CN101208108A - Gene therapy for spinal disorders - Google Patents
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- CN101208108A CN101208108A CNA200680023365XA CN200680023365A CN101208108A CN 101208108 A CN101208108 A CN 101208108A CN A200680023365X A CNA200680023365X A CN A200680023365XA CN 200680023365 A CN200680023365 A CN 200680023365A CN 101208108 A CN101208108 A CN 101208108A
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Abstract
This disclosure provides methods and compositions for treating disorders or injuries that affect motor function and control in a subject. In one aspect, the invention provides a method to deliver a transgene to a subject's spinal cord by administering a recombinant neurotropic viral vector containing the transgene. The viral vector delivers the transgene to a region of the deep cerebellar nuclei region of the brain. Also provided are compositions and methods to deliver a transgene to a subject's spinal cord by administering a recombinant neurotropic viral vector containing the transgene to the motor cortex region of the subject's brain.
Description
[01] according to United States code 35 volume 119 (e) bar regulation, the application requires the U.S. Provisional Application No.60/677 of application on May 2nd, 2005, the U.S. Provisional Application No.60/790 of application on April 8th, 213 and 2006, the priority of 217 claim, the content of above-mentioned application is incorporated herein by reference.
Technical field
[02] the present invention relates to treatment influences experimenter's motor function, particularly is subjected to the compositions and the Therapeutic Method of the motor function imbalance of brain and/or diseases of spinal cord or damage influence.
Background technology
[03] gene therapy is the treatment pattern that a kind of emerging treatment influences central nervous system's (CNS) deficiency disorder.The CNS gene therapy has been subjected to effectively infecting the promotion of neuronic viral vector development after division stage.The central nervous system is made up of spinal cord and brain.The sensory information of spinal cord conduction from the peripheral nervous system to the brain, and the movable information of conduction from brain to various effectors.Can be about being used for that gene is delivered to the summary of central nervous system's viral vector referring to people such as Davidson, the article of (2003) Nature Rev.4:353-364.
[04] adeno-associated virus (AAV) carrier is considered to useful to the CNS gene therapy, because it has favourable toxicity and immunogenicity characteristic, the neuronal cell of transduceing, and can in CNS, mediate secular expression (people such as Kaplitt, (1994) Nat.Genet.8:148-154; People such as Bartlett, people such as (1998) Hum.Gene Ther.9:1181-1186 and Passini, (2002) J.Neurosci.22:6437-6446).
[05] the AAV carrier useful characteristic depends on some AAV carrier and can stand reverse in neuronal cell and/or the ability of transportation forward.Interconnect and arrive the brain region of far-end by aixs cylinder at the neuron of a brain region, therefore the transmission for carrier provides transportation system.For example, the AAV carrier may or give near the position of neuron axon tip.Neuron is included the AAV carrier in and it is transported with the direction of mode along aixs cylinder to cyton of driving in the wrong direction.Adenovirus, HSV and pseudorabies virus have also demonstrated the similar characteristic that gene is delivered to distal structure in the brain.(people such as Soudas, (2001) FASEB is J.15:2283-2285; People such as Breakefield, people such as (1991) New Biol.3:203-218 and deFalco, (2001) Science, 291:2608-2613).
[06] several groups are reported that the brain transduction of carrying out with AAV serotype 2 (AAV2) is defined in intracranial injection site (people such as Kaplitt, (1994) Nat.Genet.8:148-154; People such as Passini, people such as (2002) J.Neurosci.22:6437-6446 and Chamberlin, (1998) Brain Res.793:169-175).Recent report shows, the reverse aixs cylinder transportation of neurophilic viral vector also can occur in the loop of selected normal mouse brain (people such as Kaspar, (2002) Mol.Ther.5:50-56 (AAV carrier); People such as Kasper, people such as (2003) Science 301:839-842 (slow virus carrier) and Azzouz, (2004) Nature 429:413-417 (slow virus carrier).People such as Roaul, (2005) Nat.Med.11 (4): people such as 423-428 and Ralph, (2005) Nat.Med.11 (4): 429-433 report, the slow virus of intramuscular injection expression silencing people Cu/Zn superoxide dismutase (SOD1) RNA interfering have hindered and (ALS) the disease generation of the ALS of relevant rodent models of treatment amyotrophic lateral sclerosis (amytrophic lateral sclerosis).
[07] by the transgene product of the cell of AAV carrier transduction possibility express therapeutic,, mediates useful born of the same parents' internal effect such as enzyme or neurotrophic factor.These cells also may be secreted these curative transgene products, and these products may be absorbed by the far-end cell subsequently, and it may mediate its useful effect in these far-end cells.This process has been described to intersect to proofread and correct (cross-correction) people such as (, (1970) Science 169:141-146) Neufeld.
[08] yet, treatment causes the unusual compositions of the spinal cord of patient's motion function forfeiture and the demand of method still to exist.The present invention has satisfied this demand and relevant advantage is provided.
Summary of the invention
[09] the invention provides and transmit the method and composition of transgenic, the containing the neurophilic viral vector of genetically modified reorganization and can realize this transmission of at least one zone in deep cerebellar nuclei (DCN) zone by being administered into experimenter's brain to experimenter's spinal cord and/or brain stem zone.Viral is carried out under the situation that transgenic expresses in spinal cord and/or brain stem zone helping.
[010] on the other hand, the invention provides and transmit the method and composition of transgenic, containing the neurophilic viral vector of genetically modified reorganization and can realize this transmission by administration experimenter brain nervus motorius cortex zone to experimenter's spinal cord.Being delivered in of viral vector helps carrying out under the situation that transgenic expresses in spinal cord.The viral vector passive movement neuron that is administered into nervus motorius cortex zone is included in by its cyton zone, and transgenic obtains expressing then.The transgenic of expressing may experience the axon terminal part of anterograde transport to motor neuron then, and this terminal portion branch is present in the spinal cord.Because the character of nervus motorius cortex is administered into this regional viral vector of brain and also may the neuronic axon ends of passive movement includes in.Viral vector also may experience along the antiport of the aixs cylinder of motor neuron and express in the cyton of motor neuron.
[011] further provides and transmit the method and composition of transgenic, the containing the neurophilic viral vector of genetically modified reorganization and can realize this transmission of at least one zone in the cerebellar nuclei zone, deep by administration experimenter brain to experimenter's motor neuron.Being delivered in of carrier helps transgenic and carries out under situation about expressing in the motor neuron that is positioned at administration site far-end.
[012] also provides the method and composition of transmission transgenic to experimenter's motor neuron, containing genetically modified neurophilic viral vector and can realize this transmission by administration experimenter brain nervus motorius cortex zone, and, here, administration is being positioned under the situation about expressing in the motor neuron of administration site far-end and is carrying out helping transgenic.
[013] on the other hand, the invention provides the method and composition of treatment experimenter motor neuron deficiency disorder, the containing the neurophilic viral vector of genetically modified reorganization and can realize this treatment of at least one zone in the cerebellar nuclei zone, deep by administration experimenter brain.Administration is carried out under situation about expressing at least one subregion in spinal cord and/or brain stem zone at the transgenic that helps treating effective dose.
[014] aspect further, the invention provides compositions and the method for improving experimenter's motor neuron deficiency disorder symptom, containing the neurophilic viral vector of the genetically modified reorganization of therapeutic and can realize this improvement effect by administration experimenter brain nervus motorius cortex zone, and administration is carried out under situation about expressing at least one subregion in spinal cord and/or brain stem zone at the transgenic that helps treating effective dose.
[015] should be appreciated that above-mentioned describe, in general terms and detailed description subsequently all only are exemplary and indicative, do not have restricted to the present invention.
Description of drawings
[016] Fig. 1: how diagram DCN is used to transport therapeutic virus to spinal cord.The lines that originate from the black surround of drawing out the DCN profile have been represented the axon ends that originates from cyton (arrow), and these cytons are positioned at spinal cord.
[017] Fig. 2: reproduced the cross section of organizing, shown three zones of DCN by pons oblongata joint and cerebellum.
[018] Fig. 3: block the schematic views of the cerebellum that flattens then along the boundary line (vowing the shape tangent plane) of vermis, and by the horizontal section of spinal cord and the figure of skeletal musculature.It has shown main importing into (input) approach.
[019] Fig. 4: the figure that shows the main efferent pathway (outgoing route) of DCN.
[020] Fig. 5: the sketch map that connects the neural circuit that is input to output in the cerebral cortex.Climbing fiber is initial in inferior olive, and these fibers itself can be accepted from cerebral cortex, spinal cord and special senses (vision and audition) and the input (signal) that comes.MF input is imported into initially from every other, and for example vestibule imports into, spinal column imports into, muscle-spindle, neural chordae tendineae, joint receptor, skin receptor and cerebral cortex.Three kinds of suppressor relay cells are also arranged in built-in system, comprise basket cells, Golgi cells and sternzellen.These all relate to the lateral inhibition and the fine setting of nervus motorius meta function.
[021] Fig. 2 is from people such as Williams to Fig. 5, duplicate among (2005) The Human Brain:Chaper 3:The Cerebellum, from network address:
Www.vh.org/adult/provider/Anatomy/BrainAnatomy/Ch3Text/Section07.html can obtain these contents.
[022] Fig. 6 A is to Fig. 6 E: shown the different AAV serotype carriers [(A) 2/1 at the people ASM that will encode, (B) 2/2, (C) 2/5, (D) 2/7 and (E) 2/8] be expelled to the deep cerebellar nuclei of ASMKO mice after, vow the immune positive staining of the acid lipid sphyngomyelin (" hASM ") of people in the shape cerebellum part.
[023] Fig. 7 A is to 7E: shown acid lipid sphyngomyelin (" the hASM ") protein transport of people from the deep cerebellar nuclei to spinal cord.This result is from using AAV2/2-ASM, AAV2/5-ASM, AAV2/7-ASM﹠amp; AAV2/8-ASM (A) hASM, 10 times of amplifications; (B) hASM, 40 times of amplifications; (C) laser co-focusing hASM; (D) laser co-focusing ChAT; (E) laser co-focusing hASM﹠amp; Observe in the mice that ChAT handles.
[024] Fig. 8: different AAV serotype carriers (2/1,2/2,2/5,2/7 and 2/8) (n=5/ group) the cerebellum tissue homogenate level behind the cerebellar nuclei of deep that illustrates injection coding people ASM.The group that connects with same letter is not significantly different (p＜.0001).
[025] Fig. 9 A is to 9G: shown that different AAV serotype carriers at injection coding people ASM [(A) 2/1, (B) 2/2, (C) 2/5, (D) 2/7 and (E) 2/8] vow the calbindin immunity positive staining of shape cerebellum part behind the deep cerebellar nuclei of ASMKO mice.
[026] Figure 10 A is to 10B: shown the acceleration of the ASMKO mice (n=8/ group) of handling with ASMKO (with AAV-β gal injection), WT and AAV-ASM and waved rotation performance (14 all ages).The group that connects with same letter is not significantly different.In quickening rotation test, demonstrated with the mice of AAV2/1-ASM and AAV2/8-ASM injection that remarkable (p＜.0009) longer delay is fallen the time than the ASMKO mice of injecting with AAV2/1-β gal.For waving rotation test, the mice of injecting with AAV2/1-ASM has demonstrated the mice of comparing with AAV2/1-β gal injection, and significantly (p＜.0001) longer delay is fallen the time.
[027] Figure 11 A and Figure 11 B: shown the rotation performance of the mice (20 age in week) that ASMKO (n=8), WT (n=8) and two-way AAV-ASM (n=5/ group) handle.For quickening and rolling test, the mice that AAV-ASM handles is than the better significantly (p＜.001) of the performance of the mice that ASMKO AAV2/1-β gal handles.Quicken and rolling test in, can not distinguish with wild-type mice with the performance of the mice of AAV2/1-ASM injection and to come.
[028] Figure 12 A: illustrate the connection between cerebellar nuclei zone, deep (inboard, centre and the outside) and spinal cord zone (cervical region, chest, waist and the sacrum portion).Figure 12 B: illustrate the connection between cerebellar nuclei zone, deep (inboard, centre and the outside) and brain stem zone (midbrain, pons and the medullary substance).These connections represent that with arrow these arrows originate in neuronic cyton zone and end in neuronic axon terminal zone.For example, each all has the neuron that has cyton three zones of DCN, and these neurons spread out of aixs cylinder and end in the neck area of spinal cord, simultaneously, the neck area of spinal cord have the inboard that spreads out of aixs cylinder and end in DCN or zone line cyton.
[029] Figure 13: after the DCN that illustrates the AAV of encoding green fluorescent protein (GFP) transmits, the distribution of green fluorescent protein in brain stem or top motor neuron.
[030] Figure 14: after the DCN that illustrates the AAV of encoding green fluorescent protein (GFP) transmitted, green fluorescent protein was in the distribution in spinal cord zone.
[031] Figure 15: illustrate and the DCN of the AAV of the GFP that the encodes result after transmitting relatively, after the DCN of the AAV of coding IGF-1 transmits, the painted minimizing of glial fibrillary acidic protein in the brain stem (GFAP).
[032] Figure 16: illustrate and the DCN of the AAV of the GFP that the encodes result after transmitting makes comparisons, after the DCN of the AAV of coding IGF-1 transmits, the painted minimizing of (nuclei quintus, nucleus of hypoglossal nerve and nucleus of facial nerve) glial fibrillary acidic protein (GFAP) in the MN nuclear.
[033] Figure 17: illustrate and the DCN of the AAV of the GFP that the encodes result after transmitting makes comparisons, after the DCN of the AAV of coding IGF-1 transmits, the painted minimizing of glial fibrillary acidic protein in the whole spinal cord (GFAP).
[034] Figure 18: illustrate and the DCN of the AAV of the GFP that the encodes result after transmitting makes comparisons, after the DCN of the AAV of coding IGF-1 transmits, the distribution of IFG-1mRNA among the central nervous system (CNS).The Beta-actin is as positive control, the level of coming more total mRNA.
[035] Figure 19: the DCN that illustrates AAV-IGF-1 transmits the survival that has promoted motor neuron.The mice of handling with the AAV of coding IGF-1 and DCN with the AAV of coding GFP transmit the mice of handling relatively, and not being both therebetween significant (the p value equals 0.01) on the statistics is as what represent with asterisk.
[036] Figure 20: illustrate and the DCN of the AAV of the GFP that the encodes result after transmitting relatively, transmit the lifting of mice on rotation performance, hind leg grip and forelimb grip of handling with the DCN of the AAV of coding IGF-1.
[037] Figure 21: illustrate and the DCN of the AAV of the GFP that the encodes result after transmitting relatively, the increase of transmitting the survival rate of mediation with the DCN of the AAV of coding IGF-1.
[038] Figure 22: shown the AAV1 carrier bi-directional of expressing GFP behind deep cerebellar nuclei (DCN), GFP is in the distribution of mouse brain.Except DCN, the GFP positive staining is also observed in olfactory bulb, cerebral cortex, thalamus, brain stem, cortex of cerebellum and spinal cord.These all zones are all accepted the projection (projections) from DCN and/or are issued to the projection of DCN.
The specific embodiment
[039], at first defines some term in order to make the present invention easier to understand.Being defined in the whole detailed description of other defines.
[040] except as otherwise noted, enforcement of the present invention will be used the technology of traditional immunology, molecular biology, microbiology, cytobiology and recombinant DNA, and these all belong to the technical ability of this area.Referring to for example, Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:A LABORATORYMANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (people such as F.M.Ausubel compiles, (1987)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2:A PRACTICAL APPROACH (MJ.MacPherson, B.D.Hames and G.R.Taylor compile, (1995)), Harlow and Lane compile, (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R.I.Freshney compiles, (1987)).
[041] as used in description and claim, singulative " a " " an " and " the " comprise the plural number relation, unless context clearly shows.For example, term " cell " comprises a lot of cells, comprises its mixture.
[042] term used herein " comprises " and is meant that compositions and method comprise the key element of having stated, but does not get rid of other key element.When being used for definitions section compound and method " basically by ... form " will mean that what get rid of any other is the implication of basic significant composition for combination.Like this, basically by the elementary composition compositions of definition in this article will can not get rid of from separate and purification process and contaminant trace species and pharmaceutically acceptable carrier, for example phosphate buffered saline, antiseptic, or the like." by ... form " will mean the composition that surpasses trace of getting rid of other compositions and the basic method steps that is used for administration compositions of the present invention.By the defined embodiment of each term of these transitional term all within the scope of the present invention.
[043] all appointments with numeral (comprising scope), for example pH, temperature, time, concentration and molecular weight all are the approximations with the change of (+) or (-) 0.1 increment.Although should be appreciated that always not clear and definite statement equal term " approximately " in addition before all are with the appointment of numeral.Although should be appreciated that also the reagent that always not clear and definite statement is described in this article only is exemplary, and the equivalent of these reagent is in common knowledge in the art.
[044] term " transgenic " is meant polynucleotide, and it is introduced into cell and can be transcribed into RNA and also can be translated in appropriate circumstances alternatively and/or express.In one aspect, it can give the desirable characteristic of cell that is introduced into it, or causes desirable treatment or diagnostic result.On the other hand, it may be transcribed into the interferential molecule of mediate rna, for example siRNA.
[045] be meant the number that contains the genomic virion of reorganization AAV DNA with reference to the used term of virus titer " genome particle (gp) " or " genome coordinate ", and no matter it is infectious or functional.The number of the genome particle in the specific carrier prepared product can by such as described in the embodiment of this paper or for example, people such as Clark, (1999) Hum.Gene Ther, 10:1031-1039; People such as Veldwijk, (2002) Mol.Ther., the method in the 6:272-278 document is measured.
[046] term " infectious unit (iu) ", " infectious particles of using with reference to virus titer " or " replicator " is meant the number of infective and reproducible reorganization AAV carrier granular, it can pass through for example people such as McLaughlin, (1988) J.Virol., the infectious center test of describing in the article of 62:1963-1973 is also referred to as the duplication centre test and measures.
[047] term " trnasducing element (tu) " that uses with reference to virus titer is meant the number of the infectivity reorganization AAV carrier granular that causes functional transgene product production, it can pass through such as in this paper embodiment or for example people such as Xiao, (1997) Exp.Neurobiol., 144:113-124; Or people such as Fisher, (1996) J.Virol., the functional experiment of describing in 70:520-532 (LFU experiment) article is measured.
[048] term " treatment ", " treatment effective dose " and its related term refer to the amount of the expression product of RNA, DNA or DNA and/or RNA; in the experimenter, produce the acquisition of the biological results of protection or morbidity delay or doing well,improving or expection; proofread and correct such as the neuro pathology, for example follow cytopathology such as the motor neuron disease of ALS.Term " treatment is proofreaied and correct " refers to produce in the experimenter protection or morbidity postpones or the degree of correction of doing well,improving.Effective dose can be measured by known empirical method.
[049] " compositions " also means the combination that comprises potent agent and other carriers, for example, chemical compound or compositions, non-activity (for example, detectable reagent or labelling) or activated, for example adjuvant, diluent, bonding agent, stabilizing agent, buffer agent, salt, lipophilic solvent, antiseptic, adjuvant or or the like.Carrier comprises that also (for example, sugar comprises monosaccharide, disaccharide, trisaccharide, tetrose and oligosaccharide for pharmaceutic adjuvant and additive protein, peptide, aminoacid, lipid and carbohydrate; Derived carbohydrate, for example alditol, glycuronic acid, esterified saccharides or the like and polysaccharide or glycopolymers), it can exist separately or with combining form, by forming separately or with weight ratio or the volume ratio of the 1-99.99% of combining form.Exemplary protein adjuvant comprises serum albumin, for example human serum albumin (HAS), recombined human albumin (rHA), gelatin, casein or the like.Representational aminoacid/antibody component, it also can work in buffer (buffering capacity), comprises alanine, glycine, arginine, betanin, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame or the like.The carbohydrate adjuvant is also included within the scope of the present invention, and its example is including but not limited to monosaccharide, for example fructose, maltose, galactose, glucose, D-mannose, sorbose or the like; Disaccharide, for example lactose, sucrose, trehalose, cellobiose or the like; Polysaccharide, for example Raffinose, melezitose, dextrin-maltose mixture, glucosan, starch or the like and alditol, for example mannitol, xylitol, maltose alcohol (maltitol), lactitol, xylitol Sorbitol (sorbitol) and inositol.
[050] the term carrier comprises that in addition buffer or pH adjust agent; Typically, buffer is to prepare and next salt from organic acid or alkali.Representational buffer comprises acylate, for example citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, aminoguanidine hydrochloride butantriol, or phosphate buffer.Other carrier comprises polymer adjuvant/additive, polyvidon, ficoll (a kind of sugar of poly), dextrates (cyclodextrin for example for example, for example the 2-hydroxypropyl-. quadrature .-cyclodextrin), polyethylene glycol, flavoring agent, antibacterial, sweetener, antioxidant, antagonist, surfactant (Polysorbate for example, for example " polysorbas20 " and " Tween 80 "), lipid (phospholipid for example, fatty acid), steroid (for example, cholesterol) and intercalating agent (for example, EDTA).
[051] as used herein, term " pharmaceutically acceptable carrier " comprises the pharmaceutical carrier of any standard, for example phosphate buffered solution, water and emulsion, for example oil/water or water/oil emulsion and various types of wetting agent.Compositions can comprise that also stabilizing agent and antiseptic can be used for intravital any top mentioned carrier with satisfying.The example of carrier, stabilizing agent and adjuvant can be referring to Martin REMINGTON ' S PHARM.SCI., 15th Ed. (Mack Publ.Co., Easton, (1975) and Williams﹠amp; Williams, (1995), " PHYSICIAN ' S DESK REFERENCE ", 52nd ed., Medical Economics, Montvale, NJ. (1998).
[052] " experimenter ", " individuality " or " patient " replaceable herein use, it refers to vertebrates, preferred mammal is more preferably human.Mammal includes, but not limited to mice, rat, ape and monkey, people, farm-animals, motion animal and house pet.
[053] " contrast " is selectivity experimenter or the sample that is used to contrast purpose in the experiment.Contrast can be " positive " or " feminine gender ".For example, experiment purpose be measure gene alteration expression during with the mutual relation between the pathology of particular type (referring to ALS, for example, hereinafter), usually more preferably use positive control (have such change and show the experimenter of the sort of disease symptoms feature or the sample that comes from the experimenter), and negative control (lacking the expression of change and the experimenter of the sort of disease Clinical symptoms or the sample that comes from the experimenter).
[054] when being used for gene, term " differential expression " refers to from the diversity production of the protein of the mRNA of genetic transcription or this gene code.Expression with normal or control cells is compared, and the gene of differential expression may be to express or owe to express.On the one hand, it refers to than high or low 1.5 times of detected expression in control sample at least, or at least 2.5 times, or selectable at least 5 times, or selectable at least 10 times difference.Term " differential expression " is the nucleotide sequence in phalangeal cell or the tissue also, or not when these sequences are expressed or expressed in control cells when reticent in control cells.
[055] used herein, term " adjustment " refers to change effect or result's quantity or intensity, for example strengthens, enlarges, reduces or reduce.
[056] " to alleviate " together be synonym to term used herein " improvement ", refers to reduce or alleviate.For example, a people may more can stand the symptom of improving disease or imbalance by disease or imbalance are become.
[057] in the gene transmission by dna viral vector, for example adenovirus (Ad) or adeno-associated virus (AAV) mediation the aspect, vector construct refers to contain a viral genome or its part and genetically modified polynucleotide.Adenovirus (Ads) is the virus of the same clan that a class is characterized relatively goodly, comprises surpassing 50 serotypes.Referring to, for example international pct application No.WO 95/27071.Ads is easy to growth, and does not need to be incorporated in the genome of host cell.The deutero-carrier of reorganization Ad, those have reduced reorganization and have produced the possible carrier of wild-type virus especially, also are fabricated out.Referring to, international pct application WO 95/00655 and WO 95/11984.Wild type AAV has very high infectivity and is incorporated into specifically in the host cell gene group.Referring to, Hermonat and Muzyczka, people such as (1984) Proc.Natl.Acad.Sci.USA 81:6466-6470 and Lebkowski, (1988) Mol.Cell.Biol.8:3988-3996.
[058] the invention provides the transmission transgenic to experimenter's the spinal cord and/or the method for brain stem, the containing the neurophilic viral vector of genetically modified reorganization and can realize this transmission of at least one zone in the cerebellar nuclei zone, deep by the administration brain, therein, be delivered in and help transgenic and under situation about expressing, carry out away from the site in administration site.Transmit and also may cause the expression of transgenic in the administration site.
[059] reverse situation of unless specifically stated otherwise, genetically modified expression not only are defined in translates into polypeptide or protein also comprises duplicating and/or transcribing of transgenic polynucleotide.
[060] on the other hand, the invention provides and transmit the method for therapeutic transgene product to the CNS target cell, these target cells are to stand for example mammiferous neuron or the neurogliocyte of motor neuron deficiency disorder such as ALS or traumatic spinal cord injury.The transgenic IGF-1 that can encode.
[061] on the other hand, the invention provides the method for transgenic of transmitting to experimenter's spinal cord, contain the neurophilic viral vector of described genetically modified reorganization by administration and can realize this transmission to the nervus motorius cortex zone of brain, described herein being delivered in helps described transgenic and carries out under situation about expressing away from the site in described administration site.
[062] on the other hand, viral vector of the present invention is administered at least one zone of cerebral deep cerebellar nuclei, and in this zone, experimenter's spinal cord and/or brain stem zone expressed and be delivered to transgene product.
[063] in another embodiment, viral vector is administered at least one zone of the cerebral deep cerebellar nuclei of interconnection brain stem and dynamoneure.These target areas have contacting directly with cell (for example relay cell and the astrocyte) of the cellular environment of forming motor neuron.The cellular environment of transgene product to motor neuron transmitted in administration, and the useful effect of cell of this cellular environment is formed in this product mediation there.
[064] in one embodiment, the invention provides and transmit transgenic to experimenter's motor neuron or adjust the method that transgenic is expressed in experimenter's motor neuron, by being administered into containing genetically modified neurophilic viral vector and can realizing this transmission of experimenter's brain nervus motorius cortex zone, wherein transgenic is expressed in the motor neuron zone away from described administration site.
[065] in another embodiment, the invention provides the method for treatment experimenter motor neuron deficiency disorder, the containing the genetically modified neurophilic viral vector of therapeutic and can realize this treatment of at least one zone by being administered into experimenter's cerebral deep cerebellar nuclei, transgenic is expressed with effective dose at least one subregion of experimenter's spinal cord therein.These subregions comprise one or more (referring to Fig. 1, Figure 12 A) of cervical region, chest, waist or sacrum portion.Transgenic also may be at brain stem, such as, for example, express with the treatment effective dose at least one zone of midbrain, pons or medullary substance (referring to Figure 12 B).It also may be expressed with the treatment effective dose at least one zone of experimenter's brain stem and at least one subregion of experimenter's spinal cord.
[066] the present invention also provides the method for improving experimenter's motor neuron deficiency disorder symptom, by being administered into containing the genetically modified neurophilic viral vector of therapeutic and can realizing this improvement of brain nervus motorius cortex, described therein transgenic is expressed with the treatment effective dose at least one subregion of experimenter's spinal cord.These subregions comprise one or more (referring to Fig. 1, Figure 12 A) of cervical region, chest, waist or sacrum portion.
[067] the suitable neurophilic viral vector of enforcement of the present invention includes, but are not limited to gland relevant viral vector (AAV), herpes simplex virus vector (U.S. Patent No. 5,672,344) and slow virus carrier.
[068] in the method for the invention, the AAV of any serotype all can use.In certain embodiments, the AAV of any serotype all can use, as long as these carriers can be stood the retrograde aixs cylinder transportation in the brain that lacks disease resistance, or the transportation of the aixs cylinder in resistive brain.The serotype of the viral vector that uses in certain embodiments of the invention is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 (referring to, people such as Gao, (2002) PNAS, 99:11854-11859 and Viral Vectors forGene Therapy:Methods and Protocols, editor Machida, Humana Press, 2003).Other serotypes except those listed herein viral vector also can be used.In addition, pseudotype AAV carrier also may be used for method described herein.Pseudotype AAV carrier is the genome that contains an AAV serotype at the housing of second AAV serotype; For example, contain the genomic AAV carrier of AAV2 capsid and AAV1 or contain the AAV5 capsid and the genomic AAV carrier of AAV2 (people such as Auricchio, (2001) Hum.Mol.Genet, 10 (26): 3075-81).
[069] the AAV carrier originates from the non-pathogenic strand of mammal (SS) DNA parvovirus group (at Muzyscka, (1992) Curr.Top.Microb.Immunol. summarizes among the 158:97-129).Briefly, the rep and the cap gene of virus genomic 96% function of removing based on the accounting for of the carrier of AAV (account for), the reverse far-end that stays two section 145 base pair (bp) repeats (ITRs), and it is used to start viral dna replication, packing and integration.When lacking helper virus, wild type AAV is incorporated into human host cell's genome, and it has the preferential locus specificity at chromosome 19q 13.3, or it may keep free the expression.One AAV granule can hold the ssDNA that reaches 5kb, therefore is the space that transgenic and controlling element have stayed about 4.5kb, and this is that the typical case is enough.Yet in U.S. Patent No. 6,544, the trans-splicing system of 785 li descriptions may nearly be the twice of this restriction as for example.
[070] in an embodiment of doing illustration, AAV is AAV2 or AAV1.The adeno-associated virus of many serotypes, particularly AAV2 are characterized into gene therapy vector by extensive studies and quilt.Those skilled in the art are familiar with the preparation based on the functioning gene treatment carrier of AAV.Countless documents about the no counting method of the AAV production, purification and the preparation that are used for the administration human experimenter can find at the publication document of enormous quantity (referring to, for example, Viral Vectors for Gene Therapy:Methods and Protocols, editor Machida, Humana Press, 2003).In addition, at the cell of CNS based on the gene therapy of AAV at U.S. Patent number Nos.6, be described in 180,613 and 6,503,888.Exemplary AAV carrier in addition is reorganization AAV2/1, AAV2/2, AAV2/5, AAV2/7 and the AAV2/8 serotype carrier of coding human protein.
[071] in some method of the present invention, carrier comprises the transgenic that can be connected to promoter.The molecule of this transgenes encoding biologically active, its expression at CNS causes neuro pathology's the correction to small part.Genomic and functional cDNA sequence of people ASM deliver (referring to, for example, U.S. Patent number No.5,773,278 and 6,541,218).Insulin-like growth factor-1 (IGF-1) gene has complicated structure, and this is known in the art.It contains the mRNA product that two selectivitys that come from gene transcripts splice at least.One is 153 amino acid whose peptides, is known by a plurality of names that comprise IGF-1A or IGF-1Ea, and one is 195 amino acid whose peptides, is known by a plurality of names that comprise IGF-1B or IGF-1Fb.The mature form of IGF-1 is 70 amino acid whose polypeptide.IGF-1Ea and IGF-1Eb contain this 70 amino acid whose mature peptides, but difference to some extent on terminal sequence that prolongs of its carboxylic and length.The peptide sequence of IGF-1Ea and IGF-1Eb is respectively by SEQ ID NOS:1 and 2 represented.Genomic and functional cDNAs of people IGF-1, and other the information about IGF-1 gene and its product can obtain at the Unigene number of logining No.NM 00618.
[072] level of transgene expression is mainly determined by the transcripting promoter in the transgene expression frame in eukaryotic cell.Demonstrate secular activity and be tissue-and even cell-special promoter use in certain embodiments.Nonrestrictive promoter example comprises, but be not limited to, cytomegalovirus (CMV) promoter (people such as Kaplitt, (1994) Nat.Genet.8:148-154), CMV/ people β 3-globin promoter (people such as Mandel, (1998) J.Neurosci.18:4271-4284), GFAP promoter (people such as Xu, (2001) Gene Ther.8:1323-1332), 1.8kb specificity neuron enolase (NSE) promoter (people such as Klein, (1998) Exp.Neurol.150:183-194), chicken beta-actin (CBA) promoter (Miyazaki, (1989) Gene 79:269-277), beta-glucuronidase (GUSB) promoter (people such as Shipley, (1991) Genetics 10:1009-1018) and for example those from people's ubiquitin A, people's ubiquitin B separates with people's ubiquitin C and next ubiquitin promoter, in U.S. Patent No. 6, in 667,174 this there is description.In order to prolong expression, other controlling element may be by extra being connected on the transgenic, such as, for example, controlling element after the woodchuck hepatitis virus (WPRE) (people such as Donello, (1998) J.Virol.72:5085-5092) or bovine growth hormone (BGH) polynucleotide site.
[073] use for some CNS gene therapy, the control transcriptional activity may be necessary.For this target, the pharmacology who carries out gene expression with viral vector regulates and control can be by comprising various controlling elements and medicaments insensitive promoter, for example, people such as Haberma, (1998) people such as Gene Ther.5:1604-16011 and Ye, (1995) Science 283:88-91 is described and obtain.
[074] in the method for the invention, viral vector can allow virion to be transported (driving in the wrong direction) by the direction of interior annexation along aixs cylinder to pericaryon in born of the same parents by contacting neuronic terminal axon terminal and administration with containing the compositions of carrying genetically modified viral vector; Allow the therapeutic transgene product to be expressed, the therapeutic transgene product alleviates experimenter's pathology by this therein.Effect may be on motor neuron, on the cell (for example relay cell and astrocyte) of component movement neuron environment, or on the two.In certain embodiments, the concentration of carrier is at least in the compositions: (a) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
12Gp/ml); (b) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
9Tu/ml); Or (c) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
10Iu/ml).
[075] in the other method of the present invention, viral vector can be by contacting neuronic cell space and administration with containing the compositions of carrying genetically modified viral vector, allow virion by endocytosis, allow the therapeutic transgene product to be expressed and the direction at end direct motion transportation in born of the same parents eventually along aixs cylinder to neuron axon, the therapeutic transgene product alleviates experimenter's pathology by this therein.Effect may be on motor neuron, on the cell (for example relay cell and astrocyte) of component movement neuron environment or on the two.In certain embodiments, the concentration of carrier is at least in the compositions: (a) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
12Gp/ml); (b) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
9Tu/ml); Or (c) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
10Iu/ml).
[076] in one aspect, transgenes encoding bioactive molecule, this molecule produce to the neuro pathology of small part in the expression of CNS and proofread and correct.In certain embodiments, the therapeutic transgene product is to suppress the RNA molecule that experimenter SOD expresses, the symptom that alleviates and prevent ALS by this.Referring to people such as Roaul, (2005) Nat.Med.11 (4): people such as 423-428 and Ralph, (2005) Nat.Med.11 (4): 429-433.
[077] aspect when these methods of enforcement, transgene expression is selected from the albumen of the therapeutic dose of insulin-like growth factor-1 (IGF-1), calbindin D28, paralbumin, HIF1-alpha, SIRT-2, VEGF, EPO (erythropoietin), CBP ([CREB] is conjugated protein for the cAMP response element binding protein), SMN-1, SMN-2 and CNTF (ciliary neurotrophic factor).
[078] alternatively, the expression of the mutant form of transgenic Profilin for example causes the expression of the sudden change SOD of ALS.Referring to top people such as Roaul, (2005) and top people such as Ralph, (2005).
[079] to differentiate the structure of human brain, referring to, for example, The Human Brain:Surface, Three-Dimensional Sectional Anatomy With MRI, and Blood Supply, 2nd ed., people such as editor Deuteron, Springer Vela, 1999; Atlas of the Human Brain, people such as editor Mai, Academic Press, 1997 and Co-Planar Sterotaxic Atlas of the Human Brain:3-Dimensional Proportional System:An Approach to Cerebral Imaging, people such as editor Tamarack, Thyme Medical Pub., 1988.Differentiate the structure of mouse brain, referring to, for example, The Mouse Brain in Sterotaxic Coordinates, 2nd ed., Academic Press, 2000.Fig. 1 has roughly shown spinal cord and its four subregions: cervical region, chest, waist and sacrum portion.
[080] the invention provides the motor function that adjustment, correction or enhancement stand the experimenter of damage of motoneurons torment.Only for illustrative purposes, the experimenter may suffer one or more in amyotrophic lateral sclerosis (ALS), spinal column bulbar muscular atrophy, Duchenne-Arandisease, spinocebellar ataxia, primary lateral sclerosis (PLS) or the traumatic spinal cord injury.
[081] be not confined to theory, the pathology of associated movement neuronal damage may comprise that motor neuron degeneration, neuroglia disease, neurofilament are unusual, the forfeiture of corticospinal tract and preceding middle Medullated nerve fibre.For example, two types morbidity is familiar with: the oblongata morbidity that influences upper motor neuron (cortex and brain stem motor neuron) influences facial muscle, language and swallows; The extremity morbidity that influences LM (dynamoneure) is reflected by spasm, asthenia universalis, amyotrophy, paralysis and respiratory failure.In ALS, the experimenter has oblongata and extremity morbidity simultaneously.In PLS, the experimenter has the oblongata morbidity.
[082] be not confined to theory, one embodiment of the invention have the therapeutic molecules of providing (for example, protein or peptide) to each sectional ability of spinal cord.This may realize in DCN by injection of AAV vectors.In addition, indivedual laminas of aiming in each spinal cord subregion may be important.Lamina is the specific territory, subprovince in brain and the spinal cord zone.In certain embodiments, the specific lamina of aiming in certain spinal cord subregion may make us expecting.Because damage of motoneurons may also can take place in upper motor neuron, provide therapeutic molecules (for example, protein or peptide) in the subregion of brain stem, also may make us expecting.In an embodiment, provide therapeutic molecules to spinal cord, comprise some or all inferior subregion, and, comprise in some or all inferior subregion and may make us expecting to brain stem.The present invention realizes the transmission of above-described therapeutic molecules to spinal cord district and/or brain stem by importing a kind of AAV carrier to DCN.Figure 12 A illustrates the connection of cerebellar nuclei zone, deep and spinal cord, and Figure 12 B illustrates the connection between cerebellar nuclei zone, deep and brain stem.
[083] tissue and the ability of carrying out complicated athletic performance depend on from the zona rolandica territory, i.e. motor cortex and the signal that comes.The cortex motion command descends from two passes.The nucleus motorius of mobile facial muscle in the corticobulbar fibers control brain stem, the dynamoneure of corticospinal fibers control domination trunk and limb muscle.Cerebral cortex also affects indirectly the spinal motor activity by acting on descending brain stem approach.
[084] primary motor cortex is positioned at the precentral gyrus (4) in Broadmann district.The aixs cylinder that projects the cortical neuron of spinal cord also turns round in the cortico bulbar fibre of a large amount of fibre bundles that contain about 100 ten thousand aixs cylinders.About 1/3 of these aixs cylinders originate from the precentral gyrus of frontal lobe.In addition 1/3 originate from the zone 6.The remaining zone of originating from somatic cortex 3,2 and 1 and regulate the transmission of afferent input by dorsal horn.
[085] the corticospinal fibers bundle with corticobulbar fibers Shu Gongtong running, arrives the abdominal part of midbrain by the hind leg of capsula interna.It is separated into the fubril bundle through pontine nucleus in pons.It is recombinated in medullary substance and forms ventripyramid.About 3/4 corticospinal fibers intersection is passed through the median line in the cone at the joint position of medullary substance and spinal cord is intersected.The fiber that intersection is passed through is descending in the part at the back of columna lateralis medullae spinalis (dorsolateral fasciculus(of Lissauer)), forms lateral pyramid tract.The uncrossed fiber that passes through is at the descending cortex oblongata veutro bundle that becomes of abdomen post.
[086] sidepiece of corticospinal tract terminates about the zone with the brain stem outside gray nucleus identical with intermediate system greatly with abdomen area.Lateral corticospinal tract mainly projects the nucleus motorius in the Outboard Sections of ventral horn and the relay cell of mesozone.The abdominal part corticospinal tract is two-way project the abdomen MCC and contain innervation axis muscle motor neuron zone line adjoin part.Fig. 3 diagram has shown main afferent (importing into) path.
[087] deep of cerebellum is definition inboard (pinnacle) nucleus, centre (between two parties) nucleus and the outside (dentation) nucleus and the grey matter that is called the deep cerebellar nuclei.Used herein, term " deep cerebellar nuclei " jointly refers to this three zones.Fig. 2 diagram has shown three zones of DCN.Fig. 4 diagram has shown main spreading out of (output) path of sending from DCN.Fig. 5 diagram has shown the neural circuit in the cerebral cortex.Figure 12 A and 12B diagram have respectively shown connection between DCN and spinal cord or brain stem.
[088] if needs are arranged, the human brain structure can connect with the analog structure of other mammal brains.For example, most mammal, comprise people and rodent, smell in having shown-the similar subregion organization of Hippocampus projection, have neuron, yet mainly originate from neuron (the Principles of Neural Science of the inside part of entorhinal cortex to the projection of abdominal part Hippocampus at the lateral part of the outside of back that projects Hippocampus or distance bar and inboard entorhinal cortex, 4th ed., people such as editor Kandel, McGraw-Hill, 1991; The Rat Nervous System, 2nd ed., editor Paxinos, Academic Press, 1995).In addition, the II confluent monolayer cells of entorhinal cortex projects dentate gyrus, and it is in outer 2/3 place of dentate gyrus molecular layer termination.Two-way cornu ammonis zone C A1 and the CA3 that projects Hippocampus of aixs cylinder that comes from the III confluent monolayer cells terminates at the strata lacunosum molecular layer.
[089] in one aspect, the method for disclosure comprises the method that the neurophilic viral vector that carries the transgenic of coding therapeutic product and allow transgenic to express with treatment level in the CNS of administration site far-end is administered into ridden experimenter's CNS.In addition, carrier may comprise the effectively polynucleotide of the bioactive molecule of treatment CNS imbalance of coding.Such bioactive molecule may comprise peptide, includes but not limited to the natural or mutant form of full length protein, the natural or mutant form of protein fragments, synthetic polypeptide, antibody and such as the antibody fragment of Fab ' molecule.Bioactive molecule also may comprise nucleotide, comprises strand or double-stranded DNA polynucleotide and strand or double-stranded RNA polynucleotide.If need to be applied to the summary of exemplary nucleic acid technique of the enforcement of the method that this paper discloses, can be referring to Kurreck, (2003) J., Eur.J.Biochem., 270,1628-1644[antisense technology]; People such as Yu, (2002) PNAS, 99 (9), 6047-6052[RNA perturbation technique] and people such as Elbashir, (2001) Genes Dev., 15 (2): the 188-200[siRNA technology].
[90] in the embodiment of an illustration, can realize administration to experimenter or patient's DCN by the high titre carrier solution of direct injection.For example, can be by in the cerebellar nuclei zone, deep that is injected directly into one or more brains of selecting in the group that be selected from inboard (pinnacle) zone, middle (between two parties) zone and the outside (dentation) zone composition and administration.Since DCN widely with brain stem and spinal cord between spread out of and import into and connect, DCN is an attracting site of injection.These cells provide a kind of effectively and the mode of minimally invasive transmit viral vector and expression transgenic to spinal cord regional and brain stem zone.Be not confined to theory, viral vector may be absorbed by the aixs cylinder tip and along aixs cylinder to the reverse transportation of direction of these pericaryons, and these neurons are in whole spinal cord zone and/or the brain stem projection.Have and end at, for example, the pericaryon of the aixs cylinder terminal tip of spinal cord neck area also exists in DCN.Viral vector is absorbed by these cell spaces or comes from the transgenic that gives expression to of viral vector or the aixs cylinder terminal tip that the two may forward be transported to the spinal cord neck area.Therefore,, only there is the viral vector of small size to be injected by using DCN as injection site, but this significant transgene expression of mediation in spreading all over of spinal cord and/or brain stem one or more zones.
[091] in certain embodiments, method comprises the medication of carrying the genetically modified neurophilic carrier of therapeutic of high titre, so that express with treatment level in transgene product second site away from first site in CNS.In certain embodiments, the titre of carrier is at least in the compositions: (a) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
12Gp/ml); (b) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
9Tu/ml); Or (c) 5,6,7,8,8.4,9,9.3,10,15,20,25 or 50 (* 10
10Iu/ml).In embodiment in addition, can finish administration by the neurophilic carrier solution of the high titre of injection in the direct brain essence to the morbidity brain, after this, transgenic at far-end, offside or the homonymy of injection site with treatment level and apart from administration site at least 2,3,5,8,10,15,20,25,30,35, express in 40,45 or 50 millimeters place.
Distance between [092] first and second site is defined as administration site (first site) and far-end site can detect minimum range zone between the border of transduction, this distance is by using operation known in the art or using in an embodiment, and for example the method for describing in the in situ hybridization is measured.Some neuron among the bigger mammal CNS may be crossed over bigger distance owing to the reason that its aixs cylinder is throwed.For example, in the people, some aixs cylinder may be crossed over 1000 mm distance or bigger.Like this, in several different methods of the present invention, carrier can be transported by aixs cylinder along the whole length of aixs cylinder with such distance, and the blast cell body arrives and transduce.
[093] site of carrier administration is selected with relating to the topology in brain loop with the target area based on desirable neuro pathology among the CNS, and site only to be administered and target area have aixs cylinder to connect.The target area can be defined, for example, and by using the three-dimensional elements of a fix of 3-D.In certain embodiments, at least 0.1,0.5,1,5 or 10% of the selected so that injection carrier cumulative volume in administration site be delivered in target area at least 1,200,500 or 1000mm by far-end
3In.An administration site may be positioned at the zone of the projection neuron domination of coupled brain remote area.For example, black substance and veutro sections district transmit dense projection to tail body and shell nuclear (being called as striatum jointly).Neuron in black substance and abdomen section tegmentum can be used as the target of transduction by the retrograde transportation of AAV after being expelled to striatum.In another embodiment, hippocampus accept to come from other zones of brain clearly, the projection of predictable aixs cylinder.Other administration site may be positioned, for example, spinal cord, brain stem (medullary substance, midbrain and pons), midbrain, cerebellum (comprising the deep cerebellar nuclei), diencephalon (thalamus, hypothalamus), akrencephalon (striatum, cerebral cortex or, in cortex, occipital lobe, temporal lobe, top, frontal lobe) or its combination.
[094] second (target) site can be positioned at contains the neuronic any zone that comprises the CNS of brain and spinal cord that projects first (administration) site.In certain embodiments, second site is in the zone of the CNS that selects from black substance, oblongata, brain stem or spinal cord.
[095] in order to transmit the specific region of carrier clearly, particularly arrive the characteristics zone of brain to the central nervous system, may administration by three-dimensional location microinjection.For example, in that day of operation, patient will have fixing on the throne (with screw tightening in skull) three-dimensional locating support substrate.Brain with three-dimensional locating support substrate of brain function (the suitable MRI that has credible labelling) will carry out imaging by using high-resolution MRI.The MRI image will be passed to the computer of the three-dimensional positioning software of operation then.The image of a series of crown, arrow-shaped and axle will be used for determining the target site and the track of vector injection.Software is directly translated into track with 3 suitable mutually dimension coordinates of three-dimensional locating support.Boring is beaten on entry site, and the fixed stereotaxic instrument device that contains pin is implanted at given depth.Carrier in pharmaceutically acceptable carrier will be injected then.Carrier is the administration by being injected directly into main target site then, and via the reverse target site that is transported to far-end of aixs cylinder.Other administration route may be used, for example, direct visual method or other non--the top layer cortex usage used during three-dimensional position application.
[096] in addition, because therefore each zone of DCN, can be passed to this by the regional transgenic of the DCN of preselected administration clearly at certain zone of CNS all at the specific region (seeing Fig. 1 and Figure 12 A and 12B) of CNS.Significantly, those technology by changing the number of location, sequence and transgenic administration, can be finished a large amount of administrations and directed the transmission as is known to persons skilled in the art.To be determined for the cumulative volume and the particulate total number of drug administration carrier of medicinal substances based on the aspect of known treatment by those skilled in the art.Treatment effectiveness and security performance detect in appropriate animal model.For example, exist in a large number at LSDs by the animal model of well-characterized, for example, described herein or people such as Watson, (2001) Methods Mol.Med., people such as 76:383-403 or Jeyakumar, (2002) Neu ropath.Appl.Neurobiol., 28:343-357 and ALS (referring to people such as Tu, (1996) P.N.A.S., 93:3155-3160; People such as Roaul, (2005) Nat.Med., 11 (4): people such as 423-428 and Ralph, (2005) Nat.Med., 11 (4): 429-433).
[097] in experiment mice, the cumulative volume of the AAV solution of injection is for example, to arrive 5ul 1.For other mammals, comprise human brain, volume and transmission ratio are suitably defined (scale).For example, verified, the 150ul volume energy be safely injected in the brain of primate (people such as Janson, (2002) Hum.Gene Ther., 13:1391-1412).If treatment may comprise each target site single injection or needs, may be repeated to carry out along the injection channel.A plurality of injection site can be used.For example, in certain embodiments, except primary administration site, contain another site that the compositions of carrying genetically modified viral vector is administered into first administration site offside or homonymy.Injection can be a single or multiple, one-sided or bilateral.
[098] high titre AAV prepared product can pass through to use technology known in the art, for example, and in U.S. Patent No. 5,658,776 and Viral Vectors for Gene Therapy:Methods and Protocols, editor Machida, Humana Press, the technology of describing in 2003 is produced.
[099] the following examples provide illustrative embodiment of the present invention.A kind of ordinary skill of this area will be discerned and countless may be implemented and do not change the modifications and variations of the spirit or scope of the present invention.Such modifications and variations comprise within the scope of the invention.These embodiment are not limitations of the present invention.
Embodiment
The titration of recombinant vector
[0100] titre of AAV carrier copies number (genome particle number in every milliliter) according to genome and measures.Genome particle concentration is based on the Taqman of carrier DNA
PCR is as previous (people such as Clark, (1999) Hum.Gene Ther., the 10:1031-1039 that reports; People such as Veldwijk, (2002) Mol.Ther, 6:272-278).In brief, the AAV-ASM of purification digests buffer (50mMTris-HCl pH8.0,1.0mM EDTA, 0.5%SDS, 1.0mg/ml E.C. 3.4.21.64) with capsid protein and handles at 50 ℃ and came release vehicle DNA in 1 hour.The DNA sample is with being annealed to the carrier DNA specific region, carries out polymerase chain reaction (PCR) such as the primer of promoter region, transgenic or poly (A) sequence.PCR result is then by real-time Taqman
Software, (Foster City, CA) the Prism7700 sequence detection system provides, and carries out quantitatively such as Perkin Elmer-Applied Biosystems.
[0101] carry the marker gene that can detect, for example the carrier of beta galactosidase or green fluorescence protein gene (GFP) can carry out titer determination by using a kind of infectivity experiment.Permissive cell (for example Hela or COS cell) is transduceed with AAV, carries out then measuring gene expression such as the dyeing of the cell of the beta galactosidase carrier transduction that carries out with X-gal (5-bromo-4 chloro-3 indole-β-D-galactoside) or at the experiment of the fluorescence microscopy of the cell of GFP transduction.For example, experiment can followingly be carried out: 4 * 10
4Individual HeLa cell inoculation is in each hole of 24 well culture plates that use normal growth medium.After absorption, after promptly about 24 hours, cell infects with infection multiplicity (MOI) 10 with the Ad5 type and transduces with the grade dilution of the carrier of packing, and hatches at 37 ℃ then.After one to three day, before cytopathic effect was observed widely, (for example, X-gal dyeing or fluorescence microscopy) carried out in appropriate experiment on cell.If used the reporter gene such as beta galactosidase, cell is fixing and carry out the dyeing of beta galactosidase with X-gal in the solution of 2% paraformaldehyde, 0.5% glutaraldehyde.Provide the carrier dilution that separates good cell to be carried out counting.Each positive cell is represented a transduced unit (tu) of carrier.
The expression of functional protein has stoped the athletic injury in the relevant mouse model of treatment.
[0102] the ASMKO mice is acceptable model (people such as Horinouchi, (1995) Nat.Genetics, the 10:288-293 of one type of A and B niemann-Pick disease; People such as Jin, (2002) J.Clin.Invest., 109:1183-1191 and Otterbach, (1995) Cell, 81:1053-1061).Niemann-Pick disease (NPD) is classified as LSD and is a kind of genetic nerve metabolism deficiency disorder, it is characterized in that the genetic defect (ASM in ASM; Lipid sphyngomyelin phosphocholine hydrolytic enzyme, EC3.1.3.12).The proteic shortage of functional ASM causes accumulating of lipid sphyngomyelin substrate in the neuron of whole brain and the neuroglial lysosome.This has caused a large amount of lysosomal formation of expanding in the perikaryon, and this is the sign characteristics of type ANPD and main cell phenotype.Expand lysosomal existence with the forfeiture of normal cell function with cause carrying out property neurodegenerative process interrelated (the TheMetabolic and Molecular Bases of Inherited Diseases of affected individuals in early childhood death, people such as editor Scriver, McGraw-Hill, New York, 2001, the 3589-3610 page or leaf).Second cell phenotype (for example additional Developmental and Metabolic Disorder) also with this disease association connection, accumulate in the lysosome organelle by remarkable high-caliber cholesterol.Lipid sphyngomyelin has the strong affinity to cholesterol, and this causes the separating of a large amount of cholesterol in ASMKO mice and patient's the lysosome (people such as Leventhal, (2001) J.Biol.Chem., 276:44976-44983; Slotte, (1997) Subcell.Biochem., people such as 28:277-293 and Viana, (1990) J.Med.Genet., 27:499-504).
[0103] Xia Mian experiment, estimate the reorganization AAV2/1 of coding people ASM (hASM), AAV2/2, AAV2/5, AAV2/7 and the proteic relative ability of AAV2/8 serotype vector expression hASM at the one-sided injection post-equalization cholesterol storage pathology of deep cerebellar nuclei, are stood transportation, functional rehabilitation in rescue Purkinje cell and the startup ASMKO mice.The recovery whether the transgene protein propagation/expression that the winding of an additional ASMKO mice is injected by the bilateral of DCN to estimate increase will improve behavioral function.
The acid sphingomyelinase of [0104] six ten six male isozygotying (/-) knocks out (ASMKO) mice and 16 and comes the male wild type littermate control Mus of (+/-) to be raised from hybridization.According to people such as Gal, (1975) N Engl J Med, the step of describing among the 293:632-636 is carried out gene type with PCR with mice.The mice that comes from initial Mus group is backcrossed with C57/B16 kind system.Animal was raised and unconfined food and the water of providing in the light and shade circulation environment at 12:12 hour.All operations are all in public organizations animals love with use under the method for permission of committee and carry out.
[0105] after with isoflurane anesthesia, mice (about 7 week ages) is used following AAV serotype carrier (n=8/ carrier): a kind of among AAV1-CMV-β gal, AAV1-CMV-ASM, AAV2-CMV-ASM, AAV5-CMV-ASM, AAV7-CMV-ASM and the AAV8-CMV-ASM proceeds to the deep cerebellar nuclei, and (A-P:-5.75 is from bregma, M-L:-1.8 is from anterior fontanelle, D-V:-2.6 is from dura mater, front tooth bar: one-sided injection 0.0).Carrier is altogether 1.86 * 10 with the 10ul Hamilton syringe that is installed on the syringe pump with each brain
10Individual genome particle is sent with 0.5ul/ minute speed.The final volume injected of each carrier is 4ul.Before one hour of operation and after 24 hours, mice is by administration Ketoprofen (5mg/kg; SC) carry out pain relieving.
[0106] puts to death (14 age in week) 7 weeks of mice after injection.When putting to death, the pentobarbital sodium (150mg/kg of mice administration overdose; IP) and rapidly broken end (n=5/ group) or through carotid artery perfusion (n=3/ group).The big capsules of brain of decapitated mice removes rapidly, and quick freezing in liquid nitrogen is cut into 3 parts (right cerebral hemisphere, left cerebral hemisphere and cerebellum), homogenate, and analyze hASM with ELASA.Brain of perfusion mice and spinal cord are verified the proteic expression of people ASM, accumulate and be used in the Purkinje cell survival detection that the calbindin dyeing in 50um vibrations (vibratone) section is carried out as the cholesterol of the detection of being dyeed by filipin.Accepting the ASMKO mice of the bilateral injection (about 7 ages in week) of AAV2/1-β gal (n=8), AAV2/1-ASM (n=5) and AAV2/2-ASM (n=5) puts to death in 20 ages in week behind the experience rotation test.By using methods known in the art, mice at Smartrod (AccuScan) by by quickening and waving rotation test and detect motor function.Exemplary method is people such as Sleat, and (2004) J.Neurosci. reproduces among the 24:9117-9126.Figure 10 and 11 diagrams have shown the result as the rotation test of motor function recovery detection means.
[0107] down, the total length people ASM cDNA that has SV40 polyadenylic acid sequence and a hybridization intron is cloned into one and is contained from AAV serotype 2 and the plasmid of next ITRs (AAV2ITR) in early stage immediately (CMV) promoter control of human cytomegalic inclusion disease virus.People such as Jin, (2002) J Clin Invest., 109:1183-1191.By using a series of triple transfections that contain the helper plasmid of the special capsid coding region of serotype except that AAV type 2 replicators, can produce hybrid plasmid.This strategy allows the AAV2ITR carrier package to go into people such as the special virion Rabinowitz of each serotype, (2002) J Virol., 76:791-801.In this way, hASM recombination group is used to produce the rAAV-hASM carrier that a series of various serotypes comprise AAV2/1, AAV2/2, AAV2/5, AAV2/7 and AAV2/8.Reorganization AAV carrier carries out purification (serotype 2/1,2/2 and 2/5) by ion exchange chromatography.People such as O ' Riordan, (2000) J Gene Med, people such as 2:444-54 or csCl centrifugation (serotype 2/8 and 2/7) Rabinowitz, (2002) J.Urrol., 76:791-801.The final titre of AAV-ASM virion (granule of anti-DNA enzyme) is measured by the TaqMan PCR of CMV sequence.People such as Clark, (1999) Hum.Gene Therapy, 10:1031-1039.
[0108] people ASM antibody be human specific and can not be with mice ASM cross reaction.(Corning, NY) 9018 flat boards carry out overnight incubation at 2-8 ℃ with (100ul/ hole) Coster that is diluted in monoclonal recombined human ASM (rhASM) antibody (2ug/ml) the bag quilt in the 50mM sodium carbonate buffer (pH9.6).Remove excessive coated antibody, (KPL, Inc. MD) were hatched 1 hour at 37 ℃ to add the sealing diluent.Dull and stereotyped with microtest plate scrubber (Molecular Devices, CA) 2 circulations of flushing.(1%HP-BSA) standard in, contrast and sample splash into for PBS, 0.05% tween, and be duplicate, and be allowed to hatch under 37 ℃ 1 hour to be diluted in standard dilution buffer liquid.Flat board washes by above-described method.The biotinylated recombined human ASM of one hectolambda (rhASM) antibody (dilutes 1: 20K) be added in each hole, and hatch 1 hour under 37 ℃, remove with the microtest plate scrubber then in standard dilution buffer liquid.(Pierce Biotechnology, Inc. IL) are added into (100ul/ hole) and at room temperature hatched 30 minutes streptavidin-HRP of dilution 1:10K then.Culture plate cleans with above-described method, and (KPL, Inc. MD) were hatched 15 minutes at 36-38 ℃ to use SureBlue TMB subsequently.(KPL, Inc. MD) stop, and (MolecularDevices CA) reads the value of absorbance at 450nm to use Spectra Max 340 culture plate readers then with stop bath in reaction.(Molecular Devices CA) finishes data analysis by using Softmax Pro 4.3 softwares.
(Pierce Biotechnology, Inc. IL) measures the protein concentration of [010g] each sample with the BCA albumen test kit that uses bovine serum albumin as standard.
[0110] fixative that contains 2% paraformaldehyde, 0.03% glutaraldehyde, 0.002% calcium chloride that is used in the 0.1M sodium-acetate buffer of pH6.5 of mice carries out through the carotid artery perfusion, afterwards the perfusion that carries out with the same fixed agent of pH8.5.Back the fixing that mouse brain and spinal cord are cut open and spend the night under 4 ℃ in the fixative of the pH8.5 that does not have glutaraldehyde.These are organized in the kaliumphosphate buffer of 0.1M of pH7.4 and are rinsed, and are embedded in 3.5% agar and are cut into the arrow shape tangent plane of 50um with the vibrations microtome.
[0111] brain and spinal cord are vowed shape vibration section with the 50um spacing.Section is handled with the anti-immunofluorescence of carrying out of anti-people ASM (1: 200).Section was hatched 1 hour in the PBS that contains 10% donkey serum, 0.3%Triton X-100, hatched 72 hours in the PBS that contains 2% donkey serum, 0.2%Triton X-100 with biotinylated mouse-anti people ASM antibody then.After washing, (PerkinElmer, Boston MA) carry out signal and amplify to amplify test kit with cheese ammonia signal.People ASM albumen is observed with the Nikon fluorescence microscope, catches image with SPOT camera and Adobe Photoshop software.
[0112] filipin becomes thing again (Sigma, St.Louis MO) at first is diluted in 100% methanol, as the storage concentration of 1mg/ml.Storage liquid can stablize for 4 weeks at-20 ℃.After with the PBS1 washing, hatched 3 hours in the dark place in the 10ug/ml filipin of section in the fresh PBS of being formulated in.Section is then with PBS washing three times.Cholesterinosis is observed with the ultraviolet filter of fluorescence microscope.
[0113] brain is handled with the direct anticalcium protein-bonded one anti-immunofluorescence of carrying out.Section is washed with kaliumphosphate buffer (KPB), uses potassium phosphate buffer saline (KPBS) to wash then.Section sealing 3 hours in the KPBS that contains 5% donkey serum, 0.25%Triton X-100 then, then contain 5% donkey serum, 0.2%Triton X-100 and mouse-anti calbindin antibody (1: 2500, sigma, St.Louis is hatched among KPBS MO).4 ℃ hatch 72 hours after, section is with the KPBS flushing that contains 0.1%Triton X-100 three times.Two is anti-, and the anti-Mus CY3 of donkey antibody (1: 333, Jackson Immunoresearch Laboratories, West Grove PA) joins in the buffer of KPBS+0.1%Triton X-100 in incubated at room 90 minutes.Section is washed with KPB, counts on the microscope slide of coating glue then.The calbindin positive cell is gone up at epifluorescence microscope (epifluorescence) and is observed.For the Purkinje cell in the quantitative cerebellum, from each animal, select the inboard little brain section of four full faces.The positive Purkinje cell fluorescence microscope of calbindin immunity, cyton is counted under the situation of 20 times of amplifications.Each blade is counted respectively.Two isolating focal planes are carried out counting in each blade.Have only focal cell to be counted, guarantee not have cell to be counted twice.
[0114] five ten (50) μ m vibration section is at first carried out the immunofluorescence processing by above-described method with the antibody of direct anti-people ASM.Section is washed in PBS then and is carried out CAT (ChAT with above-described method at calbindin; Rabbit polyclonal, 1: 500, Chemicon International, Temecula, CA) dyeing.But be to use the anti-rabbit FITC of donkey antibody (1: 200, JacksonImmunoresearch Laboratories, West Grove, PA), rather than anti-with CY3 two.At first, obtain image with Laser Scanning Confocal Microscope then with the epifluorescence microscope observation of dyeing.
[0115] filipin dyeing is quantitative with following method.Obtain the correct image of exposure with the vertical epifluorescence microscope in the full visual field of Nikon E600 that is equipped with the SPOT digital camera.At first imaging AAV2/1-β-gal organizes, and this exposure parameter is used to obtain its other all image then.By the length of each half brain, the image of each width of cloth analysis has been represented an inboard sagittal plane.(Universal ImagingCorporation) carries out morphometric analysis with Metamorph software.AAV2/1-β-gal image is as threshold value; In case set up, same threshold value is used for all images.Following zone is chosen by hand and is analyzed respectively by user: cerebellum, pons, medullary substance, midbrain, cerebral cortex, hippocampus, thalamus, hypothalamus and striatum.Bulk strength in each zone is measured, and from given group of animal and all measurements (n=3/ group) that come are used to produce average.Then, reduce and calculate reducing percentage ratio with the bulk strength of comparing through the cholesterol of treatment in the animal with the injection Mus that knocks out β-gal.Behind one-sided injection of AAV-ASM, positive hASM immunostaining all is observed in whole cerebellum (table 1), pons, medullary substance and spinal cord in the cerebellar nuclei of deep.
Table 1
| Structure | AAV1 | AAV2 | AAV5 | AAV7 | AAV8 |
| The deep cerebellar nuclei | ++++ | ++ | +++ | +++ | ++++ |
| Cerebellar lobe | ++++ | ++ | +++ | +++ | ++++ |
| Pons | ++ | ++ | ++ | ++ | + |
| Medullary substance | + | ++ | ++ | +++ | + |
| Spinal cord | +++ | +++ | ++ | + | |
| Thalamus | ＊ | ＊ | ＊ | ＊ | ＊ |
| Hypothalamus | ＊ | ＊ | ＊ | ＊ | ＊ |
| Hippocampus | ＊ | ＊ | ＊ | ＊ | ＊ |
| Striatum | ＊ | ＊ | ＊ | ＊ | ＊ |
| Cerebral cortex | ＊ | ＊ | ＊ | ＊ | ＊ |
[0116] the positive hASM pigmented section as AAV serotype function shows that positive hASM is lower than detectability, but the correction of cholesterol pathology has still taken place.
[0117] mice is handled in cerebellum with AAV2/1-ASM and have the most eurytopic (promptly distributing) hASM expression between the lobule in the same arrow shape tangent plane, yet the mice of handling with AAV2/2-ASM has the most limited people ASM protein expression level.Occupy between these two groups with the people ASM protein expression in the mice of AAV2/5-ASM, AAV2/7-ASM and AAV2/8-ASM processing.In with serotype 1 and 8 mices of handling, vow that the medial-lateral distribution between the shape tangent plane is maximum, minimum is the mice of injection serotype 2.The serotype 5 and 7 that the beginning medial-lateral is spread pattern occupy between serotype 1 and 2.Each layer (being molecule, Purkinje and granule) of cerebellum is transduceed with each AAV serotype; Yet, all be tangible to all serotypes to the affinity increase of molecular layer.In with serotype 1 and 5 mices of handling, the Purkinje cell transduction is maximum.The mice of injection serotype 7 has minimized number transduction Purkinje cell.The mice of handling with serotype 8 also almost has few transduction Purkinje cell, but when homologous serotype 1,2,5 and 7 was compared, its ASM that has in granular layer still less expressed.Healthy cellularity has appearred in the Purkinje cell with the ASM transduction.In the little brain tissue homogenate that is undertaken by ELISA AAV mediation hASM protein expression quantitative analysis support these immunohistochemistries to find.When other its mices relatively time the with all, injection serotype 1 and 8 mice have proved significantly (the cerebellum hASM protein level that p＜.0001) is higher.The mice cerebellum hASM level of injecting serotype 2,5 and 7 is not higher than WT level (being background).Just as expected, not detecting the hASM antibody that people ASM-uses in wild-type mice in ELISA is that the people is special.
[0118] the proteic shortage of functional ASM causes the lysosome of lipid sphyngomyelin to be accumulated, and causes second metabolic deficiency such as unusual cholesterol transportation subsequently.People such as Sarna, Eur.J.Neurosci., people such as 13:1873-1880 and Leventhal, (2001) J.Biol.Chem., 276:44976-4498.Free cholesterol is accumulated by using filipin in the ASMKO mouse brain, separates and next auto-fluorescence molecule from streptomyces filipinensis, observes.The wild-type mice brain is to the filipin dyeing that is not positive.In the mice (except the AAV2/1-β gal) that all AAV handle, all to produce the male area of hASM immunostaining of functional type transgene product overlapping with indicating each serotype carrier in the painted removing of filipin (table 2).
Table 2
| 2/1 | 2/2 | 2/5 | 2/7 | 2/8 | |
| Cerebellum | 96.54± 2.14 | 93.85± 1.257 | 86.75± 9.58 | 96.47± 1.93 | 99.12± .66 |
| Midbrain | 96.72± 1.73 | 53.08± 22.89 | 65.88± 24.53 | 73.39± 22.39 | 91.10± .105 |
| Pons | 91.31± 5.80 | 50.07± 21.26 | 70.96± 25.60 | 93.15± 31.20 | 96.72± 1.20 |
| Medullary substance | 93.29± 6.22 | 88.46± 3.04 | 81.55± 17.31 | 80.73± 14.99 | 97.40± 1.60 |
| Thalamus | 48.88± 25.25 | 41.21± 27.35 | 34.86± 16.67 | 48.44± 28.65 | 77.03± 12.08 |
| Hypothalamus | 82.81± 10.14 | 86.96± 12.93 | 88.46± 5.90 | 82.95± 11.46 | 99.68± .31 |
| Cortex | 27.60± 24.75 | 73.62± 14.9 | 55.65± 28.89 | 76.97± 14.27 | 98.30± .34 |
[0119] coexists in the selected brain region different AAV serotype (the n=3/ serotypes of injection coding people ASM in cerebellum; 2/1,2/2,2/5,2/7 and 2/8) the ASMKO mice of handling with AAV-β gal behind the deep cerebellar nuclei of ASMKO mice the filipin (being cholesterol) of comparing is removed percentage ratio and reduces.
[0120] as before by (people such as Passini, (2003) " Society for Neuroscience ", NewOrleans LA) proves, the removing of filipin also takes place in the zone that connects anatomically with injection site, but it is not to the hASM dyeing that is positive.Metamorph the analysis showed that the painted minimizing of filipin takes place in whole piece kiss tailing axle.In cerebellum and brain stem, filipin reduces at most in the mice of handling with AAV2/1-ASM and AAV2/8-ASM, yet in diencephalon and cerebral cortex, the mice of injecting with AAV2/8-ASM has the best aggregate level (table 2) that filipin is removed.Even so, these results show, proofreading and correct cholesterol in ASMKO mice CNS, to accumulate the required hASM level of pathology be minimum limit the quantity of (promptly being lower than hASM immunofluorescence detectability).
[0121] Histological research shows, ASMKO mice cerebellum has experienced quick deterioration.More clearly, and Purkinje cell carrying out property death between 8 and 20 ages in week (people such as Sarna, (2001) Eur.J.Neurosci., people such as 13:1813-1880 and Stewart, (2002), and " Society for Neuroscience ", Orland, FL).Calbindin is a kind of wide received Purkinje cell labelling.Positive calbindin dyeing shows that it is curative that the hASM of AAV mediation expresses in the mice that AAV-ASM handles.All our result shows, the hASM of AAV mediation expresses the death (table 3) that has stoped Purkinje cell in the ASMKO mice in the cerebellum.As desirable, the Purkinje cell survival does not take place in lobule I-III; Mice was injected in 7 ages in week, and the great majority of 8 all these cells are dead.In lobule IV/V, the Purkinje cell survival is maximum in the mice of handling with serotype 1.In lobule VI, in the mice that AAV handles, do not observe the survival of significant Purkinje cell.In lobule VII, the mice that only useful serotype 5 is handled has shown significant Purkinje cell survival.In lobule VIII, the mice of handling with serotype 5 and serotype 2 has shown significant Purkinje cell survival again.In lobule IX and X, (or between mice that AAV handles) Purkinje cell counting is significant not different between WT and KO mice.This is supposed to, because in 14 ages (i.e. the age of Chu Siing) in week, the Purkinje cell in these lobules of ASMKO mice remains great-hearted.In all lobules, be maximum with the Purkinje cell survival in serotype 1,2 and 5 mices of handling, be minimum with the Purkinje cell survival in serotype 7 and 8 mices of handling.
Table 3
| 2/1 | 2/2 | 2/5 | 2/7 | 2/8 | KO | WT | |
| I/II | 7.42± 9.80 | 4.5± 10.58 | 9.40± 11.59 | 12.33± 10.58 | 1±9.16 | 5.8± 11.59 | 113± 10.58 |
| III | 12.42± 10.32 | 11.33± 11.14 | 26.80± 12.21 | 15.33± 11.14 | 9.8± 12.21 | 2±9.65 | 147.50+ 11.14 |
| IV/ V | 60.57± 17.28 | 36.5± 18.67 | 27.80± 20.45 | 29.66± 18.67 | 6.8± 20.45 | 8±16.16 | 220.66± 18.67 |
| VI | 61.14± 11.21 | 27.5± 12.11 | 72.20± 13.26 | 31.16± 12.11 | 3.8± 13.26 | 68.5± 10.48 | 121.16± 12.11 |
| VII | 17.42± 4.15 | 37.66+ 4.49 | 40.60± 4.91 | 5.33± 4.49 | .2±4.95 | 17.37± 3.88 | 37.16± 4.49 |
| VIII | 44.14± 10.75 | 48.66± 11.62 | 82.80± 12.73 | 11.33+ 11.62 | 18.40± 12.73 | 35.12± 10.06 | 103.33± 11.62 |
| IX | 126.28± 19.17 | 102.66± 20.71 | 136.40± 22.68 | 60.16± 20.71 | 84.40± 22.68 | 108.0± 17.93 | 144± 20.71 |
| X | 89.85± 12.54 | 76.83± 13.55 | 93.80± 14.84 | 48.16+ 13.55 | 64.80± 14.84 | 87± 11.73 | 86.66± 13.55 |
[0122] different AAV serotype (the n=3/ serotypes of injection coding people ASM in cerebellum; 2/1,2/2,2/5,2/7 and 2/8) behind the deep cerebellar nuclei of ASMKO mice, the Purkinje cell counting in the subfolium I-X of WT and ASMKO mice.Black italic numeral is remarkable different p≤.01 with KO mice (i.e. the mice of handling with AAV2/1-β gal).
[0123] in quickening rotation test, proved that with the mice of AAV2/1-ASM and the one-sided injection of AAV2/8-ASM remarkable (p＜.0009) longer delay is fallen the time than the ASMKO mice of injecting with AAV2/1-β gal.Mice with serotype A AV2/1-ASM injection is not remarkable difference with wild-type mice.Demonstrated the fall trend of time of long delay more with the mice of AAV2/2-ASM and AAV2/5-ASM injection than the ASMKO mice of injecting with AAV2/1-β gal; Yet, really not so with the mice of AAV2/7-ASM injection.For waving rotation test, significantly (p＜.0001) longer delay is fallen the time to have only the mice of injecting with M2/1-β gal with the mice ratio of AAV2/1-ASM injection to prove one.In this case, wild-type mice shows significantly better than the mice with the AAV2/1-ASM injection.For quickening and rolling test, the ASMKO mice of accepting the injection of AAV2/1-ASM or AAV2/2-ASM bilateral all shows significantly (p＜.001) better than the mice that ASMKO AAV2/1-β gal handles.For these two tests, the mice performance of AAV2/1-ASM bilateral injection can be suitable with wild-type mice.
[0124] measuring the approach whether AAV produces among the ASMKO CNS hASM have functional activity is to estimate it cholesterol to be accumulated the secondary metabolism defect influence of pathology-NPA disease.In the mice that all AAV handle (except AAV2/1-β gal), all to produce the male area of hASM immunostaining of functional type transgene product overlapping with indicating each serotype carrier in the correction that cholesterol is accumulated pathology.As what prove above, cholesterol metabolism is proofreaied and correct in the zone that also connects on injection site anatomy and is taken place unusually, but also takes place in the zone that hASM dyeing is not positive, this hint, and it is bottom line that the correction cholesterol is accumulated the required hASM level of pathology.With these hASM histochemistries and biochemistry result consistently, proved that with serotype 1 and 8 mices of handling cholesterol accumulates the remarkable reduction of pathology.The mices of handling with serotype 2,5 and 7 have shown that also cholesterol accumulates the reduction of pathology, but degree is with different with 8 mices of handling with serotype 1.
The treatment correlation model of amyotrophic lateral sclerosis (ALS)
[0125] amyotrophic lateral sclerosis (ALS) is fatal neurodegenerative disease, is characterized in the selectivity forfeiture of motor neuron in cortex, brain stem and the spinal cord.The progress of this disease can cause the amyotrophy of extremity, axle and respiratory muscle.The motor neuron cell death is accompanied by reactive neurogliosis, neurofilament is unusual and corticospinal tract and abdomen root
1-6In the remarkable loss of huge Medullated nerve fibre.Although the nosetiology to ALS is known little, the evidence of accumulation shows that (SALS) that distributes has many similar pathological characters with familial (FALS) ALS; Like this, provide the wherein research of one type of disease will cause a kind of hope of co-therapy method
7FALS accounts for 10% of diagnosed SARS case greatly, and it 20% follows Cu/Zn superoxide dismutase (SOD1)
8Dominant inheritance sudden change.Express the proteic transgenic mouse of mutant human SOD1 (SOD1 for example
G93AMice) having reproduced many pathological characters of ALS, is a kind of effective animal model of research ALS
9For SALS, countless pathomechanisms has involved following reason, the forfeiture of comprise that the contact of excitotoxicity, toxin, the proteasome of glutamate induction is unusual, mitochondrial injury, neurofilament division and neurotrophy being supported
10,11
[0126] so far, the effective Therapeutic Method that does not still have ALS.Neurotrophic factor such as insulin-like growth factor 1 (IGF-1) is widely studied owing to its potentially useful in the ALS treatment.Viral vector provides administration potential medicine to the intracranial transmission in the CNS zone that connects brain stem and dynamoneure (can carry out the aixs cylinder transportation), such as IGF-1, is difficult to the method in the zone that arrives to those technical methods by formerly.
[0127] be not confined to theory, these target areas may needn't need the direct connection with motor neuron; That is, these target areas have with the direct connection of the cell of component movement neuronal cell environment (for example, relay cell and spider cell) only just enough.This is guessed by the institute support of the chimeric Mus of normal and SOD1 mutant cell mixture.These test demonstration, and the non-neuronal cell of not expressing sudden change SOD1 has postponed to degenerate and enlarged markedly the survival of the motor neuron of mutation expression
13In addition, test in addition is verified, the cell of the neuronic cellular environment of component movement (for example spider cell and microgliacyte) is the important source of neurotrophic factor, and these cells injury (taking place as the pathologic in ALS) have been one of latent factor that causes motor neuron degeneration by hint
11
[0128] possibility Supporting Therapy's sexually transmitted disease (STD) poisonous carrier and/or expressing protein are the deep cerebellar nuclei (DCN) of cerebellum to the zone of the CNS of the transportation of motor neuron cellular environment.DCN has importing in a large number and spread out of the connection (see figure 1) with brain stem and spinal cord
14-19Suffer from the DCN of the mouse model of nerve metabolism disease with the viral vector targeting that can carry out aixs cylinder transportation, in brain stem and spinal cord, all detected transgene protein
20Ironically, transgene protein at those to choline acetyltransterase (ChAT), a nervus motorius meta-tag, be positive and negative cell in all detect.
[0129] mistake of superoxide dismutase-1 (SOD1) gene mutation is expressed summary and has been reappeared the clinical of human ALS and pathology characteristics in mice and the rat.The patient that the reactive compound that delays this model symptom has been shown suffering from ALS has predictable clinical effectiveness, is a kind of treatment correlation model of this disease therefore.Such mouse model is before people such as Tu, (1996) P.N.A.S., 93:3155-3160; People such as Kaspar, (2003) Science, 301:839-842; People such as Roaul, (2005) Nat.Med., 11 (4): people such as 423-428 and Ralph, (2005) Nat.Med., 11 (4): be described among the 429-433.
[0130] current experiment, therefore, the bilateral DCN that attempts to study AAV-IGF-1 transmits Symptomatic (promptly 90 day age) SOD1
G93AThe influence of mouse disease progress.Especially, main target is that can transmission that determine AAV-IGF-1 cause (1) carrier and/or albumen transmission to brain stem and spinal cord; (2) minimizing of the europathology in brain stem and spinal cord; (3) the significant life-span of the raising of motor behavior function and (4) prolongs.The result shows that the CNS zone that viral vector is expelled to interconnected brain stem and spinal cord is to transmit a kind of effective method of potential therapeutic transgenic to brain stem and spinal cord.And our result supports to design the development by the Therapeutic Method of the molecule milieu therapy motor neuron degeneration of modifying it.
(SOD1 is carried out in [0131] two research in G93A SOD1
G93AMutant mice is represented with the SOD1 mice herein).This model has closely been simulated human ALS.The carrying out property motor neuron degeneration that the hind leg movement defect when the about 90 day age of mice, can occur following.When death occurs in about 120-122 days.Each research all has four treatment groups: the mice of 1) accepting the AAV serotype 1 (AAV-IGF-1) of coding IGF-1; 2) accept the mice of the AAV serotype 1 (AAV1-GFP) of encoding green fluorescent protein; 3) accept the mice and 4 of the AAV serotype 2 (AAV2-IGF-1) of coding IGF-1) accept the mice of the AAV serotype 2 (AAV2-GFP) of encoding green fluorescent protein.
[0132] be not confined to theory, IGF-1 is owing to have many benefits to the neuraxon of varying level, therefore be treatment ASL human cytokines (referring to people such as Dore, Trends Neurosci, 1997,20:326-331).In brain: it is considered to reduce neuron and neuroglial programmatic death, and neuroprotective unit avoids the toxicity of being brought out by ferrum element, Colchicine, calcium destabiliser, superoxides and cytokine.It also is considered to regulate the release of neurotransmitter acetylcholine and glutamic acid.It also is considered to induce the expression of neurofilament, tublin and myelin basic protein.In spinal cord: the forfeiture that IGF-1 is considered to regulate the ChAT activity and weakens the cholinergic phenotype, strengthen the motor neuron rudiment, increase myelin and form, suppress demyelination, the stimulus movement neuron is somatic breeding and differentiation in the past, and promotes neurilemma cell division, ripe and growth.In muscle: IGF-1 is considered to induce myoneural junction place acetylcholinergic receptor pack to form and strengthens neuromuscular function and muscle strength.In this experiment, used this proteic IGF-1Ea form.
[0133] green fluorescent protein is as reference protein, and it also makes the expression energy that is mediated by the AAV vector injection observed.
[0134] the birth back is 90 days, and the SOD1 mice is expelled to DCN with AAV recombinant vector bilateral.In a research, the dosage in each site approximately is 2.0e10gc/ml.Some mice was condemned to death after birth in about 110 days, the analyzed GFP dyeing of its brain and spinal cord then, via the IGF-1 of SABC express, via the IGF-1 of ELISA express, via the IGF-1 of RT-PCR express, via ChAT location, the glial fibrillary acidic protein (GFAP) of SABC express, motor neuron counting, the functional test on turntable as described above (wave and quicken), front and back limb grip and survival rate by using the grip dosimeter to carry out.
[0135] again it oneself " just " can not be come in 30 seconds after animal is placed in its oneself back or animal when being looked after the technician and find death by animal, " death incident " is transfused to." death incident " is sorted in the time of evaluation and carried out with the group (GFP is a blind test to IGF-1) of animal by two people.
[0136] the AAV carrier of expressing GFP is expelled to deep cerebellar nuclei (DCN) by bilateral after, at brain stem with spread all over GFP detected (seeing Figure 13 and 14) in each subregion of spinal cord.Figure 22 has shown the distribution of GFP in the mouse brain.Except that DCN, the GFP positive staining is also observed in olfactory bulb, cerebral cortex, thalamus, brain stem, cerebellar cortex and spinal cord.All these zones are also accepted to project DCN from the projection of DCN and/or transmission.In addition, GFP positive fiber and/or cell also are observed in the place near the ChAT positive cell.
[0137] IGF-1mRNA detects in each subregion of mice brain stem of handling with AAV1-IGF-1 or AAV2-IGF-1 and spinal cord, and this proof carrier has experienced reverse transportation (referring to Figure 18).IGF-1 albumen detects in mice brain stem of handling with AAV1-IGF-1 or AAV2-IGF-1 and spinal cord.In the mice of handling, detect (referring to Figure 15-17) in the dyeing of the GFAP in each subregion of mouth nucleus (oromotor nuclei) (for example, motion nuclei quintus, nucleus of facial nerve and nucleus of hypoglossal nerve) and spinal cord minimizing with AAV1-IGF-1 or AAV2-IGF-1.GFAP is the labelling of neurogliosis (it is the pathology sign of ALS).The transmission of AAV1-IGF-1 or AAV2-IGF-1 causes rotating the remarkable functional promotion (referring to Figure 20) with the grip task.The transmission of AAV1-IGF-1AAV2-IGF-1 has also prolonged the life-span of SOD1 mice significantly (referring to Figure 21, median survival is increased to 133.5 or 134 days in the mice that AAV-IGF-1 handles, by comparison, in the mice that AAV-GFP handles, it is 121 or 120 days).Figure 19 shows that the DCN of AAV-IGF-1 transmits the survival rate that has improved motor neuron.Not being both on the statistics between the DCN of mice of handling with the AAV of coding IGF-1 and the AAV of coding GFP transmits is significant, and p value=0.01 is represented with asterisk.
[0138] no matter serotype, AAV-IGF-1 handles the survival rate that has improved motor neuron significantly, has promoted the athletic performance of rotation and grip experiment and significant prolongation the life-span.By using PCR and ELISA to detect, IGF-1 is expressed in whole brain stem and the spinal cord and detects.
[0139] this description can be understood according to the instruction of the list of references of quoting in this description the most up hill and dale.Enforcement division in this manual provides an illustration of the invention process division, should not be interpreted as the restriction to scope of the present invention.Those skilled in the art easily obtain many other enforcement divisions and also be the present invention includes.All publications, patent and the biological sequence of quoting in the disclosure all introduced as document with its whole integral body.If material that document comprises and the application's description are conflicting or inconsistent with this description, then are as the criterion with this description.Quoting of any document do not admit that such document is a technology formerly of the present invention herein.
[0140] except as otherwise noted, comprise that in this description all numerals of the quantity of the expression composition that uses in the claim, cell culture, treatment situation or the like should be understood that all to be modified by term " approximately " in all cases.Correspondingly, unless specify in addition, with the parameter of numeral the character of approximation and may very the depend on the present invention expectation being attempted to obtain.Unless show in addition, the term before series of elements " at least " should be understood to mean each element in this series.Only by using routine experiment, those those of skill in the art will discern the many equivalents that maybe can determine the specific embodiments described among the present invention.Same valency thing so also is considered as being included in the following claim.
List of references
1.Leigh，P.N.&Swash，M.Cytoskeletal pathology in motor neurondiseases.Adv Neurol 56，115-24(1991).
2.Carpenter，S.Proximal axonal enlargement in motor neuron diseaseNeurology 18841-51(1968).
3.Gonatas，N.K.et al.Fragmentation of the Golgi apparatus of motorneurons in amyotrophic lateral sclerosis.Am J Pathol 140，731-7(1992).
4.Hirano，A.et al.Fine structural study of neurofibrillary changes in afamily with amyotrophic lateral sclerosis.J Neuropathol Exp Neurol 43，471-80(1984).
5.Leigh，P.N.et al.Ubiquitin-immunoreactive intraneuronal inclusions inamyotrophic lateral sclerosis.Morphology，distribution，and specificity.Brain 114(Pt 2)，775-88(1991).
6.Delisle，M.B.&Carpenter，S.Neurofibrillary axonal swellings andamyotrophic lateral sclerosis.J Neurol Sc/63，241-50(1984).
7.Hirano，A.Neuropathology of ALS：an overview.Neurology 47，S63-6(1996).
8.Rosen，D.R.et al.Mutations in Cu/Zn superoxide dismutase gene areassociated with familial amyotrophic lateral sclerosis.Nature 362，59-62(1993).
9.Gurney，M.E.et al.Motor neuron degeneration in mice that express ahuman Cu1Zn superoxide dismutase mutation.Science 264，1772-5(1994).
10.Rowland，L.P.&Shneider，N.A.Amyotrophic lateral sclerosis.N Engl JMed 344，1688-700(2001).
11.Bruijn，L.I.，Miller，T.M.&Cleveland，D.W.Unraveling the mechanismsinvolved in motor neuron degeneration in ALS.Annu Rev Neurosci 27，723-49(2004).
12.Cleveland，D.W.&Rothstein，J.D.From Charcot to Lou Gehrig：deciphering selective motor neuron death in ALS.Nat Rev Neurosci 2，806-19(2001).
13.Lindsay，R.M.Neurotrophic growth factors and neurodegenerativediseases：therapeutic potential of the neurotrophins and c iiiary neurotrophicfactor.Neurobiol Aging 15，249-51(1994).
14.Kaspar，B.K.，Llado，J.，Sherkat，N.，Rothstein，J.D.&Gage，F.H.Retrograde viral delivery of IGF-1prolongs survival in a mouse ALS model.Science 301，839-42(2003).
15.Clement，A.M.et al.Wild-type nonneuronal cells extend survival ofSOD1mutant motor neurons in ALS mice.Science 302，113-7(2003).
16.Matsushita，M.Projections from the lowest lumbar and sacral-caudalsegments to the cerebellar nuclei in the rat，studied by anterograde axonaltracing.J Comp Neurol 404，21-32(1999).
17.Matsushita，M.&Gao，X.Projections from the thoracic cord to thecerebellar nuclei in the rat，studied by anterograde axonal tracing.J CompNeurol 386，409-21(1997).
18.Matsushita，M.&Xiong，G.Projections from the cervical enlargement tothe cerebellar nuclei in the rat，studied by anterograde axonal tracing.J CompNeurol 377，251-61(1997).
19.Matsushita，M.&Yaginuma，H.Afferents to the cerebellar nuclei fromthe cervical enlargement in the rat，as demonstrated with the Phaseolusvulgaris leucoagglutinin method.Neurosci Lett
13，253-9(1990).
20.Matsushita，M.&Yaginuma，H.Projections from the central cervicalnucleus to the cerebellar nuclei in the rat，studied by anterograde axonal tracing.J Comp Neurol 353，234-46(1995)；Voogd，J.The cerebellar nuclei and theirefferent pathways，in The rat nervous system (ed.Paxinos，G.)208-215(Elsevier Academic Press，San Diego，2004).
Sequence table
＜110〉Jianxin Company
＜120〉gene of spinal disorders is controlled
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Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala
35 40 45
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe
50 55 60
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly
65 70 75 80
Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys
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Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu
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Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp
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Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly
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Ser Ala Gly Asn Lys Asn Tyr Arg Met
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Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala
35 40 45
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe
50 55 60
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly
65 70 75 80
Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys
85 90 95
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu
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Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp
115 120 125
Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr
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Lys Ser Gln Arg Arg Lys Gly Trp Pro Lys Thr His Pro Gly Gly Glu
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Glu Gln Arg Arg Glu Ile Gly Ser Arg Asn Ala Glu Cys Arg Gly Lys
180 185 190
Lys Gly Lys
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Claims (17)
1. a method comprises that administration contains the neurophilic viral vector of genetically modified reorganization at least one zone to the cerebellar nuclei district, deep of brain, and wherein said administration is carried out under the situation that transgene product is delivered to spinal cord and/or brain stem helping.
2. a method comprises that administration contains the nervus motorius cortex zone of the neurophilic viral vector of genetically modified reorganization to brain, and wherein said administration is carried out under the situation that transgene product is delivered to spinal cord helping.
3. one kind is transmitted the method that transgenic arrives experimenter's motor neuron, comprise that administration contains the neurophilic viral vector of described genetically modified reorganization at least one zone to the cerebellar nuclei district, deep of brain, the motor neuron away from described administration site is expressed or be delivered to wherein said transgenic in away from the motor neuron in described administration site.
4. one kind is transmitted the method that transgenic arrives experimenter's motor neuron, comprise that administration contains the nervus motorius cortex zone of described genetically modified neurophilic viral vector to brain, the motor neuron away from described administration site is expressed or be delivered to wherein said transgenic in away from the motor neuron in described administration site.
5. treat the method that experimenter's motor neuron is lacked of proper care for one kind, comprise administration and contain the neurophilic viral vector of the genetically modified reorganization of therapeutic at least one zone to the cerebellar nuclei district, deep of brain, wherein this transgene product is delivered at least one subregion of spinal cord and/or at least one subregion of brain stem with the treatment effective dose.
6. method of improving experimenter's motor neuron diagonosis of disorder, comprise that administration contains the neurophilic viral vector of the genetically modified reorganization of therapeutic to brain nervus motorius cortical area, wherein this transgene product is delivered at least one subregion of spinal cord with the treatment effective dose.
7. the method for any one of claim 1 to 6, wherein said neurophilic viral vector is gland relevant viral vector (AAV).
8. the method for any one of claim 1 to 6, wherein said neurophilic viral vector is the gland relevant viral vector that is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.
9. claim 1,3 or 5 any one method, the zone in cerebellar nuclei district, wherein said brain deep is selected from medial area, mesozone or LHA.
10. the method for any one of claim 1 to 6, wherein said transmission is a bilateral.
11. the method for claim 5 or 6, wherein said spinal cord subregion is selected from cervical region subregion, chest subregion, waist subregion or sacrum and partly distinguishes.
12. claim 5 or 6 method, wherein said transgenic is passed to all subregions of spinal cord.
13. the method for claim 12, at least a of wherein said multiple administration is bilateral.
14. the method for any one of claim 1 to 6, wherein said transgenic are selected from insulin-like growth factor-1 (IGF-1), calbindin D28, paralbumin, HIF1-alpha, SIRT-2, VEGF, SMN-1, SMN-2 or CNTF (ciliary neurotrophic factor).
15. the method for claim 6, wherein said transgene expression have alleviated the symptom of the motor neuron imbalance that is selected from amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), spinal cord bulbar muscular atrophy, spinal cord cerebellar ataxia, Duchenne-Arandisease or traumatic spinal cord injury.
16. any one method of claim 1 to 6, wherein said experimenter is a human patients.
17. any one method of claim 1 to 6, the albumen of wherein said transgene expression therapeutic dose is selected from insulin-like growth factor 1 (IGF-1), EPO (erythropoietin), CBP ([CREB] is conjugated protein for the cAMP response element binding protein), calbindin D28, paralbumin, HIF1-alpha, SIRT-2, VEGF, SMN-1, SMN-2 or CNTF (ciliary neurotrophic factor).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US67721305P | 2005-05-02 | 2005-05-02 | |
| US60/677,213 | 2005-05-02 | ||
| US60/790,217 | 2006-04-08 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610153759.2A Division CN105770909A (en) | 2005-05-02 | 2006-05-02 | Gene Therapy For Neurometabolic Disorders |
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| Publication Number | Publication Date |
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| CN101208108A true CN101208108A (en) | 2008-06-25 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA200680023365XA Pending CN101208108A (en) | 2005-05-02 | 2006-05-02 | Gene therapy for spinal disorders |
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| CN (1) | CN101208108A (en) |
| SI (1) | SI1879623T1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113302202A (en) * | 2018-07-17 | 2021-08-24 | 脑若脉仁股份有限公司 | Treatment of neuropathy using deoxyribonucleic acid constructs expressing insulin-like growth factor 1 isoforms |
| CN113587811A (en) * | 2021-07-24 | 2021-11-02 | 中交四公局（北京)公路试验检测科技有限公司 | Bridge measuring point positioning method, control device, system and medium |
| WO2024021431A1 (en) * | 2022-07-28 | 2024-02-01 | 深圳先进技术研究院 | Use of gene delivery system in retrograde delivery of gene from brain to spinal neuron |
-
2006
- 2006-05-02 CN CNA200680023365XA patent/CN101208108A/en active Pending
- 2006-05-02 SI SI200631484T patent/SI1879623T1/en unknown
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113302202A (en) * | 2018-07-17 | 2021-08-24 | 脑若脉仁股份有限公司 | Treatment of neuropathy using deoxyribonucleic acid constructs expressing insulin-like growth factor 1 isoforms |
| CN113302202B (en) * | 2018-07-17 | 2024-01-30 | 赫利世弥斯株式会社 | Treatment of neuropathy with DNA constructs expressing insulin-like growth factor 1 isoforms |
| CN113587811A (en) * | 2021-07-24 | 2021-11-02 | 中交四公局（北京)公路试验检测科技有限公司 | Bridge measuring point positioning method, control device, system and medium |
| WO2024021431A1 (en) * | 2022-07-28 | 2024-02-01 | 深圳先进技术研究院 | Use of gene delivery system in retrograde delivery of gene from brain to spinal neuron |
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| SI1879623T1 (en) | 2013-01-31 |
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