Splice modulation strategy applied to deep intronic variants in COL7A1 causing recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1. One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa.

Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of COL7A1 leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the COL7A1 exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the COL7A1 target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced COL7A1 transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).

Therapeutic base editing and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). For fibroblasts from two patients (Pat1 and Pat2), we applied adenine base editors (ABEs) to correct the pathogenic mutation of COL7A1 or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the COL7A1 mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that transfer of edited patient-derived skin equivalents (i.e., RDEB keratinocytes and PE-corrected RDEB fibroblasts from the RDEB patient) into the skin of immunodeficient mice led to C7 deposition and anchoring fibril formation within the dermal-epidermal junction, suggesting that base editing and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

COL7A1 Editing via RNA Trans-Splicing in RDEB-Derived Skin Equivalents.

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.

Analysis of COL7A1 pathogenic variants in a large cohort of dystrophic epidermolysis bullosa patients from Argentina reveals a new genotype-phenotype correlation.

Dystrophic epidermolysis bullosa (DEB) is a clinically heterogeneous heritable skin disorder, characterized by blistering of the skin and mucous membranes following minor trauma. Dominant (DDEB) and recessive (RDEB) forms are caused by pathogenic variants in COL7A1 gene. Argentina's population has a heterogeneous genetic background, and little is known about the molecular basis of DEB in our country or in native South American populations. In this study, we present the prevalence and geographical distribution of pathogenic variants found in 181 patients from 136 unrelated families (31 DDEB and 105 RDEB). We detected 95 different variants, 59 of them were previously reported in the literature and 36 were novel, nine of which were detected in more than one family. The most prevalent pathogenic variants were identified in exon 73 in DDEB patients and in exon 3 in RDEB patients. We also report a new phenotype-genotype correlation found in 10 unrelated families presenting mild blistering and severe mucosal involvement. Molecular studies in populations with an unexplored genetic background like ours revealed a diversity of pathogenic variants, and we hope that these findings will contribute to the definition of targets for new gene therapies.

5'RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa.

Mutations within the COL7A1 gene underlie the inherited recessive subtype of the blistering skin disease dystrophic epidermolysis bullosa (RDEB). Although gene replacement approaches for genodermatoses are clinically advanced, their implementation for RDEB is challenging and requires endogenous regulation of transgene expression. Thus, we are using spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutations in COL7A1 at the mRNA level. Here, we demonstrate the capability of a COL7A1-specific RNA trans-splicing molecule (RTM), initially selected using a fluorescence-based screening procedure, to accurately replace COL7A1 exons 1 to 64 in an endogenous setting. Retroviral RTM transduction into patient-derived, immortalized keratinocytes resulted in an increase in wild-type transcript and protein levels, respectively. Furthermore, we revealed accurate deposition of recovered type VII collagen protein within the basement membrane zone of expanded skin equivalents using immunofluorescence staining. In summary, we showed for the first time the potential of endogenous 5' trans-splicing to correct pathogenic mutations within the COL7A1 gene. Therefore, we consider 5' RNA trans-splicing a suitable tool to beneficially modulate the RDEB-phenotype, thus targeting an urgent need of this patient population.

Epidermolysis Bullosa in Chinese Patients: Genetic Analysis and Mutation Landscape in 57 Pedigrees and Sporadic Cases.

Epidermolysis bullosa encompasses a group of inherited blistering skin disorders. The pathogenic mutations in 10-25% of patients with epidermolysis bullosa have not been identified by Sanger sequencing. The aims of this study were to identify the pathogenic sequence alterations in a large cohort of Chinese patients with epidermolysis bullosa and to clarify the relationship between clinical phenotypes and genotypes. Whole-exome sequencing was performed on 44 pedigrees and 13 sporadic cases. The results were further confirmed by Sanger sequencing. In total, 52 mutations, comprising 19 novel and 33 previously reported mutations, were identified in 5 genes, with a mutation detection rate of 100%. A relationship between subtypes and pathogenic genes was established: 12 cases of epidermolysis bullosa simplex were associated with mutations in KRT5/14 and PLEC; one case of junctional epidermolysis bullosa carried mutations in ITGB4; and 44 cases of dystrophic epidermolysis bullosa were caused by mutations in COL7A1. The results of this study support whole-exome sequencing as a promising tool in the genetic diagnosis of epidermolysis bullosa.

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.

Signatures of Dermal Fibroblasts from RDEB Pediatric Patients.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a debilitating disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Mutations in the COL7A1 gene induce multiple abnormalities, including chronic inflammation and profibrotic changes in the skin. However, the correlations between the specific mutations in COL7A1 and their phenotypic output remain largely unexplored. The mutations in the COL7A1 gene, described here, were found in the DEB register. Among them, two homozygous mutations and two cases of compound heterozygous mutations were identified. We created the panel of primary patient-specific RDEB fibroblast lines (FEB) and compared it with control fibroblasts from healthy donors (FHC). The set of morphological features and the contraction capacity of the cells distinguished FEB from FHC. We also report the relationships between the mutations and several phenotypic traits of the FEB. Based on the analysis of the available RNA-seq data of RDEB fibroblasts, we performed an RT-qPCR gene expression analysis of our cell lines, confirming the differential status of multiple genes while uncovering the new ones. We anticipate that our panels of cell lines will be useful not only for studying RDEB signatures but also for investigating the overall mechanisms involved in disease progression.

Apparent Missense Variant in COL7A1 Causes a Severe Form of Recessive Dystrophic Epidermolysis Bullosa via Effects on Splicing.

Dystrophic epidermolysis bullosa is an inherited skin disorder characterized by fragile skin that is prone to blistering. We report here a consanguineous Pakistani family with two siblings, in whom a severe recessive dystrophic epidermolysis bullosa was suspected. Using whole-exome sequencing for one sibling, the homozygous base substitution c.7249C>G in COL7A1 was identified, and could be confirmed in the other sibling by Sanger sequencing. In our exome data, this mutation was annotated as a missense substitution (p.Gln2417Glu), but in silico tools indicated a possible effect on splicing. Using the ExonTrap vector it was verified that the mutation leads to activation of a cryptic donor splice site, which leads to loss of 26 nucleotides, and a frame-shift event predicted to result in a truncated protein (p.Q2417Sfs*57). The present report de-scribes an apparent COL7A1 missense substitution with an unexpected consequence on splicing that leads to a severe recessive dystrophic epidermolysis bullosa phenotype.

Dominant pretibial dystrophic epidermolysis bullosa in an Italian family.

We describe a case of pretibial dystrophic epidermolysis bullosa in a 5-year-old girl, her mother, and maternal great aunt. All subjects had trauma-induced blisters and erosions, with scarring, on the knees and lower legs, and nail dystrophy of variable severity. Genetic analysis in all three patients showed a 6849del18 mutation in the COL7A1 gene, causing the production of shortened collagen VII polypeptides and resulting in a mild phenotype, with localized acral blisters and nail involvement.

Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1.

The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the COL7A1 pre-mRNA.

Identification of novel enriched recurrent chimeric COL7A1-UCN2 in human laryngeal cancer samples using deep sequencing.

BACKGROUND: As hybrid RNAs, transcription-induced chimeras (TICs) may have tumor-promoting properties, and some specific chimeras have become important diagnostic markers and therapeutic targets for cancer. METHODS: We examined 23 paired laryngeal cancer (LC) tissues and adjacent normal mucous membrane tissue samples (ANMMTs). Three of these pairs were used for comparative transcriptomic analysis using high-throughput sequencing. Furthermore, we used real-time polymerase chain reaction (RT-PCR) for further validation in 20 samples. The Kaplan-Meier method and Cox regression model were used for the survival analysis. RESULTS: We identified 87 tumor-related TICs and found that COL7A1-UCN2 had the highest frequency in LC tissues (13/23; 56.5%), whereas none of the ANMMTs were positive (0/23; p < 0.0001). COL7A1-UCN2, generated via alternative splicing in LC tissue cancer cells, had disrupted coding regions, but it down-regulated the mRNA expression of COL7A1 and UCN2. Both COL7A1 and UCN2 were down-expressed in LC tissues as compared to their paired ANMMTs. The COL7A1:ß-actin ratio in COL7A1-UCN2-positive LC samples was significantly lower than that in COL7A1-UCN2-negative samples (p = 0.019). Likewise, the UCN2:ß-actin ratio was also decreased (p = 0.21). Furthermore, COL7A1-UCN2 positivity was significantly associated with the overall survival of LC patients (p = 0.032; HR, 13.2 [95%CI, 1.2-149.5]). CONCLUSION: LC cells were enriched in the recurrent chimera COL7A1-UCN2, which potentially affected cancer stem cell transition, promoted epithelial-mesenchymal transition in LC, and resulted in poorer prognoses.

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

Identical COL71A1 heterozygous mutations resulting in different dystrophic epidermolysis bullosa phenotypes.

Dystrophic epidermolysis bullosa is a rare blistering condition caused by mutations in the COL7A1 gene. Different clinical variants have been described, with dominant and recessive inheritance, but no consistent findings have been elucidated to establish a genotype-phenotype correlation. We present three unrelated patients with two identical pathogenic compound heterozygous mutations in the COL7A1 gene that developed different clinical forms of dystrophic epidermolysis bullosa-epidermolysis bullosa pruriginosa and mild recessive non-Hallopeau-Siemens-raising the possibility of other genetic or environmental modifying factors responsible for the phenotype of the disease.

A nonsense mutation in the COL7A1 gene causes epidermolysis bullosa in Vorderwald cattle.

BACKGROUND: The widespread use of individual sires for artificial insemination promotes the propagation of recessive conditions. Inadvertent matings between unnoticed carriers of deleterious alleles may result in the manifestation of fatal phenotypes in their progeny. Breeding consultants and farmers reported on Vorderwald calves with a congenital skin disease. The clinical findings in affected calves were compatible with epidermolysis bullosa. RESULTS: Pedigree analysis indicated autosomal recessive inheritance of epidermolysis bullosa in Vorderwald cattle. We genotyped two diseased and 41 healthy animals at 41,436 single nucleotide polymorphisms and performed whole-genome haplotype-based association testing, which allowed us to map the locus responsible for the skin disease to the distal end of bovine chromosome 22 (P = 8.0 × 10-14). The analysis of whole-genome re-sequencing data of one diseased calf, three obligate mutation carriers and 1682 healthy animals from various bovine breeds revealed a nonsense mutation (rs876174537, p.Arg1588X) in the COL7A1 gene that segregates with the disease. The same mutation was previously detected in three calves with dystrophic epidermolysis bullosa from the Rotes Höhenvieh cattle breed. We show that diseased animals from Vorderwald and Rotes Höhenvieh cattle are identical by descent for an 8.72 Mb haplotype encompassing rs876174537 indicating they inherited the deleterious allele from a recent common ancestor. CONCLUSIONS: Autosomal recessive epidermolysis bullosa in Vorderwald and Rotes Höhenvieh cattle is caused by a nonsense mutation in the COL7A1 gene. Our findings demonstrate that deleterious alleles may segregate across cattle populations without apparent admixture. The identification of the causal mutation now enables the reliable detection of carrier animals. Genome-based mating strategies can avoid inadvertent matings of carrier animals thereby preventing the birth of homozygous calves that suffer from a painful skin disease.

Identification of Two Homozygous Sequence Variants in the COL7A1 Gene Underlying Dystrophic Epidermolysis Bullosa by Whole-Exome Analysis in a Consanguineous Family.

Dystrophic epidermolysis bullosa (DEB) is an inherited skin disorder with variable severity and heterogeneous genetic involvement. Diagnostic approaches for this condition include clinical evaluations and electron microscopy of patients' skin biopsies, followed by Sanger sequencing (SS) of a large gene (118 exons) that encodes the alpha chain of type VII collagen (COL7A1) located on Chromosome 3p21.1. However, the use of SS may hinder diagnostic efficiency and lead to delays because it is costly and time-consuming. We evaluated a 5-generation consanguineous family with 3 affected individuals presenting the severe generalised DEB phenotype. Human whole-exome sequencing (WES) revealed 2 homozygous sequence variants: the previously reported variant p.Arg578* in exon 13 and a novel variant p.Arg2063Gln in exon 74 of the COL7A1 gene. Validation by SS, performed on all family members, confirmed the cosegregation of the 2 variants with the disease phenotype. To the best of our knowledge, 2 homozygous COL7A1 variants have never been simultaneously reported in DEB patients; however, the upstream protein truncation variant is more likely to be disease-causing than the novel missense variant. WES can be used as an efficient molecular diagnostic tool for evaluating autosomal recessive forms of DEB.

[Recessive epidermolysis bullosa due to composite heterozygote mutations in the COL7A1 gene]. / Épidermolyse bulleuse héréditaire dystrophique par hétérozygotie composite COL7A1.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a genodermatosis characterized by various abnormalities of anchoring fibrils, composed mainly of type VII collagen, at the dermal-epidermal junction. These changes are induced by mutations in the type VII collagen gene (COL7A1). PATIENTS AND METHODS: A new-born boy was diagnosed with recessive DEB on the basis of typical skin lesions composed of multiple blisters with erosions on trauma-exposed body sites, including the hands and feet and the navel. Diagnosis was confirmed by pathology examination and irregular immunofluorescence staining of type VII collagen. Genomic DNA from the patient and parents were subjected to direct sequencing for the COL7A1 gene. Two heterozygous mutations were detected in the affected child. Each parent was a carrier of one heterozygous mutation. DISCUSSION: Over 730 mutations of the COL7A1 gene have been identified as responsible for phenotypic polymorphism of EBD. The relatively mild phenotype seen in our patient, known as "non-Hallopeau-Siemens" or "mitis" EBD, is due to residual synthesis of collagen VII. The mutation present on the maternal allele that prevents synthesis of collagen VII is compensated by the mutation on the paternal allele, which enables more or less functional collagen VII synthesis.

The COL7A1/PI3K/AKT axis regulates the progression of cholangiocarcinoma.

Background: The role and molecular mechanisms of collagen type VII (COL7A1) in cholangiocarcinoma (CCA) remain unknown. Methods: We analyzed the expression of COL7A1 in CCA and its relationship with patient prognosis using bioinformatic techniques. Expression levels of COL7A1 in CCA cells and tissues were detected using reverse transcription-quantitative PCR, western blotting, and immunohistochemistry. The effects of COL7A1 expression on the proliferation, migration, and invasion of CCA cells were assessed using CCK-8, colony formation, and Transwell assays. Bioinformatics and luciferase reporter gene assays were performed to examine the binding of KLF4 to COL7A1, and cytological experiments further verified the role of KLF4 in regulating the CCA phenotype through COL7A1. Xenograft mouse models were established to investigate the effects of COL7A1 on CCA tumor growth in vivo. Results: CCA tissues exhibited higher COL7A1 expression than normal bile duct tissues. There was no significant correlation between high or low COL7A1 expression and the survival time of patients with CCA. COL7A1 knockdown inhibited CCA cell proliferation, migration, and invasion. Furthermore, COL7A1 knockdown suppressed the activation of the PI3K/AKT signaling pathway. KLF4 can bind to COL7A1 and regulate COL7A1 expression, which in turn regulates the PI3K/AKT signaling pathway and impacts the proliferation and metastasis of CCA cells. Conclusion: Our findings suggest that KLF4 regulates CCA cell proliferation, migration, and invasion via the COL7A1/PI3K/AKT axis.