Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.

Use of adenine base editing and homology-independent targeted integration strategies to correct the cystic fibrosis causing variant, W1282X.

Small molecule drugs known as modulators can treat ~90% of people with cystic fibrosis (CF), but do not work for premature termination codon variants such as W1282X (c.3846G>A). Here we evaluated two gene editing strategies, Adenine Base Editing (ABE) to correct W1282X, and Homology-Independent Targeted Integration (HITI) of a CFTR superexon comprising exons 23-27 (SE23-27) to enable expression of a CFTR mRNA without W1282X. In Flp-In-293 cells stably expressing a CFTR expression minigene bearing W1282X, ABE corrected 24% of W1282X alleles, rescued CFTR mRNA from nonsense mediated decay and restored protein expression. However, bystander editing at the adjacent adenine (c.3847A>G), caused an amino acid change (R1283G) that affects CFTR maturation and ablates ion channel activity. In primary human nasal epithelial cells homozygous for W1282X, ABE corrected 27% of alleles, but with a notably lower level of bystander editing, and CFTR channel function was restored to 16% of wild-type levels. Using the HITI approach, correct integration of a SE23-27 in intron 22 of the CFTR locus in 16HBEge W1282X cells was detected in 5.8% of alleles, resulting in 7.8% of CFTR transcripts containing the SE23-27 sequence. Analysis of a clonal line homozygous for the HITI-SE23-27 produced full-length mature protein and restored CFTR anion channel activity to 10% of wild-type levels, which could be increased three-fold upon treatment with the triple combination of CF modulators. Overall, these data demonstrate two different editing strategies can successfully correct W1282X, the second most common class I variant, with a concomitant restoration of CFTR function.

Mutation-class dependent signatures outweigh disease-associated processes in cystic fibrosis cells.

BACKGROUND: The phenotypic heterogeneity observed in Cystic Fibrosis (CF) patients suggests the involvement of other genes, besides CFTR. Here, we combined transcriptome and proteome analysis to understand the global gene expression patterns associated with five prototypical CFTR mutations. RESULTS: Evaluation of differentially expressed genes and proteins unveiled common and mutation-specific changes revealing functional signatures that are much more associated with the specific molecular defects associated with each mutation than to the CFTR loss-of-function phenotype. The combination of both datasets revealed that mutation-specific detected translated-transcripts (Dtt) have a high level of consistency. CONCLUSIONS: This is the first combined transcriptomic and proteomic study focusing on prototypical CFTR mutations. Analysis of Dtt provides novel insight into the pathophysiology of CF, and the mechanisms through which each mutation class causes disease and will likely contribute to the identification of new therapeutic targets and/or biomarkers for CF.

Development of novel therapeutics for all individuals with CF (the future goes on).

Despite the major advances and successes in finding and establishing new treatments that tackle the basic defect in Cystic Fibrosis (CF), there is still an unmet need to bring these potentially curative therapies to all individuals with CF. Here, we review aspects of what is still missing to treat all individuals with CF by such approaches. On the one hand, we discuss novel holistic (high-throughput) approaches to elucidate mechanistic defects caused by distinct classes of mutations to identify novel drug targets. On the other hand, we examine therapeutic approaches to correct the gene in its own environment, i.e., in the genome.

CFTR RNA- and DNA-based therapies.

This review provides an update on recent developments of RNA- and DNA-based methodologies and their intracellular targets in the context of cystic fibrosis (CF) lung disease. Ultimately, clinical success will require a suitable delivery system, but since the cargo for all these strategies is nucleic acid, it should hopefully be possible to exploit delivery breakthroughs from one study and apply these innovations to other experiments in order to identify the best strategy for everyone with CF. Ultimately, it may be the same approach for everyone, or possibly a number of different strategies tailored to particular mutations or classes/groups of mutations. And whilst the current focus is on CF lung disease, in the longer term the goal is to treat all affected organs in people with CF such as the pancreas, gut, and liver.

Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation.

BACKGROUND: W1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations. METHODS: We compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors. RESULTS: Cas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR. CONCLUSION: Altogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.

CRISPR Deletion of a SVA Retrotransposon Demonstrates Function as a cis-Regulatory Element at the TRPV1/TRPV3 Intergenic Region.

SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3' end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1 and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.

In Vitro and In Vivo Models to Study Nephropathic Cystinosis.

The development over the past 50 years of a variety of cell lines and animal models has provided valuable tools to understand the pathophysiology of nephropathic cystinosis. Primary cultures from patient biopsies have been instrumental in determining the primary cause of cystine accumulation in the lysosomes. Immortalised cell lines have been established using different gene constructs and have revealed a wealth of knowledge concerning the molecular mechanisms that underlie cystinosis. More recently, the generation of induced pluripotent stem cells, kidney organoids and tubuloids have helped bridge the gap between in vitro and in vivo model systems. The development of genetically modified mice and rats have made it possible to explore the cystinotic phenotype in an in vivo setting. All of these models have helped shape our understanding of cystinosis and have led to the conclusion that cystine accumulation is not the only pathology that needs targeting in this multisystemic disease. This review provides an overview of the in vitro and in vivo models available to study cystinosis, how well they recapitulate the disease phenotype, and their limitations.

Polycystic Kidney Disease Ryanodine Receptor Domain (PKDRR) Proteins in Oomycetes.

In eukaryotes, two sources of Ca2+ are accessed to allow rapid changes in the cytosolic levels of this second messenger: the extracellular medium and intracellular Ca2+ stores, such as the endoplasmic reticulum. One class of channel that permits Ca2+ entry is the transient receptor potential (TRP) superfamily, including the polycystic kidney disease (PKD) proteins, or polycystins. Channels that release Ca2+ from intracellular stores include the inositol 1,4,5-trisphosphate/ryanodine receptor (ITPR/RyR) superfamily. Here, we characterise a family of proteins that are only encoded by oomycete genomes, that we have named PKDRR, since they share domains with both PKD and RyR channels. We provide evidence that these proteins belong to the TRP superfamily and are distinct from the ITPR/RyR superfamily in terms of their evolutionary relationships, protein domain architectures and predicted ion channel structures. We also demonstrate that a hypothetical PKDRR protein from Phytophthora infestans is produced by this organism, is located in the cell-surface membrane and forms multimeric protein complexes. Efforts to functionally characterise this protein in a heterologous expression system were unsuccessful but support a cell-surface localisation. These PKDRR proteins represent potential targets for the development of new "fungicides", since they are of a distinctive structure that is only found in oomycetes and not in any other cellular organisms.

Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis.

BACKGROUND: Mutations in CTNS-a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments. METHODS: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls. RESULTS: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities. CONCLUSIONS: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.

Gene and Base Editing as a Therapeutic Option for Cystic Fibrosis-Learning from Other Diseases.

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the CFTR gene. There are at least 346 disease-causing variants in the CFTR gene, but effective small-molecule therapies exist for only ~10% of them. One option to treat all mutations is CFTR cDNA-based therapy, but clinical trials to date have only been able to stabilise rather than improve lung function disease in patients. While cDNA-based therapy is already a clinical reality for a number of diseases, some animal studies have clearly established that precision genome editing can be significantly more effective than cDNA addition. These observations have led to a number of gene-editing clinical trials for a small number of such genetic disorders. To date, gene-editing strategies to correct CFTR mutations have been conducted exclusively in cell models, with no in vivo gene-editing studies yet described. Here, we highlight some of the key breakthroughs in in vivo and ex vivo gene and base editing in animal models for other diseases and discuss what might be learned from these studies in the development of editing strategies that may be applied to cystic fibrosis as a potential therapeutic approach. There are many hurdles that need to be overcome, including the in vivo delivery of editing machinery or successful engraftment of ex vivo-edited cells, as well as minimising potential off-target effects. However, a successful proof-of-concept study for gene or base editing in one or more of the available CF animal models could pave the way towards a long-term therapeutic strategy for this disease.

Minigene assay to Evaluate CRISPR/Cas9-based excision of Intronic mutations that Cause Aberrant Splicing in Human Cells.

The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing signals in the vector and endogenous splicing signals in a cloned genomic DNA fragment that contains one or more introns and exons. Minigene-based assay has been used extensively to test the effect of mutations in the splicing of a target sequence. They can also be used to test the ability of CRISPR/Cas9 and one or more associated gRNAs to target specific sequences in the minigene, and determine the effect of these editing events on splicing. As an example, it is shown that CRISPR/Cas9-based, targeted excision of short intronic sequences containing mutations which create cryptic splice signals, can restore normal splicing in a CFTR Hybrid minigene.

The Regulation of Monoamine Oxidase A Gene Expression by Distinct Variable Number Tandem Repeats.

The monoamine oxidase A (MAOA) uVNTR (upstream variable number tandem repeat) is one of the most often cited examples of a gene by environment interaction (GxE) in relation to behavioral traits. However, MAOA possesses a second VNTR, 500 bp upstream of the uVNTR, which is termed d- or distal VNTR. Furthermore, genomic analysis indicates that there are a minimum of two transcriptional start sites (TSSs) for MAOA, one of which encompasses the uVNTR within the 5' untranslated region of one of the isoforms. Through expression analysis in semi-haploid HAP1 cell lines genetically engineered in order to knockout (KO) either the uVNTR, dVNTR, or both VNTRs, we assessed the effect of the two MAOA VNTRs, either alone or in combination, on gene expression directed from the different TSSs. Complementing our functional analysis, we determined the haplotype variation of these VNTRs in the general population. The expression of the two MAOA isoforms was differentially modulated by the two VNTRs located in the promoter region. The most extensively studied uVNTR, previously considered a positive regulator of the MAOA gene, did not modulate the expression of what it is considered the canonical isoform, while we found that the dVNTR positively regulated this isoform in our model. In contrast, both the uVNTR and the dVNTR were found to act as negative regulators of the second less abundant MAOA isoform. The haplotype analysis for these two VNTRs demonstrated a bias against the presence of one of the potential variants. The uVNTR and dVNTR differentially affect expression of distinct MAOA isoforms, and thus, their combined profiling offers new insights into gene-regulation, GxE interaction, and ultimately MAOA-driven behavior.

A beginner's guide to gene editing.

NEW FINDINGS: What is the topic of this review? This review summarizes the development of gene editing from early proof-of-concept studies in the 1980s to contemporary programmable and RNA-guided nucleases, which enable rapid and precise alteration of DNA sequences of almost any living cell. What advances does it highlight? With an average of one clustered regularly interspaced short palindromic repeat (CRISPR) Cas9 paper published every 4 h in 2017, this review cannot highlight all new developments, but a number of key improvements, including increases in efficiency, a range of new options to reduce off-target effects and plans for CRISPR to enter clinical trials in 2018, are discussed. ABSTRACT: Genome editing enables precise changes to be made in the genome of living cells. The technique was originally developed in the 1980s but largely limited to use in mice. The discovery that a targeted double-stranded break at a unique site in the genome, close to the site to be changed, could substantially increase the efficiency of editing raised the possibility of using the technique in a broader range of animal models and, potentially, human cells. But the challenge was to identify reagents that could create targeted breaks at a unique genomic location with minimal off-target effects. In 2005, the demonstration that programmable zinc finger nucleases (ZFNs) could perform this task led to a number of proof-of-concept studies, but a limitation was the ease with which effective ZFNs could be produced. In 2009, the development of TAL effector nucleases (TALENs) increased the specificity of gene editing and the ease of design and production. However, it was not until 2013 and the development of the clustered regularly interspaced short palindromic repeat (CRISPR) Cas9/guide RNA that gene editing became a research tool that any laboratory could use.

Genetic therapies for cystic fibrosis lung disease.

Gene therapy for cystic fibrosis (CF) has been the subject of intense research over the last twenty-five years or more, using both viral and liposomal delivery methods, but so far without the emergence of a clinical therapy. New approaches to CF gene therapy involving recent improvements to vector systems, both viral and non-viral, as well as new nucleic acid technologies have led to renewed interest in the field. The field of therapeutic gene editing is rapidly developing with the emergence of CRISPR/Cas9 as well as chemically modified mRNA therapeutics. These new types of nucleic acid therapies are also a good fit with delivery by non-viral delivery approaches which has led to a renewed interest in lipid-based and other nanoformulations.