Chapter 3 - Engineering Sequence-Specific DNA Binding Proteins for Antiviral Gene Editing

Abstract

Customizing DNA-binding proteins to inactivate viral gene expression has advanced at a rapid pace and the technology has considerable potential for treating viral infections. Viruses that cause serious public health problems, and which are amenable to therapeutic effects of DNA-binding proteins, include human immunodeficiency virus (HIV)-1, hepatitis B virus, herpes simplex virus, and human papilloma virus. The four main classes of DNA-targeting proteins are derivatives of zinc finger proteins, transcription activator-like effectors (TALEs), homing endonuclease (HEs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) systems. Engineered proteins may be functionalized by addition of nuclease moieties or through the coupling of transcriptional suppressing domains, such as the Krüppel-associated box. Site-specific cleavage of target DNA may induce targeted mutation as a result of error-prone repair by nonhomologous end joining or through homology-directed repair. The CRISPR/Cas system has the significant advantage of being dependent on the use of an RNA guide to direct the Cas protein to cleave targets at specific sites. The RNA-guided nucleases are easier to engineer than are the proteins that interact directly with DNA sequences, and this has been the main reason for the enormous popularity of CRISPR/Cas systems. Concerns about off-target effects of engineered CRISPR/Cas nucleases are currently being addressed by ingenious methods that improve guide RNA specificity and also through engineering of two “nickases” that only cleave both DNA strands when juxtaposed on a larger cognate. Zinc finger nucleases (ZFNs) have been studied thoroughly and are now at an advanced stage of clinical development for the disabling of CCR5 to treat HIV-1 infection. However, a difficulty with engineering ZFNs has been the poor efficacy that results from context-dependent positioning of the finger modules. TALENs do not suffer from this problem, they are easier to generate than ZFNs, and they have been reported to act with greater target specificity than ZFNs. Advances with tailoring HEs to achieve target-specific cleavage have also been significant, but the coupled sequence-binding and enzymatic cleavage functions restrict the ease with which HEs may be engineered. The small size of HEs is a distinct advantage for their delivery in a therapeutic context. The gene editing function of designer DNA binding proteins is powerful and will no doubt have therapeutic application for viral infections that have DNA replication intermediates.