Using an approach that combines gene therapy with aromatic L-amino acid decarboxylase (AADC) gene and a pro-drug (L-dopa), dopamine, the neurotransmitter involved in Parkinson's disease, can be synthesized and regulated. Striatal neurons infected with the AADC gene by an adeno-associated viral vector can convert peripheral L-dopa to dopamine and may therefore provide a buffer for unmetabolized L-dopa. This approach to treating Parkinson's disease may reduce the need for L-dopa/carbidopa, thus providing a better clinical response with fewer side effects. In addition, the imbalance in dopamine production between the nigrostriatal and mesolimbic dopaminergic systems can be corrected by using AADC gene delivery to the striatum. We have also demonstrated that a fundamental obstacle in the gene therapy approach to the central nervous system, i.e., the ability to deliver viral vectors in sufficient quantities to the whole brain, can be overcome by using convection-enhanced delivery. Finally, this study demonstrates that positron emission tomography and the AADC tracer, 6-[ 18 F]fluoro-Lm-tyrosine, can be used to monitor gene therapy in vivo. Our therapeutic approach has the potential to restore dopamine production, even late in the disease process, at levels that can be maintained during continued nigrostriatal degeneration.

Dopamine, the major neurotransmitter depleted in Parkinson disease, can be synthesized and regulated in vivo with a combination of intrastriatal AAV-hAADC gene therapy and administration of the dopamine precursor l-Dopa. When tested in MPTP-lesioned monkeys, this approach resulted in long-term improvement in clinical rating scores, significantly lowered l-Dopa requirements, and a reduction in l-Dopa-induced side effects. Positron emission tomography with [(18)F]FMT confirmed persistent AADC activity, demonstrating for the first time that infusion of AAV vector into primate brain results in at least 6 years of transgene expression. AAV-hAADC restores the ability of the striatum to convert l-Dopa into dopamine efficiently. Introduction of this therapy into the clinic holds promise for Parkinson patients experiencing the motor complications that result from escalating l-Dopa requirements against a background of disease progression.

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1␣ promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL͞6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2-10 g͞ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200 -400 ng͞ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

Adeno-associated virus type2 (AAV-2) binds to heparan-sulfate proteoglycans on the cell surface. In vivo, attachment of viral particles to cells adjacent to the injection tract limits the distribution of AAV-2 when infused into the CNS parenchyma and heparin co-infusion might decrease the binding of AAV-2 particles to cells in the vicinity of the infusion tract. We have previously shown that heparin co-infusion combined with convection enhanced delivery enhances distribution of the GDNF family trophic factors (heparin-binding proteins) in the rat brain. In this work we show that heparin co-infusion significantly increases the volume of distribution of AAV-2 as demonstrated by immunoreactivity to the transgene product 6 days after infusion into the rat striatum.

We tested the hypotheses that initial immunization of rats with rAAV might limit subsequent transduction by rAAV-hAADC when stereotaxically infused into the striatum and that the level of inhibition would correlate with AAV neutralizing antibody titers. Immunohistochemical detection of AADC and analysis by stereology revealed that the control group (no immunization) had the greatest volume of distribution of AADC (20.32 +/- 2.03 mm3) (+/-SD). There was a 58% decrease in spread (8.46 +/- 3.67 mm3, P < 0.008) in the high-dose immunization group (5 x 10(10) vg rAAV-null). Transduction weakly correlated with preexisting titer levels of neutralizing antibody at the time of intrastriatal rAAV-hAADC infusion. Only rats with neutralizing antibody titers of 1:1208 +/- 332 had significantly decreased AADC transgene expression compared to the unimmunized control group. Immunohistochemistry on serial sections for inflammatory markers including GFAP, CD11b, CD4, and CD8a revealed normal morphology and no cellular infiltration, suggesting little immune reaction in the CNS. We conclude that rAAV vectors can transduce brain tissue in the context of preexisting immunity, but that efficiency of transduction declines significantly in the presence of very high titers of neutralizing antibodies. These results have important implications for gene therapy for CNS disorders.