On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.

Emerging Gene Therapeutics for Epidermolysis Bullosa under Development.

The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient's skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin's basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.

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).

MicroRNA-200b-mediated reversion of a spectrum of epithelial-to-mesenchymal transition states in recessive dystrophic epidermolysis bullosa squamous cell carcinomas.

BACKGROUND: Cutaneous squamous cell carcinoma (SCC) is the leading cause of death in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, the survival time from first diagnosis differs between patients; some tumours spread particularly fast, while others may remain localized for years. As treatment options are limited, there is an urgent need for further insights into the pathomechanisms of RDEB tumours, to foster therapy development and support clinical decision-making. OBJECTIVES: To investigate differences in RDEB tumours of diverging aggressiveness at the molecular and phenotypic level, with a particular focus on epithelial-to-mesenchymal (EMT) transition states and thus microRNA-200b (miR-200b) as a regulator. METHODS: Primary RDEB-SCC keratinocyte lines were characterized with respect to their EMT state. For this purpose, cell morphology was classified and the expression of EMT markers analysed using immunofluorescence, flow cytometry, semi-quantitative reverse transcriptase polymerase chain reaction and Western blotting. The motility of RDEB-SCC cells was determined and conditioned medium of RDEB-SCC cells was used to treat endothelial cells in an angiogenesis assay. In addition, we mined previously generated microRNA (miRNA) profiling data to identify a candidate with potential therapeutic relevance and performed transient miRNA transfection studies to investigate the candidate's ability to reverse EMT characteristics. RESULTS: We observed high variability in EMT state in the RDEB-SCC cell lines, which correlated with in situ analysis of two available patient biopsies and respective clinical disease course. Furthermore, we identified miR-200b-3p to be downregulated in RDEB-SCCs, and the extent of deregulation significantly correlated with the EMT features of the various tumour lines. miR-200b-3p was reintroduced into RDEB-SCC cell lines with pronounced EMT features, which resulted in a significant increase in epithelial characteristics, including cell morphology, EMT marker expression, migration and angiogenic potential. CONCLUSIONS: RDEB-SCCs exist in different EMT states and the level of miR-200b is indicative of how far an RDEB-SCC has gone down the EMT path. Moreover, the reintroduction of miR-200b significantly reduced mesenchymal features.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

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.

A Novel Fluorescence-Based Screen of Gene Editing Molecules for Junctional Epidermolysis Bullosa.

Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP-COL17A1mut-expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.

Molecular Research and Treatment of Skin Diseases.

The intention of this Special Issue is to highlight current treatment options to target the cause, as well as disease-associated complications, of skin diseases, including a group of monogenetic skin disorders referred to as genodermatoses [...].

Transcriptome-Guided Drug Repurposing for Aggressive SCCs.

Despite a significant rise in the incidence of cutaneous squamous cell carcinoma (SCC) in recent years, most SCCs are well treatable. However, against the background of pre-existing risk factors such as immunosuppression upon organ transplantation, or conditions such as recessive dystrophic epidermolysis bullosa (RDEB), SCCs arise more frequently and follow a particularly aggressive course. Notably, such SCC types display molecular similarities, despite their differing etiologies. We leveraged the similarities in transcriptomes between tumors from organ transplant recipients and RDEB-patients, augmented with data from more common head and neck (HN)-SCCs, to identify drugs that can be repurposed to treat these SCCs. The in silico approach used is based on the assumption that SCC-derived transcriptome profiles reflect critical tumor pathways that, if reversed towards healthy tissue, will attenuate the malignant phenotype. We determined tumor-specific signatures based on differentially expressed genes, which were then used to mine drug-perturbation data. By leveraging recent efforts in the systematic profiling and cataloguing of thousands of small molecule compounds, we identified drugs including selumetinib that specifically target key molecules within the MEK signaling cascade, representing candidates with the potential to be effective in the treatment of these rare and aggressive SCCs.

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.

Evaluating a Targeted Cancer Therapy Approach Mediated by RNA trans-Splicing In Vitro and in a Xenograft Model for Epidermolysis Bullosa-Associated Skin Cancer.

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.

Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases.

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.

Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa.

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

Online Videos Provide Poor Information Quality, Reliability, and Accuracy Regarding Rehabilitation and Return to Sport After Anterior Cruciate Ligament Reconstruction.

PURPOSE: To investigate the information quality available on YouTube regarding rehabilitation and return to sport (RTS) after anterior cruciate ligament reconstruction (ACLR). METHODS: By use of The Onion Router software and predefined search terms, 140 YouTube videos regarding rehabilitation and RTS after ACLR were systematically included. Three scoring systems were used to analyze the included videos: (1) Journal of the American Medical Association (JAMA) benchmark criteria; (2) Global Quality Score (GQS); and (3) self-developed scores for rehabilitation after ACLR and RTS after ACLR, following American Academy of Orthopaedic Surgeons guidelines and current evidence. RESULTS: The vast majority of the included videos offered poor information quality, reliability, and accuracy. Videos that were uploaded by medically trained professionals showed significantly higher information quality regarding rehabilitation (P = .006 for JAMA score, P < .001 for GQS, and P = .001 for rehabilitation score) and regarding RTS (P < .001 for JAMA score, P < .001 for GQS, and P < .001 for RTS score) compared with commercial videos or personal-testimony videos. Multivariate linear regression also revealed medically trained professionals as significant predictors of higher information quality regarding rehabilitation (ß = 0.496 [P < .001] for JAMA score, ß = 1.3 [P < .001] for GQS, and ß = 3.7 [P < .001] for rehabilitation score) and RTS (ß = 0.754 [P < .001] for JAMA score, ß = 1.3 [P < .001] for GQS, and ß = 5.3 [P < .001] for RTS score). CONCLUSIONS: The average information quality, reliability, and accuracy of YouTube videos regarding rehabilitation and RTS after ACLR are poor. The information quality of related YouTube videos from medically trained professionals is significantly higher compared with commercial videos or personal-testimony videos. CLINICAL RELEVANCE: Current YouTube videos regarding rehabilitation and RTS after ACLR do not meet the necessary quality standards. Physicians should also be able to provide alternative sources of high-quality information.

Ramp lesions are frequently missed in ACL-deficient knees and should be repaired in case of instability.

PURPOSE: The aim of the current study was (1) to provide an overview of common definitions and classification systems of ramp lesions (RL) and (2) to systematically review the available literature with regard to the diagnosis and treatment of RLs in anterior cruciate ligament (ACL)-deficient knees. METHODS: Following the PRISMA guidelines, MEDLINE and Scopus were searched for articles (1) reporting on acute or chronic ACL injuries, (2) with concomitant medial meniscus injury, (3) located at the posterior meniscocapsular attachment site (and red-red zone). Ex vivo studies, reviews and technical notes were excluded. RESULTS: Twenty-seven studies were included based on the criteria mentioned above. RLs are common in ACL-deficient knees with a prevalence ranging from 9 to 24%. RLs should especially be suspected in younger patients, patients with an increased meniscal slope and in patients with prolonged time from injury to surgery. The sensitivity of MRI for the detection of RLs ranges from 48 to 86% at a specificity of 79-99%. For arthroscopy, RLs are easily missed through standard anterior portals (sensitivity 0-38%). RL repair leads to a significant improvement of subjective knee scores, regardless of the specific fixation technique. For stable RLs, the literature suggests equivalent postoperative stability for trephination and abrasion compared to surgical RL repair. CONCLUSION: Ramp lesions are frequently missed in ACL-deficient knees on standard arthroscopy with anterior portals only. If a RL is suspected, exploration via an additional posteromedial portal is indicated. In case of instability, RL repair should be performed. LEVEL OF EVIDENCE: IV.

Sport and Physical Activity Following Primary Total Knee Arthroplasty: A Systematic Review and Meta-Analysis.

BACKGROUND: This systematic review and meta-analysis aims to analyze the sport habits of patients before and after primary total knee arthroplasty (TKA) by answering the following questions: (1) Is there a postoperative improvement of sport activity based on validated activity scores? (2) Does age influence the postoperative improvement of sport activity based on validated activity scores? (3) What are the preoperative and postoperative sport participation rates and the return to sport rates (RTS)? (4) What are the sport disciplines and sport patterns? METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, MEDLINE and Scopus were searched for studies reporting the physical activity level and sport habits of patients before and after primary TKA based on validated activity scores or an activity questionnaire. Random effect models were implemented to pool the mean differences (MDs) of activity score values and the difference between preoperative and postoperative sport participation rates. RESULTS: Twenty-five studies were included reporting on 6035 TKAs. Physical activity levels improved significantly according to the University of California, Los Angeles (UCLA) activity score (MD 1.55, 95% confidence interval [CI] 0.35-2.76, n = 1239, I2 = 99%, P < .01) and the Tegner score (MD 1.14, 95% CI -0.48 to 2.76, n = 483, I2 = 93%, P < .01). Younger patients (≤55 years) had the highest improvement in the UCLA activity scores following primary TKA (MD 3.12, 95% CI -1.79 to 8.04, n = 67, I2 = 96%, P < .01). Sport participation decreased slightly but not significantly (incidence rate difference -8%, 95% CI -0.14 to -0.2, n = 2673 patients, I2 = 38%, P = .09). The median RTS was 71.2%. Patients predominantly engaged in low-impact sports, especially walking, cycling, and swimming. CONCLUSION: According to validated activity scores, the level of physical activity significantly increases following primary TKA. Young patients (≤55 years) had the highest gain in physical activity according to the UCLA activity score following primary TKA. Sport participation shows a slight but nonsignificant decrease; intermediate and high-impact sports were abandoned to a large degree while participation rates for low-impact sports predominantly increased. RTS varied, although approximately 70% resume sport activities. LEVEL OF EVIDENCE: IV (review including case series).

[Ex vivo stem cell gene therapy of the skin : Ready for clinical use?] / Ex-vivo-Stammzellgentherapie an der Haut : Reif für klinische Anwendungen?

BACKGROUND: Use of ex vivo stem cell gene therapy enables the correction of the genetic cause of a monogenetic skin disease. OBJECTIVES: The procedure and choice of gene therapy method in the course of ex vivo gene therapy of the skin are presented. MATERIALS AND METHODS: Current gene therapeutic applications focus on the addition or targeted correction of the respective gene within the genome. RESULTS: So far, gene replacement therapy has been successfully used in patients suffering from the blistering skin disease epidermolysis bullosa. Designer nuclease-based gene therapy approaches are at the preclinical stage. CONCLUSIONS: The selection of the gene therapy method depends on its safety profile, the target genodermatoses and the genetic mutation to correct.

An RNA-targeted therapy for dystrophic epidermolysis bullosa.

Functional impairment or complete loss of type VII collagen, caused by mutations within COL7A1, lead to the severe recessive form of the skin blistering disease dystrophic epidermolysis bullosa (RDEB). Here, we successfully demonstrate RNA trans-splicing as an auspicious repair option for mutations located in a wide range of exons by fully converting an RDEB phenotype in an ex vivo pre-clinical mouse model based on xenotransplantation. Via a self-inactivating (SIN) lentiviral vector a 3' RNA trans-splicing molecule, capable of replacing COL7A1 exons 65-118, was delivered into type VII collagen deficient patient keratinocytes, carrying a homozygous mutation in exon 80 (c.6527insC). Following vector integration, protein analysis of an isolated corrected single cell clone showed secretion of the corrected type VII collagen at similar levels compared to normal keratinocytes. To confirm full phenotypic and long-term correction in vivo, patches of skin equivalents expanded from the corrected cell clone were grafted onto immunodeficient mice. Immunolabelling of 12 weeks old skin specimens showed strong expression of human type VII collagen restricted to the basement membrane zone. We demonstrate that the RNA trans-splicing technology combined with a SIN lentiviral vector is suitable for an ex vivo molecular therapy approach and thus adaptable for clinical application.

Modern cemented total knee arthroplasty design shows a higher incidence of radiolucent lines compared to its predecessor.

PURPOSE: To prevent early failure it is necessary to evaluate modern TKA system for possible shortcomings during implantation. The aim of this study was to evaluate the radiographic outcome and short-term survival of a modern cemented primary TKA system compared to its predecessor. METHODS: The authors reviewed 529 primary cemented TKAs [276 Attune (ATT) and 253 PFC Sigma (PFC)], which were implanted between 2014 and 2017 concerning the radiographic outcome and short-term survival. Radiographs were taken before discharge, 6 weeks, 6 months and 12 months postoperatively. Radiographic analysis was performed by two independent assessors using the Modern Knee Society Radiographic Evaluation System. RESULTS: The incidence of radiolucent lines was significantly higher in the ATT group compared with the PFC group 12 months postoperatively (35.1%; n = 97 TKAs vs. 7.5%; n = 19 TKAs; p < 0.001). Survival analysis could not show any differences in revision-free survival or revision rate. CONCLUSION: The modern primary TKA system shows an increased number of radiolucent lines, especially on the tibial component in this short-term analysis and may mostly be due to technique-related issues. Patients with those radiolucent lines even though they show no clinical evidence for loosening should be closely monitored at regular intervals. These findings are of vital clinical importance because surgeons should be aware of particular challenges in preparation and cementing technique once they are using this TKA-system. LEVEL OF EVIDENCE: Retrospective cohort study, Level III.