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Molecular optimization of laser/electrotransfer DNA administration into muscle and skin for gene therapy

Final Report Summary - MOLEDA (Molecular optimization of laser/electrotransfer DNA administration into muscle and skin for gene therapy)

For gene therapy, the use of non-viral DNA offers the advantage of lack of immunogenicity, absence of size limit for the therapeutic expression cassette, simpler GMP production, and improved safety / toxicity profiles. However, the efficient, precise and safe delivery of plasmids or other forms of non-viral DNA remains to be improved. Two different in vivo local tissue plasmid delivery techniques were recently introduced, and are currently the most efficient in terms of gene expression level: electrotransfer using mild Electric pulses (ET) and laser beam gene transfer using femtosecond infrared titanium sapphire laser energy (LBGT).

MOLEDA aimed to determine the optimal conditions for precise and selective plasmid transfer into skeletal muscle and skin, in the frame of a head-to-head comparison between the two most promising in vivo plasmid DNA delivery technologies at present, and to introduce molecular strategies to improve these technologies. Optimised conditions were then used on animals with four different therapeutic applications. The overall objective was to develop non-viral DNA technology into a preclinical phase. Consequently, MOLEDA addressed safety issues (tissue damage, inflammation, etc.) to ensure the success of its efforts.

The project identified the optimal conditions for plasmid delivery into muscle and skin. This optimisation was accompanied by a head-to-head comparison, using the same DNA preparations, between ET and LBGT. This optimisation / comparison was performed in two different tissues: muscle and skin.

An assessment for each tissue and each model of delivery (ET or LBGT), to determine which is the best promoter: cytomegalovirus (CMB) or two different tissue-specific promoters. For muscle, two strong skeletal muscle promoters were examined. For skin vaccination, one strongly active keratinocyte promoter, and one dendritic cell specific promoter was used.

In addition, MOLEDA has made great progress by constructing a new generation of high efficiency plasmids devoid of antibiotic resistance genes and their corresponding genetically modified bacterial host.