CN101213305A - Regulated expression of transgenes in the central nervous system of mammals - Google Patents

Abstract

Recombinant adeno-associated virus (rAAV) vectors are provided for delivery of regulatable transgenes to the central nervous system (CNS) of a mammal. Also provided are methods of treatment of subjects with neurodegenerative disorders using the vectors, and kits for constructing or using the vectors or performing the methods of the invention. Transgene sequences are expressed from a promoter/enhancer region comprising one or more binding sites for a transcription factor that is responsive to a small molecule inducer. Both the transgene construct and a construct comprising the transcription factor are delivered to the target cells. The regulatable transgene may be delivered on the same rAAV vector as the transcription factor, or on a separate vector. The transcription factor may comprise two polypeptide chains, e.g. a DNA binding domain and a transcription activation domain, that form an active dimer in the presence of a dimerizer such as rapamycin or a non-immunogenic analog thereof. Vectors, methods and kits of the invention may be used to deliver genes such as AADC or GDNF to the brain of a subject with a neurodegenerative disorder, such as Parkinson's disease, where the expression of AADC or GDNF in the brain can subsequently be regulated by treatment of the subject with rapamycin or a rapamycin analog.

Description

The regulatable type of transgenosis in mammalian central nervous system expressed

Technical field

The present invention relates to the regulation and control of the gene of gene therapy importing, regulation and control transduction specifically enters the genetically modified expression of mammalian central nervous system (CNS).

Background technology

Many human diseasess are to be caused by gene abnormal expression.When genetic expression is not enough, or when the defectiveness of gene product own, the disappearance of corresponding function gene product can be delivered to the patient and treat by the gene product that will lose.Yet proteinic sending usually had any problem, and only gives benefit in the limited time, and the meaning refers to that described protein must repeat administration more termly, perhaps needs irregular administration, as existing under the situation of chronic disease.The repeat administration expense is very high, inconvenient, and may make suffer owing to patient's conformability of difference.And time behind dosage of administration just and the time before another dosage of administration, the level of gene product can change significantly.The variability of this level is to therapeutical agent with poor pharmacokinetics (for example, transformation period short) or low therapeutic index problem particularly.

Gene therapy can be used for gene, rather than gene product is delivered to the cell that demonstrates suboptimal those genes.Except replenishing the insufficient or defective native gene of expression, gene therapy also can be sent other gene, described other gene may be worked as can have beneficial effect when it is expressed in target cell, for example the protein of cytokine, hormone, antibody or genetic modification.In this article, the gene of sending by gene therapy is meant transgenosis.When transgenosis in target cell during stably express, gene therapy has the potential of the gene product of irregularly sending maintenance level.Self compare with the repeating delivery gene product, gene therapy only needs to implement once, and is perhaps rare at least.As the method for delivery treatments protein to brain, gene therapy is particularly preferred, because because hemato encephalic barrier, the protein that is administered systemically can not enter brain, and direct three-dimensional locating injection to enter brain be unpractical for repeat administration.

Gland relative virus mediated gene therapy

Studied and be used to assist the efficient transgenosis of sending to target cell virus vector, this method is called transduction in this article.A kind of viral system that has been used to gene delivery is adeno-associated virus (AAV).AAV is the parvovirus that belongs to dependovirus.AAV has some undiscovered attractive feature in other virus.At first, AAV can infect a large amount of host cells, comprises Unseparated Cell.The ability that infects Unseparated Cell makes the AAV CNS that becomes to be used to transduce organize for example good especially selection of brain.The second, AAV can infect from different types of cell.As if the 3rd, AAV has nothing to do with any mankind or Animal diseases, and can not change the biological characteristics of the host cell in integration.Even, there is the people to be exposed to this virus according to the human crowd of meter 80-85%.At last, AAV is stable under a large amount of physics and electrochemical conditions, and it is convenient to generate, stores and transportation.

The AAV genome is linear single strand dna, and length is about 4681 Nucleotide (nt).The AAV genome generally includes the non-duplicate genes group in inside that connects by the reverse repetition (ITRs) at each end.ITRs is long for about 145nt.ITRs plays the dna replication dna source and is used for virus genomic packaging signal.

For duplicating for (rep) and housing (cap) gene of AAV, the non-repeating part in genomic inside comprises two main open reading-frame (ORF)s.Those protein that make virus replication and viral genome is packaged into virosome of rep and cap genes encoding.Especially, expressed from least four virus protein families in AAV rep zone: Rep78, Rep 68, Rep 52 and Rep 40, according to their apparent molecular weight name.At least three protein of AAV cap regional code: VP1, VP2 and VP3.

AAV is auxiliary dependovirus (helper dependent virus), that is, in order to form the AAV virosome, it need infect simultaneously with helper virus (for example adenovirus, simplexvirus or vaccinia virus) usually.Do not exist under the situation about infecting simultaneously with helper virus, AAV has formed latent state, and wherein viral genome continues as episome or inserts host cell chromosome, but can not generate infective virosome.The genome that the infection of helper virus subsequently " relief " is integrated makes its replicator group and is packaged into infective AAV virosome.Although when AAV can infect from different types of cell, helper virus must have identical kind, as host cell.Therefore, for example, human AAV will duplicate in the Canidae cell that infects simultaneously with the Canidae adenovirus.

The AAV carrier through design by deleting the non-repeating part in the genomic inside of AAV (being rep and cap) and between ITRs, inserting heterologous gene (" transgenosis) send interested gene.Transgenosis may be relevant with allogeneic promoter.Also can comprise termination signal, for example the polyadenylation site.

In order to produce the original seed (stock) that comprises genetically modified infective reorganization AAV, with the suitable production clone of AAV expression vector transfection, described expression vector comprises the transgenosis that is clipped between the AAV ITRs.AAV subsidiary function and auxiliary function are also expressed in producer's cell.When wild-type AAV (wtAAV) duplicates in the cell by helper virus (for example adenovirus) co-infected, those functions that subsidiary function provides for the gene (for example rep and cap) of being encoded by AAV usually, auxiliary function are those functions that provided by helper virus usually.The gene of coding subsidiary function must provide respectively in wtAAV, because they are transferred to abdicate the space to transgenic sequence in the structure of rAAV carrier.Under the situation that has subsidiary function and auxiliary function, comprise that the carrier structure of transgenosis and AAVITRs is replicated and packs formation reorganization AAV virosome (rAAV).The generation of rAAV original seed further describes in United States Patent(USP) Nos. 5,622,856; 6,001,650; 6,027,931; 6,365,403; 6,376,237; 5,945,335; In 6,004,797 and 6,482,633, wherein disclosed content all is incorporated herein by reference with it in this article.

Therefore, so the rAAV original seed of preparation can be by being used for transgenosis is introduced external target cell with the rAAV target cell infection not existing under the helper virus, or in the introducing patient body.Because patient's cell lacks rep and cap gene and auxiliary function gene, so in target cell, the rAAV carrier is defective for duplicating; That is, they can not further duplicate and pack their genome.Similarly, do not having in patient's cell, can not to form wtAAV under rep and the cap GENE SOURCES.

The regulation and control of transgene expression

In case a possible shortcoming of AAV mediated gene therapy and common gene therapy be its by administration, just reverse the ability of handling or adjusting its effect.Send differently with traditional medicine, whenever it can end as required, and successful gene therapy provides stable long-term genetic expression behind drug administration carrier.In the treatment human patients, the ability that stops transgene expression is important security consideration.And the ability of downward modulation transgene expression will be controlled in (administration) useful by the suitable pharmacology of transgene product in guaranteeing the patient.

After deliberation use the small molecules inductor to regulate and control the various systems of transgene expression.Rivera etc. (1996) Nat.Med.2:1028-32; No etc. (1996) Proc.Natl.Acad.ScL USA, 93:3346-51; Gossen and Bujard (1992) Proc.Natl.Acad.Sci USA 89:5547-51;

System (Valentis, Inc.Burlingame, California).These systems are based on transcription factor and the transgenosis of utilizing design, and the activity of the transcription factor of described design can be controlled by small-molecule drug, and described genetically modified expression drives by the transcription factor of regulation and control.A kind of such system induces (being referred to herein as " dipolymer (dimerizer) system ") based on rapamycin, comprises from two synthetic property fused proteins forming functional transcription factor, depends on the rapamycin of adding.Rivera etc. (1996) Nat.Med.2:1028-32. rapamycin is the available small-molecule drug of a kind of oral biology, itself and natural product immunosuppressor FK506 are closely related, it is bonded to the protein FKBP12 of cell with high affinity (200pM), and then, mixture is in conjunction with FRAP.Although rapamycin has interior inherent immunosuppressive activity, after deliberation non-immunosuppression analogue, it can use the startup in the dipolymer system transcribe with the FRAP gene order of modification, and does not have undesired immunosuppression.Pollock etc. (2000) Proc.Natl.Acad.Sci USA 97:13221-26.

Be used for being described in greater detail among (2000) Proc.Natl.Acad.Sci.USA 97:13221-26 of (1996) Nat.Med.2:1028-32 of Rivera etc. and Pollock etc. of genetic regulation in the body based on the dipolymer system.The dipolymer system is suitable for using with delivery of gene to muscle (Ye etc. (1999) Science 283:88-91 with virus vector; Rivera etc. (1999) Proc.Natl.Acad.Sci USA 96:8657-62; Johnston etc. (2003) Mol.Ther.7:493-7), liver (Auricchio etc. (2002) Gene Ther.9:963-71) and eye (Auricchio etc. (2002) Mol.Ther.6:238-42).Described dipolymer system also is ARIAD Pharmaceuticals, Inc. (Cambridge, the component of ARGENT transcription technique platform Massachusetts).This technology further is disclosed in the U.S. Patent No. 6 of Crabtree etc., in 043,082 and the U.S. Patent No. 6,649 of Clackson etc., in 595, at No.6, generality has been described ARGENT based on the dipolymer system in 043,082, at No.6, the dipolymer system that uses based on rapamycin or forms of rapamycin analogs has been described, the immunosuppressant rapalogs that it comprises synthetic DNA binding domains, p65 transcriptional activation domain and has reduction in 649,595.In this article with United States Patent (USP) .Nos.6, disclosed content all is incorporated herein by reference with it in 043,082 and 6,649,595.

Described dipolymer system has the advantage of full-length human, and wherein the sequence of the functional component of transactivator fusion protein matter all derives from the human protein, thus reduced in the mankind disadvantageous immunne response may.Rivera etc. (1999) Proc.Natl.Acad.Sci.USA 96:8657-62.

Parkinson's disease (PD)

Parkinson's disease (PD) is for may receptor gene treating the particularly example of the disease of AAV gene therapy.In the U.S., PD is second kind of modal neurodegenerative disease, has influenced to surpass a million people.PD is characterized as autokinetic movement minimizing, difficulty in walking, posture unstable, stiff and vibration.These clinical symptom are the direct result that the painted neurone (being dopaminergic neuron) in the black substance in the basal ganglion zone of brain is degenerated.The gradual degeneration of black substance causes the efficient reduction of Dopamine HCL, because the painted neurone of black substance is the position of synthetic this important catecholamine neurotransmitter.Dopamine HCL is at the terminal synthetic of the terminal nerve of the dopaminergic neuron of black substance, particularly at the black substance compact part.Plan enters striatal dopaminergic nerve, arranges shell nuclear and caudate putamen especially.Three kinds of enzymes are that to be used for effective biosynthesizing of Dopamine HCL necessary: tyrosine hydroxylase (TH), GTP cyclohydrolase I (GCH), and aromatic l-amino acid decarboxylase (AADC).Tyrosine hydroxylase adds to amino acid tyrosine with hydroxyl, has made up L-dihydroxyphenylanaline (levodopa).GCH catalysis BH ₄The biosynthetic the first step and rate-limiting step, it is to be used for the active essential cofactor of TH.At last, AADC removes the terminal carboxyl(group) of levodopa, generates Dopamine HCL.

At present, the treatment of PD comprises the combination of the periphery inhibitor of common oral administration levodopa and AADC..Along with the development of PD, Most patients is standing the minimizing of the infected area AADC content (being black substance) of brain.Because AADC is that to be used for that levodopa is changed into Dopamine HCL needed, the levodopa of the dosage that progressively raises is that result of treatment is needed, but this can cause that usually side effect increases.In addition, along with black substance is little by little rotten, the AADC disappearance exists, and reaches the level that the treatment benefit of administration L levodopa is not reentried usually.

AADC

A kind of PD of treatment based on gene therapy methods for provide coding one or more participate in the gene of the biosynthetic enzyme of Dopamine HCLs, for example AADC.The AADC that replenishes has recovered the effect of levodopa treatment.AAV deutero-carrier and be described in U.S. Patent Application Publication No.2002/0172664 by the method for sending and express AADC treatment PD in the brain of mammalian subject, wherein disclosed content all is incorporated herein by reference with it in this article.

Parkinson's disease (PD) is characterised in that the serious minimizing of Dopamine HCL in the gradual loss of the dopaminergic neuron in the black substance and the striatum.Hornykiewicz(1975)Natl.Inst.Drug Abuse Res.Monogr.Ser.13-21。The 6-OHDA model extends striatal medial forebrain bundle by chemical damage from black substance and produces, and histopathology is similar to PD.Ungerstedt (1971) Acta Physiol.Scand.Suppl.367:69-93.It is active that the rat of one-sided 6-OHDA infringement has produced the quick bilateral rotation that the levodopa or the dopamine-receptor stimulant of therapeutic dose are replied on a hemisphere.Ungerstedt(1971)。In Parkinson's disease (PD) animal model, existing studies show that out can be recovered Dopamine HCL to level of significance with reduce the levodopa demand with the gene delivery of the human AADC of coding (hAADC) to rat or non-human primates striatum and the exogenous levodopa of sending.Bankiewicz etc. (2000) Exp.Neurol.164:2-14; Sanchez-Pernaute etc. (2001) Mol.Ther.4:324-30.

GDNF

Another that is used for the treatment of PD is to send the gene that can block or delay ongoing deterioration process based on gene therapy methods.Glial cell line deutero-neurotrophic factor (GDNF) is effective neurotrophic factor, and it has demonstrated in external and PD animal model has increased the DA neuron survival rate.(Brain Res. (2000) 886:82-98 such as Bjorklund; Bohn, M.C.Mol.Ther. (2000) 1:494-496; Deng J.Neural Transm.Suppl. (2000) 58:181-191), wherein disclosed content all is incorporated herein by reference with it in this article.AAV deutero-carrier and be described in U.S. Patent Application Publication No.2003/0050273 by send and express the method that GDNF treats PD in the brain of mammalian subject, wherein disclosed content all is incorporated herein by reference with it in this article.

Although AADC or the GDNF expression in the disturbances in patients with Parkinson disease brain may be useful, the overexpression of this gene can cause dangerous side effect.The typical carrier that uses in gene therapy is to be used for introducing strong constitutive promoter (strong constitutive promoters) to guarantee result of treatment, and described promotor is the comprehensive transgene expression that is used for maximizing in the patient.The expression of those a few cell of being transduceed of maximization is attempted in researchs before many, when the expection transduction efficiency is low relatively, and when transgene product be to enter the body circulation time by the transducer cell secretion, it may be a reasonable target.Research has related to these secreted proteins before the transgene expression of some dipolymer regulation and control.Rivera etc. (1996) Nat.Med.2:1028-32; Rivera etc. (1999) Proc.Natl.Acad.Sci.USA 96:8657-62; Ye etc. (1999) Science 283:88-91; Pollock etc. (2000) Proc.Natl.Acad.Sci.USA 97:13221-26; Auricchio etc. (2002) Gene Ther.9:963-71; Johnston etc. (2003) Mol.Ther.7:493-7; Auricchio etc. (2002) Mol.Ther.6:238-42.

Different with the secreted protein that product is built up in the recycle system, if the non-secretory transgene product is crossed expression at some cell, and when in other cell, lacking, can not obtain to treat benefit.The level of utilizing the proteinic gene therapy of non-secretory regulating transgene expression can be wanted to each transfectional cell, it makes that such protein is regulatable this particularly important after transduction.

The gene therapy of neurodegenerative disease and other CNS obstacle is the field (Tinsley and Eriksson (2004) Acta Neurol.Scand.109:1-8) of an expansion, and transgenic regulation may be necessary for dosed administration control and purpose of safety.Though regulate gene expression may be optional under the situation of AADC treatment, because can control by exogenous its precursor levodopa of sending on the generation composing type of Dopamine HCL, but having to benefit, regulation and control control sending or causing that treatment stops of doses enzyme exactly, if necessary.Regulation and control may be the prerequisites that is used for the gene delivery of neurotrophic factor (for example GDNF), and wherein said neurotrophic factor is crossed expression can have deleterious effect, can determine the control degree of needs by special applications.

Exist be used for regulating and control by gene therapy be incorporated into the patient CNS (for example brain) expression of gene method and start the needs of the carrier of this regulation and control.Particularly, exist and to be used for transgenosis at the needs of suffering from the neurodegenerative disease method that for example regulatable type is expressed in the patient's of PD the brain.Preferably, this regulator control system has low transgene expression basal level under the condition that suppresses, and demonstrates high induction ratio.Most preferred system also will comprise the functional component of special derived from human proteinoid, and described component can make and reverse (adverse) immunoreactive probability minimum during the human gene therapy.

Summary of the invention

In this area these and other needs to realize that it is provided for carrier, method and the test kit of AAV mediated gene therapy by the present invention that wherein the expression in the target cell of transgenosis in neural system can use inductor to regulate and control.

In one aspect, AAV (rAAV) carrier that the present invention relates to recombinate, wherein transgenosis was entered patient's CNS (for example brain) by transduction after, transgene expression can be regulated and control.

In one embodiment, regulation and control are to use the transcription factor that comprises two peptide species components to realize, only when two kinds of components are interosculated, described transcription factor is only activated, wherein exists under the situation of inductor, and described component is distinguished combination independently.In one embodiment, described two peptide species comprise that the DNA binding domains merges and activation domain merges.A kind of exemplary DNA binding domains fusion comprises two DNA binding domainss from human transcription factor Zif268, the homeodomain that is derived from Oct-1 and three medicine binding domainss from the FK506 acceptor of kytoplasm.Exemplary activation domain fusion comprises the binding domains of fusion to the human FRAP rapamycin of the transcriptional activation domain of the p65 subunit that is derived from NFKB.

In one embodiment, a rAAV carrier comprises transgenosis, and the 2nd rAAV carrier comprises the sequence of transcription factor component.In another embodiment, single rAAV carrier comprises the sequence and the transgenosis of transcription factor component.

In one embodiment, regulation and control realize by the administration inductor.In another embodiment, inductor is a dipolymer, for example rapamycin or its non-immunosuppression analogue, for example AP21967.

In yet another aspect, the present invention relates to the method with rAAV vehicle treatment patient, wherein transgenosis was entered patient's CNS (for example brain) by transduction after, transgene expression can be regulated and control.In one embodiment, methods of treatment comprises the administration inductor, and dipolymer for example is such as rapamycin or its non-immunosuppression analogue, for example AP21967.

In yet another aspect, the test kit that the present invention relates to make up carrier of the present invention or implement method of the present invention.In one embodiment, this test kit comprises having polylinker zone the one rAAV carrier, and it is used to clone the interested transgenosis of user.In another embodiment, described test kit further comprises the 2nd rAAV carrier of the encoding transcription factor, and it can regulate and control the genetically modified expression that the clone enters a rAAV carrier.In another embodiment, test kit comprises a kind of inductor, and dipolymer for example is such as rapamycin or its non-immunosuppression analogue, for example AP21967.

In one embodiment, the present invention relates to treat for example Parkinson's disease (PD) of human nerve's degenerative disease.

In certain embodiments, the regulatable type transgenosis is AADC or GDNF, although the regulatable type transgenosis can be any gene of wanting for its expression in target tissue usually.

For those of ordinary skills, according to disclosed content in this article, these and other embodiment of the present invention will be carried out at an easy rate.

Description of drawings

The synoptic diagram of the activation of the transcription factor that Figure 1A is used to regulate and control transgene expression for encoding and the rAAV carrier (AAV-CMV-TF) of DNA binding domains.In this article, such carrier is called the transcription factor carrier.

Figure 1B is the synoptic diagram of the reorganization AAV carrier of coding hAADC (AAV-Z12 hAADC), and the expression of described carrier can be regulated and control by adding the rapamycin or derivatives thereof.In this article, such carrier is called expression vector.

Fig. 1 C is the synoptic diagram of the reorganization AAV carrier of coding hAADC (AAV-CMV-hAADC), and the expression of described carrier is by the CMV promoters driven of composing type.

Fig. 2 has shown the result of following test: wherein with multiple rAAV construction transduction D7-4 cell, subsequently with 0,5 or the forms of rapamycin analogs AP21967 processing of 25nM.Also shown non-transduction contrast." OD " value refers to use by the relative AADC that calculates with the typical curve that obtains with reference to the cell of measuring the AAV-CMV-hAADC2 transduction to be expressed (as pass through OD in AADC expression ELISA ₄₀₅Detect).

Fig. 3 is the time line of following test: wherein the rotation of levodopa being replied is the function of the rapamycin treatment measured in the rat of 6-OHDA infringement.

Fig. 4 has shown that the rat of 6-OHDA infringement replys the rotation of 5mg/kg levodopa, the expression in the rotation of offside per hour, and it is the function along time of the time line that shows in Fig. 3.Rat is with vehicle independent (contrast) or dabbling with carrier and the corresponding transcription factor of the adjustable hAADC of coding.First the week and second week, data did not appear.Infusion carried out at the 0th day, and as diagrammatic in Fig. 3, it is served as reasons, and it begins to measure the date (for example " 3 week " refer to perfusion back the 21st day) of the quantity in week.The group of injecting 4 rapamycins (" rap ") in Fig. 3 the every day that shows for the sake of simplicity, is expressed as single arrow in Fig. 4.

Fig. 5 A has shown the immunohistochemical result of AADC in the striatum of the rat of using rapamycin treatment with 7 weeks of AAV carrier perfusion back, and described AAV carrier is loaded with the rapamycin regulatable type that is used for hAADC and expresses necessary sequence.The scale bar is represented 75 μ m.

Fig. 5 B shown be similar to the result of experiment that shows in Fig. 5, but rat rapamycin treatment of no use wherein.

Fig. 6 has shown the immunohistochemistry result from AADC in whole embedding brain (mounted brain) part of the representative rat in three treatment group that 7 weeks obtained after perfusion: A: carrier-dabbling (+) rap; B: carrier dabbling (-) rap; C: vehicle-dabbling (+) rap.As shown in Figure 4, rat is separately with excipient (Fig. 6 C) or dabbling with carrier and the corresponding transcription factor (Fig. 6 A and 6B) of the adjustable hAADC of coding.Subsequently, with the animal (as shown in FIG. 3) of rapamycin treatment in Fig. 6 A and 6C, still do not handle the animal in Fig. 6 B.Left cerebral hemisphere is 6-OHDA infringement and the dabbling position of intrastriatal carrier (or vehicle), and right cerebral hemisphere shows the intrinsic staining from complete rat AADC positive fiber.

Fig. 7 demonstrates in three treatment group discussing in reference Fig. 6, and the expression of 7 all AADC after perfusion is as measuring by western blot analysis.Top collection of illustrative plates is the image of observed AADC and β Actin muscle band in from the gel electrophoresis of protein in the representative animal brain in each of three treatment group, and the β Actin muscle is included as contrast.Histogram has shown image intensity (average integrated image intensities) and the standard deviation that the mean value of the protein belt of the AADC band that is used for three animals of each treatment group is gathered.

Fig. 8 A is rAAV carrier (AAV-TF-Z8-hGDNF) figure, described carrier comprises the transcription product that activation domain merges and the DNA binding domains merges of the encoding transcription factor, the adjustable transcription product of coding transgenosis hGDNF, and wherein hGDNF expresses and can regulate and control by adding the rapamycin or derivatives thereof.

Fig. 8 B is the figure that is similar to the carrier that shows in Fig. 8 A, difference is that the expression of the DNA binding domains of transcription factor is driven by SV40 promotor on the transcription product independently rather than activation domain, and activation domain is to be expressed by opposite strand (in the opposite direction promptly).

Fig. 8 C is driven by the CMV promotor (AAV-CMV-hGDNF) of composing type for the synoptic diagram of the reorganization AAV carrier of coding hGDNF, the expression of described carrier.

Fig. 9 has shown that the GDNF with the HeLa D7-4 cell of CMV-GDNF plasmid (AAV-CMV-hGDNF) transient transfection of regulatable type TF-GDNF plasmid (AAV-TF-Z8-hGDNF) or composing type expresses, in pico-gram (pg), it is 0,5 or the function (function) of 25nM rapamycin treatment.

Embodiment

Except as otherwise noted, protein chemistry, biological chemistry, recombinant DNA technology and the pharmacological ordinary method in the art technology used in enforcement of the present invention.Such technology is what prove absolutely in the literature.Referring to, for example, T.E.Creighton, Proteins:Structures and Molecular Properties (W.H.Freeman and Company, 1993); A.L.Lehninger, Biochemistry (Worth PublishersInc.current addition); Sambrook, et al.Molecular Cloning:A LaboratoryManual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N.Kaplan eds.Academic Press, Inc.); Remington ' s Pharmaceutical Sciences, 18th Edition (Easton, Pa.Mack Publishing Company, 1990).

The I definition

Except as otherwise noted, all scientific terminologies that use in this application and technical term have the conventional implication of using in this area.As using in this application, following word or phrase have specified implication.

Must be pointed out that as using, singulative " " and " described " comprise plural implication in this specification sheets and accessory claim book, unless content has regulation clearly in addition.Therefore, for example, " carrier " comprises the mixture of the carrier that two or more are such etc.

Term " transgenosis " refers to can be delivered to arbitrarily the gene of target cell as used herein, and does not consider genetically modified source, comprises the other copy or the sequence variant of native gene in the target cell Already in certain embodiments.Transgenosis can be the genetic engineering varient or synthetic (non-natural existence) dna sequence dna of the gene order that obtains from any organism or portion gene sequence, this gene.Transgenosis can directly be generated by the messenger RNA(mRNA) s of coded protein or codified biologic activity RNA molecule, and described biologic activity RNA molecule is antisense, ribozyme, formation triple helical, RNAi or other RNA sequence for example.

" adjustable type gene (regulatable) " is that its expression can change by the administration inductor after gene transfer was entered the gene of target cell as used herein.The regulation and control of therapeutic transgene expression provide the pharmacology control of transgene product level.

" expression cassette (Expression cassette) " refers to instruct the assembly of interested sequence or genetic expression.Expression cassette comprises the promotor or the promotor/enhanser of its be operably connected (transcribing so that instruct) interested sequence or gene, also comprises the polyadenylic acid sequence usually.In certain embodiments of the invention, the expression cassette of describing in this article can be included in the adeno-associated virus construction.

That " reorganization " that is used to describe nucleic acid molecule as this paper refers to is genomic, the polynucleotide of cDNA, virus, semisynthetic or synthetic source, and according to its source and operation, all or part polynucleotide of itself and its natural combination are irrelevant.Refer to the polypeptide that common express recombinant polynucleotide produce as the term " reorganization " relevant with protein and peptide that uses, usually, clone interested gene, then, the biological expression in vivo transforming further describes as following.Host organisms is expressed can produce proteinic foreign gene under expression condition.

The sequence of " encoding sequence " or " coding " selectivity polypeptide is when under the control that is placed on suitable regulating and controlling sequence (or " controlling elements "), is transcribed (under the DNA situation) in vivo and translation (under the mRNA situation) is the nucleic acid molecule of polypeptide.The border of encoding sequence is by the initiator codon that is positioned at 5 ' (amino) end and be positioned at the initiator codon decision that 3 ' (carboxyl) held.Encoding sequence can include, but are not limited to from the cDNA of virus, prokaryotic organism or Eukaryotic mRNA, from the genomic dna sequence of virus or prokaryotic organism or eukaryotic dna, even the synthetic dna sequence dna.The transduction terminator sequence is positioned at 3 ' of encoding sequence.

Typically " controlling elements " includes, but are not limited to transcripting promoter, transcriptional enhancer element, transcription termination signal, polyadenylation sequence (be positioned at 3 of translation stop codon '), is used for the sequence that the optimizing translation starts (be positioned at 5 of encoding sequence ') and translation termination sequence.

Term " nucleic acid " comprises DNA and RNA, and their analogue, for example comprises modification main chain those (for example phosphorothioates etc.) and peptide nucleic acid(PNA) (PNA) etc.The present invention includes and comprise the nucleic acid of sequence complementary sequence as mentioned above (for example be used for antisense or survey purpose).

As used herein, term " transfection " phalangeal cell absorbs external DNA, and when the DNA with external source entered in the cytolemma, cell just " transfection ".In this area, many rotaring dyeing technologies are well-known.Referring to, for example, Graham etc. (1973) Virology, 52:456, Sambrook etc. (1989) MolecularCloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis etc. (1986) Basic Methods in Molecular Biology, Elsevier, Gene 13:197 such as and Chu, 1981.Can use the DNA composition of these technology with one or more external sources, for example plasmid vector and other nucleic acid molecule are incorporated in the suitable host cells.This term refers to the absorption of the stable and moment of genetic material.

Term " transduction " expression by such as the carrier of recombined glandulae correlation viral vectors with in the dna molecular body or external being delivered in the recipient cell.

Term " allos () " refer to nucleotide sequence for example when gene order and control sequence when it, the sequence that expression does not under normal circumstances link together and/or under normal circumstances do not interrelate with specific cell.Thereby, " allos () of nucleic acid construct or carrier " zone is nucleic acid fragment, it is in another nucleic acid molecule or be additional on this nucleic acid molecule, and does not find that at occurring in nature itself and above-mentioned another nucleic acid molecule interrelate.For example, the allos district of nucleic acid construct can comprise following encoding sequence: the sequence of not finding these encoding sequence both sides at occurring in nature is related with it.Another example of allogeneic coding sequence is following construct: this does not have to find (composition sequence that for example has the codon that is different from natural gene) in occurring in nature encoding sequence wherein.Similarly, for the purposes of the present invention, by under the normal circumstances in cell non-existent construct institute cells transfected will be considered to allogenic.As used herein, the catastrophic event of allelic variation or natural generation does not produce allogenic DNA.

Term " controlling elements " jointly refers to promoter region, polyadenylation signal, transcription termination sequence, adjusted and controlled territory, upstream, replication orgin, internal ribosome entry site (" IRES "), enhanser etc., and they jointly duplicate, transcribe and translate for the encoding sequence in the recipient cell provides.As long as the encoding sequence of selecting can be replicated, transcribe and translate, all these controlling elementss of unnecessary existence in suitable recipient cell.

Its conventional sense of the term of Shi Yonging " promoter region " is meant the Nucleotide zone that comprises the DNA regulating and controlling sequence in this article, and wherein said regulating and controlling sequence is derived from can the bind rna polysaccharase and start the gene that (3-direction) encoding sequence in downstream is transcribed.

The arrangement of " (Operably Iinked) is operably connected " finger element, wherein the described component of Miao Shuing is configured as making the common function of they performances.Therefore, the controlling elements that is operably connected with encoding sequence can influence the expression of encoding sequence.It is adjacent with encoding sequence that controlling elements does not need, as long as they have the function that instructs its expression.Therefore, for example, do not translate but the intervening sequence of transcribing may reside between promoter sequence and the encoding sequence, and promoter sequence can think that still encoding sequence " is operably connected ".

When being used for nucleotide sequence, " isolating " meaning is that this molecule of expression exists under the biology polymer of other same type not having on the composing type.Therefore, " nucleic acid molecule of the specific polypeptide of separated coding " refers to not comprise on its composing type the nucleic acid molecule of other nucleic acid molecule of the target polypeptides of can not encoding; Yet this molecule can comprise that some can not influence other matrix or the part of composition composing type feature nocuously.

" carrier " can be delivered to nucleotide sequence target cell (for example virus vector, non-virus carrier, particulate carrier and liposome).Typically, " vector construct ", " expression vector " and " gene transfer vector " refer to can instruct arbitrarily the nucleic acid construct thing of interested expression of nucleic acid, and, its can the nucleic acid delivery sequence to targeted cells.Therefore, this term comprises clone and expression vector and virus vector.

" patient " refers to arbitrary Yuan animal of chordate subphylum.This term is intended to contain the arbitrary member in this subphylum, includes but not limited to the mankind and other primatess for example orangutan and other ape and monkey class; Domestic animal is ox, sheep, pig, goat and horse for example; The Mammals of raising and train, for example dog and cat; Laboratory animal comprises rodents, for example mouse, rat and cavy; Bird comprises poultry, wild fowl and hunts fowl, for example chicken, turkey and other gallinaceous bird, duck, goose etc.The specific age do not represented in this term.Therefore, adult and newborn individuality all is covered by in this term.

" the treatment effective dose or the quantity " of coding regulatable type transcription factor and/or genetically modified AAV carrier is meant when administration AAV carrier as described herein, makes the positive treatment reaction takes place for example to improve the amount that symptom or prevention neurological disorder develop.For example, the AAV carrier of drug treatment significant quantity, it causes that therapeutical agent (for example AADC, GDNF) is expressed and is used for the treatment of Parkinson's disease in the patient, can improve symptom, for example improves motor function or reduces static vibration.The accurate amount of the AAV carrier that needs will be different between the patient, depend on the particular composition of kind, age and patient's general situation, subject severity of disease and use, administering mode etc.

II. implement mode of the present invention

Before describing the present invention in detail, should be appreciated that to the invention is not restricted to special example molecule or processing parameter that certainly, it is changeable.Should be appreciated that also the proprietary term that uses in this article is the purpose that only is used to describe particular of the present invention, and do not mean that restriction the present invention.And except as otherwise noted, enforcement of the present invention will be used virusology, microbiology, molecular biology, recombinant DNA technology and immunologic ordinary method, and all these methods are all in ordinary skill.Such technology is what prove absolutely in the literature.Referring to, for example, Sambrook, et al.Molecular Cloning:A LaboratoryManual (2nd Edition, 1989); DNA Cloning:A Practical Approach, vol.I ﹠amp; II (D.Glover, ed.); Oligonucleotide Synthesis (N.Gait, ed.1984); A Practical Guide toMolecular Cloning (1984); And Fundamental Virology, 2nd Edition, vol.I ﹠amp; II (B.N.Fields and D.M.Knipe, eds.).Although those many methods described herein and class material or equivalent can be used to implement the present invention, preferred material and method are described herein.

The invention provides the vector construct, method and the test kit that are used for regulating and control the target cell transgene expression.In one embodiment, the necessary sequence of regulatable type gene in the target cell that on a large amount of isolating reorganization AAV carrier molecules, is provided for transduceing.Embodiment 1 and 2 has confirmed the structure and the purposes of of the present invention pair of carrier embodiment.In another embodiment, substance group carrier is loaded with and is used for transduceing the necessary all sequences of target cell regulatable type gene.Embodiment 3 has confirmed the structure and the purposes of a kind of single carrier embodiment so of the present invention.

In one embodiment, with the AADC gene delivery of adjustable form to target cell.Embodiment 1 and 2 has confirmed the sending of AADC gene of adjustable form.In another embodiment, the gdnf gene with adjustable form is delivered to target cell.Embodiment 3 has confirmed the sending of gdnf gene of adjustable form.

The term transgenosis refers to can be delivered to arbitrarily the gene of target cell as used herein, and does not consider genetically modified source, in certain embodiments, comprises the other copy or the sequence variant of native gene in the target cell Already in.Transgenosis can be the genetic engineering varient or synthetic (non-natural existence) dna sequence dna of the gene order that obtains from any organism or portion gene sequence, this gene.Transgenosis can directly be generated by the messenger RNA(mRNA) s of coded protein codified biologic activity RNA molecule, and described biologic activity RNA molecule is antisense, ribozyme, formation triple helical, RNAi or other RNA sequence for example.

The regulatable type gene is for after entering the gene of target cell with gene transfer as used herein, and its expression can change right by the administration inductor.The regulation and control of therapeutic transgene expression provide the pharmacology control to the transgene product level.In embodiment preferred, inductor is a small-molecule drug.A kind of such small-molecule drug is a rapamycin.

In one embodiment, tetracycline-regulated gene expression system of the present invention has utilized under the situation that has rapamycin or AP21967 (a kind of non-inhibitive ability of immunity forms of rapamycin analogs), the specificity dimerisation of immunophilin FKBP and lipid kinase homologue FRAP has produced the interactive transcriptional factor mixture of specific, activated inducible promoter.The structure of Rivera etc. (1996) Nat.Med.2:1028-32.AP21967 provides as follows:

Insert the 22nd page of figure

AP21967 (molecular weight 1017.4Da) is in U.S. Patent No. 6,649, the 7-skatole base analogue of the compound 69 in 595 (' 595 patents), and it can be as passing through with the preparation of 7-skatole substituted dimethyl oxygen base benzene of describing in embodiment 5.AP21967 is that the compound 71 in ' 595 patents is similar, and difference is that AP21967 has the 7-methyl on indole ring.

Do not deviating under the scope of the present invention, also can use other forms of rapamycin analogs.Those can or not have the possible undesired immunosuppression and the cell cycle inhibition of possibility rapamycin with the interactional forms of rapamycin analogs of endogenous FRAP among the patient.Pollock etc. (2000) Proc.Natl.Acad.Sci.USA 97:13221-26. also can set up the mutant forms of FRAP, its maintenance can be used for the FRB part of the activation domain fusion rotein of dipolymer system in conjunction with the ability of these non-inhibitive ability of immunity forms of rapamycin analogs.Referring to, for example, Pollock etc. (2000).

The regulatable type of AADC is expressed

At HeLa D7-4 cells in vitro with in the rodents model body of Parkinson's disease (PD), the genetically modified regulating and expressing experiment of the AADC that uses the carrier mediated rapamycin of AAV to rely on.The genetically modified expression of human AADC (hAADC) is that the reconstruction of the functionalization transcription factor (TF) of the carrier that further describes in embodiment 1 and Figure 1A and 1B by the utilization of dipolymer rapamycin is carried out.Transcription factor AAV carrier is used to send the DNA binding domains and the activation domain of transcription factor, expresses the AAV carrier and be used to send the hAADC transgenosis under the control of adjustable promotor.

The external regulation and control of AADC

The test that confirms that AADC that dipolymer relies on expresses in external human cell has been described in embodiment 1.The dipolymer AP21967 that uses in embodiment 1 is non-inhibitive ability of immunity forms of rapamycin analogs, and it has than the little about 3 times effect of rapamycin.The result is presented among Fig. 2, and wherein the most of data presentation in right side goes out that the dose-dependent hAADC of rapamycin expresses (from AAV-Z12-hAADC) in the cell of the carrier cotransduction of the transcription factor (AAV-CMV-TF) that relies on the coding dipolymer.The ELISA data presentation goes out to exist under the 25nMAP21967, and the AADC expression ratio does not exist under the dipolymer high 9 times.25nM is the maximum effective concentration of AP21967, has given the external transgene expression of peak level.Under 25nM AP21967, express that the chances are from half ((2004) such as Fig. 1 C and Sanftner are described) of the hAADC level under CMV promotor (AAV-CMV-hAADC2) control of composing type from the hAADC of adjustable promotor in Mol.Ther.9:403-9.

Do not exist under the AP21967, promptly system does not have under " seepage (leaky) ", and the cell that is loaded with two kinds of carriers (AAV-Z12-hAADC and AAV-CMV-TF) only demonstrates low-level AADC and produces.Not having seepage (leakiness) may be important for guaranteeing that transgenosis can stop (if necessary) fully and a large amount of pharmacology regulation and control are provided.

Regulation and control in the body of AADC

As in embodiment 2, describing, in the 6-of Parkinson's disease hydroxydopamine (6-OHDA) rat model, the denervated surgery model body of striatum, measure the identical carrier of AAV mediated by protein transduction (Figure 1A and 1B) of the hAADC that is used for external confirmation dipolymer regulation and control.Treatment plan is presented among Fig. 3 by graphic.With AAV-CMV-TF and the AAV-Z12-hAADC parkinsonian rat of transduceing, then as shown in Figure 4, after with a series of rapamycin treatment, measure its circling behavior.

With AP21967 is used for external different, rapamycin is used for testing in the body, only be because before measured the optimal dose of rapamycin.The dipolymer rapamycin has many favourable character that are used for as human gene therapy's inductor, but its inherent immunosuppression character has limited its purposes.The non-inhibitive ability of immunity analogue of rapamycin, for example AP21967 can be the preferred inductor of transgene expression, particularly in the treatment human patients.In animal and human's class patient body, the optimal dose of AP21967 can comprise clinical trial by the determination of test method of standard.

In the rat of the 6-OHDA infringement that produces the significance level Dopamine HCL, the transduction of the hAADC carrier of regulation and control has produced behavioral implications with the administration of levodopa and rapamycin.As diagrammatic in Fig. 4, reversibly increased the expression of hAADC in the striatum that damages with rapamycin treatment, as by rotation RACK response acknowledge that the 5mg/kg levodopa is changed, it is reversible after rapamycin is withdrawn from carrier dabbling (+) rap group.The 3rd, 5 and 7 weeks after with rapamycin treatment, carrier dabbling (+) rapamycin group is replied the rotation of levodopa and is higher than carrier dabbling (-) rapamycin group and the dabbling control group of vehicle (P＜0.001) significantly, but, be back near control group again in the 4th and 6 weeks.These data acknowledgements Mammals brain body in, the reversible regulation and control that genetically modified hAADC expresses can influence the behavior phenotype.

The rat of real-time quantitative PCR confirmation in carrier dabbling (+) rapamycin group and carrier dabbling (-) rapamycin group is by the transduction of the copy of the equal amount of hAADC gene, and the difference of having eliminated transduction efficiency can be used as the explanation that is used for two observed Different Results of group.

Test the result that (table 1 and Fig. 5,6 and 7) confirmed behavior at immunohistochemistry and protein expression that the research terminal point carries out, as going through following.

Fig. 5 A has confirmed in medium-sized many sour jujubes neurone of the rat striatum of dabbling (+) rapamycin group from carrier (medium spiny neurons), to strong immunohistochemical staining and the therefore effective transgene expression of AADC.And Fig. 5 B has shown that only very low-level AADC expresses in the rat of dabbling (-) rapamycin group from carrier.

Fig. 6 A-6C has presented the low-resolution image of the whole embedding brain part immunohistochemical staining that is used for AADC.The result has provided the simple visual judgement of AADC remarkable high level expression relatively in left side (processing) hemicerebrum of the left side (processing) of the rat of dabbling (+) rapamycin group (Fig. 6 A) from carrier hemicerebrum neutralization and the rat of dabbling (-) rapamycin group (Fig. 6 B) from carrier.Result similar (Fig. 6 C) from the vehicle vehicle-dabbling animal of result in the animal of carrier dabbling (-) rapamycin group and contrast.

Immunohistochemical stereological analysis confirms when carrier during with the rapamycin administration, and the expression level by positive cell counting mensuration has increased significantly.Table 1 listed the result of transgenosis deutero-immunostaining and by quantitative stereology to rapamycin inductive and the positive cell number estimated of inductive animal not.Compare with inductive animal not, rapamycin inductive animal demonstrate about twice the AADC immunostaining before or after the coating volume and the AADC positive cell number of coating, AADC immunostaining.

The total protein analysis shows protein level even bigger difference.As shown in Figure 7, compare with (-) rapamycin group, in (+) rapamycin group, after the gel electrophoresis of striatum protein example, hAADC enzyme level western blot analysis demonstrates significantly higher hAADC and expresses.When deducting in the data that endogenous AADC expression is drawn from Fig. 7, to compare with (+) rapamycin group, the level of the hAADC in (-) rapamycin group descends 88%.

The experimental result of describing in embodiment 2 shows that hAADC genetic expression is subjected to the dipolymer rapamycin and induces, and shows that some " seepage " arranged although observe low-level expression in inductive animal not in the regulator control system body.The seepage reason of system is unclear, does not plan to be limited by theory, and it may be by causing not existing in conjunction with the not expression of regulation and control from the IL-2 promotor of minimum under the transcription factor.Yet, reply in the behavior to the levodopa of inferior therapeutic dose (sub-therapeutic) of not inducing following observed low-level hAADC to be not enough to draw, it shows that a small amount of genetic expression that is produced by inducible promoter is tolerable for specific application under the situation that does not have inductor.

The optimal dose that is used to influence the rapamycin (or analogue) of the transgenic induction of wanting level can be by carrying out test determination on the basis of different cases (case-by-case basis), as common use treatment agreement.Dosage can be debugged by repetition test based on phenotype test result or surrogate marker.Also adjustable dosage is to obtain the intended target concentration of rapamycin in target tissue or in patient's blood.When introducing by intravenous injection, exemplary dosage can be for 0.01 to 50mg/kg, and preferred 0.1 to 10mg/kg, and for example 0.1,0.2,0.3,0.4,0.5,0.6,0.8,0.9,1,1.5,2,3,4,5,6,7,8,9 or 10mg/kg.Under the situation of treatment human patients, dosage can be determined with reference to the generation of clinical trial or from the animal inferred from input data.

Can introduce different site in the brain with the surgical operation AAV carrier of will recombinating, depend on the target cell of transgenosis, its mechanism of action and the purpose of sending.For example, basal ganglion is for being positioned infracortical one group of neurone.They comprise caudate putamen, shell nuclear and pallidum.Caudate putamen and shell nuclear form striatum (or simple striatum) together.Caudatum reciprocally is connected with black substance with shell nuclear, and described black substance comprises black substance compact part (SNpc) and black substance pars reticulata.SNpc is the biosynthetic normal site of Dopamine HCL, the sign that deteriorates to PD of SNpc.By with the gene of the enzyme in the coding Dopamine HCL biosynthetic pathway the transduce neurone of striatal shell nuclear or caudate putamen of AADC for example, it is synthetic to recover Dopamine HCL, thereby overcomes the cancellated functional minimizing of SNpc.

The Corpus striatal cell can use the whole bag of tricks transduction known in the art.For example, three-dimensional locating injection is a kind of common surgical method that is administered to all cpds use of CNS by the neurosurgical doctor.Also can use direct injection; If use this method, can use the anatomical images be derived from CT, PET or MRI scanning to help to select the site of injecting by the surgeon.Other method comprises that convection current increases the sending of (sub-therapeutic) (be described in detail in U.S. Patent No. 6,309, in 634, it all be incorporated herein by reference), can be applicable in the method for the present invention so that the rAAV virosome is delivered to CNS.

The experiment of describing in embodiment 1 and 2 comprises of the present invention pair of carrier embodiment, and wherein carrier of separating is used to send transcription factor fusion rotein and transgenosis.In other embodiment, use the hAADC in the different gene replacement expression vectors.The Nucleotide (nt) of maximum for about 5000-5200 can be packaged in the AAV virosome, about 4500nt is best.Dong etc. (1996) Hum.Gene Ther.7:2101-12. gives other sequence composition that needs of rAAV expression vector as diagrammatic in Figure 1B a kind of (for example ITR zone, promotor, transcription factor binding sequence etc.), and the transgenic sequence in the expression vector all can be shorter than about 2500 Nucleotide (nt).In the qualification of this size, the transgenosis of wanting arbitrarily can use the rAAV expression vector establishment thing of the two carrier embodiments of the present invention to send.Expression vector varient with optimum of short ITR, promotor or transcription factor calmodulin binding domain CaM can be constructed as the long transgenic sequence of transmissibility.

Embodiment 1 and 2 result confirm among the human cell in culture and in the rat body in the neurone, the genetically modified expression of hAADC that can use the embodiment regulation and control dipolymer of the two carrier rAAV mediations of the present invention to rely on.

GDNF regulation and control in single carrier system

As describing in embodiment 3, experiment utilizes single carrier embodiment of the present invention to carry out, and wherein single rAAV carrier is to be used to express the necessary all proteins of regulation and control that the dipolymer of GDNF transgene expression relies on.The figure of the rAAV hGDNF expression vector of regulation and control is provided in Fig. 8 A, the figure of the control vector of (not regulating and control) hGDNF expression with composing type is provided in as Fig. 8 C.(Fig. 8 B shows the another kind of scheme that is used to regulate and control the GDNF expression vector, and it does not use in embodiment 3).Plasmid vector is entered external HEK-293 cell by transient transfection.Then, with comprising 0,5 or the media processes cell of the rapamycin of 25nM.

Transfection in Fig. 9, occurs after 3 days, carry out the result of GDNF ELISAs.Data presentation goes out the GDNF rapamycin dose response expression (" TF-GDNF plasmid construction " in the construction cells transfected of using regulation and control, it refers to pAAV-TF-Z8-hGDNF), observed expression maximum is about 1/4th (they refer to pAAV-CMV-hGDNF " CMV-GDNF plasmid ") from the expression level of the GDNF of constitutive expression.GDNF from pCMV-GDNF does not express and can increase because of adding rapamycin.

The experiment confirm of describing in embodiment 3 can make up the adjustable AAV-GDNF construction of single carrier, and wherein among the human cell in culture, GDNF expresses and can regulate and control by adding small molecules inductor rapamycin.The result shows that in patient's body the expression of GDNF also can utilize adjustable AAV-GDNF carrier to regulate and control.Single carrier of embodiment 3 is carried out (fills) the transcription factor carrier in two carrier embodiments of above-mentioned discussion and the function (rolls) of expression vector.With need compare with two support methods of two kinds of carrier cotransfection target cells, have the advantage that only needs a kind of incident of transduceing is introduced adjustable GDNF with single vehicle treatment.This advantage of single carrier embodiment is for those method particularly importants that only obtains low transduction efficiency, and described method is gene therapy method for example, wherein with two kinds of carriers expection whiles only very during the transduction target cell of small proportion.For example, suppose that the transduction effect is independently, if do for oneself 5%, then with only transduce 0.25% cell of two kinds of carriers for every kind of total transduction efficiency of carrier.

Because the carrier that is packaged in the AAV virosome can not be longer than about 5000-5200 Nucleotide (nt), so the adjustable construction of single carrier is sent short relatively transgenosis with only actually.For example, use the element (as using) of the regulation and control of using in the regioselective transcription factor fusion rotein of diagrammatic and rAAV carrier in embodiment 3 in Fig. 8 A, transgenosis (GDNF) sequence is grown for about 600nt.Possible is long transgenic sequence, for example reach 850 or even 1000nt, can use the optimum adjustable AAV construction of single carrier to send, wherein said construction has the sequence composition of more effective arrangement, and wherein unnecessary Nucleotide is removed from the composition of fusion rotein and regulation and control.In the limiting demensions that is used for the AAV packing, the transgenosis of wanting arbitrarily all can use single vector construct of the present invention to send.Transgenosis can comprise the active subfragment of the gene of wanting, rather than the gene of total length, sends to help single carrier.

The dosage that needs the rAAV virosome of acquisition particular treatment effect, the dosage device in vector gene group/per kilogram of body weight (vg/kg) for example, can be depending on some factor, include but not limited to: the level of the transgene expression of the route of administration of rAAV virosome, needs acquisition result of treatment and the stability of heterologous gene products.The rAAV virosome dosage range that those skilled in the art can be identified for treating the patient who suffers from specific disease or obstacle based on above-mentioned factor and other factors well-known in the art.In certain embodiments of the invention, the optimal dose that is used for influencing the rAAV that Mammals transduces can be 1 * 10 ⁸Vg/kg to 1 * 10 ¹⁵Vg/kg, preferred 4 * 10 ⁹Vg/kg to 4 * 10 ¹²Vg/kg is although can determine the higher or lower dosage of application by test.Although AADC and GDNF are present in as in the exemplary transgenosis in embodiments of the present invention, the specific transgenic that is used to send not is the aspect that the present invention limits.Use carrier of the present invention, method and test kit to provide the part of useful (for example treatment) resultful other gene or gene to be incorporated in the brain with expecting, to be described sequence is enough short can pack the AAV vector construct that enters the AAV virosome with suitable to prerequisite.

AADC (OMIM 107930, EC 4.1.1.28, Genbank Accession No.M76180) is for participating in the biosynthetic transgenosis of Dopamine HCL as discussed above.Participating in biosynthetic other latent gene of Dopamine HCL is that (OMIM 191290 for tyrosine hydroxylase (TH), EC 1.14.16.2, Genbank Accession No.X05290) and GTP cyclohydrolase I (GCH) (OMM 600225, EC 3.5.4.16, Genbank Accession No.NM_000161).Still other potential transgenosis comprises neurotrophic factor, (OMIM 600837 to comprise GDNF, Genbank Accession No.AX713049, L19063) and the member of other gdnf protein matter family, for example (OMIM 603886 for artemin, Genbank AccessionNo.AF109401), neurturin (OMM 602018, Genbank Accession No.HSU78110) and persephin (OMIM 602921, Genbank Accession No.AF040962.).Saarma etc. (1999) Microscopy Res.Tech.45:292-302.Also have other transgenosis to comprise IL-10 (OMIM 124092, Genbank Accession No.M57627).

The OMIM numeral refers to can be obtained from the Internet at the National of www.ncbi.nlm.nih.gov/entrez Library of Medicine by network address by the online human Mendelian inheritance database (Online Mendelian Inheritance in Man database) of Johns Hopkins University maintenance.Therefore, be incorporated herein by reference with its full content with the content of all OMM clauses and subclauses of quoting herein with by the sequence that registration number is quoted.

Provide the Genbank registration number only to be used to represent complete cDNA sequence, and do not mean that and limit the scope of the invention.Especially, the potential transgenosis be included in the total length of other sequence, natural gene of the gene report among the Genbank and subfragment, from the mutant forms of the gene of the allelic variation body of the homologous gene of other kind, these sequences and naturally occurring or artificial foundation.

Transgenosis also comprises the gene that is used for the treatment of other neurodegenerative disease, and described neurodegenerative disease is alzheimer's disease, Heng Tingdunshi disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, canavan's disease, cerebral ischemia, stein-leventhal syndrome, dementia with Lewy body, Xi-De syndrome, ADDS dementia, essential tremor, dystonia, corticobasal degeneration, multiple system atrophy and retinal degeneration (for example macular degeneration, diabetic retinopathy, retinitis pigmentosa, glaucoma) for example.

Inductor is preferably and is selected from those compounds that can be delivered to human patients and not have remarkable toxicity or side effect.In the preferred embodiment that is used for the treatment of neuropathy or other CNS disease, inductor can pass hemato encephalic barrier.In a more preferred embodiment, inductor is a dipolymer, for example rapamycin or its non-immunosuppression analogue.

In certain embodiments, inductor is sent with parenteral, for example subcutaneous usefulness, muscle, intraocular or intravenous injection.In preferred embodiments, inductor is a kind of for what can send relatively easily, described send easily for example oral, local, by nasal delivery (nasal mist), by aerosol/pulmonary delivery (sucker), by ocular delivery (eye drops)) or send mode easily by any other.Inductor also can use transdermal patch, subcutaneously send continuously or semi-continuously with implant, implantable osmotic pony pump, mechanicalness infusion pump or controlled release pharmaceutical compositions.

Original seed according to rAAV carrier of the present invention can use this area to be used for some known method preparation that the AAV virosome produces.In under the situation of triple turn guiding method, wild-type AAV and helper virus can be used for being provided for producing the essential copy function of rAAV virosome, and perhaps plasmid can be used to provide subsidiary function gene (for example pHLP 19), auxiliary function gene (for example pladeno 5) or two kinds.Referring to, for example, United States Patent(USP) Nos. 5,139,941; 5,622,856; 6,001,650 and 6,004,797, wherein disclosed content all is incorporated herein by reference with it in this article.

For sending in the body, the rAAV virosome is formulated to one or more rAAV virosome that comprise optimal dose and the pharmaceutical composition of pharmaceutically acceptable vehicle.Such vehicle comprises itself can not induce any medicament of generation to the individual deleterious antibody that receives described composition, and it can administration and do not have undue toxicity.Pharmaceutically acceptable vehicle includes, but are not limited to liquid for example water, physiological saline, glycerine and ethanol.Pharmacologically acceptable salt can be included in wherein, for example for example hydrochloride, hydrobromide, phosphoric acid salt, vitriol etc. of inorganic acid salt; With organic acid salt for example acetate, propionic salt, malonate, benzoate etc.In addition, auxiliary substance is wetting agent or emulsifying agent, pH buffer substance etc. for example, can be present in such carrier.Going through of pharmaceutically acceptable vehicle can obtain in REMINGTON ' S PHARMACEUTICAL SCIENCES (Mack Pub.Co.NJ.1991).

In certain embodiments, rAAV virosome of the present invention can provide with the pharmaceutical composition of the vehicle that comprises following feature: described vehicle increased delay between the shelf lives and repeated exposure in the virus stability in freeze thaw cycle, and reduced carrier and adhered on the device for casting.In preferred embodiments, vehicle for example demonstrates hypotoxicity among the CNS at target tissue.For example, virosome can store and be provided at comprise general stream Buddhist nun restrain the buffer reagent of F-68 (BASF Corp.Mount Olive, NJ) in, content is 0.01% to 0.0001%, preferred 0.001%.Other composition and vehicle are at United States Patent(USP) Nos. 6,759, describe in 050 and 6,764,845, and wherein disclosed content all is incorporated herein by reference with it in this article.

In yet another aspect, the present invention relates to be used to make up the test kit of the AAV carrier that is used for the expression of transgenosis regulatable type.In certain embodiments, will be included in this test kit with one or more the substantially the same carriers that in Figure 1A and 1B or Fig. 8 A or 8B, show.

In other embodiments, be included in such test kit with the similar carrier that in Figure 1B, 8A or 8B, shows, difference be with cloned sequence easily for example polylinker replace AADC and GDNF encoding sequence.Can be used in this pair carrier method with those similar expression vectors that in FIG.1B, show, and can be used in single carrier method of the present invention with those similar carriers that in Fig. 8 A and 8B, show.The user of test kit of the present invention can clone interested gene or gene fragment enters expression vector, then, and the rAAV virosome that preparation is used to transduce.

The test kit that comprises expression vector that is used for using in two support methods also can comprise be substantially similar to those the transcription factor carrier that shows in Figure 1A.

Test kit can randomly comprise rapamycin or its analogue.

Test kit also can randomly comprise the plasmid that is used to prepare rAAV virosome original seed, for example plasmid pHLP 19 and pladeno 5, and as at United States Patent(USP) Nos. 6,001, more abundant description in 650 and 6,004,797.

Test kit also can comprise working instructions.

In certain embodiments, gene therapy vector of the present invention, method and test kit administration can be subjected to for example patient of Parkinson's disease puzzlement of disease, so that result of treatment to be provided.Generally speaking, " result of treatment " refers to be enough to make the level of the component of disease (or obstacle) towards one or more transgene expressions of result who wants or clinical endpoint change, so make disease of patient or obstacle show clinical improvements, usually reflect by the clinical sign of diseases related or obstacle or the improvement of symptom, under the situation of Parkinson's disease, result of treatment can be the improving of the motor function that a shows (Jian Kang motor activity (fine motor tasking) for example, for example, the improvement by manual dexterity.In addition, the minimizing of rest tremor also can be the signal of PD improvement.For the special treatment of PD, other is used for determining the generally acknowledged observable and terminal point that can measure in field of result of treatment (being curative effect) some.

In the human patients that demonstrates PD clinical symptom and symptom, the result of treatment that the unified Parkinson's disease rating scale (Unified Parkinson ' s Disease Rating Scale) that the common dependence of clinicist is known (UPDRS) is estimated severity of disease and measured the particular treatment pattern.Be similar to the UPDRS system, scientist utilizes primates Parkinson's disease rating scale (Primate Parkinsonism RatingScale) (PPRS, be also referred to as clinical rating scale (Clinical Rating Score-CRS)) estimate the PD feature in the primates model of PD, it measures healthy motor activity, rest tremor, bradykinesia, hypokinesis and myotony with further feature.Described PPRS system description is in (2000) Ann.Neurol.47:S79-89 such as Langston.

III. test

Following for implementing the embodiment of particular of the present invention.Embodiment only is used to the purpose of furnishing an explanation property, and does not mean that by any way and limit the scope of the invention.About the numeral of using (for example quantity, temperature etc.), although carried out making great efforts to guarantee precision, some experimental error and deviation should be admissible certainly.

Embodiment 1

The external controllable express of AADC

The genetically modified external adjusting of AADC is according to following acquisition:

The AAV carrier

Expression for the dipolymer that obtains hAADC:AAV-CMV-TF and AAV-Z12-hAADC relies on makes up two kinds of carriers.Figure 1A is the figure of AAV-CMV-TF, and wherein cytomegalovirus (CMV) enhancers/promoters drives the expression of the bicistronic mRNA information of coding activation structure territory and DNA integrated structure domain fusion protein.The activation structure domain fusion protein comprises human FRAP rapamycin binding domains (FRB ^*), it merges to (p65) transcription activating domain that is derived from NF κ B.When comparing with wild-type FRB sequence, the specific FRB structural domain (FRB of Shi Yonging in this embodiment _T2098L) and in Figure 1A the specific frb domain (FRB of diagrammatic ^*) be loaded with T to L at 2098 and suddenly change.Pollock etc. (2000) Proc.Natl.Acad.Sci USA 97:13221-26.FRB _T2098LCan utilize AP21967 or rapamycin dimerization to DNA binding domains to merge.

Internal ribosome enters sequence (IRES) for being derived from encephalomyocarditis virus.DNA integrated structure domain fusion protein comprises two DNA binding domainss from human transcription factor Zif268, the homeodomain that is derived from Oct-1 (ZFHDI) and three medicine binding domainss from the cytosol receptor of FK506 (3xFKBP).Figure 1B is the figure of AAV-Z12-hAADC, and the expression of wherein human AADC (hAADC) is subjected to the control of 12 fusions to the combining site of the transcription factor of minimum IL-2 promotor.Shown that also AAV2 art end oppositely repeats (ITR), hAADC encoding sequence (hAADC) and human growth hormone polyadenylic acidization (pA).

Vivoexpression is analyzed

The following test of regulating the ability of hAADC genetic expression: at external AAV-CMV TF and the AAV-Z12-hAADC cotransduction HeLa D7-4 cell that passes through with 1: 1 ratio, use subsequently 0,5 or the forms of rapamycin analogs AP21967 of 25nM (ARIAD Pharmaceuticals Inc.CambridgeMassachusetts) handles cell.The result is presented among Fig. 2, it comprises the result of each control experiment, comprise non-transducer cell, with the transcription factor carrier separately transduction cell (AAV-CMV-TF), express with composing type (constitutively) hAADC carrier transduction cell (AAV-CMV-hAADC2) and with the cell (AAV-Z12-hAADC) of the hAADC carrier transduction of adjusting, but do not comprise the transcription factor carrier.

The experimental detail of the relevant test method of using in embodiment 1 is as follows:

Vector construction

AAV-CMV-TF is (AAV-CMV-TF1Nc, Auricchio etc. (2002) Mol.Ther.6:238-42) that described in the past.AAV-Z12-hAADC is for preparing (Sanftner etc. (2004) Mol.Ther.9:403-9) by the cmv enhancer and the promotor of replacing pAAV-hAADC with the Z12-IL2-SEAP zone (Rivera etc. (1996) Nat.Med.2:1028-32) that comprises 12 binding sites of ZFHDI DNA binding domains that are positioned at minimum IL-2 promotor upstream.

The generation of recombinant vectors

Reorganization AAV carrier (serotype 2) is by three transfection HEK-293 (ATCC Accession No.CRL1573) cells and passes through the generation of CsCl density gradient centrifugation.Grimm etc. (2003) Blood102:2412-9.In brief, Micro Fluid comprises the cell harvesting thing of rAAV, by the strainer filtration of 0.2 μ m.Carrier is passed through CsCl density gradient centrifugation purifying from the cell lysates that purifies, pass through ultrafiltration and concentration, diafiltration enters in the phosphate-buffered saline that comprises 5% sorbyl alcohol, pH 7.4 (PBS-sorbyl alcohol), (BASF Corp.Mount Olive is NJ) to prevent the loss of carrier in delivery catheter to add 0.001% general stream Buddhist nun gram (pluronic) F-68.Estimate carrier purity by SDS-PAGE.The rAAV carrier of the purifying that will use in this research is by the silver dyeing of SDS-PAGE gel, and it only demonstrates VP1, VP2 and VP3 basically.Titre is to analyze the vector gene group by Q-PCR to determine.

Analyze external AADC by expressing EL/SA

Express ELISA and utilize the expression of anti-hAADC TPPA hAADC protein in the HeLa of saturatingization D7-4 cell.With rAAV carrier transduction HeLa D7-4 cell, and handle with the AP21967 of three kinds of various dose (0,5,25nM).Every group of n=3.In transduction preceding 24 hours, with the cell kind in 96 hole plates.Be used in 10 in the perfect medium of 100 μ l ⁴The MOI transducer cell of vg/ cell (when using a plurality of carrier, for each carrier).

Back 48 hours of transduction transducer cell is carried out ELISA.In brief, extract medium out, use the PBS washed cell.With 4% Paraformaldehyde 96 fixed cell, at room temperature cultivated 20 minutes.Use the PBS washed cell, jolting simultaneously, was at room temperature cultivated 60 minutes in jolting simultaneously then with blocking-up buffer reagent (the PBS solution of 3% lowlenthal serum, 0.5%TritonX-100) blocking-up.One anti-(the anti-hAADC of AB 136 rabbits, Chemicon, 1: 1000) is diluted in the washing buffer (1% lowlenthal serum, the PBS solution of 0.5%Tritonx100), at room temperature in shaking table, cultivated 60 minutes.Wash plate with washing buffer, at room temperature, (the sub-IgG-AP of goat antirabbit (Vector Labs, Burlingame, California), 1: 1000 washing buffer agent solution) cultivated 30 minutes on shaking table with two anti-.Wash plate with washing buffer.Add matrix solution [IX is right-the nitrophenyl phosphoric acid ester, IX LEVAMISOLE HCL (quencher), matrix buffer reagent (100mM NaHCO ₃, pH 10.0) (VectorLabs, Burlingame, California)], at room temperature culture dish 30-60 minute, then at OD ₄₀₅Descend reading in the plate reader.Derive from the relative optical density of data report that this hAADC expresses ELISA, it is compared with the dose response curve to the reference point (reference lots) of the AAV-CMV-hAADC2 that transduces in cell, measuring with carrier transduction.

Embodiment 2

The intravital regulated expression of AADC

Also in the body of the 6-of Parkinson's disease hydroxydopamine (6-OHDA) rat model, measure the identical carrier of AAV mediated by protein transduction (embodiment 1, Figure 1A and 1B) with the hAADC that is used for external confirmation dipolymer adjusting, as follows.In embodiment 2, use rapamycin to replace AP21667, because it has higher tiring and known pharmacokinetics as dipolymer.In vivo, rapamycin has about 10 hours transformation period, can remove fast.Gallant-Haidner etc. (2000) Ther.Drug Monit.22:31-5.

All rats of Shi Yonging are that one-sided 6-OHDA damages in the left side in this embodiment.Three test group are arranged: vehicle perfusion control group (vehicle perfusion (+) rapamycin treatment), carrier perfusion (-) rapamycin treatment control group and carrier perfusion (+) rapamycin treatment group.Carrier perfusion (-) rapamycin group plays a part do not have control hAADC genetic expression under the rapamycin,, is used for whether seepage of mensuration system that is.

With (every group 3 * 10 of AAV-CMV-TF and AAV-Z12-hAADC ¹⁰Viral genome (vg)) 1: 1 mixture homonymy pours into the rat to one-sided 6-hydroxydopamine (6-OHDA) infringement.Independent vehicle is injected vehicle perfusion control group.At specified time point, finish inducing of transgene expression, following discussion via the peritoneal injection rapamycin.

Fig. 3 has shown the tentative time line of the test of describing in this embodiment.Before rAAV handles, all groups are carried out the benchmark whirl test of administration levodopa (5mg/kg).At the 0th day carrier or vehicle are filled in the striatum.At the 17th day, induce rat or use the diluent treatment rat with rapamycin.Induce and comprise 10mg/kg/ days continuous four days of rapamycin (the administration rapamycin continuous 4 days was in order to guarantee circulation (circulating) level in brain the biglyyest) of IP injection.Arrow in Fig. 4 has pointed out that four space mine handkerchief mycin inductive begin.At the 21st day, test rat once more the rotation of 5mg/kg levodopa is replied.Rat was recovered for 1 week from rapamycin, then, reply test in the 28th day.Induced rat at the 31st day once more, reply test rotation in the 35th day.After from rapamycin, recovering for 1 week, repeated rotary test at the 42nd day.At the 45th day, rat was accepted to induce process for the third time and for the last time, subsequently, carries out last whirl test at the 49th day.At this time point, induce under the state of (+rapamycin) at them, animal is bestowed euthanasia (euthanasize), carry out immunohistochemistry and handle.

Behavior rotation to levodopa is replied

The rotation to dopamine agonist of use standard is replied and is carried out behavioural analysis.In this case, agonist is by exogenous levodopa synthetic Dopamine HCL in the one-sided 6-OHDA rat model of PD.Ungerstedt(1971)Acta Physiol.Scand.Suppl.367：69-93。As shown in Figure 4, after three weeks of transduction, animal in carrier perfusion (+) rapamycin group demonstrates strong sideway swivel is replied of levodopa (5mg/kg), it is higher than (rotating at 60 minutes clockwise directions in carrier perfusion (-) rapamycin group significantly, 215.13 ± 73.86 pairs 16.33 ± 21.92, P＜0.001).By using the significant difference of the many groups of one-way analysis of variance.

In carrier perfusion (+) rapamycin group, the 3rd, 5 and 7 weeks levodopa was excited sideway swivel is replied obviously along with perfusion before score or time match contrast (carrier perfusion (-) rapamycin group and vehicle pour into (+) rapamycin group) and increase (P＜0.001).On the contrary, time point and removing the time point of rapamycin (4 week and 6 weeks) before perfusion, carrier perfusion (+) rapamycin group is not different less than remarkable with two control groups (P＞0.05).

Carrier perfusion (-) rapamycin is put not significantly different (P＞0.05) at any time with vehicle perfusion control group.Do not plan to be bound by theory, during seven weeks, rotating the increase gradually of replying in two control groups may be to be caused by the sensitization that the multiple levodopa is handled.

In carrier perfusion (+) rapamycin group, reply and not different significantly at those time points of carrier perfusion (-) rapamycin group or vehicle perfusion (+) rapamycin group in " closing (off) " rapamycin time point, the 4th week and the rotation in the 6th week, this has confirmed to induce the reversibility of replying.That observes in three successive rapamycin treatment cycles that hAADC expresses induces and expression decreased in untreated several weeks subsequently, and this fact has been strengthened the conclusion of inducing existence of rapamycin inductive gene expression in vivo.

HAADC transgenosis copy is quantitative in the perfusion striatum

Two carrier perfusion groups of real-time quantitative PCR confirmation of carrying out on all rats are all transduceed by the hAADC gene copy of a great deal of.The copy number (222 ± 92 genome copy/20ng DNA) of hAADC gene in carrier perfusion (+) rapamycin group (± SD) do not have different with carrier perfusion (-) rapamycin group (229 ± 48 genome copy/0ng DNA).

The immunohistochemistry of expressing and quantitative

In seven weeks after perfusion, estimate the hAADC expression level with immunohistochemical analysis.Be presented among Fig. 5 A from high power enlarged image in the striatum perfusion position of the representative animal of carrier perfusion (+) rapamycin group, pour into a pictorial display of (-) rapamycin group in Fig. 5 B from carrier.As diagrammatic in Fig. 5 A, the hAADC transgene expression is positioned the medium-sized many sour jujubes neurone in the rat striatum.On the contrary, with carrier perfusion but never inductive rat (carrier perfusion (-) rapamycin group) demonstrates extremely low-level hAADC transgene expression (Fig. 5 B).

Fig. 6 has shown the immunohistochemical low power enlarged image at the AADC of whole embedding brain sections, and described brain sections is from the representational animal in each group after seven weeks, the back striatum poured into.Will be with AAV-CMV-TF+AAV-Z12-hAADC (each carrier 3 * 10 ¹⁰Vg) perfusion and induce (A) or contrast (C) with rapamycin and compare without rapamycin (B) inductive animal and rapamycin inductive vehicle.Left cerebral hemisphere is carrier (or vehicle) perfusion position in 6-OHDA infringement and the striatum.Right cerebral hemisphere is not infringement and not dabbling, and therefore, the dyeing of right cerebral hemisphere reflects the intrinsic staining from complete rat AADC positive fiber.

6-OHDA infringement causing endogenous AADC consumes, and causes the low histochemical stain of enzyme in the left cerebral hemisphere, best being diagrammatically shown among Fig. 6 C in vehicle perfusion control group.Rat in carrier dabbling (+) the rapamycin group all demonstrates hAADC transgenosis dyeing (referring to, Fig. 6 A for example) in perfusion left side.Rat in carrier perfusion (-) rapamycin group has low-level hAADC transgene expression (referring to, Fig. 6 B for example).Observing hAADC in carrier perfusion (-) rap animal of 5/6ths expresses.Compare with carrier perfusion (+) rapamycin group, in carrier perfusion (-) rapamycin group, there is the dyeing of less positive cell and more low intensive each cell, confirmation only has low-level hAADC from inducible promoter to express under the situation that does not have rapamycin.

Perfusion in striatum, seven weeks behind the administration rapamycin at once, carry out quantitative stereology in the continuous brain sections that from the rat of sentencing euthanasia, obtains.In the serial section of fixed cerebral tissue, use three-dimensional learning to estimate and quantitative AADC immunostaining.Use optical grade device stereological method (protocol), a kind of object count method of effective justice, it is the combination of the spatial sampling method of optics dissector method and statistics the best.Gundersen etc. (1988) Apmis 96:857-81; Gundersen (1986) J.Microsc.143 (Pt1): 3-45.The result is presented in the table 1, it demonstrates carrier perfusion (+) rapamycin group and has maximum painted longitudinal separation (anterior-to-posterior distance) (3,720 ± 1,276 μ m) (± SD), and in carrier perfusion (-) rapamycin group, have much lower level distribution (1,920 ± 1,577 μ m).

Table 1

The expression of AADC in rat brain

	Rapamycin inductive (n=8)	There is not inductive (n=5)	Induce ratio
				The longitudinal separation (μ m) that distributes	3,720±1,276	1,920±1,577	1.94X
The AADC positive cell quantity	75,825±30,506	31,000±25,812	2.45X
				Volume of distribution (mm 3)	15.75±8.16	7.09±5.69	2.22X

" n " is the hemicerebrum number of research.

Data value is ± 1 standard deviation of existing value

Use Student ' s t calibrating, the value of the distribution of average front and back, volume of distribution and the positive cell number in the rapamycin inductive group and " not having inductive " group has significant difference (P＜0.02).

Table 1 also shows the mean number and the intrastriatal volume of distribution of the transgenic positive cell of knowing clearly.Carrier perfusion (+) rapamycin group has the AADC positive cell (75 of maximum quantity, 825 ± 30,506 cells), the positive cell (31 that secondly low amount is arranged in carrier perfusion (-) rapamycin group, 000 ± 25,812 cells) (P＜0.01) does not have detectable expression (data do not show) in vehicle perfusion control group.Similarly, carrier perfusion (+) rapamycin group demonstrates a large amount of hAADC striatum distribution (15.75 ± 8.16mm ³), and carrier perfusion (-) rapamycin group demonstrates the striatum distribution (7.09 ± 5.69mm of 55% low amount ³).

Stereological analysis is the striatal neuron quantity of transduction and the accurate measuring method of vector particles distribution.Yet the total protein analysis is the more accurate mensuration of the hAADC enzyme content of generation, because the amount of the hAADC that each cell produces can be different between each group.For the more low intensive hAADC in not transduction group that finds by immunostaining expresses with relevant than the hAADC expression of hanging down aggregate level, from connective tissue's section, extract total protein, check with western blot analysis.

Having confirmed has lower hAADC protein concn in not inducing group.Fig. 7 has shown the image of relevant gel bands of a spectrum, band intensity figure with integral body, it is from the rat of carrier perfusion (+/-) rapamycin and the one-sided 6-OHDA infringement of vehicle perfusion (+) rapamycin, and it demonstrates the change of the protein level of hAADC in striatum (50kDa).The β Actin muscle is included as sample contrast (loading control).Compare with two control groups, the AADC bands of a spectrum density in carrier perfusion (+) rapamycin group is higher significantly, P＜0.001.In 7 weeks after perfusion, be respectively 102.04 ± 7.02 megapixels (MP) and 30.63 ± 3.47MP from the spectral intensity on the western blotting of carrier perfusion (+) rapamycin and (-) rapamycin rat.After having endogenous rat AADC horizontal adjustment, and to compare in carrier perfusion (+) rapamycin group, the average total AADC protein level in carrier perfusion (-) rapamycin group has reduced 88%.These data acknowledgements in the hAADC enzyme level, have the bigger difference that shows than with stereological analysis between two groups.

The experimental detail of the relevant test method of using in embodiment 2 is as follows:

Surgical method

The adult Sprague-Dawley rat of 6-OHDA-infringement is (for vehicle perfusion and carrier perfusion (-) rapamycin group, n=6 rat, for carrier perfusion (+) rapamycin group, n=8 rat) from TaconicFarms obtain (Germantown, NY).Under standard atmosphere conditions, rat is pressed raising of each cage: the temperature and humidity of control, 12 hours periodicity of illuminations freely obtain food and water.Send (CED) that increase by convection current pours into carrier, obtains to spread all over striatal optimum distribution.Bankiewicz etc. (2000) Exp.Neurol.164:2-14; Lieberman etc. (1995) J.Neurosurg.82:1021-9.In brief, carrier is packed into polymer pipe (OD, 1/16 "; ID, 0.030 "; Upchurch Scientific, Oak Harbor, WA) described pipe coupling is to using the pipeline of filling from the sweet oil of gastight Hamilton syringe extraction.With programmable pump (Bioanalyticai Systems, Inc, West Lafayette, IN) delivery vector.With intubate, comprise fixedly vitreosil kapillary (OD, the 164 μ m of 27-pin (fitted into a 27-gauge needle); ID, 100 μ m; Polymicro Technologies, Phoenix AZ) is connected to the far-end of polymer pipe.Use O ₂The 3% isoflurane induced anesthesia of stream in (2L/ minute), with animal place stereotactic framework (Kopf, Tujunga, CA).Then, with passing the mask that is fixed on three-dimensional positioning framework, use O ₂In 1% isoflurane keep anesthesia.In target site boring, insert in the caudate putamen (nerve) intubate is vertical by the following coordinate that relates to bregma and dura mater: AP 0.0mm, ML-3.5mm, DV-5.0mm, incisor bar (incisor bar) is set in-3.3mm.With 0.5 μ l/ minute speed, two kinds of carrier perfusion animals of 1: 1 ratio of one-sided usefulness 10 μ l.When flush phase finished in 20 minutes, speed was reduced to 0 μ l/ minute, continue 5 minute stationary phase, take out intubate at leisure.

Behavioural analysis

With the automatic rotometer (AccuScanInstruments that is connected to computer run RotoMax rotational analysis software, Inc.Columbus OH) estimates Apomorphine (0.05mg/kg, Sigma, St.LouisMO) and levodopa (methyl esters, replying Sigma).In 30 minutes, (be used for Apomorphine) and in 60 minutes, (be used for levodopa) calculating total offside and same sideway swivel.Three weeks after infringement, only have average rotation＞6 of in 30 minutes, replying Apomorphine (0.05mg/kg) to sideway swivel/minute rat be considered to (Ungerstedt (1971) the Acta Physiol.Scand.Suppl.367:69-93) of good infringement, test its levodopa and reply.The benserazide co-administered that utilization is lower than the NSC 295453 of dosage (5mg/kg) of therapeutic domain and 2.5mg/kg test these animals to the replying of levodopa (Sigma, St.Louis, Missouri).Be higher than 95% test rat can not reply and rotate the 5mg/kg levodopa.From experiment, get rid of and demonstrate the animal to sideway swivel pure these dosage.In striatum, after the perfusion, before surgical operation with at different time point (3,4,5,6 and 7 week) evaluation levodopa, reply.

Immunohistochemistry

For Histological research, animal passes aortic perfusion with physiological saline, then with the perfusion of 4% Paraformaldehyde 96 (for vehicle perfusion and carrier perfusion (-) rapamycin group, n=6 rat is for carrier perfusion (+) rapamycin group, n=8 rat).Spent the night in 4% Paraformaldehyde 96 in the fixing back of brain, with gradient sucrose solution balance, freezing in iso-pentane.In cryostat, brain is cut into the thick coronal section of 40 μ m continuously.In the section of free-floating (free-floating), carry out immunohistochemistry (Chemicon, Temecula, CA, 1: 1500) to AADC.In 3% hydrogen peroxide, cultivate section 30 minutes, with the cancellation endogenous peroxydase.After with 5% normal goats serum blocking-up non-specific binding, at room temperature, will cut into slices and an anti-overnight incubation.At room temperature cultivate, use the anti-rabbit IgG antibody (VectorLaboratories of vitamin H (acyl) change earlier, Burlingame CA, 1: 300), horseradish peroxidase (the Vector Laboratories that connects with streptavidin then, 1: 300) hatched each 1 hour, (DAB is VectorLaboratories) with hydrogen peroxide development mixture with 3-3 ' diaminobenzidine.Section is fixed on the slide glass of gelatin coating, drying is dewatered in increasing continuously the ethanol of concentration, cleans with dimethylbenzene, utilize Cytoseal-60 fix (Richard-Allen Scientific, Kalamazoo, MI).The front and back of hAADC immunostaining distribute definite by formula (n * 12 * 40 μ m), and wherein n is the number of slices with hAADC positive cell, and 40 μ m measure all the 12 sections for the thickness of section.In serial section, estimate volume of distribution and positive cell counting (each the 12), (Optical Fractionator-Optical-Optical Dissector design stereological method is being equipped with the three-dimensional software (Microbrightfield that learns of pick up camera and three-dimensional research under 63X amplifies based on optical grade device-optics dissector in utilization, Williston, Zeiss microscopically VT) dyes to AADC.Every group CEE＜5%.As mean value ± SD ecbatic.Use Student ' s t calibrating to measure statistical significance.

Real-time quantitative PCR

The carrier A AV-Z12 hAADC that uses in this research comprises human AADC target gene.Make the Q-PCR primer and be annealed to the exon 2 of AADC gene and 3 probe, therefore, generated non-existent intron in the carrier sequence, thereby minimized the amplification of genomic dna.Q-PCR (Heid etc. (1996) Genome Res.6:986-94) is with comprising the plasmid DNA stdn that carrier inserts in real time.With the described plasmid of restriction enzyme linearize, purifying, quantitative with the UV light absorption ratio, and be diluted in (10mM Tris-HCl, pH 8.0,1mM EDTA, 10 μ g/ml yeast tRNA and 0.1% tween 80s) in the agent of Q-PCR dilution buffer, obtain each reaction from three to 10 ⁶10 standards of copy.Each standard is carried out the 50 μ l reaction of three replicate(determination)s in 96 hole optics plates.The sample of the 10 μ l of each self-contained 20ng DNA is joined in the Q-PCR hole of three replicate(determination)s that comprise 40 μ l reaction mixtures.PCR carries out in Applied Biosystems 7700Sequence Detection System.Compare with typical curve and to calculate the hAADC gene copy number, the every hole copy number that obtains multiply by two, supposes that a copy of double-stranded plasmid DNA is equivalent to two single-stranded vector genomes.

Western blot analysis

Respectively with in the cracking buffer reagent of the mixture that comprises inhibitors of phosphatases and proteinase inhibitor, homogenize ten serial section (each 40 μ m) of whole brain of hand-held homogenizer.Use Bradford method quantitative protein.(4-15% gradient gel on the SDS-PAGE gel; Bio-Rad, Hercules, California) protein isolate quality sample (15 μ g) and be transferred to polyvinylidene difluoride film (Millipore, Bedford, Massachusetts).

With 3% milk closing membrane, with the anti-AADC of polyclone rabbit (1: 500 Chemicon, Temecula, California) anti-hatching 1 hour.Then, at room temperature resist (1: 3000 with two of corresponding HRP connection; Amersham Biosciences, Arlington Heights, Illinois) hatched trace 1 hour, at ECL solution (PerkinElmer Life Sciences, Emeryville, California) developed 1 minute in, be exposed to from the X-Omat film of Kodak (Rochester, New York) 1-30 minute.At last, under 50 ℃, in stripping buffer (67.5mM Tris, pH 6.8,2%SDS and 0.7% beta-mercaptoethanol), hatched trace 30 minutes, with polyclone rabbit anti-β-actin antibody (1: 1000; Alpha Diagnostics, San Antonio is Texas) as application of sample contrast reprobed.In above-mentioned research, used anti-AADC one to resist widely, observed protein spectra demonstrates identical bands of a spectrum size (50kDa), as pointing out in the antibody information table in these researchs.

The density available computers of each specificity bands of a spectrum assist the imaging analysis system (

AlphaInnotech Corporation, San Leandro California) measures.There is not significant difference in the density of β Actin muscle contrast bands of a spectrum between each group.For the difference between vehicle perfusion (+) rapamycin control group and the carrier perfusion group relatively, at first with respect to the density of each specificity bands of a spectrum of density criterionization of corresponding internal reference bands of a spectrum (for every group, n=3).After deducting the endogenous hAADC level of in vehicle perfusion rat, finding, can determine minimizing (reduction) percentage ratio in the gross protein from two kinds of carrier perfusion groups.By using the significant difference of the many groups of one-way analysis of variance.

Embodiment 3

The external regulated expression of GDNF

With the regulatable GDNF transgenosis construct transduction of dipolymer mammalian cell, as follows.

The AAV-GDNF carrier

Fig. 8 A and 8B are for being used for the genetically modified reorganization AAV of deliverer GDNF (hGDNF) vector plasmid construction figure.The control vector that is used for sending constitutive expression hGDNF (pAAV-CMV-hGDNF) is presented at Fig. 8 C.

Adjustable construction (Fig. 8 A and 8B) comprises and is loaded with coding hGDNF gene and the activation structure domain fusion protein of transcription factor and single rAAV carrier of DNA integrated structure domain fusion protein component.In two kinds of constructions, the expression of hGDNF is subjected to the minimum IL-2 promoters driven of the combining site of contiguous eight transcription factors that are used for dimerization, will describe in more detail following.

In Fig. 8 A in the diagrammatic construction (pPAAV-TF-Z8-hGDNF), the expression of the cmv enhancer/activation of the promoters driven encoding transcription factor and the single transcription product of DNA binding domains, described transcription product has internal ribosome entry site (IRES) between two binding domainss.In Fig. 8 B diagrammatic construction, on the contrary, the expression of SV40 promoters driven DNA binding domains, the expression in cmv enhancer/promoters driven activation structure territory, described binding domains or activation structure territory are the different transcription products from opposite strand (in the opposite direction promptly).

DNA integrated structure domain fusion protein comprises two DNA binding domainss from human transcription factor Zif268, the homeodomain that is derived from Oct-1 (ZFHDI) and three medicine binding domainss from the cytosol receptor of FK506 (3xFKBP).

The activation structure domain fusion protein comprises human FRAP (FRB ^*) the rapamycin binding domains, it merges the transcriptional activation domain to the p65 subunit that is derived from NFKB (p65).Diagrammatic FRB in Fig. 8 A and 8B ^*Structural domain is to describe in embodiment 1.

Terminal (ITR), MIN SV40 polyadenylic acidization (Min.SV40pA), MIN rabbit betaglobulin polyadenylic acidization (Min.RBG pA) and MIN human growth hormone 3 polyadenylations (Min hGH pA) of oppositely repeating of AAV2 have been pointed out.

In control vector pAAV-CMV-hGDNF (Fig. 8 C), CMV promotor/enhanser drives the expression of hGDNF.Pointed out terminal (ITR) and human growth hormone polyadenylic acidization (pA) sequence of oppositely repeating of AAV2.

The result of rapamycin-induction experiment is presented among Fig. 9, and wherein the expression of GDNF is the function of vector construct and rapamycin concentrations.When with the rapamycin treatment cell, pAAV-TF-Z8-hGDNF instructs the generation of GDNF in the dose response mode, and pAAV-CMV-hGDNF instructs the horizontal GDNF of composing type (height) to express, and it has nothing to do with rapamycin treatment.

The experimental detail of the relevant test ELISA assay method of using in embodiment 3 is as follows:

GDNF EL/SA

The following ELISA test of carrying out quantitative GDNF expression.

In two 6 hole plates, make HEK-293 cell (5 * 10 ⁵Cells/well) grow overnight reaches 60-70% and merges.Utilize 300 μ M CaCl ₂, with pAAV-TF-Z8-hGDNF or the pAAV-CMV-hGDNF transfection plate of 10 μ g.After six hours, replace former substratum, grew three days with new rapamycin (0nM, 5nM or 25nM, each the two parts) substratum that comprises.Gather in the crops substratum and cell respectively, suddenly freezing and be kept under-80 ℃, up to carrying out ELISA.To being lower than pH 3.0, return pH 7.6 with the 1NaOH neutralization with all sample of 1N HCl acidifying 15 minutes then.

Utilize Promega

Immunoassay system (Promega, Inc.Madison, Wisconsin) existence of mensuration media samples GDNF.The coating buffer reagent is joined in the 96 hole plates, with it 4 ℃ of following overnight incubation.Remove the coating buffer reagent, dry plate.To seal buffer reagent (200 μ l) and join in the plate, at room temperature hatch 1 hour, not need jolting.Remove the deblocking buffer reagent, dry plate.

8 hole posts of two 96 hole plates are appointed as the GDNF standard.With 1X Block ﹠amp; The B-H that SampleBuffer (100 μ L/ hole) joins standard column is capable.The A that the GDNF standard (1000pg/ml of 200 μ L) of diluting is joined standard column is capable, the capable serial dilution of carrying out 2 times of 100 μ L/ hole of G.The H behavior does not contain the independent buffer reagent contrast of GDNF.

To pAAV-TF-Z8-hGDNF test, test sample is diluted to 1: 300, to the pAAV-CMV-hGDNF experiment, test sample is diluted to 1: 1000, every kind of 100 μ l is joined in two holes, at room temperature hatch six hours, and jolting (500rpm).Washing hole five times is used the washing buffer from about 400 μ l recommendation of test kit at every turn.With 1: 500 dilution anti-hGDNF polyclonal antibody of 100 μ l (at IX Block ﹠amp; Sample Buffer) joins in each hole,, do not need jolting 4 ℃ of following overnight incubation.Wash plate as mentioned above once more.With 1: 250 dilution anti-chicken IgY of 100 μ L, HRP conjugate (at IX Block ﹠amp; Among the Sample Buffer) join in each hole, at room temperature hatch two hours, and plate is washed in jolting (500rpm) as mentioned above once more.With the room temperature TMB One solution of 100 μ L (comprise the HRP substrate, 3,3 ', 5,5 '-tetramethyl benzidine) join in each hole, at room temperature hatched 15 minutes, do not need jolting.By stopping color reaction for the 1N hydrochloric acid that adds 100 μ L in each hole.At 450nm, in adding 30 minutes of TMB, writing down light absorption ratio on the plate reader.With compare from containing the signal that obtains GDNF standard and the test sample at identical plate, determine GDNF level (pg/ml).Standard deviation is from four mensuration (the double sample on each of two plates) altogether to each sample.

Though described the preferred illustrative embodiment of the present invention, but to it is evident that those skilled in the art, do not deviating under the present invention, wherein can carry out various variations and modification, cover all such variation and modifications that fall in the real spirit and scope of the present invention by additional claim.

Therefore, all publications, patent, patent application, sequence and data base entries all are incorporated herein by reference with it.

Claims (11)

1. be used for the treatment of the patient's who suffers from neurological disorder pharmaceutical composition, comprise:

Recombinant adeno-associated virus (AAV) carrier, its coding has the adjustable transcription factor of transcripting reinforcing activity;

The genetically modified reorganization AAV carrier of encoding;

Wherein genetically modified expression is subjected to the influence of transcription factor activity.

2. the pharmaceutical composition of claim 1, wherein adjustable transcription factor and transgenosis are for encoding on independent rAAV carrier.

3. each pharmaceutical composition among the claim 1-2, wherein, under the situation that has rapamycin or forms of rapamycin analogs, the activity of adjustable transcription factor increases.

4. the pharmaceutical composition of claim 3, wherein forms of rapamycin analogs is AP21967.

5. each pharmaceutical composition among the claim 1-4, wherein neurological disorder is a Parkinson's disease.

6. each pharmaceutical composition among the claim 1-5, wherein transgenosis is the L-amino acid decarboxylase (AADC) of fragrance.

7. each pharmaceutical composition among the claim 1-5, wherein transgenosis is a glial cell line deutero-neurotrophic factor (GDNF).

8. treatment suffers from patient's the method for neurological disorder, comprises among the claim 1-7 of drug treatment effective dose each pharmaceutical composition.

9. implement the test kit of the method for claim 8, comprising:

Reorganization AAV carrier, its adjustable transcription factor of encoding;

Reorganization AAV carrier, its transgenosis of encoding; With

Rapamycin or forms of rapamycin analogs.

10. suffers from purposes in patient's the method for neurological disorder according to each composition among the claim 1-7 in treatment.

The purposes of reorganization AAV carrier in preparing the composition for the treatment of the patient who suffers from neurological disorder of genetically modified reorganization AAV carrier and the adjustable transcription factor of coding 11. encode, wherein this adjustable transcription factor has transcripting reinforcing activity, and this genetically modified expression is subjected to the influence of this transcription factor activity.

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