Elsevier

Experimental Neurology

Volume 194, Issue 2, August 2005, Pages 476-483
Experimental Neurology

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

https://doi.org/10.1016/j.expneurol.2005.03.007Get rights and content

Abstract

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 Parkinson's disease (PD) patients. Two infusion protocols were tested: a ramped procedure (slow stepwise increases in rate from 0.2 μL/min to 1 μL/min), thought to be essential for convection-enhanced delivery (CED), and a non-ramped infusion at a constant rate of 1 μL/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.

Introduction

PD is characterized in part by the progressive loss of dopaminergic neurons in the substantia nigra and a severe decrease of dopamine in the putamen (Hornykiewicz, 1975). Aromatic l-amino acid decarboxylase (AADC) is an enzyme in the dopamine biosynthetic pathway that converts l-dopa to dopamine. We have previously shown that transfer of the cDNA encoding human AADC to rat or non-human primate putamen can reduce effective l-dopa doses in animal models of PD, and thereby restore dopamine to normal levels (Bankiewicz et al., 2000, Sanchez-Pernaute et al., 2001). In human PD patients, this therapy would be expected to lower l-dopa requirements and extend the duration during which clinical benefit of the drug is observed. A challenging feature of this therapy is that vector infusion must be quantitatively and accurately delivered to the putamen and distribution should be as diffuse from the needle delivery site as possible. Focal, concentrated delivery and expression of AADC could result in localized dopamine production in the putamen that may be undesirable.

Parkinsonian rhesus macaques have been used in pharmacological studies with the AADC gene expressed from a recombinant AAV2 vector with a CMV enhancer and promoter (AAV-hAADC-2) (Bankiewicz et al., 2000). The vector was infused by CED with a ramped infusion procedure through a prototype delivery device (“preclinical”) composed of a short fused silica catheter connected to a syringe pump by Teflon tubing. Although a standardized preclinical device was proven safe for delivery of vector to the putamen of non-human primates (Bankiewicz et al., 2000), an unacceptable amount of the vector was lost by adsorption to Teflon. The relatively large hold-up volume (or internal volume) of the Teflon tubing also required that the syringe and proximal end of the tubing (end furthest from the cannula) be filled with oil to conserve vector. Thus, a new device (Clinical Device B) was designed for administration of AAV2 vector to humans.

The clinical infusion device was designed to (1) minimize vector loss to the internal surfaces of the catheter and tubing, (2) minimize the hold-up volume of the device such that vector could be dispensed from a syringe placed approximately 1.2 m away from the site of infusion, (3) strengthen and lengthen the cannula material for added stability in the larger human brain, (4) attach to a commonly used stereotaxic surgery frame, and (5) allow manufacturing of a device in compliance with quality system requirements (QSR) for medical devices. The end product of these design requirements was Clinical Device B, a stereotaxic frame-compatible surgical steel cannula and Teflon tubing that are lined with fused silica and fitted to a standard Luer lock at the proximal end for attachment to a programmable syringe pump.

Macromolecules can be administered to large areas of the brain by taking advantage of fluid convection through interstitial spaces in the brain (Bobo et al., 1994, Lieberman et al., 1995). Maximal volume of distribution is achieved by applying a pressure gradient and optimizing dose, infusion time, and volume (Kroll et al., 1996). Typically, the infusion rate is ramped up over about 30 min to avoid an initial pressure buildup at the infusion site that could result in reflux of product along the needle track. However, a non-ramped delivery procedure would allow for a simpler infusion protocol and shorter infusion times that are important considerations for clinical success. The goal of this study was to evaluate the safety and efficacy of the new infusion device (Clinical Device B) and to compare ramped vs. non-ramped infusion procedures in normal monkeys. The primary endpoints of the present study were vector distribution as measured by immunohistochemistry for hAADC expression, and safety as evaluated by clinical observations, histopathology, and antibody formation.

Section snippets

Recombinant vector production

Recombinant AAV2 was generated by a triple transfection protocol (Matsushita et al., 1998). Briefly, after expansion of cells from the HEK 293 working cell bank through a series of disposable culture ware in DMEM containing 10% fetal bovine serum and 2 mM glutamine, cells were co-transfected with 3 plasmids (pAAV-hAADC-2, pHLP19 and pladeno5). After an appropriate transfection time, the medium containing the transfection reagent was replaced with serum-free medium and the cells were incubated

Experimental design

Recombinant AAV2 vectors transduce brain tissue efficiently, but transduction levels decline significantly in the presence of high neutralizing antibody (NAb) titers (>1:1200) (Sanftner et al., 2004). Therefore, 4 male rhesus monkeys with NAb titers of ≤1:100 were selected for AAV2 infusions (Table 1). MRI scans were performed prior to AAV2 delivery to anatomically determine stereotaxic coordinates for vector administration to the putamen. Animals were bilaterally infused with 1.5 × 1011 vg of

Discussion

In this study, newly developed infusion devices intended for administration of AAV2 vector in a clinical trial for Parkinson's disease were evaluated along with a comparison of infusion conditions. Mock infusions designed to test vector delivery established that essentially 100% of the intended dose could be delivered with Clinical Device B by avoidance of all contact of vector with Teflon or steel surfaces. Stereotaxic administration of AAV-hAADC-2 into the putamen of 4 non-human primates was

Acknowledgments

The authors thank the members of the Avigen manufacturing team (Jennifer Barr-Davidson, Hoc Nguyen, Joseph Prado, Shawn Chan, Hermia Kwan, James Giang, Liza Africa, Nadia Gowahari, Maria Aguago, Tannie Lee) for production of recombinant vector and Sierra (Sparks, NV) personnel for careful implementation of the in vivo portion of this study. Cheryl Pater is acknowledged for animal resource, contract management, and technical assistance. Peter Little at PAI is acknowledged for expert

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