   and  LV- 2xZif   HIV-1   F   N,   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-2xZif   HIV-pol   F   N   and  LV- 2xZif   HIV-1   F   N   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.

Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy