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

Contributors Thais Federici Lerner Research Institute, Cleveland Clinic Foundation: NB2-126A, 9500 Euclid Ave., Clevela...

Author: Michael G. Kaplitt | Matthew During

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Contributors

Thais Federici Lerner Research Institute, Cleveland Clinic Foundation: NB2-126A, 9500 Euclid Ave., Cleveland, OH 44195, USA.

Paul D. Acton Department of Radiology, Thomas Jefferson University, Philadelphia, PA, USA. Kaveh Asadi-Moghaddam Department of Neurological Surgery, The Ohio State University Medical Center N-1017 Doan HaU 410 W, 10th Avenue Columbus, OH 43210, USA.

David J. Fink University of Michigan, 1914 Taubman Center, 1500 East Medical Center Drive Ann Arbor, MI 48109-0316, USA.

Krystof Bankiewicz Department of Neurological Surgery, University of California, San Francisco, CA 94103, USA.

Helen L. Fitzsimons NY 10032, USA.

Gerard J. Boer Netherlands Institute for Brain Research, Meibergdreef 33,1105 AZ Amsterdam, The Netherlands.

Neurologix Research, Inc., New York,

John R. Forsayeth Department of Neurological Surgery, University of California, San Francisco, CA 94103, USA.

Nicholas M. Boulis Lemer Research Institute Cleveland Clinic Foimdation: NB2-126A, 9500 Euclid Ave., Cleveland, OH 44195, USA.

Cornel Fraefel Institute of Virology, University of Zurich, Zurich, Switzerland. Justin F. Eraser Department of Neurological Surgery, New York Presbyterian Hospital - Weill Cornell Medical Center, New York, USA.

Xandra O. Breakefield Department of Neurology and Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, MA 02114, USA.

Guangping Gao Gene Therapy Program, Department of Medicine, University of Pennsylvania School of Medicine, Philadephia, PA 19104, USA.

Peter Carmeliet Center for Transgene Technology and Gene Therapy Flanders Intenmiversitary Institute for Biotechnology KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Leuven, Belgium.

Joseph C. Glorioso Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, USA.

E. Antonio Chiocca Department of Neurological Surgery, The Ohio State University Medical Center N-1017 Doan Hall 410 W, 10th Avenue Columbus, OH 43210, USA.

Steven A. Goldman Division of Cell and Gene Therapy, Department of Neurology, University of Rochester Medical Center, Rochester, NY 14580, USA.

Ronald G. Crystal Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

Thomas A. Green Department of Psychiatry and Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.

Jane Dunning Department of Neurological Surgery, Weill Medical College of Cornell University, New York, NY 10021, USA.

Neil R. Hackett Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

Matthew J. During Department of Neurological Surgery, New York Presbyterian Hospital, Weill Medical College of Cornell University New York, NY 10021, USA.

Piotr Hadaczek Department of Neurological Surgery, University of California, San Francisco, CA 94103, USA.

Marina E. Emborg Wisconsin National Primate Research Center and Department of Anatomy, University of Wisconsin - Madison, 1223 Capitol Court, Madison, WI53715, USA.

William T.J. Hendriks Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands.

IX

CONTRIBUTORS

Charles E. Inturrisi Department of Pharmacology Weill Medical College of Cornell University New York, NY 10021, USA.

Neurosurgery, Wiell Medical College of Cornell University, New York, NY 10021, USA.

Luc Jasmin Department of Anatomy and Neurological Surgery, University of California, San Francisco, CA 94143-0452, USA.

Eric J. Nestler Department of Psychiatry and Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.

Stephen M. Kaminsky Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

Francesco Noe Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20157 Milan, Italy

Michael G. Kaplitt Department of Neurological Surgery, New York Presbyterian Hospital, Weill Cornell Medical College of Cornell University New York, NY 10021, USA.

Peter T. O'Hara Department of Anatomy and W M . Keck Foimdation Center for Integrative Neuroscience, University of California, 513 Parnassus Ave., San Francisco, CA 94143-0452, USA.

Matthias Klugmann Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. Diether Lambrechts Center for Transgene Technology and Gene Therapy Flanders Interuniversitary Listitute for Biotechnology KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Leuven, Belgium. Patricia A. Lawlor Department of Molecular Medicine & Pathology, Faculty of Medical and Health Sciences, The University of Auckland, New Zealand. Claudia B. Leichtlein Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany Neal Luther Department of Neurological Surgery, New York Presbyterian Hospital, Weill Cornell Medical College of Cornell University New York, NY, USA. Marina Mata Department of Neurology, University of Michigan and VA Ann Arbor Healthcare System, Ann Arbor, MI 48109, USA. Jerry R. Mendell Ohio State University, Center for Gene Therapy, Columbus Children Research Institute, 700 Colombus Drive, Columbus, OH 43205, USA. Anne Messer Wadsworth Center, New York State Department of Health, and Department of Biomedical Sciences, University of Albany Albany, NY 12201, USA. Andra Miller The Biologies Consulting Group 6113 Walhonding Road, Bethesda, MD 20816, USA.

Sonoko Ogawa Department of Kansei and Cognitive Brain Science, University of Tsukuba, Tsukuba, Japan. Donald W. Pfaff Laboratory of Neurobiology and Behavior, The Rockefeller University New York, NY 10021, USA. Harish Poptani Department of Radiology, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Marc J. Ruitenberg Red's Spinal Cord Research Laboratory, School of Anatomy and Human Biology, & Western Australian Institute for Medical Research and UWA Centre for Medical Research, The University of Western Australia, Crawley, Perth, Western Australia. Claudia Senn Institute of Virology, University of Zurich, Zurich, Switzerland. Fraser Sim Division of Cell and Gene Therapy, Department of Neurology, University of Rochester Medical Center, Rochester, NY 14580, USA. Dolan Sondhi Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA. Mark M. Souweidane Department of Neurological Surgery, Weill Medical College of Cornell University, New York, NY 10021, USA. Qingshan Teng Lemer Research Institute, Cleveland Clinic Foundation: NB2-126A, 9500 Euclid Ave., Cleveland, OH 44195, USA. Luk H. Vandenberghe Department of Medicine, Gene Therapy Program, Katholieke Universiteit Leuven, Kapucijnenvoer 33, B-3000 Leuven, Belgium.

Todd W. Miller Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, University of Albany Albany, NY 12201, USA and Vanderbilt University 2200 Pierce Ave., PRB 618 Nashville, TN 37232, USA.

Joost Verhaagen Netherlands Institute for Brain Research, Meibergdreef 33,1105 AZ Amsterdam, The Netherlands.

Jeffrey Moirano Department of Medical Physics, University of Wisconsin - Madison, 1300 University Avenue, The Madison, WI 3706, USA.

Annamaria Vezzani Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20157 Milan, Italy

Sergei Musatov Laboratory of Neurobiology & Behavior, The Rockefeller University and Laboratory of Molecular

Charles H. Vite WF. Goodman Center for Comparative Medical Genetics and Department of Clinical Studies,

CONTRIBUTORS

School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. James M. Wilson Department of Medicine, Gene Therapy Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

XI

John Wolfe W.R Goodman Center for Comparative Medical Genetics and Department of Pediatrics, University of Pennsylvania, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

Preface

Genetic manipulation in behavioral neurobiology is among the more ambitious and complex research fields, yet the contributions here demonstrate the power of this technology to understand the molecular biology of behaviors such as drug addiction and sexual behavior. Finally, stem cells have become a major source of hope to many with debilitating diseases, yet many scientific hurdles limiting effective human therapeutic applications remain. As outlined here, the marriage of gene therapy and stem cells has the potential to facilitate translation of this important technology into clinical practice. We hope that this volume will prove valuable to anyone interested in gene therapy. To those new to the field, we have asked several authors to highlight important methodological or technical issues, which could facilitate successful application of gene therapy to various areas of basic and translational neurobiology research. But we also expect that even the most seasoned gene therapy veteran will find many new and interesting things here. Despite having worked in this field for more than 15 years, we nonetheless found that most of the chapters offered valuable insights or interesting perspectives on many issues, which we are facing in our own labs and clinical practice. This effort would not have been possible without the help and understanding of many people. First, we are extremely grateful to all of the authors who have contributed to this book. Although the number of excellent scientists in this area are far too large to be included in a single volume, we believe that the reader will find many of the pioneers of neurological gene therapy represented here. In addition, we have tried to identify those newer investigators whose creativity and energy are helping to increase the breadth and promise of this field for the future. We are also indebted to Johannes Menzel, Senior Publishing Editor

Ten years ago, an earlier version of this book, entitled "Viral Vectors: Gene Therapy and Neuroscience Applications" focused upon developing gene transfer technologies in the brain and potential applications largely in neurobiological research. The title of the current volume and the contents reflect the enormous strides made in this field over the past decade. Since the last book, several gene therapy societies have either begun or expanded around the world to become large, robust organizations. Those interested in research or clinical gene therapy applications in the nervous system are now among the largest constituencies at the annual meetings of these groups. At the time of the earlier version, the only clinical trials ongoing were in neurooncology, and those were in their infancy. While neurooncology remains an important area, which is represented here, at least three other chapters reflect ongoing or completed human clinical trials of gene therapy for Parkinson's disease. Batten disease and Canavan disease. Several other contributions outline areas which may be in clinical trial at or soon after the printing of this book, including pain and epilepsy. With the expansion of clinical gene therapy applications, new considerations have recently arisen which are comprehensively reviewed here. Among these are the role of the immune system in both the safety and efficacy of gene transfer, functional imaging to follow both gene expression and the consequences to various brain regions, and the method of gene delivery to the human brain. Although clinical trials and associated issues have become more prominent over the past 10 years, many important and fascinating basic science applications of gene transfer remain as well. Technical issues relating to the efficiency of gene packaging and delivery are addressed, but we have endeavored not simply to review well-documented issues but rather focus upon newer areas such as synthetic or chimeric viruses.

xui

XIV

at Elsevier, and his assistant, Maureen Twaig, for their outstanding work in overseeing the development and completion of this project. We and many of the chapter authors have both research and clinical responsibilities, and often we have not been the easiest group to work with, but this book would likely never have been completed without their balance of patience and diligence. Finally, this is one of the rare opportunities

PREFACE

that we have to thank our families for their support not only during the production of this book, but over the many years of training and subsequent long hours and frequent travel which have allowed us to participate in the evolution of this exciting field. Michael G. Kaplitt Matthew J. During

C H A P T E R

1 Design and Optimization of Expression Cassettes Including Promoter Choice and Regulatory Elements Helen L, Fitzsimons, 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.

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

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 Copyright © 2006, Elsevier, Inc. All rights of reproduction in any form reserved.

1. DESIGN AND OPTIMIZATION OF EXPRESSION CASSETTES INCLUDING PROMOTER CHOICE

have been carried out. Xu et al. (2001) demonstrated that a 5n 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 wildtype 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 celltype 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 aVj85 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 j^-chain (PDGF) promoter (Peel et al., 1997; Paterna et al., 2000), all of the transduced cells co-localized with the neuronal marker NeuN and failed to co-localize with the astrocytic marker glial fibrillary acidic protein (GFAP). Similarly, no glial transduction was detected when the expression cassette was under control of the cellular hybrid cytomegalovirus (CMV) immediateearly enhancer/chicken j8-actin (CBA) promoter (see Section IV.C; Klein et al., 2002b; Burger et al., 2004). Under some conditions, rAAV2-mediated transduction of glia has been observed. When the viral CMV promoter was used (see Section IV.A), approximately 1-1.5% of transduced cells were astrocytes (Klein et al., 1998). This was also observed when the CMV promoter was used in combination with the human jS-globin second intron (Scammell et al., 2003), a small

I. GENE TRANSFER TECHNOLOGY AND REGULATORY ISSUES

CHOICE OF PROMOTER

portion of human j8-globin exon two and exon three (the MD promoter, Mandel et al., 1998) or the first hGH intron (Shen et a l , 2000). The cell-type-specific tropisms of rAAVl and rAAV5 have more recently been characterized in the brain. The VPl capsid proteins of AAVl are reasonably well conserved with AAV2, sharing 83% amino acid identity; however AAV5 is more divergent, sharing 58% and S7% amino acid identity with AAVl and AAV2, respectively. The three serotypes all differ with respect to their target cell surface receptors. Neither AAVl nor AAV5 bind heparin (Chiorini et al., 1999; Rabinowitz et al., 2002). AAV5 binds a-2,3-and a-2,6-N-linked sialic acid with high affinity (Walters et al., 2001; Kaludov et al., 2001) and its receptor was recently determined to be the platelet-derived growth factor-a receptor (DiPasquale et al., 2003). The membrane receptor(s) for AAVl have not been identified. Although they enter the cell by different receptors a n d / o r pathways, rAAVl and rAAV5 also preferentially target to neurons. Passini et al. (2003) designed an expression cassette containing the human jS-glucuronidase (GUSB) promoter and transgene, the SV40 splice donor/acceptor and polyA flanked by AAV2 inverted terminal repeats (ITRs) and crosspackaged it into either the rAAVl or rAAV5 capsid. Following intraventricular injection into neonates, an analysis of the cell types transduced by either of the rAAV vectors revealed that almost all the transduced cells contained the neuron-specific marker NSE and none co-localized with an astrocytic or oligodendrocytic marker. Burger et al. (2004) carried out a comprehensive analysis of rAAVl or rAAV5mediated cell-type specific expression in many areas of the rat brain. An EGFP expression cassette driven by the CBA promoter was cross-packaged into either the rAAVl or rAAVS capsid and injected into the hippocampus, substantia nigra, striatum, globus pallidus and spinal cord. Following immunohistochemistry with cell-type specific markers, cell counts revealed that all of the transduced cells from both rAAV serotypes were neuronal. The authors calculated that based on the number of cells that were analyzed, the level of astrocytic transduction could not be higher than 0.05%. At odds with this conclusion are the results from Wang et al. (2003), who found that injection of a crosspackaged rAAVl vector containing EGFP driven by the CMV promoter into the mouse brain resulted in considerable glial expression although the majority of expression was neuronal. Astrocytic expression in the striatum and corpus callosum as well as oligodendrocytic and

mîcroglial expression within the white matter was detected. Following immunohistochemistry with celltjrpe specific markers, quantitative cell counts of transduced cells lining the ventricular space showed that approximately 36% of transduced cells were astrocytes, 8% were oligodendrocytes and 5% were microglial cells (Wang et al., 2003). A possible explanation for the differences observed in the level of glial transduction between these studies is that rAAV has the ability to transduce glia at low frequencies and the level of glial transduction observed depends on the activity of the promoter in glia. It is clear from the rAAV2-based studies on cell-specific tropism that viral promoters (see Section IV.A) such as CMV promoter or the rous sarcoma virus (RSV) promoter appears to allow a higher level of expression in glial cells whereas the neuronal NSE promoter and the CBA promoter appear to be almost silent. The method of vector administration, site of injection and the use of dissimilar titers between research groups may also account for the differences observed. In addition, rAAV produced by different purification methods may produce different results as cellular contaminants in the vector stocks can alter transduction efficiency (Tenenbaum et a l , 1999). In summary, if rAAVl, rAAV2 or rAAV5 are injected into the brain with the transgene under control of a neuronal promoter the cell-specific pattern of expression will most likely be entirely neuronal. If a viral-derived promoter is used there may be a low level (probably less than 2%) of astrocytic a n d / o r oligodendrocytic/microglial expression, depending on which area of the brain is injected and at what titer. In many instances, although neuronal expression is desired, the level of glial expression will be low enough to be considered insignificant. A promoter that drives neuronspecific expression is thus not absolutely required and in selection of a promoter, equal if not more importance should be placed on its size and the level of expression it drives in the target brain area. IV>

CHOICE OF PROMOTER

The choice of promoter often depends upon a compromise between the levels of expression required, the target cell type and the size of promoter that can be accommodated by the cassette without overstretching the 4.7 kb rAAV packaging limit. As more efficient packaging and purification technologies are developed, leading to higher vector titers, the compromise is leaning less toward achieving an optimal level of


1. DESIGN AND OPTIMIZATION OF EXPRESSION CASSETTES INCLUDING PROMOTER CHOICE

expression but more toward the ability to target particular cell types. The following is a detailed description of the expression properties of viral, neuronal and hybrid promoters that have been most commonly used in concert with rAAV for transduction of the specific brain areas and cell types. A summary of the main characteristics of the promoters described in this chapter is provided in Table 1.

A,

Viral Promoters

1. The Human Cytomegalovirus Immediate/Early Qene Promoter and Enhancer (CMV promoter) The 0.7 kb CMV promoter has been the most widely used promoter for achievement of rAAV-mediated gene expression in the brain. Almost all the original studies examining the potential of rAAV as a vector for gene transfer to the brain employed the CMV promoter. In the first published example of rAAV-mediated transduction of the brain, Kaplitt et al. (1994) used an rAAV2 expression cassette consisting of the CMV promoter driving expression of tyrosine hydroxylase. Three weeks after injection of this vector into the rat striatum, approximately 1000 transduced neurons could be detected, however expression was significantly diminished 4 months post-injection (Kaplitt et al., 1994). Although rAAV-mediated gene expression driven by the CMV promoter can be detected 12 months following administration (Lo et a l , 1999; Tenenbaum et al., 2000), many researchers have reported a reduction of gene expression over time when using the CMV promoter (McCown et al., 1996; During et al., 1998; Klein et al., 1998; Lo et al., 1999; Tenenbaum et al., 2000). It is not entirely clear why this occurs, although silencing of the CMV promoter has been observed following methylation at CpG dinucleotides (Prosch et al., 1996). 2, The Rous Sarcoma Virus Long Terminal Repeat Promoter The small size of the 0.4 kb RSV promoter is an attractive property for use in rAAV expression cassettes. This promoter has been used to direct rAAV2, rAAV4 and rAAV5-mediated lacZ expression in the rat ependyma and striatum (Davidson et al., 2000). Recombinant AAV5-mediated expression was highest in the striatum, with approximately 5000 cells transduced in comparison to fewer than 100 for rAAV2 and rAAV4. In the ependyma, rAAV4 and rAAV5 both

facilitated transduction of around 200 ependymal cells 15 weeks post-injection. Nomoto et al. (2003) compared the performance of the RSV and CMV promoters in gerbil brain. Recombinant AAV2 and rAAV5 vectors containing lacZ under control of each of the RSV and CMV promoters were injected into the mongolian gerbil hippocampus (Nomoto et al., 2003). Transgene expression was examined 5 days post-injection by Xgal staining. Expression from the rAAV5 vectors was higher than that directed by the rAAV2 vectors. A comparison of CMV- versus RSV-driven expression for each serotype showed that while transduction rAAV2/CMV-lacZ resulted in a poor level of staining clustered around the stratum oriens, rAAV2/RSV-lacZ expression was more widespread in the pyramidal and granule cell layers. Recombinant AAV5/CMV-lacZ expression was present throughout the hippocampus whereas rAAV5/ RSV-lacZ expression was concentrated at high levels in the granule cell layer. A quantitative comparison of transgene expression was not carried out, although the overall level of gene expression directed by the RSV promoter appeared to be superior to that driven by the CMV promoter (Nomoto et al., 2003). The stability of the RSV promoter over a longer time frame has not been examined. B.

Neuron^Specific Promoters

1.

The Rat 'Neuron-Specific Enolctse Promoter

The 2.2 kb NSE promoter was originally shown to drive a high level of exclusively neuronal expression in the brains of NSE-lacZ transgenic mice (Forss-Petter et al., 1990). Use of the NSE promoter to drive EGFP expression from an rAAV2 vector resulted in a high level of EGFP expression in the rat spinal cord (Peel et al., 1997). Since then, this promoter has also been demonstrated to promote robust rAAV-mediated expression in many brain areas including the striatum (Mastakov et al., 2001; Xu et al., 2001) medial septum (Klein et a l , 1998), substantia nigra (Klein et al., 1998, 2002a; Peel and Klein, 2000; Xu et al., 2001) and the hippocampus (Klein et al., 1998, 2002a; Xu et al., 2001). Klein et al. (1998) compared rAAV2/NSE-driven expression in the rat striatum and substantia nigra to that controlled by the CMV promoter and found that expression driven by CMV declined to barely detected levels by 3 months post-injection (Klein et al., 1998) whereas NSE-driven expression was eightfold higher than that of CMV at its peak and furthermore expression remained stable over the 3-month


CHOICE OF PROMOTER

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es become more refined, we may envisage the acquisition of progenitors for all major phenotypes of the human CNS. As a result, neurodegenerative diseases as diverse as Parkinson's, Huntington's, and Alzheimers's may prove feasible targets for cell-based therapy, as progenitors restricted to midbrain dopaminergic, striatal GABAergic, and forebrain cholinergic fate, respectively, become available. Indeed, as a therapeutic modality, the transplantation of neuronal progenitors would seem of greatest potential therapeutic efficacy for neurodegenerative diseases such as Parkinson's, which is largely attributable to the loss of a single neural phenotype concentrated in a single location, and the metabolic oligodendrocytic disorders, in which a relatively homogeneous phenotype is affected within a single brain compartment. On the other hand, diseases of

multiple phenotypes or multifocal pathology may be less appropriate for cell therapy, and rather more amenable to strategies intended to induce resident progenitors to replace injured or lost cell populations in situ, in response to both delivered and endogenous cues.

V. E N D O G E N O U S PROGENITOR CELLS ARE MOBILIZED BY INJURY A N D DISEASE The replacement of neostriatal neurons from resident progenitors was first identified in songbirds, in which neurons of the adult vocal control nucleus are seasonally replaced by a newly generated cohort (Goldman and Nottebohm, 1983; Nottebohm, 1985, 2002). However, neostriatal neurogenesis does not appear to occur in the normal adult mammalian brain, despite the production of new neurons along the striatal wall that migrate rostrally to join the olfactory stream (Lois and Alvarez-BuyUa, 1994; Lois et al., 1996). Yet neostriatal neuronal production has been reported in adult marmnals in response to injury (Lindvall et al., 2004), as have been limited instances of compensatory cortical neurogenesis (Magavi et al., 2000; Chen et al., 2004) (Fig. 2). Compensatory neuronal addition to the neostriatum in particular has been reported by several groups following experimental focal stroke (Arvidsson et al., 2002; Parent et al., 2002; Jin et al., 2003). Similarly, Nakafuku and co-workers described compensatory replacement of hippocampal pyramidal neurons - which, despite their apparent dissimilarity from striatal medium spiny cells, comprise another periventricular subcortical neuronal pool (Nakatomi et al., 2002). Other groups have recently reported apparent compensatory neurogenesis in the striatum of Huntington's disease patients (Curtis et al., 2003), and increased dentate neurogenesis in the hippocampus of Alzheimer's patients (Jin et al., 2004). Together, these instances of compensatory neurogenesis in the striatum and hippocampus, though quantitatively modest, suggest the potential for recruiting new neurons from endogenous progenitor cells as a therapeutic strategy in each of these subcortical structures.

VL RESIDENT PROGENITOR CELLS C A N BE MOBILIZED PHARMACOLOGICALLY A N D GENETICALLY Many studies in restorative neurology of the past decade have focused on the use of transplanted neurons


RESIDENT PROGENITOR CELLS CAN BE MOBILIZED PHARMACOLOGICALLY AND GENETICALLY

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57

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FIGURE 2 Compensatory and induced neuronal recruitment to the adult brain. This schematic illustrates both the described loci of compensatory and experimenterinduced neurogenesis in the adult rat brain, with relevant references listed for each. Loci of experimental compensatory neurogenesis include the neostriatum and hippocampal pyramidal layer following stroke. In patients, compensatory neurogenesis has similarly been reported in response to neurodegeneration in both Huntington's disease and Alzheimer's disease, in the striatum and dentate gyrus, respectively. Loci of induced neurogenesis include the neostriatum and diencephalon in response to BDNF, with potentiation of the striatal response with concurrent BMP-suppression via noggin, and the dentate gyrus of the hippocampus, in response to IGFl and VEGF, as well as to NOS inhibition and serotinergic agonists.

and glia, and more recently of stem and progenitor cells, as therapeutic agents. Relatively less effort has been devoted to utilizing or mobilizing endogenous progenitor populations. Yet several major populations of accessible progenitor populations persist in the adult brain. These pools are individually accessible, and may be mobilized through a variety of both pharmacological and gene therapeutic strategies, that result in the mitotic expansion of resident stem and progenitor cells. A number of humoral growth factors have been identified as modulating the mitotic expan-

sion and differentiated fate of neural stem cells. EGF and FGF2, which each have mitogenic effects on neuronal progenitor cells of the adult subependyma, can both potentiate neuronal replacement in the presence of permissive signals for neuronal differentiation (Kuhn et al., 1997). TGFa, a membrane-bound EGF-like ligand, has also been shown to achieve a similar effect in the adult striatum, in which TGFa exposure in the setting of catecholaminergic cell injury was found sufficient to induce the heterotopic recruitment of new dopaminergic neurons to the striatum (Fallon et a l , 2000). Yet EGF


58

5. TARGETED INDUCTION OF ENDOGENOUS NEURAL STEM AND PROGENITOR CELLS: A NEW STRATEGY FOR GENE THERAPY

and FGF2 appear to act solely as mitogens in the adult ventricular subependyma. EGF stimulation led largely to gliogenesis (Craig et al., 1996) and FGF2 infusion increased neuronal recruitment to the olfactory bulb, but nowhere else; neurons generated under the sole influence of FGF2 did not depart their tjîcal migratory paths to enter any other subcortical structures along their migratory route (Kuhn et al., 1997). A number of other ligands for receptor tyrosine kinases have been found to drive mitotic expansion by neural stem and progenitor cells, including vascular endothelial growth factor and stem cell factor, through the VEGFR2 and c-kit receptors, respectively (Jin et al., 2002a,b). Similarly, the inhibition of nitric oxide, an agent that appears to tonically suppress progenitor turnover in the adult brain, increased neuronal production in the olfactory bulb and dentate gyrus (Packer et al., 2003), doing so in a BDNF-dependent manner (Cheng et al., 2003). Yet despite these many approaches to influencing progenitor cell mobilization and neurogenesis within the forebrain subependyma, none of these strategies has been shown to be associated with heterotopic neuronal addition in vivo, i.e., the recruitment of new neurons into otherwise nonneurogenic regions of the adult brain. VIL

I N D U C E D NEUROGENESIS IN THE ADULT NEOSTRIATUM

To achieve the addition of new neurons to the mature brain, we and others have focused on delivering the trkB ligand BDNF to adult progenitor cells. BDNF had previously been shown to stimulate the production and survival of new neurons from adult precursor cells in vitro (Ahmed et a l , 1995; Kirschenbaum and Goldman, 1995; Goldman, 1997; Goldman et al., 1997). On the basis of these studies in culture, Luskin and colleagues next demonstrated that BDNF given intraventricularly could potentiate the addition of new neurons to the adult olfactory bulb (Zigova et al., 1998). On this basis, we then used adenoviral gene therapy to deliver the gene encoding BDNF to the adult rat ventricular wall, in an effort to assess the ability of BDNF to promote neurogenesis in otherwise non-neurogenic regions of the forebrain. We found that a single injection into the forebrain ventricles of replication-incompetent adenoviral BDNF induced the production of new neurons from neural progenitor cells in the ventricular subependyma (Benraiss et al., 2001). Most of the new neurons migrated to the olfactory bulb, but a large number also invaded the neostriatum.

a region of the brain that does not typically recruit new neurons in the normal, uninjured brain. Importantly, the new neurons integrated largely as medium spiny neurons, precisely the phenotype typically lost in Huntington's disease. Moreover, once integrated into the existing striatal network, the newly generated cells survived independently of periventricular BDNF overexpression (Benraiss et al., 2001). Together, these observations suggested that AdBDNF-induced neurons might directly replace the very phenotype lost in the course of Huntington's. Of note, Luskin and colleagues similarly demonstrated BDNF-associated neuronal addition to the adult neostriatum (Pencea et al., 2001), using BDNF protein infusion rather than virus. Interestingly, the high dose and sustained protein infusion used in this case led to neuronal addition to other subcortical limbic and diencephalic structures as well, the significance of which remains to be explored. VIIL THE DEVELOPMENT OF GLIAL SUPPRESSIVE STRATEGIES E N H A N C I N G HETEROTOPIC NEUROGENESIS Most neural stem cells differentiate as glia unless otherwise challenged. Chmielnicki et al. asked whether BDNF-stimulated striatal neuronal addition might be enhanced by the concurrent suppression of glial differentiation. Since gliogenesis by neural stem cells appears to be mediated by the pro-gliogenic bone morphogenetic proteins (BMPs) (Gross et al., 1996; Zimmerman et al., 1996), Chmielnicki et al. attempted to suppress adult glial production by periventricular overexpression of a potent BMP inhibitor, noggin. Noggin is a developmental inhibitor of the BMPs, and it continues to be expressed in regions of ongoing neurogenesis in vivo (Lim et al., 2000). In this study, a noggin mutein was used in which the heparin-binding domain was deleted, so as to ensure that ependymally expressed noggin would permeate the ventricular wall to achieve dissemination throughout regions of subependymal cell genesis (Economides et al., 2000). While adenovirally delivered noggin (AdNoggin) alone did not trigger the production of new striatal neurons, AdNoggin co-injected with AdBDNF strongly potentiated the latter's induction of striatal neurogenesis (Chmielnicki and Goldman, 2002). Within a month after viral injection, animals so treated added >350 confocal-confirmed new neurons per mm^, three-fold the number of new neurons observed in rats given AdBDNF alone (Chmielnicki and Goldman, 2002; Chmielnicki et al., 2004). These data indicated that the viral overexpression of noggin indeed suppressed


INDUCED NEUROGENESIS AS A RESTORATIVE STRATEGY FOR THE HIPPOCAMPAL ATROPHIES

gliogenesis in the adult subependyma, thereby expanding the BDNF-responsive pool of potentially neurogenic SVZ progenitor cells. The concurrent inhibition of glial differentiation and promotion of neuronal differentiation, via the intraventricular delivery of adenovectors overexpressing noggin and BDNF, thus appears to be an effective means of inducing progenitors to add new^ neurons to the adult forebrain. IX. INDUCED NEUROGENESIS AS A THERAPEUTIC STRATEGY IN HUNTINGTON^S DISEASE To assess the feasibility of induced neurogenesis for treating neurodegenerative diseases, we asked if AdBDNF and AdNoggin could stimulate the addition of medium spiny neurons into the neostriata of R6/2 mice. These mice were generated to include a 150 CAG-repeat polyglutamine expansion in the first exon of the Huntington gene; they display a progressive and severe behavioral phenotype, associated with striatal atrophy (Mangiarini et al., 1996), and as such provide a robust model of Huntington's disease. In preliminary studies, Cho et al. noted that R6/2 mice treated with AdBDNF and AdNoggin indeed exhibit substantial striatal neuronal addition, and recruit new medium spiny neurons throughout the medial neostriatum (Cho et al., 2004). These newly induced medium spiny neurons extend fibers to their normal efferent targets in the globus pallidus, and include both enkephalinergic and Substance P-defined striopallidal projection neurons. On this basis, we may postulate that the induction of striatal neuronal addition in the Huntington mutant brain may permit the functional replacement of those neurons lost to disease. Indeed, since BDNF may be used to stimulate human precursor cells as well as those of rodents (Pincus et al., 1998; Roy et al, 2000b), it is a real possibility that the BDNF-mediated, nogginenhanced induction of striatal neuronal recruitment from endogenous progenitor cells might prove a viable treatment strategy for Huntington's disease, as well as for such other causes of striatal neuronal loss as striatonigral degeneration and lenticulostriate stroke. X.

PROGENITOR CELL TARGETING IN PARKINSON^S DISEASE

As in Huntington's disease, nigral neurons lost to Parkinson's disease might optimally be regenerated from a patient's own store of endogenous progenitors, rather

59

than delivered as an allograft. But in regions lacking contiguity to a source of ventricular zone progenitors, it remains unclear whether an inductive approach to nigral regeneration is feasible. The mesencephalic ventricular zone continues to harbor neural stem cells, and these are especially biased toward dopaminergic neurogenesis (Sawamoto et al., 2001). Indeed, when isolated and expanded in vitro, mesencephalic neural stem cells give rise to dopaminergic neurons in sufficient numbers and proportions that they may be used to restore dopaminergic innervation to the 6-OHDA-lesioned striatum (Sawamoto et al., 2001). Nonetheless, no means of inducing the endogenous mesencephalic stem cell pool to in situ neurogenesis has yet been identified, so that no credible strategy of induced neuronal recruitm^t comparable to that established in the striatimi has yet been defined. As an alternative but still highly speculative approach, several groups have begim to focus on inducing dopaminergic neurogenesis from resident progenitors within the parenchyma of the substantia nigra. Neural progenitor cells indeed persist within the nigra (Lie et al., 2002; Zhao et al., 2003), as they do throughput much of the adult brain, and they are able to generate neurons in vitro (Palmer et al., 1997; Kondo and Raff, 2000). However, whether these parenchymal progenitor cells may be induced to generate neurqns in vivo remains unknown, and whether they may be stimulated specifically to generate dopaminergic neurons, and dopaminergic neurons competent to extend axons to the striatum no less, remains problematic. XL INDUCED NEUROGENESIS AS A RESTORATIVE STRATEGY FOR THE HIPPOCAMPAL ATROPHIES The adult dentate gyrus exhibits persistent constitutive neurogenesis throughout life in animals, and appears to do so as well in humans (Altman and Das, 1965; Eriksson et al., 1998; Roy et al., 2000a). New neurons are added to the adult dentate from progenitors in the subgranular zone (SGZ) of the hippocampus, a layer which is developmentally contiguous with the most posterior reaches of the subependymal zone of the lateral ventricle (Gage et al, 1998). The SGZ progenitors appear committed to neuronal phenotype, although they may derive from less committed multipotential progenitors (Seaberg and van der Kooy, 2002). SGZ progenitors respond to FGF2, IGFl, and VEGF with mitotic expansion (Palmer et al., 1995; Aberg et al., 2000; Jin et al., 2002a), the efficacy of which may increase in the setting of antecedent factor depletion


60


or injury. VEGF in particular has been an agent of much recent interest, in that it may act upon neural progenitors and vascular endothelial cells in concert, cementing the interaction of these phenotypes, which are frequently found dividing in co-association (Louissaint et al., 2002; Palmer, 2002; Palmer et al., 2002). In this regard, adeno-associated viral overexpression of VEGF in the adult hippocampus has been associated with both enhanced dentate neurogenesis and improved cognitive performance (Cao et al., 2004). Sonic hedgehog, a morphogen more often implicated in phenotypic induction and regionalization of the developing nervous system, also appears to regulate both the proliferation and differentiation of adult neural progenitor cells (Rowitch et al., 1999), including those of the SGZ in vivo (Lai et al., 2003). Besides the peptide growth factors, other positive regulators of hippocampal neurogenesis include environmental enrichment, exercise, and serotonin agonists (Gould, 1999; Nilsson et al., 1999; van Praag et al., 1999; Brezun and Daszuta, 2000; Malberg et al., 2000), all of which have been associated with improved performance in a variety of mood and memory-dependent tasks. The very malleability of hippocampal neurogenesis argues that SGZ progenitor cells should be especially amenable to genetic and pharmacologic modulation. The modulation of SGZ neurogenesis may thus prove beneficial not only in the affective disorders, but also in the degenerative dementias associated with hippocampal atrophy. XII. PARENCHYMAL GLIAL PROGENITORS ARE ATTRACTIVE TARGETS FOR EXOGENOUS MOBILIZATION Glial progenitor cells (GPCs) are widespread throughout the adult brain, and are competent to differentiate as both oligodendrocytes and astrocytes after transplantation. Glial pathologies may therefore lend themselves to cell-based therapy even more readily than neuronal disorders, given the relative homogeneity and accessibility of the major glial phenotypes, oligodendrocytes, and astrocytes, and the abundance of their progenitors. Predictably then, they have been used for cell-based therapy of diseases of myelin (Archer et al., 1997; Duncan et al., 1997; Windrem et al., 2002, 2003, 2004). Glial progenitors have proven competent to engraft both adult targets of acquired demyelination (Windrem et al., 2002), and perinataUy, in disorders of myelin formation or maintenance, such as the congenital leukodystrophies (Duncan et al., 1997).

A, Endogenous Glial Progenitors as Targets for Induction Given their prevalence and distribution, glial progenitor cells present one of the more exciting targets for cell-directed gene therapy in the adult CNS. As a result, a number of investigators have attempted to induce oligodendrocyte production and myelinogenesis from resident GPCs, as a means of restoring structural and functional integrity to demyelinated foci in diseases of acquired demyelination, such as multiple sclerosis. Indeed, from the standpoint of structural repair, disease targets as diverse as the vascular leukoencephalopathies in adults, and cerebral palsy in children, may prove amenable to therapies based on glial progenitor cell induction. For instance, cerebral palsy with perventricular leukomalacia appears largely due to a perinatal loss of oligodendrocytes and their precursors (Back et al., 2001; Back and Rivkees, 2004; FoUett et al., 2004; Robinson et al., 2005), which may prove amenable to replenishment from local progenitor stores, appropriately mobilized. Yet though straightforward in concept, the targeted induction of myelinogenesis by resident glial progenitors has proven difficult. To be sure, a variety of agents, delivered both as protein growth factors and as competent expression vectors thereof, have been used to stimulate endogenous glial progenitors, but to variable and generally modest effect (reviewed in Levine et al., 2001). Indeed, even those studies that have reported induced remyelination or improved functional competence have not been able to causally attribute the effect of experimental treatment to progenitor cell mobilization, so much as to broader paracrine effects on the disease environment and immune response (Franklin et al., 2001). For example, NTS and BDNF-expressing fibroblasts were reported to potentiate oligodendrocytic production and myelination in the contused rat spinal cord (McTigue et al., 1998). However, whether such effects are due to progenitor cell mobilization and attendant myelinogenesis, or rather to BDNF and NT-3-associated support of host axons, increasing their own availability and paracrine support for myelination, has proven difficult to define. Similarly, IGFl was reported to reduce lesion incidence and improve compensatory remyelination in models of experimental allergic encephalomyelitis (EAE), attendant with an IGFl-dependent increase in the number of oligodendrocytes (Yao et al., 1995). Yet IGFl's effects in EAE include attenuating vasculitic damage to the blood-brain barrier, thus acting as an immune modulator (Liu et al., 1997, 1998). Systemic


TRANSDUCED NEURAL PROGENITORS AS VECTORS FOR ENZYMATIC REPLETION IN THE STORAGE DISEASES

delivery of the neuregulin glial grov^th factor-2 (GGF2) wâs likewise associated with improved remyelination and clinical benefit in EAE, but whether this was causally dependent upon progenitor mobilization and myelinogenesis was similarly unclear (Cannella et al., 1998). Indeed, long-term GGF2 infusion improved neither the incidence nor rate of remyelination following ethidium bromide-induced demyelination in aged rats (Penderis et al., 2003). As a result, the search continues for effective means of activating resident GPCs, particularly in regards to defining those ligand-receptor interactions that may signal their mobilization and myelinogenesis. B.

Gene Delivery to Parenchymal Progenitor Cells

Besides identifying effective ligands competent to specifically activate parenchymal glial progenitor cells, we also need to identify vectors able to deliver transgenes to these cells, whether stably or transiently. Although both oligodendrocytes and their progenitors can be readily transduced with adenoviral vectors, they are relatively sensitive to viralinduced cytotoxic effects; as such adenoviruses have to be carefully titrated (Franklin et al., 1999). In contrast, lentiviral vectors have been used to successfully infect adult human GPCs in vitro, and these cells proved non-immunogenic and stably transduced after transplantation to both perinatal and adult demyelinated hosts (Windrem et al., 2002; Nunes et al., 2003). In adddition, NG2-defined GPCs were transduced with lentiviral lacZ in vivo, with stable transgene expression and little associated demyelination (Zhao et al., 2003). These promising results are offset though by the often modest efficiency of transgene transcription and restricted dispersal of lentiviral vectors in the adult white matter. To address these latter concerns, a number of investigators have used adeno-associated viruses (AAVs) as non-immunogenic vectors for stably targeting oligodendrocytes and their progenitor cells (Chen et al., 1998,1999). In particular, by taking advantage of the exquisite phenotypic specificity of different AAV serotypes, we might hope to establish vectors selectively competent to infect parenchymal progenitor cells. For instance, the identification of PDGFaR and PDGFjSR as potential receptors for AAV5 led to the observation that AAVS can stably and efficiently infect glial progenitors (Di Pasquale et al., 2003), although the promiscuity of PDGFAR expression does not yet permit the level of cell-type specificity required for selective gene delivery to GPCs. More likely, some combination of progenitor-accessible AAV serotypes

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and GPC-selective promoters, such as CNP2 (Gravel et al., 1998; Roy et al., 1999) and PLP (Mallon et al., 2002), will permit the selective delivery of therapeutic transgenes to parenchymal glial progenitor cells. XIII. T R A N S D U C E D N E U R A L PROGENITORS AS VECTORS FOR ENZYMATIC REPLETION I N THE STORAGE DISEASES Glial progenitor cells may have additional value besides structural repair and myelination, in that their widespread dispersal and efficient integration into recipient brain suggests their use as cellular delivery vehicles of wild-type or overexpressed gene products. This function may be of particular utility in the congenital metabolic diseases of the CNS, especially those due to enzyme dysfunction or depletion, such as the mucopolysaccaridoses, the gangliosidoses, and other lysosomal lipid storage disorders (Kaye, 2001; Powers, 2004). Indeed, the congenital leukodystrophies due to lysosomal storage disorders present especially attractive targets for using genetically modified progenitor cells as therapeutic vectors, since wild-type lysosomal enzymes may be released by integrated donor cells, and picked up by enzyme deficient host cells through the mannose-6-phosphate receptor pathway (Urayama et al., 2004). As a result, only a relatively small number of donor cells may be needed within a much larger volume of diseased host cells, to provide sufficient enzymatic activity to correct the underlying host catalytic deficit and storage disorder. That being said, the enzymatic activity of implanted wild-type cells may be insufficient to achieve regional correction, and the resultant shortfall in enzymatic activity may be only incompletely addressed by increased donor cell dosage. To address this problem, donor GPCs might be transduced to overexpress therapeutic transgenes, specifically those encoding enzymes deficient in the diseased host. Genomically integrating retroviruses, AAVs and lentiviruses have been developed that express genes implicated in the metabolic and hereditary leukodystrophies, and several have been assessed with regards to their ability to restore normal phenotype after intracerebral injection. Mucopolysaccharidosis VII (MPSVII) has been an especially fruitful experimental model in this regard, and feline immunodeficiency virus expressing jS-glucoronidase, the enzyme deficient in MPSVII, has been shown to


62


improve survival after intrastriatal viral injection (Brooks et al., 2002). However, the widespread nature of these diseases argues that the intracerebral injection of replication-incompetent viral vectors, whose effects are necessarily limited to their effective infection radius, may be insufficient to achieve the widespread and uniform degrees of enzymatic correction required throughout the neuraxis. As an alternative, systemic administration of hematopoietic stem cells (HSCs) transduced to overexpress arylsulfatase A has recently been reported as an approach to treatment in mouse models of metachromatic leukodystrophy (Biffi et al., 2004). This strategy depends upon the infiltration of the CNS and PNS with donor-derived microglia and endoneural macrophages, respectively, carrying the lentivirally delivered transgene. Yet despite the inherent promise of this approach, the penetration of peripheral macrophages into the adult CNS remains limited to perivascular structures, which may sharply limit the range of potential enzymatic deficiencies amenable to correction by transduced HSCs. In contrast, unlike HSCs, glial progenitors enjoy widespread dispersal in the CNS (Windrem et al., 2004). As such, implanted glial progenitors may be capable of achieving high donor: host cell ratios throughout the recipient brain parenchyma. Indeed, it is conceivable that for some enzymatic disorders, wild-type unmodified glial progenitors may be sufficient to restore enzymatic activity throughout the CNS of affected hosts. Alternatively, glial progenitors may be transduced to overexpress the deficient gene, for the purpose of engrafting the diseased host with a cell type able to both deliver its transduced gene products at high levels throughout the neuraxis, while meaningfully contributing to host cytoarchitecture.

XIV.

OVERVIEW

The use of viral expression vectors to mobilize resident neural stem and progenitor cells may prove an effective strategy for treating a wide variety of neurological disease, particularly the geographically and phenotypically restricted neurodegenerative diseases. In these disorders, the reconstruction of precise neural circuits may depend upon the development of new neurons in situ, within the local context in which they will ultimately reside, and from which they will need to both attract and extend site-specific afferents and efferents. As such, the mobilization of endogenous progenitor cells by gene therapeutic vectors, and the directed differentiation of their daughter cells into discrete

neuronal and glial phenotypes in situ, may prove an especially attractive strategy for eliciting CNS repair.

ACKNOWLEDGMENTS Work discussed in the Goldman lab is supported by NIH/NINDS, the National Multiple Sclerosis Society, the NY State Spinal Cord Research Program, the AtaxiaTelangiectasia Children's Project, The CNS Foundation, Merck Research Labs and Berlex Bioscience. References Aberg, M. et al. (2000) Peripheral infusion of IGF-1 selectively induces neurogenesis in the adult rat hippocampus. J. Neurosci., 20: 2896-2903. Ahmed, S., Reynolds, B.A. and Weiss, S. (1995) BDNF enhances the differentiation but not the survival of CNS stem cell- derived neuronal precursors. J. Neurosci., 15(8): 5765-5778. Altman, J. and Das, G.D. (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol., 124(3): 319-335. Alvarez-Buylla, A. and Garcia-Verdugo, J.M. (2002) Neurogenesis in adult subventricular zone. J. Neurosci., 22(3): 629-634. Alvarez-Buylla, A., Garcia-Verdugo, J.M. and Tramontin, A. (2001) A unified hypothesis on the lineage of neural stem cells. Nat. Rev. Neurosci., 2: 287-293. Archer, D. et al. (1997) Myelination of the canine central nervous sytem by glial cell transplantation: a model for repair of human myelin disease. Nat. Med., 3: 54-59. Arsenijevic, Y. et al. (2001) Isolation of multipotent neural precursors residing in the cortex of the adult human brain. Exp. Neurol., 170(1): 48-62. Arsenijevic, Y. et al. (2001) Isolation of multipotent neural precursors residing in the cortex of the adult human brain. Exp. Neurol., 170: 48-62. Arvidsson, A. et al. (2002) Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med., 8: 963-970. Back, S. and Rivkees, S. (2004) Emerging concepts in periventricular white matter injury. Semin. PerinatoL, 6: 405-414. Back, S.A. et al. (2001) Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury. J. Neurosci., 21(4): 1302-1312. Belachew, S. et al. (2003) Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J. Cell Biol., 161(1): 169-186. Benraiss, A. et al. (2001) Adenoviral brain-derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain. J. Neurosci., 21(17): 6718-6731. Biffi, A. et al. (2004) Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells. J. Clin. Invest., 113:1118-1129. Brezun, J. and Daszuta, A. (2000) Serotonin may stimulate granule cell proliferation in the adult hippocampus, as observed in rats grafted with foetal raphe neurons. Eur. J. Neurosci., 12: 391-396.


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6 Neurosurgical Targeting, Delivery, and Infusion of Gene Therapy Agents in the Brain Justin R Fraser, Neal Luther, Michael G. Kaplitt

Abstract: Gene delivery to the brain has until recently focused largely upon the molecular factors necessary to permit efficient transfer of therapeutic genes into target cells. However, as the ability to safely alter cellular function in a variety of settings has advanced, it has become increasingly clear that the physical mechanism of delivering gene therapeutics to the brain can limit effective clinical translation. Delivery from the vascular system to the brain is very difficult, and when focal gene expression is desirable, current molecular methods for controlling transduction and gene expression remain crude. Therefore, direct surgical delivery into the brain has been the method of choice for gene delivery in every trial of gene therapy in the brain conducted to date. Many of the techniques used in current applications derive from operations designed to either lesion portions of the brain or implant devices such as deep brain-stimulating electrodes. Stereotactic methods permit precise three-dimensional targeting of even the deepest structures, and this has been aided by advanced imaging techniques and computer-assisted reconstruction and navigation. These can be performed with either a traditional stereotaxic frame, or with frameless methods. The type of catheter and infusion parameters can also significantly influence both the efficiency of gene delivery and the area of spread, while adjuvant molecules can be added to the gene therapy solution to further influence these parameters. Finally, experiences with human trials in which a small focal area of the brain is targeted, such as Parkinson's disease, has revealed very different surgical delivery requirements compared with diseases where global delivery may be desirable, such as the genetic disorder Batten disease. As gene therapy continues to move into clinical practice, continued evolution of surgical techniques and infusion devices will aid in the safe and effective translation of biologically promising agents. Keywords: stereotactic surgery; deep brain stimulation; infusion; catheter; computer navigation

As described below, current methodology limits the clinical utility of delivering gene therapy from a peripheral administration, so proper targeting of an infusion catheter is necessary so that the intended target cell population is in fact treated. Second, the method of delivery may differ according to the surgical goal. Finally, variables that alter infusion can modify the efficacy of the final product. It is important to understand these principles as they guide current practice and future research in the emerging field of gene delivery to the human brain.

In an era of increasing research in gene therapy for neurological diseases, the delivery methodology and surgical implantation protocol can profoundly influence the ability to successfully translate a promising strategy into clinical practice. Utilization of gene therapy in the brain necessitates increasing safety, efficiency, and accuracy in stereotactic neurosurgery. Effective gene delivery depends upon several important elements. Anatomical targeting represents the first element; the tools and methods must be reliable and precise in order to facilitate accurate surgical planning.


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6. NEUROSURGICAL TARGETING, DELIVERY, AND INFUSION OF GENE THERAPY AGENTS IN THE BRAIN

I. ANATOMICAL TARGETING I N STEREOTACTIC NEUROSURGERY Targeting methodology represents a vital component in stereotactic neurosurgery. As therapeutic targets become more specific and delineated, the need for more accurate targeting tools drives continuing advances in the field. Current tools in neuroradiology provide excellent resources for evaluating brain regions and nuclei, and for their localization within a stereotactic plane. A review of the current techniques in stereotactic planning emphasizes the role of both these radiographic tools and novel advances in computer technology that permit real-time utilization and optimization of radiographic information. Recent studies have demonstrated roles for new targeting methodologies that will provide additional tools to improve neurosurgical stereotaxy. Stereotactic neurosurgery is a field that began before the availability of magnetic resonance imaging. Armed with only a thorough understanding of gross neuroanatomy and radiographs from such tools as aircontrast ventriculography, neurosurgeons undertook procedures to approach and impact the deep-brain nuclei (Walter and Vitek, 2004). While an understanding of these subjects is important in neurosurgical stereotaxy, current targeting methods depend largely upon radiographic anatomy from CT and MRI, and electrophysiologic monitoring. While CT and ventriculography can still be utilized for stereotactic planning, MRI has emerged as the standard for assessing neuroanatomical landmarks as it provides a high-resolution view of specific neurosurgical targets. Improvements in MR technology such as stronger magnets, faster acquisition, and employment of adjimcts such as MR angiogram have bolstered the utility of MRI as a planning tool for stereotactic neurosurgery. Within the MR environment, stereotactic targeting of deep-brain nuclei, such as the thalamus, subthalamic nucleus (STN), and globus pallidus (GP), employs two distinct methods for planning surgical approaches. Aptly named the 'direct' and 'indirect' methods, the former represents a newer method in the MR-era that relies on visual selection of the target from MR imaging, while the latter utilizes a standard set of measures from a midline landmark. The 'direct' method may be ideal for MRI, as the high resolution enhances the ability to directly visualize specific intracranial structures. Vayssierre et al., (2002) compared direct MRI selection of targets to selection based upon the Schaltenbrand and Talairach atlases (Talairach and Toumoux, 1998; Schaltenbrand and Wahren, 2002). Using the directly selected targets

as a standard verified by postoperative clinical results, the investigators found significant differences between the atlas-based coordinates and the directly selected coordinates(Vayssiere et al., 2002). As such, the direct method of targeting represents an increasingly useful tool as MRI imaging technology continues to improve. In contrast, the indirect method of targeting utilizes the line connecting the anterior and posterior commissures as a zeroing standard from which deep-brain nuclei are targeted using a set of calculations. Ventriculography, CT, and MRI can all act as foundation radiographic studies for this method, although MRI may provide the best atlas. In evaluating these options for surgical planning, Cuny et al. investigated the use of the direct and indirect methods for targeting the subthalamic nucleus (STN) in 14 patients. Using electrophysiologic guidance and functional stimulation response as a baseline standard for optimal electrode placement in patients with advanced Parkinson's disease, the investigators found that, while the indirect method was more accurate than the direct method, within the indirect method, the use of 3D MR imaging was superior to ventriculography in determining accurate, reliable, and reproducible targets for electrode placement in the STN (Cuny et al., 2002). While the specificity of the target in this study represents a caveat to its generalization in stereotactic targeting, it underscores the trend in stereotactic research toward methods that express reliable and reproducible precision in head-to-head comparisons. Thus, while surgical planning should not rely solely upon direct visual selection of targets, particularly in deep-brain nuclei, MRI represents a vital tool that allows target selection and surgical planning for both the direct and indirect method. As a radiographic tool, early MR! technology was impeded by lower resolution, slow image acquisition, and less well-defined protocols for diagnosis-specific imaging. However, recent studies have verified that current technical improvements have corrected some of these earlier deficiencies to produce relatively small targeting error with relative efficiency. In a study of MRI error among 11 patients undergoing repeat deep-brain stimulation electrode placement surgery, Simon et al. quantified targeting error through three-dimensional assessment of the distance between electrode placement and targeted coordinates (Simon et al., 2005). The investigators found that direct targeting utilizing images from a 1.5-T MRI resulted in a mean lateral-medial error of 0.09 ± 0.34 mm, a mean anterior-posterior error of 0.01 ± 0.32 mm, and a mean superior-inferior error of -0.08 ±0.33 mm (Simon et al., 2005). Thus, MRI represents a tool with demonstrated accuracy in surgical


ANATOMICAL TARGETING IN STEREOTACTIC NEUROSURGERY

planning for access to deep-brain nuclei. In addition to progress in accuracy, MRI can now be performed in relatively short times given appropriate sequence protocol designation. Hariz et al., (2003) in a study among eight medical centers utilizing predominantly 1.5-T MRI, tested a protocol using single T2-weighted nonvolumetric MRI to preoperatively and postoperatively assess deep-brain stimulation electrode placement (Hariz et al., 2003). Image acquisition varied between 3 min, 5 s and 7 min, 48 s (Hariz et a l , 2003). From such throughput studies, it is clear that, while MRI remains slower than CT, image acquisition time using high-resolution scanners continues to improve, enhancing the applicability of MRI as a practical method for preoperative stereotactic imaging. In addition to advances in MRI acquisition, quality, and processing, progress in other radiographic methodologies provides opportunities for other instruments in stereotactic neurosurgical planning. Functional MRI (fMRI) utilizes blood oxygen level contrasts to radiographically differentiate task-specific cortical activity (Ogawa et al.,1990; Atlas et a l , 1996; Krishnan et al., 2004). Krishnan et al., (2004) in a study of 54 patients with intracranial tumors near the motor cortex, found that fMRI could be used to calculate distance of the lesion/resection from the primary motor cortex, and that increasing distance is correlated with better neurological outcome if resection. Pirotte et al., (2005) found that preoperative fMRI findings correlated to intraoperative cortical mapping as a functional targeting method for epidural motor cortex stimulation in 17 of 18 patients with neuropathic pain. Chavez et al., (2005) studied three-dimensional fast imaging employing steady-state acquisition (3-D FIESTA) MRI to plan surgical intervention for trigeminal neuralgia. In 14 of 15 patients with trigeminal neuralgia, FIESTA imaging demonstrated clear anatomy of the trigeminal complex including the root entry zone, the trigeminal ganglion, and vasculature (Chavez et al., 2005). As such, fMRI and FIESTA represent variations in image acquisition, and may clearly add new dimensions to surgical planning in functional stereotaxy. Despite such advances, MRI continues to have limits as a tool for stereotactic preoperative planning. Not all institutions can utilize fast acquisition protocols with high resolution. Additionally, scanning the patient prior to the day of the procedure provides an opportunity to plan the approach before the operation begins. However, the typical headframes utilized are not fit for outpatient application and use. Due to such limitations, practitioners now routinely employ image fusion software to bring preoperative imaging and

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planning into the real-time operating environment. One method entails a preoperative MRI used to plan the surgery, an early CT on the day of the procedure with the headframe in place, followed by fusion of the two images immediately and preoperatively. The software overlays the high-resolution MRI images (containing the surgical target and proposed tract) onto the three-dimensional stereotactic grid created by the head CT. While fusion software has been used in the literature to validate targeting methods by fusion pre- and postoperative imaging, its incorporation into the operating room has created a tool for real-time targeting and coordinate adjustment (Ferroli et al., 2005; Hamid et al., 2005). Furthermore, fusion technology allows for a more efficient operative chronology: the patient is fitted with a headframe, taken to noncontrast head CT, and taken directly to the operating room with no loss of time performing preoperative targeting measurements on the day of surgery. As such, fusion technology represents an important tool in the current and future practice of functional neurosurgery. Despite recent advances in MRI accuracy, imaging protocols, and software processing, some radiographic questions remain. For example, while many stereotactic procedures such as biopsies and deep-brain stimulator implantations have emerged as frame-dependent operations, stereotactic targeting for neuro-oncology and tumor resection has thrived on frameless stereotaxy. Frameless stereotaxy involves placement of some identifying mark on the patient's head, either stickers or small screws, which can be seen on MRI or CT. These are then identified to the navigation machine, and when a sufficient number of such 'fiducials' are identified, the device can then track a pointer anywhere within a certain radius of three-dimensional space with acceptable accuracy. Recent studies have examined the true accuracy of frameless stereotaxy. Gralla et al, (2003) found a technique of frameless stereotactic biopsy for intracranial tumor to deliver accurate diagnoses in 96.5% of patients (N = 57) (Gralla et al., 2003). Future research and advancement will provide further data to improve the efficacy and accuracy of frameless and frame-based stereotaxy, particularly as frameless stereotaxy is increasingly utilized in functional procedures such as surgery for Parkinson's disease. However, each method has some clear advantages and limitations. While a headframe provides a rigid three-dimensional system that can incorporate a mechanized delivery system, frameless stereotaxy provides flexibility, freedom of movement, and adjustment in all planes. Also, while frame-based stereotaxy continues to have a slight advantage in accuracy over frameless systems, only one


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target can be accessed at a time using a frame, while multiple targets such as bilateral procedures can be performed simultaneously if desired using frameless systems. Future techniques may combine such concepts, utilizing 'fiducials' to orient the patient to a nonattached rigid frame which would provide the support needed for certain types of infusion systems. While controversies in imaging improvements and coordinate mapping exist, they are frequently compared to electrophysiological data as the gold standard for targeting. Despite the increasing availability of intraoperative MRI, many technical limits issues continue to limit MRI to use primarily as a preoperative tool. Electrophysiologic monitoring, however, allows real-time evaluation of positioning in stereotactic neurosurgery. Methods for monitoring deep-brain nuclei have been studied and validated (Macias et al., 1997; Molinuevo et al., 2003; Nowinski et al., 2004).Hamani et al, (2005) found excellent correlation between MRI and microelectrode physiology in 10 patients with Parkinson's disease. However, the electrophysiologically defined subthalamic nucleus sometimes extended more anteriorly than that appreciated on MRI . As such, electrophysiologic monitoring can be important for real-time intraoperative confirmation of a functionally relevant target, which may even differ slightly from the radiographic target. This of course depends upon understanding the physiology of the structure to be treated, and whUe this is well-understood for several deep-brain structures targeted for movement disorder or epilepsy surgery, many other areas of the human brain have not been as well studied and therefore the value of intraoperative electrophysiology may be limited by a lack of relevant information for some applications. Neurosurgical targeting has undergone exponential growth and advancement recently, and continued research and development in neuroradiological methods, imaging software, and electrophysiologic monitoring will provide better techniques for preoperative planning and perioperative targeting in stereotactic neurosurgery. These tools are vital to the implantation of gene therapy in the brain as complete accuracy must be the goal.

IL

METHODS OF ACCESSING THE CENTRAL NERVOUS SYSTEM

While target selection and surgical planning are vital steps to gene delivery, the method of delivery is as important. Several techniques for accessing the central

nervous system (CNS) have been studied, including retrograde translation via the olfactory tracts, intravascular injection, intraventricular injection, intracavitary placement, and direct intraparenchymal infusion. The technique most appropriate depends highly upon the overall goal and specific neuronal target. The olfactory route offers a direct and nonpenetrating method for CNS gene delivery. The olfactory nerve endings, penetrating through the olfactory mucosa, trace directly through the cribiform plate into the CNS. While it is unknown whether substances are taken up directly by the neurons and transported in a retrograde fashion or whether they move into the subarachnoid space via the olfactory mucosa, it is clear than many substances can access the CNS through this pathway (Begley, 2003, 2004; Davis et al., 2003; Ilium, 2003). However, most substances absorbed this way express effects throughout the CNS, suggesting a lack of defined endpoint targets of delivery. However, Jerusalmi et al.,(2003) indirectly studied intranasal injections of gene therapy for experimental autoimmune encephalomyelitis. The investigators utilized a Semliki Forest virus expression system to express IL10 and green florescent protein (GFP) in Balb/c mice, and found that protein expression was visually detected by fluorescence in the olfactory bulb (Jerusalmi et al., 2003). While this method has demonstrated some promise for treating diffuse neuropathology, much future research is required to further elucidate the capabilities and limitations of the olfactory route for delivery of gene therapy to the brain. Intravascular injection represents an important technique for delivering therapeutics in medicine. As with olfactory delivery, it is limited in its ubiquitous nature; all parts of the CNS would be exposed to the potential agent. The blood-brain barrier represents the most severe limitation for intravascular delivery of gene therapy to the brain. The blood-brain barrier severely limits penetration of particular molecules from the intravascular space, protecting the brain from systemically administered substances .(Kaplitt and Lozano, 2001). Larger molecules (>500kDa), charged substances, molecules with a high propensity to form hydrogren bonds, and molecules with significant polarity are less able to penetrate the blood-brain barrier (Bodor and Buchwald, 2003; Begley, 2004). However, transvascular gene therapy may be possible if one can access endogenous transport systems that are present within the blood-brain barrier and serve to permit selective transport under normal conditions (Pardridge, 2002; Schlachetzki et al., 2004). In a review of transvascular approaches to gene therapy, Schelachetzki et al.


METHODS FOR ENHANCING INTRACRANIAL GENE TRANSFER

hypothesized that CNS-directed gene therapy could be successfully delivered by intravascular injection through the use of selective transporter targeting in conjunction with a vector containing a CNS-specific promoter (Shi et al., 2001; 2003; Schlachetzki et al., 2004). While these methods may be feasible and have excellent potential, they are not at a translational stage to be employed as active surgical techniques for applying gene therapy to the brain. Intraventricular injection of gene therapy, while more invasive, represents a previously well-studied method for delivery of drugs to the central nervous system. In particular, the Ommaya reservoir is a chronic implant that permits injection of chemotherapy directly into the cerebrospinal fluid. However, while the Ommaya reservoir permits long-term access to the CSF space, its purpose is directed at treatment of leptomeningeal and diffuse disease. Ooboshi et al. (1995) demonstrated that adenoviral injection into the cisterna magna results in infection of overlying major arteries, adventitial cells of large blood vessels, and some smooth-muscle of smaller vessels. This diffusion pattern is supported by recent studies, most notably by Sugiura et al., (2005) who demonstrated that intraventricular administration of recombinant adenovirus expressing heparin-binding epidermal growth factorlike growth factor significantly improved functional recovery, angiogenesis, and neurogenesis (as assessed by bromodeoxyuridine injection) in Wistar rats that underwent middle cerebral artery occlusion ischemic strokes. As such, intraventricular injection of gene therapy in the brain could play an important role in diffuse cerebrovascular diseases such as ischemic stroke, as well as leptomeningeal diseases. However, the brain-CSF barrier does present an important limitation to intraventricular injection, acting to restrict the applications of intraventricular gene therapy. In addition, the immune system within the ventricular lining is different and more robust than that in the normal brain parenchyma, so there may be concerns regarding a more profound immune reaction to gene therapy delivered via an intraventricular route. Despite such limitations, directed research to further define indications and applications for intraventricularly injected gene therapy is needed. Intraparenchymal infusion of gene therapy offers a more direct method for localized delivery. Such a method avoids the BBB altogether, limits potential for systemic toxicity of the infusate, and limits the amount of virus necessary to deliver the gene to the therapeutic target area (Tang and Chiocca, 1997). This method applies to both neuro-oncological surgery

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and functional neurosurgery. Direct implantation of chemotherapeutics in tumor cavities has provided a demonstrated foundation for embedding of adjuncts in the treatment of intracranial tumors. For example, utilization of the carmustine-loaded 'Gliadel' wafer has demonstrated significant survival benefits in clinical trials for glioblastoma multiforme (Brem et al., 1995; Westphal et al., 2003; Chiocca et al., 2004). Through such pioneering innovation, a method for adjunctive gene therapy in neuro-oncology becomes clear. Retroviruses, adenovirus, and Herpes Simplex Virus have all acted as vectors in human clinical trials for gene therapy adjuncts in neuro-oncology. Implantation of such genes as thymidine kinase (often paired with gangiclovir), j8-galactosidase, p-53, and oncolytic adenovirus have been subjects of several phase I, II and even III trials (Shand et al., 1999; Rainov, 2000; Rampling et al., 2000; Chen et a l , 2001; Lang et al., 2003; Chiocca et al., 2004). Direct implantation of gene therapy has also entered a clinical phase in functional neurosurgery. We have been involved in two current trials, including a Phase I trial investigating the infusion into the subthalamic nucleus of the glutamic acid decarboxylase (GAD) gene via an adeno-associated virus (AAV) vector, as well as a trial of gene therapy for Batten's disease ((NIH) TNIoH, 2005). In the Parkinson's disease trial, the goal is to efficiently deliver vectors focally to the subthalamic nucleus, which is a structure of roughly 6mm X 5mm X 3mm in the human. Therefore, standard frame-based stereotactic techniques were used for targeting this area, similar to those used for traditional deep-brain stimulation. The vector was infused via a single injection of 50 ]x\ of solution over 100 min via a borosilicate catheter of only 140 i^m in diameter. By contrast. Batten's disease is a global pediatric neurogenetic degenerative disorder, with the goal of widely delivering the potentially corrective gene therapy to large areas of the brain. Therefore, this protocol utilized frameless stereotaxy to identify three areas on each side of the brain (six total injection sites), and then catheters were inserted into these sites along a trajectory and to a depth guided by the computerized frameless stereotactic system. This was essential in this disorder, since many of the patients have significant brain atrophy and therefore simply creating six random injection sites would likely result in infusion into the cerebrospinal fluid rather than into brain substance in most cases. Similar infusion parameters were utilized to the Parkinson's disease trial, although two infusions were performed per site, with each infusion performed at a different depth to optimize


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spread. These two trials reflect only a small sampling of the differences which may be faced in various trials depending upon the specific issues of gene delivery, target and disease state of the individual brain. Future clinical trials such as these will rely both upon previous stereotactic success with electrode implantation, such as deep-brain stimulation in the STN for Parkinson's disease, and upon animal data demonstrating reliable, accurate, and beneficial infusion of the therapeutic viral vector (Luo et al., 2002; Erola et al., 2005; Nilsson et al., 2005). Thus, intraparenchymal infusion represents a central method for delivery of gene therapy in functional and neuro-oncologic neurosurgery. Intraparenchymal infusion, intraventricular infusion, intravascular administration, and olfactory transportation combine to provide a virtual armamentarium to approach intracranial disease processes with gene transfer therapy. However, in considering these options, it is important to understand their limitations as well as variables that can alter their efficacy and applicability. III. METHODS FOR E N H A N C I N G INTRACRANIAL GENE TRANSFER While the different approaches to gene transfer in the brain have specific advantages and limitations, their value may be enhanced through alterations in technique and through the employment of specific adjuncts. Improvements in intraparenchymal injection techniques increase the potential volume of infusate, and potentiate improved dissemination of gene transfer. Coadministration of supplemental materials such as mannitol may augment absorption and delivery in several infusion methods. Finally, it is important to be mindful of the target environment at the time of gene delivery; pathological changes can alter the efficacy of gene transfer. These important elements exemplify the importance of research aimed not at comparisons of different delivery approaches, but at improving delivery techniques to maximize effect and therapeutic benefit. While different methods exist for intraparenchymal injection of gene-carrying vectors, one of the most efficacious methods is convection-enhanced delivery. Convection-enhanced delivery (CED), also known as interstitial infusion or intracerebral clysis, is a method of delivering therapeutic agents intracranially via a stereotactically positioned cannula. High-flow, continuous pressure gradients are utilized to drive the infusate through the interstitial compartment; this pressure gradient is theorized to improve overall

uniformity and volume of distribution of a therapeutic agent (Bobo et al., 1994; Morrison et al., 1994; Laske et al,. 1997). Through CED, the amount of agent injected directly affects distribution (KroU et al., 1996; Tang et al., 1997). Furthermore, High-flow microinfusion permits distribution in a mathematically predictable model (Morrison et al., 1999). Over some thresholds, rapid infusion may cause significant backflow along a catheter tract resulting in extra-target dissemination of viral vectors (Morrison et al., 1994). In one study of flow dynamics, Morrison et al. concluded that smaller flow rates (e.g., u C

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30% of the tumor mass positive for the marker gene without BTB disruption. Bradykinin infusion was able to increase further the number of transduced tumor cells to >50%. Although lipoDNA-mediated transfer resulted in increased efficacy as compared to Ad-mediated gene transfer, it was less specific since larger numbers of endothelial and glial cells also expressed the transgene. Both Ad and lipoDNA injections, in the absence and presence of bradykinin, also resulted in transduction of peripheral organs. LipoDNA transduced

parenchymal organs such as liver, lung, testes, lymphatic nodes, and especially spleen, whereas AAV displayed only its known affinity for liver and lung. B. Local Vector Delivery in Treatment of Brain Tumors Our group has also tested the use of local CED of AAV2 for treatment of brain tumors (Hadaczek et al., 2005). We transduced tumors (athymic rats bearing 87MG-derived glioblastomas) with the HSV-TK gene, which activates the nucleoside analog prodrug ganciclovir (GCV). This is one of the most effective and most commonly explored gene therapy approaches for treatment of experimental brain tumors. This therapy has the potential to selectively kill dividing cancer cells, since AAV2 selectively infects tumor cells and neurons both expressing heparan sulfate proteoglycan binding sites, leaving other cell types such as astrocytes and endothelial cells uninfected. The effectiveness of the AAV2-TK/ GCV strategy depends critically on transduction of a sufficient number of tumor cells to achieve total eradication of the tumor mass. Despite a statistically significant difference in survival between GCV-treated and control animals (25.8 compared with 21.3 days, p < 0.05), we concluded that even if an extensive tumor area (39%) was transduced with AAV2-TK vector, we were not able to eradicate tumors. Through the CED technique, AAV2 particles were delivered into the central portion of a growing neoplastic mass, locally transducing only the core of the tumor and leaving its periphery unaffected. Even if the central mass was most likely eradicated by GCV treatment, peripheral cells were still dividing because they were isolated from immediate contact with the TK-positive cells (Fig. 2). Therefore, the well-known bystander effect could not exert its function and the unaffected tumor masses had eventually overgrown the space left by the cells killed by GCV That experiment demonstrated the considerable divergence between in vivo and in vitro bystander effects. Our in vitro studies showed that, when the cell culture consisted of only 10% of TK-positive cells, the inhibition of cell growth by GCV was substantial. Such powerful action is most likely due to the fact that TK-positive cells are evenly dispersed among other untransduced cells, and thus toxic molecules can be distributed. This was not the case in vivo, where we encounter more focal patterns of transduction. Anti-tumoral efficacy depends most likely on achieving a highly diffuse transduction pattern within a tumor mass. This again emphasizes how critical it is to design efficient vector delivery system tailored for individual diseases of the CNS.


ACKNOWLEDGMENT

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FIGURE 2 Thymidine kinase immunoreactivity of the transduced U87MG-derived rat brain tumors. AAV-TK vector was used for intratumoral infusion by CED. Tumors were collected 3 days after infusion with the vector, and the transduction area of TK (arrows) within the tumor mass was calculated. In 18-day-old tumors (A), 39% of the total tumor volume was transduced with AAV2-TK; in 22-day-old tumors (B), this transduction accounted for only 18%. Through CED, AAV2 particles were delivered into the central portion of a growing neoplastic mass, locally transducing only the core of the tumor leaving the periphery unaffected.

The gene therapy of CNS diseases is particularly challenging because the delivery of drugs to the brain is often precluded by a variety of anatomical and physiological obstacles that collectively comprise the BBB or the BTB. Drug delivery directly to the brain interstitium has recently been markedly enhanced through development of methods such as CED and the optimization of its parameters. Human brain is a heterogeneous organ; therefore, all aspects of its organization (anatomical, physiological, and biochemical) should be taken into consideration in designing techniques of drug delivery. Even the most potent drug will not be effective until administered properly. Thus, formulating new vectors and molecular therapeutics should be undertaken in parallel with devising and optimizing their delivery and distribution within the CNS.

ACKNOWLEDGMENT The authors would like to thank Dr. Michal Krauze for contributing digital reconstruction figure. References Bankiewicz, K.S. et al. (2000) Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using prodrug approach. Exp. Neurol., 164: 2-14.

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10. DELIVERY OF MOLECULAR THERAPEUTICS INTO THE CNS AND THEIR DISTRIBUTION WITHIN THE BRAIN

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ACKNOWLEDGMENT Ren, H. et al. (2003) Immunogene therapy of recurrent glioblastoma multiforme with a liposomally encapsulated replication-incompetent Semliki forest virus vector carrying the human interleukin-12 gene - a phase I/II clinical protocol. J. NeurooncoL, 64: 147-154. Rennels, M.L. et al. (1985) Evidence for a 'paravascular' fluid circulation in the mammalian central nervous system, provided by the rapid distribution of tracer protein throughout the brain from the subarachnoid space. Brain Res., 326: 47-63. Rennels, M.L. et al. (1990) Rapid solute transport throughout the brain via paravascular fluid pathways. Adv. Neurol, 52: 431^39. Rubin, L.L. and Staddon, J.M. (1999) The cell biology of the bloodbrain barrier. Annu. Rev. Neurosci., 22: 11-28. Saito, R. et al. (2004) Distribution of liposomes into brain and rat brain tumor models by convection-enhanced delivery monitored with magnetic resonance imaging. Cancer Res., 64: 2572-2579. Sanovich, E. et al. (1995) Pathway across blood-brain barrier opened by the bradykinin agonist, RMP-7. Brain Res., 705: 125-135. Segovia, J. et al. (1998) Astrocyte-specific expression of tyrosine hydroxylase after intracerebral gene transfer induces behavioral recovery in experimental parkinsonism. Gene Ther., 5: 1650-1655. Shi, N. et al. (2001a) Receptor-mediated gene targeting to tissues in vivo following intravenous administration of pegylated immunoliposomes. Pharm. Res., 18: 1091-1095.

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Shi, N. et al. (2001b) Brain-specific expression of an exogenous gene after i.v. administration. Proc. Natl. Acad. Sci. USA, 98: 12754-12759. Shi, N. and Pardridge, W.M. (2000) Noninvasive gene targeting to the brain. Proc. Natl. Acad. Sci. USA, 97: 75^7-7572. Siegal, T. et al. (2000) In vivo assessment of the window of barrier opening after osmotic blood-brain barrier disruption in humans. J. Neurosurg., 92: 599-605. Sun, N. et al. (1996) Anterograde, transneuronal transport of herpes simplex virus type 1 strain H129 in the murine visual system. J. Virol., 70: 5405-5413. Ueda, K. et al. (1992) Human P-glycoprotein transports Cortisol, aldosterone, and dexamethasone, but not progesterone. J. Biol. Chem., 267: 24248-24252. Wolf, D.C. and Horwitz, S.B. (1992) P-glycoprotein transports corticosterone and is photoaffinity-labeled by the steroid. Int. J. Cancer, 52: 141-146. Yang, M. et al. (1999) Retrograde, transneuronal spread of pseudorabies virus in defined neuronal circuitry of the rat brain is facilitated by gE mutations that reduce virulence. }. Virol., 73: 4350-4359. Zhang, Y. et al. (2003a) Intravenous non-viral gene therapy causes normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism. Hum. Gene Ther., 14: 1-12. Zhang, Y. et al. (2003b) Global non-viral gene transfer to the primate brain following intravenous administration. Mol. Then, 7:11-18.


C H A P T E R

11 Gene Therapy for CNS Diseases Using Intrabodies Todd W, Millery Anne Messer

Abstract: Single-chain Fv and single-domain antibodies retain the binding specificity of full-length antibodies, but they can be expressed as single genes in phage or yeast surface-display libraries, thus allowing efficient in vitro selection from a naive human repertoire using standard molecular and cellular techniques. Candidate genes can then be expressed intracellularly as intrabodies, with the potential for alteration of the folding, interactions, modifications, or subcellular localization of their targets. These reagents have already been developed as therapeutics against cancer and HIV. The misfolded and accumulated proteins that characterize a wide range of neurodegenerative disorders provide a novel class of potential intrabody targets. Here, we review the extension of intrabody technology to the nervous system, where studies of Huntington's disease have been used to develop the approach, and antisynuclein, anti-jS-amyloid, and anti-prion strategies are under development. Research on several other neurodegenerations suggests that intrabodies directed against specific targets, or possibly against more common downstream targets, might be developed as novel genetic therapeutics, and as drug discovery tools, to further unravel disease pathways. Keywords: intrabody; antibody; scFv; DAB; therapeutics; Alzheimer's; Parkinson's; poly-glutamine; Huntington's; prion; tauopathy; synucleinopathy; amyloid; ALS

L

Alzheimer's disease (AD), and prion diseases, and we discuss potential targets in other prominent neurological disorders. Table 1 summarizes the current literature on intrabodies and antibodies for neurodegenerative protein targets as of early 2005.

INTRODUCTION

Intrabodies are intracellularly expressed antibodies (Abs) or Ab fragments that target intracellular antigens. As several neurological disorders are mediated by abnormal proteins, intrabodies may serve as genetic therapeutics to selectively target such proteins, and as drug discovery and validation tools. Intrabodies have already been investigated as treatments for a variety of conditions (Stocks, 2004), including HIV infection (Marasco et al., 1999; Tewari et al., 2003), tumor growth (Wheeler et al., 2003a, b), and tissue transplantation (Beyer et al., 2004). They are also being tested in clinical trials for cancer (Alvarez et a l , 2000; Leath et al., 2004). Here, we highlight the progress made with intrabodies for Huntington's disease (HD), synucleinopathies.


A,

Intrabody Generation and Selection

Antibody engineering has facilitated the generation of small Ab fragments that can recapitulate the binding properties of a full Ab while using a much smaller gene coding sequence. A single-chain Fv (scFv; sometimes also abbreviated as sFv) is generated by cloning of the variable domains of an Ab, and then joining the single domain cDNA sequences with DNA encoding a flexible linker (Bird et al., 1988; Huston et a l , 1988), thereby allowing a scFv (--250 amino acids.

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11. GENE THERAPY EOR CNS DISEASES USING INTRABODIES

TABLE 1

Current Literature on the Utilization of Intrabodies and Antibodies for Diseases of the Central Nervous System

Neurological disorder

Protein target

Publications

Parkinson's disease and synucleinopathies

a-synuclein

Emadi et al. (2004), Maguire-Zeiss et al. (2004), Zhou et al. (2004a)

Huntington's disease

Huntingtin

Marks et al. (1991), Lecerf et al. (2001), Khoshnan and Patterson (2002), Colby et al. (2004a, b). Murphy and Messer (2004), Miller and Messer (2005)

Alzheimer's disease Tauopathies"

j5-amyloid Tau

Rangan et al. (2003), Liu et al. (2004a, b), Paganetti et al. (2005) Visintin et al. (2002)

Prion disease

Prion diseases

Leclerc et al. (2000), Heppner et al. (2001), Wuertzer et al. (2004), Cardinale et al. (2005)

Amyloidogenic diseases^

Amyloid conformation

O'Nuallain and Wetzel (2002), Kayed et al. (2003) "Disorders for which scFvs or DABs have been selected, but for which efficacy in disease model systems has not been reported. Modified from Miller and Messer (2005). ''Amyloidogenic diseases include Alzheimer's, Huntington's, Parkinson's, and prion diseases.

29 kDa) to be expressed from a single gene. This technique may be performed using the cDNA from a single cell line to create an intrabody of known specificity (e.g., hybridoma, Orlandi et al., 1989), or from popu-lations of cells (e.g., naive spleens, peripheral blood lymphocytes) to generate phage or yeast surface-display libraries (Marks et a l , 1991). Single-variable domains of an Ab (DAB) can similarly be utilized (Tanaka et a l , 2003). Figure 1 summarizes the different selection approaches which are described in more detail below. The advantage of starting with a monoclonal Ab is that the capacity to target a defined epitope is retained, taking advantage of previous work, as shown in recent publications (Khoshnan et al., 2002; Cardinale et a l , 2005; Paganetti et al., 2005). The advantages of scFv or DAB libraries, which can represent the entire repertoire of potential Abs from a human pool, include a reduced immunogenic potential for clinical use (since they are derived from a human, rather than a mouse), and an enhanced probability of finding an initial candidate with acceptable intracellular folding (Lecerf et al., 2001; Colby et al., 2004b). Synthesis of an scFv or DAB is not guaranteed to yield a functional intrabody, primarily due to misfolding of the fragment intracellularly, with an additional contribution from unformed intrachain disulfide bonds in the reducing cytoplasmic environment (Worn et al., 2000; Mossner et a l , 2001). This is less of a problem if the target protein is within the endoplasmic reticulum, as discussed in Sections II.A.4 and II.D. An scFv or DAB library incorporated into a phage (Marks et a l , 1991) or yeast surface-display system (Boder and Wittrup, 1997; Kieke et al., 1997) for in vitro

biopanning against an antigen of interest can partially overcome this pitfall by initially providing several high-affinity candidates for functional intracellular screening. Yeast libraries provide a eukaryotic expression context, although these Ab fragments are generally not glycosylated, so a bacterial environment is sufficient in most cases. Typically, the antigen will consist of a peptide sequence found in the target protein; however, selection against discontinuous conformational epitopes may require biopanning against larger protein fragments, and/or altering selection conditions. Strategies for secondary screening for intrabody expression and stability, following initial selection from phage or yeast libraries, include in vivo selection in a yeast two-hybrid strategy (yeast ratracellular Ab capture) (Tse et a l , 2002; Visintin et a l , 2002), and direct phage to intrabody screening (DPIS) in mammalian cells, starting with pools of intrabodies selected from phage ELISA screens (Gennari et al., 2004). Antibody fragments may be further engineered to produce derivatives with higher affinity, lower dissociation rate, or improved stability through targeted mutagenesis of complementarity-determining regions (CDRs), light-chain shuffling (Osbourn et al., 1996), DNA shuffling for molecular evolution (Stemmer, 1994), BIAcore-driven selection (Schier and Marks, 1993), error-prone PCR (Colby et a l , 2004c), or grafting of CDRs onto a stable framework (Ewert et al., 2004). Stability improvement could be critical, since Zhu et al. (1999) strongly linked half-life to efficacy. The true intracellular affinity of an intrabody for its antigen cannot be absolutely determined by current methods, although it can be approximated using in vitro

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INTRODUCTION

scFv

VH-DAB

RT/PCR and clone variable domains / yeast surface-display

/

yeast

\

VLDAB

yeast intracellular antibody capture

\ phage surface-display phag(

Functional testing in disease models FIGURE 1 Intrabody selection. Intrabodies may exist as single-chain Fvs (scFvs), where the DNA encoding both the VH (red) and VL (blue) domains of an immunoglobulin are cloned and joined with DNA encoding a flexible linker. Alternatively, single-variable domain antibodies (DABs) may also be used as intrabodies. ScFv and DAB genes are cloned and expressed on the surface of bacteriophage or yeast to create a surface display library. The library is then used for biopanning against an antigen of interest, where yeast or phage expressing an scFv/DAB specific for the antigen will bind more strongly. Candidate scFvs/DABs can then be further screened in vivo by the yeast intracellular antibody capture technique; this approach helps ensure that the selected scFvs/DABs will fold properly and bind antigen in an intracellular environment. The candidate intrabody genes are fused to a VP16 transcriptional activation domain (yellow). The antigen of interest (blue star) is fused to a Lex A DNA-binding domain (pink). The histidine-deficient yeast genome has a LexA binding element (green) just upstream of the His3 gene (orange). Therefore, binding of a specific intrabody to the antigen will bring the VP16 domain into close proximity with the DNA upstream of the His3 gene, allowing transcription and production of histidine, and hence survival on a histidine-deficient medium, only in yeast expressing an antigen-specific intrabody

binding or yeast intracellular Ab capture techniques. Primary selection against an antigen of interest is not currently being performed intracellularly in mammalian cells due to the large size of the libraries. Hence, selection of desirable candidates does not

guarantee the same antigen-binding properties when they are expressed as intrabodies in mammalian cells. However, iterative in situ testing of candidate intrabodies and further engineering can be readily accomplished.

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136 B,

11. GENE THERAPY FOR CNS DISEASES USING INTRABODIES

The Power of Intrabodies

Antibodies and Ab fragments possess some distinct advantages over other classes of therapeutics for neurodegenerative diseases, which include random peptides, small molecules, and RNA interference (RNAi). Random peptides are the most similar to an intrabody approach. They can be selected from libraries with biopanning similar to that for scFvs. Nagai et al. (2000) have shown that a peptide against the expanded polyglutamine poly-Q can rescue some toxic effects of ataxin3 in cultures and Drosophila models, and Kazantsev et al. (2002) have also isolated a peptide with anti-aggregation effects in cultures. Intrabodies may offer a greater degree of antigen-binding specificity and stability, and may also prove to be less immunogenic than other protein/peptide therapeutics, particularly when selected from a human library. Small molecule drugs, chosen for their established effects on processes in the pathogenic cascade, or from high-throughput screens using aggregation suppression as an assay (Bates, 2003; Marsh and Thompson, 2004; Zhang et al, 2005), are likely to be much less specific than intrabodies. Intrabody genes may also be delivered to and stably expressed by target cells, providing a one-time procedure with long-term effects; small molecules would likely require repeated administrations and hence be more expensive. RNAi, which includes the advantages of gene therapy, provides protection by reducing the amount of substrate available to form toxic protein species (Xia et al., 2004; Harper et al., 2005). However, the proteomic approach provided by intrabodies offers the potential to utilize conformational specificities, and blockage of post-translational modifications and proteolytic inhibition. Offtarget effects may also be reduced. Use of combination therapies of small molecules, which can utilize doses of individual compounds that are below the threshold for toxicity, has improved outcomes in HD models (Hersch and Ferrante, 2004; Agrawal et al, 2005). Combinations that include intrabodies which act on early stages of the degenerative process may prove especially effective when combined with low doses of agents that reduce pathology globally due to downstream parts of the pathogenic cascade.

IL

MECHANISMS OF INTRABODY APPROACHES TO NEURODEGENERATION

The binding of an intrabody to its target protein may elicit any of several possible effects. The intrabody

may sterically prevent interactions of the target with other protein partners. The intrabody may stabilize or destabilize the target, thus preventing or facilitating turnover, respectively. Intrabody binding may alter folding of the target, possibly leading to altered stability or interaction capacity. The intrabody may also act as a signal to facilitate degradation, should the cellular machinery recognize this intrabody-target complex as a misfolded or foreign protein. Intrabodies may also be labeled with cellular localization sequences to re-target antigens to select subcellular compartments (e.g., lysosome, endoplasmic reticulum). The genetic manipulability of intrabodies and the rich diversity of intrabody libraries, provide a convenient platform from which to generate candidate therapeutics. Several prominent neurological diseases involve aberrantly modified proteins that can serve as unique targets. Here, we discuss three major categories of intrabodies which may affect the target by (1) altering protein folding a n d / o r interactions, (2) altering post-translational modifications, or (3) preventing pathogenic proteolysis. The major pathways through which intrabodies may act are depicted in Fig. 2. Each of these intrabody classes may be applicable to a spectrum of neurological disorders, due to both common disease mechanisms and common targets, and there is some overlap. A, Influencing Target Protein Folding and Interactions Using Intrabodies Abnormally folded proteins are a major theme among neurological diseases, including HD, AD, Parkinson's disease (PD), and prion diseases. The proteins implicated in such disorders are thought to misfold and assume abnormal conformations, thus rendering them susceptible to aberrant protein-protein interactions and formation of oligomers and aggregates. Binding of an exogenous molecule to the vulnerable protein could prevent misfolding a n d / o r sterically prevent aberrant interactions, thus blocking subsequent pathogenesis. 1 • Modifying the Folding and Interactions of Huntingtin in Huntington^s Disease A growing group of neurodegenerative disorders, including HD, several spinocerebellar ataxias, and spinobulbar muscular atrophy are attributable to the expansion of a CAG repeat in the coding region of a gene, leading to extension of a poly Q tract in the disease protein (Michalik and Van Broeckhoven, 2003). Proteins containing an expanded poly-Q tract are



137

Extracellular matrix Cytoplasm pathogenic ^ proteolysis^

benign proteolysis

l l l | post-transiationa! ^ modification

i1 V ^ Intrabodies

ipftiiiSiiiif

Wf-PO. M

proteasomal degradation

scFv VH-DAB VL-D

'^^m^

ITIB US

•

pathogenic misfolding/ aggregation

FIGURE 2 Mechanisms of intrabody action. Intrabodies may alter target proteins through one or a combination of a variety of actions. Depicted here are several of the more likely possible mechanisms, and steps at which intervention with intrabodies could block a pathogenic cascade. Black arrows indicate transitions and modifications of a protein, including misfolding, proteolytic processing, proteasomal degradation, post-translational modification, and transport between organelles and subcellular compartments. Green arrows indicate pathways where stimulation by intrabodies could be therapeutic. Red perpendicular lines indicate stages where inhibitory intrabodies could be useful. Intrabodies may be beneficial by stimulating or inhibiting protein transport between subcellular compartments depending on the compartment involved, as depicted by the dual green/red arrows. Additionally, several of the protein modifications such as proteasomal degradation, here depicted in the cytoplasm, may occur within organelles such as the nucleus, so intrabody targeting to subcellular compartments to alter protein modifications could also be favorable.

prone to misfolding (Ross et a l , 2003), aggregation (Onodera et al., 1997; Scherzinger et a l , 1997), and aberrant protein-protein interactions. Based on the hypothesis that the intrabody technology already in clinical trials for cancer and HIV therapies can be applied to neurodegenerative diseases, a collaboration between the Messer and Huston labs used a large naive human spleen sFv phage-display library (Marks et al., 1991) to examine the behavior of intrabodies against both the expanded poly-Q region and the amino-terminal flanking region. Intrabodies selected against mutant poly-Q showed both a lack of specificity and a degree of generalized toxicity. However, one intrabody, anti-HD-C4, selected against

amino-terminal residues 1-17 adjacent to the poly-Q of huntingtin, successfully counteracted in situ lengthdependent huntingtin aggregation in three different cell lines (Lecerf et al., 2001), as well as in organotypic slice-culture models (Murphy and Messer, 2004). Functional protection against mutant huntingtin-specific malonate toxicity was also demonstrated in the latter study. Binding intrabodies on either side of the mutant poly-Q sequence appears to have beneficial effects. The Patterson lab showed that mouse intrabodies to the poly-proline region flanking the poly-Q on the carboxyl-terminal side (generated from mouse monoclonal Abs) also reduce aggregation and apoptosis, while intrabodies to the expanded poly-Q itself

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138


were toxic (Khoshnan et al., 2002). These results also suggest that it may be possible to flank the poly-Q with intrabodies, which could prevent misfolding that starts at a region distant from the binding site of a single intrabody. Recently, complete rescue of the eclosion deficit in a Drosophila model of HD, < 25 to 100% was achieved by transgenic expression of the anti-HD-C4 intrabody. Partial rescue of lifespan and delay of neuronal degeneration were also observed. This is the first demonstration of intrabody protection in an intact nervous system (Wolfgang et a l , 2005). The anti-HD-C4 intrabody has also been stably expressed in mouse brain using an equine infectious anemia virus (EIAV) vector (Mazarakis et a l , 2001). Further studies of functional protection continue to show promise, with no apparent toxicity (Fig. 3). Additional intrabodies were isolated using a human scFv yeast surface-display library. A scFv specific for the amino-terminal 20-amino acid residues of huntingtin, and a smaller version consisting of a single-variable light-chain domain ( V L - D A B ) , inhibited aggregation and decreased yeast and cell culture toxicity, demonstrating the potential of further antibody engineering (Colby et a l , 2004a, b). By removing the

cysteine residues using site-directed mutagenesis, and increasing affinity via rounds of random mutations, an intrabody with stronger cytoplasmic effects on huntingtin aggregation has been selected. Analysis of mutant huntingtin aggregation in the presence of severe overexpression of huntingtin exon 1 fragments may test both homo- and heterophilic interactions. Successful intrabodies may sterically block aberrant interactions a n d / o r aggregation, or alter the folding of expanded poly-Q; however, the mechanism of intrabody action remains undefined. The intrabodies described above can also bind to wild-type huntingtin protein. Evidence suggests that the soluble fraction of proteolytically cleaved mutant huntingtin is a toxic species (Kim et al., 1999, 2001; Wellington et a l , 2000). Huntingtin fragments from the wild-type protein are apparently normal breakdown products. Encouragingly, the intrabody that rescued phenotypes in a Drosophila model of HD selectively binds soluble huntingtin fragments, rather than full-length huntingtin protein (Miller et al., 2005). Neurons have the ability to slowly clear huntingtin inclusions in vivo, resulting in a reversal of behavioral dysfunction, if expression of the mutant protein is silenced (Yamamoto et al., 2000; Martin-Aparicio et al., 2001). Therefore, targeting the

FIGURE 3 In vivo expression of an anti-huntingtin scFv intrabody in mouse brain. Four-week old mice were stereotactically injected intrastriatally into the left hemisphere with 10^ lU (2 pL of 10^ lU/mL) of rabies virus glycoprotein-pseudotyped equine infectious anemia virus (Mitrophanous et al,, 1999) encoding a cytomegalovirus promoter-driven anti-huntingtin scFv, anti-HD-C4 (Lecerf et al., 2001). Brains were removed at 3 months postinjection for immunostaining. C4 scFv expression was robust in striatum and cortex in the left hemisphere, suggesting that both striatal and corticostriatal neurons were transduced. The right hemisphere, injected with control virus, did not show C4 immunoreactivity. Ctx- cortex; Str- striatum; V-ventricle. Inset (lower right) shows a C4-expressing cell at higher magnification (40X). Adapted from Miller and Messer (2005).

:L GENE THERAPY FOR DEGENERATIVE AND FUNCTIONAL DISORDERS


soluble pathogenic fragment of mutant huntingtin may be beneficial. 2* Influencing cn-'Synuclein Protein Folding and Interactions in Parkinson^s Disease and Synucleinopathies A similar strategy was utilized in the development of anti-a-synuclein intrabodies for synucleinopathies, a group of neurodegenerative disorders characterized by the intracellular accumulation of a-synuclein-positive fibrillar aggregates (Lewy bodies). Synucleinopathies include PD, dementia with Lewy bodies, pure autonomic failure, multiple system atrophy, Lewy body variant of AD, and neurodegeneration with brain iron accumulation type 1 (Lee et al., 2004). The roles of a-synuclein and Lewy body formation in pathogenesis remain controversial, but evidence indicates that a soluble (possibly protofibril) oligomeric a-synuclein intermediate may be a toxic species (Uversky and Fink, 2002; Voiles and Lansbury, 2003). a-Synuclein is believed to exist in an equilibrium between monomeric, jS-sheet oligomeric, and aggregated forms (Maguire-Zeiss and Federoff, 2003), where dominantly transmitted pathogenic mutations (Vila and Przedborski, 2004), oxidative insults (Dawson and Dawson, 2003), and proteasomal inhibition (Giasson and Lee, 2003; McNaught et al., 2004; Zhou et al., 2004b) shift the balance toward toxic oligomer formation. Peptide mimics of a-synuclein have been shown to prevent oligomer/aggregate formation and toxicity in cell culture (El-Agnaf et al., 2004). However, such peptides may interact with other cellular proteins, and they are generally less stable than Ab fragments. Emadi et al. (2004) reported that an scFv selected against monomeric a-synuclein prevents the formation of high-molecularweight oligomers, protofibrils, and aggregates in vitro. Moving into mammalian cells, Zhou et al. (2004a) similarly demonstrated that an anti-monomeric-a-synuclein scFv preferentially binds to and stabilizes monomeric a-synuclein, preventing incorporation into a-synuclein oligomers and increasing the level of monomeric a-synuclein, while decreasing dimer and trimer formation. Such an approach may block the generation of toxic a-synuclein oligomers. The latter intrabody also rescued a cell-adhesion phenotype linked to a-synuclein overexpression in cultured cells, indicating functional correction. Maguire-Zeiss et al. (2004) have begun testing conformation-specific anti-a-synuclein scFvs, which could help to identify the toxic a-synuclein species. Should a-synuclein prove non-essential in adults, targeting of monomeric a-synuclein for degradation

139

may also be therapeutic. An alternative strategy could utilize intrabody-mediated targeting of the toxic aS3niuclein species for degradation. Parkinson's disease is a particularly attractive target for intrabody gene therapy, since the crucial cells are initially limited to a distinct brain region, which should be accessible for gene therapy either directly or via retrograde transport from the striatum (Mazarakis et al., 2001; Martinov et al., 2002). 3. Altering Protein Folding and Interactions in Alzheimer^s Disease and Tauopathies Alzheimer's disease is the most prominent neurodegenerative disorder associated with aging, with rare early-onset disease occurring in younger individuals. This disorder is characterized by the accumulation of extracellular and intracellular fibrils, respectively, containing jS-amyloid and tau (Citron, 2004). A fraction of early-onset familial AD cases are associated with dominantly inherited missense mutations in the gene encoding amyloid precursor proteins (APP) (Goldgaber et al., 1987). jS-Amyloid, a secreted 40- or 42-amino acid peptide, arises from sequential proteolytic processing of APP (Citron, 2004). The jS-amyloid^_42 species is more prone to formation of neurotoxic fibrils (Barrow and Zagorski, 1991; Pike et al., 1991; Lorenzo and Yankner, 1994), and it is a major component of amyloid plaques in AD brains (Glenner and Wong, 1984; Masters et al., 1985; Kang et a l , 1987). Supporting evidence that implicates jSamyloid in AD pathology comes from Down's syndrome cases due to trisomy 21, leading to triplication of the APP gene, AD-like pathology, and formation of j8-amyloid plaques (Wisniewski et al., 1985; Oliver and Holland, 1986). Therefore, intrabodies that block the formation of jS-amyloid plaques or prevent misfolding may be protective. Should j5-amyloid be proven non-essential, targeting it for degradation may be beneficial. Although jS-amyloid plaques accumulate extracellularly, )8-amyloid production likely occurs intracellularly (Shoji et al., 1992; Busciglio et al., 1993; Haass et al., 1993), so intrabody binding and re-targeting of jS-amyloid prior to secretion may be feasible. Indeed, recent findings by the Molinari group indicate that a scFv intrabody targeting an epitope adjacent to the j5-secretase cleavage site of APP can be combined with an endoplasmic reticulum retention signal (ER-retention signal) to prevent jS-amyloid secretion (Paganetti et al., 2005). (This is also a proteolysis inhibition strategy, see Section ILD) Liu et al. (2004a, b) have also demonstrated that an anti-j8-amyloid scFv can prevent


140


aggregation in a cell-free system, inhibiting subsequent toxicity in neuronal cells. Manoutcharian et al. (2004) have recently reported synthetic anti-jS-amyloidi_42 peptides based on the V^-CDRS sequences, which can bind and offer protection when delivered extracellularly to primary rat hippocampal cultures. Binding of scFv or DAB protein or fragments to extracellular ^-amyloid, so as to prevent deposition, plaque formation, and pathogenesis, may also be beneficial, although such a strategy lies outside the realm of intracellular Abs. A 35-amino acid fragment of a-synuclein, the nonamyloid component (NAC), is found in the j5-amyloid plaques of AD brains (Ueda et al., 1993), suggesting that a-synuclein is proteolytically cleaved to release the NAC fragment. NAC may stimulate j5-amyloid aggregation, and vice versa (Han et al., 1995; Yoshimoto et al., 1995). Fibrils of both NAC and a-synuclein are neurotoxic (Conway et al., 1998; El-Agnaf et al., 1998). Bodies et al. (2001) proposed that a-synuclein residues 68-76 are critical for NAC aggregation. Therefore, an intrabody that targets this region of NAC could be more broadly useful, serving to reduce NAC, a-synuclein, and jS-amyloid fibril assembly. Since many neurological disorders show common markers, they may also have common mechanisms of pathogenesis. Such commonalities could permit the development of widely applicable therapeutics. The most frequently occurring feature in neurological diseases is, arguably, neurofibrillary tangles composed primarily of filaments of the microtubule-associated protein, tau (Lewis et al., 2000; Zhang et al., 2004); such tangles have been correlated with neurodegeneration (Braak and Braak, 1991). However, it has not been explained whether tangle formation is itself pathogenic, or whether the tangles appear concomitantly with neuropathology caused by another mechanism. Should the former be the case, blockage of tangle formation could be protective for an array of conditions. Several tauopathies have been linked to dominant missense mutations in tau, including frontotemporal dementia with Parkinsonism linked to chromosome 17, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration (Lee et a l , 2001). Once the domains of tau that are implicated in fibril formation have been thoroughly elucidated, intrabodies could theoretically be selected against these regions and would bind to prevent the regions' self-interactions. There are six isoforms of tau in brain, generated by alternate mRNA splicing (Lee et al., 2001). These isoforms have differing microtubule-binding abilities, and possibly differing roles in disease, so intrabody

selection against particular isoforms could be valuable to an understanding of fibril assembly and the disease process in general. Anti-tau intrabodies have been selected by yeast intracellular Ab capture technology (Visintin et al., 2002), but their efficacy in disease applications remains unknown. 4» Influencing Prion Protein Folding and Interactions In contrast to the disorders described above, prion diseases are caused by an infectious protein. There are several forms of prion diseases that occur in humans, including Creutzfeld-Jakob disease, Gerstmann-Straussler disease, familial fatal insomnia, and the transmissible forms, kuru and new variant Creutzfeld-Jakob disease. Most prion disease cases are idiopathic, but approximately 15% are caused by dominantly inherited mutations in the PRNP gene, encoding prion protein (Prusiner, 1998). The disease mechanism put forth by S.B. Prusiner identifies an abnormal conformation of the prion protein (PrP^^) as the toxic species, where this infectious protein can induce normal prion protein (PrP^) to change conformation and adopt the same abnormal features, eliciting a cascade effect and accumulation of PrP^^ (Prusiner, 1982). The altered conformation shows increased j8-sheet structure and protease resistance (Caughey et al., 1991; Prusiner, 1991; Pan et al., 1993), and it has been detected in both intracellular and extracellular prion amyloid (Kitamoto et al., 1991; Tagliavini et al., 1994; Ma and Lindquist, 2002; Ma et al., 2002). PrP^^ causes neurotoxicity through an undefined mechanism, although roles in proteasomal dysfunction, endoplasmic reticulum stress (Castilla et al., 2004), and astrocyte-mediated damage (Jeffrey et al., 2004) due to altered copper homeostasis (Brown, 2004) have been proposed. In a model of passive immunization, Heppner et al. (2001) demonstrated that transgenic expression of secreted anti-PrP Abs protected mice against PrP^^ infection. This mode of rescue likely utilized extracellular Ab-PrP interaction to prevent disease. Since prion amyloid has also been detected in the cytosol (Ma and Lindquist, 2002; Ma et a l , 2002), and since the disease mechanism may involve intracellular pathways (Brown, 2004; Castilla et al., 2004; Jeffrey et al., 2004), intracellular Ab expression may also be effective. Additionally, an scFv version of the therapeutic Ab was generated (Heppner et al., 2001), although the efficacy of this scFv is unknown. Cardinale et al. (2005) have generated two anti-PrP scFvs from monoclonal Abs, and fused them to secretory leader or ER-retention signals. Retention in the endoplasmic reticulum



of PC12 neuronal cells prevented the appearance of the PrP^ on the cell surface, and also prevented PrP^'^ accumulation. This provides evidence that the toxic misfolding occurs on the cell surface, thus illustrating the use of an intrabody as a tool to study cellular trafficking and molecular pathology, as well as a potential therapy. Other anti-PrP scFvs that may bind PrP and block the pathogenic PrP^'^ cascade have been selected from phage-display libraries (Leclerc et al., 2000; Wuertzer et a l , 2004), and could also prove useful. PrP may be an essential protein. Knock-out mice appear to develop normally, but they show subclinical demyelination with age (Weissmann and Flechsig, 2003). Therefore, anti-PrP'^ intrabody therapy to block Pj.pc ^ pj-psc conversion could require selection of an agent that binds PrP^ but does not interfere with essential functions. Alternatively, a PrP^^-specific intrabody, which would not bind PrP^, could remove PrP^^ or block its interaction with PrP"^. PrP^^ is known to be protease-resistant (Prusiner, 1998); however, Luhr et al. (2004) have recently demonstrated that neuronal cells can degrade PrP^^ when endogenous PrP expression is silenced. This suggests that the pathogenic cellular pathway requires a continuous supply of PrP^, and that interference with PrP^ -> PrP^^ recruitment could similarly enable cells to clear themselves of PrP^^. B.

Targeting Common Amyloid Structures

There appear to be common amyloid protein conformations within inclusions observed in AD, PD, poly-Q, and prion diseases; these conformations share structural features, including j8-sheets, j8-strands, and j8-turns (Ross and Poirier, 2004). The Wetzel group has found that Abs specific for such amyloid conformations can bind inclusions composed of various disease proteins (O'Nuallain and Wetzel, 2002), suggesting shared structural characteristics among disorders. Another Ab described by the Glabe lab recognizes a common conformation-dependent structure that appears to be unique to soluble oligomeric forms of APP, a-synuclein, prion, poly-Q, and some non-neuronal prefibrils, regardless of amino acid sequence. This latter Ab can also inhibit the in vitro toxicity of soluble oligomers (Kayed et al., 2003). Such structural similarities may permit the development of a generic, conformation-specific intrabody for misfolded, pathogenic proteins. However, such conformation-specific intrabodies may also stabilize the abnormal conformations; therefore, extensive testing under multiple conditions will be required. Conformation-dependent intrabodies could be selected from scFv libraries using

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the colloidal gold bead-coupled-Ab molecular mimic of soluble toxic oligomers, as originally described (Kayed et al., 2003). The candidate intrabodies might then be expressed intracellularly to identify common features of toxic species of a series of proteins, thus making them useful in target identification and validation (Glabe, 2004). C.

Altering Post-Translational Modifications

Proteins implicated in several neurological diseases have been found to be abnormally phosphorylated or oxidatively damaged. Whether these modifications are a cause or effect of the disease process remains a matter of debate. However, substantial evidence suggests that the aberrant modifications of a-synuclein (Fujiwara et al., 2002; Ischiropoulos, 2003), tau (Geschwind, 2003; Horiguchi et al., 2003), superoxide dismutase-1 (Rakhit et al., 2004), neurofilaments (Ackerley et al., 2004), and ataxin-1 (Emamian et al., 2003) contribute to pathogenesis. Intrabody-mediated alteration of the post-translational modifications of disease proteins, either by binding unaltered proteins and directly blocking modifications, or by targeting modified proteins and removing them, could theoretically prevent disease and should be considered an avenue for therapeutic development. !• Intrabody Targeting of Post-Translationally Modified Tau A fraction of tauopathy cases are caused by mutations in tau (Lee et al., 2001). Such mutations can result in altered phosphorylation states of tau (Crowther and Goedert, 2000), decreased microtubule-binding and assembly abilities, and susceptibility to insoluble filament formation (Lee et al., 2004). However, most tauopathies are not related to tau mutations, and hyperphosphorylated, non-mutated tau has been found in neurofibrillary tangles (Drewes, 2004), suggesting that abnormal phosphorylation is linked to pathogenesis. Hyperphosphorylated tau exhibits decreased microtubule-binding ability and is arguably involved in insoluble fibril formation (Lee et al., 2004). Tau may play a role in axonal microtubule organization, although knock-out mice appear phenotypically normal (Harada et al., 1994). Haploinsufficiency has not been reported to result in human disease, suggesting that a single normal allele is adequate. Therefore, therapeutic antitau intrabodies could theoretically target abnormally phosphorylated tau for degradation, or to prevent its incorporation into fibrils, without adversely affecting essential levels or functions of tau.


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2. Targeting of Post'Translationally Modiêd (X-Synuclein Using Intrabodies Synucleinopathy lesions similarly contain a-synuclein selectively phosphorylated at S129, and phosphorylation at this residue accelerates fibril formation in vitro (Fujiwara et al., 2002; Kahle et a l , 2002). This phosphorylated species is ubiquitinated in humans (Hasegawa et a l , 2002), implying that it has been targeted for proteasomal degradation. Targeting of S129-phosphorylated a-synuclein using intrabodies may prevent accumulation and fibril formation. Alternatively, an anti-a-synuclein intrabody that blocks phosphorylation could prevent pathogenesis; however, the normal role for the phosphorylated species must first be established. Increased nitration of a-synuclein has also been observed in synucleinopathies (Duda et al., 2000; Giasson et a l , 2000), although in inverse correlation to phosphorylation in mutant mice (Papay et a l , 2002). Nitrotyrosine residues stabilize a-synuclein oligomers (Souza et a l , 2000; Takahashi et al., 2002), suggesting a mechanism for oxidative damage-induced Lewy body formation. Intrabodies that selectively target nitrotyrosinated a-synuclein could remove this damaged species from the cell. 3. Potential for Intrabody Therapeutics in Amyotrophic Lateral Sclerosis Familial amyotrophic lateral sclerosis type 1 (ALS) is caused by mutations in SODl, the gene encoding superoxide dismutase-1. Mutant SODl is prone to aggregation (Bruijn et al., 1997,1998; Elam et al., 2003) and may prevent the formation of functional homodimers (Fridovich, 1986). It is theoretically possible to select an intrabody that will specifically target the mutant form for degradation, thus preventing aggregation, and possibly facilitating nornial dimer formation. However, given the large number of different mutations involved in this disorder (see www.alsod. org), it may be more reasonable to carefully examine the SODl structural data to determine the sites of interaction, and then try to block these sites. In the more common sporadic ALS, however, it is less clear that the accumulating material is SODl itself. Rather, there appears to be a more general breakdown of cellular pathways, leading to deposition of a range of other proteins, which may represent markers rather than primary pathogenic species. If further studies continue to support hypotheses that oxidative stress is a causal agent in ALS (Agar and Durham, 2003), it may be possible to intervene with intrabodies designed to identify abnormally modified proteins (nitrated

or phosphorylated), and to establish a more general approach to easing the toxic burden on neurons or support cells. 4* Intrabody Targeting of Post-Translationally Modified Ataxin-1 Phosphorylation of S776 of ataxin-1, the poly-Qcontaining protein involved in spinocerebellar ataxia type 1, was shown to be important for pathogenesis in transgenic mice (Emamian et a l , 2003). Accordingly, an anti-ataxin-1 intrabody targeting this phosphorylation site could block modification and prevent disease. Alternatively, intrabody-mediated removal of S776-phosphorylated ataxin-1 may be another therapeutic approach. Interestingly, this carboxyl-terminal region implicated in phosphorylation-dependent pathogenesis is also essential for ataxin-1 interaction with an ubiquitin-specific protease, USP7 (Hong et al., 2002); this link further supports the role of aberrant proteolytic processing in the disease pathway. D . Preventing Pathogenic Proteolysis of Disease Proteins A series of neurological diseases are believed to involve abnormal proteolytic cleavage, resulting in the liberation of a pathogenic protein fragment; notable among these are the cleavage of APP to release j5-amyloidi_42 in AD (Goldgaber et al., 1987; Kang et al., 1987; Konig et a l , 1992), and the cleavage of proteins to release the expanded poly-Q-containing fragments that exacerbate disease (Tarlac and Storey, 2003). Proteolytic processing of a-synuclein has also been hypothesized as a factor in PD and other synucleinopathies (Mishizen-Eberz et al., 2003; Li et al., 2005; Liu et al., 2005). Such sites of proteolysis present attractive targets for therapeutic intrabody development, as blockage of the cleavage of disease proteins may prevent the generation of pathogenic fragments and subsequent disease. While blockage of the proteolytic enzymes has been proposed as a treatment for such diseases, an alternative strategy, the use of engineered Ab fragments, could theoretically offer specificity against a sequence that is restricted to the target protein, while leaving the enzymes themselves free to participate in other crucial reactions in the cell. The intrabodies in this application would need to have higher affinities a n d / o r lower off-rates than those in the aforementioned paradigms, since the enzymatic modification of the substrate is an irreversible process. They could also be valuable as part of a multiplex strategy to reduce the toxic load.

. GENE THERAPY FOR DEGENERATIVE AND FUNCTIONAL DISORDERS

POTENTIAL INTRABODY TOXICITY

Paganetti et al. (2005) recently described inhibiting the generation of j8-amyloid using scFvs derived from monoclonal Abs to an epitope adjacent to the APP j8-secretase cleavage site. The intrabodies were targeted to the endoplasmic reticulum, where they attached to the newly formed APP chains, and prevented the abnormal cleavage. One construct also retained the protein in the endoplasmic reticulum, which has the advantage that it also deals with the form of jS-amyloid that escapes from intrabody shielding against cleavage, but at the cost of forcing endoplasmic reticular disposal of excess APP, which could lead to eventual toxicity itself. A potentially beneficial proteolytic cleavage event unique to the treatment of AD is a-secretase-mediated cleavage of APP within the j8-amyloid sequence. Interestingly, an anti-jS-amyloid DAB has shown a-secretase-like activity through internal cleavage of jSamyloid and prevention of cytotoxicity (Rangan et al., 2003; Liu et al., 2004a, b), suggesting that this strategy may also be advantageous in select disease contexts. IIL IMPROVING INTRABODY GENE DELIVERY, EXPRESSION, A N D FUNCTION One of the most powerful aspects of the combination of genetic and proteomic approaches to protein deposition is the capability to further engineer intrabodies via directed or random mutagenesis. As noted in Section LA, the environment used for selection does not currently mimic the intracellular environment in which the intrabody protein must fold and interact. The affinity and stability of effective intrabodies would be relative to the disease or condition under study, and such parameters are only moderately predictable with current selection techniques. The data from several studies have confirmed that while binding to the correct epitopes is valuable for partial correction of a disease, further improvements will be necessary to optimize these reagents. One recent study used a strategy of site-directed elimination of the cysteine residues in a DAB selected against the amino-terminal 20 residues of huntingtin, followed by random mutagenesis to revitalize high-binding affinity. This generated an intrabody whose efficacy has been increased by a factor of 5- to 10-fold in an aggregation assay, with significant protection against mutant huntingtin toxicity in two functional assays (Colby et a l , 2004a). These data suggest that the use of such an iterative mutagenesis

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and testing strategy with future intrabodies that show promising preliminary properties in cellular assays could be worthwhile, since it would facilitate the more efficient utilization of intrabodies in vivo. However, such mutagenesis techniques may also run the risk of increasing the immunogenicity of an intrabody. Delivery of exogenous genes to the central nervous system poses perhaps the greatest obstacle to intrabody therapeutics for neurological disorders. We will briefly discuss the potential limitations of such an approach, as the reader can find detailed reviews of this subject elsewhere in this book. There are three foreseeable options for intrabody expression in the nervous system: (1) intrabody gene delivery to neurons in vivo using viral vectors, (2) such delivery using intrabody genes tagged with a protein transduction domain (PTD) (Dietz and Bahr, 2004), and (3) ex vivo transduction of cells with PTD-tagged intrabody transgenes followed by cell implantation into the nervous system. Option (1) is currently the most viable, since the ability of PTD-tagged proteins to leave a producer cell and then transduce neighboring cells' cytoplasm in a properly folded configuration is controversial. However, should a reliable PTD system be created, options (2) and (3) might allow intrabodies to treat larger populations of cells, and hence provide more widespread protection. Diseases that are most readily amenable to intrabody therapy will be those in which correction of neurons in a discrete region will be beneficial. It is possible that in diseases such as HD, where there is both a dramatic focus of initial damage in the striatum, and evidence of significant pathology elsewhere, combinations of intrabody therapy in the most affected regions, plus small molecules that can act more ubiquitously, would be most efficacious. IV

POTENTIAL INTRABODY TOXICITY

Many of the intrabodies already developed or proposed for the above studies target sequences that are shared by normal and mutant proteins; this is particularly true for those that have been designed to prevent misfolding or abnormal interactions, rather than to remove altered proteins. The full-length forms of many such proteins may be essential. Therefore, an intrabody targeting a proteolytic fragment may be more therapeutically effective than one specific for a much larger full-length protein in a setting in which both species exist. This situation is especially advantageous in HD, where proteolytic cleavage of poly-Q


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disease proteins is thought to result in the release of toxic, expanded poly-Q-containing fragments with no known essential functions (Tarlac and Storey, 2003). Expression of virally delivered anti-HD-C4 intrabody (Lecerf et a l , 2001; Murphy and Messer, 2004; Miller et al., 2005; Wolfgang et al., 2005) in mouse brain over several months did not elicit any obvious toxicity (Miller and Messer, 2005). Careful toxicity testing will be required prior to clinical use, and levels of intrabody that reduce — rather than eliminate — the abnormal protein load may strike a balance between preservation of adequate normal function and removal of the offending species. It is also possible that short-term, or pulsed/periodic expression of intrabody genes could act to clear neurons of toxic material that appears to take many years to accumulate, thereby "resetting the clock." This would require regulatable delivery systems, which are under development.

V-

PERSPECTIVE

These new classes of intrabody reagents that can be highly engineered, both as direct therapeutics and as tools for further drug discovery, are theoretically applicable to a wide range of neurological diseases, most of which currently have very few viable treatment options. The obstacles to intrabody applications in neurological disorders reside in (1) the delivery of intrabody-encoding genes to sufficient numbers of target cells in the nervous system, and (2) the need for further understanding of disease mechanisms to enable the optimal targeting of antigens. Engineered Abs may themselves be very valuable in elucidating disease mechanisms. As fusions with capsid or envelope proteins, Ab fragments may also help to direct delivery of viral vectors to cells with specific surface properties. Recombinant viral vectors are obviously promising vehicles for intrabody gene delivery to the nervous system, and the crucial regulatable delivery systems are the subject of intense investigation. The rapidly advancing fields of virus-mediated gene therapy and neurological diseases hold great promise for intrabody therapy for the nervous system in the near future.

ACKNOWLEDGMENTS We thank the current and past members of the Messer lab (Thomas Shirley, William Wolfgang, Jack

Webster, Kevin Manley, Robert Murphy, and Chun Zhou), as well as Drs. James Huston, Dane Wittrup, David Colby, Valerie Bolivar, Kyri Mitrophanous, and Michael Sierks for many helpful discussions of aspects of the work reported here. The EIAV vector used in Fig. 3 was generously provided by Dr. Nicholas Mazarakis of Oxford Biomedica, Ltd. Support for intrabody work in the Messer lab has been provided by NIH, Hereditary Disease Foundation, Cure HD Initiative, Huntington's Disease Society of America, High Q Foundation, and National Parkinson Foundation. References Ackerley, S., Grierson, A.J., Banner, S., Perkinton, M.S., Brownlees, J., Byers, H.L., Ward, M., Thomhill, P., Hussain, K., Waby, J.S., Anderton, B.H., Cooper, J.D., Dingwall, C , Leigh, PN., Shaw, C.E. and Miller, C.C. (2004) p38alpha stress-activated protein kinase phosphorylates neurofilaments and is associated with neurofilament pathology in amyotrophic lateral sclerosis. Mol. Cell. Neurosci., 26: 354-364. Agar, J. and Durham, H. (2003) Relevance of oxidative injury in the pathogenesis of motor neuron diseases. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 4: 232-242. Agrawal, N., Pallos, J., Slepko, N., Apostol, B.L., Bodai, L., Chang, L.W., Chiang, A.S., Thompson, L.M. and Marsh, J.L. (2005) Identification of combinatorial drug regimens for treatment of Huntington's disease using Drosophila. Proc. Natl. Acad. Sci. USA, 102: 3777-3781. Alvarez, R.D., Barnes, M.N., Gomez-Navarro, J., Wang, M., Strong, T.V., Arafat, W., Arani, R.B., Johnson, M.R., Roberts, B.L., Siegal, G.P. and Curiel, D.T. (2000) A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial. Clin. Cancer Res., 6: 3081-3087. Barrow, C.J. and Zagorski, M.G. (1991) Solution structures of beta peptide and its constituent fragments: relation to amyloid deposition. Science, 253: 179-182. Bates, G. (2003) Huntington aggregation and toxicity in Huntington's disease. Lancet, 361:1642-1644. Beyer, W.E., Palache, A.M., Luchters, G., Nauta, J. and Osterhaus, A.D. (2004) Seroprotection rate, mean fold increase, seroconversion rate: which parameter adequately expresses seroresponse to influenza vaccination? Virus Res., 103:125-132. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. (1988) Single-chain antigen-binding proteins [erratum appears in Science, 1989 Apr 28; 244(4903): 409]. Science, 242: 423-426. Boder, E.T. and Wittrup, K.D. (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat. BiotechnoL, 15: 553-557. Bodies, A.M., Guthrie, D.J., Greer, B. and Irvine, G.B. (2001) Identification of the region of non-Abeta component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity. J. Neurochem., 78: 384-395. Braak, H. and Braak, E. (1991) Neuropathological stageing of Alzheimer-related changes. Acta NeuropathoL, 82: 239-259. Brown, D.R. (2004) Role of the prion protein in copper turnover in astrocytes. Neurobiol. Dis., 15: 534-543. Bruijn, L.I., Becher, M.W, Lee, M.K., Anderson, K.L., Jenkins, N.A., Copeland, N.G., Sisodia, S.S., Rothstein, J.D., Borchelt, D.R.,


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Tanaka, T., Chung, G.T., Forster, A., Lobato, M.N. and Rabbitts, T.H. (2003) De novo production of diverse intracellular antibody libraries. Nucleic Acids Res., 31: E23. Tarlac, V. and Storey, E. (2003) Role of proteolysis in polyglutamine disorders. J. Neurosci. Res., 74, 406-416. Tewari, D., Notkins, A.L. and Zhou, P (2003) Inhibition of HIV-1 replication in primary human T cells transduced with an intracellular anti-HIV-1 p l 7 antibody gene. J. Gene Med., 5:182-189. Tse, E., Lobato, M.N., Forster, A., Tanaka, T., Chung, G.T. and Rabbitts, T.H. (2002) Intracellular antibody capture technology: application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein. J. Mol. Biol., 317: 85-94. Ueda, K., Fukushima, H., Masliah, E., Xia, Y, Iwai, A., Yoshimoto, M., Otero, D.A., Kondo, J., Ihara, Y and Saitoh, T. (1993) Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA, 90:11282-11286. Uversky, V.N. and Fink, A.L. (2002) Amino acid determinants of alpha-synuclein aggregation: putting together pieces of the puzzle. FEBS Lett., 522: 9-13. Vila, M. and Przedborski, S. (2004) Genetic clues to the pathogenesis of Parkinson's disease. Nat. Med., 10 (Suppl): S58-S62. Visintin, M., Settanni, G., Maritan, A., Graziosi, S., Marks, J.D. and Cattaneo, A. (2002) The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies. J. Mol. Biol., 317: 73-83. Voiles, M.J. and Lansbury, P.T., Jr. (2003) Zeroing in on the pathogenic form of alpha-synuclein and its mechanism of neurotoxicity in Parkinson's disease. Biochemistry, 42: 7871-7878. Weissmann, C. and Flechsig, E. (2003) PrP knock-out and PrP transgenic mice in prion research. Brit. Med. Bull., 66: 43-60. Wellington, C.L., Singaraja, R., EUerby, L., Savill, J., Roy, S., Leavitt, B., Cattaneo, E., Hackam, A., Sharp, A., Thomberry, N., Nicholson, D.W, Bredesen, D.E. and Hayden, M.R. (2000) Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J. Biol. Chem., 275: 19831-19838. Wheeler, YY, Chen, S.Y and Sane, D.C. (2003a) Intrabody and intrakine strategies for molecular therapy. Mol. Ther., 8: 355-366. Wheeler, YY, Kute, T.E., Willingham, M.C., Chen, S.Y. and Sane, D.C. (2003b) Intrabody-based strategies for inhibition of vascular endothelial growth factor receptor-2: effects on apoptosis, cell growth, and angiogenesis. FASEB J., 17:1733-1735. Wisniewski, K.E., Wisniewski, H.M. and Wen, G.Y (1985) Occurrence of neuropathological changes and dementia of Alzheimer's disease in Down's syndrome. Ann. Neurol., 17: 278-282. Worn, A., Auf der Maur, A., Escher, D., Honegger, A., Barberis, A. and Pluckthun, A. (2000) Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors. J. Biol. Chem., 275: 2795-2803. Wuertzer, C.A., Sullivan, M. and Federoff, H.J. (2004) Single-chain antibodies for viral-based passive immuno-therapy for prion disease: linear epitope mapping and clearance from thalamic injection. Mol. Ther., 9: S208. Xia, H., Mao, Q., Eliason, S.L., Harper, S.Q., Martins, I.H., Orr, H.T., Paulson, H.L., Yang, L., Kotin, R.M. and Davidson, B.L. (2004) RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med., 10: 816-820. Yamamoto, A., Lucas, J.J. and Hen, R. (2000) Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease.[see comment]. Cell, 101: 57-66. Yoshimoto, M., Iwai, A., Kang, D., Otero, D.A., Xia, Y and Saitoh, T. (1995) NACP, the precursor protein of the non-amyloid beta/A4


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CHAPTER

12 Gene Therapy for Epilepsy Francesco Noe, Matthew J. During, Annamaria Vezzctni

Abstract: Gene therapy techniques provide a realistic therapeutic approach for intractable focal epilepsies not responding to conventional antiepileptic drugs. These techniques involve the transfer and expression of a "therapeutic" gene into the ictogenic brain area(s), thus permitting long-term central nervous system expression of neuromodulatory molecules with potential anticonvulsive and antiepileptogenic properties. This chapter will review the selection of the "therapeutic" genes delivered into the rodent brain for studying their ability to inhibit seizures and delay epileptogenesis in vivo experimental models of epilepsy. Important aspects that contribute to determine the success or failure of a gene therapy approach are also described, such as the methods of gene delivery, strategies for improving cell transfection and neuronal expression, regulation of gene expression, and possible host tissue reactions to the transgene. Preclinical studies focused on the antiepileptic efficacy of gene therapy in pathological brain tissue, and on its possible side-effects are instrumental for establishing a proofof-principle of the applicability of gene transfer technologies in epilepsy. Keywords: antiepileptic; cell transplantation; viral vectors; neuroprotection; neuropeptides; glutamate receptors; seizures; non viral delivery

I.

properties. In this context, gene therapy techniques, involving the transfer and expression of a therapeutic gene, offer the possibility of delivering specific genes directly into the brain area where seizures originate, thus permitting long-term CNS expression of specific proteins with neuromodulatory actions. Other forms of generalized epilepsies, for example, those of genetic origin, such as the channelopathies, are less suitable for this therapeutic approach because of the need of transfecting cells all over the brain. Ultimately, once global gene transfer technology becomes further developed, such genetic epilepsies will be a primary target for gene therapy, but ironically, today focal epilepsies are a better indication by nature of the localized pathology. Clinical applications for gene transfer to the CNS have been developed so far for some CNS neurodegenerative disorders like Parkinson's disease and

INTRODUCTION

Epilepsy affects about 1% of the population and the current medical therapy is largely symptomatic, thus it is aimed at controlling seizures in affected individuals. However, about 30% of patients are not responsive to available antiepileptic drugs in spite of appropriate treatment (Perucca, 1998). If antiepileptic drugs fail, surgical resection of epileptogenic tissue provides an alternative treatment; however, this choice is amenable only for patients where the epileptogenic area can be adequately defined and removed without major functional impairment (Foldvary et al., 2001). In principle, patients with intractable seizures of focal onset may benefit from therapeutic strategies that are an alternative to resective surgery, such as the delivery of molecules into the seizure focus with potential anticonvulsive and antiepileptogenic


151


152

12. GENE THERAPY FOR EPILEPSY

Alzheimer's disease, in cancer, inherited monogenic disorders (cystic fibrosis) and genetic diseases such as Canvan and Batten's disease or infectious diseases (HIV). The selection of the "therapeutic" gene and the method of its delivery are two crucial aspects that determine the success or failure of a gene therapy approach, and their choice depends on the specific pathology under investigation.

11.

THERAPEUTIC TARGETS I N EPILEPSY

At least two nonmutually exclusive endpoints can be considered when devising a gene therapy approach to epilepsy, namely to suppress seizures and to spare or rescue neurons from otherwise irreversible damage (Table 1). These two therapeutic outcomes should lead to sparing of function at the level of synaptic physiology and plasticity, as well as at the level of behavior and cognition.

As discussed in more detail in Section IV, gene therapy has been successful in suppressing seizures both in rat models of acutely induced focal seizures with or without secondary generalization, and in rats with congenital audiogenic seizures or showing primarily generalized absence-like seizures and tonic convulsions. It is important to note that seizure control in models of focal vs. generalized seizures was achieved using different delivery methods (viral vs. nonviral delivery) and route of administration (intraparenchymal vs. intraventricular). Limited information is available on the efficacy of gene therapy approaches in delaying epileptogenesis, thus preventing the occurrence of spontaneous seizures by early intervention after an epileptogenic insult. Two pieces of evidence so far have been provided on the effect of transgenes on the recurrence of spontaneous seizures in rats in which epilepsy has been already established (Thompson and Suchomelova, 2004; Noe' et al., 2005). B.

A.

Anticonvulsant Activity

Therapeutic strategies using conventional antiepileptic drugs have shown that a reduction of excitatory glutamatergic neurotransmission, enhancement of gamma-aminobutyric acid (GABA)-mediated inhibitory effects and blockade of Na^ and Ca^^ channels can provide effective seizure control. The mechanims of action of antiepileptic drugs and their molecular targets are compatible with the widely supported hypothesis that neuronal hyperexcitability underlying the epileptic state depends on an imbalance in the excitatory and inhibitory transmission in CNS (Rogawski and Loscher, 2004). In addition, the fortuitous occurrence of seizures in both mutant and transgenic mice has enabled the discovery of a large array of genes that can directly or indirectly affect neuronal excitability (Noebels, 1996). Experimental approaches to gene therapy in rodent models of seizures have characterized several possible targets to suppress seizures in epilepsy, namely some neuropeptides (galanin, cholecystokinin (CCK) and neuropeptide Y (NPY)), GABA and adenosine (see Section IV of this review). Furthermore, the delivery of an antisense sequence against the N-methyl-Daspartate (NMDA) receptor has been proven successful in suppressing seizures of focal onset (Haberman et al., 2002). A proof-of-principle for the possibility of sparing cognitive function and rescue neurons post seizure has also been provided using the glucose transporter or antiapoptotic genes (McLaughlin et al., 2000).

Neuroprotection

Neuronal damage is at least in part due to ongoing and uncontrolled seizure activity, therefore, suppression of seizures may in turn stop progression of cell death. However, cell damage may precede in some instances the onset of seizures and contribute to epileptogenesis, as it has been proposed for those cases of symptomatic epilepsies where brain damage can evolve to active epilepsy. Rescue of neurons at the morphological and functional level is more likely at the early stages of damage and the latency between the onset of damage and the gene delivery probably impacts the likelihood of ultimate therapeutic success. One possible solution highlighted in the current literature is to develop vectors that can be introduced into the CNS where they remain transcriptionally quiescent until a proper injurydependent stimulus is provided. In this respect, neuroprotection has been achieved in vitro against an acute necrotic insult using vectors containing a synthetic glucocorticoid-responsive promoter, which would exploit the high levels of adrenal stress hormones secreted in response to this insult (Ozawa et al., 2000). Overexpression of antinecrotic and antiapoptotic genes, neurotrophins, Glut-1 glucose transporter and neuropeptides are among the candidate targets studied in gene therapy strategies using in vitro and in vivo models of cell death (Table 1). Transgene expres-sion has been shown to reduce neurodegeneration and, in some instances, also provides recovery from physiological and behavioral dysfunction (Dumas and Sapolsky, 2001).


153

DELIVERY METHODS FOR GENE THERAPY

TABLE 1 Target

Route

Molecular Targets for Gene Therapy in Epilepsy

Method of delivery

Eperimental model

Functional effect

Jlllerences

Anticonvulsive Adenosine GAD-65

Anti-NMDA

ICV

Grafting of fibroblasts

Hippocampal kindling

Suppression of generalized seizures

Huber et al. (2001)

Anterior SN

Immortalized mouse cortical neurons and glia

Entorhinal kindling

Delayed rate,of kindling

Thompson et al. (2000)

Posterior SN

Entorhinal kindling

Faster rate of kindling

Thompson et al. (2000)

Anterior SN

Status epilepticus

Reduced spontaneous seizures

Thompson and Suchomelova (2004)

Piriform cortex

Amygdala kindling

Increased threshold seizures

Gemert et al. (2002)

Focal electrical stimulation

Increased seizure threshold

Haberman et al. (2002)

Decreased seizure threshold






Collicular cortex

AAV-CMV vector AAV-TET-off vector

Galanin

Collicular cortex

AAV-CMV vector

Focal electrical stimulation

AAV-TET-off vector Hippocampus

Neuropeptide Y

Hippocampus

AAV-TET-off vector

Status epilepticus

Neuroprotection


AAV-NSE vector

Ictal activity

Seizure inhibition

Lin et al. (2003)

AAV-NSE vector

Ictal activity. Status epilecticus Hippocampal kindling

Seizure inhibition

Richichi et al. (2004)

Increased threshold and delayed rate of kindling

Richichi et al. (2004)

ASPA

ICV

AV-CAG vector

Spontaneous seizure in SER

Reduced incidence of tonic seizure

Seki et al. (2004)

Cholecystokinin

ICV

Lipofectin

Audiogenic seizures

Seizure inhibition

Zhang et al. (1997)

Hippocampus Cell cultures

Viral vectors

Necrotic or apoptotic injury

Neuronal survival

Dumas and Sapolsky (2001), Haberman et al. (2003), McLaughlin et al. (2000), Ozawa et al. (2000)

Neuroprotective Glut-1 Anti-apoptotic Neuropeptides Neurotrophins Calbindin

AAV, adeno-associated viral; AV, adenoviral; GAG; cytomegalovirus enhancer, chicken j&-actin promoter; GMV, cytomegalovirus; GAD-65, glutamic acid decarboxylase; Glut-1, glucose transporter; IGV, intracerebroventricular; NMDA, N-methyl-D-aspartate; NSE, neuron-specific enolase ; SER, spontaneously epileptic rats; TET off, tetracycline-off regulatable promoter.

III.

DELIVERY METHODS FOR GENE THERAPY

There are two main types of gene delivery methods, which consist of in vivo gene transfer using viral vectors, naked DNA or cation-lipid DNA complexes and

ex vivo gene transfer using cells previously transfected in vitro with the transgene of interest (Costantini et al., 2000; Mountain, 2000). Some of these strategies have been used to study the therapeutic effect of gene transfer in experimental models of seizures. At the present stage, viral vectors


154

12. GENE THERAPY FOR EPILEPSY

generally give the most efficient transfection in vivo, although their main disadvantage concerns size-limitation of the transgene and the potential immunogenicity. Among the viral vector-delivery systems, adeno-associated virus (AAV) vectors, lentivirus and herpes simplex virus (HSV) vectors have specific tropism for post-mitotic neurons of the CNS, with AAV having the best safety profile. Viral vectors derived from retrovirus are not adequate for gene transduction in neurons since they transfect only proliferating cells (Janson et al., 2001). Physical methods of gene delivery, such as lysosomes carrying plasmids have the potential advantage over viral vectors to allow systemic, thus noninvasive, delivery of genes, which then successfully penetrate into the brain parenchyma, providing widespread gene expression in the brain. This method of delivery is impaired when using vector-mediated gene transfer since the blood-brain barrier prevents the vectors to get into the brain parenchyma. However, noninvasive gene targeting to the brain is extremely inefficient and allows a very short expression of the transgene limited to a few days; in addition, gene transfer will occur also in peripheral tissues, thus requiring that the specific brain expression is regulated at the promoter level. In the context of ex vivo gene transfer, the production and release of a protein from a transduced gene using cell transplantation and grafting of in vitro engineered cells, will strongly depend on the survival of these cells in the transplanted tissue. A. Vector^Mediated Gene Delivery: The Focus on AAV Vectors Neurotropic AAV represent the most often used tool for gene delivery in experimental models of epilepsy. They present many advantages over the other available delivery methods both for brain functional studies and the brain expression of transgenes for therapeutic purposes. Thus, they can efficiently express single or multiple transgenes together with a wide range of regulatory elements; they permit long-term gene expression and can be engineered at the capsid and promoter level to preferentially target specific populations of neurons in a controllable manner, and very importantly they are nonpathogenic and appear to be innocuous on normal brain physiology (Monahan and Samulski, 2000). Figure 1 depicts the expression of green fluorescent protein (GFP) used as a reported gene in AAV vectors engineered with the neuron-specific enolase (NSE)

promoter. The type of transduced neurons and the spread of the transgene expression around the injection site is determined by the serotype of the viral capsid (Davidson et al., 2000). After intrahippocampal delivery, the AAV-2-mediated GFP expression is mainly observed in intemeurons for about 1.5 mm around the site of injection, while AAV-1/2 (a chimeric with 50/50 capsid proteins from both serotypes 1 and 2) mediates the transgene expression also in granule cells and in pyramidal neurons for about 2.5 mm around the injection site (Xu et al., 2001). The mechanisms determining these differences are still unresolved, but it is clear that the choice of the serotype affects the type of cell populations, which will express the transgene (Davidson et al., 2000). This aspect has functional relevance since the neuronal population preferentially transfected will determine the subsequent changes in synaptic transmission and, at a more general level, the physio-logy of the brain area targeted by the vector. Table 2 reports the relative transduction efficiency and the duration of transgene expression of AAV vectors with different promoters and post-transcriptional regulatory elements, as exemplified using AAV2-mediated gene transfer into the hippocampus. Transgene expression driven by the hybrid cytomegalovirus (CMV)- Ji a> 0(b)

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AAV.H1.Luc

efficacy of siRNA-mediated ERa silencing in vivo. As can be seen in Fig. 3, injections into the hypothalamus resulted in an efficient transduction of VMN neurons. Double labeling for ERa (purple nuclear staining) and EGFP (brown cytoplasmic staining) revealed that virtually, all EGFP-positive cells in the area were also ERa-negative in mice injected with AAV.Hl.ERl. Concomitantly, no change in ERa staining was observed in control animals treated with AAV.Hl.Luc. Of importance is the fact that AAV.Hl.ERl-infected neurons produced EGFP, retained normal morphology and were as abundant as AAV.Hl.Luc-infected cells, suggesting that lack of ERa immunoreactivity was not due to cell loss caused by ERa siRNA toxicity but was rather a result of specific inhibition of ERa expression. To ensure that ERa knockdown is specific, we examined the expression of a homologous gene, ERjS, which is also present in the ventral part of the VMN and has some overlapping functions with ERa. In fact, both genes share high sequence similarity in the DNA- as well as the ligand-binding domains (Mosselman et al., 1996). Furthermore, our ERa-specific siRNA sequence ERl (GGCATGGAGCATCTCTACA) was also similar to a corresponding sequence of ERjS with only four mismatches (GGCATGGAACATCTGCICA, mismatched nucleotides are underlined). Although it

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FIGURE 3 AAV-mediated knockdown of ERa expression in the brain. (Top panel) Double-label immunostaining for EGFP (brown) and ERa (purple) in the VMN of female mice 8 weeks after stereotaxic surgery (top two panels). (Bottom panel) expression of ERj5 in the VMN of the same animals. III. PSYCHIATRIC AND BEHAVIORAL GENE THERAPY

SILENCING ESTROGEN-RECEPTOR EXPRESSION IN MOUSE HYPOTHALAMUS USING AAV VECTORS

AAV,H1.Luc

199

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FIGURE 4 Knockdown of ERa in the VMN prevents estrogen-induced upregulation of PR immunoreactivity. Note silencing of ERa by AAV.Hl.ERl (top panel) and concomitant inhibition of PR induction in the VMN but not the ARC (bottom panel).

has been demonstrated that a single base pair substitution in the antisense strand of siRNA duplex would prevent RNAi in vitro (Brummelkamp et al., 2002), the fidelity of this process in vivo is not well characterized. Results presented in Fig. 3 demonstrate that transduction of VMN neurons with AAV.Hl.ERl did not diminish ERjS immunoreactivity, as evident from a similar number of ERjS-positive cells and staining intensity To characterize the effects of ERa silencing in the brain, the mice were injected bilaterally into the VMN with either AAVHl.Luc or AAV.Hl.ERl and then treated with jS-estradiol 3-benzoate (EB). Consistent with the previous experiment, infection with AAV. Hl.ERl resulted in a complete loss of ERa immunoreactivity in the VMN compared to AAV.Hl.Lucinjected mice (Fig. 4, top panel). In addition, we did not observe any decrease in the ERa immunoreactivity in other ERa-positive brain regions, such as the juxtaposed arcuate nucleus (ARC, Fig. 4, top panel) as well as amygdala (Fig. 5, top panel), where projections of EGFP-positive fibers from the hypothalamus can be readily detected (Fig. 5, bottom panel). These results demonstrate the power of this technology as a vehicle for highly focal, highly specific silencing of gene expression in the brain of a normally developed adult animal.

One of the major targets of ERa signaling pathway in the brain following estrogens exposure is upregulation of progesterone receptor (PR) transcription. We and others have previously shown that upregulation of PR following estrogens surge is critical for female reproductive behavior, as inhibition of PR translation by antisense oligonucleotides significantly reduced female proceptive and receptive responses (PoUio et al., 1993; Mani et al., 1994; Ogawa et a l , 1994). We therefore set out to determine if ERa silencing would suppress activation of PR expression after estrogen administration. As anticipated, in mice injected with AAVHl.Luc, estrogen treatment resulted in a robust PR immunoreactivity in the VMN and ARC (Fig. 4, bottom panel), as well as other brain regions such as medical preoptic area MPOA (data not shown). In contrast, in AAV.Hl.ERl-treated mice detectable PR expression in the VMN was completely eliminated, yet it was unaffected in the ARC (Fig. 4, bottom panel). As indicated earlier, this is in distinction to the ERKO transgenic knockout mice, which retained PR induction by estrogens in the VMN presumably due to the presence of the aberrant transcript (see below). These findings demonstrate that rAAV-mediated siRNA delivery can be used to achieve a precise, region-specific silencing of ERa to a level, sufficient to suppress the normal physiological signaling cascade of this nuclear receptor

HI. PSYCHIATRIC AND BEHAVIORAL GENE THERAPY

200

15. USE OF VIRAL VECTORS TO INFLUENCE BEHAVIOR

AAV. H1.Luc

AAV.H1.ER1

FIGURE 5 Downregulation of ERa is region-specific as expression of ERa in the amygdala is not affected by AAV.Hl.ERl (top panel). Note EGFP-positive projections from the hypothalamus in both animals (bottom panel).

in neurons, and unequivocally establishes ERa as the mediator of estrogen induction of PR expression in the VMN in vivo. Having characterized the effects of ERa knockdown at a histological level, we examined these effects on complex behaviors. As expected, after priming with estrogen, mice in the control group injected with AAV. HI.Luc became sexually receptive, displaying proceptive still posture (Fig. 6a) and the lordosis response (Fig. 6b). In addition, they demonstrated very few rejections toward male mounting (Fig. 6c). This was equivalent to the response seen in naive, untreated females, confirming that neither the AAV vector nor expression of EGFP or RNAi inherently causes a reduction in female sexual responses to estrogens. In female mice treated with AAV.Hl.ERl, however, sexual receptivity toward males was completely abolished (Fig. 6a, b). Instead, these female mice showed vigorous rejection such as kicking and defensive fight back toward male approach and attempted mounts (Fig. 6c). Since the female rejections were very strong, stud males could hardly show normal mounts or intromissions. It thus appears that silencing of ERa restricted to the VMN of normal adult mice confers a behavioral response very similar to that of transgenic ERKO mice. It was also found that ERa silencing in the VMN had a profound

effect on body weight. Female mice injected with AAV. Hl.ERl in the VMN gained significantly more weight over a period of several weeks after surgery compared to those treated with AAV.Hl.Luc (Fig. 7). The degree of weight gain following ERa knockdown in the VMN was similar to that reported for ERKO mice (Heine et al., 2000). Several conclusions can be drawn from these data regarding estrogen signaling in the VMN specifically, as well as regarding viral vector-mediated siRNA in general. As indicated above, this can clearly be a highly focused and efficient method, in this case completely silencing ERa expression only in the VMN and not in surrounding regions, without influencing local expression of the highly homologous ERjS gene. Furthermore, this silencing was physiologically significant since this completely blocked estrogen induction of PR expression. This is in opposition to the ERKO transgenic knockout which did not demonstrate this phenotype, thereby raising some questions regarding the true cause of behavioral changes seen in those animals. The similarly profound behavioral change, which we observed now conclusively confirms without such ambiguity prior suggestions that ERa in the VMN is the major mediator of estrogen action on female sexual behavior. Furthermore, the change in

III. PSYCHIATRIC AND BEHAVIORAL GENE THERAPY

RESTORATION OF ESTROGEN RECEPTOR EXPRESSION IN TRANSGENIC KNOCKOUTS USING AAV VECTORS

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C H A P T E R

22 Prodrug-Activation Gene Therapy Kaveh Asadi-Moghaddarriy E. Antonio Chiocca

Abstract: The gene therapy approach most commonly used in clinical trials for brain tumors is gene-directed enzyme-prodrug therapy also known as suicide gene therapy. This approach is comprised of three components; the prodrug to be activated, the enzyme used for activation, and the delivery system for the corresponding gene (Anderson, 2000). With this strategy, the systemically administered prodrug is ideally converted to the active chemotherapeutic agent only in cancer cells, thereby allowing a maximal therapeutic effect while limiting systemic toxicity. Several suicide gene therapy approaches are being explored: (i) herpes simplex virus type 1 thymidine kinase/ganciclovir; (ii) cytosine deaminase/5-fluorocytosine; (iii) cytochrome P450/cyclophosphamide or ifosfamide; (iv) guanine phosphoribosyl-transferase/6-thioxantine; (v) nitroreductase/CB1954; (vi) carboxylesterase/CPT-11; (vii) Escherichia coli purine nucleoside phosphorylase/purine analogs. Keywords: suicide gene therapy; HSV thymidine kinase; XGPRT; PNP; cytosine deaminase; cytochrome P450; nitroreductase; carboxylesterase

L

an acyclic analog of the natural nucleoside 2'-deoxyguanosine (Faulds and Heel, 1990) and is a specific substrate of the HSVtk, which is multiple magnitudes more efficient than human nucleoside kinase at monophosphorylating GCV (see Fig. 1) (Elion et al., 1977). The resulting GCV-monophosphate is then converted by cellular kinases into the toxic GCV-triphosphate. GCV-triphosphate's structural resemblance to 2'-deoxyguanosine triphosphate makes it a substrate for DNA polymerase. Once bound to DNA polymerase, GCVtriphosphate inhibits the enzyme or is incorporated into DNA, causing DNA chain elongation to terminate. This causes cell death by inhibition of incorporation of dGTP into DNA, and also by prevention of chain elongation (Mesnil and Yamasaki, 2000). GCV metabolites target replicating cells much like the S-phase specific chemotherapeutics. Glioma cells, which were transduced and selected to express HSVtk were 5000 times more sensitive to GCV than nontransduced cells (Shewach et al., 1994), and

HERPES SIMPLEX VIRUS TYPE 1 THYMIDINE KINASE (HSVtk)/ GANCICLOVIR

The most widely studied suicide gene strategy is the herpes simplex virus thymidine kinase (HSVtk) enzyme approach. The HSVtk system was developed in 1986 (Moolten, 1986) and was the first approach used in patients with malignant brain tumors in 1992 (Oldfield et al., 1993). This approach has been conducted in combination with guanosine-based prodrugs, such as ganciclovir and acyclovir, originally developed as antiviral agents (De Clercq, 2000). HSVtk converts these nontoxic nucleoside analog prodrugs into phosphorylated compounds. Consequently, these compounds directly inhibit DNA polymerase and render the formed DNA molecule unstable, leading to DNA synthesis arrest and cell death. The most commonly used prodrug is ganciclovir (GCV). GCV is


291

Copyright© 2006, Elsevier, Inc. All rights of reproduction in any form reserved.

293

HERPES SIMPLEX VIRUS TYPE 1 THYMIDINE KINASE (HSVtk)/GANCICLOVIR

TABLE 1 Selection of Closed HSVtk/GCV Gene Therapy Trials in Human Brain Tumors Phase

Tumor type

Application

Vector

Patients, N

Median survival

I

Recurrent GBM or metastasis

Stereotactic injection into tumor of RV-producer cells

RV

15

8 mo[Ram et aL, 1997 ]

I

Recurrent GBM

Stereotactic injection of adenoviral vector

AV

13

4 mo[Trask et aL, 2000]

I/II

Recurrent GBM

Ommaya reservoir injection of RV-producer cells

RV

30

8 mo[Pardos et a l , 2003]

I/II

Recurrent GBM

Freehand injection into resected tumor cavity of RV-producer cells

RV

12

7 mo[Klatzmann et aL, 1998]

I/II

Recurrent GBM

Freehand injection into resected tumor cavity of RV-producer cells

RV

48

9 mo[Shand et aL, 1999]

I/II

Primary/recurrent GBM

Freehand injection into resected tumor cavity of RV-producer cells or adenoviral vector

RVorAV

21 (7 RV, 7AV, 7 LacZ)

7 mo (RV) 15 mo (AV) 8 mo (LacZ)[Sandmair et aL, 2000]

II/III

Primary / recurrent GBM

Freehand injection into resected tumor cavity of adenoviral vector

AV

36(17AV, 19 control)

16 mo (AV) 9 mo (control) [Immonen et aL, 2004]

III

Primary GBM

Freehand injection into resected tumor cavity, followed by radiotherapy

RV

248 (124 RV, 124 control)

365 d (RV) 354 d (control)[Rainov, 2000]

Abbreviations: AV, adenovirus; d, days; GBM, glioblastoma multiforme; GCV, ganciclovir; HSVtk, herpes simplex virus thymidine kinase; LacZ, E. coli jS-galactosidase gene; mo, months; RV, retrovirus.

Amulticenter uncontrolled study including 48 patients with recurrent GBIVI showed a median survival time of 8.6 months. Tumor recurrence was absent on IVIRI in seven patients for at least 6 months, in two patients for at least 12 months, and one patient remained recurrence free at 24 months (Shand et al., 1999). A similar phase-I study with 12 children between ages 2 and 15 years with recurrent malignant supratentorial brain tumors showed a disease progression at a median time of 3 months after treatment (Packer et al., 2000). A large controlled phase-III study was conducted for an ultimate confirmation of the efficacy of the retroviral HSVt/c/GCV approach. This study used an adjuvant gene therapy protocol to the standard therapy of maximum surgical resection and irradiation for newly diagnosed GBIVI. After 4 years of follow-up of 248 patients, who were divided in a gene therapy and a control arm, survival analysis showed no advantage of gene therapy in terms of tumor progression and overall survival (Rainov, 2000). Several Phase-I trials were conducted in order to identify the effectiveness and safety of adenoviral vectors bearing the HSVtk gene. In one study, 13 malignant brain tumor patients were treated with a single intratumoral injection of a replication-defective adenoviral vector, followed by GCV treatment. Patients who received the highest vector dose showed central nervous system toxicity (confusion, seizures). Two patients survived for 2 years before lethal tumor

progression occurred and one patient survived 2.5 years after treatment and remained in stable condition (Trask et al., 2000). Retroviruses were compared with adenoviruses in another phase-I/II trial, including 21 patients with primary or recurrent GBJVI. Seven randomly selected patients with a total of eight tumors were treated with the retrovirus vector, and adenoviruses were used for seven tumors in seven patients. At the time of surgical resection, the two experimental groups were treated with either retrovirus VCP or adenoviruses expressing HSVtk (AdvHSVffc), whereas the control group received either adenovirus or retrovirus VPC expressing the marker gene lacZ (E. coli jS-galactosidase). Four patients with adenovirus injections had a significant increase in anti-adenovirus antibodies and two of them had a short-term fever reaction. Frequency of epileptic seizures increased in two patients. After subsequent GCV treatment, the adenovirus group had significant improvement in mean survival time, with 15 month compared to 7.4 (retrovirus group), and 8.3 month (control group). The assumed mechanism contributing to the greater efficacy of the adenovirus group were greater titer, a benefit from the inflammatory reaction to adenoviruses, and the ability of adenovirus to infect nonreplicating cells. In the retrovirus group, all treated gliomas showed progression by MRl at the 3-month time point, whereas three of the seven patients treated with AdvHSVfA: remained stable (Sandmair et al., 2000). On the basis of these results, a

V. GENE THERAPY FOR BRAIN TUMORS AND NEUROGENETIC DISEASES

294

22. PRODRUG'ACTIVATION GENE THERAPY

5-FC

5-FU

OH 5-FdUMP

FIGURE 2 Cytosine deaminase (CD); 5-fluorocytosinde (5-FC); 5-fluorouracil (5-FU); 5-fluorodeoxyuridine monophosphate (5-FdUMP).

randomized controlled trial involving 36 patients with operable primary or recurrent malignant glioma was conducted. Seventeen patients received AdvHSVffc by local injection into the wound bed after tumor resection, followed by intravenous GCV administration. The control group of 19 patients received standard care consisting of radical tumor resection followed by radiotherapy in those patients with primary tumors. AdvHSVfA: treatment produced a significant increase in mean survival from 39 to 70.6 weeks. The median survival time increased from 37.7 to 62.4 weeks. Six patients had increased anti-adenovirus antibody titers, without adverse effects (Immonen et al., 2004).

II.

CYTOSINE DEAMINASE (CD)/ S^FLUOROCYTOSINE (5^FC)

The cytosine deaminase (CD)/5-fluorocytosine (5-FC) approach is the next most widely studied suicide gene therapy approach. CD is uniquely expressed in certain fungi and bacteria and it converts the prodrug 5-FC (used to treat infections by fungi such as Candida albicans and Cryptococcus neoformans) into the active agent 5-fluorouracil (5-FU) (see Fig. 2). While 5-FC is nontoxic to human cells because of the lack of CD, 5-FU is used to treat cancers like colon, pancreatic, and breast cancer. The cytotoxic effects of 5-FU occur following its conversion to 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP). 5-FdUMP is an irreversible inhibitor of thymidylate synthase and thus inhibits DNA synthesis by deoxythymidine triphosphate (dTTP) deprivation and causes DNA strand breakage, leading to cell death (Grem, 1996). Rodent gliosarcoma cells expressing the E. coli CD gene in vitro become 77 times more sensitive to 5-FC (Aghi et a l , 1998). In addition, tumor cells expressing CD may present CD peptides on MHC class I,

where they could lead to an immune response (Mullen et al., 1996). In contrast to the HSVffc/GCV approach, 5-FU metabolites do not require cell-cell contact for a bystander effect. On cell lysis, 5-FU is released into the medium and is thus likely to be responsible for the bystander effect, and, indeed, the 5-FU levels in the medium correlated well with the degree of cytotoxicity (Kuriyama et al., 1998). Animal studies of C D / 5-FC using adenoviral vectors for rodent and human glioma cell lines showed an increase in survival time compared to controls (Miller et al., 2002). In order to improve tumor cell killing a therapeutic vector has been developed that encompasses two suicide genes to sensitize cells doubly to GCV and 5-FC (Aghi et a l , 1998; Desaknai et al., 2003). From the CD/5-FC clinical trials underway none is for brain tumors.

III. CYTOCHROME P450 (CYP)/ CYCLOPHOSPHAMIDE (CPA) OR IFOSFAMIDE (IFA) The large number of different cytochrome P450 (CYP) isozymes, and the fact that many drugs are metabolized by them, makes the choice of prodrugs quite wide (Waxman et al., 1999). But to date, this area has been dominated by two prodrugs, cyclophosphamide (CPA) and ifosfamide (IFA). Most of CYPs are expressed in the liver rather than in tumor cells, so the goal of this strategy is to selectively increase tumor cell exposure to cytotoxic drug metabolites by targeting expression of enzymes to tumor cells. CPA is a prodrug that is activated by liver-specific enzymes of the CYP family (see Fig. 3). The active form of CPA, phosphoramide mustard, is an alkylating agent that generates DNA cross-links and consecutively DNA strand breaks and cell death. The efficacy of CPA in treating brain tumors


CYTOCHROME P450 (CYP)/CYCLOPHOSPHAMIDE (CPA) OR IFOSFAMIDE (IFA)

295

HO.

.OH CYP

spontaneous — >-

O

CI CI

| N.

CI

CI

4-hydroxy CPA

Phosphoramide Mustard

FIGURE 3 Cytochrome P450 (CYP); cyclophosphamide (CPA),

has been limited by the fact that although CPA crosses the blood-brain barrier, its active metabolites can be generated only by liver P450, and these metabolites are poorly transported across the blood-brain barrier (Wei et al., 1994). Gene therapy using the rat cytochrome P450 2B1 (CYP2B1) which activates CPA with high efficiency (Clarke and Waxman, 1989) was designed primarily for the use in brain tumors since other malignancies have already access to CPA's active metabolites. The implantation of CYP2B1 expressing retroviral vectors was shown to induce regression of intracerebral rat glioma cells after intratumoral or intrathecal CPA administration (Manome et al., 1996). Like the CD/5-FC approach, CPA metabolites do not require cell-cell contact for a bystander effect, distributing by passive diffusion (Wei et al., 1995). Clinical trials of cancer gene therapy commonly involve inoculation of a replication-defective vector. When inoculated tissue was studied, very little diffusion away from the needle tract was noted (Puumalainen et al., 1998). In order to improve this hurdle several strategies have been used. One approach uses an oncolytic virus (OV) to deliver the CYP2B1 cDNA into the tumor (Chase et al., 1998). OVs are genetically altered viruses with deletions that restrict viral replication in normal cells but permit it in tumor cells (Smith and Chiocca, 2000). It has been reported that nucleoside analogs are synergistic in their anticancer action with alkylating agents (Andersson et al., 1996). While CPA metabolites are alkylating agents, GCV metabolites are nucleoside analogs. One approach uses the replacement of the large subunit of the HSV-1 genome with the CYP2B1 gene to generate a HSV-1 vector (rRp450) that is able to kill tumor cells through three modes: (1) using viral oncolysis and rendering infected cell sensitive to (2) CPA and (3) GCV (Chase et al..

1998). Subcutaneous tumors established from glioma cell lines in immunodeficient mice regress only when they are treated with rRp450, CPA, and GCV (Aghi et al., 1998). This has led to the hypothesis that after DNA chain alkylation by CPA metabolites, DNA repair mediated by DNA polymerases, is affected by GCV metabolites. The transient immunosuppression provided by activated CPA metabolites has also been shown to favor viral replication and anticancer effects in vivo (Ikeda et al., 1999). The cytochrome P450 system actually comprises two polypeptide components, the P450 and the P450 reductase (RED). RED expression is required to provide full catalytic activity of the rat CYP2B1 for gene therapy. Studies performed with rat gliosarcoma cells stably transfected with CYP2B1, a n d / o r RED cDNA, showed that further supplementation of RED by gene transfer enhanced the CYP/CPA efficiency, although tumor cells express enough RED to fulfill the transferred CYP gene's capability of CPA conversion (Chen et al., 1997). The addition of RED not only improves CYP2Bl-mediated conversion of CPA, but also provides the ability to convert other prodrugs such as tirapazamine into active anticancer agents (Jounaidi and Waxman, 2000). Another way to improve the therapeutic index of CYP/CPA is the inhibition of hepatic metabolism of CPA (Huang et al., 2000). This might decrease systemic toxicity of CPA metabolites as well as increasing prodrug availability to tumor cells expressing CYP. One of the essential steps in tumorigenesis is the active recruitment of a neovascular supply by the neoplasm. A modified CPA regime showed an anti-angiogenic effect, which also increased the therapeutic index of the CYP/CPA approach (Browder et al., 2000; Jounaidi and Waxman, 2001). Three CYP/CPA clinical trials are underway, none of them on brain tumors.


296

22. PRODRUG'ACTIVATION GENE THERAPY

IV. G U A N I N E PHOSPHORIBOSYL^ TRANSFERASE/6^THIOXANTHINE The E. coli gpt gene encodes for the enzyme xanthine/guanine phosphoribosyl-transferase (XGPRT). Mammalian cells do not efficiently use xanthine for purine nucleotide synthesis. Therefore, cells producing XGPRT after transfection can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis (Mulligan and Berg, 1980, 1981). XGPRT transforms a xanthine analog, 6-thioxanthine (6-TX) to a toxic form for mammalian cells (Besnard et al., 1987). The weakly toxic purine analog 6-TX is phosphorylated to 6-thioxanthine monophosphate (6-XMP) by XGPRT (see Fig. 4). 6-XMP is subsequently converted to the highly toxic 6-thioguanine monophosphate (6-GMP). Rat glioma cells were infected with a retroviral vector expressing the gpt gene and a clonal line exhibited significant 6-TX susceptibility in vitro. In a 'l^ystander"

6-TX 6-TXRMP FIGURE 4 Xanthine / guanine phosphoribosyl-transferase (XGPRT); 6-thioxanthine (6-TX); 6-thioxanthine riboso-monophosphate (6-TXRMP).

assay, tumor cells from the clonal line efficiently transferred 6-TX sensitivity to uninfected tumor cells. This in vitro bystander effect was abrogated when transduced and untransduced cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6-TX and 6-thioguanine (6-TG) significantly inhibited the growth of subcutaneously transplanted XGPRT expressing clonal tumor cells. In an intracerebral model, both 6-TX and 6-TG exhibited significant antiproliferative effects against transduced clonal tumors cells (Tamiya et al., 1996). In a nude mouse model retrovirus-mediated transfer of the gpt gene into rat glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon treatment with 6-TX (Ono et al., 1997). There is no GPT/6-TX clinical trial for brain tumors underway.

V.

NITROREDUCTASE/CB1954

The E. coli enzyme nitroreductase (NTR) is used in combination with the prodrug CB1954 [5-(aziridinl-yl)-2,4-dinitrobenzamide] as a suicide gene therapy approach. CB1954 is a synthesized, weak alkylating agent (Khan and Ross, 1969, 1971). The activating enzyme for CB1954 in mammals is DT-diaphorase (NAD(P)H dehydrogenase). This enzyme converts CB1954 to its 4-hydroxylamino derivative (see Fig. 5) (Knox et a l , 1988). After acetylation via thioesters such as acetyl coenzyme A (CoA), an alkylating agent is produced in a further activation step. The activated prodrug is then capable of forming poorly reparable DNA cross-links. The E. coli ISTTR is sensitized to CB1954 whereas human DT-diaphorase is poorly capable of performing this conversion, thereby limiting toxicity to transformed cells (Boland et al., 1991). The advantage of the NTR/CB1954 approach is that killing mediated by activated CB1954 is not dependent on the cell

CONH

CONH, NTR HO—NH

DNA CB1954 FIGURE 5

Nitroreductase (NTR).


E. COLI PURINE NUCLEOSIDE PHOSPHORYLASE (PNP)/PURINE ANALOGS

CPT-11

297

SN-38

FIGURE 6 Carboxylesterase (CE).

cycle phase, potentially allowing quiescent tumor cells to be killed. In vitro studies using a retroviral vector expressing the E. coli NTR gene in human colorectal and pancreatic cancer cell lines showed selective killing of NTR-expressing cells following CB1954 administration (Green et al., 1997). To improve the delivery of the NTR gene a replication-defective adenoviral vector expressing NTR was constructed (Grove et al., 1999). In vivo studies with the adenoviral vector in nude mice bearing human ovarian carcinoma showed promising results (Weedon et al., 2000). One study also demonstrated a synergistic effect when cells expressing both NTR and HSVtk were treated with a combination of CB1954 and GCV (Bridgewater et al., 1995). Furthermore, a significant bystander effect was seen with the NTR/CB1954 approach analogous to the HSVtk/GCV system (Grove et a l , 1999). A very effective NTR vector is an oncolytic adenovirus. The combination of viral oncolysis and NTR expression resulted in significantly greater sensitization of colorectal cancer cells to the prodrug CB1954 in vitro. In vivo, the oncolytic adenoviral vector was shown to replicate in subcutaneous colorectal cancer tumor xenografts in immunodeficient mice, resulting in more NTR expression and greater sensitization to CB1954 than with replicationdefective viruses (Chen et al., 2004). From the four NTR/CB1954 clinical trials underway, none is for brain tumors.

VL

CARBOXYLESTERASE (CE)/CPT^ 11

The enzyme-prodrug combinations described so far illustrate the concept of introducing a viral or bacterial enzyme to provide an activity that is almost absent in mammalian cells, whereas in the carboxylesterases (CE) approach a mammalian enzyme is used. CE converts the prodrug CPT-11 (irinotecan.

7-ethyl-10-[4-(l-piperidino)-l-piperidino] carbonyloxycamptothecin) to its active moiety, SN-38 (see Fig. 6). In this process, CPT-11 undergoes hydrolysis or deesterification to form the active metabolite SN-38, which is 100-1,000 times as potent as CPT-11 as an inhibitor of topoisomerase I (Rothenberg, 1997). In addition, SN38 freely passes though cell membranes, increasing the likelihood for a bystander effect. The activation of CPT-11 in humans has been thought to be mediated by the hepatic (hCE2) and human intestinal (hiCE) CEs. Evidence of in vivo activation of CPT-11 by endogenous human CEs of 2 X 10^^ p u / m l we were limited to a total dose of 3.6 X 10^^ particle units. All clinical trials have many challenging issues related to ethics and design. A particularly difficult decision for this clinical trial design was whether to treat LINCL patients with AAV2cuhCLN2 at the mild or severe phase of the disease. For gene therapy this issue is an ongoing dilemma. Due to the risk of direct surgical administration of vector into the brain, we decided to first treat patients with advanced LINCL for whom the impact of possible adverse events would have less impact on life expectancy. This decision is complicated by the fact that advanced LINCL patients have impairment in several organ systems and therefore there is a chance of serious adverse events possibly related to underlying disease but indistinguishable from effects the drug or surgery. In addition, it may be argued that the potential for benefit of gene transfer diminishes as the neurodegeneration in the brain increases. As a compromise, the trial was designed to start with five subjects with advanced disease (designated Group A) and, pending review by the regulatory

agencies, to proceed to six subjects with moderate disease (designated Group B; Crystal et al., 2004). Administration of AAV2(ûhCLN2 to subjects with advanced LINCL also complicates the choice of safety endpoints. The toxicology studies in rats and nonhuman primates provided no evidence to anticipate likely adverse effects. Therefore, aside from the brain, there is no obvious anatomical site of interest for safety assessments. Moreover, the neurological tests and EEC normally used as sensitive tests of drug safety in normal children provide no insight in LINCL patients with severely degraded neurological function. Therefore, a set of standard medical and hematological assessment parameters were used to assess vector safety with the caveat that their significance is better understood in normal subjects than in children with LINCL. Secondary to safety assessment are the efficacy endpoints, which provide additional challenges. The LINCL clinical rating scale in which the broad loss of brain function is the primary readout provided a useful starting point. This clinical rating scale, modified from an original model by Steinfeld et al. (2002), was constructed to provide an integrated sense of each patient's disease severity. The scale is comprised of three functional categories: motor function, seizures and language, each of which are rated


330

24. GENE THERAPY FOR THE LATE INFANTILE FORM OF BATTEN DISEASE

on a scale of 0-3 allowing for a total CNS disability score between 0 and 9. Patients receiving total disability ratings between 0 and 4 are categorized in the gene transfer protocol as "severe" or Group A, while those who receive total disability scores of 5-6 are classified as "moderate" or Group B. This scale was modified from an original design by Steinfeld et al. (2002), which included a categorization for vision. We eliminated this category for the following reasons: (1) most patients are blind by the time of enrollment in the trial, largely due to the rapid progression of the disease; and (2) the central nervous system vector delivery route selected for this trial proscribes the possibilities for visual improvement in subjects post vector administration, since retinal cells are not targeted for gene transfer. In addition, the Steinfeld scale categorized patients as "severe" if they received total CNS disability scores between 0 and 3. This was broadened in our modification to account for the fact that for most LINCL patients, seizure activity is adequately controlled by anti-convulsants, and subjects are therefore expected to receive seizure scores of " 3 " (indicating no grand mal seizures in the last 3 months). Unfortunately, the modified LINCL clinical rating scale is relatively insensitive as an efficacy parameter. A favorable outcome for this trial might simply be a decrease in the rate of deterioration of this parameter but given variability among subjects and the relative insensitivity of the scale, it may take a long time and multiple subjects to see this impact. Although the rating scale is the most important tool, we also attempted to use magnetic resonance spectroscopy (MRS) to assess the status of the brain in LICNL patients. Previous studies have shown that there are decreases in N-acetylaspartate (NAA) concentration and increase in lactate concentration in the brains of LINCL patients (Brockmann et al., 1996; Jarvela et al., 1997; Seitz et al., 1998; Vanhanen et al., 2004). These assessments were made by the use of single voxel magnetic resonance spectrometers, but the advent of high magnetic field spectrometers has improved the resolution of these metabolites, thus local effects due to gene therapy may be evident from a lack of decrease in NAA concentration by the expected rate/amount. Therefore, the clinical protocol included both pre-surgical MRS spectroscopy as well as repeated assessments at 6 and 18 months post-therapy. A full clinical protocol based on the considerations above was accepted by the local regulatory agencies (Institutional Review Board and Institutional Biosafety Committee). In conjunction with the efficacy data and toxicology data presented above, it was also reviewed

by the National Institutes of Health Recombinant DNA Advisory Committee, who opted for no detailed review and the Food and Drug Administration. Therefore, subject accrual and gene transfer in those enrolled in the trial is ongoing.

X.

CONCLUSION

The program described above for commencing a clinical trial for LINCL by direct administration of AAV2 vector expressing CLN2 to the brain is a typical example of translational research in the academic setting. While missing some of the stringency that may be applied to a commercial entity in the development of a marketed drug, the program still consumed a substantial amount of resources and time. The overall cost is several million dollars and identification of support on the scale needed for such pre-clinical programs is difficult even in the context of the NIH roadmap for translational research. To maximize the return on the effort invested, other neurological lysosomal storage diseases may be subsequently identified that may benefit from a similar approach and the knowledge learned from the manufacturing program and toxicology studies for LINCL could be applied to these other diseases.

ACKNOWLEDGMENTS We thank N. Mohamed for help in preparing this manuscript. These studies were supported, in part, by UOl NS04758, NIH M01RR00047 from The NIH and The Will Rogers Memorial Foundation, Los Angeles, CA, and Nathan's Battle Foundation, Greenwood, IN. References Arruda, V.R., Fields, RA., Milner, R., Wainwright, L., De Miguel, M.R, Donovan, RJ., Herzog, R.W., Nichols, T.C., Biegel, J.A., Razavi, M., Dake, M., Huff, D., Flake, A.W., Couto, L, Kay, M.A. and High, K.A. (2001) Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males. Mol. Ther., 4: 586-592. Arruda, V.R., Schuettrumpf, J., Herzog, R.W., Nichols, T.C., Robinson, N., Lotfi, Y., Mingozzi, R, Xiao, W., Couto, L.B. and High, K.A. (2004) Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1. Blood, 103: 85-92. Birch, D.G. (1999) Retinal degeneration in retinitis pigmentosa and neuronal ceroid lipofuscinosis: an overview. Mol. Genet. Metab., 66: 356-366. Bohn, M.C., Choi-Lundberg, D.L., Davidson, B.L., Leranth, C , Kozlowski, D.A., Smith, J.C, O'Banion, M.K. and Redmond, D.E.


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b-glucuronidase-deficient mice after FIV vector-mediated gene transfer to brain. Mol. Ther., 1: A688. Dawson, G. and Cho, S. (2000) Batten's disease: clues to neuronal protein catabolism in lysosomes. J. Neurosci. Res., 60: 133-140. Dhar, S., Bitting, R.L., Rylova, S.N., Jansen, P.J., Lockhart, E., Koeberl, D.D., Amalfitano, A. and Boustany, R.M. (2002) Flupirtine blocks apoptosis in batten patient lymphoblasts and in human postmitotic CLN3- and CLN2-deficient neurons. Ann. Neurol., 51: 448-466. During, M.J., Kaplitt, M.G., Stern, M.B. and Eidelberg, D. (2001) Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation. Hum. Gene Ther., 12:1589-1591. Elliger, S.S., EUiger, C.A., Aguilar, C.R, Raju, N.R. and Watson, G.L. (1999) Elimination of lysosomal storage in brains of MPS VII mice treated by intrathecal administration of an adeno-associated virus vector. Gene Ther., 6: 1175-1178. Ezaki, J., Tanida, I., Kanehagi, N. and Kominami, E. (1999) A lysosomal proteinase, the late infantile neuronal ceroid lipofuscinosis gene (CLN2) product, is essential for degradation of a hydrophobic protein, the subunit c of ATP synthase. J. Neurochem., 72: 2573-2582. Flotte, T.R. (2004) Immune responses to recombinant adeno-associated virus vectors: putting preclinical findings into perspective. Hum. Gene Ther., 15: 716-717. Frisella, WA., O'Connor, L.H., Vogler, C.A., Roberts, M., Walkley, S., Levy, B, Daly, T.M. and Sands, M.S. (2001) Intracranial injection of recombinant adeno-associated virus improves cognitive function in a murine model of mucopolysaccharidosis type VII. Mol. Ther., 3: 351-358. Gao, G., Lebherz, C , Weiner, D.J., Grant, R., Calcedo, R., McCuUough, B., Bagg, A., Zhang, Y. and Wilson, J.M. (2004) Erythropoietin gene therapy leads to autoimmune anemia in macaques. Blood, 103:3300-3302. Grimm, D., Kern, A., Rittner, K. and Kleinschmidt, J.A. (1998) Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum. Gene Ther., 9: 2745-2760. Goebel, H.H., Mole, S.E. and Lake, B.D. (1999) The Neuronal Ceroid Lipofuscinoses (Batten Disease). lOS Press, Amsterdam. Hackett, N.R. and Crystal, R.G. (2000) Adenovirus vectors for gene therapy. In: Smyth Tempelton N. and Lasic D.D., (Eds.), Gene Therapy. Marcel Dekker, New York, pp. 17-40. Haltia, M. (2003) The neuronal ceroid-lipofuscinoses. J. Neuropathol. Exp. Neurol., 62: 1-13. Harvey, B.-G., Maroni, J., O'Donoghue, K.A., Chu, K.W, Muscat, J.C, Pippo, A.L., Wright, C.E., HoUmarm, C , Wisnivesky, J.R, Kessler, R, Rasmussen, H., Rosengart, T.K. and Crystal, R.G. (2002) Safety of local delivery of low and intermediate dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions. Hum. Gene Ther., 13; 15-63. Haskell, R.E., Hughes, S.M., Chiorini, J.A., Alisky J.M. and Davidson, B.L. (2003) Viral-mediated delivery of the late-infantile neuronal ceroid lipofuscinosis gene, TPP-I to the mouse central nervous system. Gene Ther. 10: 34-42. Herzog, R.W., Fields, PA., Arruda, V.R., Brubaker, J.O., Armstrong, E., McClintock, D., Bellinger, D.A., Couto, L.B., Nichols, T.C. and High, K.A. (2002) Influence of vector dose on factor IX-specific T and B cell responses in muscle-directed gene therapy. Hum. Gene Ther., 13: 1281-1291. loannou, Y.A., Zeidner, K.M., Gordon, R.E. and Desnick, R.J. (2001) Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase. A replacement in enzyme-deficient mice. Am. J. Hum. Genet., 68:14-25.


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Janson, C , McPhee, S., Bilaniuk, L., Haselgrove, J., Testaiuti, M., Freese, A., Wang, D.J., Shera, D., Hurh, P., Rupin, J., Saslow, E., Goldfarb, O., Goldberg, M., Larijani, G., Sharrar, W., Liouterman, L., Camp, A., Kolodny, E., Samulski, J. and Leone, P. (2002) Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum. Gene Then, 13: 1391-1412. Jarvela, I., Autti, T., Lamminranta, S., Aberg, L., Raininko, R. and Santavuori, P. (1997) Clinical and magnetic resonance imaging findings in Batten disease: analysis of the major mutation (1.02kb deletion). Ann. Neurol., 42: 799-802. Kaspar, B.K., Erickson, D., Schaffer, D., Hinh, L., Gage, EH. and Peterson, D.A. (2002) Targeted retrograde gene delivery for neuronal protection. Mol. Then, 5: 50-56. Kho, S.T., Pettis, R.M., Mhatre, A.N. and Lalwani, A.K. (2000) Safety of adeno-associated virus as cochlear gene transfer vector: analysis of distant spread beyond injected cochleae. Mol. Then, 2: 368-373. Kniesel, U. and Wolburg, H. (2000) Tight junctions of the bloodbrain barrien Cell. Mol. NeurobioL, 20: 57-76. Kurachi, Y, Oka, A., Mizuguchi, M., Ohkoshi, Y, Sasaki, M., Itoh, M., Hayashi, M., Goto, Y and Takashima, S. (2000) Rapid immunologic diagnosis of classic late infantile neuronal ceroid lipofuscinosis. Neurology, 54: 1676-1680. Lane, S.C., Jolly, R.D., Schmechel, D.E., Alroy, J. and Boustany, R.M. (1996) Apoptosis as the mechanism of neurodegeneration in Batten's disease. J. Neurochem., 67: 677-683. Lin, L. and Lobel, P. (2001) Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis. Biochem. J., 357: 49-55. Lin, L., Sohar, I., Lackland, H. and Lobel, P (2001) The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH. J. Biol. Chem., 276: 2249-2255. Mastakov, M.Y, Baer, K., Kotin, R.M. and During, M.J. (2002a) Recombinant adeno-associated virus serotypes 2- and 5-mediated gene transfer in the mammalian brain: quantitative analysis of heparin co-infusion. Mol. Then, 5: 371-380. Mastakov, M.Y, Baer, K., Symes, C.W., Leichtlein, C.B., Kotin, R.M. and During, M.J. (2002b) Immunological aspects of recombinant adeno-associated virus delivery to the mammalian brain. J. Virol., 76: 8446-8454. Mole, S.E. (1999) Batten's disease: eight genes and still counting? Lancet, 354: 443-445. Monahan, P.E., Jooss, K. and Sands, M.S. (2002) Safety of adenoassociated virus gene therapy vectors: a current evaluation. Expert Opin. Drug Safety, 1: 79-91. Nguyen, J.B., Sanchez-Pemaute, R., Cunningham, J. and Bankiewicz, K.S. (2001) Convection-enhanced delivery of AAV-2 combined with heparin increases TK gene transfer in the rat brain. Neuroreport, 12: 1961-1964. Niwa, H., Yamamura, K. and Miyazaki, J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108: 193-199. Pachori, A.S., Melo, L.G., Zhang, L., Loda, M., Pratt, R.E. and Dzau, VJ. (2004) Potential for germ line transmission after intramyocardial gene delivery by adeno-associated virus. Biochem. Biophys. Res. Commun., 313: 528-533. Palmer, D.N., Fearnley, I.M., Walker, J.E., Hall, N.A., Lake, B.D., Wolfe, L.S., Haltia, M., Martinus, R.D. and Jolly R.D. (1992) Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease). Am. J. Med. Genet., 42: 561-567. Passini, M.A., Lee, E.B., Heuer, G.G. and Wolfe, J.H. (2002) Distribution of a lysosomal enzyme in the adult brain by axonal transport

and by cells of the rostral migratory stream. J. Neurosci., 22: 6437-6446. Qui, J.P, Mendez, B.S., Crystal, R.G. and Hackett, N.R. (2002) Construction and verification of an Ad/AAV helper plasmid designed for manufacturing recombinant AAV vectors for human administration. Mol. Then, 5: S47-S48. Seitz, D., Grodd, W, Schwab, A., Seeger, U., Klose, U. and Nagele, T. (1998) MR imaging and localized proton MR spectroscopy in late infantile neuronal ceroid lipofuscinosis. Am. J. NeuroradioL, 19: 1373-1377. Sferra, T.J., Qu, G., McNeely D., Rennard, R., Clark, K.R., Lo, W D . and Johnson, PR. (2000) Recombinant adeno-associated virusmediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type VII mouse. Hum. Gene Then, 11: 507-519. Skorupa, A.E, Fisher, K.J., Wilson, J.M., Parente, M.K. and Wolfe, J.H. (1999) Sustained production of beta-glucuronidase from localized sites after AAV vector gene transfer results in widespread distribution of enzyme and reversal of lysosomal storage lesions in a large volume of brain in mucopolysaccharidosis VII mice. Exp. Neurol., 160: 17-27. Sleat, D.E., Donnelly, R.J., Lackland, H., Liu, C.G., Sohar, I., Pullarkat, R.K. and Lobel, P. (1997) Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. Science, 277: 1802-1805. Sleat, D.E., Gin, R.M., Sohar, I., Wisniewski, K., Sklower-Brooks, S., Pullarkat, R.K., Palmer, D.N., Lerner, T.J., Boustany, R.M., Uldall, P, Siakotos, A.N., Donnelly, R.J. and Lobel, P (1999) Mutational analysis of the defective protease in classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorden Am. J. Hum. Genet., 64: 1511-1523. Sleat, D.E., Wiseman, J.A., El Banna, M., Kim, K.H., Mao, Q., Price, S., Macauley, S.L., Sidman, R.L., Shen, M.M., Zhao, Q., Passini, M.A., Davidson, B.L., Stewart, G.R. and Lobel, P (2004) A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration. J. Neurosci., 24: 9117-9126. Smith, J.G., Raper, S.E., Wheeldon, E.B., Hackney, D., Judy, K., Wilson, J.M. and Eck, S.L. (1997) Intracranial administration of adenovirus expressing HSV-TK in combination with ganciclovir produces a dose-dependent, self-limiting inflammatory response. Hum. Gene Then, 8: 943-954. Sondhi, D., Hackett, N.R., Apblett, R.L., Kaminsky, S.M., Pergolizzi, R.G. and Crystal, R.G. (2001) Feasibility of gene therapy for late neuronal ceroid lipofuscinosis. Arch. Neurol., 58:1793-1798. Sondhi, D., Peterson, D.A., Giannaris, E.L., Sanders, C.T., Mendez, B.S., Bishnu, D., Rostkowski, A., Blanchard, B., Bjugstad, K., Sladek, J.R.J., Redmond, D.E., Leopold, PL., Kaminsky, S.M., Hackett, N.R. and Crystal, R.G. (2005) AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL. Gene Then, 12: doi: 10.1038/sj.gt.3302549. Song, S., Scott-Jorgensen, M., Wang, J., Poirier, A., Crawford, J., Campbell-Thompson, M. and Flotte, T.R. (2002) Intramuscular administration of recombinant adeno-associated virus 2 alpha-1 antitrypsin (rAAV-SERPINAl) vectors in a nonhuman primate model: safety and immunologic aspects. Mol. Then, 6: 329-335. Stein, C.S., Ghodsi, A., Derksen, T. and Davidson, B.L. (1999) Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice. J. Virol., 73: 3424-3429.


ACKNOWLEDGMENTS Steinfeld, R., Heim, P., von Gregory, H., Meyer, K., Ullrich, K., Goebel, H.H. and Kohlschutter, A. (2002) Late infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutations. Am. J. Med. Genet., 112: 347-354. Turner, C.T., Hopwood, J.J. and Brooks, D.A. (2000) Enzyme replacement therapy in mucopolysaccharidosis I: altered distribution and targeting of alpha-L-iduronidase in immunized rats. Mol. Genet. Metab., 69: 277-285. Umehara, R, Higuchi, I., Tanaka, K., Niiyama, T., Ezaki, J., Kominami, E. and Osame, M. (1997) Accumulation of mitochondrial ATP S5mthase subunit c in muscle in a patient with neuronal ceroid lipofuscinosis (late infantile form). Acta Neuropathol., 93: 628-632. Vanhanen, S.L., Puranen, J., Autti, T., Raininko, R., Liewendahl, K., Nikkinen, P., Santavuori, P., Suominen, P., Vuori, K. and Hakkinen, A.M. (2004) Neuroradiological findings (MRS, MRI, SPECT) in infantile neuronal ceroid-lipofuscinosis (infantile CLNl) at different stages of the disease. Neuropediatrics, 35: 27-35.

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Williams, R.E., Gottlob, I., Lake, B.D., Goebel, H.H., Winchester, E.G. and Wheeler, R.B., CLN 2. (1999) Classic late infantile NCL. In: Goebel H.H., Mole S.E. and Lake B.D., (Eds.), The Neuronal Ceroid Lipofuscinoses (Batten Disease). lOS Press, Amsterdam, pp. 37-55. Wisniewski, K.E., Kida, E., Golabek, A.A., Kaczmarski, W , Connell, F. and Zhong, N. (2001a) Neuronal ceroid lipofuscinoses: classification and diagnosis. Adv. Genet., 45:1-34. Wisniewski K. and Zhong N. (Eds.) (2001) Batten Disease: Diagnosis, Treatment and Research. Academic Press, New York. Wisniewski, K.E., Zhong, N. and Philippart, M. (2001b) Pheno/genotypic correlations of neuronal ceroid lipofuscinoses. Neurology, 51: 576-581. Zolotukhin, S., Byrne, B.J., Mason, E., Zolotukhin, I., Potter, M., Chesnut, K., Summerford, C , Samulski, R.J. and Muzyczka, N. (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther., 6: 973-985.


C H A P T E R

25 Molecular Imaging of Gene Therapy for Neurogenetic Diseases ]ohn H. Wolfe, Paul D. Acton, Harish Poptani, Charles H. Vite

Abstract: Gene transfer and stem cell transplantation hold great promise for treating neurogenetic diseases. Animal studies rely on post-mortem analysis of experimentally treated brains at intervals after the treatment is started. For human clinical application, it will be necessary to develop non-invasive methods to monitor the function of the transferred gene or stem cells, as well as the therapeutic effects, longitudinally in individual patients. Imaging modalities being developed to monitor gene therapy in the central nervous system include raagnetic resonance imaging and spectroscopy, radioisotope probes, optical methods, and others. Each modality has specific strengths and limitations imposed by the biological properties or processes being monitored, as well as those imposed by the chemistry and physics associated with the instrumentation and the functions being measured. Rapid progress is being made on developing novel reagents, which promises to expand the applicability for imaging modalities for neurogenetic disease studies. Keywords: non-invasive imaging; nuclear magnetic resonance (NMR); magnetic resonance imaging (MRI); magnetic resonance spectroscopy (MRS); diffusion-weighted imaging (DwI); diffusion-weighted spectroscopy (DwS); magnetization transfer imaging (MTI); radiotracer; positron emission tomography (PET); single photon emission computed tomography (SPECT); stem cell tracking; superparamagnetic iron oxide (SPIO) particles; reporter genes; pathologic imaging; optical imaging; bioluminescence; nearinfrared; animal models

L

blood-brain barrier (BBB), and the regional specialization of functions within the CNS. Experiments in mouse models have shown that if the gene is delivered to the target cells it can mediate the needed change. Treating the human brain is, however, a much more formidable challenge because it is about 2000 times larger in a 1-year-old child than in an adult mouse. Studies in some large animal models (cats, dogs, and non-human primates) have shown that it should be possible to scale up treatments to childrens' brains for certain types of diseases. This must be accompanied by the ability to monitor the function of the transferred gene and the therapeutic effects over the course of the disease, in individual patients using non-invasive methods.

G E N E T I C DISEASES OF THE C N S

A large number of single-gene deficiency diseases involve the central nervous system (CNS) and have generally been refractory to treatment (Scriver et a l , 2001). Most genetic diseases involve metabolic deficiencies, which typically affect cells throughout the brain, thus they require global correction. Effective treatment will need either widespread delivery of gene transfer vectors, widespread dissemination of the therapeutic gene product, or a combination. Gene therapy approaches for the brain require special methods due to the physical inaccessibility imposed by the skull, the isolation from the circulation by the


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25. MOLECULAR IMAGING OF GENE THERAPY FOR NEUROGENETIC DISEASES

IL

IMAGING MODALITIES

A number of imaging modalities are being used and developed for monitoring gene therapy in the CNS, including nuclear magnetic resonance (NMR), positron emission tomography (PET), and optical methods. Each modality has specific strengths and limitations imposed by the biological properties or processes being monitored, as well as imposed by the chemistry and physics associated with each method (also see the following recent reviews, Gelovani, Tjuvajev and Blasberg, 2003; Massoud and Gambhir, 2003; Herschman, 2004; Min and Gambhir, 2004). Imaging modalities are generally used in three ways: to monitor activity of the gene transfer vector; to track transplanted cells; or to assess progression or resolution of neuropathology. The detection method must be sensitive enough to distinguish between normal and diseased brain, a n d / o r to identify transferred cells or gene activity against a background of endogenous tissue. The most advanced imaging methods for detecting reporter gene activity at the present time use radioisotope tracers. PET and the closely related single photon emission computed tomography (SPECT) measure the three-dimensional distribution of a radioactively labeled tracer in vivo. The main advantage of radioisotope detection is its high sensitivity, which can measure nanomolar (

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