Limitations of rAAV Vectors for Muscle Gene Therapy

Gene transfer of rAAV vectors to rodents has rarely been associated with elicitation of a cellular immune response against the AAV capsid proteins (Gregorevic et al. 2004a; Jooss and Chirmule 2003; Snyder et al. 1997; Xiao et al. 1996). In contrast, studies using IM injection of rAAV2 and 6 capsids in dogs elicited a strong cellmediated immune response against the vector (Wang et al. 2007a; Yuasa et al. 2007). However, a brief course of immune suppression was found sufficient to allow long-term expression of microdystrophin when delivered to the canine model of DMD using rAAV6 (Wang et al. 2007b). Several studies have used rAAV vectors for systemic gene transfer to muscles of dogs (Arruda et al. 2005; Greelish et al. 1999; Ohshima et al. 2009; Yue et al. 2008; Chamberlain et al. unpublished observations). A recent study used hydrodynamic limb vein injection of rAAV8 vectors to achieve widespread expression of the canine myostatin propeptide in limb musculature. Nevertheless, cardiomyocytes and diaphragmatic muscle were not targeted (Bartoli et al. 2007; Qiao et al. 2008). A single intravenous injection of rAAV9 into neonatal dogs resulted in whole body skeletal muscle transduction, although canine cardiac muscle, unlike rodent muscle, was not transduced by rAAV9 (Yue et al. 2008). Also, it appears that skeletal muscle transduction was not maintained in the absence of immune suppression when rAAV was administered to more mature dogs (Wang et al. 2007b; Yue et al. 2008; Ohshima et al. 2009), although some studies suggested that this may depend on serotype and/or route of administration (Arruda et al. 2005). More importantly, it has become clear from several human clinical trials involving systemic and IM injection of rAAV vectors that a cellular immune response can be generated against both rAAV2 and rAAV1 (Manno et al. 2006; Mingozzi and High 2007; Stroes et al. 2008). These cellular immune responses are presumably dose dependent and could be influenced by vector pseudotype. While the best choice of AAV serotype for systemic skeletal muscle therapy is not clear from animal studies, clinical studies could potentially benefit from the use of multiple serotypes or modified capsids to escape prior immunity or to enhance transduction of multiple muscle types and groups. Nonetheless, it appears increasingly likely that systemic gene transfer to human patients will require careful safety studies and the use of transient immune suppression to avoid immune mediated destruction of transduced tissues (Hasbrouck and High 2008; Wang et al. 2007b).

Several other limitations will influence the use of rAAV vectors for systemic gene transfer to muscle. As noted earlier, the ~5 kb cloning capacity of these vectors hinders transfer of large genes such as dystrophin, dysferlin, and laminin (Blankinship et al. 2006). Whether the mini-gene strategies being developed for dystrophin can be applied to other large genes will need to be addressed individually for each gene (Warner and Chamberlain 2002). An alternative approach to delivering mini-genes is to use multiple vectors to carry portions of a larger gene. Such a dual vector approach has led to surprisingly efficient generation of larger expression cassettes that were generated in vivo using either a trans-splicing method (Ghosh et al. 2007, 2008; Lai et al. 2005) or a homologous recombination (Duan et al. 2000; Halbert et al. 2002). Vector dose and production issues will also complicate systemic delivery to humans. While an entire mouse can be transduced with 1-4 x 1012 vector genomes (vg; ~4-16 x 1013 vg/kg) (Gregorevic et al. 2004a; Grimm et al. 2003b; Salva et al. 2007; Wang et al. 2005), the same dosage, when scaled for humans application would require ~1,000 times more vector. Further, for large-scale clinical trials production yield and vector batch quality need to be improved. At present rAAV is typically produced by direct transfection of cells with plasmids (Grimm et al. 2003a; Xiao et al. 1998), a tedious and difficult to scale procedure. In contrast, baculovirus-based production methods have much greater potential for high titer production in smaller volumes and may be amenable to use in bioreactors (Merten et al. 2005; Negrete et al. 2007).

The high vector doses needed for systemic gene delivery also present safety issues beyond those associated with immune rejection of transduced tissues. It will be critical to monitor any potential innate immune responses and acute or chronic toxicity resulting from high dose rAAV administration, which will require careful dose escalation studies in animal models and in the clinic (Raper et al. 2003). As vector genomes transduce multiple tissues, muscle gene transfer will likely be facilitated by the use of muscle-specific promoters (Goehringer et al. 2009; Gregorevic et al. 2004a; Mori et al. 2006). Finally, while rAAV vectors integrate into transduced cells at a very low frequency, the high doses needed for systemic gene transfer will likely lead to a significant number of integration events in both muscle and nonmuscle tissues (Chamberlain et al. 2004; Donsante et al. 2007; Inagaki et al. 2007). Despite these various limitations, rAAV vector remains the most efficient vector for muscle gene therapy, as evidenced by the number of ongoing preclinical and clinical studies for muscle gene therapy (Mueller and Flotte 2008).

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