31.8 Gene therapy for DMD includes

31.8.1 Transfer of new dystrophin gene: Transfer of new dystrophin gene uses high capacity gutless adenovirus (into the nuclei of dystrophic muscle cell). Since the size of the dystrophin cDNA is very large (14Kbp) and in addition the size of the vector (adenovirus) is also large thus it cannot easily enter the mature myofibre. The myofibre also lacks adenovirus receptors. Adeno-associated virus (AAV) has a smaller size and can be used instead of adenovirus. Limitation of using AAV is that it cannot carry the dystrophin cDNA (since size of dystrophin cDNA is very large). Thus, the truncated cDNA has to be used. It is known that the dystrophin protein consists of N-terminus, C-terminus, cysteine rich region and central rod region. Cysteine rich region, N and C terminus are essential for maintaining the integrity of the DGC thus; the truncated cDNA may be shortened at the central rod shaped domain while retaining the whole N-terminus, C-terminus, cysteine rich region and some portion of the central rod region. The reduced cDNA produced as a result of deleted portions of central rod region is known as the dystrophin mini-gene . The dystrophin mini-gene can be easily inserted into the AAV which has good transfection efficiency as a result the mini dystrophin gene can be successfully targeted to the muscle cells. To overcome the lack of receptors for adenovirus on the myofibre, monoclonal antibodies with specific binding at one end for the adenovirus and another end to various receptors (like integrin) on myofibre cells can be used.

31.8.2 Complete gene as DNA: The whole dystrophin cDNA can be inserted into the plasmid and injected into the blood stream. The limitation of this method is low transfection efficiency and high dose requirement for successful transfection. Electroporation, ultrasound as well as non-viral vectors may be used for delivery of the target gene to the target tissue.

31.8.3 Chimeraplasty: Chimeraplasty utilizes the endogenous repair mechanism, which may be used for repairing the defective dystrophin gene.

31.8.4 Supressing premature termination: Gentamycin, an amino glycoside, has been reported to suppress the premature termination (due to stop codon generated as a result of mutation) and enable the synthesis of 20% functional dystrophin protein in mdx mouse. Its efficacy in human subjects has not been ascertained till date.

31.8.5 Exon skipping : Exon skipping is another technique which may be employed to elevate the effects of DMD by making it milder like BMD. In DMD dystrophin is absent as ribosome encounters premature stop codon thus by skipping the mutated exon the rest of the shorter functional truncated dystrophin can be synthesized. The purpose of exon skipping is to induce the splicing mechanism and remove the introns from the pre mRNA and exons before and after the point mutation or deletion and thus enable read through the rest of the exon as in normal condition.

In order to perform exon skipping antisense oligoribonucleotides (AONs) have to be inserted into the target cell. The AON should target either the exon splice enhancer (ESE) region or the introns-exon splicing region lying within the particular exon that has to be skipped in the pre mRNA. On binding the AON blocks the normal formation of spliceosome preventing the splicing of the exon correctly. Thus the exon skipping takes place. As a result of exon skipping (exons are lost) shorter mRNA, and truncated dystrophin protein is synthesized. In case the skipped exon is not critical and belongs to the central region then the truncated protein can function and stabilize the muscle cell membrane.

The method of production of AONs in vivo involves the modification of the component of spliceosome (U7-snRNAs) which enables the recognition of target binding site rather than the wild type binding site. The U7-snRNAs function by binding to the splice sites at specific regions in the pre mRNA thus impeding the splicing of the specific exon. As a result several protein of varying length is produced from a single gene. The genetic alteration in the U7-snRNA would enable the binding to the splice region of the exon in the dystrophin gene. In this way the exon skipping would be easily performed in the dystrophin gene.