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

Abstract

BackgroundInfection 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 ResultsFirst, 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.ConclusionLV-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.

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…However, the results from in vitro studies are very promising [223] , with CRISPR editing able to excise the provirus from infected cells, and thus able to target latent proviruses [224] . ZFNs have also been used to target the provirus, using lentivirus to achieve stable expression of the nucleases [225] . However, the abovementioned ZFN-related clinical trials used adenovirus vectors.…”

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…Recently, several research groups have successfully applied the type II CRISPR systemSpCas9 protein from Streptococcus pyogenes with guided RNA (gRNA)-for targeted genome editing in diverse cell types and organisms, including human cells [12][13][14] . Most recently, a couple of studies have demonstrated the excision of the HIV-1 provirus from the host cell genome using gene-editing tools [15][16][17][18] . Here we apply the CRISPR/Cas9 system to directly target and disrupt the reverse-transcribed products of the lentiviral RNA genome during their life cycle within host cells.…”

MOLECULAR ANALYSIS OF A COMPLETE HIV-1 Pol GENE ISOLATED IN CENTRAL JAVA, INDONESIA

“…Protein physicochemical properties, like polarity, hydropathicity, charge, volume, aromaticity, aliphaticity and hydrogenation are shown to play a role in determining the rate and pattern of protein evolutions (Xia and Li, 1998).In present study, physicochemical profiling was perfomed using ProtParamserver to measure the molecular weight (Mw), estimated half-life, instability index (II), aliphatic index, and grand average of hydropathicity (GRAVY) of each protein encoded by our sequence (Table 2). Moreover, these datawere relevant for estimating the in-vivo ideal temperatures of function, solubility patterns in aqueous solution, and life-expectancies of the functional protein in vivo (Wayengera, 2011). Table 2.…”