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).

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.

Leveraging immune memory against measles virus as an antitumor strategy in a preclinical model of aggressive squamous cell carcinoma.

Viral antigens are among the strongest elicitors of immune responses. A significant proportion of the human population already carries pre-existing immunity against several childhood viruses, which could potentially be leveraged to fight cancer. We sought to provide proof of concept in mouse models that a pre-existing measles virus (MeV) immunity can be redirected to inhibit tumor growth by directly forcing expression of cognate antigens in the tumor. To this end, we designed DNA vaccines against known MeV cytotoxic and helper T epitopes, and administered these intradermally to mice that were subsequently challenged with syngeneic squamous cancer cells engineered to either express the cognate antigens or not. Alternatively, established wild-type tumors in vaccinated animals were treated intratumorally with in vitro transcribed mRNA encoding the cognate epitopes. Vaccination generated MeV cytotoxic T lymphocyte (CTL) immunity in mice as demonstrated by enhanced interferon gamma production, antigen-specific T cell proliferation, and CTL-mediated specific killing of antigen-pulsed target cells. When challenged with syngeneic tumor cells engineered to express the cognate antigens, 77% of MeV-vaccinated mice rejected the tumor versus 21% in control cohorts. Antitumor responses were largely dependent on the presence of CD8+ cells. Significant protection was observed even when only 25% of the tumor bulk expressed cognate antigens. We therefore tested the strategy therapeutically, allowing tumors to develop in vaccinated mice before intratumoral injection with Viromer nanoparticles complexed with mRNA encoding the cognate antigens. Treatment significantly enhanced overall survival compared with controls, including complete tumor regression in 25% of mice. Our results indicate that redirecting pre-existing viral immunity to fight cancer is a viable alternative that could meaningfully complement current cancer immune therapies such as personalized cancer vaccines and checkpoint inhibitor blockade.