Long-Term Benefit of Adeno-Associated Virus/Antisense-Mediated Exon Skipping in Dystrophic Mice

Abstract

Many mutations and deletions in the dystrophin gene, responsible for Duchenne muscular dystrophy (DMD), can be corrected at the posttranscriptional level by skipping specific exons. Here we show that long-term benefit can be obtained in the dystrophic mouse model through the use of adeno-associated viral vectors expressing antisense sequences: persistent exon skipping, dystrophin rescue, and functional benefit were observed 74 weeks after a single systemic administration. The therapeutic benefit was sufficient to preserve the muscle integrity of mice up to old age. These results indicate a possible long-term gene therapy treatment of the DMD pathology.

Long-Term Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Golden Retriever Muscular Dystrophy

“…The majority of genetherapy investigations have focused on strategies aimed at restoring dystrophin expression or increasing muscle mass and have been performed in either the mdx mouse (Sicinski et al, 1989) or canine X-linked muscular dystrophy (Valentine et al, 1988;Sharp et al, 1992) models of DMD. Restoration of dystrophin expression has been accomplished by gene transfer of mini-and microdystrophin constructs in dystrophic mice (Harper et al, 2002;Gregorevic et al, 2004;Liu et al, 2005), dogs (Wang et al, 2007;Kornegay et al, 2010), and humans (Miyagoe-Suzuki and Takeda, 2010), as well as by induction of exon skipping in dystrophic mice (Goyenvalle et al, 2004;Denti et al, 2006Denti et al, , 2008, dogs (Yokota et al, 2009), and humans (Miyagoe-Suzuki and Takeda, 2010;Moulton and Moulton, 2010;Partridge, 2010). Proof-of-principle studies demonstrating successful gene transfer of microdystrophin (Rodino-Klapac et al, 2010) and successful induction of exon skipping (Moulton and Moulton, 2010) have also been performed in healthy nonhuman primates (NHPs).…”

Accurate quantification of dystrophin mRNA and exon skipping levels in Duchenne Muscular Dystrophy

“…This can and has been measured using different techniques during the past decade. 5,9,11,18,19 The most commonly used method is a two-round amplification (nested PCR) of cDNA obtained from an RT reaction using different amounts of total RNA (100 to 800 ng 8,11 ) as template. To assess the optimal method to determine exon-skipping percentages, we reviewed the most used methods and compared them using AON-treated mouse muscle-derived RNA as input material.…”

“…[5][6][7][8][9][10][11] As dystrophin expression is rather low, cDNA synthesis followed by two rounds of PCR amplification (primary and nested PCR) is the method used most to detect and quantify the exon-skipping percentage, but protocols greatly differ and the total number of amplification cycles can vary from B50 to 70. 5,[7][8][9][10][11] Only one group has recently used a single round amplification 12 to assess exon 23 skipping in the mdx mouse, which is a dystrophin-negative mouse model with a point mutation in the in-frame exon 23 and which has been instrumental to optimize the exon-skipping approach in vivo. Although RT-PCR-based methods are inexpensive and allow high throughput, there is a possibility that the shorter skipped fragment is amplified more efficiently than the larger unskipped fragment, thus leading to an overestimation of exon-skipping percentages.…”

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy

“…Adeno-associated viruses (AAVs) are very efficient at transferring genes into skeletal muscles. Injection of AAV vectors expressing U7 or U1 snRNPs targeting mouse exon 23 resulted in sustained production of functional dystrophin in the mdx mouse after intramuscular injection and body-wide dystrophin expression and reduced muscle wasting after systemic treatment (Denti et al, 2008;Goyenvalle et al, 2004). However serious problems with the use of AAV vectors are the possibility of an immune response against the viral capsid and the difficulty to produce them on a large scale under good manufacturing practice (GMP), necessary for implementation in the clinic.…”