Remyelination: Cellular and gene therapy

Remyelination: Cellular and Gene Therapy Lori L. Billinghurst, Rosanne M. Taylor, and Evan Y. Snyder Dysfunctional myelination or oligodendroglial abnormalities play a prominent role in a vast array of pediatric neurological diseases of genetic, inflammatory, immunological, traumatic, ischemic, developmental, metabolic, and infectious causes. Recent advances in glial cell biology have suggested that effective remyelination strategies may, indeed, be feasible. Evidence for myelin repair is accumulating in various experimental models of dysmyelinating and demyelinating disease. Attempts at remyelination have either been directed towards creating myelin de novo from exogenous sources of myelin-elaborating cells or promoting an intrinsic spontaneous remyelinating process. Ultimately, some disorders of myelin may require multiple repair strategies, not only the replacement of dysfunctional cells (oligodendroglia) but also the delivery or supplementation of gene products (ie, growth factors, immune modulators, metabolic enzymes). Although primary oligodendrocytes or oligodendroglial precursors may be effective for glial cell replacement in certain discrete regions and circumstances and although various genetic vectors may be effective for the delivery of therapeutic molecules, multipotent neural stem cells may be most ideally suited for both gene transfer and cell replacement on transplantation into multiple regions of the central nervous system under a wide range of pathological conditions. We propose that, by virtue of their inherent biological properties, neural stem cells possess the multifaceted therapeutic capabilities that many diseases characterized by myelin dysfunction in the pediatric population may demand. Copyright 9 1998 b y W.B. Saunders Company

LTHOUGH MULTIPLE sclerosis (MS) is classically regarded as the most prevalent white matter disease, many other neurological conditions, including a great number effecting the pediatric population, are also characterized by disorders of myelination and of oligodendrocytes, the neuroglial cell type that elaborates myelin. These include disorders of traumatic, ischemic, genetic, developmental, metabolic, immunological, infectious, and inflammatory origin. 1,2 Therapies directed towards promoting remyelination and oligodendrocyte replacement must target a broad range of primary developmental disorders of myelin formation ("dysmyelination") as well as defects in the preservation and maintenance of already formed myelin ("demyelination"). To use an overly simplistic distinction, dysmyelination typically is of genetic origin; demyelination typically denotes an acquired pathology. For example, Pelizaeus-Merzbacher disease is an example of dysmyelination. Demyelination and oligodendrocyte cell death are consequences of such acquired problems as head and spinal cord trauma, underlie a significant portion of the pathology following perinatal asphyxia, and may play a role in various infections, including those that are retroviral-mediated. Of course, certain processes probably straddle this distinction, for example, the leukodystrophies and other neurodegenerative processes. And some "classical" white matter diseases involve "gray matter" as well, for example, MS, some leukodystrophies, such as adrenoleukodystrophy (ADL), some inborn metabolic errors, such as [3-mannosidosis. Present-day approaches to remyelination fall

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into two classes: (1) promotion or stimulation of spontaneous, endogenous myelin repair mechanisms and (2) creation of myelin repair de novo. We review both classes of therapy in various animal models of dysmyelinating and demyelinating disease. Furthermore, we will highlight one promising avenue of treatment that uses the transplantation of exogenous multipotent neural stem cells (NSCs) into the myelin-disordered central nervous system (CNS), particularly in situations where the diseases are extensive, multifocal, or even global. The inherent biological properties of these cells make them ideally suited for neural cell replacement (oligodendrocytes as well as neurons) and gene transfer--both of which are important strategies for remyelination and CNS repair, particularly in the range of diseases that affect the pediatric population. NSC transplantation (as well as the mobilization and recruitment of endogenous NSCs) may provide valuable, novel therapeutic options for the treatment of white matter disease, including not

From the Department of Neurology, Division of Neuroscience, and the Department of Pediatrics, Division of Newborn Medicine, Harvard Medical School, Children's Hospital, Boston, MA; and the Department of Animal Science, Faculty of Veterinary Science, University of Sydney, New South Wales, Australia. Address reprint requests to Evan Y. Snyder, MD, PhD, Departments of Neurology (Division of Neuroscience) and Pediatrics (Division of Newborn Medicine), Harvard Medical School & Children 's Hospital, Boston, 300 LongwoodAve, 248 John F. Enders Pediatric Research Laboratories, Boston, MA 02115. Copyright 9 1998 by W.B. Saunders Company 1071-9091/98/0503-000758.00/0

Seminars in Pediatric Neurology, Vol 5, No 3 (September), 1998: pp 211-228

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only MS but also those resulting from inborn errors of metabolism; brain and spinal cord injury; hypoxic-ischemic damage; and other inflammatory, immune, and infectious insults to CNS. EXPERIMENTAL MODELS OF MYELIN DISORDERS The myelin sheath is a specialized membranous outgrowth of oligodendrocytes in the CNS and of Schwann cells in the peripheral nervous system (PNS). It has a compact periodic structure and is enriched with both protein and lipid. Functioning as an axon insulator, myelin permits fast saltatory conduction and improves metabolic efficiency. Damage to the myelin sheath, therefore, results in a decrease in conduction velocity. It may well also play a role in insuring the health and survival of neuronal fibers and the neurons from which they emanate. Myelination of the CNS is a complex process; requiring that oligodendrocytes efficiently colonize the brain, differentiate, and express a variety of specific genes necessary for myelin synthesis, alignment, and compaction. Animal models, many of them naturally occurring mutants, have been useful for understanding and addressing both dysmyelinating and demyelinating diseases. Animals with inherited myelin disorders (dysmyelination) express a generalized failure of myelination, sometimes following a stereotypic progression. In severely affected mutants, the life span of the animal may be limited, thus precluding long-term studies. However, because these models have dysmyelinated regions that are diffusely distributed throughout the CNS, they are ideal for testing remyelinating strategies for widespread disease through the global repletion of oligodendrocytes or gene products. For example, the homozygote shiverer (shi) mouse suffers from extensive, CNS-wide white matter disease, and serves as an excellent experimental model of human dysmyelinating conditions. The shi phenotype results from a deletion of 5 out of 7 exons comprising the gene encoding myelin basic protein (MBP), which is essential for proper myelination by oligodendroglia. 3-7 Therapy for this cell autonomous defect, therefore, requires widespread replacement with MBP-expressing oligodendrocytes. The myelin deficient (md) rat and thejimpy mouse are dysmyelinated mutants with a defect in proteolipid protein (PLP), a gene product that plays a role in maintaining oligodendrocyte health and function. Whether

toxicity in these mutations is purely intrinsic or extrinsic to the oligodendrocyte is still an active area of investigation. Certain inborn errors of metabolism are characterized by mutations in lysosomal enzymes in which demyelination or death of oligodendrocytes (sometimes due to the accumulation of toxic metabolites) is a prominent neuropathological manifestation (eg, the twitcher mutant mouse model of Krabbe's disease [galactocerebrosidase deficiency] or the caprine, bovine, and canine models of [3-mannosidosis [[3-mannosidase deficiency]). CNS demyelination can be experimentally induced by one of several ways. One approach is to use either an autoimmune (eg, experimental autoimmune encephalomyelitis [EAE] 8) or virus-induced (eg, corona virus9; Theiler's murine encephalomyelitis (TMEV) 1~ model of demyelination. EAE is a T-cell-mediated inflammatory model of CNS demyelination induced by immunizing animals with autologous CNS tissue, purified myelin antigens, or encephalitogenic myelin peptides. H Classically, it has been used to appraise therapies that prevent or suppress the progression of autoimmune disease, rather than examine myelin repair. On the other hand, animals infected with TMEV display a chronic progressive immune-mediated disease that is similar both pathologically and clinically to MS. 12As such, this model is useful for identifying agents that promote remyelination. Another approach is to create focal areas of demyelination by the injection of myelinotoxic chemicals, such as lysolecithin, 13 ethidium bromide, 14 and Cuprizone. 13 As these demyelinated lesions are usually spontaneously remyelinated by host oligodendrocytes or Schwann cells, self-repair is inhibited by prior focal irradiation that kills the glial cell population at the experimental site. One disadvantage to this technique is that other tissue components may be damaged by the irradiation, including endogenous glia and endothelial cells. However, the reproducibility of the method and the ability to control the environment are characteristics useful for remyelination studies. THERAPEUTIC APPROACHES TO REMYELINATION

A variety of therapeutic approaches have been devised to promote myelin repair in dysmyelinating and demyelinating diseases. Considerable effort has been directed toward creating myelin repair de novo, using cells of both CNS and PNS origin to

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remyelinate lesions present in various animal models. Grafts of exogenous Schwann cells, cells of the oligodendrocyte lineage (both mature and immature), cell lines (both naturally occurring and experimentally created), and NSCs have been effective following transplantation into dysmyelinated and demyelinated lesions. Other studies have exploited endogenous myelin repair mechanisms. For example, it has recently been reported that immature, cycling cells endogenous to the subcortical white matter of adult rats can respond to a focal lysolecithin-induced demyelinating lesion by differentiating into myelinating oligodendrocytes engaging in apparent repair of the lesion. 15 Ultimately, it may be necessary to combine a number of therapeutic approaches--for example, supplementing natural endogenous repair processes with exogenous cells and trophic factors--interceding along several interrelated pathways that might halt myelin destruction or promote myelin repair.

Promoting Endogenous Remyelination Although demyelination was once thought to be irreversible, spontaneous CNS remyelination was demonstrated in 1961.16 Over the subsequent 37 years, many studies have reported remyelination after experimentally-induced toxic, 14-17 inflammatory,8 and viral9 myelin injury. Importantly, histopathological and ultrastructural studies have confirmed that remyelination occurs, as well, in the adult human CNS affected by MS. 18-2~ These observations provide hope for therapies directed towards promotion of this spontaneous remyelinating process. In fact, recent studies have suggested that oligodendrocyte precursor cells persist locally even in chronic stages of MS; they are not destroyed but rather appear simply limited in their ability to proliferate and differentiate. Identification of ways of more efficiently stimulating this endogenous oligodendrocyte precursor pool may represent an effective method for re-expanding the oligodendrocyte population and for promoting more efficient myelin repair in MS. 21 Providing such gene products (when identified)--perhaps through viral- or cell-mediated gene transfer techniques-may set the stage for gene therapy approaches for the generation of remyelinating cells.

Growth Factors Growth factors are expressed during recovery from several kinds of CNS injury, including cere-

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bral ischemia, 22 spinal cord (SC) injury,23 and demyelination.24 Many in vitro experiments have shown that growth factors, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), insulinlike growth factor-1 (IGF-1), and ciliary neurotrophic factor (CNTF) promote the proliferation, differentiation, and survival of cells in the oligodendroglial lineage. 25 Furthermore, IGF-1 treatment has been shown to reduce lesion severity and promote myelin regeneration in the EAE model of demyelinating disease. 26,27 In addition to upregulating expression of MBP, IGF-1 reduces some of the blood-brain barrier (BBB) defects and inflammatory responses in this model. Although in vitro observations suggest that PDGF, bFGF, and CNTF may similarly effect immune responses and myelin regeneration, 25 studies have not yet directly examined their ability to stimulate remyelination in vivo.

Immunoglobins Recent work suggests that manipulating the immune system may also be beneficial for myelin repair. For instance, in the TMEV (immune) model of demyelination, CNS remyelination can be promoted via global immunosuppression 28or by stimulation of the immune system by passive transfer of immunoglobins. 1~ The favorable effect of immunoglobins is not limited to the TMEV model; another study has demonstrated that intravenous polyclonal human IgG (IVIG) inhibits the active induction of EAE. 3~ In another model of EAE, 31 which was induced by injection into mice of a T-cell clone directed against a specific epitope of MBP, both the inflammatory infiltrates and the animal's paralysis regressed following subsequent intraperitoneal treatment with an altered peptide that was analogous to the MBP epitope and that, as a partial agonist, silenced the pathogenic T cells. Such treatment resulted in a reduction in tumor necrosis factor-a (TNF-~) (the cytokine critical for recruiting inflammatory cells and T cells with a variety of specificities to the lesion site) and an increase in interleukin 4 (IL-4) (associated with recovery from inflammatory demyelinating diseases, including probably MS). In fact, the presence of IL-4 (a potent inhibitor of TNF-a) was a sina qua non for the therapeutic efficacy of the peptide analogue. That this approach may have general applicability comes from the observation that a similar rescue was

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observed in cases of EAE initiated by T cells specific to epitopes of PLP when an altered peptide analogous to that epitope was administered. This study provides insight into a number of molecular "handles" for future immune modulation that might lead to myelin repair, including some that might be amenable to gene-based therapy. Spontaneous remyelination can be promoted in the lysolecithin (toxin) model of demyelination by a naturally occurring autoantibody (mAB SCH 94.03). 32 This study suggests that the beneficial effects of immunosuppression and immune stimulation are not restricted to immune models of demyelinating disease, and that immune modulation may also have a role in remyelinating therapies directed towards a broader range of neurological conditions characterized by demyelination. Two placebo-controlled, double-blind clinical trials examining the effects of IVIG therapy in MS patients are presently underway. 33 Their purpose is to determine whether the administration of IVIG is able to reverse the motor and visual deficits characteristic of MS, resulting in clinical improvement. Trials such as these will provide new insight into both the pathogenesis of demyelinating disease as well as myelin repair mechanisms intrinsic to the nervous system.

Gene Therapy Ideally, one would want a source within the brain or spinal cord of therapeutic genes (perhaps encoding some of the factors or immunomodulators discussed earlier) that could forestall damage to myelin promote its self-repair. The idea of transferring therapeutic genes (or their end metabolic products) to the CNS has attracted considerable recent attention. Many methods of gene transfer are under study.34 The delivery of genes directly into a host's own neural cells in situ--typically via genetically altered viruses--has been hampered by an inability to achieve sustained, regulated expression efficiently, safely, selectively in the specific cell types and regions most in need of correction. Although progress is being made in targeting post-mitotic neural tissue with viral vectors (eg, lentivirus, adenovirus, adeno-associated virus, herpes virus), even these may not address the widespread, extensive lesions characteristic of many neurodegenerative diseases, particularly those of genetic, perinatal, metabolic, inflammatory, infectious, or traumatic origin. Furthermore, such strate-

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gies depend on relaying new genetic information through established endogenous neural populations and circuits, which in fact, may have degenerated or failed to develop. Gene therapy has been attempted for certain underlying diseases that are characterized by a variety of visceral and neurological manifestations, including white matter degeneration and mental retardation. Typically, these have been inherited errors of metabolism (eg, the leukodystrophies, such as ADL, metachromatic leukodystrophy, Krabbe's disease). The approach has typically been either by pharmacological infusion of enzymes systemically or by somatic cell-mediated gene transfer, usually hematopoietic cells, administered via bone marrow transplantation (BMT). 35-41 As discussed later (w and w although these approaches have often been successful for treating the peripheral and visceral manifestations of these various diseases, they have generally made little, if any, impact on the CNS manifestations, including disordered white matter. However, recent encouraging data have just been reported following peripheral allogeneic hematopoietic stem cell transplantation in four patients with the late-onset, more slowly progressive form of globoid-cell leukodystropy (Krabbe's disease), a form whose tempo of progression due to slightly higher residual levels of galactocerebrosidase allows intervention via the BMT route of enzyme supplementation. 42 In these patients, CNS deterioration appeared to be reversed or arrested with a decrease in white matter signal intensity on T2 magnetic resonance imaging and an improvement in cerebrospinal fluid total protein levels in 3 of the 4. Some progress may also result from an ongoing gene therapy trial directed towards treating some of the CNS manifestation of children with Canavan's disease, an autosomal-recessive leukodystrophy characterized by mutations in the aspartoacylase (ASPA) gene resulting in elevated levels of N-acetylaspartate (NAA). A novel, nonviral vector termed " L P D " (liposome-encapsulated, polymer-condensed DNA complex) is used to transfer a plasmid containing human ASPA cDNA directly to the brains of rats and primates and now a few children following disruption of the BBB with mannitol and intraventricular administration of the vector.43 (LPD is a mixture of the neutral lipid DOPE with a cationic liposome, DC-Chol, together with poly-Llysine to form virus-sized, nonaggregating par-

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ticles). In the two children studied to date, there was a suggestion of improvement in psychometric testing and neurological functioning at 6 weeks post-gene transfer with NAA levels within or close to the normal range in several brain regions (including frontal cortex, parietal white matter, and the anterior occipital lobe) on NMR proton spectroscopy. Whether myelination has been improved and long-term neurological gains are achieved will require further evaluation. Eight additional children have received the vector, though their outcome remains to be studied. To date, there have been no specific gene transfer approaches using noncellular vectors directed specifically toward repairing or replacing myelin. However, the use of neural stem cells (NSCs), as detailed below, has begun, indirectly, to approach this issue and provide some novel gene therapeutic approaches that might be relevant to remyelination. Although even the most effective viral or liposome or hematopoietic cell vector, used in isolation, will likely face the difficulty discussed previously of forestalling damage but being unable to restore lost neural elements, if used in combination with neural cell-mediated techniques (eg, NSCs, as discussed later), genuine permanent gains in even aggressive diseases might be possible.

Remyelination By Transplantation Transplantation of cells into the CNS of human patients with neurodegenerative disorders offers an alternative approach to previously incurable diseases. The safety and survival of grafts of fetal neural tissue transplanted into the brains of patients with Parkinson's disease has provided encouragement that glial cell transplantation may prove useful for human dysmyelinating and demyelinating conditions. Though not yet attempted in patients, both inherited and acquired disorders of myelin might theoretically be treated by cell transplantation; transplanted cells could be used either to replace damaged or degenerated glia or to modify the endogenous glial population of the host. Indeed, in a number of experimental protocols using a wide variety of donor cells and recipients, there is increasing evidence that transplantation may lead to persistent remyelination of large areas of the CNS with focal functional repair. 44 The logical candidates for graft material are Schwann cells and cells of the oligodendrocyte lineage; both have been capable of myelinating axons on trans-

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plantation into pathological environments, including focal areas of demyelination in the CNS of myelin mutants and toxin-induced lesions.

Cells From the Oligodendroglial Lineage Cells from all stages of the oligodendrocyte lineage have been used in transplantation experiments, from immature bipotential progenitors ("O-2A progenitors") to mature differentiated oligodendrocytes. The O-2A progenitor is defined by its ability to differentiate into oligodendrocytes as well as into fibrous (type 2) astrocytes. These immature cells exhibit plasticity and possess the capacity to proliferate and migrate locally on transplantation--characteristics necessary for effective repair of the CNS.44-52As such, they are the cell of the oligodendrocyte lineage, which has become favored for glial cell transplantation. Recent reports have outlined the promising remyelinating capacity of these cells. 45,49"52 Glial cell lines can also be used for transplantation. In such an approach, cells from various stages in the development of an oligodendrocyte--from most primordial to most mature have been expanded or perpetuated by growth factors or by genes that interact with cell cycle proteins. 44 Cell lines offer the advantage of an unlimited supply of well-characterized, well-tested, more-or-less homogeneous donor cells for transplantation. However, results from studies using these techniques have been variable. Some studies suggest that such cell lines may actually produce little myelin49 (possibly representing the propagation of less mature cells in the oligodendrocyte lineage). Other studies suggest that the cell lines yielding the most differentiated donor-derived oligodendrocytes migrate minimally from the injection site (a limitation for the treatment of extensive or multifocal demyelinated lesions), whereas cell lines generating the most migratory cells prove unfortunately to be the least differentiated along the oligodendroglial lineage. 53 As might be anticipated, expansion of the O-2A progenitor cell--both experimentally via epigenetic and genetic means 45,49and as a spontaneously emerging cell line (known as the "CG4" cell lineS~ shown some therapeutic promise, at least when applied to relatively focal demyelinated lesions in the SC. Epidermal growth factor (EGF) has also been used to expand mixed populations of cells from the murine brain--presumably of oligodendrocyte lineage based on the available data

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(probably also O-2A cells)--that appear capable of local myelin formation following transplantation into focal regions of demyelination in the md rat SO. 54

Schwann Cells Schwann cells have proven to be a viable alternative to the use of oligodendrocytes for transplantation. Schwann cells have been found to migrate spontaneously into the CNS and ensheathe axons with PNS myelin in MS 55 as well as in animal models, such as EAE, 56TVI, 57 and lysolecithin-induced demyelination. 58Endogenous Schwann cell remyelination in MS has been shown to restore central conduction 59 and can also restore normal conduction properties to the demyelinated rodent SC. 6~An advantage to using human Schwann cells is that they are considerably more accessible than human oligodendrocytes: they can be cultured from adult peripheral nerve biopsies and then purified and exlSanded to generate large populations. 61,62 Autologous Schwann cell harvesting, expansion, and transplantation in patients with demyelinating diseases could offer a considerable advantage with respect to availability and would avoid the immunosuppression that would necessarily accompany allografts. However, before they can be used clinically, evidence is needed that Schwann cells do not form tumors in vivo, a hazard consistently described when rodent Schwann cells expanded and immortalized by growth factors are transplanted. 63 This could be an obvious impediment to the clinical application of Schwann cell transplants.

Neural Stem Cells NSCs are postulated to be immature, uncommitted cells that exist in the developing and even adult nervous system and are responsible for giving rise to the vast array of more specialized cells of the mature CNS. They are operationally defined by their ability to self-renew and to differentiate into cells of most (if not all) neuronal and glial lineages, and to populate developing or degenerating CNS regions. An unambiguous demonstration of clonality is imperative to the definition. 64-77With the first recognition that NSCs, propagated in culture, could be reimplanted into mammalian brain where they could reintegrate appropriately and stably express foreign genes, 78,79 gene therapists and restorative neurobiologists began to speculate how such a phenomenon might be harnessed for therapeutic

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advantage. These, and the studies that they spawned 66,68,73,75-77provided hope that the use of N S C s - - b y virtue of their inherent biology--might sidestep some of the limitations of presently available graft material and gene transfer vehicles (eg, non-neural cells, viral vectors), and make feasible a variety of novel therapeutic strategies, including against white matter disease (Table 1). NSC clones have been maintained in a proliferative state by several equally effective and safe strategies: through manipulation of "internal commands" (eg, transduction of genes that interact in a self-regulated manner with cell cycle proteins) or through such epigenetic means as chronic exposure to mitogens or co-culture on various cellular membrane substrates. 64-85 Such manipulations do not subvert the ability of NSCs to respond to normal microenvironmental cues--to withdraw from the cell cycle, interact with host cells, differentiate. This point has been nicely illustrated by the prototypical NSC clone C17.2, a clone with which we have had a great deal of experience. 78,86-99 When transplanted into various germinal zones throughout the brain, these cells participate in the normal development of multiple regions at multiple stages (from fetus to adult) along the murine neuraxis, differentiating appropriately into diverse neuronal and glial cell types. They intermingle nondisruptively with endogenous neural progenitor/stem cells, responding to the same spatial and temporal cues in a similar manner: donor-derived neurons receive appropriate synapses and possess appropriate ion channels; the BBB remains intact where donorderived astroglia put foot processes onto cerebral vasculature; donor-derived oligodendroglia express MBP and myelinate neuronal processes. Crucial for therapeutic considerations, the structures to which they contribute develop normally. Thus, their use as graft material can be considered almost analogous to hematopoietic stem-cellmediated reconstitution. Indeed, in their earliest use as a therapeutic tool (discussed later), they delivered a missing gene product throughout the brain of a mouse in which the gene was mutated in all cells, cross-correcting host neurons and glia by creating virtually chimeric regions of brain. As one of the best studied of NSC clones in a number of animal models of neurologic disease, C17.2 cells have been useful for illustrating the range of therapeutic possibilities of NSCS for gene therapy and repair. On transplantation in these

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REMYELINATION: CELLULAR AND GENE THERAPY Table 1. Properties of Neural Stem Cells That Make Them Appealing Vehicles for CNS Gene Therapy and Repair

Genetic manipulability Progenitor/stem cells easily transduced ex vivo by most viral and nonviral gene transfer methods Facile engraftability following simple implantation procedures From engraftment in germinal zones (as well as into parenchyma), can broach blood-brain barrier unimpeded; no requirement for conditioning regimens (eg, irradiation as in BMT or opening of blood-brain barrier) Sustained foreign (therapeutic) gene expression Throughout CNS, from fetus to adult, following technically simple and safe reimplantation procedures; CNS levels rise immediately Potential for normal reintegration into host cytoarchitecture and circuitry Differentiate along all CNS cell-type lineages; important for diseases in which neurons and glia both affected (eg, asphyxia, trauma, multiple sclerosis); not only allows direct, stable, and perhaps regulated delivery of therapeutic molecules, but also enables replacement of range of dysfunctional neural cells and possible reconstruction of connections and networks. Ability to migrate Particularly within germinal zones, enabling replacement of genes and cells to be directed not only to discrete sites but to widely disseminated lesions as well for diseases of a more global nature; for more focal implants, ability of cells to intermingle with host cells rather than clump at injection track insures homogeneous distribution of therapeutic molecules throughout target tissue Plasticity Ability to accommodate to region of engraftment and assume array of phenotypes; obviates necessity for obtaining donor cells from many specific CNS regions, or imperative for precise targeting of donor cells during reimplantation, or need for tissue-specific promoters for foreign gene expression Compensatory of transgene nonexpression Low levels of normal neural products expressed intrinsically by progenitor/stem cells (lysosomal enzymes; neurotrophic, matrix, adhesion and homeodomain molecules; myelin) helps safeguard against transgene inactivation; neural cells may sustain expression of foreign neural genes longer than non-neural vehicles; ability to integrate multiple copies of a transgene into its genome (eg, following repeated sequential retroviral infection) helps thwart loss of expression; may also provide as-yet-unrecognized beneficial neural-specific substances One stem cell may carry multiple transgenes Following multiple transfection events, one cell can transfer multiple gene products simultaneously Minimization of side-effects Distribution of gene products restricted to CNS; although proteins may be disseminated by stem cells throughout brain for diseases of global nature, by altering mode of administration, cells can be selectively integrated in proximity to neurons that require given factor without affecting cells for which the molecule might be problematic; conditioning regimes not required before transplantation as in BMT Ability to serve as producer cells for the in vivo dissemination of viral vectors May help amplify distribution of virus-mediated genes to large CNS regions and numbers of cells Tropism for and trophism within regions of CNS degeneration When confronted with neurodegenerative environments, stem cells alter their migration and differentiation patterns towards replacement of dying cells; probably a vestigial developmental strategy with therapeutic value Immunotolerance In rodent transplant studies, multiple recipients and mouse strains can integrate the same murine stem cell clone without immunosuppression, suggesting a need for generating very few effective clones (one clone used by many)

various neurodegenerative conditions, they (and presumably most NSCs) are successful in replacing degenerated or dysfunctional neural cells and in expressing therapeutic gene products that are either intrinsic to the NSC or are the result of ex vivo engineering or both). 86-98

Remyelination Via CeU and Gene Replacement. NSCs, in many ways, have helped to broaden the paradigmatic scope of transplantation and gene therapy in the CNS. Traditionally, these approaches have been reserved for neuropathologies that are quite localized anatomically (eg, Parkinson's disease), grafts and vectors (viral or cellular) directed

to discrete neural regions (eg, lesioned striatum, hippocampus, septum, and spinal cord). 2A~176 However, most neurodegenerative diseases (particularly those of genetic, infectious, immunological, metabolic, traumatic, or ischemic etiology that seem to affect white matter so prominently) are characterized not by focal but rather by large, extensive, multifocal, or even global neuropathology. These are therapeutic challenges conventionally regarded as beyond the purview of neural transplantation or gene therapy and consigned (albeit frequently ineffectively) to pharmacological interventions or to BMT. However, the uniqueness of NSCs as a

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vehicle for gene transfer and their potential for providing treatment options for heretofore refractory CNS diseases was actually enunciated with its very first use in an experimental therapeutic paradigm 86 in which its biological attributes were harnessed to address genetic mouse models of neurodegenerafion requiring disseminated and extensive enzyme or cell replacement. For the delivery of therapeutic molecules, the feasibility of a NSC-based strategy was first affirmed by correcting the widespread neuropathology of a murine model of the genetic neurodegenerative lysosomal storage disease mucopolysaccharidosis type VII (MPS VII) caused by an inherited deletion mutation of the [3-glucuronidase (GUSB) gene, a condition that causes mental retardation in humans. Exploiting their ability to engraft diffusely and become integral members of structures throughout the host CNS, retrovirally engineered GUSBoverexlSressing NSCs (clone 17.2) were implanted at birth (using a simple, rapid intracerebroventricular implantation technique developed for readily integrating donor cells into germinal zones). The NSCs permanently corrected lysosomal storage in neurons and glia throughout the brains of mutants devoid of that secreted enzyme. 86 This approach is being extended to other untreatable neurodegenerative diseases characterized by absence of discrete gene products or accumulation of toxic metabolites, for example, the gangliosidoses, 8vmannosidosis, 9~ Gaucher's disease, and Neimann-Pick disease. It was recognized that, although the ability of NSCs to engraft diffusely was exploited for widespread delivery of a lysosomal enzyme, a similar tact could be used for dissemination of other diffusible (eg, synthetic enzymes, neurotrophins, viral vectors) and nondiffusible (eg, myelin, extracellular matrix) therapeutic factors, as well as for the distribution of "replacement" neural cells. Many neurological diseases, even those that can be ameliorated by replacement of a gene product, are characterized by the degeneration of neural cells or circuits. These structures may need to be replaced in a functional manner and be resistant to residual toxic processes. Mutants characterized by CNSwide white matter disease provide an ideal model for testing hypotheses that NSCs might also be useful in neuropathologies requiring widespread

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(indeed, almost "global") replacement of degenerated or dysfunctional neural cells. As previously noted [w the oligodendroglia of the dysmyelinated shi mouse are dysfunctional because they lack MBP essential for effective myelination. 3-7 Therapy for this cell-intrinsic defect, therefore, requires widespread replacement with oligodendrocytes encoding and expressing a full-length, functional MBP gene. (In a sense both replacement of an abnormal neural cell type and of a dysfunctional gene are entailed). The shi cellular and behavioral phenotype can be entirely rescued genetically by introducing the wild type MBP gene into the germline of shi m i c e . 4 Although it suggests that gene therapy might be efficacious in a dysmyelinating disorder of this type, this approach is certainly not applicable to human therapies. By cellular methods, the shi phenotype has been treated in discrete regions by injecting a fragment of primary CNS tissue containing normal, mature, MBP-expressing oligodendrocytes6 in much the same way as other discrete demyelinated regions have been addressed by cells of the oligodendrocyte lineage as mentioned previously [w However, this approach will not correct the large expanses of CNS demanded not only by shi but also by such human myelin disorders as MS and the leukodystrophies. NSCs are capable of differentiating into oligodendrocytes and of expressing MBP both in vitro and in vivo. 78,86,87,94Furthermore, their integration into germinal zones of fetuses and newborns 86,87 insures their distribution throughout a mutant's affected brain, an important requirement for the treatment of widespread white matter diseases. Therefore, NSCs (clone C17.2) were transplanted at birth, using the same intracerebroventricular implantation technique devised for diffuse engraftment of enzyme-expressing NSCs to treat global metabolic lesions, s6,s7 The result was widespread engraftment throughout the MBP-deficient shi brain at maturity (including within white tracts) with repletion of significant amounts of wholebrain MBP on immunoelectrophoretic and immunocytochemical analysis. 8s Accordingly, of the many donor cells that differentiated into oligodendroglia (based on morphologic, ultrastructural, and immunocytochemical criteria), a subgroup myelinated up to 40% of host neuronal processes with healthier, better compacted myelin that now possessed major dense lines (a sign of good compaction), had a

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Fig 1.

Neural stem cells (NSCs) may help in the Twitcher In Krebbe's disease and the twi mouse that models it, oligodendrocytes and myelin degenerate owing to an inherited lack of ~-galactocerebroside, a major component of myelin. In very preliminary studies, Taylor and Snyder transplanted NSCs (clone C17.2) into a major germinal zone of twitcher mouse mutants at birth. At maturity, the animals were analyzed ultrastructurelly for remyelination. The histochemical reaction product of the cells' lacz reporter gene creates an electron dense precipitate, which enables identification of donor-derived cells. The NSCs appeared to differentiate into remyelinating replacement oligodendrocytes throughout the twi brain. This electron micrograph shows an overview of an engrafted region of a twi brain demonstrating an enormous number of host axons enwrapped by donor-derived, well-compacted, thick myelin wraps (arrowheads). An ollgodendrocyte derived from a donor NSC (labeled by the dark X-gal histochemical precipitate) is clearly seen, along with its membranous process extending along many axons (arrows). A single ollgodendrocyte could myelinate up to 50 host axons. Such findings were present throughout the engrafted twibrain.

(twi) mutant mouse model of Krabbe's Disease.

periodicity (regularity of myelin wraps) closer to normal, and was of a thickness that approximated normal myelin's. In fact, there was even a suggestion that these multipotent NSCs shifted their differentiation fate towards the oligodendroglial lineage to compensate for the dearth in functional oligodendrocytes. In some recipient animals (60% of those tested), the symptomatic tremor decreased. Therefore, "global" cell replacement seems feasible for some pathologies if cells with stemlike features are used. Specifically, NSCs appear efficacious for the widespread reversal of shi neuropathology and perhaps that of other dysmyelinating and demyelinating conditions.

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This approach is being extended to other poorly myelinated mutants both at birth and after the onset of disease. For example, in preliminary studies with Taylor and Snyder, extensive engraftment and myelin repair adjacent to transplant-derived cells has been achieved in brains of the twitcher mouse model of the virulent infantile-onset form of globoid cell leukodystrophy by again employing the paradigms described for the MPS VII and shi mice above (Fig 1).89 The twitcher mouse, like humans, is characterized by a spontaneous mutation of [3-galactocerebrosidase, a lysosomal enzyme required to degrade galactocerebroside, which makes up 12% of myelin. 9-41Undegraded galactocerebroside is converted to the toxic intermediate psychosine, which directly damages oligodendrocytes and causes progressive demyelination and neurologic deficits throughout the neural axis resulting in early death. (BMT in this mutant has made little impact on the progression of neurological symptoms or short life span of these mice. 9-4j) This ability of NSCs not only to supply therapeutic molecules immediately to the CNS but also to differentiate into cells that can remyelinate is significant because dysmyelination/demyelination-typically of an extensive nature--plays an important role in many other genetic (eg, leukodystrophies, inborn metabolic errors) and acquired (traumatic, infectious, asphyxial, ischemic, inflammatory) neurodegenerative processes. Contemporaneously with these experiments described using clone C17.2 NSCs, murine brain cells propagated with E G F - - a method that has been effective for propagating NSCs although it is unclear for lack of clonal analysis or cell type analysis whether it was true in this case--were effective in promoting myelin formation in the focal regions of spinal cord in the md rat. 54 Complementation studies in mutants, such as shi, twitcher, and md, help support an NSC-based approach--whether with exogenous NSCs or with appropriately mobilized endogenous NSCs--for compensating for myelin disorders of many etiologies.

Promotion of Endogenous Remyelination. Few studies, to date, have directly examined growth factor stimulation of remyelination in vivo. Indeed, the factors limiting the degree of spontaneous remyelination in demyelinating disease are poorly

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understood. NSC transplantation may soon address this question by providing a unique vehicle for the delivery of growth factors to demyelinating lesions. Previous work from our laboratory and others has demonstrated that NSCs can be successfully used for diffuse or localized gene transfer into brain. 66,73,75-77 These have included not only lysosomal enzymes as described earlier, but also such neurotrophic agents as nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived growth factor (BDNF), and L1 adhesion molecule. As suggested earlier, growth factors that are either intrinsic to the NSC or that are secreted following genetic engineering before transplantation, could be delivered to demyelinated CNS regions in a sustained, direct, and perhaps regulated fashion via NSCs. A better understanding of the determinants of glial differentiation and proliferation in vivo will certainly have implications for future remyelinating strategies of this type. Exploring the Etiology of Diseases. The ability of NSCs to disseminate foreign gene products throughout the brain has prompted their use in a somewhat unexpected context. NSCs have proven useful for actually creating models of CNS disease where none presently exist via the distribution of "disease-producing" agents throughout normal CNS--a "poor-man's" transgenic mouse. Recently, a model of "spongiform myeloencephalopathy" was created in this way through the diffuse engraftment of genetically engineered NSCs. 99 Importantly, this technique not only created a model of this retrovirally induced neurodegeneration but permitted a systematic dissection of its underlying pathophysiology by using NSCs variously genetically manipulated to deliver isoforms of molecules and viral strains. We suspect that an approach such as this may be useful in attempting to understand the pathobiology underlying various demyelinating conditions, including the most enigmatic of all, MS. For example, NSCs might prove useful for investigating the role of immune modulation. Although the presence of increased oligoclonal immunoglobulins in the cerebrospinal fluid is a hallmark of MS, some investigators have suggested that a subset of autoantibodies may be produced in an attempt actually to promote recovery.33 In other words, antibodies expressed in autoimmuneinduced demyelination may actually play a secondary role, perhaps by stimulating mature or immature oligodendrocytes to differentiate, divide, and

BILLINGHURST, TAYLOR, AND SNYDER

produce myelin. By engineering NSCs to express different classes of antibodies in demyelinated lesions, the role of specific antibodies in this pathologic environment might be elucidated.

Specific Diseases ldeaUy Suited for NSCs. Multiple Sclerosis. Multiple sclerosis (MS) is characterized pathologically by focal demyelinated lesions found throughout the CNS, with a predilection for the optic nerves, brainstem, spinal cord, and periventricular white matter. Although the pathogenesis of the disease is still not entirely clear, both cellular and humoral immune mechanisms have been implicated. The current consensus is that MS results from an autoimmune inflammatory mechanism that targets myelin and oligodendrocytes. 101 Clinically, treatment strategies in MS have focused almost exclusively on limiting inflammation and subsequent demyelination. The absence of an effective treatment for the underlying pathogenesis of MS has represented a significant obstacle to the clinical application of remyelination strategies. Indeed, it may be of little value to supplement myelin repair in the face of ongoing inflammat i o n - t h e transplanted neural cells may simply become targets for subsequent demyelinating attacks. However, suppression of the inflammatory process responsible for demyelination (if that indeed proves to be the fundamental pathogenic process) coupled with NSC transplantation may prove to be an effective strategy to promote lasting myelin repair. Although it remains possible that MS derives from a defect intrinsic to the particular patient's oligodendrocytes, the most popular hypothesis of MS pathogenesis is that the disease results from factors extrinsic to the oligodendrocyte, that is, in an inhospitable, toxic CNS environment. These factors include circulating inflammatory cells (T cells, macrophages) and antibodies, as well as immune mediators (cytokines, complement). Although NSCs clearly can replace dysfunctional oligodendrocytes, it may be possible to use the same NSCs, genetically engineered ex vivo before transplantation, to deliver protective or neutralizing substances simultaneously to the oligodendrocytemyelin unit in vivo and to defend against cytotoxins released at sites of both antigen specific and nonspecific immune responses. It may also be important to modify donor NSCs, through genetic manipulation, so that they avoid antigen specific insult (such that antibodies do not interact with

REMYELINATION: CELLULAR AND GENE THERAPY

their surface components or myelin processes). Furthermore, they could be engineered to secrete trophic substances for the enhanced growth and support of endogenous oligodendrocyte progenitors as well as for their own benefit in an autocrine/ paracrine fashion. Classically, MS has been defined as a purely demyelinating disease. Recently, however, it has been demonstrated that, in addition to demyelination, axonal transection is also evident in the brains of patients with this disease. 102,103 This result has important clinical and therapeutic implications. The aim of most MS therapies is to promote endogenous remyelination while limiting immunemediated damage to myelin. Despite aggressive treatment, many patients experience a chronic progression of their disease. This progression may be related to the destruction of axons, in addition to demyelination, by mechanisms that are still unknown. Because axonal transection is irreversible, multipotent NSC transplantation could be used not only to replace oligodendrocytes but also to provide the neurons whose axonal processes can be remyelinated. Furthermore, because the most potent stimulus to oligodendrocyte survival and differentiation and one of the most effective signals for myelination is the presence of neuronal processes, the provision of healthy axons may also promote a reparative remyelination process. Future treatment strategies might also be directed at the delivery of agents protective of neurons and axons as well as of oligodendrocytes and myelin) ~ As noted previously, these can also be conveyed via NSC transplantation at the same time, with the same cells, in the same host, and via the same implantation procedure. These agents could improve the survival of the axon-oligodendroglial unit during active demyelination by fortifying diseased axons and shielding them from proteolytic enzymes, cytokines, and free radicals produced by activated immune and astroglial cells within the demyelinated lesion. Leukodystrophies. The inherited disorders of myelin metabolism are an important subgroup of demyelinating diseases, particularly for pediatricians and pediatric neurologists. They include the leukodystrophies, a group of progressive white matter diseases affecting both the central and peripheral nervous systems. These diseases are characterized by various enzymatic and biochemical defects that disturb myelin metabolism and

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result in a global depletion of normal myelin throughout the CNS. X-linked and neonatal adrenoleukodystrophy, Alexander's disease, Canavan's disease, cerebrotendinous xanthomatosis, globoid cell leukodystrophy (Krabbe's disease), metachromatic leukodystrophy, Pelizaeus-Merzbacher disease, Refsum disease, and Zellweger syndrome are all members of this broad group of white matter diseases of different underlying etiologies. 38A~176 The pattern of inheritance in leukodystrophy is either autosomal recessive or X-linked, and though the onset of disease is usually in infancy, some of the disorders have both childhood and adult forms. Leukodystrophies with onset in infancy are usually characterized by progressive mental retardation, muscular flaccidity and weakness, impaired psychomotor development, spasticity, and seizures, followed by death in early childhood. Unfortunately, the only available treatment for many of the leukodystrophies is symptomatic and supportive care. Other methods of therapy currently under investigation include dietary therapy, BMT, and immunosuppression. 38,1~ BMT has been successful in addressing visceral manifestations but has been generally disappointing in reversing or forestalling damage to the CNS, presumably because of restrictions imposed by the BBB to entry of therapeutic molecules and cells emanating from a peripheral, non-neural source. Furthermore, BMT entails conditioning regimens (including irradiation) that may be detrimental to the developing CNS; there are also significant risks for systemic complications such as graft-versushost disease. In addition, in the few cases where biologically significant levels of an enzyme can be achieved in the CNS via BMT, attainment of that level is often slow--as long as a year--suggesting that the balance between ongoing destruction and prevention of disease may not be optimal in the most virulent, progressive, or early onset versions of the diseases. 3542 The limitations of viral and nonviral vectors for gene transfer to host neural tissue was discussed previously (w (For example, for both noncellular vectors and cellular vectors of a non-neural nature, transmission of new genetic information to or via old, endogenous neural elements that may have already degenerated remains a common limitation). The delivery of gene products to the CNS via NSC transplantation-analogous to the use of hematopoietic stem cell transplantation for cell replacement and gene trans-

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BILLINGHURST, TAYLOR, AND SNYDER

fer to the periphery--might circumvent these problems. As detailed earlier, the feasibility of this strategy has been affirmed by demonstrating diffuse engraftment and immediate therapeutic gene expression by lysosomal enzyme-expressing NSCs that slipped unimpeded through the BBB and integrated seamlessly throughout the brain following intracerebroventricular injections at birth or in utero. 86,87 The feasibility of widespread neural cell replacement was demonstrated by oligodendrocyte replacement in the shi mutant via NSCs similarly administered. That NSCs can replace not only glia but also neurons has been proven in both localized94 and more extensive92 regions of abnormal CNS. In the not-so-distant future, the leukodystrophies may become excellent candidates for gene therapy-perhaps via NSC transplantation in concert with other techniques such as BMT because many can be diagnosed prenatally or shortly after birth, and there is often a short period in early infancy before neurological deficits are manifest. This may provide a "window of opportunity" for therapeutic intervention. As mentioned previously (w experiments are now ongoing in the use of N S C s , 89 some engineered to over-express galactocerebrosidase, in the treatment the twitcher mouse model of the virulent, unrelenting infantile-onset form Krabbe's disease, a prototype for the class of leukodystrophies that have been generally refractory to other types of intervention, including BMT. 35-4I The human trials ongoing on Canavan disease (described previously in w in which liposomal vectors are administered intraventricularly to children provide assurance that such a method of administration of therapeutic substances to the human brain (the manner in which the NSCs are transplanted) is also, indeed, safe. 43 Ultimately, it may well prove that combined interventions--intracranial vectors, BMT, and NSC transplantation, orchestrated and administered at various times in the diagnosis and course of the most virulent and aggressive diseases (including prenatal, postnatal, and ongoing maintenance treatment--all quite feasible with present technology) may prove to be the most efficacious interventions for forestalling, salvaging, repairing, and regaining neurological function.

"Acquired" Demyelinating Conditions.

A

number of acquired diseases that figure prominently in the pediatric population are accompanied

by insults to oligodendroglia and loss of functional myelin. Because these defects, however, often do not stand in isolation and are accompanied by abnormalities in neurons, axons, and other cell types, the focus on white matter is often diffused or obfuscated. However, it is precisely in instances such as this that the use of NSCs may be most effective. Oligodendroglial pathology is prominent in all hypoxic-ischemic conditions and may account for a significant proportion of the neurological handicap seen in asphyxiated newborns (certainly in periventricular leukomalacia, but also in other ischemic conditions as well. 1~ During phases of active neurodegeneration, as-yet-unidentified factors seem to be transiently elaborated to which NSCs may respond by migrating to degenerating regions and differentiating towards replacement of dying neural cells. 91'94 When NSCs (clone C17.2) are transplanted into brains of postnatal mice subjected to experimental unilateral hypoxic-ischemic (HI) brain injury (particularly within a specified window of time following HI), donor-derived cells migrate preferentially to and integrate extensively within the large ischemic areas that typically span the injured hemisphere. 95 A subpopulation of donor NSCs, particularly in the penumbra of the infarct, "shift" their differentiation fate towards neurons and oligodendrocytes, the neural cell types typically damaged following asphyxia/stroke and the cell types least likely to regenerate spontaneously in the postnatal CNS. In the injured neocortex, there was a fivefold increase in donor-derived oligodendrocytes compared with an intact neocortex. When NSCs engineered to express the neuronpromoting neurotrophic agent, NT-3, were implanted into asphyxiated mouse brain, the percentage of donor-derived neurons was substantially increased from 5% to 80%. This observation demonstrated that NSCs may be capable of simultaneous gene therapy and neural cell replacement with the same cells, during the same transplantation procedure, in the same transplant recipient. With such feasibility established, this approach can quite plausibly be applied to other neural cell types in this and other examples of acquired and genetic CNS degeneration and injury. Specifically, NSCs may be similarly engineered ex vivo to express trophic factors particularly suited to the induction

REMYELINATION: CELLULAR AND GENE THERAPY

of oligodendrocyte differentiation (eg, insulinlike growth factor- 1, thyroid hormone).7~ It is now generally accepted that persistent demyelination is a prominent component of spinal cord (SC) injury. 1~ It is conceivable that demyelinated axons might be rendered functional with appropriate transplantation strategies using NSCs (which possess neuronal-glial multipotency). As in the asphyxiated brain, a tropism and trophism for NSCs in neurodegenerative environments appears evident in the SC. Induction of segmental o~-motor neuron (MN) degeneration in postnatal SC by sciatic axotomy is a classic experimental model of spinal neuron degeneration and spinal dysfunction/ injury. SC involvement results not only from trauma but also accompanies many neurogenetic, metabolic, infectious, ischemic, and inflammatory diseases. Although MNs are normally born only in the fetus, if NSCs are implanted during active degeneration, a significant proportion will engraft, migrate toward and throughout the segments of a-MNimpoverished ventral horn, and approximately 20% will differentiate into cells that resemble the lost ct-MNs. However, the remainder are non-neuronal cells--precisely the same cells which, as noted previously, engrafted throughout the dysmyelinated shi mutant mouse brain and were capable of differentiating into oligodendrocytes88 and similar to the cells that could differentiate into oligodendrocytes in a demyelinated SC. 45,54 Therefore, presumably, a subpopulation of these multipotent NSCs in the axotomized (or otherwise injured) SC will differentiate spontaneously, or with proper induction, into oligodendroglia that might successfully elaborate myelin and help redress problems resulting from demyelination. Engrafted NSCs continue to express foreign reporter genes in this abnormal environment, suggesting that, as in the asphyxiated brain, implantation of genetically engineered NSCs expressing trophic agents, cytokines, or other factors might enhance not only neuronal differentiation, neurite outgrowth, and proper connectivity, but also promote oligodendrocyte differentiation and remyelination--all in the same transplant intervention. Although the focus in SC dysfunction has traditionally been in the restoration of neurons and neuronal connection, it is quite plausible that, in accomplishing that goal, remyelination may also be achieved concurrently from the same cells when

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NSCs are used. Enthusiasm in this regard, of course, will need to be tempered by evidence that oligodendrocytes and their membranes may produce factors inhibitory to axonal regeneration,I~176 which, however, can be neutralized by monoclonal antibodies against these factors.117,172 Both asphyxial brain injury and SC dysfunction, by virtue of their complexity and multiple pathological processes and cell type involvement, may not only demand all of the therapeutic capabilities of NSCs, but may also serve as a prototype for a wide range of degenerative, developmental, or acquired CNS injuries possibly amenable to a strategy of NSC intervention. Summary. In summary, multipotent NSCs may concurrently replace injured oligodendrocytes and neurons; they may accomplish this while simultaneously providing---either intrinsically or following ex vivo genetic engineering--neurotrophins, growth factors, neuroprotective factors, and "support structures" (eg myelin, "bridges," cell-to-cell contact signals, extracellular matrix, adhesion molecules) that might facilitate regeneration of the injured host's own lost or dysfunctional neural cells, both glial and neuronal, or forestall ongoing degeneration resulting from the insufficiency of a trophic factor or enzyme in the milieu. In other words, because, in gene therapy paradigms, NSCs have the ability to pass unimpeded through the BBB and incorporate into the host cytoarchitecture in a functional manner, they are more than simple vehicles for passive delivery of gene products. FUTURE DIRECTIONS

Although NSC transplantation seems to be ideally suited for a range of processes in which dys/demyelination is prominent, particularly those with a heterogeneous pathogenesis and variable degrees of oligodendrocyte pathology I~ (eg, MS, trauma, some of the leukodystrophies), these theoretical possibilities need to be actually tested in each condition individually. The success of NSC transplantation as a remyelinating therapy will ultimately be determined by its ability to achieve myelination significant enough for functional improvement. It may even be important to identify subtypes of myelin disorders to ensure that stimulation of remyelination is a rational therapeutic approach. For example, as noted earlier, myelin

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BILLINGHURST, TAYLOR, AND SNYDER

formation inappropriately in SC injury might actually inhibit neurite outgrowth and prevent connectivity, not promote it. 1~176 In other disorders of myelin, the CNS milieu may vary from phase to phase of the disease. The migratory capacity of remyelinating cells may vary depending on the degree of inflammation, cell death, gliosis, and demyelination; at later stages of a demyelinating disease, when pronounced oligodendrocyte loss is present, dense astroglial scarring might profoundly impair the success of cellular transplantation, 1~ suggesting that the intervention was too late. Most of the results described in this review-both with cells in the oligodendrocyte lineage as well as with NSCs---have been derived from observations in idealized animal models. How this biology may be translated to treatment strategies in genuine clinical situations will require an even better understanding of fundamental oligodendrocyte biology. Some challenges include: determining the parameters that optimize engraftment and oligodendrocyte differentiation in various developing and degenerating environments as well as identifying mechanisms for regulating the efficiency and amount of foreign gene expression by engrafted cells. In the long run, such knowledge may allow NSC and oligodendrocyte cell lines with a similar repertoire of characteristics to be generated expeditiously from human neural tissue and transplanted efficaciously and without concern for recipient safety. Progress in this regard is already being made by a small group of laboratories, including ours. 113-118 Preliminary findings in animals suggest that human NSCs can similarly engraft and respond to normal developmental cues in vivo, can express foreign genes throughout the recipient brain, and can replace degenerating or underdeveloped neural cells, 114 suggesting that the behaviors observed in both mouse and human systems may reflect basic biological properties of NSCs and laying the groundwork for potential human therapies. It also should go without saying that future challenges must include optimization of extant interventions such as BMT and a better understanding and identification of the gene products that are pivotal in both the destruction and repair of myelin in such neurodegenerative diseases as MS and the leukodystrophies.

CONCLUSION

Most neurological diseases, particularly those affecting myelin, are characterized by widespread and multifocal pathology. Therapies directed towards effecting remyelination may also require multiple repair strategies. We hypothesize that, by virtue of their inherent biologic properties, NSCs may possess--better than other cell types the multiple capabilities that disorders of myelin may demand, particularly in the developing nervous system. Such therapeutic strategies against white matter disease might include: replacement of dysfunctional oligodendroglia with effective myelinproducing cells; engineering of donor cells to be resistant to immune-mediated attack or for the expression, delivery, or supplementation of missing or therapeutic metabolic enzymes, growth factors, and immunomodulators; and, also important and but often not considered in white matter disease, replacement of neurons and reconstitution of degenerated neural connections and circuitry. Some of these approaches have been successful in several mouse models of neurodegeneration and are being extended to other toxin-induced, autoimmune, viralmediated, or genetic animal models that emulate human dysmyelinating and demyelinating disease. These studies are not only enhancing our understanding of oligodendrocyte and NSC biology but are bringing us closer to clinical use of these novel cellular and genetic therapeutic vehicles. Ultimately, it may well prove that combined interventions--intracranial vectors, BMT, enzyme administration, dietary manipulation, and NSC transplantation, administered in a coordinated fashion at various stages in the course of the most virulent diseases of white matter--may prove to be the most efficacious approach for forestalling damage and regaining neurological function. ACKNOWLEDGMENTS

The authors wish to thank Kevin Hall for his helpful suggestions in the preparationof this manuscript.We also thank members of the Snyder lab, particularly Booma D. Yandava, Shaoxiang Liu, KookIn Park, and JonathanD. Flax for someof the data describedin this review.Someof the workdescribedin this review was supportedby grants from the National Institute of NeurologicDiseases and Stroke (NS33852, NS34247), from the AmericanParalysisAssociation, the ParalyzedVeterans of America and by Mental Retardation Research Center grant NIH-P30-HD18655.

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