Phillip Pivirotto

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.

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.

Overlesioned hemiparkinsonian non human primate model: correlation between clinical, neurochemical and histochemical changes.

Monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have been widely used as animal models of Parkinsons disease (PD). Depending on the method of administration different PD models can be developed. Systemic (iv, sc.) MPTP administration can induce an advanced parkinsonian syndrome. However, systemic administration may require intensive animal care after neurotoxin administration, as well as repeated high doses of MPTP to avoid spontaneous recovery. Unilateral intracarotid artery (ICA) MPTP administration induces a stable hemiparkinsonian syndrome, with the advantage of allowing the animal to groom and feed itself and having a control side in the same animal. However, this unilateral syndrome lacks the bilateral characteristics of advanced PD. Bilateral ICA administration can induce a reliable bilateral syndrome but inherent is the risk of severely impairing the animals and leaving them unable to maintain themselves. This report analyzed the PD model induced by administration of unilateral ICA and subsequent intravenous injections of MPTP in rhesus monkeys. The combined method of MPTP administration induces an advanced stable parkinsonian syndrome, in which the ICA injection of MPTP initiates the parkinsonian syndrome primarily in one hemisphere and the subsequent iv. doses (administered as needed) further deplete the dopamine (DA) system to induce a bilateral lesion in a shorter period of time, with fewer side effects. We studied the relationships between the behavioral, biochemical and histochemical changes related to the combined MPTP treatments to further characterize this model. The monkeys were categorized as presenting mild (stage 2) or moderate (stage 3) parkinsonism based on a parkinsonian rating scale. Postmortem biochemical analysis showed massive DA reduction equally in the caudate nucleus and putamen ipsilateral to ICA MPTP infusion, with varying degrees of DA preservation in the contralateral striatum. Differences between stage 2 and stage 3 were attributed to DA concentrations in the caudate nucleus and putamen of the contralateral hemisphere. Tyrosine hydroxylase immunohistochemistry revealed that the midbrain DA neurons of the group A8, A9, and A10 showed differential vulnerability for MPTP. This finding was similar to that observed in idiopathic PD with significant relationships between the clinical stages and cell losses in the group A9 (substantia nigra pars compacta). Positron emission tomography (PET) using [18F] 6-fluoro-L-m- tyrosine (FMT) showed that uptake (Ki) values correlated well with the biochemical data and are good predictors of DA levels in the contralateral striatal regions. Consistent with the immunohistochemical analysis, PET data also showed significant correlations with all groups of the DA cells. Here we describe an animal model that can play an important role in understanding the symptoms and therapeutic basis of PD since different severities of parkinsonian symptoms can be mimicked.

A novel MPTP primate model of Parkinson's disease: neurochemical and clinical changes

Positron emission tomography (PET) and the dopamine (DA) metabolism tracer, [18F]6-fluoro-L-m-tyrosine (FMT) were used to evaluate the relationship between DA metabolism and the clinical stage of parkinsonism monkeys following either unilateral ICA MPTP infusion or unilateral ICA MPTP infusion and subsequent varying sequential systemic doses of MPTP. Clinical stage corresponded to PET measures of striatal DA metabolism, showing the usefulness of the overlesioned hemiparkinsonian monkey as a stable model of various stages of Parkinsons disease (PD).

ADENO-ASSOCIATED VIRUS TYPE 6 IS RETROGRADELY TRANSPORTED IN THE NON-HUMAN PRIMATE BRAIN

We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.

Dopamine transporter loss and clinical changes in MPTP-lesioned primates

Single photon emission computed tomography (SPECT) and the dopamine (DA) transporter tracer, 2 beta-carboxymethoxy-3 beta-(4-iodophenyl)tropane ([123I]beta-CIT), were used to determine DA transporter density in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-lesioned monkeys with varying degrees of parkinsonism. The clinical stage of parkinsonism corresponded to SPECT measures of striatal DA transporter density suggesting that more severe parkinsonism was associated with a greater degree of dopaminergic terminal degeneration. These findings are similar to those reported earlier using positron emission tomography (PET) and the DA metabolism tracer, 6-[18F]fluoro-L-m-tyrosine (FMT), indicating that both are good methods for evaluating nigrostriatal degeneration in MPTP primate models.

Technique for bilateral intracranial implantation of cells in monkeys using an automated delivery system.

Intracerebral grafting combined with gene transfer may provide a powerful technique for local delivery of therapeutic agents into the CNS. The present study was undertaken to: (i) develop a reliable and reproducible automated cell implantation system, (ii) determine optimal implantation parameters of cells into the striatum, (iii) determine upper safe limits of cellular implantation into the neostriatum of monkeys. Autologous fibroblasts were infused into six sites of the striatum in nonhuman primates (Macaca mulatta, n = 11). Twenty-six-gauge cannulae were inserted vertically through cortical entry sites into the striatum (two sites in the caudate nucleus and four sites in the putamen) at predefined coordinates based on magnetic resonance imaging (MRI). The cannulae were guided by an electronically operated, hydraulic micropositioner and withdrawn at controlled rates, while cells (5, 10, 20, 40, or 80 μl/site) were infused simultaneously. Varying infusion rates and cell concentrations were also evaluated. Visualization and evaluation of graft placement were performed using contrast MRI at 3–5 days postsurgery. Animals were monitored for signs of clinical complications and sacrificed 2 weeks following surgery. Postimplantation MRI revealed a tissue mass effect of the implant with shifting of midline, edema, and infiltration of the white tracts at 40 and 80 μl/site. In addition, these animals developed transient hemiparesis contralateral to the implant site. MRI of animals grafted with 20 μl/site exhibited columnar-shaped implants and evidence of infiltration into white matter tracts possibly due to a volume effect. No clinical side effects were seen in this group. At 14 days postsurgery, MRI scans showed consistent columnar grafts (measuring approximately 5 mm in height) throughout the striatum in animals implanted with 5 or 10 μl/site. No signs of clinical side effects were associated with these volumes and postmortem histological examination confirmed MRI observations. Optimal surgical parameters for delivery of cells into the striatum consist of a graft volume of 10 μl/site, an infusion rate of 1.6 μl/min, a cell concentration of 2.0 × 105 cells/μl, and a cannula withdrawal rate of 0.75 mm/min. These results show that infusion of cells into the striatum can be done in a safe and routine manner.

APPLICATION OF GENE THERAPY FOR PARKINSON'S DISEASE : NONHUMAN PRIMATE EXPERIENCE

Publisher Summary Because of certain limitations of current therapy for Parkinsons disease (PD) new therapeutic approaches to PD are must needed. Direct administration of therapeutic agents into the central nervous system (CNS) eliminates the need to bypass the blood brain barrier (BBB), thereby reducing systemic side effects. However, cellular implants can overcome the need for persistent and local physical delivery of therapeutic agents into the CNS. Several alternative approaches utilizing genetic engineering have been proposed by many investigators. Primary or immortalized cells have been engineered to produce a specific protein in culture, and the cells are then implanted into the host CNS either by direct cell transplantation or by encapsulating cells into semipermeable membranes. Other approaches consist of in vivo gene transfer based on direct introduction of genetic material into the CNS, using viral or synthetic vectors. Several vehicles have been used for in vivo transfer of cDNA, including herpes simplex viral vectors, adenoviral vectors, direct plasmid DNA transfer, and, most recently, retroviral vectors Ex vivo methods using primary fibroblasts transduced with retroviral vectors and the in vivo method utilizing adeno-associated virus (AAV) are also described in this chapter as possible means of chronic delivery of therapeutic agents into CNS. However, further technological advances are required to optimize gene delivery, regulation of gene expression, and testing in appropriate functional models before gene therapy can be used extensively.

The immunophilin ligand GPI-1046 does not have neuroregenerative effects in MPTP-treated monkeys.

Nonimmunosuppressant immunophilin ligands have been shown to have neurotrophic properties in rodent models of Parkinsons disease (PD), although little is known about the effects of these ligands in primates. The immunophilin ligand, GPI-1046, promotes the regeneration of dopamine (DA) cells in association with functional recovery in rodent models. We explored the regenerative effects of GPI-1046 in an MPTP primate model of PD. We used single photon emission computed tomography (SPECT) and the DA transporter tracer (DAT), [(123)I]beta-CIT, to evaluate DAT density and clinical recovery before and after treatment with GPI-1046 or vehicle. Subsequent histological studies were also performed. No effects of GPI-1046 were found on any of these measures. These findings show that GPI-1046 does not have regenerative effects in MPTP-treated primates and suggest that there may be species differences with respect to the trophic effects of GPI-1046 on nigrostriatal DA neurons.

Safety and tolerability of MRI-guided infusion of AAV2-hAADC into the mid-brain of nonhuman primate

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal-recessive neurological disorder caused by mutations in the DDC gene that leads to an inability to synthesize catecholamines and serotonin. As a result, patients suffer compromised development, particularly in motor function. A recent gene replacement clinical trial explored putaminal delivery of recombinant adeno-associated virus serotype 2 vector encoding human AADC (AAV2-hAADC) in AADC-deficient children. Unfortunately, patients presented only modest amelioration of motor symptoms, which authors acknowledged could be due to insufficient transduction of putamen. We hypothesize that, with the development of a highly accurate MRI-guided cannula placement technology, a more effective approach might be to target the affected mid-brain neurons directly. Transduction of AADC-deficient dopaminergic neurons in the substantia nigra and ventral tegmental area with locally infused AAV2-hAADC would be expected to lead to restoration of normal dopamine levels in affected children. The objective of this study was to assess the long-term safety and tolerability of bilateral AAV2-hAADC MRI-guided pressurized infusion into the mid-brain of nonhuman primates. Animals received either vehicle, low or high AAV2-hAADC vector dose and were euthanized 1, 3, or 9 months after surgery. Our data indicate that effective mid-brain transduction was achieved without untoward effects.