Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease.
To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.
Mandel
R J
RJ
Department of Gene Therapy Applications, Cell Genesys Inc., Foster City, California 94404, USA.
Rendahl
K G
KG
Spratt
S K
SK
Snyder
R O
RO
Cohen
L K
LK
Leff
S E
SE
D000818:Animals; D001058:Apomorphine; D001522:Behavior, Animal / Q000187:drug effects; D003342:Corpus Striatum / Q000166:cytology / Q000201:enzymology; D000229:Dependovirus; D004195:Disease Models, Animal; D015259:Dopamine Agents / Q000378:metabolism; D018491:Dopamine Agonists; D006136:GTP Cyclohydrolase / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D006801:Humans; D007980:Levodopa / Q000235:genetics / Q000378:metabolism; D008297:Male; D017551:Microdialysis; D009474:Neurons / Q000201:enzymology; D010302:Parkinson Disease, Secondary / Q000201:enzymology / Q000628:therapy*; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000235:genetics; D012399:Rotation; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D015259:Dopamine Agents; D018491:Dopamine Agonists; D011994:Recombinant Proteins; D007980:Levodopa; D014446:Tyrosine 3-Monooxygenase; D006136:GTP Cyclohydrolase; D001058:Apomorphine