A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.
There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders.
Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, 97006, USA.
Michelle M
MM
Gomes
Oregon Hearing Research Center, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR, 97239, USA.
Lingyan
L
Wang
Oregon Hearing Research Center, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR, 97239, USA.
Han
H
Jiang
Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, 97006, USA.
Christoph A
CA
Kahl
Oregon Hearing Research Center, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR, 97239, USA. brigande@ohsu.edu.
John V
JV
Brigande
D000759:Adjuvants, Anesthesia / Q000008:administration & dosage; D000818:Animals; D003638:Deafness / Q000235:genetics / Q000628:therapy*; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D007758:Ear, Inner / Q000196:embryology*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000191:economics*; D015316:Genetic Therapy; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D010424:Pentobarbital / Q000008:administration & dosage
D000759:Adjuvants, Anesthesia; D010424:Pentobarbital
Fetal gene transfer; Mouse experimental embryology; Recombinant adeno-associated virus (rAAV) vectors; Transuterine microinjection; Virus-mediated gene transfer