Optimisation of gene editing for cystic fibrosis

Abstract

Cystic Fibrosis (CF) is a recessive genetic disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. To date, 352 variants in the CFTR gene have been shown to be CF-causing. CF is the most common genetic disease in Caucasian population, with an estimation of about 70,000 to 100,000 people living with CF worldwide. The disease results in premature death at a median age of 44 years old, with patients dying mostly from end-stage lung disease as a consequence of chronic lung infections. There is no cure for CF, but there are a range of drugs to treat CF symptoms. Over the last nine years, some small molecule drugs called modulators, were designed to improve the processing and function of the CFTR protein slowing the progression of the disease for more than 90% of CF-patients. Even though those modulators revolutionised CF treatment, the cost for those treatments are expensive, cumbersome and there are still 10% of patients with no specific drug. Indeed, some CF-causing mutations, classified as Class I variants, result in expression of little or no CFTR protein; protein modulator therapies are ineffective for patients suffering from such mutations. The variant W1282X is one of them. The W1282X variant is the 6th most common CF-causing variant, concerning 2.5% of CF patients, moreover, it is the 2nd most common class I variant. Since the discovery of the CFTR gene in 1989, it was expected that being able to treat the genetic problem, could lead to a treatment for CF. Since then, multiple clinical trials for CFTR cDNA addition have been performed, unsuccessfully. However, since the discovery of programmable nucleases, for gene editing, new hopes for CF gene therapy emerged. Indeed, some clinical trials are in process for other diseases such as Leber’s congenital amaurosis, haemophilia B or mucopolysaccharidosis I and II. The goal of this project was to compare four different techniques to correct the W1282X mutation, either by itself using homology-directed repair (HDR) and base editing, or as a superexon to correct this mutation and all the ones downstream. The purpose was to determine if there was one technique that was optimal for CF correction. Targeting single mutations, the results showed that high correction efficiencies (around 20% with SpCas9 HDR and base editing and 8% with AsCas12a HDR) could be achieved, and the corrections led to accumulation of corrected mRNA (50% for AsCas12a HDR and Base editing to 60% for SpCas9 HDR). In addition, CFTR protein expression could also be observed in AsCas12a-edited samples. However, using HDR, a large amount of indels could be detected, disrupting the CFTR gene in non-corrected alleles. Moreover, base editing showed formation of by-stander modifications within the window of editing. Using a superexon for CFTR correction, the homology-independent targeted integration (HITI) technique showed an intermediate level of correction efficiency of about 6% in 16HBE14o- cells after selection, leading to about 8% of corrected mRNA. Using HDR to replace a large DNA sequence, the efficiency without selection appeared to be low with about 0.02% of mRNA correction; editing at DNA level could not be determined for this technique in the cell lines available. Even though the efficiencies appeared to be lower using a superexon, the systems seemed to be safer with indels localised in introns. Using those data, it could be possible to have a clear understanding of different gene editing techniques to correct the W1282X mutation. Those techniques could be used for other mutations as well as for other genetic diseases. With further optimisation, one or many of these techniques could be tested on CF animal models to provide safety data for a potential future use in the clinic for CF-patients.