**5. Repairing the** *CFTR* **mutation: Gene correction with genome-editing nucleases**

With residual limitations in optimizing gene and transcript supplementation therapies for CF lung disease, a new field has begun to emerge: aiming to correct, rather than supplement, the defective gene. Compared to gene or transcript replacement approaches, "gene correction" aims to replace the defective portion of the CFTR gene with a normal allele at its natural chromosomal location (Figure 3). The repair of a mutant gene directly at its original locus has two major advantages. Most importantly, the corrected gene remains under control of its endogenous promoter, hence assuring life-long expression and native regulation in the cell. Moreover, depending on the delivery vehicle(s) used, gene correction has the potential to avoid the involvement of foreign DNA, thus reducing the risk of insertional mutagenesis.

**Figure 3. Gene correction approaches aim to repair the** *CFTR* **gene mutation directly at the endogenous chromoso‐ mal locus.** The use of mRNA-encoded site-specific nucleases (SSE) can introduce double-strand breaks (DSB) near the genetic defect(s). If a donor vector with corrected sequence is also delivered to the cell, it can be utilized as a template for homologous repair (HR) as the cell works to repair the DSB (**A**). Successful homology-directed repair (HDR) will result in a corrected chromosomal locus, even after the SSE and donor template delivery vehicles are no longer ex‐ pressed (**B**).

Several lines of investigation into viable gene correction approaches have been pursued. These involve the use of genome-editing nucleases, such as ZFNs, TALENs, or CRISPR-based systems, to take advantage of the cell's natural damage repair pathways. In this strategy, delivery of a site-specific-endonuclease (SSE) or SSE pair elicits a double-strand break (DSB) in the defective gene near the site of an unwanted mutation or sequence, initiating cellular repair mechanisms including homologous recombination (HR) and non-homologous endjoining (NHEJ).

NHEJ, an error-prone process, can be utilized to initiate mutations that essentially disrupt or knock out an undesirable gene. As NHEJ repairs the DSB by ligating the broken strands together, this process commonly results in small insertions or deletions of base pairs, known as indels. The generation of indels at the repair site can cause frame-shift mutations that prevent the protein from being properly transcribed and translated. This concept was dem‐ onstrated in 2008, when investigators utilized ZFNs and NHEJ to disrupt the HIV co-receptor, *CCR5*, rendering human CD4+ T cells more difficult for the HIV virus to transfect [32]. Through this mechanism, multiple nuclease pairs can also be utilized to create two DSBs, where NHEJ may completely cut out large segments of unwanted genomic sequence [33,34].

Alternatively, a donor template can be delivered to the cell in addition to the nuclease(s), and used as a guide for directing HR, in a process referred to as homology-directed repair (HDR). In HDR, an extra-chromosomal donor fragment or "repair template" contains regions of significant homology up- and downstream of the DSB site. In between the homology arms, the repair template houses the desired, corrected sequence. Once the nuclease has cleaved, the regions of homology will be used as a template for rebuilding the site. As a result, the mutationfree sequence housed between homology arms is incorporated into the chromosome (Figure 3).

In cases where the patient is homozygous for the target allele, the uncleaved copy of the allele may be favored as a template for HR, decreasing efficiency. As such, it is important to pro‐ vide the repair template in excess, to ensure that the target cell favors the repair template over the sister chromatid. It is also important to note that even when a repair template is provided in excess for HR, SSE binding and cleavage can also occur at off-target sites, which may initiate NHEJ.In the case of anoff-target cutting event, NHEJ cancauseunexpectedmutations thatmay beharmfultothe cell.Asaresult,potentialoff-targetbindingsitesoftheSSEshouldbepredicted *in silico* and subsequently sequenced to monitor for deleterious off-target effects. Overall, HDR can be utilized to correct individual point mutations, as well as to insert larger fragments, such as complete copies of functional cDNA, into desired sites. To date, gene correction has been achieved *in vitro*, *ex vivo*, as well as directly in murine liver and lung [35–37].

Each of the available nuclease technologies utilizes a different method for recognizing specific sites and initiating DSB cleavage. In the following section, we will discuss the various mech‐ anisms of action, as well as the pros and cons of each technology. It is important to note that in order to be safe for use in a clinical setting, nuclease technology must meet the following criteria:

**•** Minimal off-target activity:

aims to replace the defective portion of the CFTR gene with a normal allele at its natural chromosomal location (Figure 3). The repair of a mutant gene directly at its original locus has two major advantages. Most importantly, the corrected gene remains under control of its endogenous promoter, hence assuring life-long expression and native regulation in the cell. Moreover, depending on the delivery vehicle(s) used, gene correction has the potential to avoid

**Figure 3. Gene correction approaches aim to repair the** *CFTR* **gene mutation directly at the endogenous chromoso‐ mal locus.** The use of mRNA-encoded site-specific nucleases (SSE) can introduce double-strand breaks (DSB) near the genetic defect(s). If a donor vector with corrected sequence is also delivered to the cell, it can be utilized as a template for homologous repair (HR) as the cell works to repair the DSB (**A**). Successful homology-directed repair (HDR) will result in a corrected chromosomal locus, even after the SSE and donor template delivery vehicles are no longer ex‐

Several lines of investigation into viable gene correction approaches have been pursued. These involve the use of genome-editing nucleases, such as ZFNs, TALENs, or CRISPR-based systems, to take advantage of the cell's natural damage repair pathways. In this strategy, delivery of a site-specific-endonuclease (SSE) or SSE pair elicits a double-strand break (DSB) in the defective gene near the site of an unwanted mutation or sequence, initiating cellular repair mechanisms including homologous recombination (HR) and non-homologous end-

NHEJ, an error-prone process, can be utilized to initiate mutations that essentially disrupt or knock out an undesirable gene. As NHEJ repairs the DSB by ligating the broken strands together, this process commonly results in small insertions or deletions of base pairs, known as indels. The generation of indels at the repair site can cause frame-shift mutations that prevent the protein from being properly transcribed and translated. This concept was dem‐

pressed (**B**).

364 Cystic Fibrosis in the Light of New Research

joining (NHEJ).

the involvement of foreign DNA, thus reducing the risk of insertional mutagenesis.


### **5.1. Zinc finger nucleases**

Zinc fingers are a common DNA-binding protein that can be found in nearly half of all transcription factors in the human genome [38]. These naturally abundant proteins can be reengineered to recognize and bind specific target sequences. ZFN technology takes advantage of this by attaching a DNA-cleaving nuclease to the zinc finger-binding domain. The result is a site-specific binding protein that can cleave a strand of DNA at a precise location.

ZFNs utilize the non-specific DNA cleavage domain from the *Fok*I restriction endonuclease to confer their cleavage activity [39]. Their design traditionally incorporates the wild-type *Fok*I cleavage domain; however, more recent studies have utilized variants with improved cleavage activity or specificity [40–42]. Functioning as a dimer, the *Fok*I domain requires two ZFN constructs, working together as a pair: one ZFN binds to a sequence immediately upstream of the intended cut site, while the other targets sequence immediately downstream of the cut site on the complementary strand (Figure 4A). This alignment places the C-terminal nucleases at a desired distance apart across the cut site, where they dimerize and create a DSB. Proper spacing of binding and cleavage domains is critical for optimal DSB induction.

**Figure 4. Genome-editing nucleases: Mechanisms of action.** An overview of the mechanisms of DNA binding and cleavage used by ZFNs (**A**), TALENs (**B**), CRISPR/Cas9 (**C**), the dimeric CRISPR RNA-guided *Fok*I system (**D**), and meganucleases (**E**). PAM, protospacer adjacent motif; sgRNA, single guide RNA; RuvC, nuclease domain in Cas9 that cleaves the non-target strand of DNA; HNH, nuclease domain in Cas9 that cleaves the target strand of DNA.

Numerous evidence has supported the use of ZFNs for targeted gene editing in multiple species, including mice, rats, rabbits, pigs, plants, and zebrafish [38]. The use of this platform has also extended to the manipulation of stem cell populations *ex vivo*. In one seminal study, ZFNs facilitated targeted disruption of *CCR5*, a co-receptor involved in HIV entry [32]. By introducing the *CCR5*-∆32 mutation into *ex vivo* expanded CD4+ T cells, followed by engraft‐ ment into HIV-1 infected mice, these target cells no longer expressed functional CCR5 entry receptors, making them more resistant to infection. Further studies utilized a dual strategy to target both of the HIV entry co-receptors, CCR5 and CXCR4 [43]. The use of ZFNs was also successfully demonstrated *in vivo* in murine liver, to correct a model of hemophilia B, or factor IX (FIX) deficiency [36].

Despite this, ZFNs are not without their limitations. First, they are relatively difficult to engineer and expensive to purchase commercially, leaving them inaccessible to the majority of investigators. Secondly, not all sequences can be targeted by ZFNs, restricting their use in certain applications. And lastly, the specificity of ZFN pairs is not 100%, resulting in the potential for off-target cleavage events and related damage to occur. In the event of low specificity, off-target DSB induction may overwhelm cellular repair machinery leading to chromosomal rearrangements and/or cell death. These instances may also support random integration of donor DNA into undesirable locations, which has the potential to interfere with tumor suppressors, proto-oncogenes, or other actively transcribed genes [24,25].
