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.

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.

Trans-splicing improvement by the combined application of antisense strategies.

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

A novel screening system improves genetic correction by internal exon replacement.

Trans-splicing is a powerful approach to reprogram the genome. It can be used to replace 5', 3' or internal exons. The latter approach has been characterized by low efficiency, as the requirements to promote internal trans-splicing are largely uncharacterized. The trans-splicing process is induced by engineered 'RNA trans-splicing molecules' (RTMs), which target a selected pre-mRNA to be reprogrammed via two complementary binding domains. To facilitate the development of more efficient RTMs for therapeutic applications we constructed a novel fluorescence based screening system. We incorporated exon 52 of the COL17A1 gene into a GFP-based cassette system as the target exon. This exon is mutated in many patients with the devastating skin blistering disease epidermolysis bullosa. In a double transfection assay we were able to rapidly identify optimal binding domains targeted to sequences in the surrounding introns 51 and 52. The ability to replace exon 52 was then evaluated in a more endogenous context using a target containing COL17A1 exon 51-intron 51-exon 52-intron 52-exon 53. Two selected RTMs produced significantly higher levels of GFP expression in up to 61% assayed cells. This novel approach allows for rapid identification of efficient RTMs for internal exon replacement.

Diffusion process with two reflecting barriers in a time-dependent potential.

We consider a Brownian particle which is driven by a harmonically oscillating force, the motion of which is restricted by two reflecting boundaries. We solve the Fokker-Planck equation using the finite-element method and focus on the dynamics of the mean position of the particle in the time-asymptotic regime. As a function of the strength of the external force, the response of the system, i.e., the amplitude of the mean position and the dynamical shift, in the stationary limit shows a resonancelike behavior as a function of the diffusion coefficient for certain parameter regimes. We explain these numerical results heuristically and give some qualitative estimates.

How to Avoid a Learning Curve in Stapedotomy: A Standardized Surgical Technique.

OBJECTIVE: To evaluate, whether a learning curve for beginners in stapedotomy can be avoided by using a prosthesis with thermal memory-shape attachment in combination with a standardized laser-assisted surgical technique. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Fifty-eight ears were operated by three experienced surgeons and compared with a group of 12 cases operated by a beginner in stapedotomy. INTERVENTION: Stapedotomy. MAIN OUTCOME MEASURES: Difference of pure-tone audiometry thresholds measured before and after surgery. RESULTS: The average postoperative gain for air conduction in the frequencies below 2 kHz was 20 to 25 dB and decreased for the higher frequencies. Using the Mann-Whitney-U test for comparing mean gain between experienced and inexperienced surgeons showed no significant difference (p = 0.281 at 4 kHz and p > 0.7 for the other frequencies). A Spearman rank correlation of the postoperative gain for air- and bone-conduction thresholds was obtained at each test frequency for the first 12 patients consecutively treated with a thermal memory-shape attachment prosthesis by two experienced and one inexperienced surgeon. This analysis does not support the hypothesis of a "learning effect" that should be associated with an improved outcome for successively treated patients. CONCLUSION: It is possible to avoid a learning curve in stapes surgery by applying a thermal memory-shape prosthesis in a standardized laser-assisted surgical procedure.

A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair.

RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

Spliceosome-mediated RNA trans-splicing facilitates targeted delivery of suicide genes to cancer cells.

Patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a hereditary blistering disease of epithelia, show susceptibility to develop highly aggressive squamous cell carcinoma (SCC). Tumors metastasize early and are associated with mortality in the 30th-40th years of life in this patient group. So far, no adequate therapy is available for RDEB SCC. An approach is suicide gene therapy, in which a cell death-inducing agent is introduced to cancer cells. However, lack of specificity has constrained clinical application of this modality. Therefore, we used spliceosome-mediated RNA trans-splicing technology, capable of replacing a tumor-specific transcript with one encoding a cell death-inducing peptide/toxin, to provide tumor-restricted expression. We designed 3' pre-trans-splicing molecules (PTM) and evaluated their efficiency to trans-splice an RDEB SCC-associated target gene, the matrix metalloproteinase-9 (MMP9), in a fluorescence-based test system. A highly efficient PTM was further adapted to insert the toxin streptolysin O (SLO) of Streptococcus pyogenes into the MMP9 gene. Transfection of RDEB SCC cells with the SLO-PTM resulted in cell death and induction of toxin function restricted to RDEB SCC cells. Thus, RNA trans-splicing is a suicide gene therapy approach with increased specificity to treat highly malignant SCC tumors.