RU2801511C1 - Adeno-associated virus-mediated gene transfer into the central nervous system - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the treatment of symptoms of type II mucopolysaccharidosis. A method of preventing, inhibiting, or treating one or more symptoms of type II mucopolysaccharidosis (MPS II) in a human includes intrathecal administration of a composition containing an effective amount of a recombinant adeno-associated virus (rAAV) vector containing an open reading frame encoding iduronate- 2-sulfatase to prevent, inhibit or treat one or more symptoms of MPS II, wherein rAAV is either rAAV9 or rAAVrh10.

EFFECT: expansion of the arsenal of agents for a specific purpose.

17 cl, 20 dwg, 3 ex

Description

Link to related applications

This application claims priority according to the filing date of U.S. Provisional Application Serial No. 61/823757, filed May 15, 2013, the disclosure of which is incorporated herein by reference.

Government's claim of rights

The present invention was made with government support according to HD032652 and DK094538 provided by the National Institutes of Health. The government has certain rights to the present invention.

Background of the Invention

Mucopolysaccharidoses (MPS) are a group of 11 storage diseases caused by disturbances in the catabolism of glycosaminoglycan (GAG), leading to its accumulation in lysosomes (Muenzer, 2004; Munoz-Rojas et al., 2008). Manifestations of varying severity include organomegaly, skeletal dysplasia, cardiac and pulmonary obstruction, and neurological deterioration. For MPS I, iduronidase deficiency (IDUA), severity ranges from mild (Scheie's syndrome) to moderate (Gurler-Scheie) to severe (Hurler's syndrome), the latter leading to neurological degradation and death by 15 years of age (Muenzer, 2004; Munoz - Rojas et al., 2008). The treatments for MPS have been mostly palliative. However, there are several MPS diseases, including Hurler syndrome, for which allogeneic hematopoietic stem cell transplantation (HSCT) has been effective (Krivit, 2004; Orchard et al., 2007; Peters et al., 2003). In addition, enzyme replacement therapy (ERT) is becoming available for more and more MPS diseases (Brady, 2006). In general, HSCT and ERT lead to clearance of accumulated substances and improvement of peripheral conditions, although some problems persist after treatment (skeletal, cardiac, corneal opacity). A major problem with these cellular and enzyme therapies is their effectiveness in targeting neurological manifestations, since the peripherally administered enzyme does not cross the blood-brain barrier, and HSCT has been found to be beneficial for some but not all MPS.

MPS I has been one of the most widely studied MPS diseases for the development of cellular and molecular therapies. The efficacy of allogeneic HSCT is most likely the result of metabolic cross-correction, whereby the missing enzyme is released from donor-derived cells and subsequently taken up by host cells and transferred to lysosomes, where the enzyme promotes lysosomal metabolism (Fratantoni et al., 1968). Subsequently, there is clearing of accumulated GAG substances in peripheral organs such as the liver and spleen, relief from cardiac obstruction, and improvement in corneal opacity (Orchard et al., 2007). Of particular importance is the effect of allogeneic stem cell transplantation on the occurrence of neurological manifestations in MPS diseases. In this regard, for several MPS diseases, there is evidence that individuals treated with allogeneic stem cells have improved outcomes compared with patients without transplantation (Bjoraker et al., 2006; Krivit, 2004; Orchard et al., 2007; Peters et al. ., 2003). The main hypothesis explaining the neurological advantage of allogeneic hematopoietic stem cell transplantation is the penetration of donor-derived hematopoietic cells (most likely microglia) (Hess et al., 2004; Unger et al., 1993) into the central nervous system, where the missing enzyme is expressed by the introduced cells, from which the specified enzyme penetrates into the tissues of the central nervous system and is involved in the release of accumulated substances. The content of the enzyme obtained by CNS tissues is thus limited to this amount expressed and released from donor-derived cells injected into the brain. While such administrations are of great benefit to MPS I, recipients nevertheless continue to exhibit sub-normal IQs. and impaired neurocognitive ability (Ziegler and Shapiro, 2007).

The phenomenon of metabolic cross-correction also explains the effectiveness of ERT for several lysosomal storage diseases (Brady, 2006), especially MPS I. However, due to the requirement to cross the blood-brain barrier (BBB) of an enzyme that is absent in a particular lysosomal storage disease (LSD) in order to In order to effectively reach the CNS, the efficacy of enzyme therapy for the neurological manifestations of lysosomal storage disease (LSD) has not been observed (Brady, 2006). Enzymes are almost always too large and usually too charged to efficiently cross the BBB. This has prompted research into invasive intrathecal enzyme administration (Dickson et al., 2007), which has been shown to be effective in a canine model of MPS I (Kakkis et al., 2004) and human clinical trials are starting for MPS I (Pastores, 2008). ; Munoz-Rojas et al., 2008). The main disadvantages of enzyme therapy are its high cost (>$200,000 per year) and the need for repeated infusions of recombinant protein. Current intrathecal IDUA clinical trials are designed to administer the enzyme only once every three months, so the effectiveness of this dosing regimen remains unclear.

Brief summary of the present invention

Described are methods for preventing, inhibiting and/or treating one or more symptoms associated with a central nervous system (CNS) disease in a mammal in need thereof. The methods include delivering to the CNS, to a 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 can be encoded by the vector rAAV include, without limitation, 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, without limitation, mucopolysaccharidosis type I disorder, mucopolysaccharidosis type II disorder, or mucopolysaccharidosis type VII disorder. The AAV vector can be administered in various forms to ensure that it enters the CNS/brain and that the transgene is successfully transduced in the CNS/brain of the subject. CNS/brain delivery routes include, without limitation, intrathecal administration, intracranial administration, eg, intracerebroventricular administration or administration to the lateral ventricles of the brain), intranasal administration, endovascular administration, and intraparenchymal administration.

In one embodiment, the methods include delivering to the CNS of an adult mammal in need of treatment a composition comprising an effective amount of a rAAV-9 vector containing an open reading frame encoding a gene. In one embodiment, the methods include delivering to the CNS of an adult mammal in need of treatment a composition comprising an effective amount of a rAAV-9 vector containing an open reading frame encoding IDUA. These methods are based in part on the discovery that the AAV-9 vector can efficiently transduce a therapeutic transgene in the brain/CNS of adult subjects, restoring the enzyme to wild-type levels (see FIG. 15, below). The results achieved using AAV-9 are surprising in view of previous work that showed that intravascular delivery of AAV-9 in adult mice did not achieve broad direct neuronal targeting (see Foust et al., 2009) and additional data showing that direct injection of AAV8-IDUA into the CNS of adult IDUA-deficient mice resulted in poor transgene expression (FIG. 18). As proof of principle, the operating examples described herein use a preclinical model for the treatment of MPS1, an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). Actual examples show 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 IDUA enzyme expression and activity in wild-type adult mice (Fig. 15, below). ).

According to a further embodiment of the present invention, the Actual Examples also show that co-therapy to induce immunosuppression or acquire immunological tolerance or treat immunocompromised animals can achieve even higher levels of IDUA enzyme expression and activity. In one embodiment, patients with genotypes that promote an immune response that neutralizes enzyme activity (see e.g. Barbier et al., 2013) are exposed to an immunosuppressant in addition to an rAAV vector containing an open reading frame encoding a gene product such as IDUA.

Newborn mice IDUA ^-/- are characterized by a lack of immunocompetence. However, administration of IDUA-expressing AAV-8 to neonatal IDUA mice results in IDUA expression (Wolf et al., 2011), thus inducing animal tolerance to IDUA. As described herein, the applicability of AAV-mediated gene transfer to adult (immunocompetent) mice by direct infusion of AAV into the central nervous system has been shown using various routes of administration. For example, AAV-IDUA serotype 9 was administered by direct injection into the lateral ventricles of adult IDUA-deficient mice that were either immunocompetent, immunodeficient (NODSCID/IDUA-/-), immunosuppressed with cyclophosphamide (CP), or immunotolerant by weekly injections. human iduronidase protein (aldurazyme) from birth. CP immunosuppressed animals were also treated with AAV9-IDUA by intranasal infusion, by intrathecal administration, and by endovascular infusion with and without mannitol to disrupt the blood-brain barrier. Animals were sacrificed 8 weeks after vector administration and brains were harvested and microprepared to assess IDUA enzymatic activity, tissue glycosaminoglycans and IDUA vector sequences compared to normal and exposed control mice. The results of these studies indicate that numerous routes can be used to introduce the AAV vector directly into the CNS, for example, in order to achieve a higher content of delivered proteins and/or enzymatic activity in the CNS. In addition, although the brain is an immune-privileged site, the administration of an immunosuppressant or the provision of immunological tolerance may increase the activity found in the brain following AAV administration. Higher levels of expression per administration and/or minimally invasive routes of administration are clinically more acceptable to patients.

Thus, the present invention contemplates the use of recombinant AAV vectors (rAAV) that encode a gene product with a therapeutic effect when expressed in the mammalian CNS. In one embodiment, the mammal is an immunocompetent mammal with a disease or disorder of the central nervous system (neurological disease). As used herein, the term "immunocompetent" mammal is a mammal at an age where both cellular and humoral immune responses occur upon exposure to an antigenic stimulus by upregulating Th1 functions or producing IFN-gamma in response to polyclonal stimuli, as opposed to a newborn that is characterized by innate immunity and immunity received from the mother, for example, during pregnancy or breastfeeding. An adult mammal that is not immunodeficient is an example of an immunocompetent mammal. For example, an immunocompetent human is typically a human at least 1, 2, 3, 4, 5, or 6 months of age and includes non-immunocompromised adults. In one embodiment, the AAV is administered intrathecally. In one embodiment, the AAV is administered intracranially (eg, intracerebroventricularly). In one embodiment, the AAV is administered intranasally, with or without a penetration enhancer. In one embodiment, the AAV is administered endovascularly, eg, through the carotid artery, with or without a permeation enhancer. In one embodiment, the AAV-administered mammal is an immunocompromised or immunotolerized or immunosuppressed mammal, e.g., to induce higher expression levels of the therapeutic protein relative to the corresponding AAV-administered mammal but not immunotolerized or immunosuppressed. In one embodiment, an immunosuppressive agent is administered to induce immune suppression. In one embodiment, the mammal to which the AAV is administered is not subjected to immunological tolerization or immunosuppression (eg, administration of the AAV alone provides a therapeutic effect).

The present invention provides 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 includes intrathecal, for example, in the lumbar region, or intracerebroventricular, for example, in the lateral ventricle, administration to a mammal of a composition containing an effective amount of a rAAV vector containing an open reading frame encoding a gene product, the expression of which in the central nervous system of a mammal prevents, inhibits or treats one or more symptoms. In one embodiment, the gene product is a storage-associated lysosomal enzyme. In one embodiment, the mammal is an immunocompetent adult mammal. 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 doses are administered. In one embodiment, the composition is administered weekly, monthly, or every two or more months.

In one embodiment, the method comprises intrathecal, e.g., in the lumbar region, administering to a mammal a composition comprising an effective amount of an rAAV vector containing an open reading frame encoding a gene product whose expression in the mammalian central nervous system prevents, inhibits, or treats one or more symptoms, and optionally introducing a penetration enhancer. In one embodiment, the penetration enhancer is administered prior to the administration of the composition. According to one embodiment, the composition comprises a penetration enhancer. In one embodiment, the penetration enhancer is administered after administration of the composition. In one embodiment, the gene product is a storage-associated lysosomal enzyme. In one embodiment, the mammal is an immunocompetent adult mammal. 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 doses are administered. In one embodiment, the composition is administered weekly, monthly, or every two or more months. In one embodiment, the mammal receiving intrathecal AAV does not undergo immunological tolerization or immunosuppression (eg, administration of AAV alone provides a therapeutic effect). In one embodiment, the mammal receiving intrathecal AAV is an immunocompromised mammal or is undergoing immunological tolerization or immunosuppression, e.g., to induce higher levels of expression of the therapeutic protein, compared to a corresponding mammal receiving intrathecal AAV but not immunologically tolerization or immunosuppression.

In one embodiment, the method comprises intracerebroventricular, e.g., in the lateral ventricle, administering to an immunocompetent mammal a composition comprising an effective amount of an rAAV containing an open reading frame encoding a gene product whose expression in the mammalian central nervous system prevents, inhibits, or treats one or more symptoms. In one embodiment, the gene product is a storage-associated lysosomal enzyme. In one embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 vector , AAV-8, AAV rh10 or AAV-9. In one embodiment, the rAAV vector is not a rAAV-5 vector. In one embodiment, the mammal is a human. In one embodiment, multiple doses are administered. In one embodiment, the composition is administered weekly, monthly, or every two or more months. In one embodiment, the mammal to which AAV is intracerebroventricularly administered does not undergo immunological tolerization or immunosuppression (eg, administration of AAV alone provides a therapeutic effect). In one embodiment, the mammal receiving intracerebroventricular AAV is an immunodeficient mammal or is subjected to immunological tolerization or immunosuppression, e.g., to induce higher levels of expression of the therapeutic protein, compared to the corresponding mammal receiving intracerebroventricular AAV but not subjected to immunological tolerization or immunosuppression. In one embodiment, the mammal acquires immunological tolerance to the gene product prior to administration of the AAV-containing composition.

Additionally, the present invention provides 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 includes endovascular administration to a mammal of a composition containing an effective amount of a rAAV vector containing an open reading frame encoding a gene product, the expression of which in the central nervous system of a mammal prevents, inhibits or treats one or more symptoms, and an effective amount of a permeability enhancer. According to one embodiment, the composition comprises a 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-lauryl ether, or EDTA. In one embodiment, the gene product is a storage-associated lysosomal enzyme. In one embodiment, the mammal is an immunocompetent adult mammal. 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 a rAAV-5 vector. In one embodiment, the mammal is a human. In one embodiment, multiple doses are administered. In one embodiment, the composition is administered weekly. In one embodiment, the composition is administered weekly, monthly, or every two or more months. In one embodiment, the mammal to which AAV is endovascularly administered does not undergo immunological tolerization or immunosuppression (eg, administration of AAV provides a therapeutic effect). In one embodiment, the mammal to which AAV is endovascularly administered is an immunodeficient mammal or is subjected to immunological tolerization or immunosuppression, e.g., to induce higher levels of expression of the therapeutic protein, compared to the corresponding mammal to which AAV is endovascularly administered but not immunologically administered. tolerization or immunosuppression.

In one embodiment, the method comprises intranasally administering to a mammal a composition comprising an effective amount of a rAAV-9 vector containing an open reading frame encoding a gene product whose expression in the mammalian central nervous system prevents, inhibits, or treats one or more symptoms, and optionally administering an enhancer permeability. In one embodiment, intranasal delivery can be achieved as described in US Pat. No. 8,609,088, the disclosure of which is incorporated herein by reference. In one embodiment, the penetration enhancer is administered prior to administration of the composition. According to one embodiment, the composition comprises a penetration enhancer. In one embodiment, the penetration enhancer is administered after administration of the composition. In one embodiment, the gene product is a storage-associated lysosomal enzyme. In one embodiment, the mammal is an immunocompetent adult mammal. In one embodiment, the mammal is a human. In one embodiment, multiple doses are administered. In one embodiment, the composition is administered weekly, monthly, or every two or more months. In one embodiment, the mammal receiving intranasal AAV does not undergo immunological tolerization or immunosuppression. In one embodiment, a mammal receiving intranasal AAV is immunotolerized or immunosuppressed, e.g., to induce higher levels of IDUA protein expression, compared to a corresponding mammal administered intranasally with AAV, but not immunotolerized or immunosuppressed.

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 includes administering to a mammal a composition containing an effective amount of an rAAV vector containing an open reading frame encoding a gene product whose expression in the mammalian central nervous system prevents, inhibits or treats 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, including an alkylating agent, or an antimetabolite such as methotrexate, azathioprine, mercaptopurine, or a cytotoxic antibiotic, antibody, or agent active against an immunophilin. In one embodiment, the immunosuppressant comprises nitrogen mustard, nitrosourea, a platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 (CD25-) or CD3 receptor-directed antibodies, antibodies to IL-2, cyclosporine, tacrolimus, sirolimus, IFN-β, interferon-γ, opioid, or TNF-α (tumor necrosis factor-alpha) binders 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 administration of the immunosuppressant. In one embodiment, the immunosuppressant is administered prior to administration of 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 storage-associated lysosomal enzyme. In one embodiment, the mammal is an adult mammal. 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 doses are administered. In one embodiment, the composition is administered weekly. In one embodiment, the composition is administered weekly, monthly, or every two or more months.

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 with acquired immunological tolerance to a gene product that is associated with a disease is administered a composition containing an effective amount of an rAAV vector containing an open reading frame encoding a gene product whose expression in the mammalian central nervous system prevents, inhibits, or treats one or more symptoms. In one embodiment, the gene product is a storage-associated lysosomal enzyme. In one embodiment, the mammal is an adult mammal. 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 doses are administered. According to one embodiment, the composition is administered weekly.

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 growth factor (BDGF), neuroturin, glial growth factor (GDGF), tyrosine hydroxylase, dopamine decarboxylase, or glutamic acid decarboxylase.

Diseases that are characterized by one or more neurological symptoms that can be prevented, inhibited, or treated using the methods described herein include, without limitation, the following: adrenoleukodystrophy, Alzheimer's disease, amyotrophic lateral sclerosis, Angelman's syndrome, ataxia telangiectasia syndrome, Charcot's syndrome -Marie-Tooth, Cockayne syndrome, deafness, Duchenne muscular dystrophy, epilepsy, essential tremor, fragile X syndrome, Friedreich's ataxia, Gaucher disease, Huntington's disease, Lesch-Nyhan syndrome, maple syrup urine disease, Menkes syndrome, myotonic dystrophy, narcolepsy, neurofibromatosis, Niemann-Pick disease, Parkinson's disease, phenylketonuria, Prader-Willi syndrome, Refsum disease, Rett syndrome, spinal muscular atrophy, spinocerebellar ataxia, Tangier disease, Tay-Sachs disease, tuberous sclerosis, Hippel-Lindau syndrome, Williams syndrome, Wilson's disease or Zellweger's syndrome. In one embodiment, the disease is a lysosomal storage disease, such as the absence or deficiency of a storage-associated lysosomal enzyme. Lysosomal storage diseases include, but are not limited to, mucopolysaccharidosis (MPS) disease, eg mucopolysaccharidosis type I, eg Hurler's syndrome and variants of Schie's syndrome and Hurler-Scheie's syndrome (alpha-L-iduronidase deficiency); Hunter's syndrome (deficiency of iduronate-2-sulfatase); mucopolysaccharidosis type III, such as Sanfilippo syndrome (A, B, C, or D; deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl-CoA:alpha-glucosaminide-N-acetyl transferase, or N-acetylglucosamine-6 -sulfatase sulfate); mucopolysaccharidosis type IV such as, for example, Morquio's syndrome (deficiency of galactosamine-6-sulphate sulfatase or beta-galactosidase); mucopolysaccharidosis type VI, for example, Maroteau-Lami syndrome (arylsulfatase B deficiency); mucopolysaccharidosis type II; mucopolysaccharidosis type III (A, B, C, or D; deficiency of heparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetyl-CoA: alpha-glucosaminide-N-acetyltransferase, or N-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV (A or B; deficiency of galactosamine-6-sulfatase and beta-galactosidase); mucopolysaccharidosis type VI (arylsulfatase B deficiency); mucopolysaccharidosis type VII (beta-glucuronidase deficiency); mucopolysaccharidosis type VIII (glucosamine-6-sulphate sulfatase deficiency); mucopolysaccharidosis type IX (hyaluronidase deficiency); Tay-Sachs disease (deficiency in the alpha subunit of beta-hexosaminidase); Sandhoff's disease (deficiency in both the alpha and beta subunits of beta-hexosaminidase); gangliosidosis GM1 (type I or type II); Fabry disease (alpha-galactosidase deficiency); metachromatic leukodystrophy (aryl sulfatase A deficiency); Pompe's disease (lack of acid maltase); fucosidosis (fucosidase deficiency); alpha-mannosidosis (deficiency of alpha-mannosidase); beta-mannosidosis (beta-mannosidase deficiency), neuronal waxy lipofuscinosis and Gaucher disease (types I, II and III; glucocerebrosidase deficiency), as well as disorders such as Hermansky-Pudlak syndrome; amavrotic idiocy; Tangier disease; aspartylglucosaminuria; congenital glycosylation disorder, type Ia; Chediak-Higashi syndrome; macular degeneration of the cornea, 1; cystinosis, nephropathic; Fanconi-Bickel syndrome; Farber's lipogranulomatosis; fibromatosis; heliophysical dysplasia; glycogen storage disease I; glycogen storage disease Ib; glycogen storage disease Ic; glycogen storage disease III; glycogen storage disease IV; glycogen storage disease V; glycogen storage disease VI; associated with the accumulation of glycogen disease VII; glycogen storage disease 0; immuno-osteoid dysplasia, Shimke's type; lipidosis; lipase b; mucolipidosis II; mucolipidosis II, including a variant form; mucolipidosis IV; neuraminidase deficiency with beta-galactosidase deficiency; mucolipidosis I; Niemann-Pick disease (deficiency of sphingomyelinase); Niemann-Pick disease without sphingomyelinase deficiency (deficiency of the npc1 gene encoding the cholesterol-metabolizing enzyme); Refsum's disease; blue histiocyte disease; infantile form of sialic acid accumulation disease; sialuria; multiple sulfatase deficiency; triglyceride storage disease with impaired long-chain fatty acid oxidation; Winchester's disease; Wolman's disease (deficiency of cholesterol ester hydrolase); deoxyribonuclease I-like disorder 1; violation of arylsulfatase E; violation of ATPase, transport of H +, lysosomal, subunit 1; glycogen storage disease IIb; disruption of the Ras-associated protein rab9; punctate epiphyseal dysplasia 1, an X-linked recessive disorder; glycogen storage disease VIII; disruption of lysosome-associated membrane protein 2; Menkes syndrome; congenital malformation of glycosylation, type Ic; and sialuria. Replacing less than 20%, such as less than 10% or about 1% to 5%, of the storage-associated lysosomal enzyme found in disease-free mammals can prevent, inhibit, or treat neurological symptoms such as neurological degenerations in mammals.

In one embodiment, the methods described herein include delivering to the CNS of an immunocompetent human in need of such treatment a composition comprising an effective amount of a rAAV-9 vector containing an open reading frame encoding IDUA. Routes of administration to the CNS/brain include, without limitation, intrathecal administration, intracranial administration, eg, intracerebroventricular administration or administration to the lateral ventricles of the brain, intranasal administration, endovascular administration, and intraparenchymal administration.

Other viral vectors may be used in the methods of the present invention, for example, viral vectors such as retrovirus, lentivirus, adenovirus, Semliki forest virus, or herpes simplex virus vectors.

Brief description of graphic materials

Figure 1. Experimental design for iduronidase deficient mice administered IDUA-AAV either intracerebroventricularly (ICV) or intrathecally. To prevent immune response, animals were either immunosuppressed with cyclophosphamide (CP), immunologically tolerated at birth by intravenous administration of the human iduronidase protein (aldurazyme), or injected into immunocompromised NOD-SCID mice, which were also iduronidase deficient. Animals were sacrificed at the indicated time after exposure, the brain was subjected to micropreparation, and iduronidase activity was analyzed in the extracts.

Figure 2. IDUA activity in immunodeficient, IDUA-deficient animals.

Figure 3. IDUA activity in immunosuppressed animals injected with the AAV vector by ICV.

Figure 4. IDUA activity in immunosuppressed animals injected with the AAV vector by IT.

Figure 5. IDUA activity in animals with acquired immunological tolerance, which was injected with the AAV ICV vector.

Figure 6. Comparison of all average levels of IDUA activity for comparison.

Figure 7. Data grouped according to brain region.

Figure 8. Analysis of the GAG accumulation substance in different parts of the brain for all four study groups.

Figure 9. Scheme of the experiment.

Figure 10. Intracranial infusion of AAV9IDUA into immunodeficient mice with MPS I. Adult animals were injected with 10 ¹¹ vector genomes and brain iduronidase expression was assessed after 10 weeks. The levels of enzymatic activity in the brain were significantly higher than in the brain of wild-type animals, and varied in the range, which was from 30 to 300 times higher than in wild-type animals.

Figure 11. Intracranial administration of AAV9IDUA to immunocompetent, IDUA-deficient mice. Adult animals were injected with 10 ¹¹ vector genomes and subjected to immunosuppression by weekly injection of cyclophosphamide (CP). CP injections were discontinued 6 weeks after vector injection due to poor health and animals were sacrificed 8 weeks after injection. The brain was subjected to micropreparation and analyzed the activity of the enzyme IDUA.

Figure 12. Intracranial infusion of AAV9IDUA into MPS I mice with acquired immunological tolerance. Mice with MPS 1 were tolerated with either a single dose of aldurazyme at birth or multiple doses administered weekly from birth. Mice were injected with the vector at 4 months and sacrificed 11 weeks after injection. The brain was subjected to micropreparation and analyzed the expression of iduronidase. Enzyme activities varied in a range that was on average 10 to 1000 times higher than levels in wild-type animals.

Figure 13. Intrathecal administration of AAV9IDUA to immunocompetent, IDUA deficient animals. MPS I adult mice were treated with AAV9IDUA intrathecally followed by a weekly cyclophosphamide immunosuppressive regimen. Animals were sacrificed 11 weeks after injection, and then IDUA enzyme activity was analyzed in the brain and spinal cord.

Figure 14. Intrathecal infusion of AAV9IDUA in MPS I mice with acquired immunological tolerance. IDUA-deficient animals were induced at birth with a single dose of aldurazyme or multiple doses administered weekly from birth. At the age of 4 months, the animals were intrathecally injected with the AAV9IDUA vector and 10 weeks after the injection, the animals were sacrificed, the brain was subjected to micropreparation and iduronidase activity was analyzed. Restoration of enzyme activity was observed in all parts of the brain, with activity in the cerebellum ranging from 200 to 1500 times higher than levels in wild-type animals. Enzyme activity levels in the olfactory bulb and cerebellum (to the right of the dotted line) correspond to the right y-axis.

Figure 15. Intrathecal infusion of AAV9IDUA into immunocompetent MPS I animals. Control MPS I animals were injected with the AAV9IDUA vector but were neither immunosuppressed nor immunotolerized. Animals were sacrificed 11 weeks after vector injection and then iduronidase activity was analyzed in their brains. Enzyme levels were restored to wild-type levels in all parts of the brain, but were significantly lower than in animals that had either been immunosuppressed or immunotolerized.

Figure 16. Normalization of the content of glycosaminoglycans (GAG) after intracranial or intrathecal infusion of AAV9. AAV9IDUA was administered intracranially or intrathecally to immunocompromised MPS I mice subjected to immunosuppression or immunological tolerization as indicated. Animals were sacrificed 8-11 weeks after injection, and then the brain was subjected to micropreparation and analyzed for GAG content. The accumulation of GAGs was restored to the content corresponding to wild-type animals or close to wild-type animals in all analyzed groups.

Figure 17. Vector copies of IDUA in the brain. IDUA vector sequences were analyzed in microprepared brains by quantitative PCR. The copy number in mice administered intracranially and intrathecally correlated with the level of enzyme activity depicted in FIG. 11 and 13.

Figure 18. AAV8-MCI ICV infusion in adult animals.

Figure 19. Intranasal administration of AAV9/IDUA to immunocompetent, IDUA deficient animals. MPS I adult mice were injected with AAV9/IDUA intranasally followed by a weekly cyclophosphamide immunosuppressive regimen. Animals were sacrificed 12 weeks after injection and IDUA enzyme activity was analyzed in the brain.

Figure 20. Vector copies of IDUA in the brain. IDUA vector sequences were analyzed in microprepared brains by quantitative PCR. The copy number in mice administered intranasally correlated with the enzyme content in FIG. 19.

Detailed description of the present invention

Definitions

As used herein, the term "individual" (as in a subject of treatment) means a mammal. Mammals include, for example, humans, non-human primates, such as monkeys and marmosets; and non-primates, such as dogs, cats, rats, mice, cattle, horses, sheep, and goats. Animals other than mammals include, for example, fish and birds.

The terms "disease" or "disorder" are used interchangeably and are used to refer to diseases or conditions in which the absence or reduction of a particular gene product, such as that associated with lysosomal enzyme accumulations, plays an important role in the disease, such that a therapeutically beneficial effect may be achieved by supplementing, for example, at least 1% of the normal content.

As used herein, the term "substantially" means wholly or almost wholly; for example, a composition that is "substantially free" of a component either contains no component or contains such a minor amount that any relevant functional property of the composition is not dependent on the presence of trace amounts, or the compound is "substantially pure" if present. only slight traces of impurities present.

"Treatment" or "treatment" as used herein refers to the alleviation of symptoms associated with a disorder or disease, "inhibition" means inhibition of further progression or worsening of symptoms associated with a disorder or disease, and "prevention" refers to the prevention of symptoms associated with with a disorder or disease.

As used herein, an "effective amount" or "therapeutically effective amount" of an agent of the present invention, such as a recombinant AAV encoding a gene product, refers to an amount of the agent that alleviates, in whole or in part, the symptoms associated with the disorder or condition, or stops or retards the further progression or worsening of these symptoms, or prevents or prevents a disorder or condition, for example, an amount that is effective to prevent, inhibit or treat one or more neurological symptoms in an individual.

In particular, "therapeutically effective amount" refers to an amount effective at the 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 deleterious effects of the compounds of the present invention are outweighed by the therapeutically beneficial effects.

As used herein, a "vector" refers to a macromolecule or complex of macromolecules that contains or is linked to a polynucleotide and that can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The delivered polynucleotide, sometimes referred to as a "target polynucleotide" or "transgene", may contain a gene therapy coding sequence of interest (such as a gene encoding a protein of therapeutic interest) and/or a selectable or detectable marker.

"AAV" is an adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term includes all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where otherwise required. As used herein, the term "serotype" refers to AAV. which is identified with and distinguished from other AAVs based on its binding properties, e.g., there are eleven AAV serotypes, AAV-1-AAV-11, including AAV-2, AAV-5, AAV-8, AAV-9 and AAV rh10 and the term covers pseudo-types with the same binding properties. Thus, for example, AAV-5 serotypes include AAVs with AAV-5 binding properties, e.g., a pseudotyped AAV containing an AAV-5 capsid and an rAAV genome that does not originate from or is not derived from AAV-5, or whose genome is chimeric. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as recombinant AAV vector (or "rAAV vector").

The term "AAV virus" refers to a viral particle consisting of at least one AAV capsid protein and a polynucleotide enclosed in a capsid. If the particle contains a heterologous polynucleotide (ie, a polynucleotide other than the wild-type AAV genome, such as a transgene for delivery to a mammalian cell), then it is generally referred to as "rAAV". AAV "capsid protein" includes the wild-type AAV capsid protein as well as modified forms of the AAV capsid protein that are structurally and/or functionally capable of packaging the rAAV genome and bind to at least one specific cellular receptor, which may be different from the receptor wild-type AAV used. The modified AAV capsid protein includes a chimeric AAV capsid protein, such as one that contains amino acid sequences from two or more AAV serotypes, e.g. AAV-2, and an AAV capsid protein containing a tag or other detectable non-AAV capsid protein or a protein fused or linked to an AAV capsid protein, e.g., an antibody moiety that binds to the transferrin receptor can be recombinantly fused to an AAV capsid protein -2.

A "pseudotyped" rAAV is an infectious virus containing any combination of the AAV capsid protein and the AAV genome. Capsid proteins from any AAV serotype can be used with an rAAV genome that is derived from the wild-type AAV genome of another serotype or that is a chimeric genome, i. formed from AAV DNA from two or more different serotypes, for example a chimeric genome containing 2 inverted terminal repeats (ITRs), each ITR from different serotypes or chimeric ITRs. The use of chimeric genomes, such as those containing ITRs from two AAV serotypes or chimeric ITRs, can lead to directed recombination, which can further enhance the production of transcriptionally active intermolecular concatemers. Thus, the 5' and 3' ITRs within the rAAV vector of the present invention may be homologous, ie. from one serotype, heterologous, i.e. from different serotypes, or chimeric, i.e. ITR that contains ITR sequences from more than one AAV serotype.

Vectors rAAV

Adeno-associated viruses of any serotype are suitable for rAAV production, since different serotypes are functionally and structurally related even at the genetic level. All AAV serotypes appear to have similar replication properties mediated by homologous rep genes; and all typically carry three interconnected capsid proteins, such as those expressed in AAV2. The degree of relatedness is further confirmed by heteroduplex analysis, which shows extensive cross-hybridization between serotypes along the length of the genome; and having similar self-annealing segments at the ends that match the ITR. Similar infectivity patterns also suggest that replication functions in each serotype are under similar regulatory control. Among the various AAV serotypes, AAV2 is the most commonly used.

The AAV vector of the present invention typically contains a polynucleotide that is heterologous to AAV. A polynucleotide is generally of interest because of its ability to provide a function to a target cell in the context of gene therapy, such as upregulating or downregulating the expression of a particular phenotype. Such a heterologous polynucleotide or "transgene" is generally characterized by a length sufficient to provide the desired function or coding sequence.

Where transcription of a heterologous polynucleotide is desired in the intended target cell, it may be operably linked to a self or heterologous promoter, depending, for example, on the desired level and/or specificity of transcription within the target cell, as is known in the art. Various types of promoters and enhancers are suitable for use in this context. Constitutive promoters provide a constant level of transcription of a gene and may be preferred when it is desired that a therapeutic or prophylactic polynucleotide be expressed on a constant basis. Inducible promoters are generally low in activity in the absence of an inducer and are activated in the presence of an inducer. They may be preferred when expression is desired only at certain times or locations, or when titration of the expression level with an inducing agent is desired. Promoters and enhancers can also be tissue-specific: i.e. have activity only in certain cell types, presumably due to gene regulatory elements found only in these cells.

Illustrative examples of promoters are the OB40 monkey virus 40 late promoter, the baculovirus polyhedron promoter/enhancer element, herpes simplex virus thymidine kinase (HSV tk), the cytomegalovirus (CMV) immediate early promoter, and various retroviral promoters, including LTR elements. Inducible promoters include heavy metal ion inducible promoters (eg, 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. By way of illustration, examples of tissue-specific promoters include various surfactin promoters (for lung expression), myosin promoters (for muscle expression), and albumin promoters (for liver expression). A wide variety of other promoters are known and publicly available in the art, and the sequences of many such promoters are available from sequence databases such as the GenBank database.

Where translation is also desired in the intended target cell, the heterologous polynucleotide will preferably also contain control elements that facilitate translation (eg, a ribosome binding site or "RBS" and a polyadenylation signal). Accordingly, a heterologous polynucleotide typically 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 poly-A signal. A heterologous polynucleotide may contain one coding region or more than one coding region under the control of the same or different promoters. The entire block containing the combination of control elements and coding region is often referred to as an expression cassette.

The heterologous polynucleotide is inserted by recombinant techniques into or at the site of the AAV genomic coding region (ie, at the site of the AAV rep and cap genes), but is typically flanked by AAV inverted terminal repeat (ITR) regions. This means that the ITR appears both upstream and downstream of the coding sequence, or in direct contact, for example (but not necessarily) without any intermediate sequence of AAV origin, in order to reduce the likelihood of recombination , which can regenerate AAV genome replication. However, a single ITR may be sufficient to perform the functions normally associated with configurations containing two ITRs (see e.g. WO 94/13788) and vector constructs with only one ITR, thus can be used in conjunction with packaging methods. and production according to the present invention.

Natural promoters for rep are self-regulating and may limit the amount of AAV particles produced. The rep gene can also be operably linked to a heterologous promoter, whether rep is provided as part of a vector construct or separately. Any heterologous promoter that is not heavily repressed by rep gene expression is suitable; but inducible promoters may be preferred since constitutive expression of the rep gene may have a negative effect on the host cell. A wide variety of inducible promoters are known in the art; including, by way of illustration, inducible heavy metal ion promoters (eg, metallothionein promoters); inducible steroid hormone promoters (such as the MMTV promoter or growth hormone promoters); and promoters such as those from T7 phage which are active in the presence of T7 RNA polymerase. One subclass of inducible promoters are those promoters that are induced by a helper virus that is used to complement the replication and packaging of the rAAV vector. A number of helper virus-inducible promoters have also been described, including an adenovirus early gene promoter that is induced by the adenovirus E1A protein; adenovirus major late promoter; a herpes virus promoter that is induced by herpes virus proteins such as VP16 or 1CP4; as well as vaccinia or poxvirus inducible promoters.

Methods for identifying and testing virus-inducible promoters have been described (see, for example, WO 96/17947). Thus, methods are known in the art for determining whether candidate promoters are inducible by a helper virus and whether or not they will be useful in obtaining high efficiency packaging cells. Briefly, one such method involves replacing the p5 promoter of the AAV rep gene with a putative helper virus-inducible promoter (either known in the art or identified using well-known techniques such as binding to "reporter" genes without promoters). The AAV rep-cap genes (replaced by p5), for example associated with a positive selectable marker such as an antibiotic resistance gene, are then stably integrated into a suitable host cell (such as HeLa or A549 cells, examples of which are given below). Cells that are able to grow relatively well under selection conditions (eg, in the presence of an antibiotic) are then tested for their ability to express the rep and cap genes when a helper virus is added. As a first test for rep and/or cap expression, cells can be easily screened using immunofluorescence to detect Rep and/or Cap proteins. Confirmation of the feasibility and efficiency of packaging can be determined by functional studies on replication and packaging of incoming rAAV vectors. Using this technique, a helper virus-inducible promoter is derived from the mouse metallothionein gene, which has been identified as a suitable replacement for the p5 promoter and is used to produce high titers of rAAV particles (as described in WO 96/17947).

Deletion of one or more AAV genes is in any case undesirable to reduce the likelihood of obtaining replication-competent AAV ("RCA"). Accordingly, the coding or promoter sequences for rep, cap, or both can be deleted, since the functions provided by these genes can be envisioned in trans.

The resulting vector is referred to as "defective" with respect to these features. In order to replicate and package the vector, the missing functions are completed by a packaging gene, or a plurality of them, which together encode the required functions for various lost rep and/or cap gene products. The packaging genes or gene cassettes of one embodiment are not in the AAV ITR environment and one embodiment does not share any significant homology with the rAAV genome. Thus, in order to minimize homologous recombination during replication between a vector sequence and separately provided packaging genes, it is desirable to avoid overlap between two polynucleotide sequences. The level of homology and the corresponding frequency of recombination increases with the length of homologous sequences and with the level of their general identity. The level of homology that will present a problem in a given system can be determined theoretically and verified experimentally, as is known in the art. Generally, however, recombination can be substantially reduced or eliminated if the overlapping sequence is less than about a 25 nucleotide sequence if it is at least 80% identical throughout its length, or less than about a 50 nucleotide sequence if it is at least 70% identical along its entire length. Of course, even lower levels of homology are preferred, as they will further reduce the likelihood of recombination. It appears that even without overlapping homology, there are some residual RCA production frequencies. Even further reduction in the frequency of RCA production (eg, by non-homologous recombination) can be obtained by "splitting" the replication and encapsulation of AAV functions, as described by Allen et al., WO 98/27204).

The rAAV vector construct and complementary gene packaging constructs may be implemented in the present invention in a number of different forms. Viral particles, plasmids, and stably transformed host cells can all be used to introduce such constructs into a packaging cell, either transiently or stably.

In some embodiments of the present invention, the AAV vector and complementary packaging gene(s), if any, are provided as bacterial plasmids, AAV particles, or any combination thereof. In other embodiments, either the AAV vector sequence, the packaging gene(s), or both, are presented as genetically modified (preferably genetically modified) eukaryotic cells. Host cell development that is genetically altered to express an AAV vector sequence, AAV packaging genes, or both, includes an established source of material that is expressed at a reliable level.

A variety of different genetically modified cells can thus be used in the context of the present invention. By way of illustration, a mammalian host cell can be used with at least one intact copy of a stably integrated rAAV vector. An AAV packaging plasmid containing at least the AAV rep gene operably linked to a promoter can be used to provide replication functionality (as described in US Pat. No. 5,658,776). In addition, a stable mammalian cell line with an AAV rep gene operably linked to a promoter can be used to provide a replication function (see, for example, Trempe et al., WO 95/13392); Burstein et al. (WO 98/23018) and Johnson et al. (US No. 5656785). The AAV cap gene, which provides protein encapsulation as described above, can be provided together with the AAV rep gene or separately (see, for example, the applications and patents mentioned above, as well as Allen et al. (WO 98/27204). Other combinations are possible and included in the scope of the present invention.

Shipping Ways

Despite the vast network of the cerebral vasculature, systemic drug delivery to the central nervous system (CNS) is not effective for more than 98% of small molecules and almost 100% of large molecules (Partridge, 2005). The lack of effectiveness is due to the presence of the blood-brain barrier (BBB), which prevents most foreign substances, even many useful drugs, from entering the brain from the circulating blood. While some systemically administered small molecule, peptide, and protein therapeutics reach the brain parenchyma by crossing the BBB (Banks, 2008), high systemic doses are generally required to achieve therapeutic levels, which can lead to adverse effects in the body. Drugs can be administered directly to the CNS by intracerebroventricular or intraparenchymal injections. Intranasal delivery bypasses the BBB and directs drugs directly to the CNS using the olfactory and trigeminal nerve pathways that innervate the nasal passages (Frey II, 2002; Thorne et al., 2004; Dhanda et al., 2005).

Any route of administration of rAAV may be used, provided that the route and amount administered is prophylactically or therapeutically useful. In one example, routes of administration to the CNS include intrathecal and intracranial routes. Intracranial administration can be carried out in the cerebellopontine cistern or ventricle. The term "cerebellar cistern" is intended to include access to the space around and below the cerebellum through the opening between the skull and the top of the spine. The term "cerebral ventricle" is intended to include cavities in the brain that are in continuous communication with the central canal of the spinal cord. Intracranial administration is by injection or infusion, and suitable dose ranges for intracranial administration typically range from about 10 ³ to 10 ¹⁵ infectious units of viral vector per microliter delivered at a dose of 1 to 3000 μl per injection volume. For example, viral genomes or infectious vector units per microliter typically contain approximately 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ^{8 , 10 9 ,} 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ^{13 ,} or 10 ¹⁴ viral genomes or infectious units of a viral vector delivered in approximately 10, 50, 100, 200, 500, 1000, or 2000 μl. It should be understood that the above dosage is only an illustrative dosage, and those skilled in the art will appreciate that this dosage may vary. Effective doses can be extrapolated from dose response curves obtained from in vitro or in vivo test systems.

The AAV delivered by the intrathecal treatments of the present invention may be administered by any convenient route commonly used for intrathecal administration. For example, intrathecal administration may be by slow infusion of the formulation over about an hour. Intrathecal administration is by injection or infusion, and the applicable dose range for intrathecal administration typically varies from 10 ³ to 10 ¹⁵ viral vector infectious units per microliter, delivered, for example, in a volume of 1 to 3000 microliters or 0.5 to 15 milliliters of injected volume. For example, viral genomes or vector infectious ^units per microliter will typically contain approximately 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 8 , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ^{13 ,} or 10 ¹⁴ viral genomes or infectious units of a viral vector.

Therapy, if a storage-related lysosomal enzyme such as IDUA is expressed, results in normalization of lysosomal storage granules in neuronal and/or meningeal tissue of subjects as described above. It is hypothesized that the deposition of storage granules is reduced in neurons and glial tissue, thereby reducing the developmental delay and regression seen in individuals suffering from lysosomal storage disease. Other effects of therapy may include the normalization of lysosomal storage granules in the meninges near the arachnoid granulation, the presence of which in lysosomal storage disease leads to high pressure hydrocephalus. The methods of the present invention can also be used in the treatment of spinal cord compression that results from the presence of lysosomal accumulation granules in the cervical membranes near the spine at the C1-C5 position or elsewhere in the spinal cord. The methods of the present invention are also directed to the treatment of cysts that are caused by perivascular accumulation of lysosomal storage granules around cerebral vessels. In other embodiments, therapy may also advantageously result in normalization of liver volume and urinary glycosaminoglycan excretion, reduction in spleen size and apnea/hypopnea events, increased height and growth rate in prepubertal subjects, increased shoulder flexion and elbow and knee extension, and decreased tricuspid regurgitation. or pulmonary regurgitation.

Intrathecal administration according to the present invention may include administering the composition to the lumbar region. Any such administration may be by bolus injection. Depending on the severity of the symptoms and the subject's responsiveness to therapy, the bolus injection may be administered once a week, once a month, once every 6 months, or once a year. In other embodiments, intrathecal administration is achieved by using an infusion pump. Those skilled in the art are aware of devices that can be used to effect intrathecal administration of the composition. The composition may be administered intrathecally, for example, by a single injection or by continuous infusion. It should be understood that dosage treatment may be in the form of a single dose or multiple doses.

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 including injection into the lateral ventricles of the brain through a burr hole or by cisternal or lumbar puncture, or the like. The term "lumbar region" is intended to include the region between the third and fourth lumbar (lower back) vertebrae, and more specifically, the L2-S1 region of the spine.

The introduction of the composition in accordance with the present invention in any of the above places can be achieved by direct injection of the composition or using infusion pumps. For injection, the composition may be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution, Ringer's solution or phosphate buffer. In addition, the enzyme may be formulated in solid form and re-dissolved or suspended just prior to use. Lyophilized forms are also included. Injections can be carried out, for example, by bolus injection or continuous infusion (eg, using infusion pumps) of the enzyme.

According to one embodiment of the present invention, rAAV is administered by injection into the lateral ventricles of the subject's brain. The injection can be performed, for example, through a burr hole made in the subject's skull. In another embodiment, the enzyme and/or other pharmaceutical formulation is administered through a shunt surgically inserted into the subject's cerebral ventricle. For example, injections can be made into the larger lateral ventricles, although injections can also be made into the smaller third and fourth ventricles. According to another embodiment, the compositions used in the present invention are administered by injection into the cerebellopontine cistern or lumbar region of the subject.

Although the exact mechanisms underlying intranasal drug delivery to the CNS are not fully understood, a body of evidence suggests that pathways involving the nerves connecting the nasal passages to the brain and spinal cord are important. In addition, pathways including the vascular system, cerebrospinal fluid, and lymphatic system have been implicated in the transport of molecules from the nasal cavity to the CNS. It is likely that a combination of these routes is responsible, although one route may predominate, depending on the properties of the drug used, the characteristics of the formulation and the delivery device.

Drugs can quickly access the CNS after intranasal administration via the olfactory nerve pathways leading from the nasal cavity directly to the CNS. The olfactory nerve pathways are a major component of intranasal delivery, as evidenced by the fact that fluorescently labeled substances are associated with the olfactory nerves as they pass through the cribriform plate (Jansson et al., 2002), drug concentrations in the olfactory bulbs tend to be one of the highest concentrations observed in the CNS (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 there is a strong, positive correlation between concentrations in the olfactory epithelium and olfactory bulbs (Dhuria et al., 2009a).

The olfactory pathways ascend at the top of the nasal passages, in the olfactory region, where olfactory receptor neurons (ORNs) are punctuated by supporting cells, microvillar cells, and basal cells. ORNs mediate olfaction by transmitting sensory information from the peripheral environment to the CNS (Clerico et al., 2003). Beneath the epithelium, the lamina propria contains mucus from Bowman's secreting glands, axons, blood vessels, lymphatics, and connective tissue. ORN dendrites extend into the mucosal layer of the olfactory epithelium, while the axons of these bipolar neurons extend centrally through the lamina propria and through foramina in the lamina cribrosa that separates the nasal and cranial cavities. ORN axons travel through the subarachnoid space containing CSF and stop at mitral cells in the olfactory bulbs. From there, neural projections spread to several areas of the brain, including the olfactory tract, anterior olfactory nucleus, piriform cortex, amygdala, and hypothalamus (Buck, 2000). In addition to the ORN, chemosensory neurons located in the anterior distal nasal cavity in the Grüneberg ganglion lead to the olfactory bulbs (Fuss et al., 2005; Koos et al., 2005).

The unique characteristics of ORNs promote a dynamic cellular environment for critical intranasal delivery to the CNS. Due to direct contact with toxins in the external environment, ORNs regenerate every 3-4 weeks from basal cells found in the olfactory epithelium (Mackay-Sim, 2003). Special Schwann cell-like cells called olfactory sheath cells (OMCs) surround ORN axons and play an important role in axonal regeneration, regrowth, and remyelination (Field et al., 2003; Li et al., 2005a; Li et al., 2005b) . OOCs create continuous, fluid-filled perineural canals that, interestingly, remain open despite ORN degeneration and regeneration (Williams et al., 2004).

Given the unique environment of the olfactory epithelium, it is possible for intranasally administered drugs to reach the CNS via extracellular or intracellular transport mechanisms along the olfactory nerves. Extracellular transport mechanisms involve the rapid movement of molecules between cells in the nasal epithelium, taking only a few minutes to 30 minutes for the drug to reach the olfactory bulbs and other areas of the CNS after intranasal administration (Frey II, 2002; Balin et al., 1986) . Transport likely involves mass movement mechanisms (Thorne et al., 2004; Thorne et al., 2001) in channels created with OOC. Drugs can also be promoted in these channels by structural changes that occur during depolarization and axonal propagation of the action potential in neighboring axons (Luzzati et al., 2004). Intracellular transport mechanisms include the uptake of molecules into the ORN by passive diffusion, receptor-mediated endocytosis or adsorptive endocytosis, followed by slower axonal transport taking the drug from hours to days to appear in the olfactory bulbs and other brain regions (Baker et al. al., 1986; Broadwell et al., 1985; Kristensson et al., 1971). Intracellular transport at the ORN has been demonstrated for small, lipophilic molecules such as gold particles (de Lorenzo, 1970; Gopinath et al., 1978), aluminum salts (Perl et al., 1987), as well as for substances with receptors on the ORN, such as WGA-HRP (Thorne et al., 1995; Baker et al., 1986; Itaya et al., 1986; Shipley, 1985). Intracellular mechanisms, while important for some drugs, may not be the predominant mode of transport in the CNS. While some large molecules, such as galanin-like peptide (GALP), exhibit saturable CNS transport pathways (Nonaka et al., 2008), other large molecules, such as NGF and insulin-like growth factor-I (IGF-I ), intranasal delivery to the brain is non-saturable and non-receptor mediated (Thorne et al., 2004; Chen et al., 1998; Zhao et al., 2004).

An often overlooked but important pathway connecting the nasal passages to 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 part of the trigeminal nerve also terminates in the olfactory bulbs (Schaefer et al., 2002). The cellular composition of the respiratory region of the nasal passages differs from that of the olfactory region, with ciliated epithelial cells distributed among mucus-secreting goblet cells. These cells contribute to mucociliary clearance mechanisms that remove mucus along with foreign matter from the nasal cavity into the nasopharynx. The trigeminal nerve transmits sensory information from the nasal cavity, oral cavity, eyelids, and cornea to the CNS via the ophthalmic branch (V1), maxillary branch (V2), or mandibular branch (V3) of the trigeminal nerve (Clerico et al., 2003; Gray, 1978). Branches from the ophthalmic branch of the trigeminal nerve supply the posterior and anterior nasal mucosa, while branches of the maxillary branch supply the lateral walls of the nasal mucosa. The mandibular branch of the trigeminal nerve extends to the mandible and teeth without direct nerve inputs to the nasal cavity. The three branches of the trigeminal nerve come together to the trigeminal ganglion and continue centrally to enter the brain at the level of the pons, ending at the spinal trigeminal nuclei in the brainstem. The unique feature of the trigeminal nerve is that it enters the brain from the respiratory epithelium of the nasal passages in two places: (1) through the anterior laceration at the pons and (2) through the cribriform plate near the olfactory bulbs, creating entry points, both in the caudal and rostral regions of the brain after intranasal administration. It is also likely that other nerves that innervate the face and head, such as the facial nerve, or other sensory structures in the nasal cavity, such as the Grueneberg ganglion, may provide CNS entry points for intranasally administered drugs.

Traditionally, the intranasal route of administration has been used to deliver drugs into the systemic circulation by absorption into capillary blood vessels underlying the nasal mucosa. The nasal mucosa is highly vascularized, receiving its blood supply from branches of the maxillary, ophthalmic, and facial arteries that originate 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 the large arterial branch of the maxillary artery (DeSesso, 1993). The relative density of blood vessels is greater in the respiratory mucosa compared to the olfactory mucosa, making the aforementioned region an ideal site for uptake into the blood (DeSesso, 1993). The vascular system in the respiratory region contains a mixture of continuous and fenestrated endothelium (Grevers et al., 1987; Van Diest et al., 1979), allowing both small and large molecules to enter the systemic circulation after nasal administration.

Delivery to the CNS after absorption into the systemic circulation and subsequent transport across the BBB is possible, especially for small, lipophilic drugs that enter the blood stream and cross the BBB more easily than large, hydrophilic drugs such as peptides and proteins.

A growing body of evidence suggests that mechanisms involving channels associated with blood vessels, or perivascular channels, are involved in intranasal drug delivery to the CNS. The perivascular spaces are connected by the outer layer of blood vessels and the basement membrane of the surrounding tissue (Pollock et al., 1997). These perivascular spaces act as a lymphatic system for the brain, where neuron-derived substances are removed from the intercellular fluid of the brain by entering the perivascular channels associated with the blood vessels of the brain. Perivascular transport occurs through volumetric flow mechanisms, as opposed to solitary diffusion (Cserr et al., 1981; Groothuis et al., 2007), and arterial pulsations are also the driving force for perivascular transport (Rennels et al., 1985; Rennels et al., 1985). Intranasally administered drugs may move into the perivascular spaces in the nasal passages or after reaching the brain, and the wide distribution observed in the CNS may be due to perivascular transport mechanisms (Thorne et al., 2004).

The pathways connecting the subarachnoid space containing cerebrospinal fluid, the perineural spaces containing the olfactory nerves, and the nasal lymphatic system are important for CSF drainage, and these same pathways provide access for intranasal drugs to the CSF and other areas of the CNS. Several studies suggest that labeled substances injected into the cerebrospinal fluid in the cerebral ventricles or subarachnoid space flow to the underside of the olfactory bulbs in channels associated with olfactory nerves passing through the cribriform plate of the cribriform bone 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). Drugs can access the CNS through these same pathways after intranasal administration, moving from the nasal passages to the cerebrospinal fluid to the interstitial spaces and perivascular spaces of the brain for distribution throughout the brain. These drainage pathways are important in a number of 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). The pathways between the nasal passages and the CSF are still important and functional in humans, as evidenced by the fact that drugs are delivered to the CSF after intranasal delivery without entering the blood to a significant extent (Born et al., 2002). A number of intranasal studies show that drugs gain direct access to the CSF from the nasal cavity with subsequent distribution to the brain and spinal cord. Many intranasally administered molecules rapidly enter the CSF and this transport depends on the lipophilicity, molecular weight and degree of ionization of the molecules (Dhanda et al., 2005; Born et al., 2002; Kumar et al., 1974; Sakane et al., 1995 ; Sakane et al., 1994; Wang et al., 2007). Assessment of distribution in the CSF can provide information on the mechanism of intranasal delivery.

Optimal delivery to the CNS along the neural pathways is associated with the delivery of the agent to the upper third of the nasal cavity (Hanson et al., 2008). While the supine position can be used, another position for targeting the olfactory region is the praying Mecca posture with the head down and forward. The supine position with a head angle of 70° or 90° may be suitable for efficient delivery to the CSF using a tube inserted into the nostrils to deliver the drug by intranasal administration (van den Berg et al., (2002)).

For intranasal drug administration, nasal drops can be administered for 10-20 minutes alternately in each nostril every 1-2 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 non-invasive method does not involve inserting a device into the nostril. Instead, the drops are placed in the opening of the nostrils, which allows the person to inhale the drop into the nasal cavity. Other methods of administration to an anesthetized individual include sealing the esophagus and inserting a breathing tube into the trachea to prevent ingestion of the nasal composition and eliminate problems associated with respiratory failure (Chow et al., 1999; Chow et al., 2001; Fliedner et al., 2006; Dahlin et al., 2001). A flexible tube can be inserted into the nostrils for localized delivery of a small volume of drug solution 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).

Nasal delivery devices such as sprays, nasal droppers, or syringes without needles can be used to target different areas of the nasal cavity. The OptiMist™ is a breath-activated device that delivers liquid or powder nasal formulations into the nasal cavity, including the olfactory region, without deposition in the lungs or esophagus (Djupesland et al., 2006). The ViaNase™ device can also be used to target the nasal spray to the olfactory and respiratory epithelium of the nasal cavity. Nasal drops tend to settle to the floor of the nasal cavity and undergo rapid mucociliary clearance, while nasal sprays are distributed in the middle part of the nasal passage of the nasal mucosa (Scheibe et al., 2008).

Immunosuppressants or immunologically tolerant agents may be administered by any route, including parenteral. In one embodiment, the immunosuppressant or immune tolerant agent can be administered by subcutaneous, intramuscular, or intravenous injection, orally, intrathecally, intracranially, or intranasally, or by sustained release, for example, using a subcutaneous implant. The immunosuppressant or immune tolerant agent may be dissolved or dispersed in a carrier liquid. For parenteral administration, the active material may be suitably admixed with a suitable carrier, for example a vegetable oil such as peanut oil, cottonseed oil, and the like. Other parenteral carriers may also be used, such as organic formulations using Solketal, glycerin, formal and aqueous parenteral formulations. For parenteral administration by injection, the compositions may contain an aqueous solution of a water-soluble pharmaceutically acceptable salt of the active acids according to the present invention, preferably at a concentration of 0.01-10%, and optionally also a stabilizing agent and/or buffering agents in aqueous solution. The dosage units of the solution may advantageously be enclosed in ampoules.

The composition, for example, a composition containing an rAAV, a composition containing an immunosuppressant, or an immunologically tolerant composition, may be in the form of an injectable unit dose. Examples of carriers or diluents suitable for the preparation of 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 pyrosulfite, EDTA, thioglycolic acid and thiolactic acid, isotonic agents such as sodium chloride and glucose, local anesthetics such as procaine hydrochloride and lidocaine hydrochloride. In addition, conventional solubilizing agents and analgesics may be added. Injections may be prepared by adding such carriers to the enzyme or other biologically active compounds following procedures well known to those skilled in the art. A detailed discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991). Pharmaceutically acceptable formulations can be readily suspended in aqueous vehicles and administered using conventional hypodermic needles or using infusion pumps. The formulations may be sterilized prior to administration, preferably by gamma irradiation or electron beam.

When an immunosuppressant or immunotolerant agent is administered as a subcutaneous implant, the compound is suspended or dissolved in a slowly dispersing material known to those skilled in the art, or introduced into a device that releases the active agent slowly by using a constant driving force, such as an osmotic pump. In such cases, administration over a long period of time is possible.

The dosage at which a composition containing an immunosuppressant or immunological tolerance-inducing agent is administered may vary over a wide range and will depend on various factors such as the severity of the disease, the age of the patient, etc., and may need to be adjusted individually. A possible range of the amount that can be administered per day is from about 0.1 mg to 2000 mg or from about 1 mg to 2000 mg. doses within the ranges indicated, either as single dosage units or as multiple dosage units. In addition to the immunosuppressant, contemplated formulations may contain one or more rAAV-encoding therapeutic gene products.

The compositions described herein may be used in combination with another drug. The compositions may appear in conventional forms, such as aerosols, solutions, suspensions, or for topical use, or in lyophilized form.

Typical compositions include an rAAV, an immunosuppressant, a penetration enhancer, or a combination thereof, and a pharmaceutically acceptable excipient, which may be a carrier or diluent. For example, the active agent(s) may be mixed with a carrier, or diluted with a carrier, or enclosed in a carrier. When the active agent is mixed with a carrier, or when the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active agent. Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, kaolin, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin. , agar, pectin, gum arabic, stearic acid or cellulose lower alkyl esters, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, fatty acid pentaerythritol esters, polyoxyethylene, hydroxymethylcellulose, and polyvinylpyrrolidone. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The formulations may be mixed with adjuvants which do not react in an adverse manner with the active agent(s). Such additives may include wetting agents, emulsifiers and suspending agents, osmotic salts, buffers and/or colorants, preservatives, sweeteners or flavors. The compositions can also be sterilized if necessary.

If a liquid carrier is used, the formulation may be in the form of a liquid, such as an aqueous liquid suspension or solution. Acceptable diluents or carriers include sterile water, Ringer's solution, or isotonic saline in water.

The agent(s) may be presented as a powder suitable for reconstitution with an appropriate solution as described above. Examples of this include, without limitation, freeze-dried, tumble-dried, or spray-dried powders, amorphous powders, granules, precipitates, or particles. The composition may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations thereof. The dosage unit form may be single containers or multi-dose containers.

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

Polymeric nanoparticles, for example, consisting of a hydrophobic polylactic acid (PLA) core and a hydrophilic methoxy-poly(ethylene glycol) (MPEG) shell, may have improved solubility and CNS targeting. Regional differences in targeting between microemulsion and nanoparticle formulations may be due to due to differences in particle size.

Liposomes are very simple structures consisting of one or more lipid bilayers of amphiphilic lipids, i.e. phospholipids or cholesterol. The lipophilic fragment of the bilayers is rotated with respect to one another and creates an internal hydrophobic environment in the membrane. Liposomes are suitable drug carriers for certain lipophilic drugs that can be associated with non-polar portions of lipid bilayers if they are of the right size and geometry. The size of liposomes varies from 20 nm to several microns.

Mixed micelles are effective surfactant-containing structures that are composed of bile salts, phospholipids, tri-, di-, and monoglycerides, fatty acids, free cholesterol, and fat-soluble micronutrients. Since long chain phospholipids are known to form bilayers when dispersed in water, the preferred phase of the short chain analogs is the spherical micellar phase. A micellar solution is a thermodynamically stable system formed spontaneously in water and organic solvents. The interaction between micelles and hydrophobic/lipophilic drugs results in the formation of mixed micelles (CMs), which are often also referred to as swollen micelles. In the human body, they contain hydrophobic compounds with low water solubility and act as a reservoir for digestion products such as monoglycerides.

Lipid microparticles include lipid nano- and microspheres. Microspheres are generally defined as small spherical particles made from any material that are between about 0.2 and 100 microns in size. Smaller spheres up to 200 nm are generally referred to as nanospheres. Lipid microspheres are homogeneous oil/water microemulsions, similar to commercially available fat emulsions, and are produced by a high sonication procedure or high pressure emulsification methods (grinding methods). The natural surfactant lecithin reduces the surface tension of the liquid, thus acting as an emulsifier to form a stable emulsion. The structure and composition of lipid nanospheres is similar to that of lipid microspheres, but with a smaller diameter.

Polymeric nanoparticles serve as carriers for a wide variety of ingredients. The active components can either be dissolved in the polymer matrix or entrapped or adsorbed on the surface of the particles. Polymers useful for preparing organic nanoparticles include cellulose derivatives and polyesters such as poly(lactic acid), poly(glycolic acid) and their copolymer. Because of their small size, their large surface area/volume ratio, and the potential for interface functionalization, polymeric nanoparticles are ideal carriers and release systems. If the particle size is less than 50 nm, they are no longer recognized as particles by many biological as well as synthetic barrier layers, but act like molecular disperse systems.

Thus, the composition of the present invention may be formulated to provide fast, sustained, controlled or delayed release or any combination of releases of the active agent after administration to an individual using procedures well known in the art. In one embodiment, the enzyme is in an isotonic or hypotonic solution. In one embodiment, for enzymes that are insoluble in water, a lipid-based delivery vehicle, such as a microemulsion such as described in WO 2008/049588, the disclosure of which is incorporated herein by reference, or liposomes can be used.

According to one embodiment, the preparation may contain the agent dissolved or suspended in a liquid carrier, such as an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizing agents such as propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens. For example, in addition to solubility, effective CNS delivery after intranasal administration may depend on membrane permeability. For enzymes where paracellular transport is difficult due to size and polarity, increasing membrane permeability can improve the extracellular mechanisms of transport into the CNS via the olfactory and trigeminal nerves. One approach to modifying membrane permeability in the nasal epithelium is to use permeation enhancers such as surfactants such as lauroyl carnitine (LC), bile salts, lipids, cyclodextrins, polymers, or tight junction modifiers.

Typically, active agents are dispensed into unit dosage forms comprising the active ingredient together with a pharmaceutically acceptable carrier per unit dose. Typically, dosage forms suitable for nasal administration include from about 125 µg to about 125 mg, for example, from about 250 µg to about 50 mg or from about 2.5 mg to about 25 mg of the compounds in admixture with a pharmaceutically acceptable carrier or diluent.

Dosage forms may be administered once a day, or more than once a day, such as two or three times a day. Alternatively, the dosage forms may be administered less frequently than every day, such as every other day or once a week, as determined by the physician to be appropriate.

The present invention will be described using the following non-limiting examples.

EXAMPLES

Example I

AAV vector-mediated delivery of iduronidase genes in a mouse model of mucopolysaccharidosis type I: a 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). Systemic and abnormal accumulation of glycosaminoglycans is associated with growth retardation, organomegaly, skeletal dysplasia, and cardiopulmonary disease. Individuals with the most severe form of the disease (Hurler's syndrome) suffer from neurodegeneration, mental retardation, and early death. The two current treatments for MPS I (hematopoietic stem cell transplantation and enzyme replacement therapy) cannot effectively treat all manifestations of this disease in the central nervous system (CNS).

With regard to gene therapy, it has previously been shown that intravascular delivery of AAV-9 in adult mice did not achieve broad direct neuronal targeting (see Foust et al, 2009). Previous work also showed that direct injection of AAV8-IDUA into the CNS of adult IDUA-deficient mice resulted in low frequency or low levels of transgene expression (see FIG. 18). The following examples, which use a pre-clinical model for the treatment of MPS1, surprisingly show that direct injection of AAV9-IDUA into the CNS of immunocompetent adult IDUA-deficient mice resulted in IDUA enzyme expression and activity that is equal to or higher than the expression and activity of the IDUA enzyme. IDUA in wild-type adult mice (Figure 15 below).

Ways

Getting AAV9-IDUA. The AAV-IDUA (MCI) vector construct has been described previously (Wolf et al., 2011) (mCags promoter). AAV-IDUA plasmid DNA was packaged into AAV9 virions at the University of Florida vector core, resulting in a titer of 3×10 ¹³ vector genomes per milliliter.

ICV infusions. Adult Idua −/− mice were anesthetized with a ketamine and xylazine cocktail (100 mg ketamine + 10 mg xylazine per kg) and placed on a stereotaxic frame. Ten μl of AAV9-IDUA was injected into the right lateral ventricle (stereotactic coordinates AP 0.4, ML 0.8, DV 2.4 mm from bregma) using a Hamilton syringe. Animals were returned to their cages on heating pads for recovery.

intrathecal infusions. Infusions into young adult mice were performed by injecting 10 μl of the AAV vector-containing solution between the L5 and L6 vertebrae 20 minutes after an intravenous injection of 0.2 ml of 25% mannitol.

immunological tolerance. Newborn IDUA-deficient mice were injected via the facial temporal vein with 5 μl of a solution containing 5.8 μg of the recombinant iduronidase protein (aldurazyme), and then the animals were returned to the cages.

Immunosuppression with cyclophosphamide. For immunosuppression, the animals were administered cyclophosphamide once a week at a dose of 120 mg/kg, starting one day after the AAV9-IDUA vector infusion.

Animals. Animals were anesthetized with ketamine/xylazine (100 mg ketamine + 10 mg xylazine per kg) and transcardially perfused with 70 ml PBS before sacrifice. The brain was harvested and microprepared on ice for the cerebellum, hippocampus, striatum, cortex, and brainstem/thalamus ("rest"). Samples were frozen on dry ice and stored at -80°C. Samples were thawed and homogenized in 1 ml PBS using automatic pestle trituration and permeabilized by adding 0.1% Triton X-100 solution. IDUA activity was determined by fluorimetric assay using 4MU-iduronide as a substrate. Activity is expressed in units (percent of substrate converted to product per minute) per mg of protein as determined by Bradford analysis (BioRad).

Fabrics. Tissue homogenates were clarified by centrifugation for 3 min at 13,000 rpm using an Eppendorf model 5415D benchtop centrifuge (Eppendorf) and incubated overnight with proteinase K, DNase1, and RNase. GAG concentration was determined using the Blyscan sulfated glycosaminoglycan assay (Accurate Chemical) according to the manufacturer's instructions.

results

In FIG. 1 shows the results for iduronidase deficient mice treated with AAV either intracerebroventricularly (ICV) or intrathecally (IT). To prevent immune response, animals were either immunosuppressed with cyclophosphamide (CP), immunotolerized at birth by intravenous injection of human iduronidase protein (aldurazyme), or NOD-SCID injections were given to immunocompromised mice that were also iduronidase deficient. Animals were sacrificed at the indicated time after exposure and the brain was subjected to micropreparation, and iduronidase activity was analyzed in the extracts.

In FIG. 2 shows data for immunodeficient, IDUA-deficient animals treated with ICV with the AAV-IDUA vector. These animals showed high levels of IDUA expression (10 to 100 times higher than in wild-type animals) in all areas of the brain, with the highest level observed in the brainstem and thalamus ("the rest").

Immunosuppressed animals injected with the AAV vector by ICV had relatively low levels of the enzyme in the brain compared to immunodeficient animals (FIG. 3). Note that immunosuppression may be abnormal in these animals because CP was canceled 2 weeks prior to sacrifice due to poor health.

In FIG. 4 shows data for immunosuppressed animals administered with the AAV vector by IT. Immunotolerant animals injected with the AAV vector by ICV exhibited broad IDUA activity in all parts of the brain (FIG. 5), similar to that seen in immunocompromised animals, demonstrating the effectiveness of the immunotolerization procedure.

Fig. 6 is a summary of all average IDUA activity levels for comparison, and FIG. 7 is data grouped according to brain region.

GAG accumulation was analyzed in different brain regions for all four study groups. For each group, the average of each brain region is shown on the left, the values for each individual animal are shown on the right (Fig. 8). IDUA-deficient animals (leftmost) had high GAG levels compared to wild-type animals (magenta bar). The content of GAG was at the level of wild type or lower than in wild type animals for all areas of the brain in all groups of animals treated with AAV. The content of GAGs was small, although not significantly higher than in wild-type animals, in the cerebral cortex and brainstem of animals injected with AAV9-IDUA intrathecally.

conclusions

The results show a high and wide distribution of IDUA in the brain irrespective of the delivery route (ICV or IT), although IDUA expression in the striatum and hippocampus was lower in IT-treated animals compared to the ICV pathway. There appears to be an immune response as immunocompromised mice have higher levels of expression than immunocompetent mice. With respect to ICV injection, when CP was withdrawn early, IDUA expression was lower. In addition, the acquisition of immunological tolerance was effective in restoring high enzyme activity. Also, the GAG content was restored to normal in all experimental groups of exposed mice.

Example II

Ways

Getting AAV9IDUA. AAV-IDUA plasmids were packaged into AAV9 virions in either the University of Florida vector core or the University of Pennsylvania vector core, resulting in a titer of 1-3×10 ¹³ vector genomes per milliliter.

ICV infusions. See example I.

intrathecal infusions. See example I.

immunological tolerance. As in Example I, except for the following: for several tolerizations, neonatal IDUA-deficient mice were given the first dose of aldurazim via the facial temporal vein, followed by 6 weekly intraperitoneal injections.

Immunosuppression with cyclophosphamide. See example I.

Animals. Animals were anesthetized with ketamine/xylazine (100 mg ketamine + 10 mg xylazine per kg) and transcardially perfused with 70 ml PBS before sacrifice. The brain was harvested and microprepared on ice for the cerebellum, hippocampus, striatum, cortex, and brainstem/thalamus ("rest"). Samples were frozen on dry ice and stored at -80°C.

Tissue IDUA activity. Tissue samples were thawed and homogenized in saline in a tissue homogenizer. Tissue homogenates were clarified by centrifugation at 15,000 rpm using an Eppendorf benchtop centrifuge at 4°C for 15 min. Tissue lysates (supernatant) were collected and analyzed for IDUA activity and GAG accumulation content.

GAG content in tissues. Tissue lysates were incubated overnight with proteinase K, RNase and DNase. GAG content was analyzed using the Blyscan sulfated glycosaminoglycan assay (Accurate Chemical) according to the manufacturer's instructions.

Copies of the IDUA vector. Tissue homogenates were used for DNA isolation and subsequent quantitative PCR as described in Wolf et al. (2011).

results

In FIG. 9 shows the setup of the experiment and the group. Animals were injected with the AAV9IDUA vector by either intracerebroventricular (ICV) or intrathecal (IT) infusion. The vector was administered to NOD-SCID mice with immunodeficiency (ID) that were also IDUA deficient, or mice with IDUA deficiency that were either immunosuppressed with cyclophosphamide (CP) or immunotolerized at birth with single or multiple injections of human iduronidase protein (aldurazyme ). The time of exposure to the vector and killing is as shown in FIG. 9. All vector injections were performed in adult animals aged 3-4.5 months. Animals were injected with 10 μl of vector at a dose of 3×10 ¹¹ vector genomes per 10 μl.

In FIG. 10 shows IDUA enzymatic activity in immunocompromised and IDUA-deficient mice with intracranial infusion. High levels of enzyme activity were observed in all areas of the brain, ranging from 30 to 300 times higher than in wild-type animals. The highest expression of the enzyme was observed in the thalamus and brainstem, as well as in the hippocampus.

Animals subjected to intracranial administration and immunosuppression with cyclophosphamide (CP) showed significantly lower levels of enzyme activity compared to other groups (FIG. 11). However, the administration of CP in this case had to be canceled 2 weeks before sacrifice due to the poor health of the animals.

The content of the IDUA enzyme in animals tolerized at birth with the IDUA protein (aldurazyme) and subjected to intracranial administration of the vector is shown in FIG. 12. All animals showed high levels of the enzyme in all parts of the brain, which ranged from 10 to 1000 times higher than the content in wild-type animals, similar to the content achieved in animals with immunodeficiency, indicating the effectiveness of the immunological tolerization procedure .

In FIG. 13 shows the content of the IDUA enzyme in mice that were subjected to intrathecal administration and injected with CP on a weekly basis. Elevated levels of IDUA were observed in all parts of the brain, especially in the cerebellum and spinal cord. The enzyme content was lowest in the striatum and hippocampus with activity at the level of wild-type animals.

IDUA-deficient mice were subjected to aldurazyme tolerization as described above and intrathecal vector administration (FIG. 14). There was ubiquitous activity of the IDUA enzyme 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 FIG. 13, the lowest levels of enzyme activity were observed in the striatum, cortex, and hippocampus.

IDUA-deficient control immunocompetent animals were intrathecally infused with the vector without immunosuppression or immunological tolerization (FIG. 15). The results show that although the enzyme activity was at the wild type level or slightly higher, the enzyme content was significantly lower than that observed in immunomodulated animals. The decrease in enzyme levels was particularly significant in the cerebellum, olfactory bulb and thalamus, and brainstem, areas that expressed the highest levels of the enzyme in immunomodulated animals.

Animals were examined for the presence of GAG storage substance as shown in FIG. 15. All groups showed clearing of GAG accumulation, with GAG levels close to those observed in wild-type animals. Animals that were immunosuppressed and intrathecally injected with the AAV-IDUA vector had GAG content in the cortex that was slightly higher than in wild-type animals, but still significantly lower than in untreated IDUA-deficient mice.

The presence of the AAV9IDUA vector in animals that had been subjected to immunological tolerization and either intrathecal or intracranial vector administration was assessed by quantitative PCR as shown in FIG. 16. IDUA copy number per cell was higher in intracranially infused animals compared to intrathecally infused animals, consistent with the higher level of enzyme activity shown in intracranially injected animals.

conclusions

High, widespread and therapeutic levels of IDUA were observed in all areas of the brain after the intracerebroventricular and intrathecal route of administration of AAV9IDUA in adult mice. Enzyme activities were restored to levels in wild-type animals or slightly higher in IDUA-deficient immunocompetent animals that received an intrathecal infusion of AAV-IDUA. Significantly higher levels of the IDUA enzyme were observed for both routes of vector injection in animals subjected to immunological tolerization, starting from birth with the introduction of the IDUA protein.

Example III

Adult immunocompetent IDUA deficient mice (12 weeks of age) were anesthetized with ketamine/xylazine and then intranasally infused with the AAV9IDUA vector. The vector was administered by injecting eight drops of 3 μl with a micropipette into the intranasal cavity, alternating nostrils, with 2-minute intervals between each application. In total, each adult animal was injected with 2.4-7×10 ¹¹ vector genomes, depending on the source of the vector. Animals were immunosuppressed with a weekly injection of 120 mg/kg of cyclophosphamide, starting the day after the introduction of the vector. Mice were sacrificed 12 weeks after vector infusion, animals were examined for IDUA enzyme expression and vector copies in the brain.

Links

Al-Ghananeem et al., AAPS Pharm. sci. Tech. 3:E5 (2002).

Bagger et al., Eur. J Pharm. Sci. 21:235-242 (2004b).

Bagger et al., Int. J. Pharm., 269:311-322 (2004a).

Baker et al., Exp. Brain Res. 63:461 (1986).

Balin et al., J. Comp. Neurol., 251:260-280 (1986).

Banks et al., J. Drug Target, 17:91-97 (2009).

Banks et al., J. Pharmacol. Exp. Ther., 309:469 (2004).

Banks, Biopolymers, 90:589 (2008).

Barakat et al., J. Pharm. Pharmacol. 58:63 (2006).

Barbier et al., Mol. Genet. Metab., 110:303 (2013).

Baumgartner et al., Neuron. 58:639 (2008).

Benedict et al., Neuroendocrinology, 86:136 (2007b).

Benedict et al., Neuropsychopharmacology, 32:239 (2007a).

Benedict et al., Psychoneuroendocrinology, 29:1326 (2004).

Bjoraker et al., J. Dev. behavior. Ped. 27:290 (2006).

Blits et al., J. Neuros. Methods, 185:257 (2010).

Born et al., Nat. Neurosci. 5:514 (2002).

Boulton et al., Am. J. Physiol., 276:R818 (1999).

Boulton et al., Neuropathol. Appl. Neurobiol. 22:325 (1996).

Bradbury et al., Am. J. Physiol., 240:F329 (1981).

Bradbury et al., J. Physiol., 339:519 (1983).

Brady, Ann. Rev. Med. 57:283 (2006).

Broadwell et al., J. Comp. Neurol. 242:632 (1985).

Broekman et al. Neuroscience 138:501 (2006).

Buck, In: Kandel ER, Schwartz JH, Jessell TM, editors. principles of neural science. 4th edition. New York: McGraw-Hill Companies, pp. 625-652 (2000).

Buxer et al., J. Neurochem., 56:1012 (1991).

Cai et al., Sichuan Da Xue Xue Bao Yi Xue Ban, 39:438 (2008).

Capsoni et al., Proc. Natl. Acad. sci. USA 99:12432 (2002).

Carare et al., Neuropathol. Appl. Neurobiol. 34:131 (2008).

Cauna, In: Proctor DF, Andersen I, editors. Amsterdam: Elsevier Biomedical Press, pp. 45-69 (1982).

Charlton et al., Int. J. Pharm., 338:94 (2007b).

Charlton et al., J. Drug Target, 15:370 (2007a).

Charlton et al., Pharm. Res. 25:1531 (2008).

Chen et al., J. Alzheimers Dis., 1:35 (1998).

Chen et al., J. Pharm. Sci. 95:1364 (2006).

Chow et al., J. Pharm. Sc. 88:754 (1999).

Chow et al., J. Pharm. Sc. 90:1729 (2001).

Clerico et al., In: Doty R.L., editor. Handbook of olfaction and gustation. 2nd edition. New York: Marcel Dekker, Inc. pp. 1-16 (2003).

Costantino et al., Int. J. Pharm., 337:1 (2007).

Cserr et al., Am. J. Physiol., 240:F319 (1981).

Cserr et al., Brain Pathol. 2:269 (1992).

Dahlin et al., Eur. J Pharm. Sc. 14:75 (2001).

Danhof et al., American Association of Pharmaceutical Scientists Annual Meeting, Atlanta, GA (2008).

Danielyan et al., Eur. J. Cell. Biol., 88:315 (2009).

Davis et al., Clin. Pharmacokinet., 42:1107 (2003).

de Lorenzo, In: Wolstenholme GEW, Knight J, editors. Taste and smell in vertebrates. London: Churchill, pp. 151-175 (1970).

De Rosa et al., Proc. Natl. Acad. sci. USA 102:3811 (2005).

DeSesso, Qual. Assur. 2:213 (1993).

deSouza et al., Eur. Neuropsychopharmacol., 19:53 (2009).

Dhanda et al., Drug Del. Tech. 5:64 (2005).

Dhuria et al., J. Pharm. Sci. 98:2501 (2009b).

Dhuria et al., J. Pharmaceutical Sciences, 99:1654 (2010).

Dhuria et al., J. Pharmacol. Exp. Ther., 328:312 (2009a).

Diano et al., J. Clin. Invest., 118:26 (2008).

Dickson et al., Mol. Gen. Metab. 91:61 (2007).

Djupesland et al., Laryngoscope, 116:466 (2006).

Domes et al. Biol. Psychiatry, 61:731 (2007b).

Domes et al. Biol. Psychiatry, 62:1187 (2007a).

Dufes et al., Int. J. Pharm., 255:87 (2003).

Einer-Jensen et al., Exp. Brain Res., 130:216 (2000b).

Einer-Jensen et al., Pharmacol. Toxicol., 87:276 (2000a).

Einer-Jensen et al., Reproduction, 129:9 (2005).

Ellinwood et al., Mol. Genet. Metab., 91:239 (2007).

Fehm et al., J. Clin. Endocrinol. Metab., 86:1144 (2001).

Field et al., J. Neurocvtol., 32:317 (2003).

Fliedner et al., Endocrinology., 17:2088 (2006).

Foust et al., Nat. Biotech., 27:5 (2009)).

Francis et al., Brain, 131:3311 (2008).

Fratantoni et al. Science 162:570 (1968).

Frey et al., Drug Delivery, 4:87 (1997).

Frey II, Drug Del. Tech. 2:46 (2002).

Fuss et al., Eur. J. Neurosci., 22:2649 (2005).

Gao et al., Biomaterials, 27:3482 (2006).

Gao et al., Int. J. Pharm., 340:207 (2007a).

Gao et al., J. Control Release, 121:156 (2007b).

Gopinath et al., Current Ther. Res. 23:596 (1978).

Gozes et al., Curr. Alzheimer Res. 4:507 (2007).

Graff et al. Pharm. Res. 20:1225 (2003).

Graff et al. Pharm. Res. 22:235 (2005a).

Graff et al. Pharm. Res. 22:86 (2005b).

Gray, 15th revised edition (Classic Collectors edition). New York: Bounty Books (1978).

Grevers et al., Arch. Otorhinolaryngol. 244:55 (1987).

Groothuis et al., J. Cereb. Blood Flow Metab., 27:43 (2007).

Gross et al., J. Anat. 135:83 (1982).

Guastella et al. Biol. Psychiatry, 63:3 (2008).

Hadaczek et al., Mol. Ther. 14:69 (2006).

Hallschmid et al., Regul. Pert. 149:79 (2008).

Han et al., J. Mol. Med. 85:75 (2007).

Hanson et al., BMC Neurosci., 9:S5 (2008).

Hanson et al., Drug Del. Tech. 4:66 (2004).

Hanson et al., San Diego, CA: Society for Neuroscience (2007).

Hanson et al., In: EPO, editor. Biopharm and HealthPartners Research Foundation (2008).

Hartung et al., J. Am. soc. Gene Ther. 9:866 (2004).

Hartung et al., Mol. Thera. 9:869 (2004).

Hashizume et al., Neuro. Oncol. 10:112 (2008).

Hatterer et al., Blood, 107:806 (2006).

Herati et al., J. Gene Med., 10:972 (2008).

Hess et al., Exp. Neuro. 186:134 (2004).

Horvat et al., Eur. J Pharm. Biopharm. 72:252 (2009).

Hussar et al., Chem. Senses, 27:7 (2002).

Ilium, J. Control Release, 87:187 (2003).

Ilium, J. Pharm. Pharmacol. 56:3 (2004).

Itaya et al. Brain Res. 398:397 (1986).

Jansson et al., J. Drug Target, 10:379 (2002).

Jogani et al., Alzheimer Dis. Assoc. Disord., 22:116 (2008).

Johnston et al., Cerebrospinal Fluid Res., 1:2 (2004).

Kakkis et al., Mol. Gen. Met. 83:163 (2004).

Kandimalla et al., J. Pharm. Sci. 94:613 (2005b).

Kandimalla et al., Pharm. Res. 22:1121 (2005a).

Kida et al., Neuropathol. Appl. Neurobiol. 19:480 (1993).

Kirsch et al., J. Neurosci., 25:11489 (2005).

Klein et al., J. Am. soc. Gene Ther. 13:517 (2006).

Koos et al., Neuroreport, 16:1929 (2005).

Kosfeld et al., Nature 435:673 (2005).

Kristensson et al., Acta Neuropathol (Berl), 19:145 (1971).

Krivit, Springer Seminars in Immunopathology, 26:119 (2004).

Kumar et al. Curr. Sc. 43:435 (1974).

Kumar et al., Int. J. Pharm., 358:285 (2008).

Li et al., Chin. J. Physiol., 48:7 (2005c).

Li et al., Glia, 52:245 (2005a).

Li et al., J. Neurocvtol., 34:343 (2005b).

Loftus et al. Neuroscience 139:1061 (2006).

Luzzati et al., J. Mol. Biol., 343:199 (2004).

Mackay-Sim, In: Doty RL, editor. Handbook of olfaction and gustation. 2nd edition. New York: Marcel Dekker, Inc. pp. 93-113 (2003).

Martinez et al. Neuroscience 157:908 (2008).

Minn et al., J. Drug Target, 10:285 (2002).

Miragall et al., J. Comp. Neurol., 341:433 (1994).

Muenzer, J. Pediatrics, 144:S27 (2004).

Munoz-Rojas et al., Am. J. Med. Gen. 146A:2538 (2008).

Neufeld and Muenzer, In A.L.B.C.R. Scriver, W.S. Sly, et al (ed.), McGraw Hill, NY, pg. 3421 (2001).

Nonaka et al., J. Pharmacol. Exp. Ther., 325:513 (2008).

Ohlfest et al., Blood, 105:2691 (2005).

Orchard et al., J. Pediatrics, 151:340 (2007).

Owens et al., Diabetes. Med. 20:886 (2003).

Pan et al. Brain Res. 1188:241 (2008).

Pardridge, NeuroRx, 2:3 (2005).

Parker et al., Psvchoneuroendocrinology, 30:924 (2005).

Pastores, Exp. Opin. Biol. Ther. 8:1003 (2008).

Perl et al., Lancet, 1:1028 (1987).

Peters et al., Bone Marrow Transpl. 31:229 (2003).

Pollock et al., J. Anat., 191:337 (1997).

Raghavan et al., J. Laryngol. Otol. 114:456 (2000).

Reger et al., J. Alzheimers Pis., 13:323 (2008a).

Reger et al., Neurobiol. Aging, 27:451 (2006).

Reger et al., Neurology, 70:440 (2008b).

Rennels et al., Adv. Neurol. 52:431 (1990).

Rennels et al. Brain Res. 326:47 (1985).

Reolon et al. Brain Res. 1076:225 (2006).

Rimmele et al., J. Neurosci., 29:38 (2009).

Ross et al., J. Neuroimmunol., 151:66 (2004).

Ross et al., Neurosci. Lett., 439: 30 (2008).

Sakane et al., J. Pharm. Pharmacol. 46:378 (1994).

Sakane et al., J. Pharm. Pharmacol. 47:379 (1995).

Sarkar, Pharm. Res. 9:1 (1992).

Schaefer et al., J. Comp. Neurol. 444:221 (2002).

Scheibe et al., Arch. Otolaryngol. Head Neck Surg. 134:643 (2008).

Schley et al., J. Theor. Biol., 238:962 (2006).

Schulz et al., Endocrinology, 145:2696 (2004).

Scott et al., Am. J. Hum. Genet. 53:973 (1993).

Shimizu et al., Int. J. Obes. (Lond). 29:858 (2005).

Shipley, Brain Res. Bull. 15:129 (1985).

Skipor et al., Reprod. Biol. 3:143 (2003).

Steen et al., J. Alzheimers Pis., 7:63 (2005).

Stefanczyk-Krzymowska et al., Exp. Physiol., 85:801 (2000).

Takano et al., J. Histochem. Cytochem. 53:611 (2005).

Thorne et al. Brain Res. 692:278 (1995).

Thorne et al., Clin. Pharmacokinet., 40:907 (2001).

Thorne et al. Neuroscience 127:481 (2004).

Thorne et al. Neuroscience 152:785 (2008).

Thorne, R.G. 2002. The nasal pathways for drug delivery to the central nervous system: Studies with protein tracers and therapeutics. Poctoral Pissertation, University of Minnesota.

Unger et al., J. Neuropath. Exp. Neuro. 52:460 (1993).

van den Berg et al., Eur. J Pharm. Biopharm. 58:131 (2004b).

Van den Berg et al., J. Prug Target, 11:325 (2003).

van den Berg et al., J. Neurosci. Methods, 116:99 (2002).

van den Berg et al., Pharm. Res. 21:799 (2004a).

Van Piest et al., J. Anat., 128:293 (1979).

Vyas et al., AAPS PharmSciTech., 7:E1 (2006c).

Vyas et al., Crit. Rev. Ther. Prug Carrier Svst. 23:319 (2006b).

Vyas et al., J. Prug Target, 13:317 (2005).

Vyas et al., J. Pharm. Sc. 95:570 (2006a).

Walter et al., Arch. Histol. Cvtol. 69:37 (2006a).

Walter et al., Neuropathol. Appl. Neurobiol. 32:388 (2006b).

Wang et al., Cancer Chemother. Pharmacol. 57:97 (2006a).

Wang et al., Eur. J Pharm. Biopharm. 70:735 (2008).

Wang et al., Int. J. Pharm., 317:40 (2006b).

Wang et al., Int. J. Pharm., 341:20 (2007).

Watson et al., Gene Therapy, 16 February 2006, doi:10.1038/sj.gt.3302735.

Weller et al., Neurol. Res. 25:611 (2003).

Westin et al., Eur. J Pharm. Sc. 24:565 (2005).

Westin et al., Pharm. Res. 23:565 (2006).

Williams et al., J. Comp. Neurol. 470:50 (2004).

Wioland et al., J. Histochem. Cvtochem. 48:1215 (2000).

Wolf et al., Neurobio. Pis. 43:123 (2011).

Xu et al., J. Clin. Invest., 118:272 (2008).

Yamada et al., Am. J. Physiol., 261:H1197 (1991).

Yang et al., J. Pharm. Sc. 94:1577 (2005).

Zhang et al., Acta Neuropathol. (Berl), 83:233 (1992).

Zhang et al., Acta Pharmacol. Sin. 25:522 (2004a).

Zhang et al., Int. J. Pharm., 275:85 (2004b).

Zhang et al., J. Prug Target, 14:281 (2006).

Zhao et al., Acta Pharmacol. Sin. 28:273 (2007).

Zhao et al., Chin. Med. sci. J. 19:257 (2004).

Zheng et al., Mol. Genet. Metab., 79:233 (2003).

Ziegler and Shapiro, In J. Ponders and S. Hunter (ed.), Cambridge University Press, p. 427 (2007).

All publications, patents and patent applications are incorporated herein by reference. While the present invention has been described in the foregoing description with respect to certain preferred embodiments thereof and many of the details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present invention is capable of further embodiments and that some of the details in this document may vary significantly without departing from the basic principles of the present invention.

Claims (18)

1. A method for preventing, inhibiting or treating one or more symptoms of mucopolysaccharidosis type II (MPS II) in a human, comprising: intrathecal administration to a person in need of a composition containing an effective amount of a recombinant adeno-associated virus (rAAV) vector containing an open reading frame, encoding iduronate-2-sulfatase to prevent, inhibit or treat one or more symptoms of MPS II, wherein rAAV is rAAV9 or rAAVrh10. 2. The method of claim 1, wherein the human is an adult. 3. The method of claim 1, further comprising administering to the subject an effective amount of a penetration enhancer. 4. The method of claim 3 wherein the composition comprises a penetration enhancer. 5. The method of claim 3 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-lauryl ether, or EDTA. 6. The method of claim 1 further comprising administering an immunosuppressant to the human. 7. The method of claim 6 wherein the immunosuppressant comprises cyclophosphamide. 8. The method according to claim 6, wherein the immunosuppressant contains a glucocorticoid, cytostatic agents, including an alkylating agent, an antimetabolite, a cytotoxic antibiotic, an antibody, or an immunophilin active agent. 9. The method of claim 8 wherein the immunosuppressant comprises nitrogen mustard, nitrosourea, a platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin directed to the IL-2 receptor (CD25-) or CD3 antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioid, or TNF-α (tumor necrosis factor-alpha) binding agent. 10. The method of claim 6 wherein the rAAV and the immunosuppressant are co-administered or the immunosuppressant is administered after the rAAV is administered. 11. The method of claim 6 wherein the immunosuppressant is administered intrathecally. 12. The method of claim 6 wherein the immunosuppressant is administered intracerebroventricularly. 13. The method of claim 1, wherein the human is immunologically tolerant of iduronate-2-sulfatase. 14. The method according to any one of paragraphs. 1-13, in which iduronate-2-sulfatase is damaged or deficient in humans. 15. The method according to any one of paragraphs. 1-13, wherein the human is an immunocompetent adult mammal. 16. The method according to any one of paragraphs. 1-13, wherein the rAAV vector is the rAAV-9 vector. 17. The method according to any one of paragraphs. 1-13, wherein the rAAV vector is the rAAV rh10 vector. 18. The method according to any one of paragraphs. 1-17, in which rAAV is injected into the cerebellopontine cistern.