Janet Cunningham

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.

Convection-enhanced delivery of AAV-2 combined with heparin increases TK gene transfer in the rat brain

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.

AAV2-mediated gene delivery to monkey putamen: Evaluation of an infusion device and delivery parameters

In this study, a modified infusion procedure and a novel infusion device designed for use in humans (Clinical Device B) were evaluated for delivery of recombinant adeno-associated virus (AAV2) to brain. The device is composed of 1.2 m of fused silica inserted through a 24.6-cm surgical steel cannula designed to fit a standard Leksell clinical stereotaxic frame and micro-infusion syringe pump. AAV2 encoding the human aromatic l-amino acid decarboxylase gene (AAV-hAADC-2) was infused into the putamen of 4 normal rhesus monkeys as a supportive study for a clinical trial in Parkinsons disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 muL/min to 1 muL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 muL/min. The primary endpoints were safety evaluation of the infusion procedures and assessment of transgene expression at 5.5 weeks post-infusion. Clinical observations after vector infusions revealed no behavioral abnormalities during the study period. No differences in gross pathology with either the ramped or non-ramped infusion procedure were observed. Histopathology of the putamen was comparable with both procedures, and revealed only minimal localized inflammatory tissue reaction along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed throughout the putamen, with no significant difference in volume of immunostaining with either infusion procedure. Serum antibody levels against AAV2 vector exhibited a minor increase after infusion. These results validate the clinical utility of this new infusion device and non-ramped infusion conditions for intraputamenal gene therapy, and have the potential to impact a number of human diseases in which delivery of therapeutics to brain is indicated.

Distribution of AAV-TK following intracranial convection-enhanced delivery into rats.

Adeno-associated virus (AAV)-based vectors are being tested in animal models as viable treatments for glioma and neurodegenerative disease and could potentially be employed to target a variety of central nervous system disorders. The relationship between dose of injected vector and its resulting distribution in brain tissue has not been previously reported nor has the most efficient method of delivery been determined. Here we report that convection-enhanced delivery (CED) of 2.5 × 108, 2.5 × 109, or 2.5 × 1010 particles of AAV-thymidine kinase (AAV-TK) into rat brain revealed a clear dose response. In the high-dose group, a volume of 300 mm3 of brain tissue was partially transduced. Results showed that infusion pump and subcutaneous osmotic pumps were both capable of delivering vector via CED and that total particle number was the most important determining factor in obtaining efficient expression. Results further showed differences in histopathology between the delivery groups. While administration of vector using infusion pump had relatively benign effects, the use of osmotic pumps resulted in notable toxicity to the surrounding brain tissue. To determine tissue distribution of vector following intracranial delivery, PCR analysis was performed on tissues from rats that received high doses of AAV-TK. Three weeks following CED, vector could be detected in both hemispheres of the brain, spinal cord, spleen, and kidney.

Biodistribution of adeno-associated virus type-2 in nonhuman primates after convection-enhanced delivery to brain.

A combination treatment of AAV2-hAADC with oral levodopa is a novel therapeutic approach that is being developed for late-stage Parkinsons disease. Biodistribution of AAV2-hAADC was assessed over a wide range of vector dose in 12 monkeys with parkinsonian syndrome, 6 months after intraputamenal infusion. Quantitative PCR (Q-PCR) from all the major neuroanatomical regions of the brain indicated a dose-dependent increase in vector DNA, with 99% being detected in the target site and other basal ganglia tissues. Within these tissues, the distribution varied widely between the putamen (PT) and the globus pallidus, and this was attributed to differences in vector transport. Q-PCR and immunocytochemistry were consistent with results reported earlier for various measures of transgene expression including aromatic L-amino acid decarboxylase (AADC) activity assays, behavioral response, and in vivo imaging with positron emission tomography (PET). Outside of the brain, trace amounts of vector DNA were detected in the spleens of animals in the two highest dose groups, but not in any other peripheral tissue, blood, or cerebrospinal fluid. Some increase in neutralizing antibody titers to adeno-associated virus type-2 (AAV2) capsid protein was observed in monkeys that received high doses of AAV2-hAADC or control AAV2-GFP. This study further validates convection-enhanced delivery (CED) as the preferred method of viral vector delivery to the brain, and supports a Phase I clinical testing of AAV2-hAADC in humans with Parkinsons disease.

Basic Fibroblast Growth Factor Enhances Transduction, Distribution, and Axonal Transport of Adeno-Associated Virus Type 2 Vector in Rat Brain

The ubiquitous expression of cell surface heparan sulfate proteoglycan, a binding receptor for adeno-associated virus type 2 (AAV-2), may account for the broad host range of this vector. Because the fibroblast growth factor receptor type 1 has been postulated to be a coreceptor for successful AAV-2 entry into host cells, we designed a strategy to investigate whether coadministration of this virus with basic fibroblast growth factor (bFGF) can enhance AAV-2-mediated gene delivery. We injected AAV-2-thymidine kinase (AAV-2-TK) vector into rat striata and checked whether coinjection with bFGF enhanced transduction and/or enlarged the area of transgene expression. Immunostaining confirmed the tropism of AAV-2-TK for neurons. The previous injection (7 days before vector delivery) of bFGF had no major impact on vector distribution area. However, when the vector was coinjected with bFGF, the right striatum showed an average viral transduction volume of 5 mm(3), which was more than 4-fold larger when compared with the left side (AAV-2-TK plus phosphate-buffered saline). This result clearly indicates that simultaneous injection of bFGF with AAV-2-TK can greatly enhance the volume of transduced tissue, probably by way of a competitive block of AAV-2-binding sites within the striatum. Robust TK immunoreactivity was also observed in the globus pallidus, which receives anterograde projections from the striatum. We propose that postsynaptic transport of recombinant particles was likely responsible for the distribution of TK in the globus pallidus on both bFGF-treated and untreated sides. In summary, we found that bFGF acts as an adjuvant for distribution of AAV-2 in rat brain.

658. Development and Evaluation of an Infusion Device for AAV-Mediated Gene Delivery to the Brain

A custom infusion device was developed for delivery of recombinant AAV2 to human putamen in a phase I/II clinical trial for treatment of Parkinsons disease. The device was designed to 1) minimize vector loss to the internal surfaces of the cannula and tubing, 2) minimize the hold-up volume of the device such that vector could be dispensed efficiently from a syringe placed approximately four feet away from the site of infusion, 3) attach to a commonly used stereotactic surgical frame, 4) connect to a programmable syringe pump, and 5) allow manufacturing in compliance with quality system requirements (QSR) for medical devices.