Cystic Fibrosis: ZFN Mediated Gene Editing Results in Functional CFTR Correction
By Ishani Dasgupta, Ph.D.
Over the last few years, gene editing has revolutionized the field of biomedical research, making it possible for scientists to target and modify desired genomic sequences in almost all eukaryotes. Gene editing primarily involves genetic alterations like insertion, deletion, or correction at the target genomic site, rendering it useful for treating a wide array of genetic diseases.
Several engineered nucleases like zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases act like molecular scissors to generate directed double stranded breaks (DSBs) in the host genomic DNA. The DSBs are then repaired by two DNA repair pathways present in all organisms. These include the non-homologous end joining (NHEJ) repair, resulting in nucleotide insertions and deletions (Indels) at the DSB and the more precise homology directed repair (HDR) pathway, enabling single nucleotide correction with high fidelity.
How ZFNs Act as Molecular Scissors for Gene Editing?
In addition to TALENs and CRISPR, ZFNs have emerged as a promising tool for genome engineering. In 1985, the site-specific DNA binding zinc-finger protein was first discovered in Xenopus oocytes as part of a transcription factor.
The synthetic Zinc finger nucleases (ZFNs) are assembled by fusing a zinc-finger domain that binds the target DNA and a DNA cleavage domain. The former is engineered to recognize the desired locus in the genome. Once this is accomplished, the cleavage domain, which contains a restriction endonuclease forms a dimer and snips the DNA at the specified target site, which is then repaired by either NHEJ or HDR.
ZFNs can, therefore, be custom designed to trigger a site-specific DSB in the genome, resulting in desired gene correction. Plasmids encoding customized ZFNs can be used to manipulate a specific gene locus in human cells, highlighting its potential to deliver therapeutic genes to a predetermined genomic site.
Several biotech companies and researchers are working towards selecting a therapeutic target, developing the appropriate specific ZFN, and optimizing delivery techniques to implement this approach in model organisms, human cells as well as in plants. Sangamo Therapeutics has been instrumental in developing a ZFN based gene editing platform for treating HIV, hemophilia, and other genetic diseases. SB-913, a ZFN-based in vivo gene editing therapy developed by Sangamo, entered the first ground-breaking clinical trial for treating Hunter’s syndrome. This approach, if successful, can be extended to introduce a therapeutic gene directly into a patient at a precise genomic location for treating several genetic diseases.
Gene Editing in Cystic Fibrosis Patients
Cystic fibrosis (CF) is one such progressive genetic disease where gene editing based treatments are explored by scientists. A faulty cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in a dysfunctional CFTR protein causes mucus accumulation in the lungs, clogging the airways and subsequent respiratory failure.
A recent study led by Hans Clevers at the Hubrecht Institute, Netherlands, and Jeffrey Beekman, UMB Utrecht used a technique called base editing to fix the mutation of the CFTR gene. This study was published in Cell Stem Cell journal in February 2020 and the coauthor of the study, Maaten Geurts mentioned: “Instead of creating a cut and replacing the faulty DNA, the mutation is directly repaired on site, making this a more effective genome editing tool.”
Also, since this technique does not induce any DSBs on the DNA, there is no further need to provide a donor template for the repair mechanism. He further added, “Here, we describe a cystic fibrosis intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations”.
ZFN Based Gene Editing to Correct CF Gene Mutation
Still, a lot remains unexplored in cell therapeutic approaches for cystic fibrosis wherein the airway basal cells with the corrected CFTR gene are transplanted into CF patients’ lungs. To this end, a recent study published in Molecular Therapy journal in April 2020 employed a dual approach to successfully correct the endogenous CFTR gene and obtain expression of the corrected gene close to physiological levels.
The authors first conducted a site-specific replacement of the endogenous mutated CFTR gene with the corrected version, restoring normal CFTR protein function. A sufficiently high level of editing was achieved for the correction of F508del, one of the most common mutations in the CFTR gene. A 30% correction frequency when both F508del CFTR alleles were restored per cell and a 60% if one F508del allele per cell was modified was observed during mutation-specific correction with AAV6 donor delivery. A similar correction efficiency was observed in CRISPR/Cas9 based F508del correction with AAV6 in bronchial primary epithelial cells.
A fundamental drawback to this approach is there are around 2000 known mutations implicated in cystic fibrosis. Although this approach accounts for correcting the most common F508del mutation, designing editing experiments for all the other variants is challenging.
To circumvent this, the authors devised a second approach of targeted integration of a splice acceptor, a partial CFTR cDNA, and a polyadenylation sequence into the endogenous CFTR intron. This homologous recombination-based method will be able to correct all CFTR mutations downstream of the CFTR cDNA site in introns 7 and 8 besides attaining a regulated expression pattern of endogenous CFTR.
This targeted integration of partial CFTR cDNAs reveals a more generalized strategy to correct primary airway basal cells and can be extended to CF patient induced pluripotent stem cells. Diseases like CF pose a challenge for delivering gene editing tools to airways and lung cells. However, the in vitro approaches on airway basal cells we discussed here, if modified accordingly, have the potential to be implemented for in vivo CFTR correction in the future.
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