MX2008010124A - Gene therapy for niemann-pick disease type a - Google Patents

Abstract

This disclosure pertains to methods and compositions for tolerizing a mammal's brain to exogenously administered acid sphingomyelinase polypeptide by first delivering an effective amount of a transgene encoding the polypeptide to the mammal's hepatic tissue and then administering an effective amount of the transgene to the mammal's central nervous system (CNS).

Description

GENE THERAPY FOR NIEMANN-PICK TYPE A DISEASE Cross-referencing related applications This application claims priority according to 35 U.S.C. § 119 (e) of the United States provisional application serial number 60 / 771,628, filed on February 8, 2006 and the United States provisional application serial number 60 / 772,360 filed on February 9, 2006, the contents are incorporated by reference in the present description. Field of the Invention The present invention relates to compositions and methods for treating disorders affecting the brain and viscera, for example, Niemann-Pick Type A disease. SUMMARY OF THE INVENTION A group of metabolic disorders known as arthritis diseases. Lysosomal storage (LSD) includes more than forty genetic disorders many of which involve genetic defects in various lysosomal hydrolases. Table 1 lists representative lysosomal storage diseases and the associated defective enzymes. Table 1 or Involvement of the CNS The characteristic feature of LSD is the accumulation of abnormal metabolites in lysosomes that leads to the formation of large numbers of distended lysosomes in the pericarion. A major challenge in treating LSD (as opposed to treating a liver-specific enzyme disease) is the need to reverse the lysosomal storage pathology in multiple separate tissues. Some LSD can be effectively treated by intravenous infusion of the absent enzyme, which is known as enzyme replacement therapy (ERT). For example, Gaucher type 1 patients have only visceral disease and respond favorably to ERT with recombinant glucocerebrosidase (Cerezyme®, Genzyme Corp.). However, patients with metabolic disease that affects the CNS (for example, Gaucher disease type 2 or 3) do not respond completely to intravenous ERT, since the substitution enzyme is prevented from entering the brain by the blood-brain barrier (BHE). In addition, the first attempts to introduce a substitution enzyme into the brain by direct injection of the protein have been limited in part due to the cytotoxicity of the enzyme at high local concentrations and limited parenchymal diffusion rates (Pardridge, Peptide Drug Delivery to the Brain, Raven Press, 1991). In addition, antibodies against the infused enzymes used in enzyme replacement therapy can be developed. Such antibodies may lack clinical significance or may lead to hypersensitivity reactions or decrease the bioavailability of the therapeutic proteins. Hunley, T.E. et al. (2004) Pediatrics 114 (4): 532-e535. For example, Kakkis E. et al. (2004) PNAS 101 (3): 829-834 describe that adverse effects of antibodies have been observed in enzyme substitution therapy of lysosomal storage disorders in the canine model of mucopolysaccharidosis I (MPS I). The authors also describe similar results in other animal models of MPS disorders, including MPS I, MPS VI and MPS VII. The decrease in the immune response generated by these proteins may be desirable. The induction of antigen-specific tolerance is a possible method to decrease such an immune response, but it has been described as difficult to achieve. Gene therapy is an emerging treatment modality for disorders affecting the CNS, including LSD. Promising results have been achieved in an accepted animal model using gene therapy for the treatment of Niemann-Pick Type A (NPA) disease. Dodge et al. (2005) PNAS 102 (49): 18722-17827. NPA is a lysosomal storage disorder caused by a deficiency in the activity of acid sphingomyelinase (ASM). The loss of ASM activity results in the accumulation of lysosomal sphingomyelin (SPM), secondary metabolic effects such as abnormal cholesterol metabolism and subsequent loss of cellular function in organ systems including the central nervous system (CNS). Schuchman and Desnick, The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, p. 3589-3610 and Horinouchi et al. (1995) Nat. Gener. 10: 288-293. This invention provides a method comprising the steps of administering an effective amount of a viral vector comprising a transgene that encodes an immunogen to the liver tissue of the mammal and subsequently administering an effective amount of a second viral vector comprising a transgene encoding an immunogen. to the brain of the mammal. A method for treating Niemann-Pick Type A disease in a mammal is also provided comprising the steps of administering an effective amount of a viral vector comprising a transgene encoding an acid sphingomyelinase polypeptide to the liver tissue of the mammal and administering then an effective amount of a second viral vector comprising a transgene encoding an acid sphingomyelinase polypeptide to the brain of said mammal, thereby treating Niemann-Pick Type A disease in the mammal. The invention also provides methods and compositions for the brain of a mammal to tolerate a pre-selected immunogen by first providing systemically an effective amount of the immunogen via a transgene and then administering an effective amount of the immunogen to the central nervous system (CNS). of the mammal. It also provides methods and compositions for causing the brain of a mammal to tolerate an acid sphingomyelinase polypeptide by first supplying an effective amount of a transgene encoding the polypeptide to the liver of the mammal and then administering an effective amount of the transgene for the polypeptide to the system. central nervous system of the mammal (CNS). The invention also provides methods and compositions for alleviating the symptoms associated with Niemann-Pick Type A (NPA) disease in a mammal suffering from NPA by transducing to the mammalian brain tissue an effective amount of a transgene encoding a sphingomyelinase polypeptide. acid after transduction to the liver of the mammal with the same transgene.

Additional advantages of the invention will be set forth in the following description and will be understood in part from the description or will be learned by practicing the invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 graphically shows various sites of administration of the transgene to the central nervous systems of mice. Figure 2 graphically shows the body weight of the treated mice as a measure of the physical state. The measurements started at the age of 6 weeks (treatment started at week four). During the time period of 6 to 36 weeks (2 to 32 weeks after the systemic injection of the transgene) all groups were compared with untreated ASMKO mice. Legend of the figure: * p < 0.05; ** p < 0.01; *** p < 0.001; ns (not significant). Figure 3 graphically shows the results of a Rotarod acceleration test as a measure of recovery of motor function. Measurements started at the age of 10 weeks (treatment started at week four). During the time period between weeks 10 and 36 (6 to 32 weeks after the systemic injection of the transgene), all groups were compared with untreated ASMKO mice. Legend of the figure: * p < 0.05; ** p < 0.01; *** p < 0.001; ns (not significant). Figure 4 graphically shows the results of a Rotarod acceleration test as a measure of the recovery of motor function. Measurements started at the age of 10 weeks (treatment started at week four). During the time period between weeks 10 and 36 (6 to 32 weeks after the systemic injection of the transgene) all groups were compared with untreated ASMKO mice. Legend of the figure: * p < 0.05; ** p < 0.01; *** p < 0.001; ns (not significant). Figure 5 graphically shows the results of a Barnes maze assay as a measure of the recovery of cognitive function. The measurements started at the age of 17 weeks (treatment started at week four). During the time period between week 17 and 33 (13 to 29 weeks after the systemic injection of the transgene), all groups were compared with untreated ASMKO mice. Legend of the figure: * p < 0.05; ** p < 0.01; *** p < 0.001; ns (not significant). Figure 6 demonstrates graphically that ASM combination gene therapy prolongs the life of ASMKO mice. The half-life of ASMKO mice was 34 weeks. The half-life of mice receiving the systemic transgene was 45 weeks (p < 0.0001). The half-life for mice receiving the transgene intracranially was 43 weeks (p <0.0001). Life for mice receiving intracranial and systemic transgenic therapy was 54 weeks for 100% of the mice. Figures 7A to 7D graphically show the levels of anti-hASM antibodies in circulation for treated and untreated mice. Legend of the figure: * p < 0.05; ** p < 0.01; *** p < 0.001. Figure 7E shows levels of human ASM protein in blood serum over time. Figures 8A to 8F graphically show levels of sphingomyelin in the brain of treated and untreated mice. Figures 9A to 9D graphically show sphingomyelin levels in various viscera of treated and untreated mice. Detailed Description of the Invention Throughout this description reference is made by means of an identification appointment to various publications, patents and published patent descriptive reports. The descriptions of those published publications, patents and published patent specifications are incorporated by reference in the present description to describe in more detail the state of the art to which this invention pertains. Definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill in the art. See, for example, Sambrook, Fritsch and Maniatis,, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (F. M. Ausubel et al., Eds., (1987)); the Methods In Enzymology series (Academic Press, Inc.): Per 2: A Practical Approach (M. J. Macpherson, B. D. Hames and G. R. Taylor eds (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL AND ANIMAL CELL CULTURE (R. Freshney, ed. (1987)).

As used herein, certain expressions have the following defined meanings. As used in the specification and claims, the singular form "a", "an" and "the" and "the" include plural references unless the context clearly dictates otherwise. For example, the expression "a cell" includes a plurality of cells, including mixtures thereof. The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (eg, a probe, a primer, EST or SAGE label), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA , recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, probes and nucleic acid primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after the polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide includes both the double-stranded form and each of the two complementary single-chain forms that are known or predicted to establish the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for guanine when the polynucleotide is RNA. Therefore, the term "polynucleotide sequence" is the alphabetic representation of a molecule of the polynucleotide. This alphabetical representation can be entered into a database on a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology search. A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after it has been transcribed and translated. Any of these polynucleotide sequences that are described herein can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods for isolating larger fragment sequences are known to those skilled in the art. A "gene product" or, alternatively, a "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

The term "polypeptide" is used interchangeably with the term "protein" and in its broadest sense refers to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics. The subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, for example, ester, ether, etc. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and the two optical isomers D and L, amino acid analogs and peptide mimetics. A peptide of three or more amino acids is commonly referred to as an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly referred to as a polypeptide or a protein. "Under transcriptional control" is an expression well understood in the art and indicates that the transcription of a polynucleotide sequence, usually a DNA sequence, depends on whether it is functionally linked to an element that contributes to the initiation of or promotes the transcription. "Functionally united" refers to a juxtaposition in which the elements are in a disposition that allows them to function. As used herein, the term "comprising" is intended to indicate that the compositions and methods include the elements listed, but do not exclude others. "Essentially consisting of" when used to define compositions and methods, means that other elements of some essential meaning for the combination are excluded. Therefore, a composition consisting essentially of the elements defined herein will not exclude small amounts of contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "Consisting in" means that more than small amounts of elements of other ingredients and substantial steps of the method for administering the compositions of this invention are excluded. Embodiments defined by each of these transition expressions belong to the scope of this invention. The term "isolated" means separated from constituents, cellular and others, with which the polynucleotide, the peptide, the polypeptide, the protein, the antibody or fragment (s) thereof are normally associated in nature. In one aspect of this invention, an isolated polynucleotide is separated from the contiguous 3 'and 5' nucleotides with which it is normally associated in its natural native environment, for example, on the chromosome. As is apparent to those skilled in the art, a polynucleotide, peptide, polypeptide, protein, antibody or fragment or fragments thereof of non-natural origin does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, a polynucleotide, a peptide, a polypeptide, a protein, an antibody or a fragment or fragments thereof "concentrated", "separated" or "diluted" can be distinguished from its homologue of natural origin because the concentration or the number of molecules per volume is greater than the "concentrated" or lower of the "separated" compared to its homologous of natural origin. A polynucleotide, a peptide, a polypeptide, a protein, an antibody or a fragment or fragments thereof, which differs from the homologue of natural origin in its primary sequence or, for example, in its glycosylation pattern, does not have to be present in its isolated form since it can be distinguished from its homologue of natural origin by its primary sequence or, alternatively, by another characteristic such as the glycosylation pattern. Therefore, a polynucleotide of non-natural origin is provided as a separate embodiment of the polynucleotide of isolated natural origin. A protein produced in a bacterial cell is provided as a separate embodiment of the naturally occurring protein isolated from a eukaryotic cell in which it occurs in nature. "Gene administration", "gene transfer", and the like, as used herein, are expressions that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, for example, viral infection / transfection or various protein-based or lipid-based gene delivery complexes) as well as techniques that facilitate the delivery of polynucleotides. "naked" (such as electroporation, administration by "gene guns" and various different techniques used for the introduction of polynucleotides). The introduced polynucleotide can be stable or can be maintained transiently in the host cell. Stable maintenance typically requires that the introduced polynucleotide contain an origin of replication compatible with the host cell or is integrated into a replicon of the host cell such as an extrachromosomal replicon (eg, a plasmid) or a nuclear or mitochondrial chromosome. Several vectors known in the art are capable of mediating gene transfer to mammalian cells. A "gene delivery vehicle" is defined as any molecule that can carry polynucleotides inserted into a host cell. Examples of lysosome gene delivery vehicles are biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; recombinant yeast cells, metal particles; and bacteria or viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as for the simple expression of proteins. A "viral vector" is defined as a recombinantly produced virus or a viral particle comprising a polynucleotide that has to be administered to the interior of a host cell in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as vectors based on the Semliki Forest virus and vectors based on the Sindbis virus, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying et al. (1999) Nat. Med. 5 (7): 823-827. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof and a therapeutic gene. As used herein, "retroviral mediated gene transfer" or "retroviral transduction" means the same and refers to the process by which a gene or nucleic acid sequence is stably transferred to the interior of the host cell by the entry of the virus into the cell and the integration of its genome into the genome of the host cell. The virus can enter the host cell by its normal mechanism of infection or it can be modified in such a way that it binds to a receptor or surface ligand of the different host cells to enter the cell. As used herein, "retroviral vector" refers to a viral particle capable of introducing an exogenous nucleic acid into a cell through a viral or virus-like entry mechanism. In aspects where gene transfer is mediated by a viral DNA vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a viral construct refers to the polynucleotide comprising the viral genome or part thereof and a transgene . Adenoviruses (Ad) are a relatively well characterized, homogeneous group of viruses, which includes more than 50 serotypes. See, for example, WO 95/27071. The Ad are simple to develop and do not require integration into the genome of the host cell. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the potential for recombination and generation of wild type viruses. See, for example, WO 95/00655 and WO 95/11984. The wild-type AAV has high infectivity and specificity to integrate into the genome of the host cell. See Hermonat and Muzyczka (1984) Proc. Nati Acad. Sci. USA 81: 6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8: 3988-3996. Recombinant AAV vectors are also known in the art. See, for example, WO 01/36620 A2. Vectors that contain both a promoter and a cloning site in which a polynucleotide can be functionally linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). To optimize in vitro expression and / or transcription, it may be necessary to remove, add or alter 5 'and / or 3' untranslated portions of the clones to eliminate extra, potentially inappropriate alternative translation start codons or other sequences that may interfere with or diminish the expression, at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5 'from the start codon to enhance expression. The gene delivery vehicles also include several non-viral vectors, including DNA / liposome complexes, recombinant yeast cells and targeted viral-DNA protein complexes. In the methods of this invention, liposomes which also comprise a targeting antibody or a fragment thereof can be used. To improve administration to a cell, the nucleic acid or proteins of this invention can be conjugated with antibodies or one or more binding fragments thereof that bind to cell surface antigens, eg, TCR, CD3 or CD4. The terms "genome particles (gp)" or "genome equivalents", as used with reference to a viral titer, refer to the number of virions that contain the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by methods such as those described in the examples herein, or for example in Clark et al. (1999) Hum. Gene Ther. 10: 1031-1039 and Veldwijk et al. (2002) Mol. Ther. 6: 272-278. The terms "infection unit (ui)", "infectious particle" or "unit of replication", as used with reference to a viral titer, refer to the number of infectious vector particles measured by the infectious center assay, also known as assay replication centers, described, for example, in McLaughlin et al. (1988) J. Viral. 62: 1963-1973. The term "transduction unit (ut)", as used with reference to a viral titer, refers to the number of infectious vector particles that result in the production of a functional transgenic product as measured in functional assays such as described in Xiao the al. (1997) Exp. Neurobiol. 144: 113-124 or in Fisher et al. (1996) J. Viral. 70: 520-532 (LFU assay). The terms "therapeutic", "therapeutically effective amount" and its cognates refer to the amount of a compound that results in the prevention or delay of onset or improvement of symptoms in a subject or obtaining a desired biological result, such as the correction of a neuropathology, for example, a cellular pathology associated with a lysosomal storage disease such as that described herein or in Walkley (1998) Brain Pathol. 8: 175-193. The term "therapeutic correction" refers to the degree of correction that results in the prevention or delay of onset or improvement of symptoms in a subject. The effective amount can be determined by methods well known in the art and as described in the subsequent sections. It is understood that a "composition" means a combination of active agent and another compound or composition, inert (eg, detectable agent or marker) or active, such as an adjuvant. It is understood that a "pharmaceutical composition" includes the combination of an active agent with a vehicle, inert or active, which makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. As used herein, the term "pharmaceutically acceptable carrier" includes any of the conventional pharmaceutical carriers, such as phosphate buffered saline, water and emulsions, such as an oil / water or water / oil emulsion and various types of wetting agents. The compositions may also include stabilizers and preservatives. As examples of vehicles, stabilizers and adjuvants; see Martin, Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)). An "effective amount" is an amount sufficient to produce beneficial or desired results such as prevention or treatment. An effective amount can be administered in one or more administrations, applications or dosages. The terms "subject", "individual" or "patient" are used interchangeably herein, and refer to a vertebrate, preferably a mammal, more preferably a human being. Mammals include, but are not limited to, mice, apes, humans, farm animals, sport animals and pets. A "control" is an alternative subject or sample used in an experiment for comparative purposes. A control can be "positive" or "negative". For example, when the objective of the experiment is to determine a correlation of an altered expression level of a gene with a disease, it is generally preferable to use a positive control (a subject or a sample of a subject, which carries such alteration and which presents syndromes). characteristic of that disease) and a negative control (a subject or a sample of a subject lacking the altered expression and clinical syndrome of that disease) The administration of proteins by gene therapy uses viral vectors capable of infecting post-mitotic neurons. As a review of viral vectors for administering genes to the CNS, see Davidson et al (2003) Nature Rev. 4: 353-364 Adeno-associated virus vectors (AAV) are useful for CNS gene therapy because they are substantially non-toxic, non-immunogenic, neurotropic and can maintain CNS expression (Kaplitt et al. (1994) Nat. Genet 8: 148-154; Bartlett et al. (1998) Hum. Gene Ther. 91181- 1186; and Passini et al. (2002) J. Neurosci., 22: 6437-6446). As demonstrated herein, the expression of a transgene in the brain of an animal resulted in an immune response to the transgenic product. See figure 7A. Applicants have discovered that administration of a transgene to the CNS of a mammal that has become tolerant to the transgene increases the efficacy of the treatment. Thus, in one aspect, this invention provides a method for causing the brain of a mammal to tolerate a preselected immunogen by means of the systemic administration of an effective amount of the preselected immunogen to the mammal prior to the administration of an effective amount of the immunogen. pre-selected to the mammal's CNS. As used herein, the term "make tolerant" is intended to refer to reducing the immune response to an immunogen. The term "immunogen" will include any agent that initially induces an immune response (mediated by T cells or by B cells). As demonstrated by Ziegler et al. (2004), the expression of a transgene in the liver after administration of a recombinant AAV vector encoding the transgene under the control of an enhancer / promoter with liver specificity resulted in a lower antibody response against the expressed transgene. The method is suitable for any mammal, and as such, a "mammal" includes, but is not limited to, mice, apes, humans, farm animals, sport animals and pets. In a particular aspect, the mammal is the mouse ASMKO which is the accepted animal model of types A and B of Niemann-Pick disease. Horinouchi et al. (1995) Nat. Genetics 10: 288-293; Jin et al. (2002) J. Clin. Invest. 109: 1183-1191; and Otterbach (1995) Cell 81: 1053-1061.

Niemann-Pick A disease (NPA) is classified as a lysosomal storage disease and is a hereditary neurometabolic disorder characterized by a genetic deficiency of acid sphingomyelinase (ASM, sphingomyelin, choline phosphohydrolase, EC 3.1.3.12). The lack of functional ASM protein produces the accumulation of the sphingomyelin substrate within the lysosomes of the neurons and the glia throughout the brain. This leads to the formation of large numbers of distended lysosomes in the pericarion, which are an indisputable feature and the main cellular phenotype of type A NPD. The presence of distended lysosomes correlates with the loss of a normal cellular function and a course progressive neurodegenerative that leads to the death of the affected individual in the first phase of childhood (The Metabolic and Molecular Bases of Inherited Diseases, eds. Scriver et al., McGraw-Hill, New York, 2001, pp. 3589-3610). Secondary cell phenotypes (for example, additional metabolic abnormalities) are also associated with this disease, notably the accumulation of a high level of cholesterol in the lysosomal compartment. Sphingomyelin has a strong affinity for cholesterol, which results in the complexation of large amounts of cholesterol in the lysosomes of ASMKO mice and human patients. Leventhal et al. (2001) J. Biol. Chem., 276: 44976-44983; Slotte (1997) Subcell. Biochem. 28: 277-293; and Viana et al. (1990) J. Med. Genet. 27: 499-504. In a specific aspect, the systemic site of administration is the liver of mammals. Any method of administration can be used, examples of such administration being provided below.

After systemic administration of the transgene, it is administered to the CNS of the mammal and, in particular, intracranially directly in the brain of the mammal and more particularly at a site selected from the group consisting of the brain stem, the hippocampus, the striatum, the medulla, the bridge, the mesencephalon, the cerebellum, the midbrain, the thalamus, the hypothalamus, the cerebral cortex, the occipital lobe, the temporal lobe, the parietal lobe, and the frontal lobe. In one embodiment, the administration is specific in the deep cerebellar nuclei of the cerebellum. As indicated above, the transgene encoding the polypeptide or protein is administered to the human to ultimately administer the polypeptide or protein using any appropriate gene transfer method, examples being those described above and in the U.S. Pat. No. 6,066,626. In one aspect, the transgene encodes ASM. The genomic and functional cDNA sequences of human ASM have been published, for example, in U.S. Patent Nos. 5,773,278 and 6,541,218. Table 1 identifies other suitable protein immunogens that have involvement in the CNS. Viral vectors are useful gene transfer vectors. In a particular embodiment, the viral vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), vaccinia, herpes virus, baculovirus and retrovirus. US Pat. No. 6,066,626 and PCT Publication No. WO 01/36620 A2 describe suitable AAV vectors. Modified vectors, such as those described in column 9, lines 14 to 66 can also be used. Suitable serotypes include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8. The AAV vector can be a recombinant or hybrid AAV vector, for example, one selected from the group consisting of the serotype vectors AAV2 / 1, AAV2 / 5, AAV2 / 7, AAV2 / 8, AAV1 / 2, AAV1 / 3, AAV1 / 5, AAV1 / 7 and AAV1 / 8, where the nomenclature refers to the serotype from which the ITR / serotype from which the capsid is generated is generated. For example, an AAV2 / 5 vector comprises the ITRs of the AAV2 serotype and the capsid of the AAV5 serotype. The mammal is administered an effective amount of the polypeptide, by means of the administration of a viral vector comprising a transgene from which the polypeptide is generated. In one aspect, a viral vector comprising a transgene is administered to the liver and, immediately after administration to the liver, a viral vector comprising said transgene is administered to the CNS. In an alternative embodiment, the subsequent administration of the viral vector to CNS occurs after the polypeptide has been expressed in the mammal for an effective period of time to generate tolerance of the mammal to said polypeptide. This may vary with the patient to be treated, the polypeptide administered and the desired therapeutic effect. The appropriate time course and the number of administrations subsequent to the CNS is determined by the physician in charge of the case. To determine if a mammal is tolerant to a polypeptide, the mammal can be exposed to the polypeptide to determine whether said exposure generates an immune response against the polypeptide. The immune response can be determined by measuring the antibody titer against the polypeptide after exposure. A reduced or insignificant antibody titer, as compared to appropriate controls, after exposure may indicate a state of tolerance. Means for measuring and generating antibody responses in mammals, for exposing a mammal to an antigen or immunogen and for determining antibody titers are well known in the art. The appropriate period of time for generating mammalian tolerance can also be selected by determining the effective period of time for expression to generate said tolerance in a test subject. The selected period of time after may be applied to the methods of the present invention without the need to test each patient individually. In one embodiment, subsequent administration of the viral vector to the CNS occurs after detecting the expression of the polypeptide encoded by the viral vector administered to the liver. The detection of polypeptide expression can be carried out by any method known in the art, including examples, but without limitation, molecular (by mRNA detection), immunological (by detection of protein expression) or biochemical (by detection of the activity of a polypeptide, such as enzymatic activity, when said activity exists).

Alternatively, subsequent administration may be carried out hours, days or weeks after the first administration. The use of multiple administrations in the SNC and in multiple administration sites of the SNC is also within the scope of this invention. Thus, in a specific aspect of this invention, there is provided a method for causing the brain of a mammal to tolerate an acid sphingomyelinase polypeptide. The method provides systemically an effective amount of a transgene encoding the polypeptide in the liver of the mammal prior to the administration of an effective amount of the transgene encoding the polypeptide in the tissue of the central nervous system (CNS) of the mammal.

In another particular aspect of this invention, a method for improving the symptoms associated with Type A Niemann-Pick disease (NPA) in a mammal suffering from NPA is provided. This method requires administering to the mammalian central nervous system (CNS) an effective amount of a transgene encoding an acid sphingomyelinase polypeptide and wherein said administration is subsequent to the systemic administration of an effective amount of the transgene in the liver tissue of the mammal, such that the ability of the mammal to induce an immune response with antigen specificity to the polypeptide is null or significantly reduced prior to administration in the CNS. In another aspect, the invention provides a method for improving symptoms associated with Niemann-Pick Type A (NPA) disease in a mammal suffering from NPA. These symptoms include, but are not limited to, weight loss or cachexia, loss of motor function, loss of cognitive function, and premature death. The method requires administering to the mammalian CNS an effective amount of a viral vector comprising a transgene encoding an acid sphingomyelinase polypeptide after administration of an effective amount of a viral vector comprising said transgene in the liver tissue of the mammal. In another embodiment, the area of the CNS in which the viral vector is administered is the brain. In another aspect, the invention provides a method for treating Niemann-Pick Type A (NPA) disease in a mammal suffering from NPA by administering an effective amount of a viral vector AAV comprising a transgene encoding a acid sphingomyelinase polypeptide in the liver tissue of the mammal and subsequently the administration of an effective amount of an AAV vector comprising a transgene encoding an acid sphingomyelinase polypeptide in the mammalian CNS, thereby treating the NPA in the mammal. In another embodiment, the area of the CNS in which the viral vector is administered is the brain. As used herein, the terms "treat", "treatment" and the like are used herein to refer to obtaining a desired therapeutic, pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or a sign or symptom thereof, and / or may be therapeutic in terms of a partial or complete cure of the disorder and / or adverse effect attributable to the disorder. "Treatment" also includes any treatment of a disorder in a mammal, and includes: preventing the occurrence of a disorder in a subject who may be predisposed to a disorder, but who has not yet been diagnosed, for example, a patient who has tested positive for the genetic marker of the disease; inhibit a disorder, that is, stop its development; or alleviating or improving the disorder, for example, causing the regression of the disorder, for example, the NPA disease. As used herein, "treating" also includes the systemic improvement of the symptoms associated with the pathology and / or a delay in the onset of symptoms. The clinical and subclinical evidence of "treatment" will vary with the pathology, the individual and the treatment. In some embodiments, the method comprises administering an AAV vector carrying a pre-selected immunogen or transgene such that the transgenic product is expressed at a therapeutic level at the selected site. In some embodiments, the viral titer of the composition is at least: (a) 1, 5, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 4 , 0, 4.5, 5, 6, 7, 8, 8.4, 9, 9.3, 10, 15, 20, 25 or 50 (x 1011 genome copies per injection (ge); (b) 1 , 5, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 4.0, 4.5, 5, 6, 7, 8, 8.4 , 9, 9.3, 10, 15, 20, 25 or 50 (x109 ut / ml), or (c) 1, 5, 2.0, 2.25, 2.5, 2.75, 3, 0, 3.25, 3.5, 4.0, 4.5, 5, 6, 7, 8, 8.4, 9, 9.3, 10, 15, 20, 25 or 50 (x 1010 infectious units [ui] / ml) Intracranial administration can be performed in any region of the brain and may include multiple regions when more than one intracranial administration is performed.These sites include, for example, the brainstem (medulla and bridge), mesencephalon, brain middle, cerebellum (including deep cerebellar nuclei), diencephalon (thalamus, hypothalamus), telencephalon (striatum, midbrain, cerebral cortex or, within the cortex, the occipital, temporal, parietal lobes) rontal.) Figure 1 shows specific examples of intracranial injection sites.

For the identification of structures in the human brain, see, for example, The Human Brain: Surface, Three-Dimensional Sectional Anatomy With MRI, and Blood Sypply, 2nd ed., Eds. Deuteron et al., Springer Vela, 1999; Atlas of the Human Brain, eds. Mai et al., Academic Press; 1997; and Co-Planer Sterotaxic Atlas of the Human Brain: 3-Dimensional Proportional System: An Approach to Cerebral Imaging, eds. Tamarack et al., Thyme Medical Pub., 1988. For the identification of structures in the mouse brain, see, for example, The Mouse Brain in Sterotaxic Coordinates, 2nd ed., Academic Press, 2000. If desired, the structure The human brain can be correlated with similar structures in the brain of another mammal. For example, most mammals, including humans and rodents, show a similar topographical organization of the entorinal-hippocampal projections, with neurons in the lateral part of the lateral and medial entorinal cortex projecting to the dorsal or septal area of the hippocampus, while the projection in the ventral hippocampus comes mainly from neurons in the middle parts of the entorinal cortex (Principies of Neural Science, 4th ed., eds Kandel et al., McGraw-Hill, 1991, The Rat Nervous System, 2nd ed., Ed. Paxinos, Academic Press, 1995). In addition, the cells of layer II of the entorinal cortex project to the dentate gyrus, and end in the outer two thirds of the molecular layer of the dentate gyrus. The axons of the cells of layer III project bilaterally to the areas of the Amón horn CA1 and CA3 of the hippocampus, ending in the molecular layer of the lacunae stratum. To administer the vector specifically in a particular region of the central nervous system, especially in a particular region of the brain, it can be administered by stereotactic microinjection. For example, on the day of the surgical operation, patients will have the base of the stereotaxic frame fixed in place (screwed into the skull). The brain will be imaged with the base of the stereotactic frame (compatible in MRI with fiduciary marks) using high resolution MRI. The MRI images will then be transferred to a computer running a stereotaxic software. A series of corona, sagittal, and axial images will be used to determine the target vector injection site, and the trajectory. The software directly translates the trajectory into appropriate three-dimensional coordinates for the stereotactic frame. Drill trephine holes above the entry site and the stereotaxic apparatus is placed with the needle implanted at the given depth. The vector will then be injected into a pharmaceutically acceptable vehicle. The AAV vector is then administered by direct injection into the primary target site and transported retrograde to the distal target sites through axons. Other routes of administration may be used, for example, superficial cortical application under direct visualization, or other non-stereotaxic application. The total volume of material to be administered and the total number of vector particles to be administered will be determined by those skilled in the art based on known aspects of gene therapy. In an appropriate animal model, efficacy and therapeutic safety can be tested. For example, there is a variety of well-characterized animal models for LSD, for example, as described herein or in Watson et al. (2001) Methods Mol. Med., 76: 383-403; or Jeyakumar et al. (2002) Neuropath. Appl. Neurobiol., 28: 343-357; or in Metabolic and Molecular Bases of Inherited Disease, 8th edition (2001), published by McGraw-Hill. In experimental mice, the total volume of AAV solution injected is, for example, a volume comprised between 1 and 5 μ? or about 3 μ? per injection site, or alternatively a volume comprised between 10 and 400 μ? or, alternatively, between 100 and 400 μ ?, as an alternative between 150 and 250 μ? or, as an alternative, a volume of approximately 200 μ ?. In the case of other mammals, including the human brain, volumes and proportions of administration are scaled up appropriately. For example, it has been shown that volumes of 150 μ? they can be safely injected into the primate's brain (Janson et al (2002) Hum. Gene Ther., 13: 1391-1412). The treatment may consist of a single injection per target site, or may be repeated throughout the injection tract if necessary. Multiple injection sites can be used. For example, in some embodiments, in addition to the first administration site, a composition comprising AAV carrying a transgene is administered at another site that may be contralateral or ipsilateral with respect to the first administration site. In the methods of the invention, AAV of any serotype can be used. In one embodiment of the invention, AAV vectors capable of undergoing retrograde axonal transport can be used. The serotype of the viral vector can be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 (see, for example, Gao et al. (2002) PNAS, 99: 1 1854-1 1859; and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Other serotypes other than those indicated in this document may be used. Pseudotyped AAV vectors, which are those comprising the ITRs of an AAV serotype in the capsid of a second AAV serotype, can also be used; for example, an AAV vector containing an AAV2 capsid and the AAV1 genome or an AAV vector containing the AAV5 capsid and the AAV2 genome (Auricchio et al. (2001) Hum. Mol. Gener., 10 (26 ): 3075-81). AAV vectors are derived from single-stranded DNA parvoviruses (me) which are non-pathogenic for mammals (reviewed in Muzyscka (1992) Curr. Top, Microb. Immunol., 158: 97-129). In summary, the viral genes rep and cap, which make up 96% of the viral genome, have been eliminated in the AAV-based vectors, leaving the two inverted terminal repeats (ITRs) of 145 base pairs (pb) flanking, which are used to initiate viral DNA replication, packaging and integration. In the absence of helper viruses, the wild-type AAV is integrated into the genome of the human host cell with a preferential site specificity on chromosome 19q 13.3 or may remain episomally expressed. A single AAV particle can accommodate up to 5 kb of mDCNA, thereby leaving about 4.5 kb of a transgene and regulatory elements, which is typically sufficient. However, the trans-splicing systems described, for example, in U.S. Patent No. 6,544,785, can almost double this limit. In an illustrative embodiment, the AAV is AAV2 or AAV8. Adeno-associated viruses of many serotypes, such as AAV2, have been studied and extensively chterized as gene therapy vectors. Those skilled in the art will be familiar with the prepion of gene therapy vectors based on functional AAVs. Numerous references to various methods of production, purification and prepion of AAV for administration to humans can be found in the large amount of published literature (see, for example, Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). In addition, gene therapies based on AAV directed to CNS cells have been described in U.S. Patent Nos. 6,180,613 and 6,503,888. The level of transgenic expression in eukaryotic cells can be regulated by the promoter and / or enhancer or transcription enhancers within the transgene expression cassette. In some embodiments tissue-specific promoters, such as promoters with liver specificity, can be used. In some embodiments tissue-specific enhancers such as enhancers with liver specificity can be used. In the practice of the present invention, combinations of tissue-specific promoters and tissue-specific enhancers, such as promoters and enhancers with liver specificity, can be used. Non-limiting examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter (Kaplitt et al (1994) Nat. Genet 8: 148-154), the human CMV / 3-globin promoter (Mandel et al. al. (1998) J. Neurosci.18: 4271-4284), the GFAP promoter (Xu et al. (2001) Gene Ther., 8: 1323-1332), the neuron-specific enolase (NSE) promoter. of 1, 8-kb (Klein et al. (1998) Exp. Neurol. 150: 183-194), the promoter of chicken beta actin (CBA) (Miyazaki (1989) Gene 79: 269-277) and the ß-glucuronidase (GUSB) promoter (Shipley et al. (1991) Genetics 10: 1009-1018), the human serum albumin promoter, and the alpha-1-antitrypsin promoter. In order to improve expression, other regulatory elements can be functionally linked to the transgene, such as, for example, the post-transcriptional regulatory element of the hepatitis B virus of the North American marmot (WPRE) (Donello et al. (1998)). J. Virol. 72: 5085-5092) or the polyadenylation site of bovine growth hormone (BGH). Other promoters that are suitable for the present invention can be any strong constitutive promoter that is capable of promoting the expression of an associated coding DNA sequence in the liver. These strong constitutive promoters include the human and murine cytomegalovirus promoter, promoters of truncated CMV, the promoter of human serum albumin [HSA] and the promoter of alpha-1-antitrypsin. The liver specificity enhancing elements useful for the present invention can be any liver specific enhancer that is capable of enhancing tissue-specific expression of an associated coding DNA sequence in the liver. These liver specific enhancers include one or more human serum albumin (HSA) enhancers, human prothrombin enhancers (HPrT), alpha-1 microglobulin (A1 MB) enhancers and intronic aldolase enhancers. Multiple enhancer elements can be combined to achieve greater expression. For example, two identical enhancers can be combined with a promoter with liver specificity. In the practice of the present invention, viral vectors comprising the following promoter / enhancer combinations can be used: one or more HSA enhancers in combination with a CMV promoter or an HSA promoter; one or more enhancers selected from the group consisting of the human prothrombin enhancer (HPrT) and the alpha-1 microglobulin enhancer (A1MB) in combination with a CMV promoter; and one or more enhancer elements selected from the group consisting of HPrT enhancers and A1MB enhancers, in combination with an alpha-1-antitrypsin promoter. PCT Patent Application Publication No. WO 01/36620 discloses additional information regarding constructs with liver specificity. Alternatively, promoters and / or enhancers with neuronal specificity are useful for the administration and directed expression of transgenes in the CNS. In some aspects, it will be desirable to control the activity of transcription. For this purpose, pharmacological regulation of gene expression can be obtained with AAV vectors including various regulatory elements and drug response promoters as described, for example, in Haberma et al. (1998) Gene Ther., 5: 1604-16011; and Ye et al. (1995) Science 283: 88-91.

AAV preparations can be produced using techniques known in the art, for example, as described in U.S. Patent No. 5,658,776 and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003. In certain aspects, detection and / or expression level of the transgene may be desired. Methods for detecting gene expression are known in the art and can be easily applied as described below or modified by those skilled in the art. Various methods for quantifying the expression of a gene of interest are known and include, but are not limited to, hybridization assays (Northern blot analysis) and PCR-based hybridization assays. To test for an alteration in mRNA level, the nucleic acid contained in a sample is first extracted according to a conventional method in the art. For example, the mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), supra or extracted by nucleic acid binding resins following the attached instructions provided by the manufacturers. As hybridization probes or PCR primers, nucleic acid molecules having at least ten nucleotides and having sequence complementarity or homology with the expression product can be used. It is known in the art that a "perfectly matching" probe is not needed for a specific hybridization. Small changes in the probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the specificity of the hybridization. In general, up to 20% uncoupling of base pairs (when optimally aligned) can be tolerated. For example, a probe useful for detecting mRNA has an identity of at least about 80% with the homologous region of comparable size contained in a previously identified sequence, for example, the ASM sequence. Alternatively, the probe has an identity of at least 85% or even at least 90% with the sequence of the corresponding gene after alignment of the homologous region. The total size of the fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments of the gene, in general, will find use in hybridization embodiments, where the length of the complementary region can be varied, such as between about 10 and about 100 nucleotides or even an entire length according to the complementary sequences that are desired detect. Nucleotide probes having complementary sequences in stretches greater than about 10 nucleotides in length will increase the stability and selectivity of the hybrid, and thus improve the specificity of the particular hybrid molecules obtained. Nucleic acid molecules having complementary stretches of genes greater than about 25 and even more preferably more than about 50 nucleotides in length, or even longer when desired, can be designed. These fragments can be easily prepared, for example, by direct synthesis of the fragment by chemical means, by application of the nucleic acid reproduction technology, such as PCR ™ technology, with two priming oligonucleotides as described in FIG. U.S. Patent No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production. In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate medium, such as a label, to detect hybridization and therefore complementary sequences. A wide variety of appropriate indicator means is known in the art., including fluorescent, radioactive, enzymatic and other ligands, such as avidin / biotin, which can provide a detectable signal. A fluorescent label or an enzymatic label, such as urease, alkaline phosphatase or peroxidase, can also be used in place of radioactive reagents or other environmentally undesirable reagents. In the case of enzymatic labels, colorimetric indicator substrates are known which can be used to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with samples containing complementary nucleic acids. Hybridization reactions can be carried out under conditions of different "stringency". Relevant conditions include temperature, ionic strength, incubation time, the presence of additional solutes in the reaction mixture such as formamide, and the washing procedure. The conditions of greatest stringency are the conditions, such as the higher temperature and lower concentration of sodium ion, which require a greater minimum complementarity between hybridization elements so that a stable hybridization complex is formed. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art. See, Sambrook, et al. (1989) supra. The detection and quantification of the mRNA level or its expression can also be used using quantitative PCR or high throughput analysis such as serial analysis of gene expression (SAGE) described in Velculescu, v. et al. (1995) Science 270: 484-487. In summary, the method comprises isolating multiple mRNAs from samples of cells or tissues that are suspected to contain the transcript. Optionally, the transcripts of the genes can be converted into cDNA. A sample of the transcripts of the genes is subjected to an analysis with sequence specificity and quantified. These abundances of the gene transcript sequence are compared against the abundances of reference database sequences that include normal data sets for sick and healthy patients. Probes can also be attached to a solid support for use in high throughput screening assays using methods known in the art. International PCT Application number WO 97/10365 and U.S. Patent Nos. 5,405,783, 5,412,087 and 5,445,934, for example, describe the construction of high density oligonucleotide chips that may contain one or more sequences. The chips can be synthesized on a modified glass surface using the methods described in U.S. Patent Nos. 5,405,783, 5,412,087 and 5,445,934. Photoprotected nucleoside phosphoramidites can be coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask and reacted with a second protected phosphoramidite nucleoside. The coupling / deprotection process is repeated until the desired probe is completed. The level of expression of the gene can be determined by exposure of a sample suspected of containing the polynucleotide to the chip modified with the probe. The extracted nucleic acid is labeled, for example, with a fluorescent label, preferably during an amplification step. Hybridization of the labeled sample is carried out at an appropriate level of rigor. The degree of probe-nucleic acid hybridization is measured quantitatively using a detection device, such as a confocal microscope. See U.S. Patent Nos. 5,578,832 and 5,631,734. The measurement obtained correlates directly with the level of expression of the gene. Hybridized probes and nucleic acids from the sample can be detected by various methods known in the art. For example, hybridized nucleic acids can be detected by detection of one or more labels bound to the nucleic acids of the sample. The labels can be incorporated by any of several means known to those skilled in the art. In one aspect, the marker is incorporated simultaneously during the amplification step in the preparation of the nucleic acid of the sample. Thus, for example, the polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a marked amplification product. In a different embodiment, the amplification of transcription, as described above, using a labeled nucleotide (eg, CTP and / or fluorescein-labeled UTP) incorporates a label into the transcribed nucleic acids. Alternatively, a marker can be added directly to the original nucleic acid sample (e.g., mRNA, polyA, mRNA, cDNA, etc.) or to the amplification product after amplification is complete. Those skilled in the art know means for attaching labels to nucleic acids and these include, for example, nick translation or terminal labeling (eg, with a labeled RNA) by treatment with nucleic acid kinases and subsequent binding (linkage) of a linker. of nucleic acid that binds the nucleic acid of the sample to a marker (eg, a fluorophore). Detectable levels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Markers useful in the present invention include biotin for staining with a labeled streptavidin conjugate, magnetic beads (e.g. Dynabeads ™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein and the like), radiolabels (eg. example, 3H, 125l, 35S, 14C or 32P), enzymes (e.g. horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA) and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g. polystyrene , polypropylene, latex, etc.). Patents that teach the use of these markers include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Those skilled in the art know means to detect these markers. In this way, for example, radiolabels can be detected using a photographic film or scintillation counters, and the fluorescent labels can be detected using a photodetector to detect the emitted light. Enzymatic markers are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by means of a simple visualization of the colored label. Patent Publication WO 97/10365 describes methods for adding the label to the target nucleic acid (s) (acid) or acids before or, alternatively, after hybridization. These are detectable labels that bind directly or are incorporated into the target nucleic acid (sample) prior to hybridization. In contrast, "indirect markers" join the hybrid duplex after hybridization. Often, the indirect marker binds to a binding moiety that has bound to the target nucleic acid prior to hybridization. Thus, for example, the target nucleic acid can be biotinylated prior to hybridization. After hybridization, an avidin-conjugated fluorophore will bind to the biotin carried by the hybrid duplexes providing a marker that is easily detected. As a detailed review of nucleic acid labeling methods and detection of labeled hybridized nucleic acids, see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization with Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y. (1993). The nucleic acid sample can also be modified prior to hybridization with the high density probe array to reduce sample complexity thereby reducing the interference signal and improving the sensitivity of the measurement using the methods described in the PCT Application International number WO 97/10365. The results of the chip assay are typically analyzed using a computer software program. See, for example, EP 0717 13 A2 and WO 95/20681. Hybridization data are read in the program, which calculates the level of expression of the gene or genes targeted. This figure is compared with the existing data series of gene expression levels for sick and healthy individuals. A correlation between the data obtained and those of a series of sick individuals indicates the appearance of a disease in the patient. Also known immunoassays can be modified to detect and quantify expression. The determination of the gene product requires measuring the amount of immunospecific binding that occurs between an antibody reactive with the gene product. To detect and quantify immunospecific binding, or signals generated during hybridization or amplification procedures, digital image analysis systems may be employed including, but not limited to, those that detect radioactivity from probes or chemiluminescence. Expression of a polypeptide product can also be detected using biochemical means such as those known in the art for the particular polypeptide expressed. The following examples provide illustrative embodiments of the invention. A person of ordinary skill in the art will recognize the numerous modifications and variations that may be made without altering the spirit or scope of the present invention. These modifications and variations are included within the scope of the invention. The examples do not limit the invention in any way. EXAMPLES Titration of recombinant vectors Titers of the AAV vector can be measured according to the number of copies of the genome (genome particles per milliliter). The concentrations of genome particles can be based on the Taqman® PCR of the vector DNA as previously indicated (Clark et al. (1999) Hum. Gene Ther.10: 1031-1039; Veldwijk et al. (2002) Mol. Ther. 6272-278). In summary, the AAV vector is treated with a DNase solution to remove any contaminating DNA that may interfere with the accurate measurement of the viral DNA. The AAV vector is then treated with capsid digestion buffer (50 mM Tris-HCl, pH 8.0, 1.0 mM EDTA, 0.5% SDS, 1.0 mg / ml proteinase K) at 50 ° C for 1 hour to release the vector DNA. DNA samples are subjected to a polymerase chain reaction (PCR) with primers that hybridize to specific sequences in the vector DNA, such as the promoter region, the transgene or the polyA sequence. The PCR results are then quantified by real-time Taqman® software, such as that provided by the Prism 7700 sequence detector system from Perkin Elmer-Applied Biosystems (Foster City, CA). Vectors carrying a testable marker gene such as the β-galactosidase gene or the green fluorescent protein (GFP) gene can be titrated using an infectivity assay. Susceptible cells (e.g. HeLa or COS cells) are subjected to transduction with the AAV and an assay is performed to determine gene expression such as staining of the cells transduced with the β-galactosidase vector with X-gal (5-bromo- 4-chloro-3-indolyl-D-galactopyranoside) or fluorescence microscopy for cells transduced with GFP. For example, the assay is performed as follows: 4 × 10 4 HeLa cells are cultured in each well of a 24-well culture plate using normal growth medium. After binding, ie approximately 24 hours later, the cells are infected with Ad 5 at a multiplicity of infection (MOI) of 10 and transduced with serial dilutions of the packaged vector and incubated at 37 ° C. One to three days later, before observing extensive cytopathic effects, the appropriate assay is performed on the cells (for example, X-gal staining or fluorescence microscopy). If a reporter gene such as the β-galactosidase gene is used, the cells are fixed in 2% paraformaldehyde, 0.5% glutaraldehyde and stained for β-galactosidase activity using X-gal. The dilutions of the vector that provide well separated cells are counted. Each positive cell represents a transduction unit (ut) of vector. The full length human ASM cDNA was cloned into a plasmid containing ATR ITR of serotype 2 and 8. Jin et al. (2002) J Clin Invest. 109: 1183-1191. AAV8-hASM contained inverted terminal repeats (ITR) of serotype 2 and cDNA of human acid sphingomyelinase (hASM) under the control of the liver-restricted promoter DC-190 [Ziegler et al. (2004). Mol. Ther. 9: 231-240]. AAV2-hASM contained inverted terminal repeats (ITR) of serotype 2 and the cDNA of human acid sphingomyelinase (hASM) under the control of the CMV enhancer and the chicken β-actin promoter. The two recombinant vectors were produced by cotransfection of triple plasmids of human 293 cells and purified on a column. The final titers of the AAV8-hASM and AAV2-hASM preparations were in 5.0 x 1012 genome copies (ge) per me, as determined by TaqMan PCR of the bovine growth hormone polyadenylation signal sequence containing each vector. Hybrid vectors can be produced by triple transfection using a series of helper plasmids containing capsid coding domains with serotype specificity in addition to the replication genes of the AAV serotype. This strategy allows the packaging of AAV ITR vectors in each virion with serotype specificity. Rabinowitz, et al. (2002) J Virol. 76: 791-801. With this strategy, the recombinant hASM genome can be used to generate a series of pseudotyped rAAV-hASM vectors. The recombinant AAV vectors can be purified by ion exchange chromatography. O'Riordan, et al. (2000) J Gene Med 2: 444-54. Recombinant AAV vectors can also be purified by centrifugation in CsCI. Rabinowitz et al. (2002) J. Urrol. 76: 791-801. The final titer of AAV-ASM virion particles (DNase resistant particles) can be determined by TaqMan PCR of the CMV sequence. Clark et al. (1999) Hum. Gene Therapy 10: 1031-1039. Combination of brain and systemic gene therapy in ASMKO mice Mice treated with acid sphingomyelinase (ASMKO) were treated, K. Horinouchi et al., Nal Genet. 10 (1995), pp. 288-292, as follows: in mice of group 1) 3 x 1011 ge of an AAV2 / 8 vector comprising two HPrT enhancers, a human serum albumin promoter and a human ASM transgene was injected through a vein injection in the tail at 4 weeks of age; in mice of group 2) 2 x 1011 ge comprising an enhancer, promoter and a transgene of human ASM were injected by means of a brain injection divided among 8 sites in the brain at 6 weeks of age; in mice of group 3) 3 x 1011 ge of AAV2 / 8 vector comprising two HPrT enhancers, a human serum albumin promoter, and a human ASM transgene were injected via tail vein injection at 4 weeks of age, and 2 weeks later in the same mice, 2 x 1011 ge of an AAV2 vector comprising an enhancer, promoter and a transgene of human ASM were injected through brain injections divided among 8 sites in the brain; and mice of group 4) did not receive injections or received simulated injections only with vehicle. The mice of group 5) that were not ASMKO, or wild-type, did not receive injections. All the ASMKO mice that underwent stereotactic surgery received injections in 8 regions of the brain with 3 μ? of AAV2-hASM (1.5 x 1010 ge) per site up to a total of 24 μ? (1, 2 x 101 ge) per brain. The sites injected in the right hemisphere were the hypothalamus (-0.50, -1.00 mm, -3.50 mm), hippocampus (-2.00 mm, -1, 75 mm, -1, 75 mm), marrow (-6.00 mm, -1, 50 mm, -3.75 mm) and cerebellum (-6.00 mm, -1, 50 mm, -2.25 mm); and the sites injected in the left hemisphere were the striatum (0.50 mm, 1.75 mm, -2.75 mm), the motor cortex (0.50 mm, 1.75 mm, -1, 25 mm), the middle brain (-4.50 mm, 1.00 mm, -3.50 mm) and the cerebellum (-6.00 mm, 1.50 mm, -2.25 mm). Injections were performed with a Hamilton syringe (Hamilton USA, Reno, NV) at a speed of 0.5 μm / min and the needle was left in place for 2 minutes after each injection to minimize the upward flow of viral solution after raising the needle. All the untreated ASMKO mice and the ASMKO mice of the groups only treated with cerebral AAV2 injection and only treated with systemic injection of AAV8 finally reached a moribund state. In contrast, ASMKO mice treated by cerebral and systemic combination injections did not reach a moribund state, but were sacrificed at 54 weeks for a comparative analysis. The animals were perfused extensively to extract all the blood and divided into biochemical and histological cohorts. In the biochemical cohort, the liver, lung, spleen and skeletal muscle were cut into two pieces; one piece was analyzed with respect to the hASM levels and the other with respect to the storage of sphingomyelin. In the brain, the left and right hemispheres were separated and each hemisphere was further cut into five 2-mm slices along the A-P axis. The left hemisphere cuts were analyzed with respect to the hASM and anti-hASM protein. Acid sphingomyelinase (ASM) serum levels and anti-ASM antibody serum levels were measured throughout the study. The mice were subjected to various tests throughout the study: body weight assessments, acceleration rotarod test, rolling rotarod test and Barnes maze assay. We also collected data on the survival curve throughout the study period. After the mice died, tissues were collected and sphingomyelin levels were measured in the brains, livers, lungs, spleens and muscle tissue of each mouse. The expression of human ASM protein in the brain and liver was also evaluated qualitatively using immunohistochemistry. The study was completed at week 54; all the mice in group 3 (combo) were alive and healthy when the study ended.

Serum sphingomyelinase acid levels were quantified by an enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies that had been specifically generated against the human enzyme. Figure 7 shows graphically the levels of ASM protein in blood serum over time. ASM was also measured by immunohistochemistry in the brains and livers of the mice once killed. Positive staining for ASM was observed in the brains of mice of groups 2 and 3, showing a qualitatively brighter stain in the mice of group 3. Staining was observed in the striatum, hippocampus, midbrain and cerebellum. No ASM staining was seen in the brains of group 1 mice or in untreated ASMKO mice. Positive staining for ASM was observed in the livers of mice of groups 1 and 3. No ASM staining was observed in the livers of the group 2 mice or in the untreated ASMKO mice. The tissue levels of sphingomyelin were quantified as indicated below. Tissue extracts were prepared by homogenization of 10 to 50 mg of tissue in chloroform: methanol (1: 2) and incubation at 37 ° C for 1 hour. After removing the cell debris by centrifugation, the homogenates were extracted twice with water and the organic phase (containing the lipids) was transferred to clean glass tubes and then dried under a nitrogen atmosphere with heating at 37 ° C. The amount of sphingomyelin present in the extracts was determined indirectly using the Amplex Red sphingomyelinase assay kit (Molecular Probes). The extracts were treated with a fixed amount of exogenous bacterial sphingomyelinase from Bacillus cereus (Sigma-Aldrich, St. Louis, MO, USA) in the Amplex Red working solution. Sphingomyelin was hydrolyzed by the bacterial enzyme to produce ceramide and phosphorylcholine. The latter was further hydrolyzed to give choline, which in turn was oxidized to betaine and hydrogen peroxide. The liberated hydrogen peroxide was quantified by reaction with Amplex Red to generate a highly fluorescent resorufin which could be detected by fluorescence emission at 590 nm. Normal sphingomyelin levels in the tissues of C57BL / 6 mice are approximately 5-10% of those seen in ASMKO mice of similar age. Figures 8 and 9 graphically show sphingomyelin levels in the brain and visceral organs. The levels of specific antibodies against human sphingomyelinase in serum were determined by means of an ELISA. See Figure 7 E. Serial dilutions of serum were added to wells of a 96-well plate coated with the enzyme or heat-inactivated AAV particles. Bound antibodies were detected using goat anti-mouse IgG immunoglobulin G (IgG), IgM and IgA conjugated with horseradish peroxidase (Zymed, San Franciso, CA, USA). Plates were incubated with substrate (Sigma-Aldrich) for 20 minutes at room temperature for color to appear. Titers were defined as the inverse of the maximum serum dilution that produced an OD450 equal to or less than 0.1. Figures 7 A to 7 D graphically show the levels of anti-hASM antibodies in circulation for the treated and untreated mice. Antibodies within the brain parenchyma were measured with a modified assay as presented below. Tissue lysates 1: 20 were diluted in antibody dilution buffer and applied in duplicate in a 96-well plate coated with 100 ng of hASM. The chromogenic substrate and conjugate reactions of HRP with secondary antibody were performed as described above. The antibody concentration specific for bound hASM must directly correlate with the color intensity of the HRP reaction of the conjugate. In this manner, the final data are presented as the absolute change in DO450 generated by the dilution of the 1: 20 lysate to provide a more sensitive determination of the antibody levels compared to the titration method used for the serum. The brain sections were analyzed for hASM expression using a biotinylated anti-hASM monoclonal antibody at a dilution of 1: 200, and were visualized with a secondary antibody conjugated with streptavidin-Cy3 under red fluorescence [Passini, M.A. et al. (2005). Mol. Ther. 11: 754-762]. The cholesterol substrate in the brain was detected using a philipin staining protocol as indicated [Passini, M.A. et al. (2005). Mol. Ther. 11: 754-762]. Briefly, filipin (Sigma, St. Louis, MO) was dissolved in 100% methanol at a working concentration of 10 mg / ml. The cerebral sections were incubated for 3 hours at room temperature (RT) in the dark, followed by 3 washes at RT with PBS, and examined with blue fluorescence. Staining with lisenin was performed to determine the sphingomyelin substrate pattern in situ, as indicated [Shihabuddin, S.L. et al. (2004). J. Neurosci. 24: 10642-10651]. Briefly, lisenine (Peptides International, Louisville, Kentucky) was dissolved at 10 mg / ml in PBS containing 0.5% BSA, 0.02% saponin (Sigma) and 5% normal donkey serum . The cerebral sections were incubated with lisenin at 4 ° C overnight, followed by an overnight incubation of a 1: 250 dilution of anti-lisenin rabbit antibody (Peptides International). Lisenin positive cells were visualized with a 1: 250 dilution of FITC anti-rabbit antibody (Jackson ImmunoResearch, West Grave, PA) and examined using green fluorescence.

Each mouse was tested by means of a rotarod of acceleration and rotarod of rocking with respect to motor function in the Smartrod (AccuScan) using methods known in the art and reproduced in Sleat et al. (2004) J. Neurosci. 24: 9117-9126. Mice were tested on the acceleration and rolling rotarod using the Smartrod Rotarod Program (AccuScan Instruments, Columbus, OH) to determine motor function. The speed of rotation of the cylinder in the acceleration rotarod was programmed to accelerate at a constant speed of 0-30 rpm for 60 seconds, and the rolling rotarod was programmed to accelerate with a forward and backward movement every 2 seconds. , 5 seconds to a final speed of 25 rpm for 54 seconds. Four tests were performed with each animal at each time point and latency was recorded until the mice fell off the platform. A high latency score is equivalent to good behavior. The individual tests were separated by at least 15 minutes to allow a period of rest to the animal. Figures 3 and 4 graphically show the results of the rotarod tests as a measure of recovery of motor function. Each mouse was tested using a Barnes maze assay. The mice were trained to locate a dark tunnel hidden behind one of 20 holes placed around the perimeter of a large flat plastic disc that was illuminated with bright light by four halogen lamps at the top. The time during which the mouse moved through the maze to locate the correct dark hole correlates with cognitive function inversely - a short displacement time indicates a better cognitive function. Figure 5 graphically shows the results of the Barnes maze test as a measure of the recovery of cognitive function. The survival curves are shown graphically in Figure 6. A combination injection protocol of AAVhASM that targets both the brain and the viscera was evaluated to assess functional abnormalities and the sequelae of ASMKO mouse disease. In the combination group (n = 11), 4-week-old ASMKO mice received 3.0 x 1011 genome copies (ge) of AAV8-hASM through a vein injection in the tail. Two weeks later, at 6 weeks of age, the same mice were injected with AAV2-hASM in the motor cortex, striatum, middle brain and cerebellum of the left hemisphere, and in the hypothalamus, hippocampus, medulla and cerebellum of the right hemisphere. In each structure, 1.5 x 1010 ge were injected for a total of 1.2 x 1011 ge per brain. The treated control groups received only systemic injections of AAV8-hASM at 4 weeks of age (n = 12) or only cerebral injections of AAV2-hASM at 6 weeks of age (n = 14), and the control groups did not treated included ASMKO (n = 23) and wild-type mice (n = 10). Periodic blood extractions were performed to measure the levels of hASM and anti-hASM antibodies in circulation. Serum analysis of ASMKO mice treated by systemic injection alone and by combination injections presented the highest level of circulating hASM. Transduction and subsequent expression by AAV8-hASM was mediated mainly by the liver due to the tropism of this viral serotype and the selection of a promoter restricted to the liver (DC190) in the design of the expression cassette. The two groups reached maximum levels of hASM at two weeks after the injection, which were subsequently reduced up to 10 times during the study period. Serum from untreated ASMKO groups and injection only into AAV2 brain did not present detectable levels of hASM. Further analysis of blood serum demonstrated that anti-hASM antibody levels in mice treated by combination or by systemic injections of AAV8 alone were similar to the low baseline level observed in untreated ASMKO mice. Conversely, mice from the group treated with only AAV2 brain injection had a rapid and solid induction (200-fold increase) of the anti-hASM antibody titers. Therefore, mice treated by systemic injection of AAV8-hASM appear to be immunotolerant to the expressed hASM. High levels of the enzyme in liver, lung, spleen and muscle of mice treated by the combination or by systemic single injections of AAV8 were evident, presumably after an endocytosis mediated by the mannose 6-phosphate receptor of the enzyme in the circulation. The levels of human ASM in the visceral organs of the group treated with only AAV2 brain injection did not rise significantly above those observed in the untreated ASMKO control mice. The analysis of the brain of the combination groups and with treatment only with cerebral injections of AAV2 showed high levels of hASM throughout the neuroaxis. However, the combination group had significantly higher levels of hASM compared to the cerebral only treatment despite the use of the same recombinant AAV2 vector in both cohorts. The levels of hASM in the brains of mice treated by systemic injection alone were low and comparable to those of untreated ASMKO mice. This indicated that the hASM of hepatic origin in the circulation could not cross the blood-brain barrier to enter the CNS. Interestingly, the anti-hASM antibody titer in brain homogenates was approximately 10-fold higher in the group treated with only brain injections of AAV2 compared to the other groups, including the combination group. In this way, the levels of expression in the brain were an inverse of the antibody titers; the combination group showed high levels of hASM and low levels of anti-hASM antibodies, while the group treated with only cerebral injections of AAV2 showed low levels of hASM and high levels of anti-hASM antibodies. The effect of hASM expression on the correction of storage pathology in the viscera and brain of ASMKO mice was determined. There was a complete correction of sphingomyelin storage in all tissues of viscera examined from the combination groups and treated only by systemic injection of AAV8. In contrast, animals that received only brain injections contained high levels of sphingomyelin in the viscera that were similar to untreated ASMKO mice. The analysis of sphingomyelin levels in the brain of the ASMKO mice treated by combination injections showed an overall reduction of the substrate up to wild type levels. This was an improvement over the group treated only with cerebral injections of AAV2, which showed a significant reduction of sphingomyelin only in the brain slices corresponding to an injection site. In this way, the degree of correction in the group with brain injections was only significantly lower and never reached the efficacy observed in the combination group. A less effective reduction in the storage of sphingomyelin in the group treated only with cerebral injections of AAV2 correlated with the lower enzyme levels observed in this group. A high level of cerebral sphingomyelin was observed in the group treated only with systemic injections of AAV8 that was similar to that of untreated ASMKO mice. The brain sections were also analyzed histologically with respect to the expression of hASM, and with respect to storage of sphingomyelin and cholesterol in situ. The expression pattern of hASM and the storage elimination of sphingomyelin overlapped in the group treated only with cerebral injections of AAV2. In contrast, sphingomyelin storage correction extended beyond the injection sites in the animals receiving the combination therapy. This produced both an overlapping pattern and a non-overlapping reversal pattern of the pathology compared to the areas of transduction with the combination therapy. This relationship was also observed with the cholesterol marker known as philippine. In large regions of the brain cholesterol storage was eliminated in the combination group, whereas only a local cholesterol elimination was observed and more limited in the group treated only with cerebral injections of AAV2. Therefore, the ability of hASM to diffuse from injection sites to correct storage pathology in distal regions of the brain was significantly better in mice treated by combination injections compared to mice that received only brain injection. As can be expected from the biochemical data, the group treated only with systemic injections of AAV8 did not show any measurable correction of the storage of sphingomyelin or cholesterol in the brain. Beginning at 10 weeks of age, the mice were tested every two weeks for motor function in the acceleration and rocking rotarods. The animals of the combination group showed significant improvements in motor behavior in the two rotational tests at all time points examined (p <0.001). Mice treated only with cerebral injections of AAV2 showed a modest improvement in motor behavior in the accelerating rotarod at the first time points compared to untreated ASMKO mice. However, their behavior worsened at later time points demonstrating that injections in the brain alone were not sufficient to sustain correction in motor function. With the rolling rotarod, which is a more rigorous test of motor function and coordination, the group treated only with cerebral injections of AAV2 behaved poorly throughout the study. The group treated only with systemic injections of AAV8 showed a beneficial effect from small to null in the rotarod assay. Beginning at 17 weeks of age, the mice were also tested for cognitive function every 4 weeks in the Barnes labyrinth. The mice used memory-mediated spatial shift keys to escape from a labyrinth under the adverse light stimulus. The group treated with only AAV2 brain injections performed better than the untreated ASMKO mice, but never reached the level of behavior observed in the wild-type mice. Mice treated by systemic injections only showed a marginal improvement compared to untreated controls. In contrast, the combination group behaved in the Barnes labyrinth with a skill that was similar to that of the wild-type mice (p >; 0.05). These data considered together with the data of the rotarod, indicate that to obtain the highest functional result it was necessary to correct the pathology both in the brain and in the viscera. The mice were weighed every two weeks to assess their general fitness and their survival was analyzed by a Kaplan-Meier chart. The combination group had a weight gain profile that was similar to that of the wild type animals, which was significantly better than that of the group treated only with cerebral injections of AAV2 and the group treated only with systemic injections of AAV8 (p < 0.001). Moribund animals, defined as animals with severe ataxia, inability to walk in a straight line without wobbling, inability to groom themselves, loss of 20% of body weight and dehydration, were sacrificed for humanitarian purposes. The combination groups, brain injection of AAV2 alone and systemic injection of AAV8 only had a higher survival compared to untreated ASMKO mice, which had an average life span of 34 weeks. However, all ASMKO mice treated by combination therapy survived at 54 weeks of age and showed no signs of ataxia. This was a significant improvement over the groups treated only with AAV2 brain injections and with systemic injections of AAV8, in which all the animals finally reached a moribund state with average life durations of 48 and 47 weeks (p < 0.0001). None of the animals in the single injection groups survived at 54 weeks. Thus, although the treatment of only the brain or the viscera provided a significant beneficial effect on survival, it was less effective than the treatment of the two body compartments with the combination therapy. The descriptive memory is understood in more detail in light of the teachings of the references cited therein. The embodiments within the specification provide an illustration of embodiments of the invention and should not be considered as limiting the scope of the invention. The person skilled in the art will readily recognize that the invention includes many other embodiments. All publications, patents and biological sequences cited in this description are incorporated by reference in their entirety. If the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will prevail over said material. The citation of any reference herein is not an admission that such references are prior art for the present invention. Unless otherwise indicated, all numbers expressing amounts of ingredients, cell culture, treatment conditions and the like used in the specification, including the claims, should be considered modified in all cases by the term "approximately". Accordingly, unless otherwise indicated, the numerical parameters are approximations and may vary depending on the desired properties that are intended to be achieved by means of the present invention. Unless otherwise indicated, the expression "at least" before a series of elements should be considered to refer to each element of the series. Those skilled in the art will recognize or may find out using only routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents should be considered included in the following claims.

Claims (24)

1. A method comprising the steps of: a) administering an effective amount of a viral vector comprising a transgene encoding an immunogen to the liver tissue of a mammal; and b) subsequently administering an effective amount of a second viral vector comprising a transgene encoding an immunogen to the brain of said mammal.

2. The method of claim 1, wherein said second vector is administered after the expression of the transgene in said mammal is detected.

3. The method of claim 1, wherein the transgene encodes a protein or polypeptide of a lysosomal storage disorder.

4. The method of claim 3, wherein the protein or polypeptide is an acid sphingomyelinase protein or polypeptide.

5. The method of claim 1, wherein the mammal is a human being.

6. The method of claim 1, wherein the administration to the brain of the mammal is at a site selected from the group consisting of the brainstem, the midbrain, the hippocampus, the striatum, the medulla, the bridge, the mesencephalon, the cerebellum, the thalamus, the hypothalamus, the cerebral cortex, the occipital lobe, the temporal lobe, the parietal lobe and the frontal lobe.

7. The method of claim 1, wherein the administration to the brain of the mammal is in the deep cerebellar nuclei of the cerebellum.

8. The method of claim 1, wherein the viral vector is an adeno-associated virus (AAV).

9. The method of claim 8, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

10. The method of claim 9, wherein the AAV is a recombinant AAV vector.

11. The method of claim 10, wherein the recombinant AAV vector is selected from the group consisting of the serotype vectors AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 7 and AAV2 / 8.

12. The method of claim 10, wherein the recombinant AAV vector comprises enhancer elements and promoters with liver specificity.

13. The method of claim 1, wherein step b) is repeated.

14. A method for treating Type A Niemann-Pick disease in a mammal, comprising the steps of: a) administering an effective amount of a viral vector comprising a transgene encoding an acid sphingomyelinase protein or polypeptide in liver tissue of the mammal; and b) subsequently administering an effective amount of a second viral vector comprising a transgene encoding an acid sphingomyelinase protein or polypeptide to the brain of said mammal, thereby treating Niemann-Pick Type A disease in the mammal.

15. The method of claim 14, wherein step b) is repeated.

16. The method of claim 14, wherein said second vector is administered after detecting the expression of the transgene in said mammal.

17. The method of claim 14, wherein the mammal is a human being.

18. The method of claim 14, wherein the administration to the brain of the mammal is at a site selected from the group consisting of the brainstem, the midbrain, the hippocampus, the striatum, the medulla, the bridge, the mesencephalon, the cerebellum, the thalamus, the hypothalamus, the cerebral cortex, the occipital lobe, the temporal lobe, the parietal lobe and the frontal lobe.

19. The method of claim 14, wherein the administration to the brain of the mammal is in the deep cerebellar nuclei of the cerebellum.

20. The method of claim 1, wherein the viral vector is an adeno-associated virus (AAV).

21. The method of claim 20, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

22. The method of claim 20, wherein the AAV is a recombinant AAV vector.

23. The method of claim 22, wherein the recombinant AAV vector is selected from the group consisting of the serotype vectors AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 7 and AAV2 / 8.

24. The method of claim 20, wherein the recombinant AAV vector comprises enhancer elements and promoters with liver specificity.