Gene therapy is the most promising treatment for recessive dystrophic epidermolysis bullosa (RDEB), however genetic cargo delivery efficiency is still a technical limitation. Viruses are the traditional vector of preference for gene therapy, as virus trophism increase tissue specificity. However, drawbacks related with safety and high manufacturing costs have facilitated the expansion of non-viral vectors, such as liposomes and cationic polymers. Our group is focused on the development of highly branched cationic polymers for gene therapy to treat RDEB. Our polymers have demonstrated encapsulation and delivery of a full COL7A1 cDNA with no toxicity, performing better transfection efficiencies than commercial counterparts in RDEB keratinocytes.
In this work, we show the research progress expanding the polymer technology for mRNA, pDNA and ribonucleoprotein complex delivery for developing CRISPR/Cas9 based gene editing therapies for RDEB. Gene edition in vivo has been achieved by a single topical application, obtaining efficiencies comparable with viral vectors (Ad5). Endosomal escape, by peptide polymer decoration is being investigated to improve efficiency in vivo by avoiding endosomal retention. Storage of the nanoparticles, formed by polymers and the genetic cargo, at -20C ensures no reduction in efficiency for more than 6 months. However, in order to avoid cold chain challenges, nanoparticles have been lyophilised, increasing dose concentration and facilitating formulation with skin absorption enhancers for topical application.
Proven ability to transfect stem cells combined with high efficiencies transfecting with multiple plasmids at the same time, should contribute to the success of prime editing strategies to pursue permanent correction of potential >89% of all described EB disease-associated mutations. The developed polymer platform shows high potential to be adapted to a wide range of genetic approaches for RDEB, including the most novel ones, that can be expanded to other EB subtypes, other genodermatoses and other rare genetic disorders such as cystic fibrosis.