Gene Therapy of the Central Nervous System: From Bench to Bedside

Michael G. Kaplitt, Matthew During

• Publisher: Academic Press
• Year: 2005
• ISBN:
  9780080454375
• Topic: Biological Sciences
• Subtopic: Genetics & Genomics
• Index: Biological Sciences

Few areas of biomedical research provide greater opportunities for radically new therapies for devastating diseases that have evaded treatment so far than gene therapy. This is particularly true for the brain and nervous system, where gene transfer has become a key technology for basic research and has recently been translated to human therapy in several landmark clinical trials. Gene Therapy of the Central Nervous System: From Bench to Bedside represents the first definitive volume on this subject. Edited by two pioneers of neurological gene therapy, this volume contains contributions by leaders who helped create this field and are expanding the promise of gene therapy for the future of basic and clinical neuroscience. Drawing upon this extensive collective experience, this book provides clear and informative reviews on a variety of subjects of interest to anyone exploring or using gene therapy for neurobiological applications in research and clinical praxis.* Presents gene transfer technologies with particular emphases upon novel vehicles, immunological issues and the role of gene therapy in stem cells
* Discusses preclinical areas that are likely to translate into clinical studies in the near future, including epilepsy, pain and amyotrophic lateral sclerosis
* Includes "insider" information on technological and regulatory issues which can often limit effective translation of even the most promising idea into clinical use

Section I

Gene Transfer Technology and Regulatory Issues

Outline

Chapter 1: Design and Optimization of Expression Cassettes Including Promoter Choice and Regulatory Elements

Chapter 2: Identification of Novel Adeno-Associated Virus Serotypes for Use as Vectors

Chapter 3: HSV Amplicon Vectors for Gene Delivery to the Nervous System

Chapter 4: Influence of the Immune System on Central Nervous System Gene Transfer

Chapter 5: Targeted Induction of Endogenous Neural Stem and Progenitor Cells: A New Strategy for Gene Therapy of Neurological Disease

Chapter 6: Neurosurgical Targeting, Delivery, and Infusion of Gene Therapy Agents in the Brain

Chapter 7: Gene Transfer for Neurological Disease: Agencies, Policies, and Process

CHAPTER 1

Design and Optimization of Expression Cassettes Including Promoter Choice and Regulatory Elements

Helen L. Fitzsimons and Matthew J. During

Abstract

Pivotal to the success of studies involving recombinant adeno-associated virus (rAAV)-mediated gene transfer to the brain is the design of the rAAV expression cassette and the selection of the rAAV serotype. Many promoters have been isolated that differ in cell-type specificity, size and strength. In addition, novel AAV serotypes are continually being isolated and characterized in vivo. These will differ in their cell-type-specific tropism, the efficiency of cellular transduction and the level and spread of gene expression mediated by the recombinant vectors. To that end, this chapter provides an introduction and summary of the promoters, regulatory elements and serotypes that are available and a guide to assist in the design of rAAV cassettes and selection of the appropriate rAAV serotype for a particular application.

Keywords

adeno-associated virus

promoter

gene expression

brain

regulatory element

I INTRODUCTION

In the 10 years since recombinant adeno-associated virus (rAAV) was first used successfully to transduce neurons (Kaplitt et al., 1994) it has proved to be a very efficient vector for gene transfer to the brain. The field is moving at a cracking pace with technical improvements in production and purification, cloning of new serotypes and also the selection and characterization of new promoters and regulatory elements. These advances have enabled transgenes to be targeted to specific cell types in focal or widespread areas of the brain and have dramatically increased the number of disease targets amenable to gene therapy.

A myriad of neurological disorders including Parkinson’s disease, Huntington’s disease, epilepsy and Alzheimer’s disease may now be treatable using rAAV-mediated gene therapy. Each disorder has different requirements in terms of the specific cell type to be transduced and the level and range of therapeutic protein necessary to fall within the therapeutic window for that particular disease.

The level of transgene expression is dependent on a number of factors. The choice of rAAV serotype influences the cell-type specificity and the dose of vector combined with the transduction efficiency of that particular serotype controls the spread of rAAV transduction within the tissue.

Also critical to the success of rAAV as a gene transfer vector is the design of the expression cassette, which once delivered by the rAAV vector, maintains control over the level and duration of transgene expression within that cell.

II DESIGN OF THE rAAV CASSETTE

The minimum requirements of an rAAV expression cassette are a promoter, a transgene and a polyadenylation site flanked on either end by AAV inverted terminal repeats. The 4.7 kb wild-type AAV genome is very tightly folded into the 20 nm AAV particle. Various analyses of the maximum size of the genome that can be accommodated with the particle have been carried out. Xu et al. (2001) demonstrated that a 5.7 kilobase (kb) rAAV expression cassette containing luciferase under control of the rat preproenkephalin promoter was packaged into functional AAV particles, which facilitated luciferase expression in primary rat neuronal cultures and in the rat brain. In addition, Hermonat et al. (1997) reported that 900 bp of stuffer sequence could be inserted into the 4.7 kb wild-type AAV genome (corresponding to a total genome size of 5.6 kb) without compromising the wild-type phenotype. In contrast, however, it has also been reported that rAAV packaging is optimal between 4.1 and 4.9 kb, with a sharp reduction in packaging efficiency up to 5.2 kb (Dong, et al., 1996). Addition of further DNA sequence precluded AAV packaging. These discrepancies in the maximum size of a transgene cassette that can be packaged into a functional rAAV particle may be reconciled by the possibility that each expression cassette has different topological constraints based on the tertiary folding structure of specific DNA sequences.

Of particular interest is the finding by Mastakov et al. (2002), who showed that the use of different promoters within the AAV expression cassette altered the antigenicity of the capsid. The efficacy of re-administration of rAAV vectors was tested by re-injecting an rAAV2-luciferase vector into the rat striatum at certain time points after the first administration (into the contralateral side). If the vector was re-administered at 2 or 4 weeks post-injection, neutralizing antibodies were detected and luciferase activity was reduced by 90%; however, if the second vector was injected after an interval of 3 months, luciferase expression was not altered. An unexpected caveat to the study was the finding that if the second dose of rAAV vector contained a different transgene or promoter to the first dose, there was no decrease in expression or production of neutralizing antibodies from the second vector. These data suggest that the outer structure of the virion is influenced by the vector genome sequence (Mastakov et al., 2002). A possibility that has yet to be examined is that alterations in the capsid structure may also influence the vector tropism.

It is becoming more obvious that obtaining optimal expression in a specific cell type is not as simple as selecting a cell-type-specific promoter of the desired size and strength. In fact the major influence over cell-type expression is in many cases not the promoter being used but the inherent tropism of rAAV. It is therefore pertinent at this point to discuss the impact that the tropism of the rAAV capsid has on achieving rAAV-mediated cell- and tissue-specific expression in the brain.

III CELL-TYPE-SPECIFIC TROPISM OF rAAV

Eleven distinct AAV serotypes have been isolated to date (Atchison et al., 1965; Mayor and Melnick, 1966; Bantel-Schaal and Zur Hausen, 1984; Gao et al., 2002; Mori et al., 2004), as have hundreds of AAV cap gene sequences (each representing a unique serotype), which were amplified from human and non-human primate tissues (Gao et al., 2003, 2004). The transduction properties of the vast majority have not yet been characterized in the brain.

Recombinant AAV serotype 2 (rAAV2) was the first rAAV vector to be used in the brain and its pattern of transduction has been the most widely characterized. The primary cell surface receptors of rAAV2 are membrane-bound heparan sulfate proteoglycans (Summerford and Samulski, 1998), which are present throughout the brain and on the surface of neurons and glial cells (Fuxe, et al., 1994). Two co-receptors for rAAV2 have so far been identified, the αVβ5 integrin receptor (Summerford et al., 1999) and the human fibroblast growth factor receptor 1 (Qing et al., 1999).

Bartlett et al. (1998) demonstrated that AAV was preferentially taken up into neurons in the rat brain by fluorescently labeling the wild-type AAV particle and thereby proving that the lack of expression in glia was not due to absence of promoter activity but lack of uptake.

Many analyses of cell-type-specific expression have been performed following rAAV2-mediated transduction of enhanced green fluorescent protein (EGFP) or other reporter genes into various brain regions. When gene expression was driven by neuron-specific promoters (see Section IV.B) including the neuron-specific enolase (NSE) promoter (Peel et al., 1997; Klein et al. 1998, 2002a), the platelet-derived growth factor β-chain (PDGF) promoter (Peel et al., 1997; Paterna et al. 2000), all of the transduced cells co-localized with the neuronal marke...

Table of contents

• Cover image
• Title page
• Table of Contents
• Contributors
• Preface
• Section I: Gene Transfer Technology and Regulatory Issues
• Section II: Gene Therapy for Degenerative and Functional Disorders
• Section III: Psychiatric and Behavioral Gene Therapy
• Section IV: Gene Therapy for Pain and Spinal Cord Diseases
• Section V: Gene Therapy for Brain Tumors and Neurogenetic Disease
• Subject Index