Intravascular Injection

Plasmid DNA can transduce hindlimb muscles with high efficiency, including in large animal models (Danialou et al. 2004; Hagstrom et al. 2004; Zhang et al. 2001) and, if delivered intraarterially or intravenously under conditions of high injectate volume and pressure together with a temporary obstruction of venous outflow from the targeted limb. Liu et al (2001) have presented evidence that a similar strategy can be applied to the diaphragm of mdx mice, by clamping the vena cava immediately following injection of plasmid DNA into the tail vein.

Huard et al (1995) reported that after intracardiac injection of first-generation adenoviral vectors in 2-day old rats, there was significant transduction of the diaphragm. However, adenovirus vectors generally perform poorly for this purpose in adult skeletal muscles due to difficulties in breaching the endothelial barrier and the relatively low levels of its receptors in mature muscles (Cho et al. 2000). AAV6 and AAV8 are superior to adenoviral vectors for intraarterial delivery to mouse hindlimb muscles, and AAV8 was also found to be effective for this purpose in macaques (Rodino-Klapac et al. 2007), although the diaphragm was not evaluated.

The most dramatic examples of successful intravascular delivery of gene transfer vectors to the diaphragm and other respiratory muscles have been achieved with AAV vectors. Gregorevic et al (2004) initially reported that by combining intravenous AAV6 administration and treatment with vascular endothelial growth factor (VEGF) to increase vascular permeability, it was possible to transduce most major skeletal muscle groups of the body in adult mice, including the intercostal muscles. However, a notable exception was the diaphragm, which demonstrated poor trans-duction. This latter finding was likely related to the version of creatine kinase promoter (CK6) used in the vector, which has been shown to have poor activity in the murine diaphragm (Salva et al. 2007). Indeed, when using a constitutively active viral (CMV) promoter within AAV6 injected by the tail vein, Gregorevic et al (2008) more recently achieved high-level microdystrophin expression in the diaphragms of older (20 months) mdx mice, and also demonstrated an improved resistance of the diaphragm to contraction-induced injury under these conditions. In keeping with these results, Salva et al (2007) showed robust microdystrophin expression in the mdx mouse diaphragm after intravenous injection of AAV6 containing a muscle-specific promoter (MHCK7) that demonstrates superior activity to the CK promoter in oxidative muscle fibers. Furthermore, the latter results were achieved in adult mdx mice without the need for concomitant vascular permeabili-zation treatment with VEGF.

Several studies have examined the ability of intravenously administered AAV to correct diaphragmatic pathology in the murine model of Pompe disease. In this model, the diaphragm and other skeletal muscles exhibit an abnormal accumulation of glycogen within muscle fibers, resulting in a loss of force-generating capacity. As mentioned earlier, the disease is caused by a lack of the glycogen-metabolizing enzyme GAA in lysosomes. After intravenous delivery of AAV vectors encoding GAA, substantial correction of the diaphragmatic phenotype has been demonstrated (Cresawn et al. 2005; Mah et al. 2005; Sun et al. 2005). Interestingly, trans-duction of the diaphragm itself is not absolutely required for amelioration of the diaphragmatic pathology, as GAA secreted from other transduced organs (eg. liver) can be taken up by distant muscles (Ding et al. 2001). However, the development of neutralizing antibodies against GAA is a frequent problem which reduces this "distant cross-correction" effect over time (Cresawn et al. 2005; Sun et al. 2005), suggesting that direct transduction of the diaphragm may be a better long-term strategy. Studies in the GAA-deficient mouse model have also indicated that in addition to AAV6, other pseudo types such as AAV1 in newborns (Mah et al. 2005) and AAV7, AAV8 or AAV9 in adults (Sun et al. 2008), are able to transduce the diaphragm with reasonably high efficiency following intravenous administration.

Finally, there is currently considerable interest in the potential of antisense oli-gonucleotides to restore dystrophin expression by inducing "exon skipping" in the Duchenne muscular dystrophy. In the mdx mouse, intravenous delivery of specific 2-0-methyl phosphorothioate antisense oligoribonucleotides mixed together with the triblock copolymer F127 induced dystrophin expression in the diaphragm, intercostals, and abdominal muscles at low levels (Lu et al. 2005). The same group later showed that morpholino phosphorodiamidate antisense oligonucleotides can be delivered repeatedly by the intravenous route in adult mdx mice, resulting in a cumulative increase in dystrophin expression in intercostal and abdominal muscles, and to a lesser extent within the diaphragm (Alter et al. 2006). More recently, cell-penetrating peptides containing arginine, 6-aminohexanoic acid, and/or b-alanine conjugated to morpholino phosphorodiamidate antisense oligonucleotides have been delivered intraperitoneally or intravenously to mice, and demonstrated a high level of efficacy in the diaphragm (Jearawiriyapaisarn et al. 2008). In mdx mice injected intravenously with these peptide-conjugated morpholinos on a daily basis for 4 days, dystrophin expression was reportedly restored to 40-50% wild-type levels for up to 17 weeks after the last treatment. This is a level of dystrophin restoration which would be expected to have major functional benefits and could even completely prevent abnormal contractile function if instituted early enough in the disease process.

Fire Up Your Core

Fire Up Your Core

If you weaken the center of any freestanding structure it becomes unstable. Eventually, everyday wear-and-tear takes its toll, causing the structure to buckle under pressure. This is exactly what happens when the core muscles are weak – it compromises your body’s ability to support the frame properly. In recent years, there has been a lot of buzz about the importance of a strong core – and there is a valid reason for this. The core is where all of the powerful movements in the body originate – so it can essentially be thought of as your “center of power.”

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