Figure 2.

Human glial-derived neurotrophic factor (hGDNF) mRNA expression following AAV-5-hGDNF delivery to the cochlea. Polymerase chain reaction (PCR) using an expression vector containing hGDNF as a template demonstrated that the mouse (m)GDNF (M) primers (6/1, 3/4, and 2/5) do not amplify the hGDNF (H) gene (A). Reverse transcription PCR using cDNA from the mouse cochlea after injection of AAV-hGDNF (B) showed expression of both hGDNF when using two different sets of human primers (H: 3/4 and 6/1) and endogenous mGDNF when using two different sets of mouse primers (M: 3/4 and 6/1). Reactions without reverse transcriptase (–) were used as negative controls. Quantitative real-time PCR (qPCR) data were obtained 2 weeks after intracochlear injections in P1–3 mice of AAV-5-hGDNF at different dilutions (undiluted, 1/10, 1/20). (C–F) Data from 1 μL of undiluted AAV-5-hGDNF injections: Mice displayed neurological symptoms (tremors, poor coordination, ataxia, malformed tails) at P12, which became lethal starting at ∼P17 (D–F). At around P17, qPCR data showed that hGDNF was expressed at 1.43 million–fold compared to mGDNF (C). (G–I) One microliter of 1:10 diluted AAV5-hGDNF: Mice showed no neurological symptoms or mortality, but did have minor tail deformities (H and I). The qPCR data revealed high levels of expression of hGDNF relative to mGDNF, around 541,040-fold amplification (G). (J–L) Data after 1 μL of 1:20 diluted AAV5-hGDNF. Mice look similar to non-injected controls (K and L). The qPCR data still show high levels of expression of hGDNF relative to mGDNF, at around 46,830-fold amplification (J).