Adeno-associated virus mediated gene transfer to central nervous system

Abstract

To provide a method for preventing, inhibiting or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof.SOLUTION: A method comprises intrathecally administering to a mammal a composition comprising an effective amount of a recombinant adeno-associated virus (rAAV) vector comprising an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits or treats one or more symptoms associated with a disease of the central nervous system, where the rAAV is not AAV-2 or a permeation enhancer is intrathecally administered to the mammal.SELECTED DRAWING: Figure 15

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Patent Application No. 61 / 823,757, filed May 15, 2013, the disclosure of which is incorporated herein by reference.

[0002] STATEMENT OF GOVERNMENT RIGHTS This invention was made with government support under awards HD032652 and DK094538 awarded by the National Institutes of Health. The United States Government has certain rights in this invention. [Background technology]

[0003] background Mucopolysaccharidoses (MPS) are a group of 11 storage disorders caused by deranged glycosaminoglycan (GAG) catabolism, leading to GAG accumulation in lysosomes (Muenzer, 2004; Muñoz-Rojas et al., 2008). Manifestations of varying severity include organomegaly, skeletal dysplasia, cardiac and pulmonary obstruction, and neurological decline. In the case of MPS I, an iduronidase (IDUA) deficiency, severity ranges from mild (Scheie syndrome) to moderate (Hurler-Scheie syndrome) to severe (Hurler syndrome), resulting in neurological failure and death by age 15 (Muenzer, 2004; Muñoz-Rojas et al., 2008). Treatment for MPS has been largely symptomatic. However, some MPS disorders, including Hurler syndrome, have benefited from allogeneic hematopoietic stem cell transplantation (HSCT) (Krivit, 2004; Orchard et al., 2007; Peters et al., 2003). Additionally, enzyme replacement therapy (ERT) is becoming available for an increasing number of MPS disorders (Brady, 2006). While HSCT and ERT generally result in clearance of stored material and peripheral improvement, some problems persist after treatment (skeletal, cardiac, and corneal opacities). The greatest challenge with these cell and enzyme therapies is their effectiveness in addressing neurological findings, as peripherally administered enzymes do not penetrate the blood-brain barrier, and HSCT has been shown to be beneficial in some, but not all, MPS cases.

[0004] MPS I is one of the most extensively studied MPS disorders for the development of cell and molecular therapies. The effectiveness of allogeneic HSCT is likely the result of metabolic cross-correction, in which missing enzymes are released from donor-derived cells, then taken up by host cells and transported to lysosomes, contributing to lysosomal metabolism (Fratantoni et al., 1968). Subsequently, clearance of GAG-storage materials is observed in peripheral organs such as the liver and spleen, leading to relief of cardiopulmonary obstruction and improvement of corneal opacity (Orchard et al., 2007). Of particular importance is the effect of allogeneic stem cell transplantation on the development of neurological findings in MPS disorders. In this context, there is evidence for several MPS disorders that individuals engrafted with allogeneic stem cells experience improved outcomes compared with non-transplanted patients (Bjoraker et al., 2006; Krivit, 2004; Orchard et al., 2007; Peters et al., 2003). A central hypothesis explaining the neurological benefits of allogeneic hematopoietic stem cell transplantation is the penetration of donor-derived hematopoietic cells (presumably microglia) into the central nervous system (CNS) (Hess et al., 2004; Unger et al., 1993), where the engrafted cells express the missing enzymes, which then diffuse into CNS tissues and participate in the clearance of accumulated material. Therefore, the levels of enzymes provided to CNS tissues are limited to those expressed and released by donor-derived cells engrafted in the brain. Although such engraftment is highly beneficial in MPS I, recipients still continue to exhibit subnormal IQ and neurocognitive impairments (Ziegler and Shapiro, 2007).

[0005] The phenomenon of metabolic cross-compensation also explains the effectiveness of ERT for some lysosomal storage diseases, particularly MPS I (Brady, 2006). However, the effectiveness of enzyme therapy for the neurological manifestations of lysosomal storage diseases (LSDs) has not been observed because the enzyme missing in that particular LSD must penetrate the blood-brain barrier (BBB) ​​to effectively reach the CNS (Brady, 2006). Enzymes are, in most cases, too large and generally too charged to cross the BBB effectively. This has prompted research into invasive intrathecal enzyme administration (Dickson et al., 2007), which has demonstrated efficacy in a canine model of MPS I (Kakkis et al., 2004) and has begun human clinical trials for MPS I (Pastores, 2008; Muñoz-Rojas et al., 2008). The major drawbacks of enzyme therapy are its significant cost (over $200,000 per year) and the need for repeated injections of recombinant protein. Current clinical trials of intrathecal IDUA administration involve enzyme injections administered only once every three months, leaving the efficacy of this administration regimen uncertain. Summary of the Invention

[0006] Methods are described for preventing, inhibiting, and / or treating one or more symptoms associated with a central nervous system (CNS) disorder in a mammal in need thereof. The methods involve delivering to the CNS of the mammal in need of treatment a composition comprising an effective amount of a recombinant adeno-associated virus (rAAV) vector containing an open reading frame encoding a gene product, e.g., a therapeutic gene product. Target gene products that the rAAV vector may encode include, but are not limited to, alpha-L-iduronidase, iduronate-2-sulfatase, heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, or glucocerebrosidase. Diseases that can be prevented, inhibited, or treated using the methods disclosed herein include, but are not limited to, mucopolysaccharidosis type I disorder, mucopolysaccharidosis type II disorder, or mucopolysaccharidosis type VII disorder. AAV vectors can be administered in various ways to ensure that they are delivered to the CNS / brain and that the transgene is successfully transduced into the CNS / brain of a subject. Routes of delivery to the CNS / brain include, but are not limited to, intrathecal administration, intracranial administration, for example, intraventricular administration or lateral ventricle administration, intranasal administration, intravascular administration, and intraparenchymal administration.

[0007] In one embodiment, the method involves delivering a composition comprising an effective amount of an rAAV-9 vector containing an open reading frame encoding a gene to the CNS of an adult mammal in need of treatment. In one embodiment, the method involves delivering a composition comprising an effective amount of an rAAV-9 vector containing an open reading frame encoding IDUA to the CNS of an adult mammal in need of treatment. These methods are based in part on the discovery that AAV-9 vectors can efficiently transduce therapeutic transgenes into the brain / CNS of adult subjects, restoring enzyme levels to wild-type levels (Figure 15, see below). The results achieved using AAV-9 are surprising given previous work demonstrating that intravascular delivery of AAV-9 in adult mice does not achieve widespread, direct neural targeting (see Foust et al., 2009), and additional data showing that direct injection of AAV8-IDUA into the CNS of adult IDUA-deficient mice did not result in sufficient transgene expression (Figure 18). As proof of principle, the Examples described herein use a preclinical model for the treatment of MPS1, a genetic metabolic disorder caused by a deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). Surprisingly, these Examples demonstrate that direct injection of AAV9-IDUA into the CNS of immunocompetent adult IDUA-deficient mice resulted in IDUA enzyme expression and activity that was equal to or greater than that in wild-type adult mice (Figure 15, see below).

[0008] In a further embodiment of the present invention, the Examples also demonstrate that even higher levels of IDUA enzyme expression and activity can be achieved by combination therapy or treatment of immunocompromised animals to induce immunosuppression or immune tolerization. In one embodiment, patients with a genotype that promotes an immune response that neutralizes enzyme activity (e.g., Barbier et al., 2013) are treated with an immunosuppressant drug in addition to an rAAV vector containing an open reading frame encoding a gene product such as IDUA.

[0009] Neonatal IDUA- / - Mice are not immunocompetent. However, IDUA-expressing AAV-8 was administered to neonatal IDUA mice. - / - Administration to mice results in IDUA expression (Wolf et al., 2011), tolerizing the animals to IDUA. As described herein, the applicability of AAV-mediated gene transfer to adult (immunocompetent) mice via direct injection of AAV into the central nervous system was demonstrated using various routes of administration. For example, AAV-IDUA serotype 9 was administered by direct injection into the lateral ventricle of adult IDUA-deficient mice that were either immunocompetent, immunodeficient (NODSCID / IDUA- / -), immunosuppressed with cyclophosphamide (CP), or immunotolerized by weekly injections of human iduronidase protein (Aldurazyme) starting at birth. CP immunosuppressed animals were also administered AAV9-IDUA by intranasal, intrathecal, and intravascular infusion, with and without mannitol to disrupt the blood-brain barrier. Eight weeks after vector administration, animals were sacrificed, and brains were harvested and microdissected to assess IDUA enzyme activity, tissue glycosaminoglycans, and IDUA vector sequences compared with normal and diseased control mice. The results of these studies indicate that numerous routes can be used to directly administer AAV vectors to the CNS, for example, to achieve higher levels of protein delivery and / or enzyme activity in the CNS. Additionally, although the brain is an immune-privileged site, administration of immunosuppressants or immune tolerization can increase the activity found in the brain after AAV administration. Routes of administration that result in higher expression levels per administration and / or less invasive routes may be clinically preferable for patients.

[0010] Thus, the present invention encompasses the use of recombinant AAV (rAAV) vectors encoding gene products that have a therapeutic effect when expressed in the CNS of a mammal. In one embodiment, the mammal is an immunocompetent mammal with a CNS disease or disorder (neurological disease). As used herein, an "immunocompetent" mammal refers to a mammal of an age at which, following exposure to antigenic stimuli, both cellular and humoral immune responses are elicited through Th1 function or upregulation of IFN-γ production in response to polyclonal stimuli, as opposed to a newborn infant, which possesses both innate immunity and immunity acquired from its mother, e.g., during pregnancy or through lactation. An example of an immunocompetent mammal is an adult mammal without an immunodeficiency disorder. For example, an immunocompetent human is typically at least 1, 2, 3, 4, 5, or 6 months of age and includes an adult without an immunodeficiency disorder. In one embodiment, the AAV is administered intrathecally. In one embodiment, the AAV is administered intracranially (e.g., intracerebroventricularly). In one embodiment, the AAV is administered intranasally, with or without a penetration enhancer. In one embodiment, AAV is administered intravascularly, for example, by carotid administration, with or without a penetration enhancer. In one embodiment, the mammal to which AAV is administered is immunodeficient or is subjected to immunotolerization or immunosuppression, such that a higher level of therapeutic protein expression is induced, for example, compared to a corresponding mammal to which AAV is administered without immunotolerization or immunosuppression. In one embodiment, an immunosuppressant is administered to induce immunosuppression. In one embodiment, the mammal to which AAV is administered is not subjected to immunotolerization or immunosuppression (e.g., a therapeutic effect is achieved by administration of AAV alone).

[0011] The present invention provides a method for preventing, inhibiting, and / or treating one or more symptoms associated with a disease or disorder of the central nervous system in a mammal in need thereof. The method comprises administering to the mammal intrathecally (e.g., into the lumbar region) or intracerebroventricularly (e.g., into the lateral ventricle) an effective amount of a composition comprising an rAAV vector containing an open reading frame encoding a gene product whose expression in the central nervous system of the mammal prevents, inhibits, or treats the one or more symptoms. In one embodiment, the gene product is a lysosomal storage enzyme. In one embodiment, the mammal is an immunocompetent adult. In one embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In one embodiment, the mammal is a human. In one embodiment, multiple administrations are performed. In one embodiment, the composition is administered weekly, monthly, or at intervals of two or more months.

[0012] In one embodiment, the method comprises intrathecally administering (e.g., into the lumbar region) to a mammal a composition comprising an effective amount of a rAAV vector comprising an open reading frame encoding a gene product whose expression in the central nervous system of said mammal prevents, inhibits, or treats said one or more symptoms, and optionally administering a penetration enhancer. In one embodiment, the penetration enhancer is administered before said composition. In one embodiment, said composition comprises a penetration enhancer. In one embodiment, the penetration enhancer is administered after said composition. In one embodiment, the gene product is a lysosomal storage enzyme. In one embodiment, the mammal is an immunocompetent adult. In one embodiment, the rAAV vector is an AAV-1, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In one embodiment, the mammal is a human. In one embodiment, multiple administrations are performed. In one embodiment, the composition is administered weekly, monthly, or at intervals of two months or more. In one embodiment, the mammal receiving AAV intrathecally is not subjected to immunotolerization or immunosuppression (e.g., administration of AAV alone provides a therapeutic effect). In one embodiment, the mammal receiving AAV intrathecally is immunodeficient or is subjected to immunotolerization or immunosuppression so as to induce a higher level of therapeutic protein expression, e.g., compared to a corresponding mammal receiving AAV intrathecally without immunotolerization or immunosuppression.

[0013] In one embodiment, the method comprises administering intracerebroventricularly (e.g., into a lateral ventricle) to an immunocompetent mammal a composition comprising an effective amount of an rAAV vector comprising an open reading frame encoding a gene product whose expression in the central nervous system of the mammal prevents, inhibits, or treats one or more symptoms. In one embodiment, the gene product is a lysosomal storage enzyme. In one embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV rh10, or AAV-9 vector. In one embodiment, the rAAV vector is not an rAAV-5 vector. In one embodiment, the mammal is a human. In one embodiment, multiple administrations are performed. In one embodiment, the composition is administered weekly, monthly, or at intervals of two or more months. In one embodiment, the mammal receiving intracerebroventricularly administered AAV is not subject to immune tolerization or immune suppression (e.g., administration of AAV alone provides a therapeutic effect). In one embodiment, the mammal to which the AAV is administered intracerebroventricularly is immunodeficient or has been subjected to immunotolerization or immunosuppression, e.g., to induce higher levels of therapeutic protein expression compared to a corresponding mammal to which the AAV is administered intracerebroventricularly without being subjected to immunotolerization or immunosuppression. In one embodiment, the mammal is tolerized to the gene product prior to administering the composition comprising the AAV.

[0014] Also provided is a method for preventing, inhibiting, or treating one or more symptoms associated with a disease or disorder of the central nervous system in a mammal in need thereof. The method comprises intravascularly administering to the mammal a composition comprising an effective amount of an rAAV vector comprising an open reading frame encoding a gene product whose expression in the central nervous system of the mammal prevents, inhibits, or treats the one or more symptoms, and an effective amount of a penetration enhancer. In one embodiment, the composition comprises the penetration enhancer. In one embodiment, the penetration enhancer comprises mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA. In one embodiment, the gene product is a lysosomal storage enzyme. In one embodiment, the mammal is an immunocompetent adult. In one embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In one embodiment, the rAAV vector is not an rAAV-5 vector. In one embodiment, the mammal is human. In one embodiment, multiple administrations are performed. In one embodiment, the composition is administered once weekly. In one embodiment, the composition is administered once weekly, once monthly, or at intervals of two months or more. In one embodiment, the mammal receiving intravascular administration of AAV is not subject to immunotolerization or immunosuppression (e.g., administration of AAV provides a therapeutic effect). In one embodiment, the mammal receiving intravascular administration of AAV is immunodeficient or is subject to immunotolerization or immunosuppression such that a higher level of therapeutic protein expression is induced, e.g., compared to a corresponding mammal receiving intravascular administration of AAV without immunotolerization or immunosuppression.

[0015] In one embodiment, the method comprises intranasally administering to a mammal a composition comprising an effective amount of an rAAV-9 vector comprising an open reading frame encoding a gene product whose expression in the central nervous system of the mammal prevents, inhibits, or treats one or more symptoms, and optionally administering a penetration enhancer. In one embodiment, intranasal delivery can be accomplished as described in U.S. Patent No. 8,609,088, the disclosure of which is incorporated herein by reference. In one embodiment, the penetration enhancer is administered before the composition. In one embodiment, the composition includes a penetration enhancer. In one embodiment, the penetration enhancer is administered after the composition. In one embodiment, the gene product is a lysosomal storage enzyme. In one embodiment, the mammal is an immunocompetent adult. In one embodiment, the mammal is a human. In one embodiment, multiple administrations are performed. In one embodiment, the composition is administered weekly, monthly, or at intervals of two or more months. In one embodiment, the mammal receiving AAV intranasally is not subject to immune tolerization or immune suppression. In one aspect, a mammal receiving AAV intranasally is subjected to immunotolerization or immunosuppression such that higher levels of IDUA protein expression are induced, for example, compared to a corresponding mammal receiving AAV intranasally without being subjected to immunotolerization or immunosuppression.

[0016] Also provided is a method for preventing, inhibiting, or treating one or more symptoms associated with a central nervous system disease in a mammal in need thereof. The method comprises administering to the mammal a composition comprising an effective amount of an rAAV vector comprising an open reading frame encoding a gene product whose expression in the central nervous system of the mammal prevents, inhibits, or treats the one or more symptoms, and an immunosuppressant. In one embodiment, the immunosuppressant comprises cyclophosphamide. In one embodiment, the immunosuppressant comprises a glucocorticoid, a cytostatic agent such as an alkylating agent, or an antimetabolite such as methotrexate, azathioprine, mercaptopurine, or an agent active against a cytotoxic antibiotic, an antibody, or an immunophilin. In one embodiment, the immunosuppressant comprises a nitrogen mustard, a nitrosourea, a platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, an antibody to the IL2 receptor (CD25) or CD3, an anti-IL-2 antibody, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, an opioid, or a TNF-α (tumor necrosis factor alpha) binding agent, such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira). In one embodiment, the rAAV and the immunosuppressant are co-administered. In one embodiment, the rAAV is administered before and optionally after the immunosuppressant. In one embodiment, the immunosuppressant is administered before the rAAV. In one embodiment, the rAAV and the immunosuppressant are administered intrathecally. In one embodiment, the rAAV and the immunosuppressant are administered intracerebroventricularly. In one embodiment, the rAAV is administered intrathecally and the immunosuppressant is administered intravenously. In one embodiment, the gene product is a lysosomal storage enzyme. In one embodiment, the mammal is an adult. In one embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In one embodiment, the mammal is a human. In one embodiment, multiple administrations are performed. In one embodiment, the composition is administered once a week.In one embodiment, the composition is administered once a week, once a month, or at intervals of 2 months or more.

[0017] The present invention also provides a method for preventing, inhibiting, or treating one or more symptoms associated with a central nervous system disease in a mammal in need thereof. A mammal immunotolerized to a gene product associated with the disease is administered a composition comprising an effective amount of an rAAV vector comprising an open reading frame encoding a gene product whose expression in the central nervous system of the mammal prevents, inhibits, or treats the one or more symptoms. In one embodiment, the gene product is a lysosomal storage enzyme. In one embodiment, the mammal is an adult. In one embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In one embodiment, the mammal is a human. In one embodiment, multiple administrations are performed. In one embodiment, the composition is administered once a week.

[0018] Gene products that can be encoded by rAAV vectors include, but are not limited to, alpha-L-iduronidase, iduronate-2-sulfatase, heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase, fibroblast growth factor 2 (FGF-2), brain-derived growth factor (BDGF), neurturin, glial-derived growth factor (GDGF), tyrosine hydroxylase, dopamine decarboxylase, or glutamic acid decarboxylase.

[0019] Diseases with one or more neurological symptoms that may be prevented, inhibited, or treated using the methods disclosed herein include adrenoleukodystrophy, Alzheimer's disease, amyotrophic lateral sclerosis, Angelman syndrome, ataxia-telangiectasia, Charcot-Marie-Tooth syndrome, Cockayne syndrome, deafness, Duchenne muscular dystrophy, epilepsy, essential tremor, Fragile X syndrome, Friedreich's ataxia, Gaucher disease, Huntington's disease, and Lesch-Nyhan syndrome. In one embodiment, the disease includes, but is not limited to, leukemia, encephalopathy syndrome ... Lysosomal storage diseases include mucopolysaccharidoses (MPS), such as mucopolysaccharidoses type I, e.g., Hurler syndrome and its variants, Scheie syndrome and Hurler-Scheie syndrome (deficiency of alpha-L-iduronidase); Hunter syndrome (deficiency of iduronate-2-sulfatase); mucopolysaccharidoses type III, e.g., Sanfilippo syndrome (A, B, C, or D; deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl-CoA); A: alpha-glucosaminide N-acetyltransferase or N-acetylglucosamine-6-sulfate sulfatase deficiency; mucopolysaccharidosis type IV, e.g., Morquio syndrome (galactosamine-6-sulfate sulfatase or beta-galactosidase deficiency); mucopolysaccharidosis type VI, e.g., Maroteaux-Lamy syndrome (arylsulfatase B deficiency); mucopolysaccharidosis type II; mucopolysaccharidosis type III (A, B, C, or D; heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl-CoA:alpha-glucosaminide N-acetyltransferase, or N-acetylglucosamine-6-sulfate sulfatase deficiency); mucopolysaccharidosis type IV (A or B;Galactosamine-6-sulfatase and beta-galactosidase deficiency); mucopolysaccharidosis type VI (arylsulfatase B deficiency); mucopolysaccharidosis type VII (beta-glucuronidase deficiency); mucopolysaccharidosis type VIII (glucosamine-6-sulfate sulfatase deficiency); mucopolysaccharidosis type IX (hyaluronidase deficiency); Tay-Sachs disease (alpha subunit deficiency of beta-hexosaminidase); Sandhoff disease (deficiency of both the alpha and beta subunits of beta-hexosaminidase); GM1 gangliosidosis (type I or type II); Fabry disease (alpha-galactosidase deficiency); metachromatic leukodystrophy (arylsulfatase A deficiency); Pompe disease (acid maltase deficiency); fucosidosis (fucosidase deficiency); alpha-mannosidosis (alpha-mannosidase deficiency); beta-mannosidosis (beta-mannosidase deficiency), ceroid lipofuscinosis, and Gaucher disease (types I, II, and III; glucocerebrosidase deficiency), as well as disorders such as Hermansky-Pudlak syndrome; amaurotic idiopathic ... 1); Nephropathic cystinosis; Fanconi-Bickel syndrome; Farber lipogranulomatosis; fibromatosis; geleophysic dysplasia Glycogen storage disease type I; Glycogen storage disease Ib; Glycogen storage disease Ic; Glycogen storage disease type III; Glycogen storage disease type IV; Glycogen storage disease type V; Glycogen storage disease type VI; Glycogen storage disease type VII; Glycogen storage disease type 0; Immunoosseous dysplasia, Schimke type; Lipidosis; Lipase B; Mucolipidosis II; Mucolipidosis II, including subtypes; mucolipidosis IV; neuraminidase deficiency with beta-galactosidase deficiency; mucolipidosis I; Niemann-Pick disease (sphingomyelinase deficiency); Niemann-Pick disease without sphingomyelinase deficiency (deficiency of the npc1 gene, which encodes a cholesterol-metabolizing enzyme); Refsum disease; Sea-Blue histiocytosis; childhood sialic acid storage disorders;These include, but are not limited to, sialic aciduria; multiple sulfatase deficiency; triglyceride storage disease with impaired long-chain fatty acid oxidation; Winchester disease; Wolman disease (cholesterol ester hydrolase deficiency); deoxyribonuclease I-like 1 disorder; arylsulfatase E disorder; lysosomal H+ transporting ATPase subunit 1 disorder; glycogen storage disease IIb; Ras-related protein rab9 disorder; X-linked recessive chondrodysplasia punctata 1 disorder; glycogen storage disease VIII; lysosomal membrane protein 2 disorder; Menkes syndrome; congenital glycosylation disorder type Ic; and sialic aciduria. Replenishing levels of lysosomal storage enzymes at levels less than 20%, e.g., less than 10%, or about 1% to 5% of those found in unaffected mammals may prevent, inhibit, or treat neurological conditions, such as neurodegeneration, in mammals.

[0020] In one embodiment, the method described herein involves delivering a composition comprising an effective amount of an rAAV-9 vector containing an open reading frame encoding IDUA to the CNS of an immunocompetent human in need of treatment. Routes of administration to the CNS / brain include, but are not limited to, intrathecal administration, intracranial administration, such as intraventricular or lateral ventricle administration, intranasal administration, intravascular administration, and intraparenchymal administration.

[0021] Other viral vectors can also be used in the methods of the present invention, such as retroviral vectors, lentiviral vectors, adenoviral vectors, Semliki Forest virus vectors, or herpes simplex virus vectors. [The present invention 1001] 1. A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, the method comprising the step of intrathecally administering to the mammal a composition comprising an effective amount of a recombinant adeno-associated virus (rAAV) vector comprising an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms, wherein the rAAV is not AAV-2 or a penetration enhancer is administered intrathecally to the mammal. [The present invention 1002] A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, said method comprising the step of intracerebroventricularly administering to said mammal an effective amount of a recombinant adeno-associated virus (rAAV) vector comprising an open reading frame encoding a gene product whose expression in said mammal prevents, inhibits, or treats said one or more symptoms, wherein said rAAV is not AAV-8 or said mammal is an adult. [The present invention 1003] A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, comprising intravascularly administering to the mammal a composition comprising an effective amount of an rAAV vector comprising an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms, and an effective amount of a penetration enhancer. [The present invention 1004] The method of claim 1003, wherein the composition comprises a penetration enhancer. [The present invention 1005] The method of any one of claims 1001 to 1004, wherein the penetration enhancer comprises mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA. [The present invention 1006] A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, comprising administering to the mammal an effective amount of a composition comprising an rAAV vector comprising an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms, and an immunosuppressant. [The present invention 1007] 1006. The method of claim 1006, wherein the immunosuppressant comprises cyclophosphamide. [The present invention 1008] 1006. The method of claim 1006, wherein the immunosuppressant comprises a glucocorticoid, a cytostatic agent including an alkylating agent, an antimetabolite, a cytotoxic antibiotic, an antibody, or an agent active against an immunophilin. [The present invention 1009] 1007. The method of claim 1008, wherein the immunosuppressant comprises a nitrogen mustard, a nitrosourea, a platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, an antibody to the IL-2 receptor (CD25) or CD3, an anti-IL-2 antibody, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, an opioid, or a TNF-α (tumor necrosis factor alpha) binding agent. [The present invention 1010] The method of any of claims 1006 to 1009, wherein the rAAV and the immunosuppressant are administered simultaneously or the immunosuppressant is administered after the rAAV. [The present invention 1011] The method of any one of claims 1006 to 1010, wherein the rAAV and the immunosuppressant are administered intrathecally. [The present invention 1012] The method of any one of claims 1006 to 1010, wherein the rAAV and the immunosuppressant are administered intracerebroventricularly. [The present invention 1013] A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, comprising the steps of: providing a mammal that has been immunotolerized to a gene product associated with the disease; and administering to the mammal a composition comprising an effective amount of an rAAV vector that includes an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms. [The present invention 1014] The method of any of claims 1001 to 1013, wherein the gene product is a defective enzyme or is deficient in the mammal, thereby resulting in a lysosomal storage disease. [The present invention 1015] The method of any of claims 1001 to 1014, wherein the mammal is an immunocompetent adult. [The present invention 1016] The method of any of claims 1001 to 1015, wherein the rAAV vector is an rAAV-1 vector, an rAAV-3 vector, an rAAV-4 vector, an rAAV-5 vector, an rAAV rh10 vector, or an rAAV-9 vector. [The present invention 1017] The method of any of claims 1001 to 1016, wherein the gene product is alpha-L-iduronidase, iduronate-2-sulfatase, heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, or glucocerebrosidase. [The present invention 1018] The method of any one of claims 1001 to 1017, wherein the mammal is a human. [The present invention 1019] The method of any one of claims 1001 to 1018, wherein the disease is alpha-L-iduronidase deficiency. [The present invention 1020] The method of any of claims 1001 to 1018, wherein the disease is a mucopolysaccharidosis type I disorder, a mucopolysaccharidosis type II disorder, or a mucopolysaccharidosis type VII disorder. [The present invention 1021] The method of any one of claims 1001 to 1020, wherein multiple administrations are performed. [The present invention 1022] The method of any of claims 1001 to 1020, wherein the composition is administered once a week. [The present invention 1023] The method of any of claims 1001 to 1022, wherein administering prevents, inhibits or treats neurodegeneration. [The present invention 1024] The method of any of claims 1001 to 1012 or 1014 to 1023, wherein the mammal is immunotolerized to the gene product prior to administration of the composition. [Brief explanation of the drawings]

[0022] [Figure 1] Experimental design for iduronidase-deficient mice in which IDUA-AAV was administered intracerebroventricularly (ICV) or intrathecally. To prevent an immune response, animals were immunosuppressed with cyclophosphamide (CP) or tolerized at birth with intravenous administration of human iduronidase protein (aldurazyme). Injections were performed in NOD-SCID immunodeficient mice that were also iduronidase-deficient. At the indicated time points after treatment, animals were sacrificed, brains were microdissected, and extracts were assayed for iduronidase activity. [Figure 2] IDUA activity in immunodeficient IDUA-deficient animals. [Figure 3] IDUA activity in immunosuppressed animals in which AAV vectors were administered by the ICV route. [Figure 4] IDUA activity in immunosuppressed animals in which AAV vectors were administered by the IT route. [Figure 5] IDUA activity in tolerized animals administered AAV vectors ICV. [Figure 6] All mean IDUA activity levels compiled for side-by-side comparison. [Figure 7] Data grouped according to brain region. [Figure 8]Assay of GAG accumulation material in various sections of the brain for all four test groups. [Figure 9] Outline of experimental design. [Figure 10] Intracranial injection of AAV9IDUA into immunodeficient MPS I mice. Adult animals were injected with 10 vector genomes, and iduronidase expression in the brain was assessed 10 weeks later. Enzyme activity levels in the brain were significantly higher than in wild-type animals, ranging from 30- to 300-fold higher than wild-type. [Figure 11] Intracranial administration of AAV9IDUA in immunocompetent IDUA-deficient mice. Adult animals were injected with 10 vector genomes and immunosuppressed with weekly injections of cyclophosphamide (CP). CP injections were terminated 6 weeks after vector injection due to poor health, and animals were sacrificed 8 weeks after injection. Brains were microdissected and assayed for IDUA enzyme activity. [Figure 12] Intracranial injection of AAV9IDUA into immune-tolerized MPS I mice. MPS I mice were tolerized with Aldurazyme, either a single dose at birth or multiple weekly doses starting at birth. Mice were injected with the vector at 4 months of age and sacrificed 11 weeks after injection. Brains were microdissected and analyzed for iduronidase expression. Enzyme activity ranged on average from 10- to 1000-fold higher than wild-type levels. [Figure 13] Intrathecal administration of AAV9IDUA in immunocompetent IDUA-deficient animals. Adult MPS I mice were intrathecally administered AAV9IDUA followed by a weekly immunosuppressive regimen of cyclophosphamide. Eleven weeks after injection, the animals were sacrificed, and the brains and spinal cords were analyzed for IDUA enzyme activity. [Figure 14]Intrathecal injection of AAV9IDUA in immune-tolerized MPS I mice. IDUA-deficient animals were tolerized with Aldurazyme, either a single dose at birth or multiple weekly doses starting at birth. Animals were intrathecally injected with AAV9IDUA vectors at 4 months of age. Ten weeks after injection, animals were sacrificed, their brains microdissected, and assayed for iduronidase activity. Enzyme activity was restored in all brain regions, with activity in the cerebellum ranging from 200- to 1500-fold higher than wild-type levels. The levels of enzyme activity in the olfactory bulb and cerebellum (to the right of the dashed line) correspond to the right Y-axis. [Figure 15] Intrathecal injection of AAV9IDUA in immunocompetent MPS I animals. Control MPS I animals were injected with the AAV9IDUA vector but were not immunosuppressed or tolerized. The animals were sacrificed 11 weeks after vector injection, and their brains were assayed for iduronidase activity. Enzyme levels recovered to wild-type levels in all brain regions but were significantly lower than those in animals that were either immunosuppressed or tolerized. [Figure 16] Normalization of glycosaminoglycan (GAG) levels after intracranial or intrathecal AAV9 injection. Immunodeficient, immunosuppressed, or tolerized MPS I mice were injected intracranially or intrathecally with AAV9IDUA, as indicated. Animals were sacrificed 8–11 weeks after injection, and brains were microdissected and assayed for GAG levels. In all groups analyzed, GAG accumulation recovered to or near wild-type levels. [Figure 17] IDUA vector copy number in the brain. Microdissected brains were analyzed by QPCR for IDUA vector sequences. Copy numbers in intracranially and intrathecally injected mice correlate with the levels of enzyme activity as illustrated in Figures 11 and 13. [Figure 18] ICV injection of AAV8-MCI into adult animals. [Figure 19]Intranasal administration of AAV9 / IDUA in immunocompetent IDUA-deficient animals. Adult MPS I mice were intranasally instilled with AAV9 / IDUA followed by a weekly immunosuppressive regimen of cyclophosphamide. Twelve weeks after injection, animals were sacrificed and brains were analyzed for IDUA enzyme activity. [Figure 20] IDUA vector copy number in the brain. Microdissected brains were analyzed by QPCR for IDUA vector sequences. Copy number in intranasally injected mice correlates with enzyme levels in Figure 19. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention definition As used herein, "individual" (as in the subject of treatment) refers to a mammal. Mammals include, for example, humans; non-human primates, such as apes and monkeys; and non-primates, such as dogs, cats, rats, mice, cows, horses, sheep, and goats. Non-mammals include, for example, fish and birds.

[0024] The terms "disease" and "disorder" are used interchangeably and refer to diseases or conditions in which the absence or reduced amount of a particular gene product, e.g., a lysosomal storage enzyme, is involved in the disease and in which a therapeutically beneficial effect can be achieved by replenishing, e.g., to at least 1% of normal levels.

[0025] "Substantially," as used herein, means completely or almost completely. For example, a composition that is "substantially free" of a component either does not contain the component at all, or contains such a small amount of the component that its presence does not affect any relevant functional properties of the composition. Alternatively, a compound is "substantially pure" if only negligible trace amounts of impurities are present.

[0026] As used herein, "treat" or "treatment" refers to the alleviation of symptoms associated with a disorder or disease, "inhibit" means inhibiting further progression or worsening of symptoms associated with a disorder or disease, and "prevent" refers to the prevention of symptoms associated with a disorder or disease.

[0027] As used herein, an "effective amount" or "therapeutically effective amount" of an agent of the invention, e.g., a recombinant AAV encoding a gene product, refers to an amount of agent that alleviates, in whole or in part, the symptoms associated with a disorder or condition, halts or slows the further progression or worsening of those symptoms, or prevents or results in the prophylaxis of a disorder or condition, e.g., an amount effective to prevent, inhibit, or treat one or more neurological symptoms in an individual.

[0028] In particular, a "therapeutically effective amount" refers to an amount that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compounds of the invention are outweighed by the therapeutically beneficial effects.

[0029] As used herein, a "vector" refers to a polymer or polymeric assembly that contains or associates with a polynucleotide and can be used to mediate the delivery of the polynucleotide to cells in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotide to be delivered may be referred to as a "target polynucleotide" or "transgene," and may include a coding sequence of interest in gene therapy (e.g., a gene encoding a protein of therapeutic interest) and / or a selectable or detectable marker.

[0030] "AAV" refers to an adeno-associated virus and may refer to the virus itself or its derivatives. The term encompasses all subtypes, serotypes, and pseudotypes, as well as both native and recombinant forms, unless otherwise specified. As used herein, the term "serotype" refers to an AAV identified by its binding characteristics and distinguished from other AAVs based on those binding characteristics. For example, there are 11 serotypes of AAV, AAV-1 to AAV-11, including AAV-2, AAV-5, AAV-8, AAV-9, and AAV rh10. The term "serotype" encompasses pseudotypes with the same binding characteristics. Thus, for example, the AAV-5 serotype includes AAVs with the binding characteristics of AAV-5, such as pseudotyped AAVs containing an AAV-5 capsid and a rAAV genome or chimeric rAAV genome that is not derived from or derived from AAV-5. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector").

[0031] An "AAV virus" is a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "rAAV." AAV "capsid protein" encompasses not only wild-type AAV capsid proteins, but also modified AAV capsid proteins that are structurally and / or functionally capable of packaging the rAAV genome and that bind to at least one specific cellular receptor (which may be different from the receptor used by wild-type AAV). Modified AAV capsid proteins include chimeric AAV capsid proteins, e.g., those having amino acid sequences from more than one serotype of AAV, such as a capsid protein formed from a portion of the capsid protein from AAV-5 fused or linked to a portion of the capsid protein from AAV-2, and AAV capsid proteins having tags or other detectable non-AAV capsid peptides or proteins fused or linked to the AAV capsid protein (e.g., a portion of an antibody molecule that binds to the transferrin receptor can be recombinantly fused to an AAV-2 capsid protein).

[0032] A "pseudotyped" rAAV is an infectious virus having any combination of AAV capsid proteins and an AAV genome. Capsid proteins from any AAV serotype can be used with rAAV genomes derived or obtainable from wild-type AAV genomes of different serotypes, or with rAAV genomes that are chimeric genomes (i.e., formed from AAV DNA from two or more different serotypes), such as rAAVs with two inverted terminal repeats (ITRs), each ITR from a different serotype or a chimeric ITR. The use of chimeric genomes, such as those containing ITRs from two AAV serotypes or chimeric ITRs, can result in directional recombination, which can further enhance the production of transcriptionally active intermolecular concatemers. Thus, the 5' and 3' ITRs in the rAAV vectors of the present invention can be homologous (i.e., from the same serotype), heterologous (i.e., from different serotypes), or chimeric (i.e., ITRs with ITR sequences from two or more AAV serotypes).

[0033] rAAV vector Any serotype of adeno-associated virus is suitable for preparing rAAV, since the various serotypes are functionally, structurally, and even genetically related. All AAV serotypes appear to exhibit similar replication characteristics mediated by homologous rep genes, and all serotypes generally possess three related capsid proteins, such as those expressed in AAV2. This relatedness is further suggested by heteroduplex analysis, which shows extensive cross-hybridization between serotypes throughout the genome, and the presence of similar self-annealing segments at the termini corresponding to the ITRs. Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Among the various AAV serotypes, AAV2 is the most commonly used.

[0034] The AAV vector of the present invention typically contains a polynucleotide heterologous to AAV. This polynucleotide is typically of interest in the context of gene therapy because it has the ability to provide a function to the target cell, such as up-regulating or down-regulating the expression of a certain phenotype. Such heterologous polynucleotides or "transgenes" generally have a length sufficient to provide the desired function or coding sequence.

[0035] If transcription of a heterologous polynucleotide is desired in the intended target cell, the heterologous polynucleotide can be operably linked to its own promoter or to a heterologous promoter, as is known in the art, depending, for example, on the desired level of transcription and / or the desired transcription specificity in the target cell. Various types of promoters and enhancers are suitable for use in this context. Constitutive promoters provide continuous levels of gene transcription and may be preferred when continuous expression of a therapeutic or prophylactic polynucleotide is desired. Inducible promoters generally exhibit low activity in the absence of an inducer and are upregulated in the presence of an inducer. Inducible promoters may be preferred when expression is desired only at certain times or locations, or when it is desirable to titrate the level of expression using an inducer. Promoters and enhancers may be tissue-specific, i.e., they exhibit their activity only in certain cell types, perhaps due to gene regulatory elements uniquely found in those cells.

[0036] Illustrative examples of promoters include the SV40 late promoter from simian virus 40, the baculovirus polyhedron enhancer / promoter element, herpes simplex virus thymidine kinase (HSV tk), the cytomegalovirus (CMV) immediate-early promoter, and various retroviral promoters containing LTR elements. Inducible promoters include heavy metal ion-inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters) and promoters from T7 phage that are active in the presence of T7 RNA polymerase. Illustrative examples of tissue-specific promoters include various surfactin promoters (for expression in the lung), myosin promoters (for expression in muscle), and albumin promoters (for expression in the liver). A wide variety of other promoters are known and commonly available in the art, and the sequences of many of these promoters can be obtained from sequence databases such as the GenBank database.

[0037] If translation in the intended target cell is also desired, the heterologous polynucleotide will preferably also contain regulatory elements that facilitate translation (e.g., a ribosome binding site, or "RBS," and a polyadenylation signal). Thus, a heterologous polynucleotide generally contains at least one coding region operably linked to a suitable promoter, and may also contain, for example, an operably linked enhancer, a ribosome binding site, and a polyA signal. A heterologous polynucleotide may contain one coding region or two or more coding regions under the control of the same promoter or different promoters. The entire unit containing the combination of regulatory elements and coding regions is often referred to as an expression cassette.

[0038] The heterologous polynucleotide is recombinantly integrated into or replaces the AAV genome coding region (i.e., replaces the AAV rep and cap genes), typically flanked on both sides by AAV inverted terminal repeat (ITR) regions. This means that the ITRs appear immediately juxtaposed, both upstream and downstream of the coding sequence, without (but not necessarily) any intervening sequences of AAV origin, to reduce the possibility of recombination that might, for example, regenerate a replication-competent AAV genome. However, a single ITR may be sufficient to perform the functions normally associated with constructs containing two ITRs (see, e.g., WO 94 / 13788), and therefore vector constructs having only a single ITR may also be used with the packaging and production methods of the invention.

[0039] The native promoter for rep is autoregulatory and can limit the amount of AAV particles produced. Whether rep is provided as part of the vector construct or separately, the rep gene can also be operably linked to a heterologous promoter. Any heterologous promoter that is not strongly downregulated by rep gene expression is suitable, but an inducible promoter would be preferred because constitutive expression of the rep gene can have negative effects on host cells. A wide variety of inducible promoters are known in the art, including, for example, heavy metal ion-inducible promoters (such as the metallothionein promoter), steroid hormone-inducible promoters (such as the MMTV promoter or growth hormone promoter), and promoters derived from T7 phage that are active in the presence of T7 RNA polymerase. One subclass of inducible promoters is one that is induced by a helper virus used to complement rAAV vector replication and packaging. Several helper virus-inducible promoters have also been described, such as the adenovirus early gene promoter induced by the adenovirus E1 A protein, the adenovirus major late promoter, herpesvirus promoters induced by herpesvirus proteins such as VP16 or 1CP4, as well as vaccinia or poxvirus-inducible promoters.

[0040] Methods for identifying and testing helper virus-inducible promoters have been described (see, e.g., WO 96 / 17947). Accordingly, methods for determining whether candidate promoters are helper virus-inducible and whether they are useful for generating high-efficiency packaging cells are known in the art. Briefly, in one such method, the p5 promoter of the AAV rep gene is replaced with a putative helper virus-inducible promoter (known in the art or identified using well-known techniques, such as linking to a promoterless "reporter" gene). The AAV rep-cap gene (with p5 replaced) linked to a positive selectable marker, e.g., an antibiotic resistance gene, is then stably integrated into suitable host cells (e.g., HeLa or A549 cells, as exemplified below). Cells that can grow relatively well under selective conditions (e.g., in the presence of an antibiotic) are then tested for their ability to express the rep and cap genes upon addition of helper virus. As an initial test for rep and / or cap expression, cells can be readily screened using immunofluorescence to detect Rep and / or Cap proteins. Functional tests for replication and packaging of the incoming rAAV vector can then confirm packaging competence and determine packaging efficiency. Using this methodology, a helper virus-inducible promoter from the mouse metallothionein gene has been identified as a suitable alternative to the p5 promoter and has been used to produce high-titer rAAV particles (as described in WO 96 / 17947).

[0041] In any event, it may be desirable to remove one or more AAV genes to reduce the likelihood of generating replication-competent AAV ("RCA"). Thus, the coding or promoter sequences for rep or cap, or both, can be removed, since the functions provided by these genes can be provided in trans.

[0042] The resulting vector is said to be "deficient" in these functions. The missing functions are complemented by a packaging gene or genes that collectively encode the necessary functions for the various missing rep and / or cap gene products to replicate and package the vector. In one embodiment, the packaging gene or gene cassette is not flanked by AAV ITRs and, in one embodiment, shares no substantial homology with the rAAV genome. Thus, to minimize homologous recombination between the vector sequence and the separately provided packaging gene during replication, it is desirable to avoid overlap between the two polynucleotide sequences. The level of homology and the corresponding recombination frequency increases with the length of the homologous sequence and the level of identity they share. The level of homology that will raise concerns in a given system can be theoretically determined and experimentally confirmed, as is known in the art. Typically, however, overlapping sequences of less than about 25 nucleotides can substantially reduce or eliminate recombination if the overlapping sequences are at least 80% identical over their entire length, and less than about 50 nucleotides if the overlapping sequences are at least 70% identical over their entire length. Of course, even lower levels of homology are preferred, as they will further reduce the likelihood of recombination. Even in the absence of any overlapping homology, some frequency of RCA is likely to remain. Further reduction in the frequency of RCA (e.g., by non-homologous recombination) can be obtained by "splitting" the replication and encapsidation functions of AAV, as described in Allen et al., WO 98 / 27204.

[0043] The rAAV vector constructs and complementary packaging gene constructs can be realized in several different forms in the present invention: viral particles, plasmids, and stably transformed host cells can all be used to transiently or stably introduce the constructs into packaging cells.

[0044] In certain embodiments of the invention, the AAV vector and complementary packaging genes, if any, are provided in the form of a bacterial plasmid, an AAV particle, or any combination thereof. In other embodiments, the AAV vector sequences, packaging genes, or both are provided in the form of a genetically modified (preferably heritably modified) eukaryotic cell. The development of host cells heritably modified to express the AAV vector sequences, AAV packaging genes, or both, provides an established source of material that is expressed at reliable levels.

[0045] Thus, a wide variety of genetically modified cells can be used in the context of the present invention. For example, mammalian host cells stably integrating at least one complete copy of the rAAV vector can be used. AAV packaging plasmids containing at least one AAV rep gene operably linked to a promoter to provide replication functions can be used (as described in U.S. Pat. No. 5,658,776). Alternatively, replication functions can be provided using stable mammalian cell lines harboring an AAV rep gene operably linked to a promoter (see, e.g., Trempe et al. (WO 95 / 13392), Burstein et al. (WO 98 / 23018), and Johnson et al. (U.S. Pat. No. 5,656,785)). As described above, the AAV cap gene, which provides encapsidation proteins, can be provided together with the AAV rep gene or separately (see, e.g., the patent applications and patents cited above and Allen et al. (WO 98 / 27204)). Other combinations are contemplated and are within the scope of the present invention.

[0046] Route for delivery Despite the vast network of cerebral vasculature, systemic delivery of therapeutic agents to the central nervous system (CNS) is ineffective for over 98% of small molecules and nearly 100% of large molecules (Partridge, 2005). This lack of effectiveness is due to the presence of the blood-brain barrier (BBB), which prevents most foreign substances, even many beneficial therapeutic agents, from entering the brain from the circulation. While certain small molecule, peptide, and protein therapeutic agents administered systemically can reach the brain parenchyma by crossing the BBB (Banks, 2008), achieving therapeutic levels generally requires high systemic doses, which can have adverse effects. Therapeutic agents can be introduced directly into the CNS by intracerebroventricular or intraparenchymal injection. Intranasal delivery bypasses the BBB and targets therapeutic agents directly to the CNS via pathways along the olfactory and trigeminal nerves, which innervate the nasal passages (Frey II, 2002; Thorne et al., 2004; Dhanda et al., 2005).

[0047] Any route of rAAV administration can be used as long as the route and dosage are prophylactically or therapeutically useful. By way of example, routes of administration to the CNS include intrathecal and intracranial administration. Intracranial administration can be into the cisterna magna or ventricles. The term "cisterna magna" is intended to encompass access to the space around and beneath the cerebellum via an opening between the skull and the top of the spinal column. The term "ventricle" is intended to encompass cavities within the brain that are continuous with the central canal of the spinal cord. Intracranial administration is by injection or infusion, and for intracranial administration, an appropriate dose range is generally about 10 per microliter. 3 ~10 15 Infectious units of a viral vector are delivered in a single injection volume of 1 to 3,000 microliters. For example, the number of viral genomes or infectious units of a vector per microliter is generally about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 1012 , 10 13 , or 10 14 The volume of the virus vector delivered may be approximately 10, 50, 100, 200, 500, 1000, or 2000 microliters, containing the viral genome or infectious units of the viral vector. It should be understood that the aforementioned dosages are merely exemplary, and those skilled in the art will appreciate that the dosages may vary. Effective doses can be estimated from dose-response curves derived from in vitro or in vivo test systems.

[0048] The AAV delivered by the intrathecal treatment method of the present invention can be administered by any convenient route commonly used for intrathecal administration. For example, intrathecal administration can be performed by slow infusion of the formulation over about 1 hour. Intrathecal administration can be performed by injection or infusion, and for intrathecal administration, the appropriate dose range is generally about 10 per microliter. 3 ~10 15 Infectious units of a viral vector, which are delivered in a single injection volume of, for example, 1 to 3,000 microliters or 0.5 to 15 milliliters. For example, the number of viral genomes or infectious units of a vector per microliter is generally about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 It may contain the viral genome or an infectious unit of the viral vector.

[0049] This treatment results in the normalization of lysosomal storage granules in the subject's neural and / or meningeal tissues, as described above, if lysosomal storage enzymes such as IDUA are expressed. It is believed that the improved deposition of storage granules in neural and glial tissues may thereby alleviate the developmental delay and regression seen in individuals with lysosomal storage diseases. Other benefits of this treatment include the normalization of lysosomal storage granules in the cerebral meninges near the arachnoid granules, the presence of which in lysosomal storage diseases leads to high-pressure hydrocephalus. The method of the present invention can also be used to treat spinal cord compression caused by the presence of lysosomal storage granules in the cervical meninges near C1-C5 or elsewhere in the spinal cord. The method of the present invention is also intended to treat cysts caused by perivascular accumulation of lysosomal storage granules surrounding cerebral blood vessels. In another aspect, this treatment may also advantageously result in normalization of liver volume and urinary glycosaminoglycan excretion, reduction in spleen size and apnea-hypopnea events, increase in height and growth velocity in prepubertal subjects, increase in shoulder flexion and extension of elbows and knees, and reduction in tricuspid or pulmonary regurgitation.

[0050] The intrathecal administration of the present invention can include introducing the composition into the lumbar region. Any such administration can be by bolus injection. Depending on the severity of the symptoms and the subject's response to the treatment, the bolus injection can be administered once a week, once a month, once every six months, or once a year. In another embodiment, intrathecal administration is achieved using an infusion pump. Devices that can be used to achieve intrathecal administration of the composition are known to those skilled in the art. The composition can be administered intrathecally, for example, by a single injection or continuous infusion. It should be understood that the dosage treatment can take the form of a single dose or multiple doses.

[0051] As used herein, the term "intrathecal administration" is intended to include delivery of a pharmaceutical composition directly into the cerebrospinal fluid of a subject by techniques such as lateral ventricle injection through a burr hole or cisternal puncture or lumbar puncture. The term "lumbar region" is intended to include the region between the third and fourth lumbar vertebrae (vertebrae in the lower back), more generally the L2-S1 region of the spinal column.

[0052] Administration of the compositions of the present invention to any of the aforementioned sites can be achieved by direct injection of the composition or by using an infusion pump. For injection, the composition can be formulated in the form of a solution in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or phosphate buffer. In addition, the enzyme can be formulated in solid form and redissolved or resuspended immediately before use. Lyophilized forms are also included. Injection can be, for example, in the form of a bolus injection or continuous infusion of the enzyme (e.g., using an infusion pump).

[0053] In one embodiment of the present invention, rAAV is administered to the subject's brain by injection into the lateral ventricle.This injection can be carried out, for example, through a burr hole made in the subject's skull.In another embodiment, enzymes and / or other pharmaceutical preparations are administered to the subject's ventricles through a surgically inserted shunt.For example, injection can be carried out into the smaller third and fourth ventricles, but can also be carried out into the larger lateral ventricles.In yet another embodiment, the composition used in the present invention is administered by injection into the cisterna magna or lumbar region of the subject.

[0054] Although the exact mechanisms underlying intranasal drug delivery to the CNS are not fully understood, a growing body of evidence indicates that pathways involving nerves connecting the nasal passages to the brain and spinal cord are important. In addition, pathways involving the vasculature, cerebrospinal fluid, and lymphatic system have also been implicated in the transport of molecules from the nasal cavity to the CNS. A combination of these pathways is likely responsible for this delivery, although one pathway may predominate depending on the nature of the therapeutic agent, the characteristics of the formulation, and the delivery device used.

[0055] Therapeutic agents can rapidly access the CNS following intranasal administration along the olfactory nerve pathway, which leads directly from the nasal cavity to the CNS. This pathway is a key component of intranasal delivery, as evidenced by the fact that fluorescent tracers associate with the olfactory nerve upon crossing the cribriform plate (Jansson et al., 2002), drug concentrations in the olfactory bulb are among the highest CNS concentrations observed (Thorne et al., 2004, Banks et al., 2004, Graff et al., 2005a, Nonaka et al., 2008, Ross et al., 2004, Ross et al., 2008, Thorne et al., 2008), and a strong positive correlation exists between concentrations in the olfactory epithelium and the olfactory bulb (Dhuria et al., 2009a).

[0056] The olfactory pathway begins in the upper nasal passage, in the olfactory region, where olfactory receptor neurons (ORNs) are interspersed among supporting cells (also known as sustentacular cells), microvilli, and basal cells. ORNs mediate olfactory sensation by transmitting sensory information from the peripheral environment to the CNS (Clerico et al., 2003). Beneath the epithelium, the lamina propria contains mucus-secreting Bowman's glands, axons, blood vessels, lymphatic vessels, and connective tissue. ORN dendrites project into the mucus layer of the olfactory epithelium, while the axons of these bipolar neurons extend centrally through the lamina propria and through foramina in the cribriform plate of the ethmoid bone, which separates the nasal cavity from the cranial cavity. ORN axons pass through the subarachnoid space, which contains CSF, and terminate on mitral cells in the olfactory bulb. From there, neural projections extend to multiple brain regions, including the olfactory tract, anterior olfactory nucleus, piriform cortex, amygdala, and hypothalamus (Buck, 2000). In addition to ORNs, chemosensory neurons located in the Grueneberg ganglion at the anterior end of the nasal cavity also connect to the olfactory bulb (Fuss et al., 2005; Koos et al., 2005).

[0057] The unique characteristics of ORNs contribute to a dynamic cellular environment critical for intranasal delivery to the CNS. Due to direct contact with toxins in the external environment, ORNs regenerate every 3–4 weeks from basal cells present in the olfactory epithelium (Mackay-Sim, 2003). Specialized Schwann cell-like cells called olfactory ensheathing cells (OECs) encase ORN axons and play a key role in axon regeneration, regrowth, and remyelination (Field et al., 2003; Li et al., 2005a; Li et al., 2005b). OECs generate continuous, fluid-filled perineurial channels that, interestingly, remain patent despite ORN degeneration and regeneration (Williams et al., 2004).

[0058] Given the unique environment of the olfactory epithelium, it is possible that intranasally administered therapeutic drugs can reach the CNS via extracellular or intracellular transport mechanisms along the olfactory nerve. Extracellular transport mechanisms involve rapid movement of molecules between cells in the nasal epithelium, and it takes only minutes to 30 minutes for drugs to reach the olfactory bulb and other regions of the CNS after intranasal administration (Frey II, 2002; Balin et al., 1986). Transport likely involves bulk flow mechanisms within channels generated by OECs (Thorne et al., 2004; Thorne et al., 2001). Drugs can also move through these channels due to depolarization of adjacent axons and structural changes that occur during axonal propagation of action potentials (Luzzati et al., 2004). Intracellular transport mechanisms involve uptake of molecules into ORNs by passive diffusion, receptor-mediated endocytosis, or adsorptive endocytosis, followed by slow axonal transport, which can take hours to days for drugs to appear in the olfactory bulb and other brain regions (Baker et al., 1986; Broadwell et al., 1985; Kristensson et al., 1971). Intracellular transport in ORNs has been demonstrated for small lipophilic molecules such as gold particles (de Lorenzo, 1970; Gopinath et al., 1978), aluminum salts (Perl et al., 1987), and substances with receptors on ORNs, such as WGA-HRP (Thorne et al., 1995; Baker et al., 1986; Itaya et al., 1986; Shipley, 1985). Although intracellular mechanisms are important for certain therapeutic agents, they may not be the predominant mode of delivery to the CNS. While some large molecules, such as galanin-like peptide (GALP), exhibit saturable transport pathways to the CNS (Nonaka et al., 2008), for other large molecules, such as NGF and insulin-like growth factor-I (IGF-I), intranasal delivery to the brain is nonsaturable and not receptor-mediated (Thorne et al., 2004; Chen et al., 1998; Zhao et al., 2004).

[0059] An often overlooked but important pathway connecting the nasal passages with the CNS involves the trigeminal nerve, which innervates the respiratory and olfactory epithelium of the nasal passages and enters the CNS at the pons (Clerico et al., 2003; Graff et al., 2003). Interestingly, a small portion of the trigeminal nerve also terminates in the olfactory bulb (Schaefer et al., 2002). The cellular composition of the respiratory region of the nasal passage differs from that of the olfactory region, with ciliated epithelial cells distributed among mucus-secreting goblet cells. These cells contribute to the mucociliary clearance mechanism, which removes mucus along with foreign particles from the nasal cavity to the nasopharynx. The trigeminal nerve transmits sensory information from the nasal cavity, oral cavity, eyelids, and cornea to the CNS via the ophthalmic (V1), maxillary (V2), or mandibular (V3) branches of the trigeminal nerve (Clerico et al., 2003; Gray, 1978). Branches from the ophthalmic (V1), maxillary (V2), or mandibular (V3) branches of the trigeminal nerve innervate the dorsal nasal mucosa and anterior portion of the nose, while branches from the maxillary (V3) branch innervate the lateral wall of the nasal mucosa. The mandibular (V3) branch of the trigeminal nerve innervates the mandible and teeth but does not directly innervate the nasal cavity. The three branches of the trigeminal nerve unite at the trigeminal ganglion, extend centrally to enter the brain at the level of the pons, and terminate in the spinal trigeminal nucleus in the brainstem. A unique feature of the trigeminal nerve is that it enters the brain from the respiratory epithelium of the nasal passages at two sites: (1) through the anterior foramen vitae near the pons and (2) through the cribriform plate near the olfactory bulb, creating an entry point for both caudal and rostral brain regions after intranasal administration. Conceivably, other nerves that innervate the face and head, such as the facial nerve, or other sensory structures within the nasal cavity, such as the Grueneberg ganglion, may also provide entry for intranasally applied therapeutic agents into the CNS.

[0060] Traditionally, the intranasal route of administration has been used to deliver drugs to the systemic circulation via absorption into capillaries underlying the nasal mucosa. The nasal mucosa is highly vascular, receiving its blood flow from branches of the maxillary, ophthalmic, and facial arteries arising from the carotid artery (Clerico et al., 2003; Cauna, 1982). The olfactory mucosa receives blood from small branches of the ophthalmic artery, while the respiratory mucosa receives blood from large arterial branches of the maxillary artery (DeSesso, 1993). The relative density of blood vessels is greater in the respiratory mucosa compared with the olfactory mucosa, making it an ideal site for absorption into the bloodstream (DeSesso, 1993). The vasculature in the respiratory tract contains a mixture of continuous and fenestrated endothelium (Grevers et al., 1987; Van Diest et al., 1979), which allows both small and large molecules to enter the systemic circulation after intranasal administration.

[0061] Delivery to the CNS is possible via absorption into the systemic circulation and subsequent transport across the BBB, particularly for small lipophilic drugs, which can more easily enter the bloodstream and cross the BBB than larger hydrophilic therapeutic agents such as peptides and proteins.

[0062] Increasing evidence suggests that mechanisms involving vascular-associated or perivascular channels are involved in intranasal drug delivery to the CNS. Perivascular spaces are sandwiched between the outermost layer of blood vessels and the basement membrane of surrounding tissues (Pollock et al., 1997). These perivascular spaces act as a lymphatic system for the brain, where neuron-derived materials are removed from the brain interstitial fluid by entering perivascular channels associated with cerebral blood vessels. Perivascular transport, unlike diffusion alone, relies on a bulk flow mechanism (Cserr et al., 1981; Groothuis et al., 2007), and arterial pulsation is also a driving force for perivascular transport (Rennels et al., 1985; Rennels et al., 1985). Intranasally applied drugs can migrate into the perivascular space in the nasal passages or after reaching the brain, and the widespread distribution observed within the CNS may be due to a perivascular transport mechanism (Thorne et al., 2004).

[0063] The subarachnoid space containing CSF, the perineural space surrounding the olfactory nerve, and the pathways connecting the nasal lymphatics are important for CSF drainage, providing intranasally administered therapeutic agents with access to the CSF and other regions of the CNS. Several studies have documented that tracers injected into the CSF in the ventricles or subarachnoid space drain into channels associated with the olfactory nerve that cross the cribriform plate at the underside of the olfactory bulb and reach the nasal lymphatic system and cervical lymph nodes (Bradbury et al., 1983; Hatterer et al., 2006; Johnston et al., 2004a; Kida et al., 1993; Walter et al., 2006a; Walter et al., 2006b). Following intranasal administration, drugs can access the CNS through these pathways, moving from the nasal passages to the CSF and then into the brain interstitial and perivascular spaces for distribution throughout the brain. These drainage pathways are important in several animal species (sheep, rabbit, and rat), accounting for approximately 50% of CSF clearance (Bradbury et al., 1981; Boulton et al., 1999; Boulton et al., 1996; Cserr et al., 1992). That the pathway between the nasal passages and the CSF remains important and functional in humans is evidenced by the fact that therapeutic agents can be delivered directly to the CSF after intranasal delivery without appreciably entering the blood (Born et al., 2002). Several intranasal studies have demonstrated that drugs gain direct access from the nasal cavity to the CSF, with subsequent distribution to the brain and spinal cord. Many molecules applied intranasally rapidly enter the CSF, and this transport depends on the lipophilicity, molecular weight, and degree of ionization of the molecule (Dhanda et al., 2005, Born et al., 2002, Kumar et al., 1974, Sakane et al., 1995, Sakane et al., 1994, Wang et al., 2007). Assessing distribution into the CSF can provide information about the mechanism of intranasal delivery.

[0064] Optimal delivery to the CNS along nerve tracts is associated with drug delivery to the upper third of the nasal cavity (Hanson et al., 2008). Another position for targeting the olfactory region is the head down-and-forward "praying to Mecca" position, although supine positioning can also be used. A supine position with a head angle of 70° or 90° may be suitable for efficient delivery to the CSF using tubes inserted into the nostrils for intranasal drug delivery (van den Berg et al., (2002)).

[0065] For intranasal drug administration, nasal drops can be administered alternately to each nostril every 1–2 minutes for 10–20 minutes to allow the solution to be absorbed into the nasal epithelium (Thorne et al., 2004, Capsoni et al., 2002, Ross et al., 2004, Ross et al., 2008, Dhuria et al., 2009a, Dhuria et al., 2009b, Francis et al., 2008, Martinez et al., 2008). This noninvasive method does not require the insertion of a device into the nostril. Instead, droplets are placed at the nostril opening, allowing the individual to sip the droplets into the nasal cavity. Other methods of administration in anesthetized individuals involve occluding the esophagus and inserting a breathing tube into the trachea to prevent swallowing of nasal formulations and eliminate problems related to respiratory distress (Chow et al., 1999; Chow et al., 2001; Fliedner et al., 2006; Dahlin et al., 2001). Flexible tubes can be inserted into the nostrils to deliver small amounts of drug solution locally to the respiratory or olfactory epithelium, depending on the length of the tube (Chow et al., 1999; Van den Berg et al., 2003; van den Berg et al., 2004a; Banks et al., 2004; van den Berg et al., 2002; Vyas et al., 2006a; Charlton et al., 2007a; Gao et al., 2007a).

[0066] Nasal delivery devices such as sprayers, nasal drops, or needleless injectors can be used to target medications to different regions of the nasal cavity. OptiMist™ is a breath-activated device that directs liquid or powder nasal formulations to the nasal cavity, including the olfactory region, without depositing in the lungs or esophagus (Djupesland et al., 2006). The ViaNase™ device can also be used to target nasal sprays to the olfactory and respiratory epithelium of the nasal cavity. Nasal sprays tend to deposit at the base of the nostrils, where they are subject to rapid mucociliary clearance, while nasal sprays are dispersed to the middle meatus of the nasal mucosa (Scheibe et al., 2008).

[0067] The immunosuppressant or immune tolerizer can be administered by any route, including parenteral administration. In one embodiment, the immunosuppressant or immune tolerizer can be administered by subcutaneous, intramuscular, or intravenous injection; orally; intrathecally; intracranially; intranasally; or by sustained release, e.g., via a subcutaneous implant. The immunosuppressant or immune tolerizer can be dissolved or dispersed in a liquid carrier medium. For parenteral administration, the active substance can be suitably mixed with an acceptable vehicle, e.g., a vegetable oil such as peanut oil or cottonseed oil. Other parenteral vehicles, such as organic compositions using solketal, glycerol, or formal, and aqueous parenteral formulations, can also be used. For parenteral administration by injection, the composition comprises an aqueous solution of a pharmaceutically acceptable water-soluble salt of the active acid of the present invention, preferably at a concentration of 0.01 to 10%, and optionally, a stabilizer and / or buffer substance in the aqueous solution. The unit-dosage solutions may conveniently be sealed in ampoules.

[0068] Compositions, such as rAAV-containing compositions, immunosuppressant-containing compositions, or immune tolerization compositions, can be in the form of unit doses for injection. Examples of carriers or diluents that can be used to prepare such injectable doses include diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters; pH adjusters or buffers such as sodium citrate, sodium acetate, and sodium phosphate; stabilizers such as sodium metabisulfite, EDTA, thioglycolic acid, and thiolactic acid; isotonicity agents such as sodium chloride and glucose; and local anesthetics such as procaine hydrochloride and lidocaine hydrochloride. Additionally, conventional solubilizers and analgesics can be added. Injectable preparations can be prepared by adding the above-mentioned carriers to enzymes or other active ingredients according to procedures well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991). The pharmaceutically acceptable formulations can be readily suspended in aqueous media and delivered via conventional hypodermic needles or via infusion pumps. Prior to delivery, the formulations can be sterilized, preferably by gamma irradiation or electron beam sterilization.

[0069] When the immunosuppressant or immune tolerizing agent is administered in the form of a subcutaneous implant, the compound is suspended or dissolved in a slowly dispersing material known to those skilled in the art, or is administered in a device that uses a constant driving force to slowly release the active agent, such as an osmotic pump, allowing for administration over an extended period of time.

[0070] The dosage of the immunosuppressant- or immune tolerizing agent-containing composition varies widely and may need to be adjusted individually depending on various factors, such as the severity of the disease and the patient's age. A possible range of amounts that can be administered daily is about 0.1 mg to about 2000 mg, or about 1 mg to about 2000 mg. The immunosuppressant- or immune tolerizing agent-containing composition can be appropriately formulated to provide a dose within these ranges as a single dosage unit or multiple dosage units. In addition to containing an immunosuppressant, the formulation may also contain one or more rAAVs encoding therapeutic gene products.

[0071] The compositions described herein can be used in combination with other pharmaceutical agents. The compositions can take conventional forms, such as an aerosol, solution, suspension, or topical formulation, or lyophilized form.

[0072] A typical composition includes rAAV, an immunosuppressant, a penetration enhancer, or a combination thereof, and a pharmaceutically acceptable excipient, which can be a carrier or diluent. For example, the active agent can be mixed with the carrier, diluted with the carrier, or encapsulated in the carrier. When the active agent is mixed with the carrier or when the carrier functions as a diluent, the carrier can be a solid, semi-solid, or liquid substance that acts as a vehicle, excipient, or medium for the active agent. Some examples of suitable carriers are water, salt solution, alcohol, polyethylene glycol, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, gum arabic, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and fatty acid diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent can comprise any sustained-release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or in admixture with a wax.

[0073] The preparation can be mixed with an auxiliary agent that does not adversely react with the active agent. Such additives can include wetting agents, emulsifying and suspending agents, salts for influencing osmotic pressure, buffers and / or coloring substances, preservatives, sweeteners or flavoring agents. The composition can also be sterilized if necessary.

[0074] When a liquid carrier is used, the preparation may be in the form of a liquid such as an aqueous suspension or solution. Acceptable solvents or vehicles include sterile water, Ringer's solution, or isotonic saline.

[0075] The drug may be provided as a powder suitable for reconstitution with the appropriate solution described above. Examples include, but are not limited to, freeze-dried powder, rotary-dried powder or spray-dried powder, amorphous powder, granules, precipitates, or particulates. The composition may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations thereof. The unit dosage form may be contained in individual containers or in multi-dose containers.

[0076] Compositions contemplated by the present invention may comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in sustained release form using biodegradable polymers such as polylactide-polyglycolide to provide a prolonged depot and / or delivery effect. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).

[0077] Polymer nanoparticles, such as those composed of a hydrophobic core of polylactic acid (PLA) and a hydrophilic shell of methoxy-poly(ethylene glycol) (MPEG), may have improved solubility and targeting to the CNS. The difference in the targeting region between microemulsion and nanoparticle formulations may be due to particle size.

[0078] Liposomes are very simple structures consisting of one or more lipid bilayers of amphiphilic lipids, i.e., phospholipids or cholesterol. The lipophilic portions of the bilayers orient themselves toward each other, creating an internal hydrophobic environment within the membrane. Liposomes are suitable drug carriers for some lipophilic drugs, provided their size and shape are compatible with those that can associate with the nonpolar portion of the lipid bilayer. Liposome sizes vary from 20 nm to several microns.

[0079] Mixed micelles are efficient surfactant structures composed of bile salts, phospholipids, triglycerides, diglycerides, and monoglycerides, fatty acids, free cholesterol, and lipid-soluble micronutrients. While long-chain phospholipids are known to form bilayers when dispersed in water, the preferred phase for their short-chain analogs is the spherical micellar phase. Micellar solutions are thermodynamically stable systems that form spontaneously in water and organic solvents. The interaction of micelles with hydrophobic / lipophilic drugs leads to the formation of mixed micelles (MMs), often referred to as swollen micelles. In the human body, they incorporate poorly water-soluble hydrophobic compounds and act as reservoirs for digestion products, such as monoglycerides.

[0080] Lipid microparticles include lipid nanospheres and lipid microspheres. Microspheres are generally defined as small spherical particles of any substance, approximately 0.2–100 μm in size. Even smaller spheres, less than 200 nm in size, are commonly referred to as nanospheres. Lipid microspheres are homogeneous oil-in-water microemulsions similar to commercial fat emulsions and are prepared by vigorous sonication or high-pressure emulsification (fragmentation). The natural surfactant lecithin reduces the surface tension of liquids, acting as an emulsifier to form stable emulsions. Lipid nanospheres have a similar structure and composition to lipid microspheres, but their diameter is smaller.

[0081] Polymer nanoparticles serve as carriers for a wide variety of ingredients. The active ingredients are dissolved in the polymer matrix, encapsulated, or adsorbed to the particle surface. Polymers suitable for preparing organic nanoparticles include cellulose derivatives and polyesters, such as poly(lactic acid), poly(glycolic acid), and their copolymers. Polymer nanoparticles are ideal carriers and release systems due to their small size, large surface area / volume ratio, and the ability to functionalize the interface. When the particle size is less than 50 nm, they are no longer recognized as particles by many biological and synthetic barrier layers and behave similarly to molecular dispersions.

[0082] Thus, the compositions of the present invention can be formulated to achieve rapid, sustained, controlled, or delayed release of the active agent after administration to an individual, or any combination thereof, by using techniques well known in the art. In one embodiment, the enzyme is dissolved in an isotonic or hypotonic solution. In one embodiment, for enzymes that are not water-soluble, lipid-based delivery vehicles such as microemulsions or liposomes can be used, for example, as described in WO 2008 / 049588 (the disclosure of which is incorporated herein by reference).

[0083] In one embodiment, the preparation can contain an active ingredient dissolved or suspended in a liquid carrier, such as an aqueous carrier for aerosol application. The carrier can contain additives such as solubilizers, e.g., propylene glycol, surfactants, absorption enhancers, e.g., lecithin (phosphatidylcholine) or cyclodextrin, or preservatives, e.g., parabens. For example, efficient delivery to the CNS after intranasal administration can depend not only on solubility but also on membrane permeability. For enzymes whose size and polarity hinder paracellular transport, improving membrane permeability can enhance extracellular transport along the olfactory and trigeminal nerves to the CNS. One approach to modifying membrane permeability within the nasal epithelium is to use penetration enhancers, such as surfactants, e.g., lauroylcarnitine (LC), bile salts, lipids, cyclodextrins, polymers, or tight junction modifiers.

[0084] Generally, the active agent is prepared in a unit dosage form, each unit dosage containing the active ingredient together with a pharmaceutically acceptable carrier. Typically, a dosage form suitable for nasal administration contains about 125 μg to about 125 mg, for example, about 250 μg to about 50 mg, or about 2.5 mg to about 25 mg of the compound mixed with a pharmaceutically acceptable carrier or diluent.

[0085] Dosage forms can be administered once daily, or more than once daily, for example, two or three times daily, or, if the prescribing physician determines this is desirable, dosage forms can be administered less frequently than once daily, for example, every other day or once a week.

[0086] The following non-limiting examples illustrate the invention. [Example]

[0087] Example I AAV vector-mediated iduronidase gene delivery in a murine model of mucopolysaccharidosis type I: comparison of different delivery routes to the CNS Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by a deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). Abnormal systemic accumulation of glycosaminoglycans is associated with growth retardation, organomegaly, skeletal dysplasia, and cardiopulmonary disease. Individuals with the most severe form of the disease (Hurler syndrome) suffer from neurodegeneration, mental retardation, and premature death. The two current treatments for MPS I—hematopoietic stem cell transplantation and enzyme replacement therapy—do not effectively treat all central nervous system (CNS) manifestations.

[0088] In the context of gene therapy, it has previously been demonstrated that intravascular delivery of AAV-9 in adult mice does not achieve widespread, direct neural targeting (see Foust et al., 2009). Previous studies have also shown that direct injection of AAV8-IDUA into the CNS of adult IDUA-deficient mice results in low or insufficient levels of transgene expression (see Figure 18). Surprisingly, in the following examples using a preclinical model for the treatment of MPS1, direct injection of AAV9-IDUA into the CNS of immunocompetent adult IDUA-deficient mice resulted in IDUA enzyme expression and activity that was equal to or higher than that in wild-type adult mice (Figure 15, see below).

[0089] method Preparation of AAV9-IDUAThe AAV-IDUA vector construct (MCI) was previously described ( Wolf et al., 2011 ) (mCags promoter). AAV-IDUA plasmid DNA was packaged into AAV9 viral particles at the University of Florida Vector Core and delivered at 3 × 10 per milliliter. 13 Vector genome titers were obtained.

[0090] ICV injection Adult Idua- / - mice were anesthetized using a cocktail of ketamine and xylazine (100 mg ketamine + 10 mg xylazine per kg) and placed in a stereotaxic frame. Ten microliters of AAV9-IDUA was injected into the right lateral ventricle (stereotaxic coordinates, in mm from bregma, AP 0.4, ML 0.8, DV 2.4) using a Hamilton syringe. The animals were then returned to their cages on a heating pad to recover.

[0091] Intrathecal injection Injections into young adult mice were performed by intravenously injecting 0.2 mL of 25% mannitol 20 minutes prior to injection of 10 μL of the AAV vector-containing solution between the L5 and L6 vertebrae.

[0092] Immune tolerization Newborn IDUA-deficient mice were injected via the facial temporal vein with 5 μL containing 5.8 μg of recombinant iduronidase protein (Aldurazyme), after which the animals were returned to their cages.

[0093] Cyclophosphamide immunosuppression For immunosuppression, animals were administered cyclophosphamide at a dose of 120 mg / kg once a week starting the day after injection of the AAV9-IDUA vector.

[0094] animalAnimals were anesthetized with ketamine / xylazine (100 mg ketamine + 10 mg xylazine per kg) and transcardially perfused with 70 mL of PBS before sacrifice. Brains were harvested and microdissected on ice into cerebellum, hippocampus, striatum, cortex, and brainstem / thalamus ("remainder"). Samples were frozen on dry ice and then stored at -80°C. Samples were thawed, homogenized in 1 mL of PBS using a motorized pestle, and permeabilized with 0.1% Triton X-100. IDUA activity was determined by a fluorometric assay using 4 MU-iduronide as substrate. Activity is expressed as units per mg of protein (percent of substrate converted to product per minute) as determined by the Bradford assay (BioRad).

[0095] organization Tissue homogenates were clarified by centrifugation at 13,000 rpm for 3 min in an Eppendorf tabletop centrifuge model 5415D (Eppendorf) and incubated overnight with proteinase K, DNase 1, and RNase. GAG concentrations were determined using the Blyscan sulfated glycosaminoglycan assay (Accurate Chemical) according to the manufacturer's instructions.

[0096] result Results for iduronidase-deficient mice administered AAV intracerebroventricularly (ICV) or intrathecally (IT) are shown in Figure 1. To prevent an immune response, animals were immunosuppressed with cyclophosphamide (CP), or tolerized at birth with intravenous administration of human iduronidase protein (aldurazyme), or injected into NOD-SCID immunodeficient mice that were also iduronidase-deficient. At the indicated time points after treatment, animals were sacrificed, brains microdissected, and extracts assayed for iduronidase activity.

[0097] Data for immunodeficient IDUA-deficient animals injected ICV with AAV-IDUA vectors are illustrated in Figure 2. These animals exhibited high levels of IDUA expression (10-100-fold higher than wild type) in all brain regions, with the highest levels observed in the brainstem and thalamus ("remainder").

[0098] Immunosuppressed animals receiving AAV vectors via the ICV route had relatively lower enzyme levels in the brain compared to immunodeficient animals (Fig. 3). Note that immunosuppression may have been impaired in these animals because CP was discontinued 2 weeks before sacrifice due to poor health.

[0099] Data for immunosuppressed animals administered AAV vectors via the IT route are shown in Figure 4. Tolerized animals receiving AAV vectors ICV exhibited widespread IDUA activity in all parts of the brain (Figure 5), similar to the observations in immunodeficient animals, demonstrating the effectiveness of the tolerization procedure.

[0100] Figure 6 has been compiled to allow a side-by-side comparison of all mean IDUA activity levels, and Figure 7 groups the data according to brain region.

[0101] GAG accumulation was assayed in various sections of the brain for all four test groups. For each group, the average for each brain region is shown on the left, and values ​​for each individual animal are shown on the right (Figure 8). IDUA-deficient animals (far left) contained higher levels of GAG compared with wild-type animals (magenta bars). In all groups of AAV-treated animals, GAG levels were at or below wild-type levels in all brain regions. GAG levels were slightly higher in the cortex and brainstem of animals administered AAV9-IDUA intrathecally than in wild-type animals, but this was not significant.

[0102] conclusion These results demonstrate a high and widespread distribution of IDUA in the brain, regardless of the delivery route (ICV or IT). However, IDUA expression in the striatum and hippocampus was lower in IT-injected animals compared with ICV-injected animals. The higher expression levels in immunodeficient mice compared with immunocompetent mice suggest an immune response. With respect to ICV injection, IDUA expression was low when CP was discontinued early. Additionally, immune tolerization was effective in restoring high levels of enzyme activity. Furthermore, GAG levels were restored to normal in all treated experimental mouse groups.

[0103] Example II method Preparation of AAV9IDUA The AAV-IDUA plasmid was packaged into AAV9 viral particles at the University of Florida Vector Core or the University of Pennsylvania Vector Core, yielding 1–3 × 10 virions per milliliter. 13 Vector genome titers were obtained.

[0104] ICV injection See Example I.

[0105] Intrathecal injection See Example I.

[0106] Immune tolerization The procedure was the same as in Example I, except for the following: For multiple tolerization, newborn IDUA-deficient mice received a first injection of Aldurazyme into the facial temporal vein, followed by six weekly intraperitoneal injections.

[0107] Cyclophosphamide immunosuppression See Example I.

[0108] animal Animals were anesthetized with ketamine / xylazine (100 mg ketamine + 10 mg xylazine per kg) and transcardially perfused with 70 mL of PBS before sacrifice. Brains were harvested and microdissected on ice into the cerebellum, hippocampus, striatum, cortex, and brainstem / thalamus ("remainder"). Samples were frozen on dry ice and then stored at -80°C.

[0109] Tissue IDUA activity Tissue samples were thawed and homogenized in saline using a tissue homogenizer. Tissue homogenates were clarified by centrifugation at 15,000 rpm for 15 minutes at 4°C in a tabletop Eppendorf centrifuge. Tissue lysates (supernatants) were collected and analyzed for IDUA activity and GAG accumulation levels.

[0110] Tissue GAG ​​levels Tissue lysates were incubated overnight with proteinase K, RNase, and DNase. GAG levels were analyzed using the Blyscan sulfated glycosaminoglycan assay according to the manufacturer's instructions.

[0111] IDUA vector copy number Tissue homogenates were used to isolate DNA, followed by QPCR as described by Wolf et al. (2011).

[0112] result Figure 9 illustrates the experimental design and experimental groups. Animals were administered the AAV9IDUA vector by either intracerebroventricular (ICV) or intrathecal (IT) injection. Vector administration was performed in NOD-SCID immunodeficient (ID) mice that were also IDUA-deficient, in IDUA-deficient mice immunosuppressed with cyclophosphamide (CP), or in IDUA-deficient mice immunotolerized at birth with one or more injections of human iduronidase protein (Aldurazyme). Vector treatment and sacrifice time points are indicated in Figure 9. All vector administrations were performed in adult animals ranging from 3 to 4.5 months of age. Animals received 10 μL of vector at a dose of 3 × 10 per 10 microliters. 11 The vector genome was injected at a dose of 100 mg / kg.

[0113] Figure 10 shows IDUA enzyme activity in intracranially injected immunodeficient IDUA-deficient mice. High levels of enzyme activity were observed in all brain regions, ranging from 30 to 300 times higher than wild-type levels. The highest enzyme expression was observed in the thalamus, brainstem, and hippocampus.

[0114] Animals that were intracranially injected and immunosuppressed with cyclophosphamide (CP) showed significantly lower enzyme activity levels than the other groups (Figure 11), although CP administration in this case had to be discontinued 2 weeks before sacrifice due to the poor health of the animals.

[0115] IDUA enzyme levels in animals tolerized with IDUA protein (Aldurazyme) at birth and administered vector intracranially are shown graphically in Figure 12. All animals showed elevated enzyme levels in all regions of the brain, ranging from 10- to 1000-fold higher than wild-type levels, similar to those achieved in immunodeficient animals, demonstrating the effectiveness of the immune tolerization regimen.

[0116] Figure 13 shows the IDUA enzyme levels in mice injected intrathecally and treated weekly with CP. Elevated IDUA levels were observed in all parts of the brain, particularly in the cerebellum and spinal cord. The lowest enzyme levels were in the striatum and hippocampus, where activity was at wild-type levels.

[0117] IDUA-deficient mice were tolerized with Aldurazyme as described above and injected intrathecally with the vector (Figure 14). There was widespread IDUA enzyme activity in all parts of the brain, with the highest levels of activity in the brainstem and thalamus, olfactory bulb, spinal cord, and cerebellum. Similar to the data in Figure 13, the lowest levels of enzyme activity were found in the striatum, cortex, and hippocampus.

[0118] Control immunocompetent IDUA-deficient animals were injected intrathecally with the vector without immunosuppression or immune tolerization (Figure 15). These results show that enzyme activity was at or slightly higher than wild-type levels, but significantly lower than that seen in immunoconditioned animals. The reduction in enzyme levels was particularly pronounced in the cerebellum, olfactory bulb, thalamus, and brainstem, which are the regions that expressed the highest levels of enzyme in immunoconditioned animals.

[0119] Animals were assayed for GAG accumulation, as shown in Figure 15. All groups showed clearance of GAG accumulation, with GAG levels similar to those observed in wild-type animals. Animals that were immunosuppressed and intrathecally injected with AAV-IDUA vectors had slightly higher GAG levels in the cortex than wild-type, but still much lower than untreated IDUA-deficient mice.

[0120] The presence of the AAV9IDUA vector in tolerized animals and injected intracranially or intrathecally with the vector was assessed by QPCR, as illustrated in Figure 16. The IDUA copy number per cell was higher in intracranially injected animals compared to intrathecally injected animals, consistent with the higher enzyme activity levels seen in intracranially injected animals.

[0121] conclusion High and widespread therapeutic levels of IDUA were observed in all brain regions after intraventricular and intrathecal AAV9-IDUA administration in adult mice. In immunocompetent IDUA-deficient animals injected intrathecally with AAV-IDUA, enzyme activity was restored to wild-type or slightly higher levels. In animals immune-tolerized by administration of IDUA protein starting at birth, significantly higher IDUA enzyme levels were observed with both vector injection routes.

[0122] Example III Adult immunocompetent IDUA-deficient mice (12 weeks old) were anesthetized with ketamine / xylazine and then intranasally instilled with the AAV9IDUA vector. The vector was administered by intranasally applying 3 μL drops of the vector to the left and right nostrils alternately eight times with a 2-minute interval between each application using a micropipette. A total of 2.4–7 × 10 vectors were administered, depending on the vector source. 11Each adult animal received 1 vector genome. Animals were immunosuppressed with 120 mg / kg cyclophosphamide administered once a week starting the day after vector administration. Twelve weeks after vector injection, mice were sacrificed and animals were assayed for IDUA enzyme expression and vector copy number in the brain.

[0123] References TIFF2025128365000002.tif239150TIFF2025128365000003.tif239155TIFF20251283650 00004.tif239134TIFF2025128365000005.tif239150TIFF2025128365000006.tif233148

[0124] All publications, patents, and patent applications are incorporated herein by reference. While the invention has been described in the foregoing specification with respect to certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is capable of further embodiments, and that some of the details herein may be varied considerably without departing from the underlying principles of the invention.

Claims

1. 1. A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, the method comprising intrathecally administering to the mammal a composition comprising an effective amount of a recombinant adeno-associated virus (rAAV) vector comprising an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms, wherein the rAAV is not AAV-2 or a penetration enhancer is administered intrathecally to the mammal.

2. A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, said method comprising the step of intracerebroventricularly administering to said mammal a composition comprising an effective amount of a recombinant adeno-associated virus (rAAV) vector comprising an open reading frame encoding a gene product whose expression in said mammal prevents, inhibits, or treats said one or more symptoms, wherein said rAAV is not AAV-8 or said mammal is an adult.

3. A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, comprising intravascularly administering to the mammal a composition comprising an effective amount of an rAAV vector comprising an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms, and an effective amount of a penetration enhancer.

4. The method of claim 3, wherein the composition comprises a penetration enhancer.

5. 5. The method of claim 1 or 4, wherein the penetration enhancer comprises mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA.

6. A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, comprising administering to the mammal an effective amount of a composition comprising an rAAV vector comprising an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms, and an immunosuppressant.

7. 7. The method of claim 6, wherein the immunosuppressant comprises cyclophosphamide.

8. 7. The method of claim 6, wherein the immunosuppressant comprises a glucocorticoid, a cytostatic agent including an alkylating agent, an antimetabolite, a cytotoxic antibiotic, an antibody, or an agent active against an immunophilin.

9. 9. The method of claim 7 or 8, wherein the immunosuppressant comprises a nitrogen mustard, a nitrosourea, a platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, an antibody to the IL-2 receptor (CD25) or CD3, an anti-IL-2 antibody, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, an opioid, or a TNF-α (tumor necrosis factor alpha) binding agent.

10. The method of any one of claims 6 to 9, wherein the rAAV and the immunosuppressant are administered simultaneously or the immunosuppressant is administered after the rAAV.

11. The method of any one of claims 6 to 10, wherein the rAAV and the immunosuppressant are administered intrathecally.

12. The method of any one of claims 6 to 10, wherein the rAAV and the immunosuppressant are administered intracerebroventricularly.

13. A method for preventing, inhibiting, or treating one or more symptoms associated with a disease of the central nervous system in a mammal in need thereof, comprising the steps of: providing a mammal that has been immunotolerized to a gene product associated with the disease; and administering to the mammal a composition comprising an effective amount of an rAAV vector that includes an open reading frame encoding a gene product whose expression in the mammal prevents, inhibits, or treats the one or more symptoms.

14. 14. The method of any one of claims 1 to 13, wherein the gene product is a defective enzyme or is deficient in the mammal, thereby resulting in a lysosomal storage disease.

15. 15. The method of any one of claims 1 to 14, wherein the mammal is an immunocompetent adult.

16. The method of any one of claims 1 to 15, wherein the rAAV vector is an rAAV-1 vector, an rAAV-3 vector, an rAAV-4 vector, an rAAV-5 vector, an rAAV rh10 vector, or an rAAV-9 vector.

17. 17. The method of any one of claims 1 to 16, wherein the gene product is alpha-L-iduronidase, iduronate-2-sulfatase, heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, or glucocerebrosidase.

18. The method of any one of claims 1 to 17, wherein the mammal is a human.

19. 19. The method of any one of claims 1 to 18, wherein the disease is alpha-L-iduronidase deficiency.

20. 19. The method of any one of claims 1 to 18, wherein the disease is a mucopolysaccharidosis type I disorder, a mucopolysaccharidosis type II disorder, or a mucopolysaccharidosis type VII disorder.

21. 21. The method of any one of claims 1 to 20, wherein multiple administrations are administered.

22. 21. The method of any one of claims 1 to 20, wherein the composition is administered once a week.

23. 23. The method of any one of claims 1 to 22, wherein administering prevents, inhibits or treats neurodegeneration.

24. The method of any one of claims 1 to 12 or claims 14 to 23, wherein the mammal is immunotolerized to the gene product prior to administration of the composition.

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