Hearing loss (HL) is the most common sensory disorder in developed countries. Bilateral congenital sensorineural HL affects one in every 500 newborns and may lead to significant problems in speech development and educational attainment (Hildebrand et al., 2008; Kochhar et al., 2007). At least half of all congenital HL cases are hereditary. Of these, the majority (~70% of cases) are nonsyndromic hearing loss (NSHL), in which hearing impairment is the sole phenotype, and the remainder (~30% of cases) are associated with syndromes such as Pendred and Usher syndrome (Kochhar et al., 2007). Genetic HL is highly heterogeneous with autosomal recessive inheritance (ARNSHL) in ~80% of cases, autosomal dominant (ADNSHL) inheritance in ~20% of cases, and instances of X-linked (<1%) and mitochondrial (<<1%) inheritance also occurring (Hilgert et al., 2009). To date, 36 ARNSHL genes and 24 ADNSHL genes have been identified. A further 45 loci for recessive and 34 loci for dominant deafness have been mapped (Hereditary Hearing Loss Homepage: http://hereditaryhearingloss.org) (Van Camp, 2010).
The inner ear is a complex structure with a large number of unique cell types. Deafness mutations are known to affect a variety of inner ear cells and tissues including: sensory hair cells (e.g. MYO7A, KCNQ4), non-sensory supporting cells (e.g. GJB2, GJB6), and the tectorial membrane (e.g. COL11A2, TECTA) (http://www.hereditaryhearingloss.org) (Kochhar et al., 2007; Petit et al., 2001; Van Camp, 2010).
The defect leading to the most common form of ARNSHL at the DFNB1 locus is in the non-sensory supporting cells of the cochlea. The DFNB1 locus contains the deafness genes GJB2 and GJB6 that encode connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively (del Castillo et al., 2002; Kelsell et al., 1997). Connexin proteins oligomerize to form gap junction channels between adjacent cells. CX26 and CX30 form gap junction networks between supporting cells in the cochlea that are thought to facilitate transport of ions (e.g. K+) and small molecules (e.g. IP3) vital for maintenance of cochlear homeostasis (Martinez et al., 2009; Nickel et al., 2008). Mutations in GJB2 account for up to 50% of autosomal recessive forms of isolated deafness in some populations, including an estimated 30–60% in Europe and the United States (Petit et al., 2001). GJB2 mutations are also associated with ADNSHL at the DFNA3 locus as well as a number of dominant syndromic disorders in which deafness segregates with skin disease such as Keratitis-Ichthyosis-Deafness syndrome (MIM 148210), Vohwinkel syndrome (MIM 124500), and palmoplantar keratoderma with deafness (MIM 148350) (Heathcote et al., 2000; Maestrini et al., 1999; van Steensel et al., 2002).
Current treatment options for sensorineural hearing loss are limited to amplification devices such as hearing aids and cochlear implants. Gene therapy targeting the inner ear to modulate or replace endogenous genes and their products offers the promise of novel treatments for hereditary hearing loss. Given the variety of cell types in the inner ear, viral tropism is an important consideration when selecting a vector for gene transfer (Hildebrand et al., 2008). As some viruses have been shown to have detrimental effects on their targets, the other major consideration for vector selection is toxicity. To date, a variety of viral vectors have been used to target genes in the inner ear, including adenovirus (AV), adeno-associated virus (AAV), herpes simplex virus and lentivirus (Derby et al., 1999; Hildebrand et al., 2008; Izumikawa et al., 2005; Lalwani et al., 2002; Lalwani et al., 1996). A variety of delivery techniques have also been described (Bedrosian et al., 2006; Jero et al., 2001; Lalwani et al., 2002; Praetorius et al., 2002; Yamasoba et al., 1999).
Many forms of genetic deafness, including those caused by GJB2 or GJB6 mutations, have pre-lingual onset. In these cases, early intervention is required to achieve effective gene correction or replacement. Mouse models of connexin-related deafness display significant hearing loss at the time of cochlear maturation, requiring that therapeutic strategies be targeted at the developing cochlea (Ahmad et al., 2007; Cohen-Salmon et al., 2002; Hildebrand et al., 2008; Kudo et al., 2003; Teubner et al., 2003). Recently, Bedrosian and colleagues developed and optimized a method for in utero gene transfer, whereby an adeno-associated virus pseudotype (AAV2/1) vector was shown to safely and efficiently transduce sensory hair cell progenitors in the murine otocyst (Bedrosian et al., 2006).
While in utero targeting of sensory hair cells with AAV2/1 provides therapeutic potential for many types of genetic hearing loss, there are important reasons to identify a virus with tropism for supporting cells in the developing cochlea. Aside from their role in the most common form of ARNSHL, cochlear supporting cells serve as the primary targets of intervention to induce hair cell regeneration (Izumikawa et al., 2005). One significant advance in the application of gene therapy to restore auditory function was the discovery that adenoviral delivery of Atoh1 (Math1) to supporting cells in the guinea pig cochlea resulted in the formation of “hair cell-like” cells (Izumikawa et al., 2005). The promise of hair cell regeneration and the potential treatment of common connexin-related deafness disorders are compelling reasons to identify vectors with tropism for inner ear supporting cells (Ballana et al., 2008; Brigande et al., 2009).
AV and bovine adeno-associated virus (BAAV) vectors have previously been used for successful transduction of cochlear supporting cells in adult rodents, but have never been delivered to the developing cochlea in vivo (Dazert et al., 2001; Di Pasquale et al., 2005; Ishimoto et al., 2002; Izumikawa et al., 2005; Ortolano et al., 2008; Shibata et al., 2009). In this study we used the transuterine microinjection approach to examine the safety and inner ear tropism of three previously untested vectors delivered to the developing murine cochlea: Ad5.CMV.GFP (early-generation adenovirus), Adf.11D (late-generation adenovirus) and BAAV.CMV.GFP (bovine adeno-associated virus).