WO2005016250A2

WO2005016250A2 - Cell therapy for neurometabolic disorders

Info

Publication number: WO2005016250A2
Application number: PCT/US2004/022311
Authority: WO
Inventors: Lamya Shihabuddin; Suzanne Numan; Gregory Stewart
Original assignee: Genzyme Corporation
Priority date: 2003-08-08
Filing date: 2004-08-06
Publication date: 2005-02-24
Also published as: WO2005016250A3

Abstract

The disclosure pertains to methods for treating neurometabolic disorders including lysosomal storage diseases that affect the central nervous system such as, e.g., Niemann-Pick A disease. The disclosed methods involve administering nonimmortalized neural stem cells into the brain of a mammal, wherein the neural stem cells secrete a lysosomal hydrolase.

Description

CELL THERAPY FOR NEUROMETABOLIC DISORDERS

[0001] This application claims priority to the United States patent application Serial No. 60/493,652, filed on August 8, 2003, herein incorporated by reference.

Field of the Invention [0002] The present invention relates to compositions and methods for treating neurometabolic disorders of the central nervous system (CNS). The invention further relates to compositions containing cells derived from neural stem cells, and methods of administration thereof.

Background of the Invention [0003] A group of metabolic disorders known as lysosomal storage diseases (LSD) includes over forty genetic disorders, many of which involve genetic defects in various lysosomal enzymes. Representative lysosomal storage diseases and the associated defective enzymes are listed in Table 1. Table 1

Table 1 (cont'd)

CNS involvement

[0004] The hallmark feature of LSD is the abnormal accumulation of metabolites in the lysosomes which leads to the formation of large numbers of distended lysosomes in the perikaryon. A major challenge to treating LSD (as opposed to treating a liver-specific enzymopathy) is the need to reverse lysosomal storage pathology in multiple separate tissues. Some LSDs can be effectively treated by intravenous infusion of the missing enzyme, known as enzyme replacement therapy (ERT). For example, Gaucher type 1 patients have only visceral disease and respond favorably to ERT with recombinant glucocerebrosidase (Cerezyme®, Genzyme Corp.). However, patients with metabolic disease that affects the CNS (e.g., type 2 Gaucher disease or Niemann-Pick A disease) do not respond to intravenous ERT because the replacement enzyme is prevented from entering the brain by the blood brain barrier (BBB). Furthermore, attempts to introduce a replacement enzyme into the brain by direct injection have been unsuccessful in part due to enzyme cytotoxicity at high local concentrations (unpublished observations) and limited parenchymal diffusion rates in the brain (Pardridge, Peptide Drug Delivery to the Brain, Raven Press, 1991). [0005] Cell therapy, or ex vivo gene therapy, is an emerging treatment modality for LSD. This approach involves transplantation of donor-derived cells that have been transduced to secrete the deficient enzyme. In theory, following implantation, the enzyme secreted by transplanted cells may be taken up by neighboring host cells, in which it then alleviates pathology. This process is known as cross-correction. However, most neurometabolic disorders affect the entire brain and spinal cord. Therefore, to treat disease effectively, transplanted cells must be able to migrate throughout the CNS, survive long-term and sustain enzyme expression. [0006] Neural stem cells (NSCs) could be well suited for cell therapy for disorders affecting the brain. Besides being amenable to genetic manipulation, these cells can migrate within the brain and differentiate into the three major CNS cell types (neurons, astrocytes, and oligodendrocytes), which should allow their incorporation into the normal brain circuitry. Theoretically, these cells may replace or compensate for cells damaged or lost as a result of a neurodegenerative pathology. [0007] NSC-mediated therapy has been employed in only one rodent model of an LSD, i.e., Sly disease (Snyder et al. (1995) Nature, 374:367-370). However, Sly disease is not representative of the majority of LSDs. First, the diffusional capacity of the enzyme deficient in Sly disease (β- glucuronidase) is exceptionally high. Second, the protein is well tolerated at extremely high doses, even up to 1000 times above normal levels. Third, the nature of pathology in the Sly mouse is unusually mild compared to other known neurometabolic disorders, such as Niemann-Pick and Batten diseases. Finally, the Sly mouse has proven to be an animal model that is unusually amenable to a wide variety of therapeutic approaches. Thus, while the Sly mouse may provide a useful tool for studying certain aspects of neurometabolic disease, it is not an animal model which produces results which can be extrapolated to diseases other than Sly disease. [0008] Further, studies in Sly mice utilized cells that were immortalized with retroviral oncogenes such as v-myc (Hoshimaru et al. (1996) Proc. Nat. Acad. Sci. USA, 93:1518-1523). However, safety concerns associated with the cell cycle deregulation make these cells uncertain candidates for clinical application (e.g., the potential for neoplastic transformation, accumulation of genetic mutations, abnormal protein expression, etc). Additionally, results obtained with immortalized cells are not necessarily transferable to nonimmortalized cells. [0009] Culture systems for propagating NSCs without the use of immortalizing oncogenes have been recently developed. In these systems, NSCs populations are epigenetically expanded under serum-free conditions in the presence of certain mitogenic factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2) (for review, see Ostenfeld et al. (2003) Adv. Tech. Stand. Neurosurg., 28:3-89). [0010] Several groups have reported that epigenetically propagated nonimmortalized NSCs have the ability to engraft, migrate, and differentiate into neurons and glial cells in a site-specific manner upon transplantation into the normal, disease-free, rodent brain (see, e.g., Fricker et al. (1999) J. Neurosci., 19:5990-6005; Tamaki et al. (2002) J. Neurosci. Res., 69:979-986; Englund et al. (2002) Dev. Brain Res. Dev., 134:123-141 ; Flax et al. (1998) Nat. Biotech., 16:1033-1039; Buchet et al. (2002) Mol. Cell. Neurosci., 19: 389-401 ; Shihabuddin et al. (200) J. Neurosci., 20:8727-8735; Numan et al. (2002) Soc. for Neurosci. Abstracts, #726.8). However, it remains unknown what cues are responsible for differentiation of NSCs in vivo, and whether sufficient survival and migration of nonimmortalized NSCs occur in a diseased brain (Armstrong et al. (2000) Cell Transplantation, 9:55-64), particularly under conditions of substantial lysosomal storage pathology such as those present in Niemann-Pick A disease. [0011] Therefore, there is a need in the art to develop new therapeutic methods for treating neurometabolic disorders, and LSD, in particular. SUMMARY OF THE INVENTION [0012] The invention provides methods and compositions for treating neurometabolic disorders such as lysosomal storage diseases (LSD). [0013] Additional objects and advantages of the invention will be set forth in part in the following description, and in part will be understood from the description, or may be learned by practice of the invention. [0014] In the experiments leading to the present invention, acid sphingomyelinase (ASM) knockout mice (ASMKO; a model of Niemann-Pick Type A disease) were administered epigenetically expanded nonimmortalized neural stem cells (NSCs) stably transduced to overexpress ASM. The present invention is based, in part, on the discovery and demonstration that, following the direct injection into the diseased brain, the implanted cells migrate away from the injection site and survive for a prolonged time in different recipient regions. The invention is further based, in part, on the discovery and demonstration that, despite low levels of ASM expression by the transplanted cells, extensive correction of lysosomal storage pathology occurs at the injection site and distal sites to which the cells migrate. [0015] Accordingly, one aspect of the present invention provides therapeutic methods for treating neurometabolic disorders in mammals. The methods of treatment comprise administering a therapeutically effective amount of nonimmortalized NSCs into the brain of a mammal having a neurodegenerative disorder. The populations treated by the methods of the invention include mammals having an LSD, such as disorders listed in Table 1. In illustrative embodiments, the disease is Niemann-Pick type A disease. The populations treated can be neonates, juveniles, or adults. [0016] Another aspect of the invention provides methods for indirect, less invasive delivery of a transgene product to regions in the brain of an affected subject to which the passage of the transgene product by intraparenchymal diffusion is restricted or impossible. Thus, in some embodiments, the invention provides a method of delivering a lysosomal enzyme (e.g., a lysosomal hydrolase) to a target site in the CNS of a mammal having a lysosomal storage disorder. The method comprises administering nonimmortalized NSCs in the brain of a mammal, wherein the nonimmortalized NSCs intraparenchymally migrate from the administration site to a target site and secrete the lysosomal enzyme for a period of time and in the amount sufficient to alleviate the lysosomal storage pathology in the target site. In some embodiments, the administration site and the target site are at a distance of at least 1 , 2, 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mm from each other. [0017] Methods of making the compositions of the invention and methods of administration thereof are also provided. The nonimmortalized NSCs of the invention are epigenetically expanded in vitro prior to the administration. In further embodiments, nonimmortalized NSCs are genetically modified prior to the administration to oversecrete a lysosomal enzyme which is deficient in the LSD being treated. In illustrative embodiments, NSCs are genetically modified by retroviral transduction with a transgene encoding the lysosomal hydrolase ASM. Intracerebral administration of the nonimmortalized NSCs is accomplished intraventricuiarly or intraparenchymally, while the exact site(s) of administration may vary. [0018] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES [0019] FIGS. 1A-1 D depict schematic drawings of coronal sections showing the distribution of BrdU-positive mouse NSCs in the ASMKO mouse brain at 4 weeks following a 1 μl injection of NSCs (100,000 cells/μl per site) into the hippocampus and thalamus. A fraction of the transplanted cells was located as a dense cluster at the injection site (FIG. 1 B); BrdU-positive cells are represented by dots. The cells survived and presented an extensive migration pattern into the adjacent gray and white matter in the ipsilateral hemisphere of the brain. [0020] FIG. 2 depicts schematic drawings of a coronal section showing the distribution of BrdU-positive mouse NSCs in the ASMKO mouse brain at 4 weeks following a 1 μl injection of NSCs (100,000 cells/μl per site) into the hippocampus and thalamus and the corresponding area of cross- correction. BrdU-positive cells are represented by dots. The migration of cells within the hippocampus and thalamus resulted in an extensive area of reversal of pathology (cross-hatched area) in the ipsilateral hemisphere of the brain as determined by filipin staining. The area of filipin staining overlaps with the distribution of NSCs. DETAILED DESCRIPTION OF THE INVENTION [0021] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. [0022] The term "clearance" refers to reduction of a substrate of a lysosomal enzyme (e.g., lysosomal hydrolase) in the lysosomes of host cells in the region of the CNS containing transplanted cells by at least 50%, 60%, 70%, 80%, or 90% as detectable by routine microscopic examination. [0023] The terms "neural stem cells (NSCs)," "neural precursor cells," and "neural progenitor cell" refer to cells that, under appropriate in vitro or in vivo conditions, are capable of sustained proliferation (self-renewal) giving rise to cells that may differentiate within the CNS into at least one of the three major CNS cell types, i.e., neurons, astrocytes, and oligodenrocytes. As used herein, the term "NSC" also encompasses partially differentiated, committed, progenitor cells such as neuronal restricted precursor cells, glial restricted precursors, and oligodendrocyte and type II astrocyte precursor cells. [0024] The terms "nonimmortalized NSCs" and "non-oncogene- immortalized" refer to a neural stem cell which is not genetically modified with a viral oncogene to sustain self-renewal. The term "immortalization" is used in the art to describe cells that have been genetically modified to divide beyond the normal limitation with respect to their proliferative potential: that is, they no longer undergo replicative senescence after a finite number of cell divisions (usually around 40-50 divisions for normal somatic cells, known as the Hayflick limit (Hayflick (1997) Biochemistry, 62:1180-1190). Oncogene-immortalization is exemplified by transfection of cells with nontransforming oncogenes such as c-myc, v-myc, adenoviral E1A and tsA58 (the temperature-sensitive allele of SV40 Large T antigen) (Cameron et al. (1993) Neuronsci., 56:337-344; Flax et al. (1998) Nat. Biotech., 16:1033-1039; Martinez-Serrano et al. (1997) Trends Neurosci., 20:530-538; Campsi (1996) Cell, 84:497). In particular, the term "non-immortalized NSCs" refers to non-oncogene-immortalized NSCs whose capacity to proliferate was sustained or enhanced epigenetically (i.e., by means not involving genetic modification), for example, by exposure to mitogenic factors in culture as described here or known in the art. In particular, the term "non-immortalized NSCs" does not refer to the NSCs clone 17.2, HiB5, RN33B, or cells derived therefrom. It is understood, however, that nonimmortalized NSCs can be genetically modified, e.g., as described here, so long as the modification does not involve introduction of an immortalizing oncogene. [0025] The terms "therapeutic," "therapeutically effective amount," and their cognates refer to that amount of a compound that results in prevention or delay of onset or amelioration of symptoms of neurometabolic disorder in a subject or an attainment of a desired biological outcome, such as replacement of lost or damaged cells, correction of neuropathology, e.g., cellular pathology associated with a lysosomal storage disease such as that described herein or in W alkley (1998) Brain Pathol., 8:175-193. The term "therapeutic correction" refers to that degree of correction that results in prevention or delay of onset or amelioration of symptoms of neurometabolic disorder in a subject. The effective amount can be determined by methods known in the art and/or as described in the subsequent sections, including but not limited to filipin-staining procedures illustrated in the Examples. [0026] The term "transgene" refers to a polynucleotide that is introduced into a cell and is capable of being expressed under appropriate conditions and confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic outcome. [0027] In the experiments leading to the present invention, ASMKO mice were subjected to an intracerebral injection of nonimmortalized NSCs that had been previously transduced to oversecrete ASM. ASMKO mice are an accepted model of types A and B Niemann-Pick disease and are described in, for example, Horinouchi et al. (1995) Nat. Genetics, 10:288-293; Jin et al. (2002) J. Clin. Invest., 109:1183-1191 ; and Otterbach (1995) Cell, 81 :1053-1061. [0028] Niemann-Pick disease (NPD) is classified as a lysosomal storage disease and is an inherited neurometabolic disorder characterized by a genetic deficiency in acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.13.12). The lack of functional ASM protein results in the accumulation of sphingomyelin substrate within the lysosomes of neurons and glia throughout the brain. This leads to the formation of large numbers of distended lysosomes in the perikaryon, which is a hallmark feature and the primary cellular phenotype of Niemann-Pick A disease. The presence of distended lysosomes correlates with the loss of normal cellular function and a progressive neurodegenerative course that leads to death of the affected individual in early childhood (The Metabolic and Molecular Bases of Inherited Diseases, Scriver et al. (eds.), McGraw-Hill, New York, 2001 , pp. 3589-3610). Secondary cellular phenotypes (e.g., additional metabolic abnormalities) are also associated with this disease, notably the high level accumulation of cholesterol in the lysosomal compartment. Sphingomyelin has strong affinity for cholesterol, which results in the sequestering of large amounts of cholesterol in the lysosomes of ASMKO mice and human patients (Leventhal et al. (2001 ) J. Biol. Chem., 276:44976-44983; Slotte (1997) Subcell. Biochem., 28:277-293; and Viana et al. (1990) J. Med. Genet., 27:499-504.) [0029] The present invention is based, in part, on the discovery and demonstration that, following direct injection into the diseased brains of ASMKO mice, nonimmortalized NSCs migrate away from the injection site and survive for a prolonged time in different recipient regions. The invention is further based, in part, on the discovery and demonstration that despite low levels of ASM expression by the transplanted cells, extensive correction of lysosomal storage pathology occurs at the injection site and all distal sites to which the cells migrate. [0030] Accordingly, in one aspect, the present invention provides therapeutic methods for treating neurometabolic disorders in mammals. The methods of treatment comprise administering a therapeutically effective amount of nonimmortalized NSCs into the brain of a mammal having a neurodegenerative disorder. The populations treated by the methods of the invention include mammals having an LSD, such as disorders listed in Table 1. However, in certain embodiments, LSD may exclude: Batten, Hunter, Krabbe, Sandhoff, Sly, and/or Tay Sachs diseases. In illustrative embodiments, the disease is Niemann-Pick A disease. The populations treated can be neonates, juveniles, or adults. The methods of treatment comprise administering a therapeutically effective amount of nonimmortalized NSCs into the brain of a mammal. [0031] In another aspect, the invention provides methods for indirect, less invasive delivery of a transgene product to regions in the brain of an affected subject to which the passage of the transgene product by intraparenchymal diffusion is restricted or impossible. Thus, in some embodiments, the invention provides a method of delivering a lysosomal enzyme (e.g., a lysosomal hydrolase) to a target site in the CNS of a mammal having a lysosomal storage disorder. The method comprises administering nonimmortalized NSCs in the brain of a mammal, wherein the nonimmortalized NSCs intraparenchymally migrates from the administration site to a target site and secretes the lysosomal enzyme for a period of time and in the amount sufficient to alleviate the lysosomal storage pathology in the target site. In some embodiments, the administration site and the target site are at a distance of at least 1 , 2, 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mm from each other. [0032] Methods of making the compositions used in the methods of the inventions and methods of administration thereof are described below.

Methods of making nonimmortalized NSCs [0033] Neural stem cells can be obtained from the CNS of any animal, including humans, at any age (e.g., embryo or adult) or post-mortem. Examples of species that can serve as a source of NSCs include human, monkey, marmoset, pig, dog, rat, mouse, etc. NSCs can also be obtained from non-CNS-derived stem cells that have the capacity to give rise to a neuronal phenotype under appropriate in vitro or in vivo conditions. [0034] NSCs can be isolated, identified, and epigenetically propagated in vitro, using methods known in the art, for example, as described in the Examples and/or in Modern Techniques in Neuroscience Research, 1999, U. Windhorst and J. Johansson (eds.), Springer Verlag, ch. 11 ; Palmer et al. (2001 ) Nature, 411 :42-43; and United States Patent Application Publication No. 20020098584. [0035] Neural tissues are composed of both neural and nonneural cells as well as connective tissue. The term "isolated" and its cognates refer to a population of cells, e.g., stem cells, obtained by separating away other cells in the tissue. Isolated stem cells are generally free from other cell types and, under appropriate condition, have the capacity to divide symmetrically (self-renew) in culture for at least 5, 7, 10, 15, 20, 25, 30, 35, 40, or more passages (each passage 3-7 days) and to produce mature differentiated cells including but not limited to the cells of the tissue from which NSCs were isolated. NSCs, for example, have the capacity to give rise to neuron, or glial cells (oligodenrocytes and astrocytes). It is understood that it may be impractical or impossible to obtain a population of stem cells which is 100% pure and homogenous. Isolated stem cells that are at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% pure can be utilized as long as the concomitant cells do not substantially impair the function of stem cells. [0036] Isolation of NSCs involves physical separation of cells intertwined in tissue and separating NSCs from other brain cells and connective tissue debris. NSCs can be isolated from tissue surgically excised, for example, from cerebellum, cortex, thalamus, hippocampus, striatum, mesencephalon, spinal cord, or whole brain. The tissue is dissected, cut into small pieces, and subjected to digestion to separate connective tissue, for example, using the papain-protease-DNase (PPD) digestion procedure as described by Ray et al. (1995) In: P.K. Vogt and Verma (eds.), Oncogene Techniques: Methods in Enzymology, vol. 354, Academic Press, San Diego, pp. 20-37; or the tripsin-hyaluronidase-kynuretic acid digestion as described by Gritti et al. (1996) J. Neurosci., 16:1091-1100; or Reynolds et al. (1992) Science, 255:1707-1710. [0037] Following the digestion, cultures may be epigenetically propagated as adherent or non-adherent (e.g., neurospheres) cultures by providing appropriate environmental conditions, including specific nutrients and growth factors. Generally, cultures are maintained at a physiological pH (e.g., 7.2-7.6) and at the appropriate osmolarity. A skilled artisan will recognize appropriate methods for various cells and intended applications. For example, cells can be seeded onto poly-lysine/laminin-coated (S. Fedoroff and A. Richardson (eds.), Protocols for Neural Stem Culture, Humana Press, Totowas, NJ, 1992) or uncoated tissue culture plates and grown in the serum-free N2 medium (based on DMEM:F12 plus N2 supplement), containing appropriate growth factor. Cells are then fed periodically with medium containing EGF, FGF-2 (20 ng/ml), or both. For propagation of clonal cultures, cells are first seeded at lower density (1-2 cells/well multi-well plate at about 1 cells per 7 cm² for 35 mm Petri dish) and fed with serum-free medium supplemented with 50% of conditioned medium collected from high density cells. Illustrative protocols for propagation of NSCs in monolayers are described by Ray et al. (1993) Proc. Nat. Acad. Sci. USA, 90:3602-3606; Ray et al. (1995), supra; Gage (1995) Proc. Nat. Acad. Sci. USA, 92:11879-11883. Alternatively, NSCs may be propagated as free-floating EGF-responsive neurospheres as described by Reynolds et al. (1992) supra; Gritti (1996) supra; Weiss et al. (1996) J. Neurosci., 16:7599-7699. For epigenetic propagation of human stem cells, see, for example, Englund et al. (2002) Exp. Neurol., 173:1-21 , utilizing a combination of EGF, FGF-2, and leukocyte inhibitory factor (LIF; 10 ng/ml). A skilled will recognize that these and other factors, alone or in combination, can also be used, depending on the source of cells and culture conditions employed. The examples of such mitogenic factors include IGF-1 (Arsenijevic et al. (2001) J. Neurosci., 21 :7194-7202),

transforming growth factor-α, platelet-derived growth factor (PDGF; mitogenic

to oligodendrocyte precursors and type II astrocyte (O-2A) precursors) (Grinspan et al. (1990) J. Neurosci., 10:1866-1873); and the sonic hedgehog (SHH) protein (mitogenic to cerebellar granule cells; Wallace (1999) Curr. Biol., 9:445-448). [0038] NSCs can be identified and characterized by the presence (or absence) of certain intracellular or cell surface markers, e.g., nestin (neuroepithelial stem cell protein), CD133, the 5E12 antigen (described in United States Patent No. 5,843,633), CD34, or CD45. If desired, the population of NSCs may be partially differentiated and/or enriched by selection of a subpool. Examples of differentiation agents may include serum (e.g., 0.5, 2, 5, 10%), retinoic acid (1 μM), forskolin ( 5 μM), brain-derived neurotrophic factor (BDNF; 20 ng/ml), neurotrophins 3 and 4 (NT-3; NT-4; 40 ng/ml); ciliary neurotrophic factor (CNTF; 10-20 ng/ml), LIF (10 ng/ml), and thyroid hormone T3 (3 ng/ml). Selection of the subpool of NSCs that are CD133-positive can be accomplished as described, for example, in Uchida et al. (2000) Proc. Nat. Acad. Sci. USA, 97(26): 14720-1475 Tamaki et al. (2002) J. Neurosci., 69:976-986, or United States Patent Application Publications Nos. 20020031792 and 20010044122). [0039] Isolated NSCs may be genetically modified as described here or used as therapeutic agents without any genetic modification. For instance, NSCs may normally express and secrete a hydrolase deficient in an LSD in an amount that is sufficient to provide a therapeutic effect when these cells are transplanted into a diseased brain.

Methods of genetically modifying NSCs [0040] In some embodiments of the invention, nonimmortalized NSCs may be used as a delivery vehicle for genetic material. In some embodiments, nonimmortalized NSCs are genetically modified to carry a transgene, which is operably linked to a promoter and/or other regulatory elements (e.g., enhancers, suppressors, etc.) so as to cause expression of the transgene at a desired level. Expression of desired genes may also be controlled using gene activation techniques. The transgene may encode a therapeutically active molecule (e.g., a lysosomal hydrolase, etc.), or a marker (e.g., green fluorescence protein (GFP; Englund et al. (2002) Dev. Brain Res., 134:123-141), red fluorescence protein (RFP), LacZ, alkaline phosphatase, neomycin-resistance gene (Verma (1997) Nature, 389:239-242), etc.). In illustrative embodiments, NSCs are genetically modified by retroviral transduction with a transgene encoding the lysosomal hydrolase ASM. In certain methods of the invention, the cells comprise a transgene operably linked to a promoter. [0041] In certain embodiments, the transgene encodes a lysosomal hydrolase. In illustrative embodiments, the lysosomal hydrolase is ASM. The genomic and functional cDNA sequences of human ASM have been published (see, e.g., United States Patent Nos. 5,773,278 and 6,541 ,218). Other lysosomal enzymes, including lysosomal hydrolases, can be used for appropriate diseases, for example, as listed in Table 1. [0042] For specific protocols for cell transfection/transduction examples of specific vectors that can be used for preparing NSCs, see, e.g., Gene Therapy Protocols, 2nd ed., Morgan (ed.), Humana Press, Totowas, NJ, 2001 ; and Gene Transfer and Expression in Mammalian Cells: New Comprehensive Biochemistry, Makrides (ed.), Elsevier Science Ltd, 2003. [0043] Generally, both viral and nonviral vectors are suitable for transfection/transduction of NSCs. A number of vectors have been used for transfer of genetic material in the context of cell therapy and are suitable in the methods of the invention (see, e.g., Hsich et al. (2002) Hum. Gene Ther., 13:579-504; and Davidson et al. (2003) Nat. Rev., 4:353-364). For specific vectors and protocols, see, e.g., Viral Vector for Gene Therapy: Methods and Protocols, Machida (ed.), Human Press, Totowa, NJ, 2003; and Non-Viral Vectors for Gene Therapy: Methods and Protocols, Findeis (ed.), Human Press, Totowa, NJ, 2001. Examples of suitable vectors include: retroviral vectors, which include vectors derived from Moloney murine leukemia virus (MoMLC), lentiviral vectors (see, e.g., Englund (2002) Dev. Brain Res., 134:123-141; Tamaki (2002) J. Neurosci. Res., 69:979-986); and adeno-associated viral (AAV) vectors, herpes-simplex-1 viral (HSV-1) vector, and adenoviral (Ad) vectors. Naked DNA, liposomes, and molecular conjugates can also be used. [0044] The level of transgene expression in eukaryotic cells is largely determined by the transcriptional promoter within the transgene expression cassette. Promoters that show long-term activity and are tissue- and even cell-specific are used in some embodiments. Nonlimiting examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet., 8:148-154), CMV/human β3-globin promoter (Mandel et al. (1998) J. Neurosci., 18:4271-4284), GFAP promoter (Xu et al. (2001 ) Gene Ther., 8:1323-1332), the 1.8-kb neuron-specific enolase (NSE) promoter (Klein et al. (1998) Exp. Neural., 150:183-194), chicken beta actin (CBA) promoter (Miyazaki (1989) Gene, 79:269-277), and the β-glucuronidase (GUSB) promoter (Shipley et al. (1991) Genetics, 10:1009-1018). To prolong expression, other regulatory elements may additionally be operably linked to the transgene, such as, e.g., the Woodchuck Hepatitis Virus Post-Regulatory Element (WPRE) (Donello et al. (1998) J. Virol., 72:5085-5092) or the bovine growth hormone (BGH) polyadenylation site. [0045] For some CNS gene therapy applications, it will be necessary to control transcriptional activity. To this end, pharmacological regulation of gene expression can been obtained by including various regulatory elements and drug-responsive promoters as described, for example, in Habermaet al. (1998) Gene Ther., 5:1604-16011 ; and Ye et al. (1995) Science, 283:88-91.

Methods of administration [0046] Intracerebral administration of the nonimmortalized NSCs is not limited to any specific way of delivery and can be accomplished intraventricuiarly or intraparenchymally, while the exact site(s) of administration and mode of delivery may vary. A site of NSC administration within the CNS is chosen based on the desired target region of neuropathology and the topology of the brain. An administration site may be localized in the caudate nucleus and putamen (collectively known as the striatum), the hippocampus, mesencephalon, cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus striatum, cerebral cortex, or, within the cortex, the occipital, temporal, parietal or frontal lobes), or combinations thereof, intracerebroventricular and intrathecal delivery. [0047] For identification of structures in the human brain, see, e.g., The Human Brain: Surface, Three-Dimensional Sectional Anatomy With MRI, and Blood Supply, 2nd ed., Deuteron et al. (eds.), Springer Vela, 1999; Atlas of the Human Brain, Mai et al. (eds.), Academic Press; 1997; and Co-Planar Sterotaxic Atlas of the Human Brain: 3-Dimensional Proportional System: An Approach to Cerebral Imaging, Tamarack et al. (eds.), Thyme Medical Pub., 1988. For identification of structures in the mouse brain, see, e.g., The Mouse Brain in Sterotaxic Coordinates, 2nd ed., Academic Press, 2000. If desired, the human brain structure can be correlated to similar structures in the brain of another mammal. [0048] To administer cells specifically to a particular region of the brain, 3D sterotaxic microinjection can be used. For example, on the day of surgery, patients will have the sterotaxic frame base fixed in place (screwed into the skull). The brain with sterotaxic frame base (MRI-compatible with fiduciary markings) will be imaged using high resolution MRI (see, e.g., Weissleder et al. (2000) Nat. Med., 6:351-335). The MRI images will then be transferred to a computer that runs stereotaxic software. A series of coronal, sagittal and axial images will be used to determine the target site of NSC injection, and trajectory. The software directly translates the trajectory into 3-dimensional coordinates appropriate for the stereotaxic frame. Burr holes are drilled above the entry site and the stereotaxic apparatus localized with the needle implanted at the given depth. The cells in a pharmaceutically acceptable carrier will then be injected. [0049] In addition to MRI, tissue imaging on living animals can be performed by fluorescence (Hoffman (2002) Lancet Oncol., 3:546-556; Tung et al. (2000) Cancer Res., 60:4953-4958), bioluminescence (Shi (2001 ) Proc. Nat. Acad. Sci. USA, 98:12754-12759; Luke et al. (2002) J. Virol., 76:12149-12161), positron emission tomography (Liang et al. (2002) Mol. Ther., 6:73-82, near-infrared fluorescence (Tung et al. (2000) Cancer Res., 60:4953-4958), or X-ray imaging (Hemminki et al. (2002) J. Nat. Cancer Inst, 94:741-749).

[0050] The number of cells to be administered will be determined by those skilled in the art based upon known aspects of ex vivo gene therapy and will depend on the desired outcome and the system used. Generally, the amount of cells per administration site is between 10³ and 10⁷, 10³ and 10⁶, 10⁴ and 10⁶, 10⁴ and 10⁵, 10³ and 10⁵, or 10³ and 10⁴. In illustrative embodiments, cells are administered at 100,000 per site at one or more sites. In experimental mice, the total volume of injected cell suspension is for example, between 1 to 5 μl. For other mammals, including the human brain, volumes and delivery rates are appropriately scaled. For example, it has been demonstrated that volumes of 150 μl can be safely injected in the primate brain (Janson et al. (2002) Hum. Gene Ther., 13:1391-1412). [0051] Therapeutic effectiveness and safety can be determined in an appropriate animal model or clinical trials. For example, a variety of well-characterized animal models exist for LSDs, e.g., as described herein or in Watson et al. (2001 ) Methods Mol. Med., 76:383-403; or Jeyakumar et al. (2002) Neuropath. Appl. Neurobiol., 28:343-357. Procedures related to the grafting, perfusion of the animals, sectioning of the brain tissues and their characterization can be found in Suhonen et al. (1997) Curr. Prot. Hum. Gene Suppl., 11 :13.3.1.1-13.3.24. [0052] Treatment may consist of a single injection per target site, or may be repeated, if necessary. Multiple injection sites can be used. For example, in some embodiments, in addition to the first administration site, a composition comprising cells is administered to another site which can be contralateral or ipsilateral to the first administration site. [0053] NSCs cell therapy may also serve as an adjunct to another cell-based or other therapy, e.g., enzyme delivery, small molecule inhibitor, or viral gene therapies (see, e.g., Eto et al. (2002) Curr. Mol. Med., 2:83-89). [0054] The following examples provide illustrative embodiments of the invention. While the procedures in the Examples are performed in rodents, a skilled artisan will recognize that they are can be successfully performed within parameters clinically feasible in human subjects. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The examples do not in any way limit the invention.

EXAMPLES

Example 1 : Isolation of and culture of nonimmortalized NSCs [0055] Adult mice brains minus the cerebellum were cut into 1-2 mm pieces, tissue was mechanically and enzymatically dissociated with papain-protease-DNase solution, and then the digestion was stopped. The dissociated cells were partially purified from contaminating debris by using Percoll™ density gradients as described in Palmer et al. (1999) J. Neurosci., 19:8487-8497. The collected and thoroughly washed cells were grown on uncoated plates in N2 medium containing 20 ng/ml EGF, 20 ng/ml FGF-2 and 5 μg/ml heparin as described in Modern Techniques in Neuroscience Research, 1999, U. Windhorst and J. Johansson (eds.), Springer Verlag, Chapter 11. Neurosphere formation were detected 3-5 days after plating, and they increased in size over time.

Example 2: Survival, migration, and engraftment of nonimmortalized NSCs [0056] NSCs were isolated and proliferated as free-floating neurospheres as described in Example 1. Expanded NSCs were cultured to passages 7-9, made into a single-cell suspension, and labeled with 5 μM bromodeoxyuridine (brdU) prior to surgery. ASMKO mice, at different ages as indicated in Table 2, were anesthetized with isoflurane and mounted on a stereotaxic frame. An incision was made to expose the underlying skull, and a single drill hole was made over one hemisphere of each mouse. Stereotaxic injection (1 μl per injection site at 100,000 cells/μl) was performed unilaterally into one-three sites in various regions of the brain (striatum, hippocampus (HPC), thalamus (TH), cerebellum, and lateral ventricles). At various time points following injection, as indicated in Table 2, mice were anesthetized and sacrificed by cardiac perfusions and the brains were sectioned (50 μm coronal sections). [0057] Prior to administration, cells were labeled with BrdU in vitro and transplanted into cortex, striatum, hippocampus, thalamus, or cerebellum of the mouse brain (100,000 cells/site). Following a 6 to 12 week survival period, brain sections were processed with immunohistochemistry for BrdU and cell phenotypic markers. Transplanted cells migrated away from the injection sites and survived at least 12 weeks in different recipient regions. The overall distribution of the stem cells appeared to be dependent on the age of the host and the region of injection (Table 2). For example, cell migration was typically greater in neonates.

Table 2

[0058] For immunostaining, sections were pretreated for BrdU detection and stained with antibodies for BrdU (Accurate Chemical and Scientific Co., Westbury, NY), for the neuronal marker NeuN (Chemicon International, Temecula, CA), for the glial progenitor marker NG2 (Chemicon), or for the astroglial marker GFAP (DACO, Carpinteria, CA). Only few BrdU+ cells co-expressed the neuronal marker NeuN outside of the hippocampal granular cell layer. In all other regions, BrdU+ cells co-expressed GFAP (astrocytes), RIP (oligodendrocytes), or NG2 (oligodendrocyte precursors). These data indicate that, following injection into the ASMKO brain, nonimmortalized NSCs can survive for an extended period of time, spread throughout the entire subtended brain structure, and differentiate based on local environmental signals (FIGS. 1A-1 D). Example 3: Retroviral transduction of nonimmortalized NSCs [0059] The ASM retroviral vector was constructed by inserting the full-length human ASM cDNA (hASM) into the pLXIN or pDON replication-defective retroviral vectors, both containing a neomycin resistance gene Miller et al. (1989) Biotechniques, 7:980-990; Kim et al. (1998) J. Virology, 72:994-1004. Adult mouse progenitor cells were suspended and transduced by incubating with the medium containing either ASM vector for 3-4 hours as described in Pear et al. (1993) Proc. Nat. Acad. Sci. USA, 90:8392-8396. Cells were then plated and cultured in the presence of 100 μg/ml neomycin (G418). ASM activity in the cell pellets and culture media of transduced (NSCs/pDON.ASM and NSCs/pLXIN.ASM) and nontransduced cells are shown in Table 3.

Table 3

Example 4: Transplantation of nonimmortalized NSCs overexpressing ASM [0060] NSCs expressing ASM were made and cultured to passages 7-9 and labeled with brdU prior to surgery as described in Examples 1 & 3. Cells were stereotactically injected into the hippocampus and/or the thalamus of ASMKO mice as described in Example 2 and indicated in Table 4. Expression of ASM by the transplanted cells was evaluated using immunostaining with anti-human ASM antibodies (Genzyme Corp., Cambridge, MA). Total RNA was isolated from transplant site regions of the brain and the contralateral side for controls. cDNA was synthesized and used for a nested PCR using two sets of primers within exon2 of the human ASM gene (571 -bp product). Transplanted cells expressed ASM at all implantation sites as detected by the PCR, however, low levels of ASM were detectable by immunostaining.

Table 4

Example 5: Correction of lysosomal storage pathology [0061] The ability of NSCs expressing ASM to reverse the cholesterol abnormalities in the ASMKO brain was investigated using filipin staining. Filipin is an autofluorescent molecule isolated from Streptomyces filipinensis that binds to cholesterol complexes (Leventhal et al. (2001 ) J. Biol. Chem., 276:44976-44983; and Sarna et al. (2001) Eur. J. Neurosci., 13:1-9). Sphingomyelin has very strong affinity for interaction with cholesterol, which results in the sequestering of large amounts of cholesterol in the lysosome of ASMKO mice and human patients (Slotte (1997) Sucell. Biochem., 28:277-293; Viana et al. (1990) J. Med. Genet, 27:499-504). The ASMKO brains exhibit high levels of filipin staining due to overabundance of cholesterol, whereas normal mouse brains produce no filipin staining. [0062] ASMKO mice were treated as described in Example 4 and processed for filipin staining to examine the reduction of cholesterol/sphingomyelin deposits in the transplanted brain regions. The amount of fluorescent deposits visualized by filipin staining was markedly decreased at the transplantation sites (hippocampus and thalamus) compared to tissues from the untreated contralateral side. Further analysis shows clearance of deposits and distended lysosomes at the implant sites. These results indicate that correction of pathology occurred even despite the low levels of ASM expression by the transplanted NSCs (FIG. 2). [0063] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supercede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention. [0064] Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, treatment conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may very depending upon the desired properties sought to be obtained by the present invention. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS 1. A method of treating a mammal having lysosomal storage disease, the method comprising administering nonimmortalized neural stem cells into the brain of the mammal, wherein the neural stem cells secrete a lysosomal enzyme in an amount sufficient to result in clearance of abnormal accumulation of a substrate of the enzyme, thereby alleviating the lysosomal storage disease.

2. A method of treating a mammal having lysosomal storage disease, the method comprising administering nonimmortalized neural stem cells into the brain of the mammal, wherein the neural stem cells secrete a lysosomal enzyme in an amount sufficient to prevent or slow abnormal accumulation of a substrate of the enzyme, thereby alleviating the lysosomal storage disease.

3. The method of claim 1 or 2, wherein the neural stem cells are genetically modified to secrete a greater amount of the lysosomal enzyme relative to the same cells without the modification.

4. The method of claim 1 or 2, wherein the modification is by retroviral transduction.

5. The method of claim 3, wherein the modification is by lentiviral transduction.

6. The method of claim 1 or 2, wherein the lysosomal storage disease is chosen from at least one of late infantile Batten, infantile Batten, juvenile Batten, Gaucher, G I gangliosidosis, Hunter, Hurler, Krabbe, α-mannosidosis, β-mannosidosis, metachromatic leukodystrophy, Niemann-Pick, mucolipidosis ll/lll, Sandhoff, Sanfilippo, Schindler, Tay-Sachs, and Wolman diseases.

7. The method of claim 1 or 2, wherein the lysosomal storage disease is Niemann-Pick A disease.

8. The method of claim 1 or 2, wherein the lysosomal enzyme is acid sphingomyelinase.

9. The method of claim 1 or 2, wherein the mammal is human.

10. The method of claim 1 or 2, wherein the neural stem cells are human.

11. The method of claim 1 or 2, wherein the mammal is a neonate, juvenile, or adult

12. A method of delivering a lysosomal enzyme to a target site in the CNS of a mammal having a lysosomal storage disease, the method comprising: administering nonimmortalized neural stem cells to the brain of a mammal, wherein the nonimmortalized neural stem cells intraparenchymally migrate from the administration site to a target site and secrete the lysosomal enzyme for a period of time and in an amount sufficient to alleviate the lysosomal storage pathology in the target site.

13. The method of claim 12, wherein the administration site and the target site are at a distance of at least 1 mm from each other.

14. A method of treating Niemann-Pick disease in a mammal, the method comprising intracerebrally administering a therapeutically effective amount of nonimmortalized neural stem cells into the brain of the mammal, wherein the neural stem cell secrete acid sphingomyelinase in an amount sufficient to result in clearance of abnormal accumulation of a substrate of the enzyme.

15. A method of treating Niemann-Pick disease in a mammal, the method comprising intracerebrally administering a therapeutically effective amount of nonimmortalized neural stem cells into the brain of the mammal, wherein the neural stem cells secrete acid sphingomyelinase in an amount sufficient to prevent or slow abnormal accumulation of a substrate of the enzyme.

16. The method of claim 14 or 15, further comprising genetically modifying the neural stem sells to oversecrete acid sphingomyelinase prior to the administration.

17. The method of claim 16, wherein the genetic modification comprises transduction of the neural stem cells with a retroviral vector.

18. The method of claim 16, wherein the genetic modification comprises transduction of the neural stem cells with a lentiviral vector.

19. The method of claim 14 or 15, further comprising epigenetically propagating the neural stem cells in vitro prior to the administration.

20. The method of claim 19, wherein the neural stem cells are propagated in the presence of FGF-2 and EGF.

21^'. The method of claim 19, wherein the neural stem cells are propagated as adherent cultures.

22. The method of claim 19, wherein the neural stem cells are propagated as neurospheres.

23. The method of claim 14 or 15, wherein the neural stem cells migrate intraparenchymally from the administration site to a target site at a distance of least 1 mm.

24. Use of nonimmortalized neural stem cells in the manufacture of a medicament for treatment of a lysosomal storage disease, wherein the medicament is suitable for administration into the brain of a mammal having the lysosomal storage disease and the neural stem cells upon administration secrete a lysosomal enzyme in an amount sufficient to result in clearance of abnormal accumulation of a substrate of the enzyme and alleviate the lysosomal storage disease.

25. Use of nonimmortalized neural stem cells in the manufacture of a medicament for treatment of a lysosomal storage disease, wherein the medicament is suitable for administration into the brain of a mammal having the lysosomal storage disease and the neural stem cells upon administration secrete a lysosomal enzyme in an amount sufficient to prevent or slow abnormal accumulation of a substrate of the enzyme and alleviate the lysosomal storage disease.

26. The use of claim 24 or 25, wherein the neural stem cells are genetically modified to secrete a greater amount of the lysosomal enzyme relative to the same cells without the modification.

27. The use of claim 26, wherein the modification is by retroviral transduction.

28. The use of claim 24, 25, or 26, wherein the lysosomal storage disease is chosen from at least one of late infantile Batten, infantile Batten, juvenile Batten, Gaucher, GMI gangliosidosis, Hunter, Hurler, Krabbe, α-mannosidosis, β-mannosidosis, metachromatic leukodystrophy, Niemann-Pick, mucolipidosis ll/lll, Sandhoff, Sanfilippo, Schindler, Tay-Sachs, and Wolman diseases.

29. The use of claim 24, 25, or 26, wherein the lysosomal storage disease is Niemann-Pick A disease.

30. The use of claim 24, 25, or 26, wherein the lysosomal enzyme is acid sphingomyelinase.

31. The use of claim 24, 25, or 26, wherein the mammal is human.

32. The use of claim 24, 25, or 26, wherein the neural stem cells are human.

33. The use of claim 24, 25, or 26, wherein the mammal is a neonate, juvenile, or adult.

34. Use of nonimmortalized neural stem cells in the manufacture of a medicament for treatment of Niemann-Pick disease, wherein the medicament is suitable for administration into the brain of a mammal having the disease and the neural stem cells upon administration secrete a lysosomal enzyme in an amount sufficient to result in clearance of abnormal accumulation of a substrate of the enzyme and alleviate the lysosomal storage disease.

35. Use of nonimmortalized neural stem cells in the manufacture of a medicament for treatment of Niemann-Pick disease, wherein the medicament is suitable for administration into the brain of a mammal having the disease and the neural stem cells upon administration secrete a lysosomal enzyme in an amount sufficient to prevent or slow abnormal accumulation of a substrate of the enzyme and alleviate the lysosomal storage disease.

36. The use of claims 34 or 35, wherein the neural stem cells are genetically modified to oversecrete acid sphingomyelinase prior to the administration.

37. The use of claim 36, wherein the genetic modification comprises transduction of the neural stem cells with a retroviral vector.

38. The use of claim 36, wherein the genetic modification comprises transduction of the neural stem cells with a lentiviral vector.

39. The method of claim 34 or 35, wherein the neural stem cells are epigenetically propagated in vitro prior to the administration.

40. The method of claim 39, wherein the neural stem cells are propagated in the presence of FGF-2 and EGF.

41. The method of claim 40, wherein the neural stem cells are propagated as adherent cultures.

42. The method of claim 39, wherein the neural stem cells are propagated as neurospheres.

43. The use of claim 34 or 35, wherein the neural stem cells are capable of migrating intraparenchymally from the administration site to a target site at a distance of least 1 mm.