Correspondence: AD Kelleher, National Centre In HIV Epidemiology and Clinical Research, University of New South Wales, 385 Victoria Street, Darlinghurst, New South Wales 2010, Australia. E-mail: t.kelleher@cfi.unsw.edu.au; DF Purcell, Department of Microbiology and Immunology, University of Melbourne, Melbourne, Australia. E-mail: dfjp@unimelb.edu.au

The chemokine receptor CCR5, together with CXCR4, are essential co-receptors with CD4 for HIV-1 entry into target cells. The initially transmitted virus is selected for CCR5 tropism during passage of HIV across the mucosa and the overwhelming majority of circulating viral strains preferentially use CCR5. Individuals who are homozygous for a naturally occurring 32 amino acid deletion mutation of the receptor (CCR532), are highly resistant to HIV infection and those that are heterozygotes have slower disease progression. Because of its important role, CCR5 has been successfully targeted for preventative and therapeutic interventions by both small molecule inhibitors and gene knockdown strategies. In a recent issue of Nature Biotechnology Perez et al.¹ report proof of principle experiments for an alternative approach to long-lived CCR5 inhibition using Zinc Finger nucleases (ZFN) to induce mutations in this gene.

ZFN are recombinant proteins that allow highly specific targeting of DNA disruption. These engineered proteins combine several Zinc Finger domains from sequence-specific DNA-binding proteins such as transcription factors, fused to a DNA nuclease FokI.² The helix of each of the fingers bind specific DNA sequences of 3 or 4 nucleotides, with their specificity determined by their amino acid sequence.³ Multimerizing modules of three or more fingers allows high level specificity of DNA-binding.⁴ This observation indicated that two carefully designed Zinc Finger—FokI units each targeting specific juxtaposed sites in the DNA duplex would assemble a functional FokI endonuclease dimer that could cleave the intervening DNA producing a ragged double-stranded cleavage within a specific gene. DNA strand repair by non-homologous end joining would result in small insertions and deletions that would mutate gene function. The design of recombinant Zinc Finger proteins is still being optimized, but libraries of amino acid sequences and their complementary codon-binding sequences that enable precise targeting of mammalian genes are being developed⁵ (Figure 1a).

Figure 1.

(a) Action of Zinc Finger nucleases (ZFN). ZFN are recombinant proteins that consist of multimers of single Zinc Finger protein (ZFP) shown as ribbon diagrams each containing an helix and zinc ion (blue circle) fused to a restriction enzyme, FokI (red). Each of these Zinc Fingers recognize a specific set of nucleotides (3–4) on either sense or antisense strand of the DNA. Here eight different Zinc Finger proteins with differing specificities are shown. Multimerization of Zinc Fingers confers high specificity of nucleotide sequence recognition. By modular design, two complementary ZFN target FokI to the same region of the sense (light blue) and antisense (dark blue) strand of DNA. This induces ragged cleavage of both strands. Repair mechanisms result in deletion of single to multiple nucleotides from both strands. Non-homologous recombination can also result in insertions (orange and red strands). Both of these mechanisms result in disrupted gene function. (b) Adenovirus delivery of ZFN-targeting CCR5. HIV uses a combination of CD4 and CCR5 to enter the cell. A recombinant adenovirus is used to deliver the coding sequence of the ZFN. Its payload is transferred to the nucleus and transcribed to mRNA coding the ZFN, which is then synthesized in the cytoplasm. The ZFN then enters the nucleus and specifically targets and disrupts the CCR5 gene as in (a). Defective CCR5 protein is degraded in the cytoplasm and is therefore not expressed on the cell membrane so HIV entry fails.

Full figure and legend (364K)

Perez et al.¹ have recently demonstrated that ZFN technology has applicability for specific disruption of the human CCR5 gene to alter the outcome of HIV-1 infection of CD4+ T cells.¹ They designed a pair of ZFN that target a site within the CCR5 gene upstream of where the naturally occurring 32 deletion occurs. The intention was to disrupt CCR5 in a similar manner in wild type CD4+ T cells and recapitulate robust HIV resistance. Each of the paired ZFN contained four finger modules targeting four separate codons separated by five intervening nucleotides where double-stranded ragged cleavage occurred after FokI endonuclease assembled into a functional dimer.

Delivery of these constructs in vitro from plasmid or recombinant adenoviral vectors resulted in decreased surface expression of CCR5 on the cell predominantly through small frame-shifting deletions but also insertions occurring after non-homologous end joining at a frequency of 22% in one allele and 8% in both alleles (Figure 1b). This resulted in partial resistance of the treated cultures to HIV-1 infection. This was demonstrated in both T cell lines and primary human T cells. Further ex vivo transduction of human CD4+ T cells and then passage through a mouse model demonstrated preferential survival of adenoviral vector transduced and CCR5 disabled CD4+ T cells in the presence of HIV-1 infection.

The technology therefore works and this is an impressive proof of principle demonstration of ZFN disruption of an important gene. However, in terms of HIV therapy or prevention strategies, considerably more work is required. Firstly, the proportion of cells transduced is reasonably low and even under selective pressure of the virus the increase in their number is modest. This results in reductions in viral load in the order of 10-fold. However, compared to current antiretroviral therapies which result in 10 to 100-fold reductions as single agents, these decreases are modest and it remains to be seen if the long-term protective benefits from genetic mutation of CCR5 are therapeutically competitive. These recombinant proteins will require considerable further development. All CCR5 therapies have the potential to select for CXCR4 tropic virus that may have higher pathogenicity. As acknowledged by the authors,¹ only acute infection models are employed in these studies. The effectiveness of this type of intervention in chronic infection requires further evaluation; however, these assessments will be difficult in the absence of small animal models of chronic HIV infection. Furthermore, HIV disease involves many cell types in addition to circulating CD4⁺ T lymphocytes, such as gut-associated T cells, tissue macrophage and brain microglia and astrocytes. The ability to modify CCR5 in these cells, especially in the context of pre-existing HIV infection, needs to be addressed.

Little data are presented on the toxicity of the approach. ZFN have problems with cytotoxicity in other systems. The exact cause of this is unclear, but may be due, at least in part, to off-target effects. Increasing the number of finger modules incorporated into the ZFN can reduce these effects. The use of four fingers improves specificity in a manner that could be rigorously validated against the sequence of the entire human genome. However, others suggest that separate causes of cytotoxicity exist when the number of modules increases beyond three.⁵ Further, the more profound knock down of CCR5 may in itself have toxicities. Although it is generally accepted that those with the CCR532 mutation are essentially immunologically normal, there are now reports that these individuals have a more severe course when infected with West Nile virus and other flaviviruses including tick borne encephalitis, but may have less severe courses of infections with Chlamydia trachomatis, Hepatitis B and C and trypanosoma cruzi inflammatory cardiac disease (reviewed in⁶). Also, trials of CCR5 inhibitors for HIV-1 infection have revealed possible increased rates of upper respiratory tract infection, Herpes infections and malignancies such as lymphoma, but these observations require confirmation. Clearly, the effects of chronic knock down of CCR5 in mature organisms requires further investigation.

Despite these limitations, this report suggests that in the medium term, ZFN may have a substantive role in the delivery of specific targeted gene therapy. Although this study shows effective disruption of a gene, other studies have demonstrated that the same sort of targeted double-stranded DNA disruption can be used to substantively increase the rates of homologous recombination resulting in corrections of targeted mutations if the ZFN are delivered in conjunction with a plasmid containing the wild type gene sequence.⁷ This has the potential to increase rates of gene repair and this has been exploited to target repair of the common cytokine chain disrupted in X-linked severe combined immunodeficiency in vitro.⁷ While the full potential of ZFN in gene disruption and gene correction is yet to be realized, the article by Perez et al.¹demonstrates another potential application of this technology that could be exploited to block the action of a number of receptors or perhaps for inactivating latent forms of the HIV-1 provirus.