Zinc finger (ZF) domains mediate nucleotide-specific binding of proteins to DNA, a property that defines a large family of DNA binding proteins 58
. Each finger makes contact with a separate DNA triplet, and natural or recombinant ZFs have been created that can recognize almost any triplet 59
. The modular nature of the ZFs allows them to be joined in useful combinations. Typically, three ZFs are combined to bind to a specific 9-bp DNA sequence, and these ZFs have been coupled to various functional domains to create artificial transcription factors that can activate or repress gene transcription with remarkable promoter specificity 60
. Zinc fingers have also been fused to the nuclease domain of the restriction enzyme FokI to cleave double-stranded DNA at specific sequences 61
. The nuclease domain must dimerize to cleave DNA, and because the dimer interface is weak, two nuclease domains are typically brought into close proximity by pairs of ZFs binding to neighboring 9-bp sites, spaced 6-bp apart () 62, 63
. In this configuration, the engineered ZF nuclease (ZFN) recognizes a specific 18-bp sequence, which is long enough, by a few orders of magnitude, to be unique in the human genome. Because of this specificity, this same technology could be used to distinguish between human and virus DNA.
Several groups have used recombinant ZF proteins to control aspects of the viral life cycle. ZF proteins fused to the KOX-1 repression domain were created that targeted the HSV-1 ICP4 promoter 64
. These proteins bound the promoter with nanomolar affinity, and one was able to significantly repress VP16-activated transcription in vitro
. This ZF-KOX-1 fusion, when delivered in trans
into HSV-1 infected cells, was able to limit HSV-1 replication and reduced viral titer by 90%. In a similar strategy, recombinant ZF proteins were designed to recognize the HPV-18 replication origin 65
. When expressed in vitro
, these ZFs were able to compete with the replication protein E2 for binding to viral DNA. This competitive antagonism led to reduced HPV replication in transient replication assays in mammalian cells. By fusing the origin-targeted ZF protein to a nuclease domain this ZFN was able to cleave viral DNA and reduce viral replication in cultured cells 66
. These experiments demonstrate that ZFN can effectively target and eliminate viral DNA in mammalian cells.
It may be feasible to deliver a therapeutic virus-specific ZFN in trans to eradicate latent viral DNA. However, delivery of the ZFNs to all latently infected cells is technically challenging. Alternatively, virus-specific ZFNs could be delivered using the viral genome itself and serve as a vaccine. In the ZFN-vaccine strategy, ZFNs targeting sequences for viral replication and other essential viral processes would be introduced into the viral genome (). Following inoculation, immunogenic viral genes and virus-specific ZFNs would be expressed. While the viral proteins would stimulate a natural immune response, the ZFNs would cleave viral DNA, and limit replication. ZFN-LAVs have potential both as prophylactic vaccines, protecting against wild-type challenge, as well as therapeutic vaccines, delivering ZFNs to cells already harboring latent viral DNA.
The immunogenicity of ZFN vaccines can be controlled by temporal and spatial regulation of ZF expression to balance viral protein expression with the ability of the ZFNs to eliminate all replication-competent viral DNA. This could best be accomplished using promoters that are temporally controlled by the virus itself. For instance, herpesvirus gene transcription occurs in at least three distinct stages; immediate-early (before most of viral protein synthesis), early (before viral replication), and late (after viral replication begins) 67
. Other DNA viruses for which ZFNs would be useful are similarly regulated. There is also the potential to encode ZFNs behind inducible promoters, so that ZFN expression would commence upon the administration of a small molecule 68
. Nuclease activity can also be controlled directly by addition of small molecule-sensitive residues to the ZFN 69
. These strategies would provide an ideal way to optimize the balance between ZFN-virus replication and nuclease activity.
The ability to create a ZFN-vaccine that can prevent and eliminate persistent viral infections is a long way from being realized. As with any LAV, safety issues are always a concern. The ZFN vaccine approach would likely be limited to non-integrating, DNA viruses, as random breaks in host chromosomal DNA caused by ZFN-cleavage of integrated viral DNA could be catastrophic. There are many non-integrating human viruses, includes the herpes-, polyoma-, adeno-, and papillomaviruses, that establish a persistent infection and provide particularly difficult challenges for the treatment of their respective diseases. ZFN-based vaccines may offer a way to prevent or eliminate these hard-to-treat latent infections. Reversion to wild type is another concern, but the risk can be reduced by including ZFNs against multiple, essential viral sequences to ensure that the intrinsic mutation rate of the virus will not allow the mutation of every ZFN target site. It is also possible that DNA cleaved by ZFNs could be repaired via homologous recombination using uncleaved viral genomes. However, if the sequence were repaired accurately, it would be subject to repeated cleavage; if it is repaired inaccurately, the virus should not be viable due to mutation of an essential sequence. Ideally, we will arrive at a live virus strain that will have limited replication, not establish latency, and elicit a protective immune response. In essence, we would turn an otherwise detrimental latent infection into an asymptomatic, acute infection.