CN101405008A - 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

The gene therapy of A type Niemann-Pick disease

The mutual reference of related application

The application requires the U.S. Provisional Application the 60/771st according to the application in 8 days February in 2006 of the 119th (e) (35U.S.C. § 119 (e)) of united states patent law, No. 628, the U.S. Provisional Application the 60/772nd of application on February 9th, 2006, No. 360 priority, the content of above-mentioned application case is incorporated in the disclosure by reference.

Technical field

The present invention relates to treat the disease that influences brain and internal organs such as the compositions and the method for A type Niemann-Pick disease.

Summary of the invention

One group is called lysosomal storage disease (lysosomal storage diseases, metabolic disease LSD) comprises and surpass 40 kinds of genetic diseasess, wherein many genetic defectes that relate to different lysosomal hydrolases.Table 1 has been listed representational lysosomal storage disease and relevant defective enzyme.

Table 1

Lysosomal storage disease	The defective enzyme
		Aspartylglucosaminuria	Aspartylglycosaminidase
Fabry	The A-galactosidase A
		The infancy stage batten disease *(CNL1)	Palmitin acyl protein thioesterase
Typical case baby later stage batten disease *(CNL2)	Three peptidyl peptidases
		Juvenile batten disease *(CNL3)	The lysosome transmembrane protein
Batten disease, other forms *(CNL4-CNL8)	The several genes product
		Cystinosis	The cysteine transporter
FaberShi disease	Acid ceramidase
		Fucosidosis	Acid alpha-L-fucosidase
The Galactosidosialidosis syndrome	Protective protein/cathepsin A

Lysosomal storage disease	The defective enzyme
		1、2 *、3 *The type Gaucher disease	Acid, perhaps glucocerebrosidase
G M1Gangliosidosis *	Acid beta galactosidase
		HH *	Iduronic acid-2-sulfatase
Hurler-Scheie compound *	A-L-idose glycosides enzyme
		Krabbe *	Galactocerebrosidase
α-mannosidosis *	Acid alpha-Mannosidase
		β-mannosidosis *	Acid beta-Mannosidase
The Maroteaux-Lamy syndrome	ARB
		Metachromatic leukodystrophy *	Aryl sulfatase A
Morquio A syndrome	N-acetylgalactosamine-6-sulfate sulfatase
		Morquio B syndrome	Acid beta galactosidase
Mucolipidosis II/III *	N-acetylglucosamine-1-phosphotransferase
		Niemann-Pick disease A type *, Type B	ASM
Niemann-Pick disease C type *	NPC-1
		Pompe disease *	Acid alpha-glucosidase
Sandhoff's disease *	B-hexosaminidase B
		Sanfilippo's syndrome B A *	Heparan N-sulfatase
Sanfilippo's syndrome B B *	The A-N-acetylaminoglucosidase
		Sanfilippo's syndrome B C *	S-acetyl-coenzyme-A: alpha-amido glucoside N-acetyl-transferase
Sanfilippo's syndrome B D *	N-acetylglucosamine-6-sulfate phosphate
		The Schindler disease *	The A-N-acetylgalactosaminidase
The Schindler-Kanzaki disease	The A-N-acetylgalactosaminidase
		Sialidosis	The neural hydramine enzyme of A-
The Sly disease *	The B-glucuronidase
		The Tay-Sachs disease *.	The B-hexosaminidase A

Lysosomal storage disease	The defective enzyme
		Fertile ear mann's syndrome *	Acid lipase enzyme

^*Influence the central nervous system

The symbolic characteristic of LSD is the abnormal accumulation of metabolite in the lysosome, and it causes forming a large amount of expansible lysosomes in perikaryon.Main difficulty (with respect to the treatment of liver specificity enzymophathy) for the LSD treatment is to reverse the lysosomal storage disease change in a plurality of independent tissues.Some LSD can effectively treat by the enzyme of venoclysis disappearance, be called algucerase (enzyme replacement therapy, ERT).For example, only there is the dagger-axe of splanchnopathy to thank to 1 type patient to reorganization glucocerebrosidase (Cerezyme

Genzyme Corp.) ERT has sound response.Yet the patient's (for example 2 types or 3 type Gaucher diseases) who suffers from the metabolic disease that influences CNS also not exclusively responds to intravenous ERT, because alternative enzyme is stoped by blood brain barrier (BBB) and can not enter brain.In addition, attempting will to substitute enzyme by direct injection albumen in early days, to introduce the behavior part of brain limited, and this is because diffusibility limited (Partridge, Peptide Drug Delivery to the Brain in the toxicity of high local concentrations enzyme and the brain essence, Raven Press, 1991).

In addition, may produce the antibody of the enzyme of the infusion that uses in the antienzyme alternative medicine.This antibody may not have clinical meaning, may cause allergy yet or reduce the proteic bioavailability of treatment.Hunley，T.E.et al.(2004)Pediatrics 114(4)：e532-e535。For example, Kakkis E.et al. (2004) PNAS 101 (3): 829-834 is reported in mucopolysaccharidosis I type (mucopolysaccharidosis I, the MPS I) canine model and has observed the detrimental effect of antibody to the lysosomal storage disease algucerase.Comprise that in the MPS disease they have also reported similar result in other animal models of MPS I, MPS VI and MPS VII.It may be desirable reducing the immunne response that these albumen cause.The inducing antigen-specific tolerance is the possible method that reduces such immunne response, still it is reported to be difficult to realize.

Gene therapy is the disease that incipient treatment influences CNS, comprises the therapeutic modality of LSD.People have obtained with gene therapy treatment A type Niemann-Pick disease (Neimann-Pick Type A, result likely NPA) in the animal model of approval.Dodge et al.(2005)PNAS 102(49)：18722-17827。NPA is by ASM (acidsphingomyelinase, ASM) lysosomal storage disease that causes of active defective.The active disappearance of ASM causes lysosome sphingomyelins (sphingomyelin, SPM) accumulation, the metabolic deficiency of secondary such as unusual cholesterol metabolism, and then cause comprising central nervous system (central nervoussystem, the cell function disappearance of tract CNS).Schuchman and Desnick, TheMetabolic and Molecular Bases of Inherited Disease, McGraw-Hill, NewYork, pp.3589-3610 and Horinouchi et al. (1995) Nat.Genet.10:288-293.

The invention provides the method that comprises the steps: give mammiferous hepatic tissue with the genetically modified viral vector of coding immunogen that contains of effective dose, genetically modified second viral vector of coding immunogen that contains with effective dose gives mammiferous brain then.

The present invention also provides the method for treatment mammal A type Niemann-Pick disease, comprise the steps: to give mammiferous hepatic tissue with the viral vector that contains coding ASM polypeptide transgenic of effective dose, second viral vector that contains coding ASM polypeptide transgenic with effective dose gives described mammiferous brain then, treats mammiferous A type Niemann-Pick disease thus.

The present invention also provides and makes the method and composition of mammiferous brain to the tolerance of previously selected immunogen, and its this immunogen of at first delivering effective dose by the transgenic general gives mammiferous central nervous system (CNS) with this immunogen of effective dose then and realizes.

The present invention also provides and makes the method and composition of mammiferous brain to ASM polypeptide tolerance, and it gives described mammiferous central nervous system (CNS) with the transgenic of this polypeptide of coding of effective dose then by transgenic from this polypeptide of coding of effective dose to this mammiferous liver that at first deliver and realizes.

The present invention also provides and improves the related indication method and composition of A type Niemann-Pick disease (NPA) in the mammal suffer from NPA, and it is realized by the transgenic that uses coding ASM polypeptide this transgenic of re-using effective dose behind this mammiferous liver described mammiferous cerebral tissue of transduceing of transduceing.

Other advantages of the present invention are stated part that part can be understood by this description by following explanation, maybe can learn by implementing the present invention.

Description of drawings

Fig. 1: illustrate to central nervous system of mice and give genetically modified different loci.

Fig. 2: illustrate the body weight of measuring of being treated mice as health.Measure since 6 ages in week (handle since the around).During 6 to 36 weeks (general inject this transgenic after 2 to 32 weeks) all groups be untreated the ASMKO mice relatively.Caption: ^*P＜0.05; ^*P＜0.01; ^{* *}P＜0.001; Ns (not remarkable).

Fig. 3: illustrate result as the acceleration roller of the measuring test (Accelerating Rotarod test) of motor function recovery.Measurement begins ages (handle since the around) in 10 weeks.During 10 to 36 weeks (general inject this transgenic after 6 to 32 weeks) all groups be untreated the ASMKO mice relatively.Caption: ^*P＜0.05; ^*P＜0.01; ^{* *}P＜0.001; Ns (not remarkable).

Fig. 4: illustrate the result who waves roller test (rockingrotarod test) who measures as motor function recovery.Measurement begins ages (handle since the around) in 10 weeks.During 10 to 36 weeks (general inject this transgenic after 6 to 32 weeks) all groups be untreated the ASMKO mice relatively.Caption: ^*P＜0.05; ^*P＜0.01; ^{* *}P＜0.001; Ns (not remarkable).

Fig. 5: illustrate the result that (BarnesMaze test) tested in the Barnes labyrinth of recovering to measure as cognitive function.Measurement begins ages (handle since the around) in 17 weeks.During 17 to 33 weeks (general inject this transgenic after 13 to 29 weeks) all groups be untreated the ASMKO mice relatively.Caption: ^*P＜0.05; ^*P＜0.01; ^{* *}P＜0.001; Ns (not remarkable).

Fig. 6: illustrate the life-span that the ASM combined gene therapy has prolonged the ASMKO mice.ASMKO mouse life median was 34 weeks.Accepting the genetically modified mouse life median of general is 45 weeks (p＜0.0001).The median length of life of accepting the genetically modified mice of intracranial is 43 weeks (p＜0.0001).100% accept intracranial and the genetically modified mouse life of general was 54 weeks.

Fig. 7 A to Fig. 7 D: illustrate handle and the circulation of untreated mice in anti-hASM antibody horizontal.Caption: ^*P＜0.05; ^*P＜0.01; ^{* *}P＜0.001.Fig. 7 E has shown people ASM protein level in the serum in time.

Fig. 8 A to Fig. 8 F: illustrate sphingomyelins level in processing and the untreated mice brain.

Fig. 9 A to Fig. 9 D: illustrate sphingomyelins level in processing and the various internal organs of untreated mice.

The specific embodiment

Run through the disclosure, the patent specification of many publications, patent and announcement is by quoting for your guidance of determining.The content that patent specification disclosed of these publications, patent and announcement is incorporated the disclosure by reference into, more fully to disclose the situation in field involved in the present invention.

Definition

Except as otherwise noted, enforcement of the present invention will be used the routine techniques of immunology, molecular biology, microbiology, cytobiology and recombinant DNA, and these all belong to the technology of this area.Referring to, Sambrook for example, Fritsch and Maniatis, Molecular Cloning:ALaboratory Manual, the 2nd edition (1989); Current Protocols In MolecularBiology (F.M.Ausubel et al.eds., (1987)); Methods In Enzymology series (Academic Press, Inc.): Pcr 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 CELLCULTURE (R.I.Freshney ed. (1987)).

Some terms used herein are just like undefined meaning.As used in description and the claim, unless context clearly indicates, singulative " ", " a kind of " and " described " comprise plural scope.For example, term " a kind of cell " comprises various kinds of cell, comprises its mixture.

Term " polynucleotide " and " oligonucleotide " use interchangeably, are meant the polymerized form of the nucleotide (deoxyribonucleotide or ribonucleotide or its analog) of any length.Polynucleotide can have any three dimensional structure and carry out any function, comprise known or unknown.Following is the indefiniteness example of polynucleotide: isolation of RNA, nucleic probe and the primer of gene or genetic fragment (for example, probe, primer, EST or SAGE label), exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombination of polynucleotide, side chain polynucleotide, plasmid, carrier, the DNA isolation of any sequence, any sequence.Polynucleotide can comprise modified nucleotide, as methylated nucleotide and nucleotide analog.To the modification of nucleotide structure, if any, can before or after the polymer assembling, give.Nucleotide sequence can be interrupted by the non-nucleotide composition.Polynucleotide can be after polymerization be further modified, as by engaging with marked member.This term also refers to two strands and single chain molecule.Except as otherwise noted or in case of necessity, the present invention comprises each bar of two complementary single stranded form of this double chain form of formation of double chain form and known or prediction for any embodiment of polynucleotide.

Polynucleotide are made up of the particular sequence of four kinds of nucleotide bases: adenine (A); Cytosine (C); Guanine (G); Thymus pyrimidine (T); With uracil (U) when polynucleotide are RNA at guanine.Therefore, term " polynucleotide sequence " is that form represented in the letter of polynucleotide molecule.This alphabetical representative form can be input among the data base of the computer with central processing unit and be used for bioinformatics and use as functional genomics and homology search.

" gene " is meant the polynucleotide of can encode after comprising at least one transcribes and translate specific polypeptide or proteinic opening code-reading frame (ORF).Any polynucleotide sequence described herein can be used to identify the bigger fragment or the complete encoding sequence of its related gene.Separating more, the method for larger sequence fragment is that ability is known to the skilled.

" gene outcome " or in other words " gene expression product " be meant the aminoacid (for example peptide or polypeptide) that produces in genetic transcription and translation back.

Term " polypeptide " and term " protein " use interchangeably, are meant the chemical compound of two or more subunit aminoacid, amino acid analogue or plan peptide (peptidomimetics) on its most important significance.Subunit can connect by peptide bond.In the another one embodiment, subunit can connect by other keys, for example ester bond, ehter bond etc.Term used herein " aminoacid " is meant natural and/or non-natural or synthetic aminoacid, comprises glycine and D and L type optical isomer, amino acid analogue and plan peptide.Have three or more amino acid whose peptides,, be commonly referred to oligopeptide if peptide chain is short.If the peptide chain length is commonly referred to polypeptide or protein.

" transcribing under the control " is term well known in the art, is meant polynucleotide sequence, and normally transcribing of DNA sequence depends on it and be operably connected to the element that transcription initiation is worked or promotes to transcribe." be operably connected " is finger element with the arrangement that allows its performance function put together (juxtaposition).

As used herein, term " comprises " and refers to that compositions and method comprise described composition, but do not get rid of other compositions.When being used for definitions section compound and method, term " substantially by ... form " refer to get rid of other components that combination had any basic importance.Therefore, as herein defined, the compositions of being made up of component will not got rid of trace contaminant and pharmaceutically acceptable carrier such as phosphate buffered saline (PBS), the antiseptic etc. that derive from separation and purification process substantially." by ... form " mean and get rid of other composition that surpasses trace constituent and the substantial method step that is used to give compositions of the present invention.The embodiment of using the definition of these transitional term also within the scope of the invention.

Term " isolating " meaning for cell or other components separate, wherein polynucleotide, peptide, polypeptide, protein, antibody or its one or more fragments (fragment (s)) are attached thereto under native state usually.In one aspect of the invention, isolating polynucleotide from its under natural or primal environment, usually link to each other 3 ' with 5 ' continuous nucleotide separate, for example on chromosome.It will be apparent to one skilled in the art that polynucleotide, peptide, polypeptide, protein, antibody or its one or more fragments that non-natural exists do not need " separation " to come so that it is distinguished from its naturally occurring homologue.In addition, the homologue difference naturally occurring with it of " spissated ", " isolated (separated) " or " dilution " polynucleotide, peptide, polypeptide, protein, antibody or its one or more fragments is that the molecule number of concentration or every volume is greater than " spissated " or less than the concentration of " dispersive " its naturally occurring homologue or the molecule number of every volume.With naturally occurring homologue primary sequence or for example on glycosylation pattern distinguishing polynucleotide, peptide, polypeptide, protein, antibody or its one or more fragments not necessarily exist with its isolating form, because the difference of they and naturally occurring homologue be primary sequence or, alternative, be other features, as glycosylation pattern.Therefore, the polynucleotide that non-natural exists separate with isolating naturally occurring polynucleotide, provide as (separate) embodiment independently.The protein that produces in the bacterial cell separates with isolating naturally occurring protein from the proteinic eukaryotic cell of natural generation, provides as embodiment independently.

" gene delivery (gene delivery) " used herein, " gene transfer " etc. are meant exogenous polynucleotide (are sometimes referred to as that " transgenic ") imports in the host cell, no matter and use which kind of introduction method.Introduction method comprises the multiple technology of knowing, as carrier mediated gene transfer (by, perhaps many other gene delivery complex of viral infection/transfection for example) and the technology (delivering and the multiple other technologies that are used to import polynucleotide) that promotes to deliver " naked " polynucleotide as electroporation, " particle gun " based on protein or lipid.The polynucleotide that import can stably or instantaneously be kept in host cell.Stably keep the polynucleotide that typically need this importing or comprise the replication initiation compatible or be incorporated in the replicon of host cell, as extrachromosomal replication (for example plasmid) or nuclear or m-chromosome with host cell.Many carriers of can mediated gene transferring in the mammalian cell known in the art.

" gene delivery carrier (gene delivery vehicle) " is defined as the molecule of any polynucleotide that can carry insertion in the host cell.The example of gene delivery carrier is a liposome, and biocompatible polymer comprises natural polymer and synthetic polymer; Lipoprotein; Polypeptide; Polysaccharide; Lipopolysaccharide; Artificial viral envelope; Recombinant yeast cell, metallic particles; With antibacterial or virus, as baculovirus, adenovirus and retrovirus, phage, coemid, plasmid, fungus carrier and other typical recombinant vectors used in the art, it is expressed in multiple eucaryon and prokaryotic hosts and is described, can be used for gene therapy and simple protein expression.

" viral vector " is defined as virus or the virion that comprises the reorganization generation that will be delivered to the polynucleotide in the host cell, (ex vivo's) or external (in vitro) body interior (in vivo), that exsomatize.The example of viral vector comprises retroviral vector, adenovirus vector, adeno-associated virus vector, Alphavirus carrier etc.The Alphavirus carrier as based on the carrier of Semliki Forest virus (Semliki Forest virus) with based on the carrier of sindbis alphavirus (Sindbis virus), also is developed and is used for gene therapy and immunization therapy.See Schlesinger andDubensky (1999) Curr.Opin.Biotechnol.5:434-439 and Ying et al. (1999) Nat.Med.5 (7): 823-827.Aspect shifting by the retroviral vector mediated gene, vector construction body (construct) is meant the polynucleotide that comprise reverse transcription virus gene group or its part and therapeutic gene." gene transfer of retrovirus-mediated method " used herein or " retrovirus transduction " have the identical meaning, thereby are meant by virus and enter cell and make gene or nucleotide sequence stably transfer to process in the host cell to the host cell gene group its genome conformity.Thereby virus can enter cell or is attached to different host cell surface receptors or part to enter cell by modifying by its normal infection mechanism." retroviral vector " used herein is meant and can enters machine-processed virion with the exogenous nucleic acid transfered cell by virus or similar virus.

Passing through dna viral vector, as the aspect of adenovirus (Ad) or the transfer of adeno associated virus (AAV) mediated gene, the vector construction body is meant and comprises viral genome or its part and genetically modified polynucleotide.(adenoviruses is one group of homology virus that relative detailed description is arranged Ads) to adenovirus, comprises the serotype more than 50 kinds.Referring to for example WO 95/27071.Adenovirus is easy to growth and does not need to be incorporated in the host genome.Also made up the carrier in recombinant adenovirus source, particularly those carriers that reduced the reorganization probability and produced wild-type virus.Referring to for example WO 95/00655 and WO 95/11984.Wild type AAV has high infectivity and the specificity that is incorporated in the host cell gene group.Referring to Hermonat and Muzyczka (1984) Proc.Natl.Acad.Sci.USA 81:6466-6470 and Lebkowski et al. (1988) Mol.Cell.Biol.8:3988-3996.Reorganization AAV carrier also is known in the art.Referring to for example WO 01/36620A2.

Comprise promoter and can be well known in the art the carrier of the exercisable cloning site that is connected into of polynucleotide.Such carrier can be external or body in transcribe rna and can obtaining by commercial sources, its source as Stratagene (La Jolla, CA) with Promega Biotech (Madison, WI).Express and/or in vitro transcription the best for making, 5 ' and/or 3 ' the untranslated part that may need to remove, add or change the clone is extra to get rid of, potential inappropriate alternative translation initiation codon or other can disturb or reduce the sequence of expression, no matter is at transcriptional level or in translation skill.Alternately, can and then insert conforming ribosome binding site behind start codon 5 ' expresses to improve.

The gene delivery carrier also comprises some non-virus carriers, comprises DNA/ liposome complex, recombinant yeast cell and targeting virus protein-dna complex.Comprise that also directed antibody or its segmental liposome can be used in the method for the present invention.For improving cytotropic delivery, nucleic acid molecules of the present invention or protein can be in conjunction with (conjugate) to antibody or its one or more binding fragments (binding fragment (s)) in conjunction with the cell surface antigen of for example TCR, CD3 or CD4.

When term " genome particle (gp) " or " genome equivalent (genome equivalents) " use in the place relevant with virus titer, be meant and contain the reorganization genomic virion of AAV DNA (virion) number, no matter and its infectivity or functional.Genome particle number in the specific support preparation can by as herein among the embodiment, or for example among Clark et al. (1999) the Hum.Gene Ther.10:1031-1039 and the method for describing among Veldwijket al. (2002) Mol.Ther.6:272-278 measure.

When term " infectious unit (iu) ", " infectious particles " or " replicator " are used in the place relevant with virus titer, be meant and pass through infectious centre assay, be also referred to as the duplication centre and measure measured infectious vector granule number, this mensuration is as for example describing in McLaughlin et al. (1988) J.Virol.62:1963-1973.

When term " transduced unit (tu) " uses in the place relevant with virus titer, be meant cause that the function transgene product produces as by the measured infectious vector granule number of functional examination, this is measured as in Xiao et al. (1997) Exp.Neurobiol.144:113-124 or description in Fisher et al. (1996) J.Virol.70:520-532 (LFU mensuration).

Term " treatment ", " treatment effective dose " and related term thereof are meant and cause preventing or postpone experimenter's morbidity or improve experimenter's symptom or the amount of the chemical compound of the biology effect of acquisition expectation, this biology effect such as neuro pathology correct, for example, the cytopathology relevant with lysosomal storage disease is as herein or described in Walkley (1998) the Brain Pathol 8:175-193.Term " treatment correct " refers to produce prevention or delays to fall ill or alleviate the correction degree of patient's symptom.Effective dose can be by method well known in the art with as determining of describing in the following part.

" compositions " means active agent and another kind inert (for example detectable reagent or labelling) or active as the chemical compound of adjuvant or the combination of compositions.

" pharmaceutical composition " means the combination that comprises active agent and inert or active carrier so that said composition is suitable for is external, in the body or the diagnosis or the therapeutic use that exsomatize.

As used herein, term " pharmaceutically acceptable carrier " comprises the pharmaceutical carrier of any standard, for example phosphate buffered saline (PBS), water and Emulsion, and as oil/water or water/oil emulsion, and various types of wetting agent.Compositions also can comprise stabilizing agent and antiseptic.Example for carrier, stabilizing agent and adjuvant; Referring to Martin, Remington ' s Pharm.Sci., 15th Ed. (MackPubl.Co., Easton (1975)).

" effective dose " is for being enough to reach the amount of useful or required result as prevention or treatment.Effective dose can by once or more times administration, application or dosage give.

" experimenter ", " individuality " or " patient " are used interchangeably at this paper, are meant vertebrates, preferred mammal, more preferably people.Mammal includes but not limited to Mus, ape, people, farm-animals, sports animal and house pet.

" contrast " is other experimenter or the sample that uses for correlated purpose in experiment.Contrast can be " positive " or " feminine gender ".For example, when the purpose of experiment during for the expression of determining gene alteration and disease association, general preferred use positive control (have above-mentioned change and show the experimenter of this genius morbi symptom or the sample that from this experimenter, obtains) and negative control (lacking the expression of change and the experimenter of this disease clinical symptoms or the sample that from this experimenter, obtains).

Gene therapy by viral vector gives albumen can infect neuron behind the mitosis.Viral vector is used for seeing Davidson et al. (2003) NatureRev.4:353-364 to the summary of CNS delivery gene.Adeno associated virus (AAV) is useful in the CNS gene therapy, because they are nontoxic substantially, non-immunogenicity, have neurotropism and can keep expression (Kaplitt et al. (1994) Nat.Genet.8:148-154 in CNS; Bartlett et al. (1998) Hum.Gene Ther.9:1181-1186; With Passini et al. (2002) J.Neurosci., 22:6437-6446).So the place proves, transgenic is expressed the immunne response that causes this transgene product in animal brain.See Fig. 7 A.

The applicant finds that giving this transgenic to the mammal CNS that transgenic is tolerated has strengthened curative effect.Therefore, the invention provides on the one hand the method for mammiferous brain to previously selected immunogen tolerance that make, this method is by giving this mammal with this previously selected immunogen general of effective dose before giving described previously selected immunogen to this mammiferous CNS.As used herein, term " makes ... tolerance " and means reduction to immunogenic immunne response.Term " immunogen " should comprise any reagent that causes immunne response (T cell or B are cell-mediated) for the first time.Confirm that as people such as Ziegler (2004) after the genetically modified reorganization AAV carrier under the liver specificity enhancers/promoters control of encoding, this genetically modified expression causes the genetically modified antibody response of this expression is reduced in liver.

This method is applicable to any mammal, and therefore, described " mammal " includes but not limited to Mus, ape, people, farm-animals, sports animal and house pet.One special aspect, described mammal for the approval the A type and the ASKMO mice of Type B Niemann-Pick disease animal model.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.

A type Niemann-Pick disease (NPA) ranges lysosomal storage disease, and unusual for genetic nerve metabolism, feature is genetic ASM defective (ASM, sphingomyelins phosphocholine hydrolytic enzyme, EC 3.1.3.12).The proteic shortage of functional ASM causes the accumulation of sphingomyelins substrate in whole brain neuroblastoma unit and the neuroglial lysosome.This causes a large amount of lysosomal formation of expanding in the perikaryon, is the symbolic characteristic of A type NPD and main cell phenotype.The lysosomal existence of expanding is relevant with progressive neurodegenerative process with losing of normal cell function, it causes affected individuality at early childhood death (The Metabolic and MolecularBases of Inherited Diseases, eds.Scriver et al., McGraw-Hill, New York, 2001, pp.3589-3610).The high level accumulation of secondary cell phenotype (for example other Developmental and Metabolic Disorder) cholesterol during also disease association therewith, particularly lysosome are separated.Sphingomyelins has strong affinity to cholesterol, and it causes separating of cholesterol a large amount of in ASMKO mice and the human patients lysosome.Leventhal et al. (2001) J.Biol.Chem., 276:44976-44983; Slotte (1997) Subcell.Biochem.28:277-293; With Viana et al. (1990) J.Med.Genet.27:499-504.

One concrete aspect, the site of general administration is mammiferous liver.Can use any medication, its example provides below.

After general gives transgenic, transgenic is administered to mammiferous CNS, particularly intracranial directly is administered to mammiferous brain, more particularly gives the site and is selected from brain stem, Hippocampus, striatum, oblongata, pons, midbtain vesicle (mesencephalon), cerebellum, midbrain, thalamus, hypothalamus, cerebral cortex, occipital lobe, temporal lobe, top or frontal lobe.In one embodiment, the special cerebellum deep nuclei that gives to cerebellum.

As mentioned above, give coded polypeptide or proteic transgenic to mammal, deliver this polypeptide or albumen with any suitable gene transfer method of final use, its example as mentioned above, and as United States Patent (USP) 6,066,626 is described.In one aspect, this transgenes encoding ASM.The genome of people ASM and functional cDNA sequence announce, for example at United States Patent (USP) 5,773, in 278 and 6,541,218).Identified that in table 1 other relate to the suitable protein immunogen of CNS.

Viral vector is useful gene transfer vector.In a special embodiment, viral vector is selected from adenovirus, adeno associated virus (AAV), cowpox, herpesvirus, baculovirus or retrovirus.

Suitable AAV carrier is at United States Patent (USP) 6,066,626 and PCT announce during WO 01/36620A2 in full open.The carrier of modifying as the 9th hurdle, is described in the 14th to 66 row, also can use.Suitable serotype includes but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.The AAV carrier can be reorganization or hybridization AAV carrier, for example a kind of AAV2/1, AAV2/5, AAV2/7, AAV2/8, AAV1/2, AAV1/3, AAV1/5, AAV1/7 or AAV1/8 of being selected from, the serotype carrier, wherein this name is meant the serotype in the serotype/capsid source in ITR source.For example, the AAV2/5 carrier comprises AAV2 serotype ITR and AAV5 serotype capsid.

By comprising the genetically modified viral vector that produces polypeptide, deliver this polypeptide of effective dose to mammal.In one aspect, comprise genetically modified viral vector and be delivered to liver, after being delivered to liver, will comprising described genetically modified viral vector immediately and be delivered to CNS.

In an alternate embodiment, thereby a back viral vector that gives CNS has been expressed effective time at this polypeptide this mammal is carried out after described polypeptide is produced tolerance in mammal.This can change with the patient who is treated, the polypeptide of being delivered and desired curative effect.Reasonable time process and the number of times that gives to CNS in the back are determined by the doctor in charge.For whether determining mammal to the polypeptide tolerance, this mammal of available described polypeptide counteracting toxic substances (challenge) determines whether above-mentioned counteracting toxic substances produces the immunne response of anti-this polypeptide.Immunne response can be determined by the antibody titer of measuring anti-this polypeptide behind counteracting toxic substances.When comparing with suitable contrast, antibody titer that reduce behind the counteracting toxic substances or inapparent may show tolerance status.Measuring and produce antibody response in mammal, is well known in the art with the method for antigen or immunogen counteracting toxic substances mammal and definite antibody titer.This mammal produces the appropriate time of tolerance and also can select by determining the effective expression time that produces described tolerance in the experimenter.The selected then time can be applicable to this method, and needn't test each patient respectively.

In one embodiment, the back one viral vector to CNS give be delivered to liver the viral vector encoded polypeptides detection of expression to after carry out.The detection of expression of polypeptides can be finished by any methods known in the art, the example of this method includes but not limited to (by detecting protein expression) of molecule (by detecting mRNA) or immunity or biochemical (by detecting polypeptide active, as enzymatic activity, if there are so active words).

Alternatively, once giving after can be after a few hours or a couple of days or a few weeks longer that give for the first time.Repeatedly CNS be administered to many CNS site purposes also within the scope of the invention.

Thereby, of the present invention one concrete aspect, the method for mammal brain to ASM polypeptide tolerance that make is provided.This method gave the transgenic of this polypeptide of coding of effective dose to this mammiferous liver before the transgenic of the coded polypeptide that gives effective dose to mammalian central nervous system tissue (CNS).

Of the present invention another special aspect, provide and improve the related indication method of mammiferous A type Niemann-Pick disease (NPA) suffer from NPA.This method need give the transgenic of the coding ASM polypeptide of effective dose to this mammiferous central nervous system (CNS), wherein said giving after this mammiferous hepatic tissue general gives this transgenic of effective dose, thereby before described CNS gave, mammal produced ability to this polypeptide antigen specific immune response and is eliminated or has significantly reduced.

On the other hand, the invention provides the related indication method of mammiferous A type Niemann-Pick disease (NPA) that improvement suffers from NPA.Described symptom includes but not limited to lose weight or cachexia, motor function forfeiture, cognitive function forfeiture and premature dead.This method need give the genetically modified viral vector of acidic sphingomyelinase enzyme polypeptide of encoding that comprises of effective dose to mammiferous hepatic tissue, gives this genetically modified viral vector that comprises of effective dose then to mammiferous CNS.In another embodiment, the CNS zone of delivering described viral vector is brain.

Be still in another aspect, the invention provides treatment and suffer from the method for the mammiferous A type Niemann-Pick disease (NPA) of NPA, this method is by giving the genetically modified AAV viral vector that comprises coding ASM polypeptide of effective dose to mammiferous hepatic tissue, deliver the genetically modified AAV carrier that comprises coding ASM polypeptide of effective dose then to this mammiferous CNS, thus this mammiferous NPA of treatment.In another embodiment, the CNS zone of delivering described viral vector is brain.

The meanings such as term used herein " treatment (treating) ", " treatment (treatment) " are for obtaining required treatment, pharmacological and/or physiological effect.Described effect can be a preventive effect from prevent disease or its symptom or symptom wholly or in part, and/or the illeffects from partially or completely treating disease and/or being caused by this disease, can be the effect of treatment.

" treatment " also covers the processing to any mammal disease, comprising: ill disease is tended in prevention but N determines to suffer from the generation of experimenter's disease of this disease, for example, and to the patient of the hereditary label tests positive of disease; Suppress disease, promptly stop its progress; Or alleviate or improve disease, and for example causing disappearing of disease, this disease is the NPA disease for example.

" treatment " used herein further comprises the symptom that the general improvement is relevant with pathological changes and/or delays the morbidity (onset) of symptom.Clinical and the subclinical evidence of " treatment " changes with pathological changes, individuality and treatment.

In some embodiments, this method comprises and carries previously selected immunogen or genetically modified AAV carrier, thereby transgene product is expressed with treatment level in selected site.In some embodiments, the virus titer of compositions at least: (a) 1.5,2.0,2.25,2.5,2.75,3.0,3.25,3.5,4.0,4.55,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (per injection * 10 ¹¹Genome copy (gc); (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 (* 10 ⁹Tu/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 (* 10 ¹⁰Infectious unit [iu]/ml).

The intracranial administration can comprise a plurality of districts in any district of brain when giving more than an intracranial delivery.Such site comprises, for example brain stem (oblongata and pons), midbtain vesicle, midbrain, cerebellum (comprising the cerebellum deep nuclei), diencephalon (thalamus, hypothalamus), akrencephalon (striatum, midbrain, cerebral cortex, or in the cortex pillow, temporo, top or frontal lobe).The object lesson in intracranial injection site is shown in Fig. 1.

For the evaluation of human brain structure, referring to for example The Human Brain:Surface, Three-Dimensional Sectional Anatomy With MRI, and Blood Supply, 2nded., eds.Deuteron et al., Springer Vela, 1999; Atlas of the Human Brain, eds.Mai et al., Academic Press; 1997; With Co-Planar Sterotaxic Atlas of theHuman Brain:3-Dimensional Proportional System:An Approach toCerebral Imaging, eds.Tamarack et al., Thyme Medical Pub., 1988.For the evaluation of Mus brain structure, referring to for example The Mouse Brain in Sterotaxic Coordinates, 2nded., Academic Press, 2000.If need, the analog structure of human brain structure and other mammiferous brains can be connected.For example, most of mammals, comprise the mankind and Rodents, the organizational structure (topographicalorganization) that has shown the projection of similar entorhinal area-hippocampus, have be projected to the hippocampus dorsal part or in every the entorhinal cortex outside and the neuron in the inboard, and main (the Principles of Neural Science that originates from of the neuron from the entorhinal cortex medial part of the projection neuron of the hippocampus veutro that arrives, 4th ed., eds Kandel et al., McGraw-Hill, 1991; The Rat Nervous System, 2nd ed., ed.Paxinos, Academic Press, 1995).In addition, the second layer cell in the entorhinal cortex is projected to dentate gyrus, ends at outer 2/3rds places of dentate gyrus molecular layer.The aixs cylinder both sides that derive from the 3rd confluent monolayer cells are projected to the CA1 and the CA3 cornu ammonis zone of hippocampus, end at porous layer (stratumlacunose) molecular layer.

Be the given zone of the given zone, particularly brain that carrier specificity are delivered to the central nervous system, can be by three-dimensional location microinjection administration (sterotaxic microinjection).For example, performed the operation the same day, installed patient's stereotaxic frame (being screwed into skull).Use high-resolution nuclear magnetic resonance, NMR to having the brain imaging of stereotaxic frame (the credible label that MRI is complementary).Nuclear magnetic resonance image is sent to the computer of the three-dimensional positioning software of operation.Use a series of crown, sagittals and axial image to determine the target position and the track of vector injection.This software can directly convert track to the three dimensional form that is complementary with stereotaxic frame.Boring (burrhole) above entry site, stereotaxic instrument is located with the pin that is implanted in set depth.Then, be infused in carrier in the pharmaceutically acceptable carrier.Then, the AAV carrier just is transported to the tip target site by being injected directly into that main target site gives and driving in the wrong direction by aixs cylinder.Also may use extra route of administration, the cortex top layer administering mode under for example can be directly visual, or other non-three-dimensional position application.

The total amount of the material that will give and the carrier granular number that will give are determined according to the known aspect of gene therapy by those skilled in the art.The effectiveness and the safety of treatment can be tested in appropriate animal model.For example, the various existing LSD animal models that are described in detail, for example as described herein or at Watson et al. (2001) Methods Mol.Med., 76:383-403; Or Jeyakumar et al. (2002) Neuropath.Appl.Neurobiol., 28:343-357; Or Metabolic and Molecular Bases of Inherited Disease, 8thedition (2001), the middle description that McGraw-Hill publishes.

In experiment mice, the cumulative volume of the AAV solution of being injected is, for example between each injection site 1 to 5 μ l or about 3 μ l, or alternatively between 10 to 400 μ l, or alternatively between 100 to 400 μ l, or alternatively between 150 to 250 μ l, or alternatively about 200 μ l.For other mammals, comprise human brain, volume and delivery speed are suitably proportional.For example, proved the volume of 150 μ l can inject safely the brain of primate (Janson et al. (2002) Hum.Gene Ther., 13:1391-1412).Treatment can be made up of each target site single injection, also can repeat along the injection road, if necessary.Can use a plurality of injection target position.For example, in some embodiments, except first administration target position, comprise another target position that the compositions of carrying genetically modified AAV can be administered into first target position offside or homonymy.

In the method for the invention, the AAV of any serotype can use.In one embodiment of the invention, can use the AAV carrier of the aixs cylinder transportation of can driving in the wrong direction.The serotype of viral vector can be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8 (referring to for example Gao et al. (2002) PNAS, 99:11854-11859; With ViralVectors for Gene Therapy:Methods and Protocols, ed.Machida, HumanaPress, 2003).Except list in herein, other serotypes also can be used.Also can use pseudo-type AAV carrier (Pseudotyped AAV vectors), it is those ITR's that comprise a kind of AAV serotype in the capsid of second AAV serotype; For example, contain the genomic AAV viral vector of AAV2 capsid and AAV1 or contain the AAV5 capsid and the genomic AAV carrier of AAV2.(Auricchio et al.(2001)Hum.Mol.Genet，10(26)：3075-81。)

The AAV carrier derives from that (summary is seen Muzyscka (1992) Curr.Top.Microb.Immunol., 158:97-129) to the nonpathogenic strand of mammal (ss) DNA parvovirus (DNA parvoviruse).In brief, to account for the rep and the removal of cap viral gene of viral genome 96% based on the carrier of AAV, stay that the flank of two 145 base pairs (bp) is terminal to repeat oppositely that (inverted terminal repeat, ITR), it is used for initial viral dna replication, packing and integration.When non-auxiliary virus, wild type AAV is integrated at chromosome 19q 13.3 to have in the specific human host cell's genome of preferred sites, or can keep expressing with episomal form.Single AAV granule can be packed into and be reached the ssDNA of 5kb, thereby stays about 4.5kb to transgenic and regulating element, and this generally is enough.Yet for example United States Patent (USP) 6,544, and the trans-splicing system described in No. 785 (trans-splicing system) can almost make this boundary double.

In an exemplary, AAV is AAV2 or AAV8.The adeno associated virus of many serotypes as AAV2, is widely studied and describes as Vectors in Gene Therapy.Those skilled in the art are familiar with the preparation based on the functioning gene treatment carrier of AAV.In the document of having delivered widely, can find numerous relevant administration of human experimenter AAV production, purification and preparation distinct methods list of references (referring to, Viral Vectors for GeneTherapy:Methods and Protocols for example, ed.Machida, Humana Press, 2003).In addition, United States Patent (USP) 6,180,613 and 6,503,888 to have described with the CNS cell be the gene therapy based on AAV of target.

Eukaryotic cell transfer expression of gene level can be regulated by the transcripting promoter in this transgene expression cassette and/or one or more enhancer.Tissue-specific promoter as the liver specificity promoter, can be used in some embodiments.Tissue-specific enhancer as the liver specificity enhancer, can be used in some embodiments.Tissue-specific promoter and tissue-specific enhancer as liver specificity promoter and enhancer, can unite and be used for enforcement of the present invention.

The non-limitative example of promoter includes but not limited to cytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat.Genet.8:148-154), CMV/ people β 3 globin promoteres (Mandel et al. (1998) J.Neurosci.18:4271-4284), GFAP promoter (Xuet al. (2001) Gene Ther, 8:1323-1332), 1.8-kb neuronspecific enolase (NSE) promoter (Klein et al. (1998) Exp.Neurol.150:183-194), chicken β actin (CBA) promoter (Miyazaki (1989) Gene 79:269-277) and beta-Glucuronidase (GUSB) promoter (Shipley et al. (1991) Genetics 10:1009-1018), human serum albumin's promoter, α-1-antitrypsin promoter.Express for improving, other regulating elements can be operably connected to this transgenic extraly, for example alpine marmot hepatitis virus is regulated and control back element (the Woodchuck Hepatitis Virus Post-Regulatory Element, WPRE) (Donello et al. (1998) J.Virol.72:5085-5092) or bovine growth hormone (BGH) polyadenylation site.

Be fit to other promoteres of the present invention and can be the strong constitutive promoter that any DNA sequences encoding that can promote to be correlated with is expressed in liver.Described strong constitutive promoter comprises CMV promoter, human serum albumin's promoter [HSA] and the α-1-antitrypsin promoter of people and murine cytomegalovirus promoter, truncate.

To the useful liver specificity enhancer element of the present invention can be any liver specificity enhancer that can strengthen relevant DNA sequences encoding tissue specific expression in liver.Described liver specificity enhancer comprises one or more human serum albumin's enhancers (HSA), the former enhancer of human thrombin (HPrT), α-1 microglobulin enhancer (A1MB) and intron aldolase enhancer.Can unite a plurality of enhancer elements for obtaining more high expressed.For example, two identical enhancers can with the coupling of liver specificity promoter.

The viral vector that comprises following promoter/enhancer associating can be used for enforcement of the present invention: one or more HSA enhancer associating CMV promoter or HSA promoter; One or more is selected from the enhancer of former (HprT) enhancer of human thrombin or α-1 microglobulin A1MB enhancer) associating CMV promoter; And one or more is selected from the enhancer element associating α-1-antitrypsin promoter of HprT enhancer or A1MB enhancer.

Published PCT application WO 01/36620 discloses other information about the liver specificity construct.Alternately, neuronal specificity promoter and/or enhancer are useful for directed delivery and express transgenic in CNS.

In some respects, the control transcriptional activity is desirable.For this reason, can use the pharmacy of the gene expression of AAV carrier to regulate, as for example at Habermaet al. (1998) Gene Ther., 5:1604-16011 by comprising different regulating elements and drug responses promoter; With describe among Yeet al. (1995) the Science 283:88-91.

The AAV preparation can be by technology production known in the art, as for example at United States Patent (USP) 5,658,776 and Viral Vectors for Gene Therapy:Methods and Protocols, ed.Machida, Humana Press describes in 2003.

In some aspects, detection and/or this genetically modified expression may be desirable.The method that detects gene expression is known in the art, can simply use by following discussion, or be made amendment by those skilled in the art.

The method of known a lot of quantitative required gene expressions includes but not limited to that hybridization detects the hybridization detection of (Northern engram analysis) and PCR-based.

In the change that detects the mRNA level, at first from the sample that contains this nucleic acid, extract this nucleic acid according to the standard method of this area.For example, mRNA can use according to people's such as above-mentioned Sambrook (1989) different lyases or chemical solution separates or extract according to the respective description book that manufacturer provides by the nucleic acid binding resin.

The nucleic acid molecules that contains at least 10 nucleotide and show with complementation of expression product sequence or homology can be used as hybridization probe or PCR primer.Specific hybrid known in the art does not need the probe of " coupling fully ".Do not influence the hybridization specificity by replacement, the deletion of small number base or the little change of inserting to probe sequence.In general, can tolerate and reach 20% base-pair mismatch (when optimum is compared).For example, be used to detect the probe of mRNA and be included in the sequence of identifying in the past, for example the homology region of the comparable size in the ASM sequence is identical at least about 80%.Alternately, the corresponding gene sequences at least 85% after the comparison of this probe and homology region or even at least 90% identical.Segmental total size and the complementation district size of being extended depend on the intended use or the application of this specific nucleic acid section.Gene generally be used for hybridizing embodiment than small fragment, the variable-length in wherein complementary district, as about 10 and about 100 nucleotide between, or even reach total length according to the complementary series that detects of want.

There is the nucleic probe of complementary series will increase the stability and the selectivity of crossbred with extension fragment (stretch), thereby improves the specificity of the specific cross molecule that is obtained greater than about 10 length of nucleotides.Can design and have, even preferably more than about 50 length of nucleotides more than about 25, or if desired even the segmental nucleic acid molecules of longer gene complementation.Can be by directly synthesizing this fragment, by the PCR that two initiation oligonucleotide are arranged that use nucleic acid replication technology such as United States Patent (USP) 4,603,102 to describe as chemical means ^TMTechnology, or easily prepare described fragment by the method that sequence that will be selected is introduced the recombinant vector that is used for recombinant production.

In some embodiments, use the suitable method of nucleotide sequence of the present invention associating to detect hybridization and complementary series is favourable as label.Multiple suitable indicating means known in the art comprises fluorescence, radioactive, enzyme or other part, and as avidin/biotin, it can provide detectable signal.Also can use fluorescence labels or enzyme label, as urase, alkali phosphatase or peroxidase, rather than radioactivity or other are unfavorable for the reagent of environment.Under the situation of using the enzyme label, can be used to provide human eye mode visible or spectrophotography to identify that with the colorimetric indicator substrate that contains the sample specific hybrid of complementary nucleic acid be known.

Hybridization can carry out under the condition of different " preciseness ".Relevant condition comprises the existence and the washing process of other solutes in temperature, ionic strength, temperature retention time, the reactant mixture such as Methanamide.Higher rigorous condition is meant that it need hybridize the higher minimum complementarity of interelement to form stable hybridization complex as higher temperature with than the condition of Cardia Salt concentration.The condition that increases the hybridization preciseness is well known in the art and publishes.See above-mentioned Sambrook, wait people (1989).

Also can use quantitative PCR or high throughput analysis such as serial analysis of gene expression (SAGE) to detect and quantitative mRNA or its expression,, describe among V.et al. (1995) the Science 270:484-487 as at Velculescu.In brief, this method comprises the multiple mRNA of cell or tissue sample separation that contains this transcript from suspection.Alternatively, this gene transcripts can be exchanged into cDNA.Sequence-specific analysis and quantitative is carried out in the sampling of gene transcripts.These gene transcripts sequence abundance are compared with the reference database sequence abundance that comprises patient and healthy person normal data group.

Use the methods known in the art probe also can be attached to and be used for the solid support that high flux screening detects.For example, international pct application WO 97/10365 and U.S. Patent number 5,405,783,5,412,087 and 5,445,934 have disclosed and can contain the structure that one or more plant the high density oligonucleotide chip of sequence.This chip can use United States Patent (USP) 5,405, and the method that discloses in 783,5,412,087 and 5,445,934 is synthetic at the glass surface of derivatization.Light protection nucleoside phosphoramidites can be coupled to glass surface, carries out selectivity by photoetching with the photodissociation of mask and goes protection, and react with second shielded nucleoside phosphoramidites.Repeat coupling and join/go the protection process until finishing required probe.

The expression of gene level can be exposed to by the sample that suspection is contained these polynucleotide on the chip of probe modification to be determined.The nucleic acid that extracts is by for example fluorescence labels, preferably labelling in amplification step.The hybridization of the sample of labelling is carried out under suitable rigorous level.The degree of probe-nucleic acid hybridization is used checkout gear such as Laser Scanning Confocal Microscope quantitative assay.See United States Patent (USP) 5,578,832 and 5,631,734.The mensuration that obtains is directly related with gene expression dose.

Hybridization probe and sample nucleic acid can detect by several different methods known in the art.For example, hybrid nucleic acid can detect by one or more label that detection is attached to sample nucleic acid.This label can be incorporated into by any mode well known by persons skilled in the art.In one aspect, this label is incorporated in the amplification step of preparation sample nucleic acid simultaneously.Thereby for example the polymerase chain reaction of the nucleotide of the primer of usage flag or labelling (PCR) will provide the amplified production of labelling.In an independent embodiment, the aforesaid transcription amplification of the nucleotide of usage flag (for example, fluorescein-labeled UTP and/or CTP) is incorporated label into the nucleic acid of being transcribed.

Alternately, label can directly add in the primary sample nucleic acid (for example mRNA, polyA, mRNA, cDNA etc.) or add in the amplified production after amplification is finished.The mode that label is attached to nucleic acid is known to those skilled in the art, for example comprises (connection) is connected sample nucleic acid with label (for example fluorophor) otch translation method or terminal label (for example RNA of the usage flag) method of adhering to by phosphorylation (kinasing) reaction of nucleic acid and subsequently nucleic acid joint.

Be suitable for detectable label of the present invention and comprise any compositions that can detect by spectrographic, photochemical, biochemical, immunochemical, electric, light or chemical means.Useful label comprises the painted biotin of streptavidin conjugate that is used for labelling, magnetic bead (Dynabeads for example among the present invention ^TM), fluorescent dye (for example fluorescein, texas Red, rhodamine, green fluorescent protein and so on), radioactive labels (for example ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P), enzyme (for example enzyme of using always among horseradish peroxidase, alkali phosphatase and other ELISA) and colorimetric label such as gold colloidal or coloured glass or plastics (for example polystyrene, polypropylene, latex etc.) pearl.Instruction uses the patent of above-mentioned label to comprise United States Patent (USP) 3,817,837,3,850,752,3,939,350,3,996,345,4,277,437,4,275,149 and 4,366,241.

The method that detects above-mentioned label is well known by persons skilled in the art.Therefore, for example radioactive labels can detect by using photographic film or scintillation counter, and fluorescent labeling can be used photodetector to detect emission light and detect.Typically by substrate being provided for this enzyme and detecting enzyme the product that this substrate-function produces is detected, the colorimetric label manifests detection by making this coloured label simply to the enzyme label.

Patent is announced the method that WO 97/10365 discloses before hybridization or alternately label added to one or more kind target (sample) nucleic acid after hybridization.These are the detectable labels that directly adhere to or incorporate into target (sample) nucleic acid before hybridization.On the contrary, " indirect labels " is attached in the heteroduplex after hybridization.Indirect labels usually is attached to the bound fraction that has been attached on the target nucleic acid before hybridization.Therefore, for example target nucleic acid can be before hybridization biotinylation.After the hybridization, the bonded fluorogen of avidin will be attached to the heteroduplex that has biotin the label that is easy to detect will be provided.Detailed summary about the method for the hybrid nucleic acid molecule of marker nucleic acid molecule and certification mark, see Laboratory Techniques in Biochemistry and MolecularBiology, Vol.24:Hybridization with Nucleic Acid Probes, P.Tijssen, ed.Elsevier, N.Y. (1993).

Thereby before hybridizing to high-density probe array, can use the method modification of nucleic acids sample disclosed in the international pct application WO 97/10365 to reduce background signal and improve the sensitivity of measuring to reduce the sample complexity.

The result of the chip detection software program analysis that typically uses a computer.Referring to for example EP0717 113 A2 and WO 95/20681.The hybridization data read-in programme, this program is calculated the target gene expression level.This numeral is compared with existing ill and gene expression dose data set healthy individual.Between the data of the data that obtain and one group of diseased individuals the relevant experimenter of showing fall ill.

Also can revise existing immunoassay detects and quantitative expression.The definite of gene outcome need measure the bonded amount of immunologic opsonin that takes place between this gene outcome reactive antibody.Signal for producing in detection and quantitative immunologic opsonin combination or hybridization or the amplification procedure can use the digital image analysis system that includes but not limited to detector probe radioactivity or chemiluminescent groups.

The expression of polypeptide product also can be used for the biochemistry means detection known in the art of expressed specific polypeptide by use.

Following embodiment provides exemplary of the present invention.One of skill in the art will recognize that enforceable countless modifications and the version that does not change the spirit or scope of the present invention.These are modified and version is covered by in the scope of the present invention.Embodiment does not limit the present invention in any way.

Embodiment

The titration of recombinant vector

AAV carrier titre can be measured according to genome copy number (every milliliter genome particle).As former report, genome particle concentration is with the Taqman of carrier DNA

PCR is basis (Clark et al. (1999) Hum.Gene Ther.10:1031-1039; Veldwijk et al. (2002) Mol.Ther.6:272-278).In brief, the AAV carrier may the accurate any contaminating dna measured of viral interference DNA to remove with the DNAse solution-treated.The AAV carrier digests buffer (50mM Tris-HCl pH 8.0,1.0mM EDTA, 0.5%SDS, 1.0mg/ml E.C. 3.4.21.64) with capsid and handles 1 hour with release vehicle DNA at 50 ℃ then.The DNA sample use with carrier DNA in specific sequence such as the annealed primer of promoter region, transgenic or polyadenylic acid (polyA) sequence carry out polymerase chain reaction (PCR).PCR result is by as Perkin Elmer-Applied Biosystems (Foster City, CA) the real-time Taqman that provides of Prism 7700 sequence detection systems (Sequence Detector System) then

Software is quantitative.

The carrier that carries as the marker gene measured of beta galactosidase or green fluorescence protein gene (GFP) can come titration by infectious measure (infectivity assay).Permissive cell (for example HeLa or COS cell) is with AAV transduction and measure determining gene expression, as with the cell of X-gal (5-bromo-4 chloro-3-indole-β-D-galactopyranosides) dyeing beta galactosidase carrier transduction or for the cell fluorescence microscope of the GFP that transduces.For example, this mensuration such as following carrying out: 4 * 10 ⁴The HeLa cell is laid on each hole in the 24-well culture plate that uses the standard growth culture medium.After adherent, after promptly about 24 hours, cell usefulness Ad 5 infects with 10 infection multiplicity (MOI), with the package carrier transduction of serial dilution and 37 ℃ of cultivations.After one to three day, before observing widely cytopathic effect, pair cell carries out suitable mensuration (for example X-gal dyeing or fluorescence microscope).If used reporter gene such as beta galactosidase, cell fix with 2% paraformaldehyde, 0.5% glutaraldehyde and with X-gal dyeing to measure betagalactosidase activity.Counting produces the carrier dilution of the cell of good separation.Each positive cell is represented 1 transduced unit (tu) of carrier.

Total length people ASM cDNA is cloned in the plasmid that contains from the ITR of AAV serotype 2 and 8.Jin et al.(2002)J Clin Invest.109：1183-1191。AAV8-hASM contains terminal people's ASM (hASM) cDNA that oppositely repeats under the restricted promoter control of (ITRS) and DC-190 liver of serotype 2-.[Ziegler et al.(2004)Mol.Ther.9：231-240]。AAV2-hASM contains terminal people's ASM (hASM) cDNA that oppositely repeats under the control of (ITRS) and cmv enhancer and avian beta-actin promoter of serotype 2-.Two recombinant vectors all produce by three plasmid co-transfection people, 293 cells and pass through column purification.The final titre of AAV8-hASM and AAV2-hASM preparation is 5.0 * 10 ¹²Genome copy (gc) every ml, determined as the TaqMan PCR of the bovine growth hormone polyadenylation signal sequence that all contains by each carrier.

Hybrid vector can produce by using a series of helper plasmid that also contains serotype specificity capsid coding structure territory except containing AAV serotype replicator to carry out triple transfections.This strategy allows AAV ITR carrier package to go in the virion of each serotype specificity.Rabinowitz，et al.(2002)J Virol.76：791-801。HASM recombination group can be used for producing a series of pseudo-type rAAV-hASM carriers by this method.Reorganization AAV carrier can pass through the ion-exchange chromatography purification.O′Riordan，et al.(2000)J Gene Med 2：444-54。Reorganization AAV carrier also can pass through CsCl centrifugal purification Rabinowitz et al. (2002) J.Urrol.76:791-801.The final titre of AAV-ASM Virosome particles (anti-DNAse granule) can be determined by the TaqMan PCR of CMV sequence.Clark et al.(1999)Hum.Gene Therapy10：1031-1039。

The combined gene therapy of ASMKO mice midbrain and general

ASM deficient mice (ASMKO), K.Horinouchi et al, Nat Genet.10 (1995), pp.288-292, carry out following processing:

Group 1) mice contains the genetically modified AAV2/8 carrier of two HPrT enhancers, human serum albumin's promoter and people ASM in 4 ages in week by tail vein injection 3 e11 gc;

Group 2) mice contains the genetically modified 2 e11 gc of enhancer, promoter and people ASM in 6 ages in week by the seam of the injection of brain between 8 sites in brain (split) injection;

Group 3) mice 4 ages in week by tail vein injection 3 e11 gc contain the genetically modified AAV2/8 carrier of two HPrT enhancers, human serum albumin's promoter and people ASM, two week the back same mouse contain the genetically modified AAV2 carrier of enhancer, promoter and people ASM by the seam of the injection of brain between 8 sites in brain injection 2 e11 gc; And

Group 4) mice does not accept to inject or accept only to have the vacation injection of carrier.

Group 5) non-ASMKO mice or wild-type mice are not accepted injection.

To all 8 district every site injection 3 μ l AAV2-hASM (1.5 e10 gc) that carry out the ASMKO mouse brain of three-dimensional positioning surgical, total amount is every brain 24 μ l (1.2 e11 gc).The injection site of right hemisphere be hypothalamus (0.50 ,-1.00mm ,-3.50mm), Hippocampus (2.00mm ,-1.75mm ,-1.75mm), oblongata (6.00mm ,-1.50mm ,-3.75mm) and cerebellum (6.00mm ,-1.50mm ,-2.25mm); The site of left hemisphere injection be striatum (0.50mm, 1.75mm ,-2.75mm), motor cortex (0.50mm, 1.75mm ,-1.25mm), midbrain (4.50mm, 1.00mm ,-3.50mm) and cerebellum (6.00mm, 1.50mm ,-2.25mm).Injection Hamilton syringe (Hamilton USA, Reno NV) carry out with the speed of 0.5 μ l/min, after the per injection pin stayed original position 2 minutes so that when extracting pin virus solution the upper reaches minimum.

All untreated ASMKO mices and the ASMKO mice that only carries out injection of brain AAV2 (AAV2brain-) and only carry out general injection of AAV 8 (AAV 8 systemic-alone) group finally all are in dying state.On the contrary, the ASMKO mice of handling by brain and whole body joint injection does not reach dying state, but for doing still execution when 54 weeks of relative analysis.Animal is extensively poured into to remove whole blood and to be divided into biochemistry group (cohort) and histology's group (cohort).In the biochemistry group, liver, lung, spleen and skeletal muscle are cut into two; A slice is used to analyze the hASM level, and another part is used to analyze sphingomyelins and stores up.In brain, the two cerebral hemispheres separates, and each hemisphere further is cut into the sheet of five 2mm along the A-P axle.The sheet of left hemisphere carries out hASM albumen and anti-hASM analyzes.

ASM (ASM) serum levels and anti-ASM serum antibody level are all measured in whole research.In whole research process mice is carried out multiple test: roller test and the test of Barnes labyrinth are tested, waved to body weight evaluation, acceleration roller.In whole experiment, also collected the survival rate curve data.Behind the dead mouse, the sphingomyelins level in brain, liver, lung, spleen and the muscular tissue of each mice is measured by collection organization.People ASM protein expression is also by using the SABC qualitative evaluation in the brain regulating liver-QI.Research stopped in 54 weeks; (the joint injection group, combo) mice all survives when research stops and is healthy all groups 3.

The ASM serum levels is undertaken quantitatively by the enzyme-linked immunosorbent assay (ELISA) of using the anti-zymoid polyclonal antibody of this people of specificity.Fig. 7 illustrates serum ASM protein level in time.During dead mouse in the brain regulating liver-QI ASM also measure by SABC.In the brain of group 2 and group 3 mices, observe positive ASM dyeing, in the mice of group 3, observe brighter qualitatively dyeing.In striatum, Hippocampus, midbrain and cerebellum, observe dyeing.In the brain of group 1 mice or the ASMKO mice of being untreated, do not observe ASM dyeing.In the liver of group 1 and group 3 mices, observe positive ASM dyeing.In the liver of group 2 mices or the ASMKO mice of being untreated, do not observe ASM dyeing.

The horizontal quantitative approach of tissue sphingomylelin is as follows: by at chloroform: homogenate 10 to 50mg tissue preparation tissue extracts and hatched 1 hour at 37 ℃ in the methanol (1: 2).Behind centrifugal removal cell debris, use water extraction homogenate twice, organic facies (containing this lipid) is transferred in the clean glass tubing then 37 ℃ of logical nitrogen dryings of heating.Sphingomyelins amount in the extract is measured indirectly by using Amplex Red sphingomyelinase to measure test kit (Molecular Probes).(MO USA) handles in Amplex Red work station the outer derived bacterium sphingomyelinase from Bacillus cereus of extract usefulness fixed amount for Sigma-Aldrich, St.Louis.Sphingomyelins by the enzyme hydrolysis of antibacterial to produce ceramide and Phosphorylcholine.The latter further is hydrolyzed into choline, and choline oxidation successively produces betanin and hydrogen peroxide.The hydrogen peroxide that discharges is by reacting to produce coming quantitatively by the resorufin that 590nm place fluorescent emission detects of high fluorescence with Amplex Red.The sphingomyelins level is about the 5-10% of observed value in the ASMKO mice of Mus coupling in age in the normal C57BL/6 mouse tissue.Fig. 8 and Fig. 9 illustrate sphingomyelins level in brain and the internal organs.

Anti-people's ASM specific antibody level is measured by ELISA in the serum.See Fig. 7 E.Serial dilution serum is joined in the hole that is coated with enzyme or particulate 96 orifice plates of hot deactivation AAV.(CA USA) detects bonded antibody for Zymed, San Francisco by using the bonded sheep anti mouse immunoglobulin G of horseradish peroxidase (IgG), IgM and IgA.Plate and substrate (Sigma-Aldrich) together incubated at room temperature 20 minutes with colour developing.Titre is defined as and produces the inverse that OD450 is equal to or less than 0.1 highly diluted serum.Fig. 7 A to 7D illustrate handle and the circulation of untreated mice in anti-hASM antibody horizontal.

Antibody in the brain essence is measured by the algoscopy of following modification.Organize lysate dilution in 1: 20 in antibody dilution buffer, join in duplicate in 96 orifice plates that are coated with 100ng hASM.The combination of second antibody HRP and chromogenic substrate reaction are undertaken by preceding method.The color intensity that bonded hASM specific antibody concentration should be reacted with the HRP from conjugate is directly related.Therefore, the final result absolute change that is reported as the OD450 that 1: 20 cracking diluent produces with provide with serum in the titrimetry used compare more responsive antibody horizontal and measure.

Use is analyzed brain section hASM with the anti-hASM biotinylation monoclonal antibody of dilution in 1: 200 and is expressed, with the bonded second antibody of streptavidin-Cy3-under red fluorescence, develop [Passini, M.A.et al. (2005) .Mol.Ther.11:754-762].The cholesterol substrate detects by using filipin (filipin) dyeing scheme in the brain, as report [Passini, M.A.et al. (2005) .Mol.Ther.11:754-762].In brief, (Sigma, St Louis MO) are dissolved in 100% methanol working concentration to 10mg/ml to filipin.Brain section is hatched 3h under the room temperature (RT) darkling, washs three times with PBS under the RT then, checks under blue-fluorescence.Carry out Lysenin dyeing determining the former bit pattern of sphingomyelins substrate, as report [Shihabuddin, L.S.et al. (2004) .J.Neurosci.24:10642-10651].In brief, (Peptides International, Louisville Kentucky) are dissolved among the PBS that contains 0.5%BSA, 0.02% saponin (Sigma) and 5% standard donkey serum to 10mg/ml lysenin.Brain section and lysenin be 4 ℃ of overnight incubation, then the anti-lysenin antibody of rabbit (Peptides International) of night incubation dilution in 1: 250.(Jackson ImmunoResearch, West Grove PA) develop and check with green fluorescence the Lysenin positive cell with the anti-rabbit antibody of FITC of dilution in 1: 250.

Each mice quickens on Smartrod (AccuScan) by the use methods known in the art and waves roller and test motor function, and this method repeats in Sleat et al. (2004) J.Neurosci.24:9117-9126.(AccuScan Instruments, Columbus OH) quicken and wave roller and test and estimate motor function mice by using SmartrodRotorod Program.Quicken speed that the roller upper roller rotates and carried out program setting quickening with constant ratio from 0-30rpm, wave roller and carry out program setting to accelerate to final speed 25rpm in per 2.5 seconds forward backward at 54 seconds at 60 seconds.Every animal carries out four tests on each time point, write down from the time of staying (latency) that platform falls.High time of staying score equals good performance.Single test at least at interval 15min so that animal has the rest period.Fig. 3 and 4 illustrates the result as the roller test of measuring motor function recovery.

Each mice carries out the test of Barnes labyrinth.Mice is trained, made it to find and ensconce the tunnel of love of 20 caves under one of them that is arranged in big flat, plastic dish periphery, vinyl disc illuminates at last Halogen light by four.By the labyrinth with find correct dark cave time and cognitive function negative correlation---the shorter time of passing through shows better cognitive function.Fig. 5 illustrates the result as the Barnes labyrinth test (Barnes Maze test) of measuring the cognitive function recovery.

Survival curve is illustrated in Fig. 6.

With the dysfunction that solves (addressing) ASMKO mice and disease sequela to being that the AAVhASM joint injection scheme of target is estimated with brain and two in internal organs.In uniting group (n=11), age, the ASMKO mice accepted 3.0 * 10 by tail vein injection all around ¹¹The AAV8-hASM of genome copy (gc).After two weeks, in 6 ages in week, same injected in mice AAV2-hASM is to motor cortex, striatum, midbrain and the cerebellum of left hemisphere, and the hypothalamus, Hippocampus, oblongata and the cerebellum that arrive right hemisphere.Each structure injection 1.5 * 10 ¹⁰Gc, the total amount 1.2 * 10 of every brain ¹¹Gc.Handle matched group and only accept general injection of AAV 8-hASM (n=12) or only accept injection of brain AAV2-hASM (n=14) in 6 ages in week in 4 ages in week, the untreated control group comprises ASMKO (n=23) and wild type (n=10) mice.

Carry out eye blood sampling regularly to measure hASM level and anti-hASM antibody horizontal in the circulation.General is injected and the analysis of the ASMKO mice serum that joint injection is handled has shown the highest circulation hASM level to only carrying out.Because the tropism of this virus serotype and the restricted promoter of selecting when the design expression cassette of liver (DC190), AAV8-hASM transduction and expression subsequently are mainly the liver mediation.Two groups of peak level that all reach hASM after injection 2 weeks, it reduces during this research subsequently and reaches 10 times.From being untreated ASMKO and only carry out not showing detectable hASM level in the serum of group of injection of brain AAV2.Further serum analysis shows by associating or only to carry out in the mice that general injection of AAV 8 handles anti-hASM antibody horizontal similar to observed low baseline values in the ASMKO mice of being untreated.On the contrary, the mice that only carries out the group of injection of brain AAV2 shows the inducing of anti-hASM antibody titer of fast strong (200 times of increases).Thereby the mice that general injection of AAV 8-hASM handles be it seems as the hASM that expresses has been produced immunologic tolerance.

Associating or only carry out in Mouse Liver, lung, spleen and the muscle that general injection of AAV 8 handles high-caliber this enzyme being arranged obviously presumably is that this enzyme in circulation is after the receptor-mediated endocytosis of 6-phosphomannose.Only carry out in the internal organs of group of injection of brain AAV2 that observed not comparing significantly improves in the people ASM level and the ASMKO control mice that is untreated.Analysis to the associating and the brain of the group of only carrying out injection of brain AAV2 shows that high-caliber hASM spreads all over axon.Yet, although in two groups, all use the reorganization AAV2 carrier of knowing clearly identical, compare with the group of only carrying out the brain processing, unite group and demonstrate significantly higher hASM level.But only carry out in the mouse brain of general injection treatment the hASM level low and be in can with the level of the ASMKO mice analogy of being untreated.This shows that the hASM that comes from liver in the circulation can not stride across blood brain barrier and enter CNS.What is interesting is that anti-hASM antibody titer comprises that than other groups to unite group high about 10 times in the brain homogenate in the group of only carrying out injection of brain AAV2.Thereby expression is the inverse (reciprocal) of antibody titer in the brain; Unite group and show high-caliber hASM and low-level anti-hASM antibody, and the group of only carrying out injection of brain AAV2 shows low-level hASM and high-caliber anti-hASM antibody horizontal.

Determined that hASM expresses the effect of storing up pathological changes in ASMKO mice internal organs and the brain for correcting.Only carry out general injection of AAV 8 and unite in the viscera tissue of group deriving from of all inspections, corrected sphingomyelins fully and stored up.On the contrary, only accept to contain high-caliber sphingomyelins in the pluck of injection of brain, this and untreated ASMKO mice are similar.Analysis to sphingomyelins level in the ASMKO mouse brain of handling by joint injection shows that this substrate generally is reduced to the wild type level.This is an improvement for the group of only carrying out injection of brain AAV2, and it only demonstrates significant sphingomyelins at the corresponding brain piece of injection site and reduces.Therefore, only carry out injection of brain group the correction scope significantly less than and never reach viewed effect in the associating group.Only carry out in the group of injection of brain AAV2 than the sphingomyelins of poor efficiency store up reduce relevant with the enzyme of observed reduced levels in this group.Observe high-caliber brain sphingomyelins in the group of only carrying out general injection of AAV 8, it is similar to untreated ASMKO.

Brain section is also carried out the histologic analysis that hASM expression and original position sphingomyelins and cholesterol are stored up.The removing pattern that hASM expression pattern and sphingomyelins are stored up overlaps each other in the group of only carrying out injection of brain AAV2.On the contrary, accept the correction that sphingomyelins is stored up in the animal of therapeutic alliance and expand to far away surpassing outside the injection site.This pattern that has caused pathological changes to reverse is compared the existing overlapping non-overlapped pattern that has again with the transduction zone of therapeutic alliance.Use cholesterol labelling filipin also to observe this relation.A large amount of brain districts have removed cholesterol and have stored up in the associating group, and only carry out only observing partial and more limited cholesterol removing in the group of injection of brain AAV2.Therefore, the mice that joint injection is handled is compared with only accepting the mice that injection of brain handles, and hASM stores up ability significance better of pathological changes from the injection site diffusion to correct the brain distal region.The group of only carrying out general injection of AAV 8 does not show the correction that sphingomyelins in any measurable brain or cholesterol are stored up, and this point can predict from above-mentioned biochemical data.

Since 10 ages in week, per two weeks of mice are quickening and are waving and test motor function on the roller.The time point of associating treated animal all inspections in two roller tests all demonstrates the athletic performance (p＜0.001) of remarkable improvement.Only carrying out mice that injection of brain AAV2 handles shows and compares in early days with untreated ASMKO mice that time point quickens to show on the roller athletic performance that moderate is improved.Yet its performance of time point has descended late, and this proof is only carried out injection of brain and is not enough to keep correction to motor function.For the roller that waves of stricter test motor function and harmony, performance is poor from start to finish in whole research for the group of only carrying out injection of brain AAV2.The group of only carrying out general injection of AAV 8 shows few to be benefited (little-to-nobenefit) that does not have in any one test.

Since 17 ages in week, per 4 weeks of mice are also tested cognitive function on the Barnes labyrinth.Mice utilizes the space walk clue of memory mediation to flee from the labyrinth under unfavorable photostimulation.The group of only carrying out injection of brain AAV2 is better than untreated ASMKO performance, but never reaches observed performance level in the wild-type mice.Only carry out the mice of general injection treatment and compare demonstration improvement more or less with untreated contrast.On the contrary, unite group and on the Barnes labyrinth, show the proficiency level similar (p＞0.05) to wild-type mice.Sum up the roller data, the correction of brain and internal organs pathological changes is essential for the maximum function result.

Weigh in per two weeks of mice to estimate its general health situation, its survival rate is by the Kaplan-Meier tracing analysis.It is similar to wild type to unite group weight increase overview, than the group of only carrying out injection of brain AAV2 and general injection of AAV 8 significantly good (p＜0.001).The animal of dying state is defined as the severe ataxia, do not tumble can not carry out straight line moving, can not clean, body weight loss 20% and dehydration, for humanistic purpose is put to death it.With median length of life is that the ASMKO mice of being untreated in 34 weeks is compared, and associating, the group of only carrying out injection of brain AAV2 and only carrying out general injection of AAV 8 show that survival rate increases.Yet all ASMKO mices of handling by joint injection survive to 54 ages in week and do not show ataxic sign.This is significant improvement with only carrying out that injection of brain AAV2 compares with the group of only carrying out general injection of AAV 8, and all animals finally become and are in dying state among the latter, and median length of life is (p＜0.0001) in 48 and 47 weeks.(singly injected group's) does not have animals survived to 54 week in the group of single position injection.Therefore, provide significant survival to be benefited really although only brain or internal organs are handled, than poor to the effect of all handling between above-mentioned body region with therapeutic alliance.

Instruction according to the list of references of quoting in this description can be carried out the understanding of fullest to this description.Embodiment in this description provides the exemplary illustration to embodiment of the present invention, should not be construed as to limit the scope of the invention.The technical staff is easy to recognize that the present invention contains many other embodiments.All publications, patent and the biological sequence quoted in the disclosure are incorporated herein by reference with its integral body.For the material and this explanation contradiction or the inconsistent place that merge by reference, this description will substitute any such material.Any list of references cited herein is not to admit that this list of references belongs to prior art for the present invention.

Except as otherwise noted, this description comprises that the numeral that used all in claims are expressed as component, cell culture, treatment conditions etc. is interpreted as in all cases by word " pact " modification.Correspondingly, unless opposite explanation is arranged in addition, digital parameters is an approximation, the required qualitative change that can will obtain according to the present invention.Except as otherwise noted, the term before a series of compositions " at least " is interpreted as being meant each composition of this series.One skilled in the art will realize that or can determine the equivalents of many specific embodiments of the present invention described herein by being no more than normal experiment.These equivalents are contained by claim.

Claims (24)

1, the method that comprises the steps:

A) give to encode immunogenic genetically modified viral vector to mammiferous hepatic tissue comprising of effective dose; With

B) give subsequently to encode immunogenic genetically modified second viral vector to described mammiferous brain comprising of effective dose.

2, method according to claim 1 wherein detects genetically modified expression and gives described second carrier afterwards in described mammal.

3, method according to claim 1, wherein transgenes encoding lysosomal storage disease albumen or polypeptide.

4, method according to claim 3, wherein albumen or polypeptide are ASM polypeptide or albumen.

5, method according to claim 1, wherein mammal is behaved.

6, method according to claim 1 wherein gives to be selected from brain stem, midbrain, Hippocampus, striatum, oblongata, pons, midbtain vesicle, cerebellum, thalamus, hypothalamus, cerebral cortex, occipital lobe, temporal lobe, top or frontal lobe to the site of mammiferous brain.

7, method according to claim 1, wherein giving to the site of mammiferous brain is the cerebellum deep nuclei of cerebellum.

8, method according to claim 1, wherein viral vector is adeno associated virus (AAV).

9, method according to claim 8, wherein the AAV carrier is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.

10, method according to claim 9, wherein AAV is reorganization AAV carrier.

11, method according to claim 10, the AAV carrier of wherein recombinating are selected from AAV2/1, AAV2/2, AAV2/5, AAV2/7 or AAV2/8 serotype carrier.

12, method according to claim 10, the AAV carrier of wherein recombinating comprises liver specificity enhancer and promoter element.

13, method according to claim 1, wherein repeating step b).

14, the method for treatment mammal A type Niemann-Pick disease, it comprises the steps:

A) give the viral vector that comprises coding ASM polypeptide or protein transgene of effective dose to mammiferous hepatic tissue; With

B) second viral vector that comprises coding ASM polypeptide or protein transgene that gives effective dose is subsequently treated this mammiferous A type Niemann-Pick disease thus to described mammiferous brain.

15, method according to claim 14, wherein repeating step b).

16, method according to claim 14 wherein detects genetically modified expression and gives described second carrier afterwards in described mammal.

17, method according to claim 14, wherein mammal is behaved.

18, method according to claim 14 wherein gives to be selected from brain stem, midbrain, Hippocampus, striatum, oblongata, pons, midbtain vesicle, cerebellum, thalamus, hypothalamus, cerebral cortex, occipital lobe, temporal lobe, top or frontal lobe to the site of mammiferous brain.

19, method according to claim 14, wherein giving to the site of mammiferous brain is the cerebellum deep nuclei of cerebellum.

20, method according to claim 1, wherein viral vector is adeno associated virus (AAV).

21, method according to claim 20, wherein the AAV carrier is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.

22, method according to claim 20, wherein AAV is reorganization AAV carrier.

23, method according to claim 22, the AAV carrier of wherein recombinating are selected from AAV2/1, AAV2/2, AAV2/5, AAV2/7 or AAV2/8 serotype carrier.

24, method according to claim 20, the AAV carrier of wherein recombinating comprises liver specificity enhancer and promoter element.

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