Antisense oligonucleotide-mediated improvement of SMaRT therapy

Spliceosome-mediated RNA trans-splicing (SMaRT) has the potential to be an effective therapeutic option in inherited monogenic diseases, particularly those whose underlying causes are dominant negative mutations. As such, SMaRT represents a promising alternative in the therapy of the skin blistering disease subtype Epidermolysis bullosa simplex-Dowling Meara (EBS-DM), wherein the presence of a single mutant KRT14 allele is sufficient to trigger the disease phenotype. SMaRT exploits the cells endogenous splicing machinery to recombine two distinct pre-mRNA molecules, resulting in the generation of a new chimeric gene product. SMaRT offers several advantages in that only the mutated exons are replaced, alleviating the need to clone and package the entire target gene. The endogenous transcriptional regulation of the gene is also maintained since correction is induced at the pre-mRNA level. Importantly, the correction of mutant transcripts leads to an overall decrease in the levels of mutant proteins, and thus SMaRT can potentially overcome the effects of dominant negative mutations. We have previously demonstrated the feasibility of correcting a dominant heterozygous mutation in KRT14 using SMaRT technology which led to a partial reversion of the disease-associated phenotype of patient cells in vitro. The focus of this proposal is to improve the level of KRT14 repair, by boosting the efficiency of the trans- splicing therapy via the addition of antisense nucleotides. The use of antisense oligonucleotides (ASO) to manipulate splice site selection has recently garnered attention as a single therapeutic strategy in muscular dystrophies. We assume that the addition of an ASO that blocks competitive cis-splicing signals in the target pre-mRNA would promote the trans-splicing reaction, leading to an increased repair efficiency and full reversion of the disease-associated phenotype. Our preliminary data point out that our screening method is a promising tool to identify an ASO that increases the efficiency of trans-splicing-mediated gene repair. Importantly, with minimal modifications, our proposed screening system could be generally applicable to the field of antisense-based therapies, and could contribute to our current understanding of RNA splicing regulation. Functional correction of KRT14 will be demonstrated by the expression and correct localization of wild type K14 protein in patient cells and in 3D skin equivalents, as well as a reduction in the migratory and invasive potential that is characteristic of EBS-DM keratinocytes. If successful, the outlined project will allow us to move forward towards our long-term goal, which is the establishment of a cellular-based therapy that involves the correction of patient stem cells ex vivo, followed by their differentiation into skin sheets, and transplantation onto the highly affected skin areas of the patient.

Epidermolysis bullosa simplex (EBS) is a rare blistering skin disease caused by mutations within keratin 14 (KRT14). KRT14 codes for an intermediate filament protein, a major component of the cytoskeleton of skin keratinocytes. Loss of stability within the keratinocyte intermediate filament network leads to blistering of the skin upon minor mechanical trauma. To correct KRT14 mutations in the generalized severe subtype of EBS (EBS-gen sev) cells, we use spliceosome-mediated RNA trans-splicing (SMaRT) technology. Here, the mutated KRT14 gene is recombined with an "RTM" (RNA trans-splicing molecule) at RNA level, thereby generating a corrected gene product. The RTM carries the healthy KRT14 sequence to introduce, core elements for efficient correction and a binding sequence to target KRT14. This repair mechanism has already been applied for different other genes affected in EB, however with rather low efficiency. For this project, we delivered randomly generated antisense RNAs (asRNAs), as well as rationally designed antisense oligonucleotides (ASOs) together with a KRT14 RTM into our target cells. The asRNAs/ASOs interfere with elements within the KRT14 target region that stabilize the mutated RNA. This results in a preferential recombination with the RTM. We analyzed 74 asRNAs and 9 ASOs in respect of their functionality via a fluorescence-based screening system. As a result, we achieved a significant increase in correction efficiency which we confirmed by PCR analysis. Protein analysis of patient cell lines transfected with the most efficient ASO together with the RTM showed normalized K14 protein expression levels comparable to the levels detectable in healthy keratinocytes. In conclusion, our results suggest that the inclusion of antisense oligonucleotides to the SMaRT system can increase the trans-splicing efficiency to levels that may be sufficient to stabilize the epidermis and therefore revert the EBS phenotype.