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Zinc Finger Nuclease Treatment of HIV

Since antiretroviral therapy requires a life-long treatment regimen, research to find more permanent cures for HIV infection is currently underway. It is possible to synthesize zinc finger nucleotides with zinc finger components that selectively (almost selectively) bind to specific portions of DNA. It has also been observed that 20% of the Caucasian population possess what is referred to as the CCR5-Δ32 mutation (frequency of 0.0808 for homozygous allele) that prevents the CCR5 protein, which is the main means of viral access into the cell, from being expressed on the surface of their T-cells. Individuals who are homozygous for this mutation are immune to HIV strains that utilize the CCR5 receptor in order to gain access to the cell while those who are heterozygous for this mutation have been found to have reduced plasma viral load in addition to a delayed progression to AIDS. By combining these facts, researchers have proposed a novel method of treatment for HIV. This method attempts to treat the infection by introducing the CCR5-Δ32 mutation and consequently, resulting in the expression of nonfunctional CCR5 co-receptors on CD4+ T cells, providing immunity against infection.

The zinc finger nucleases that have been synthesized for this treatment are manufactured by combining FokI Type II restriction endonucleases with engineered zinc fingers. The number of zinc fingers attached to the endonuclease controls the specificity of the ZFN since they are engineered to preferentially bind to specific base sequences in DNA. Each ZFN is made up of multiple zinc fingers and one nuclease enzyme.

Zinc Finger Binding

The exact constitution of the ZFNs that are to be used to treat HIV is still unknown. The binding of ZFNs for the alteration of the Zif268 genelink, however has been well-studied and is outlined below in order to illustrate the mechanism by which the zinc finger domain of ZFNs bind to DNA.

The amino terminus of the alpha helix portion of zinc fingers targets the major grooves of the DNA helix and binds near the CCR5 gene positioning FokI in a suitable location for DNA cleavage.

Zinc fingers are repeated structural protein motifs with DNA recognition function that fit into the major grooves of DNA. Three zinc fingers are positioned in a semi-circular or C-shaped arrangement. Each zinc finger is made up of anti-parallel beta sheets and an alpha helix, held together by a zinc ion and hydrophobic residues.

The zinc atom is constrained in a tetrahedral conformation through the coordination of Cys3, Cys6, His19 and His23 and Zinc – Serine bond distance of 2.30 +/- 0.05 Angstroms and Zinc – Nitrogen bond distances of 2.0 +/- 0.05 Angstroms.

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 arg-gua bond is stabilized by aspartic acid from a second residue, which positions the long chain of arginine through a hydrogen bond salt bridge interaction.

In residue 3 of the second (i.e. middle) zinc finger, histidine49 forms a hydrogen bond with a co-planar guanine in base pair 6. 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.

Fingers 1 and 3 each have an arginine residue that donates a pair of charged hydrogen bonds to nitrogen 7 and oxygen 6 of the guanine residue at the 5’ end, enhancing the site recognition sequence of zinc fingers.

Contacts with DNA backbone

The histidine coordinated to the zinc atom, which is also the seventh residue in the alpha helix of the zinc fingers, coordinates the zinc ion through its Nε and hydrogen bonds with phosphodiester oxygen through Nδ on the primary DNA strand.

In addition to histidine, a conserved arginine on the second beta strand of the zinc fingers makes contact with the phosphodiester oxygen on the DNA strand.

Also serine 75 on the third finger hydrogen bonds to the phosphate between base pairs 7 and 8, as the only backbone contact with the secondary strand of DNA.

Nuclease Dimerization and Cleavage

It has been discovered that FokI has no intrinsic specificity in its cleavage of DNA and that the zinc finger recognition domain confers selectivity to zinc finger nucleases.

Specificity is provided by dimerization, which decreases the probability of off-site cleavage. Each set of zinc fingers is specific to a nucleotide sequence on either side of the targeted gene 5-7 base pair separation between nuclease components.

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. This break is repaired by the natural repair mechanisms of the cell, specifically non-homologous end joining.

Introducing the CCR5 Mutation

Introducing genome alterations depends upon either of the two natural repair mechanisms of a cell: non-homologous end joining (NHEJ) and homology-directed repair (HDR). Repair through NHEJ comes about by the ligation of the end of the broken strands and, upon the occurrence of an error, can produce small insertions and deletions. HDR, on the other hand, makes use of a homologous DNA strand in order to repair and gene and making use of this repair mechanism and providing the desired nucleotide sequence allows for gene insertion or modification.

The main DSB repair pathway in mammals, which occurs in the absence of a homologous nucleotide base sequence can be used by a homologous recombination mechanism is through non-homologous end joining (NHEJ). NHEJ, although capable of restoring a damaged gene, is error-prone. DSBs 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. In order to accelerate this process, exonucleases can be introduced to digest the ends of the strands generated at DSBs.

Limitations

Increasing the number of zinc fingers increases the specificity by increasing the number of base pairs that the ZFN can bind to. However too many zinc fingers can lead to off-target binding and thus offsite cleavage. This is due to an increased likelihood of zinc fingers binding to parts of the genome outside of the gene of interest.

No known side effects have been noted in current ZFN treatments that focus on introducing a mutation in the CCR5 gene as no known side effects result from altering CCR5. However, there are strains of HIV that are able to use CXCR4 to enter the host cell, therefore bypassing CCR5 altogether. In theory, the same gene editing technology can be applied to CXCR4, however the side effects of such an alteration remain unknown.

Several issues exist with this experimental treatment. These include unforeseen difficulties in selecting the most suitable repair pathway and ensuring that the desired repair pathway is used to repair the DSB.[18]. . In addition, another issue with the disruption of the CCR5 gene is that CXCR4-specific or dual-tropic strains are still able to access the cell. .

To employ the ZFNs in clinical settings the following criteria need to be met: i)	High specificity of DNA-binding – Correlates with better performance and less toxicity of ZFNs. Engineered ZFNs take into account positional and context-dependent effects of zinc fingers to increase specificity . ii)	Enable allosteric activation of FokI once bound to DNA in order for it to produce only the required DSB. iii)	In order 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 . These include adeno-associated virus vectors, integrase-deficient lentiviral vectors and adenovirus type 5 vectors . iv)	Transient expression of ZFNs would be preferred over permanent expression of these proteins in order to avoid ‘off-target’ effects. 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.

Administration of Treatment

The cells in which the mutations are induced ex vivo are filtered out from lymphocytes by apheresis to produce analogous lentiviral engineered CD4+ T-cells. These are re-infused into the body as a single dose of 1 X 1010 gene modified analogous CD4+ T-cells. A viral vector is used to deliver the ZFNs that will induce the desired mutation into the cells. Conditions that promote this process are carefully monitored ensuring the production of CCR5 strain HIV-resistant T cells. .

The Berlin Patient

Timothy Ray Brown, who underwent a bone marrow transplant in 2007 to treat leukemia, was also simultaneously suffering from HIV. This is a result of the bone marrow donor being homozygous for the CCR5-Δ32 mutation. This mutation conferred a resistance to HIV in the recipient, eventually leading to an almost complete disappearance of HIV levels in his body. After nearly 2 years without antiretroviral drug therapy, HIV could still not be detected in any of his tissues .Though this method has been effective in reducing the level of infection, the risks associated with bone marrow transplants outweigh its potential value as an efficient treatment for HIV.