Jamie L. Eberling

Convection-Enhanced Delivery of AAV Vector in Parkinsonian Monkeys; In Vivo Detection of Gene Expression and Restoration of Dopaminergic Function Using Pro-drug Approach

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 Parkinsons 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 Parkinsons 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-l-m-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.

Results from a phase I safety trial of hAADC gene therapy for Parkinson disease

Background: In a primate model of Parkinson disease (PD), intrastriatal infusion of an adeno-associated viral (AAV) vector containing the human aromatic l-amino acid decarboxylase (hAADC) gene results in robust gene expression. After gene transfer, low doses of systemically administered l-dopa are converted to dopamine in the transduced striatal neurons, resulting in behavioral improvement without the side effects typically associated with higher doses of l-dopa. These studies led to the initiation of a phase I safety trial. Here we report the findings for the first cohort of five patients. Methods: Patients with moderate to advanced PD received bilateral infusion of a low dose of the AAV-hAADC vector into the putamen. PET scans using the AADC tracer, 6-[18F]fluoro-l-m-tyrosine (FMT), were performed at baseline and at 1 and 6 months after infusion as an in vivo measure of gene expression. Results: PET results showed an average 30% increase in FMT uptake (Kic) in the putamen after gene transfer. Preliminary analysis of clinical data indicates a modest improvement, but absence of a control and the nonblinded analyses make interpretation difficult. Conclusions: Thus far, this gene therapy approach has been well tolerated and shows PET evidence of sustained gene expression. These initial findings demonstrate the safety of the therapy; higher doses of adeno-associated viral vector containing the human aromatic l-amino acid decarboxylase gene in the next cohort of patients may further increase dopamine production in the putamen and provide more profound clinical benefit. GLOSSARY: AADC = aromatic l-amino acid decarboxylase; AAV = adeno-associated viral; DA = dopamine; FMT =6-[18F]fluoro-l-m-tyrosine; hAADC = human aromatic l-amino acid decarboxylase; l-dopa = levodopa; PD = Parkinson disease; ROI = region of interest; UPDRS = Unified Parkinson’s Disease Rating Scale.

Safety and tolerability of putaminal AADC gene therapy for Parkinson disease

Background: In Parkinson disease (PD), the benefit of levodopa therapy becomes less marked over time, perhaps because degeneration of nigrostrial neurons causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa into dopamine. In a primate model of PD, intrastriatal infusion of an adeno-associated viral type 2 vector containing the human AADC gene (AAV-hAADC) results in robust response to low-dose levodopa without the side effects associated with higher doses. These data prompted a clinical trial. Methods: Patients with moderately advanced PD received bilateral intraputaminal infusion of AAV-hAADC vector. Low-dose and high-dose cohorts (5 patients in each) were studied using standardized clinical rating scales at baseline and 6 months. PET scans using the AADC tracer [18F]fluoro-l-m-tyrosine (FMT) were performed as a measure of gene expression. Results: The gene therapy was well tolerated, but 1 symptomatic and 2 asymptomatic intracranial hemorrhages followed the operative procedure. Total and motor rating scales improved in both cohorts. Motor diaries also showed increased on-time and reduced off-time without increased “on” time dyskinesia. At 6 months, FMT PET showed a 30% increase of putaminal uptake in the low-dose cohort and a 75% increase in the high-dose cohort. Conclusion: This study provides class IV evidence that bilateral intrastriatal infusion of adeno-associated viral type 2 vector containing the human AADC gene improves mean scores on the Unified Parkinson’s Disease Rating Scale by approximately 30% in the on and off states, but the surgical procedure may be associated with an increased risk of intracranial hemorrhage and self-limited headache.

Estrogen- and tamoxifen-associated effects on brain structure and function.

We evaluated the effects of estrogen and tamoxifen, a selective estrogen receptor modulator, on positron emission tomography (PET) measures of brain glucose metabolism and magnetic resonance imaging (MRI) measures of hippocampal atrophy. Three groups of postmenopausal women were studied, women taking estrogen (ERT+), women with breast cancer taking tamoxifen (TAM), and women not taking estrogen or tamoxifen (ERT-). All subjects received a PET scan, an MRI scan, and cognitive testing. The TAM group showed widespread areas of hypometabolism in the inferior and dorsal lateral frontal lobes relative to the other two groups. The ERT- group showed lower metabolism in the inferior frontal cortex and temporal cortex with respect to the ERT+ group. The TAM group also showed significantly lower semantic memory scores than the other two groups. Finally, the TAM group had smaller right hippocampal volumes than the ERT+ group, an effect that was of borderline significance. Both right and left hippocampal volumes were significantly smaller than the ERT+ group when a single outlier was removed. The ERT- group had hippocampal volumes that were intermediate to the other two groups. These findings provide physiological and anatomical evidence for neuroprotective effects of estrogen.

Brain structure and cognition in a community sample of elderly Latinos

BackgroundPrevious studies have found that hippocampal atrophy and white matter hyperintensities (WMH) on MRI are linked to cognitive impairment and dementia. The authors measured these variables in a population-based cohort of older Mexican Americans with a wide spectrum of cognitive ability, ranging from normal cognition to dementia. ObjectiveTo investigate whether these structural brain changes were seen in individuals prior to the development of dementia and how these changes were related to the presence of dementia. MethodsA sample of 122 subjects was selected from the Sacramento Area Latino Study on Aging, and subjects were categorized into four groups of increasing levels of cognitive impairment: normal, memory impaired (MI), cognitively impaired but not demented (CIND), and demented. Hippocampal volume was quantified using a region of interest approach. WMH was rated on a semiquantitative scale as the percent of total volume of white matter. ResultsHippocampal volume was significantly reduced in CIND and demented individuals, and WMH were significantly increased in demented subjects. MI subjects did not have any significant changes in hippocampal volume or WMH. The risk for developing dementia was significantly and comparably increased in subjects with either hippocampal atrophy or high WMH. However, the risk for dementia increased dramatically in subjects with both hippocampal atrophy and a high degree of WMH. ConclusionReductions in hippocampal volume may be present before dementia but not until cognitive impairment is relatively severe. Because there is a synergistic effect between high WMH and hippocampal atrophy, interactions between vascular and degenerative processes may be important determinants of dementia.

Human corpus callosum in aging and alzheimer's disease: a magnetic resonance imaging study

The involvement of the corpus callosum in aging and Alzheimers disease (AD) is not clear. We measured cross sectional areas of the entire corpus callosum (CC), as well as the front 20% (genu), middle 60% (body), and posterior 20% (splenium) of the structure from a midsagittal MRI slice in AD patients (N = 20), and young (N = 16) and old (N = 13) control subjects. We found that mean CC area in young controls was 570 +/- 107 mm2. Aging did not significantly affect the mean area of the CC (562 +/- 98 mm2). A small, significant reduction was seen in AD in comparison to the young control group (480 +/- 133 mm2). However, AD is accompanied by a large and statistically significant reduction in the genu area in comparison to both young and old control subjects. A trend toward an age-dependent reduction in the body area is also accentuated in AD patients who showed significantly smaller callosal bodies than young controls. We conclude that selective changes within corpus callosum accompany aging and AD pathology.

Eight Years of Clinical Improvement in MPTP-Lesioned Primates After Gene Therapy With AAV2-hAADC

This study completes the longest known in vivo monitoring of adeno-associated virus (AAV)-mediated gene expression in nonhuman primate (NHP) brain. Although six of the eight parkinsonian NHP originally on study have undergone postmortem analysis, as described previously, we monitored the remaining two animals for a total of 8 years. In this study, NHP received AAV2-human L-amino acid decarboxylase (hAADC) infusions into the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned putamen. Restoration of AADC activity restored normal response to levodopa and gene expression could be quantitated repeatedly over many years by 6-[(18)F]fluoro-meta-tyrosine (FMT)-positron emission tomography (PET) and confirm that AADC transgene expression remained unchanged at the 8-year point. Behavioral assessments confirmed continued, normalized response to levodopa (improvement by 35% over historical controls). Postmortem analysis showed that, although only 5.6 + or - 1% and 6.6 + or - 1% of neurons within the transduced volumes of the striatum were transduced, this still secured robust clinical improvement. Importantly, there were no signs of neuroinflammation or reactive gliosis at the 8-year point, indicative of the safety of this treatment. The present data suggest that the improvement in the L-3,4-dihydroxyphenylalanine (L-Dopa) therapeutic window brought about by AADC gene therapy is pronounced and persistent for many years.

Preliminary evidence that estrogen protects against age-related hippocampal atrophy

Few studies have examined gender differences in hippocampal volumes, and the potential effect of estrogen on these measures has not been well studied. We used MRI to measure hippocampal volumes in elderly Mexican American men and women subjects in order to determine if there were gender differences and if estrogen replacement therapy (ERT) had an effect on hippocampal volume in postmenopausal women. MRI measures of hippocampal volumes (normalized to intracranial volume) were compared in 59 women and 38 men. Further comparisons were made between men subjects, women subjects taking ERT, and women subjects not taking ERT. There were no significant effects of gender on normalized hippocampal volumes. However, women subjects taking ERT had larger right hippocampal volumes than women subjects not taking ERT and larger anterior hippocampal volumes than men subjects and women subjects not taking ERT. These findings suggest a neuroprotective effect of estrogen.

Real-time MR imaging of adeno-associated viral vector delivery to the primate brain.

We are developing a method for real-time magnetic resonance imaging (MRI) visualization of convection-enhanced delivery (CED) of adeno-associated viral vectors (AAV) to the primate brain. By including gadolinium-loaded liposomes (GDL) with AAV, we can track the convective movement of viral particles by continuous monitoring of distribution of surrogate GDL. In order to validate this approach, we infused two AAV (AAV1-GFP and AAV2-hAADC) into three different regions of non-human primate brain (corona radiata, putamen, and thalamus). The procedure was tolerated well by all three animals in the study. The distribution of GFP determined by immunohistochemistry in both brain regions correlated closely with distribution of GDL determined by MRI. Co-distribution was weaker with AAV2-hAADC, although in vivo PET scanning with FMT for AADC activity correlated well with immunohistochemistry of AADC. Although this is a relatively small study, it appears that AAV1 correlates better with MRI-monitored delivery than does AAV2. It seems likely that the difference in distribution may be due to differences in tissue specificity of the two serotypes.

Regeneration of the MPTP-Lesioned Dopaminergic System after Convection-Enhanced Delivery of AAV2-GDNF

Clinical studies to date have failed to establish therapeutic benefit of glial cell-derived neurotrophic factor (GDNF) in Parkinsons disease (PD). In contrast to previous nonclinical neuroprotective reports, this study shows clinically relevant and long-lasting regeneration of the dopaminergic system in rhesus macaques lesioned with 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine 3–6 months before GDNF gene delivery (AAV2-GDNF). The observed progressive amelioration of functional deficits, recovery of dopamine, and regrowth of fibers to the striatal neuropil demonstrate that high GDNF expression in the putamen promotes restoration of the dopaminergic system in a primate model of advanced PD. Extensive distribution of GDNF within the putamen and transport to the severely lesioned substantia nigra, after convection-enhanced delivery of AAV2-GDNF into the putamen, indicates anterograde transport via striatonigral connections and is anticipated to occur in PD patients. Overall, these data demonstrate nonclinical neurorestoration after putaminal infusion of AAV2-GDNF and suggest that clinical investigation in PD patients is warranted.