Riassunto analitico
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a genetic disorder caused by mutations in COL7A1, the gene encoding for Collagen VII, which is a major component of dermal anchoring fibrils. This condition results in an extreme skin fragility, chronic wounds, blistering and progressive scarring and fibrosis. The premature death of RDEB patients is usually due to squamous cell carcinoma that develop in chronic wounds sites.
The current protocol of gene therapy for RDEB envisages the use of MLV-γ-retroviral vector (RV) carrying COL7A1 cDNA. This sequence contains different repetitive sequences that can re-arrange causing the insertion of non-functional gene copies in about 20-25% of the transduced cells. Although immune response to fragments encoded by re-arranged COL7A1 cDNAs has not been seen, we propose a possible way to reduce the re-arranged proviruses in transduced primary keratinocytes using a codon-optimized COL7A1 sequence, produced after the identification of the most frequent recombinant regions and the analysis of the best codon-optimization strategy.
Primary RDEB keratinocytes were transduced with two MLV-γ-retroviral vectors (RV) carrying two different portions of the codon-optimized sequence of COL7A1 cDNA (coCol7): a first vector where the central region of COL7A1 was replaced by coCol7, and a second vector carrying both central and 3’ codon-optimized regions.
Each vector was used separately and single cells from each transduction were analyzed in order to determine the presence of re-arranged fragments.
Genomic DNA (gDNA) was extracted from clones and analyzed by PCR with seven pairs of customized primers to cover the whole 9kb sequence: each PCR product was run on 1% agarose gel to detect re-arranged fragments. Preliminary data showed that less than 10% of clones presented re-arranged sequences.
In order to verify if the correct protein is produced from the codon-optimized sequence, we performed an immunofluorescence assay with the same antibody we use to detect the wild-type protein: transduced colonies present a bright signal, indicating a strong production of Collagen VII.
In conclusion, this study provides preliminary data about the potential application of COL7A1 codon-optimized sequence in order to reduce the re-arranged proviral integrations and, possibly, reduce the risk of an immune response to genetically modified RDEB epidermal grafts.
|
