Since antiretroviral therapy requires a lifelong treatment regimen, research to find more permanent cures for HIV infection is currently underway.
It has also been observed that 20% of the Caucasian population possess a mutation, called CCR5-Δ32 (frequency of 0.0808 for homozygous allele), that prevents the CCR5 chemokine receptor protein, which is the main means of viral access into the cell, from being expressed on the surface of their CD4+ T-cells.
[13] The authors of this work have drawn their inspiration from the innate defense mechanism against bacteria-infecting-viruses called bacteriophages, present amongst those bacteria endowed with restriction modification (R-M) systems.
These bacteria secrete a restriction enzyme (REase) that recognizes and repetitively cleaves around palindromic sequences within the xenogenic DNAs of the bacteriophages or simply phages, until the same is disabled.
It should not be surprising, therefore, that the initial work leading to the application of ZFN technology in this manner revolved around and involved the isolation and testing of HIV/SIV targeting bacteria-derived REases, whose non-specificity (due to their short recognition sequences) unfortunately, rendered them toxic to the host genome.
The latter-potential host-genome toxicity posed by the raw bacteria-derived REases limited their application to ex-vivo modalities for HIV prevention, namely synthetic or live microbicides.
Similar work is ongoing for high-risk HPVs (with the intent of reversing cervical neoplasia) [14] as well as with HSV-2 (with the goal of achieving a complete cure for genital herpes) [15][16][17][18][19][20][21][22][23] The FokI catalytic domain must dimerize to cleave the DNA at the targeted site, and requires there to be two adjacent zinc finger nucleases (see picture), which independently bind to a specific codon at the correct orientation and spacing.
[26][27][28] Each zinc finger has an arginine (arg) amino acid protruding from the alpha helix, which forms a hydrogen bond with Nitrogen 7 and Oxygen 6 of the guanine (gua) that is located at the 3’ end of the binding site.
The stacking of Histidine against Thymine in base pair 5 limits the conformational ability of Histidine49 leading to increased specificity for the histidine-guanine hydrogen bond.
Each set of zinc fingers is specific to a nucleotide sequence on either side of the targeted gene 5-7 bp separation between nuclease components.
[9] The dimerization of two ZFNs is required to produce the necessary double-strand break within the CCR5 gene because the interaction between the FokI enzyme and DNA is weak.
[30] DSB are, therefore, introduced into the gene until an error in its repair occurs at which point ZFNs are no longer able to bind and dimerize and the mutation is complete.
[35] iii) To deliver two different zinc finger nuclease subunits and donor DNA to the cell, the vectors that are used need to be improved to decrease the risk of mutagenesis.
[35] v) During gene targeting, genotoxicity associated with high expression of ZFNs might lead to cell apoptosis and thus needs to be thoroughly verified in vitro and in vivo transformation assays.
[38][39] Though this method has been effective at reducing the level of infection, the risks associated with bone marrow transplants outweighs its potential value as a treatment for HIV.