Optimized adeno-associated viral vector-mediated striatal DOPA delivery restores sensorimotor function and prevents dyskinesias in a model of advanced Parkinson's disease

Abstract

Viral vector-mediated gene transfer utilizing adeno-associated viral vectors has recently entered clinical testing as a novel tool for delivery of therapeutic agents to the brain. Clinical trials in Parkinson's disease using adeno-associated viral vector-based gene therapy have shown the safety of the approach. Further efforts in this area will show if gene-based approaches can rival the therapeutic efficacy achieved with the best pharmacological therapy or other, already established, surgical interventions. One of the strategies under development for clinical application is continuous 3,4-dihydroxyphenylalanine delivery. This approach has been shown to be efficient in restoring motor function and reducing established dyskinesias in rats with a partial lesion of the nigrostriatal dopamine projection. Here we utilized high purity recombinant adeno-associated viral vectors serotype 5 coding for tyrosine hydroxylase and its co-factor synthesizing enzyme guanosine-5'-triphosphate cyclohydrolase-1, delivered at an optimal ratio of 5 : 1, to show that the enhanced 3,4-dihydroxyphenylalanine production obtained with this optimized delivery system results in robust recovery of function in spontaneous motor tests after complete dopamine denervation. We found that the therapeutic efficacy was substantial and could be maintained for at least 6 months. The tyrosine hydroxylase plus guanosine-5'-triphosphate cyclohydrolase-1 treated animals were resistant to developing dyskinesias upon peripheral l-3,4-dihydroxyphenylalanine drug challenge, which is consistent with the interpretation that continuous dopamine stimulation resulted in a normalization of the post-synaptic response. Interestingly, recovery of forelimb use in the stepping test observed here was maintained even after a second lesion depleting the serotonin input to the forebrain, suggesting that the therapeutic efficacy was not solely dependent on dopamine synthesis and release from striatal serotonergic terminals. Taken together these results show that vector-mediated continuous 3,4-dihydroxyphenylalanine delivery has the potential to provide significant symptomatic relief even in advanced stages of Parkinson's disease.

Figure 1

Experimental design. At the onset of the experiment, 60 animals received a unilateral 6-OHDA lesion of the medial forebrain bundle (MFB) in order to obtain a complete lesion of the nigrostriatal dopamine pathway. Thirty-four animals that displayed more than six full-body turns per minute, towards the lesioned side, in the d-amphetamine (Amph)-induced rotation test were selected for further analysis in a battery of motor tests. At 6 weeks post-lesion, the animals were balanced into three treatment groups on the basis of performance in stepping, cylinder and the apomorphine (Apo)- and amphetamine-induced rotation tests to receive either the two vector mix (TH + GCH1 group, n = 14) or only the control vector (GCH1 only group, n = 8), or be treated as non-vector-injected lesion controls (Les-Sham group, n = 12). A fourth group with intact animals (n = 5) was included in the experiment as a non-lesioned control group in the l-DOPA-induced dyskinesia test. Over the 6 months that followed, the animals were tested repeatedly using the different motor tests. At Week 15 post-transduction, daily peripheral l-DOPA administration was initiated, and abnormal involuntary movement (AIM) scoring was conducted on treatment Days 1, 4, 8, 12 and 16. At Week 23, sub-groups of the TH + GCH1 group (n = 5) and the Les-Sham group (n = 5) were tested in the stepping and cylinder tests, whereafter they received a unilateral injection of 5,7-DHT into the medial forebrain bundle. At week 26, the same animals were re-tested in the two tests. All animals were killed for biochemical analysis at 28 weeks after vector delivery.

Figure 2

Recovery of motor function in tests of forelimb use. Near complete lesion of the ascending dopamine innervation by injection of 6-OHDA in the medial forebrain bundle (MFB) unilaterally caused a severe impairment in sidestepping using the contralateral limb in all animals (A). After striatal injection of rAAV vectors, the affected-limb use in the TH + GCH1 treated animals gradually improved over the first 9 weeks, to reach the level of use on the intact side at 12 weeks post-injection (illustrated by the dashed line in A, also shown in photographs in panels B’–B’’’). The therapeutic efficacy of continuous DOPA delivery was superior to the effect of peripheral l-DOPA injection, which improved the stepping performance in the two control groups (Les-Sham and GCH1-injected animals) only partially and did not have any detrimental effect in the TH + GCH1 group (C). Similar results were obtained in the cylinder test, where the animals in the TH + GCH1 group performed significantly better than the lesion and vector control groups at 12 weeks post-rAAV injection (D, see also photographs in E′ and E′′). This difference was diminished after peripheral administration of l-DOPA. In the staircase test (H), during baseline testing prior to vector treatment, all animals displayed severe impairment on the contralateral side in the number of sugar pellets eaten as compared with the ipsilateral side (dashed line in F). At 12 weeks post-rAAV, the animals in the TH + GCH1 group improved to a significantly greater extent than the lesion and vector control groups (F, G). In contrast to the other tests, peripheral l-DOPA administration had no impact on number of pellets eaten in the control groups (F). Statistics: (A). Repeated measures ANOVA time F(4,88) = 22.8, P < 0.001; time × group F(8,88) = 24.13, P < 0.001; one-way ANOVA for each time point baseline: F(2,22) = 1.01, P = 0.35; 6 weeks: F(2,22) = 5.70, P = 0.01; 9 weeks: F(2,22) = 10.04, P < 0.001; 12 weeks: F(2,22) = 27.48, P < 0.001; 26 weeks: F(2,22) = 45.74, P < 0.001, followed by Bonferroni-corrected Tukey’s HSD post hoc test. (C) Repeated measures ANOVA time F(1,22) = 5.52, P < 0.05; time × group F(2,22) = 2.95, P = 0.073; one-way ANOVA 12 weeks: F(2,22) = 58.28, P < 0.001, + l-DOPA at 15 weeks: F(2,22) = 19.92, P < 0.001, followed by Bonferroni-corrected Tukey’s HSD post hoc test and paired t-tests for within-group comparisons, P = 0.20 and P = 0.036 for GCH1-only and Les-Sham groups, respectively. (D) Repeated measures ANOVA time, F(1,22) = 0.28, P = 0.6; time × group, F(2,22) = 13.96, P < 0.001; group, F(2,22) = 6.94, P = 0.005; pre-test one-way ANOVA F(2,22) = 0.3, P = 0.997; 12 weeks post-AAV one-way ANOVA F(2,22) = 15.57, P < 0.001, followed by Bonferroni-corrected Tukey’s HSD post hoc test, + l-DOPA at 15 weeks one-way ANOVA F(2,21) = 2.85, P = 0.08. (F) Repeated measures ANOVA time F(1,21) = 33.41, P < 0.001; time × group F(2,21) = 5.65, P = 0.011; pre-score two-way ANOVA side F(1,138) = 717.20, P < 0.05; group F(2,144) = 2.25, P = 0.11; side × group F(2,138) = 2.10, P = 0.13; 12 weeks post-AAV two-way ANOVA side F(1,138) = 346.01, P < 0.05; group F(2,144) = 10.33, P < 0.05; side × group F(2,138) = 0.082, P = 0.082, followed by one-way ANOVA on the lesioned side, F(2,138) = 6.75, P < 0.05 and Tukey’s HSD post hoc test. After l-DOPA one-way ANOVA F(2,21) = 2.72, P = 0.09. (G) One-way ANOVA F(2,21) = 6.92, P < 0.05 and Tukey’s HSD post hoc test. Asterisk indicates different from Les-Sham group, hash indicates different from GCH1-only group.

Figure 3

Prevention of l-DOPA-induced dyskinesias. At Day 16 of daily l-DOPA treatment, 80% of the animals in the Les-Sham and GCH1-only groups displayed severe dyskinesias. In the TH + GCH1 group, the dyskinesias remained lower than in the control group during the entire peak of the l-DOPA 20–120 min post-l-DOPA injection (A). The dyskinesias were significantly lower in magnitude in this group when assessed as integrated scores (the sum of the individual scores for each assessed time point multiplied by the assessment interval in minutes, i.e. 20 min) presented as either total abnormal involuntary movements (AIM; axial + limb + orolingual) (B), or as individual axial (C), limb (D) and orolingual (E) components. Statistics: (A) Friedman test (non-parametric repeated measure); time effect P < 0.001; group effect P < 0.001. (B–E) Kruskal–Wallis (non-parametric one-way analysis) P ≤ 0.001. Kolmogorov–Smirnov post hoc analysis applied in all panels corrected for multiple comparisons (n = 6) using false discovery rate (FDR) compensation. Error bars in A represent 50% confidence interval (CI); boxes in B–E represent 50% CI, and whiskers 95% CI. Asterisk indicates different from Les-Sham group [P < FDR(0.05)], and hash indicates different from GCH1-only group [P < FDR(0.05)].

Figure 4

Impact of serotonin innervation in stepping and cylinder tests. Intact forebrain serotonin innervation is critical for recovery in some of the behavioural test paradigms. At 23 weeks post-rAAV injection, all animals in the Les-Sham and TH + GCH1 groups were re-tested in the cylinder and stepping tests. Thereafter, a sub-group of the animals received a unilateral injection of 5,7-DHT into the medial forebrain bundle and were re-assessed behaviourally 2 weeks later. In the cylinder test, the improvement caused by the rAAV-TH + GCH1 delivery was abolished (A), whereas the recovery in stepping performance was maintained (B). Statistics: Repeated measures ANOVA (A) Time, P = 0.027, F(1,6) = 8.51; time × group, P = 0.016, F(1,6) = 11.08; group, P = 0.015, F(1,6) = 11.27. (B) Time, P = 0.64, F(1,6) = 0.25; time × group, P = 0.42, F(1,6) = 0.76; group, P < 0.001, F(1,6) = 420. Post hoc paired t-test (A) TH + GCH1 pre-post P = 0.009; Bonferroni-corrected P = 0.018 (B) TH + GCH1 pre-post P = 0.48. Asterisk indicates significantly different from pre-5,7-DHT lesion.

Figure 5

Measurement of tetrahydrobiopterin (BH4) (A) and DOPA (B) levels in striatal tissue using HPLC with electrochemical detection after rAAV-mediated TH + GCH1 gene transfer. In the intact striatum, BH4 levels averaged ∼1 pmol/mg in the intact striatum (open bars in A). Depletion of the dopamine afferents to the striatum alone reduced BH4 levels by 85%, while the combination with a 5,7-DHT lesion caused a >98% reduction. TH + GCH1 delivery resulted in complete recovery to normal levels (solid bars in A). Striatal DOPA levels were below detection limit (<0005 pmol) in the intact rat striatum and just at the detection range in the lesioned striatum (open bars in B). The rAAV TH + GCH1 gene transfer induced an accumulation of ∼8 pmol/mg, which was not affected by the 5,7-DHT lesion. Statistics: Two-way ANOVA, (A) Side F(1,68) = 23.37, P < 0.001; group F(5, 68) = 39.3, P < 0.001; side × group F(5,68) = 36.15, P < 0.001. (B) Side F(1,68) = 57.54, P < 0.001; group F(5,68) = 26.29, P < 0.001; side × group F(5,68) = 26.29, P < 0.001. One-way ANOVA contralateral side not significant. Ipsilateral side: (A) F(5,34) = 38.340, P < 0.001; (B) F(5,34) = 26.292, P < 0.001, followed by Dunnett’s T3 post hoc test and Bonferroni-corrected paired t-tests for within-group comparisons Asterisk indicates different from contralateral side, hash indicates different from ipsilateral side of intact control group. 5-HT = serotonin; DA = dopamine.

Figure 6

Measurement of dopamine (A), 3,4-dihydroxyphenylacetic acid (DOPAC) (B) and homovanillic acid (HVA) (C) levels in striatal tissue and assay of aromatic L-amino acid decarboxylase (AADC) enzyme activity (D), quantified using HPLC-EC detection. The 6-OHDA medial forebrain bundle lesion removes >99.7% of dopamine input to the striatum and consequently results in 97% reduction in its metabolites. Following TH + GCH1 gene transfer, there is a small but clearly detectable increase in tissue dopamine levels (A). The reinstitution of 3,4-dihydroxyphenylacetic acid (B) and homovanillic acid (C) levels, in the latter case to normal level, better reflects the dopamine synthesis capacity after gene therapy. Lesioning of the serotonin system has no detectable impact on any of the measures (right-most section in A–C). The vast majority of striatal AADC activity is located in the dopamine terminals, while the remaining fraction around half appears to be located in serotonin terminals. Statistics: Two-way ANOVA, (A) Side F(1,68) = 1405.17, P < 0.001; group F(5,68) = 72.75, P < 0.001; side × group F(5,68) = 56.76, P < 0.001. (B) Side F(1,68) = 291.84, P < 0.001; group F(5,68) = 12.55, P < 0.001; side × group F(5,68) = 19.37, P < 0.001. (C) Side F(1,68) = 114.64, P < 0.001; group F(5,68) = 9.87, P < 0.001; side × group F(5,68) = 15.29, P < 0.001. (D) Side F(1,68) = 400.73, P < 0.001; group F(5,68) = 15.86, P < 0.001; side × group F(5,68) = 17.04, P < 0.001. One-way ANOVA contralateral side not significant. Ipsilateral side: (A) F(5,34) = 493.00, P < 0.001; (B) F(5,34) = 73.97, P < 0.001; (C) F(5,34) = 22.12, P < 0.001; (D), F(5,34) = 99.35, P < 0.001, followed by Dunnett’s T3 post hoc test and Bonferroni-corrected paired t-tests for within-group comparisons. Asterisk indicates different from contralateral side, hash indicates different from ipsilateral side of Les-Sham group. 5-HT = serotonin; DA = dopamine.

Figure 7

Measurement of serotonin (5HT) (A) and 5-hydroxyindolacetic acid (5-HIAA) (B) levels in striatal tissue quantified using HPLC-EC detection. The 6-OHDA lesion paradigm used here spared the serotonin projections to the striatum, and the TH + GCH1 delivery did not alter the tissue 5-hydroxytryptamine levels, whereas the 5,7-DHT lesion efficiently depleted both serotonin and 5-hydroxyindolacetic acid in the striatum in both vector-treated and lesion control animals. Statistics: two-way ANOVA, (A) Side F(1,68) = 1.18, P = 0.282; group F(5,68) = 15.44, P < 0.001; side × group F(5,68) = 17.46, P < 0.001. (B) Side F(1,68) = 55.83, P < 0.001; group F(5,68) = 7.64, P < 0.001; side × group F(5,68) = 14.52, P < 0.001. One-way ANOVA contralateral side not significant in (A); (B), F(5,34) = 3.389, P = 0.014. Ipsilateral side: (A), F(5,34) = 19.81, P < 0.001; (B), F(5,34) = 14.92, P < 0.001 followed by Dunnett’s T3 post hoc test, and Bonferroni-corrected paired t-tests for within-group comparisons. Asterisk indicates different from contralateral side; hash indicates different from corresponding side of Les-Sham group. DA = dopamine.