Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors

Abstract

Background: Herpes simplex type II (HSV-2) is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers) associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent) course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M) system, we previously identified the Escherichia coli restriction enzyme (REase) EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T) are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs) with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs) and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome.

Methods and results: Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,), and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class), 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00) at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the 5' and 3' ends of the HSV-2 genome (genomic context coordinates 0.02 and 0.98, respectively) were specificity sites of ZFNs suited for the complete excision of over 60% of HSV-2 genomic material from within infected human cells, through the process of non-homologous end joining (NHEJ). Furthermore, a model concerning a recombinant (ICP10-PK mutant) replication competent HSV-2 viral vector for delivering and transducing a diploid copy (or pair) of the HSV-2-genome-specific ZFN genotype within neuronal tissue, is presented.

Conclusion: ZFNs with specificity to HSV-2 genomic DNA that are precursors of novel host-genome expressed HSV-2 gene-therapeutics or vaccines were identified.

Figure 1

Schematics of HSV-II genome- cleavage by ZFN. This figure graphically illustrates the sites and frequency of ZFN cleavage of the HSV-2 genome by the 684 ZFNs identified. Cleavage was present at almost all positions within the HSV-2 genome. Note, however, that the highest incidence of HSV-2 genomic cleavage is situated within the last 30,950 base-pairs (154,784/5: ~between the genomic context coordinates 0.80 and 1.00) at the 3' end of the HSV-2 genome. That said, at approximately 3,094.9 bp (30,950/10) before and after the 5' and 3' ends of the HSV-2 genome (genomic context coordinates 0.02 and 0.98, respectively), there is another array of specificity sites for ZFNs that are potentially best suited for the complete excision of over 60% of HSV-2 genomic material from within infected human cells.