阅读说明：本技术 Aav衣壳设计 (The design of AAV capsid ) 是由 高光平 徐光超 P·戴 魏宇全 罗丽 于 2017-10-13 设计创作，主要内容包括：在一些方面,本公开涉及具有不同组织靶向能力的重组腺相关病毒。在一些方面,本公开涉及使用重组腺相关病毒的基因转移方法。在一些方面,本公开涉及分离的AAV衣壳蛋白和编码其的分离的核酸。(In some respects, this disclosure relates to the recombinant adeno-associated virus with different tissues targeting ability.In some respects, this disclosure relates to using recombinant adeno-associated virus gene transfer method.In some respects, this disclosure relates to isolated AAV capsid protein and encode its isolated nucleic acid.)

1. a kind of recombinant expression carrier, it includes the nucleic acid of coding polypeptide, the polypeptide has sequence selected from the following: SEQ ID Any of NO:1 to 409,435-868 or 1726-1988 or its segment, do not encode with SEQ ID NO:869,870 or 871 The identical peptide of sequence.

2. a kind of isolated AAV capsid protein, it includes amino acid sequences selected from the following: SEQ ID NO:1 to 409,435- 868 and 1726-1988 or its segment.

3. a kind of isolated AAV capsid protein, it includes amino acid sequences selected from the following: SEQ ID NO:1-409,837- 852 or 1726-1814, wherein by amino different to the corresponding amino acid of sequence shown in SEQ ID NO:869 in the sequence Acid replaces with conservative substitution.

4. a kind of isolated AAV capsid protein, it includes amino acid sequences selected from the following: SEQ ID NO:435-628 or 1815-1988, wherein by the sequence to sequence shown in SEQ ID NO:869 or 870 the different amino of corresponding amino acid Acid replaces with conservative substitution.

5. a kind of isolated AAV capsid protein, it includes amino acid sequences selected from the following: SEQ ID NO:629-836 or 853-868, wherein being by amino acid substitution different to the corresponding amino acid of sequence shown in SEQ ID NO:871 in the sequence Conservative substitution.

6. the peptide fragment of isolated AAV capsid protein according to any one of claim 2 to 5, and SEQ ID NO: 869, any of 870 or 871 sequence is different.

7. a kind of isolated AAV capsid protein, it includes peptide fragments as claimed in claim 6.

8. a kind of recombinant expression carrier, it includes the isolated AAV capsid proteins described in any one of coding claim 2 to 5 Nucleic acid sequence.

9. a kind of composition, it includes the isolated AAV capsid proteins described in any one of claim 2 to 5.

10. a kind of composition it includes the isolated AAV capsid protein described in any one of claim 2 to 5 and pharmaceutically may be used The carrier of receiving.

11. a kind of recombination AAV (rAAV), it includes the isolated AAV capsid proteins described in any one of claim 2 to 5.

12. a kind of composition, it includes the recombination rAAV described in claim 11.

13. composition according to claim 12 also includes pharmaceutically acceptable carrier.

14. a kind of host cell, contain the nucleic acid of the coded sequence comprising polypeptide selected from the following: SEQ ID NO:1-409, 435-868 and 1726-1988, is operably connected with promoter.

15. a kind of composition, it includes the host cells and sterile cell maintenance medium described in claim 14.

16. a kind of composition, it includes the host cells and cryoprotector described in claim 15.

17. a kind of method for by transgene delivery to subject comprising:

RAAV described in claim 11 is applied to subject, wherein the rAAV includes at least one transgenosis, and wherein The rAAV infects the cell of the target tissue of the subject.

18. a kind of method for generating body cell transgenic animal model comprising applied described in claim 11 to non-human animal Recombination rAAV, wherein the rAAV includes at least one transgenosis, and wherein the rAAV infects the non-human animal's The cell of target tissue.

19. according to the method for claim 17, wherein at least one transgenosis is protein coding gene.

20. according to the method for claim 17, wherein at least one transgenes encoding small RNA.

21. according to the method for claim 20, wherein the small RNA is miRNA.

22. according to the method for claim 20, inhibiting wherein the small RNA is miRNA sponge or TuD RNA The activity of at least one of the subject or animal miRNA.

23. according to the method for claim 22, wherein expressing the miRNA in the cell of the target tissue.

24. according to the method for claim 17, wherein the target tissue is liver, central nervous system (CNS), eye, stomach Intestines, breathing, breast, pancreas, the urinary tract or uterine tissue.

25. according to the method for claim 18, wherein the transgene expression includes at least one miRNA binding site Transcript, wherein the miRNA inhibits the transgenosis in the tissue other than target tissue by hybridizing with the binding site Activity.

26. a kind of method for generating body cell transgenic animal model comprising applied described in claim 23 to non-human animal RAAV, wherein the rAAV includes at least one transgenosis, wherein the transgene expression includes at least one knot of miRNA The transcript of coincidence point, wherein the miRNA inhibits the transgenosis in target by hybridizing with the binding site of the transcript The activity in tissue other than tissue.

27. according to the method for claim 26, wherein the transgenosis includes that tissue-specific promoter or induction type open Mover.

28. according to the method for claim 27, wherein the tissue-specific promoter is liver specificity thyroxine Haptoglobin (TBG) promoter, insulin promoter, glucagon promoter, growth hormone release inhibiting hormone promoter, mucoprotein -2 open Mover, pancreatic polypeptide (PPY) promoter, synapsin -1 (Syn) promoter, retinoschisis element promoter, K12 promoter, CC10 promoter, surfactant protein C (SP-C) promoter, PRC1 promoter, RRM2 promoter, uroplakin2 (UPII) are opened Mover or lactoferrin promoter.

29. according to the method for claim 17, wherein the rAAV passes through intravenous, transdermal, intraocular, intrathecal, oral, flesh Meat is interior, subcutaneously, intranasally or by sucking applies.

30. according to the method for claim 17, wherein the subject is selected from mouse, rat, rabbit, dog, cat, sheep, pig And non-human primate.

31. according to the method for claim 17, wherein the subject is people.

32. a kind of body cell transgenic animal model generated by method of claim 18.

33. a kind of kit for producing rAAV, the kit includes:

Accommodate the container of isolated nucleic acid, the isolated nucleic acid encode with SEQ ID NO:1 to 409,435-868 or The polypeptide of the sequence of any of 1726-1988.

34. kit according to claim 33 also includes the specification for producing the rAAV.

35. kit according to claim 34 also includes the container that at least one accommodates recombination AAV carrier, wherein The recombination AAV carrier includes transgenosis.

36. a kind of kit, it includes:

Container accommodates the recombination AAV with isolated AAV capsid protein described in any one of claim 2 to 5.

37. kit according to claim 36, wherein the container is syringe.

38. the isolated AAV capsid protein according to any one of claim 2-5, wherein the capsid protein is VP1 clothing Glutelin.

39. the isolated AAV capsid protein according to any one of claim 2-5, wherein the capsid protein is VP2 clothing Glutelin.

40. the isolated capsid protein according to any one of claim 2-5, wherein the capsid protein is VP3 capsid Albumen.

41. a kind of vacation type AAV, it includes the capsid proteins described in any one of claim 2 to 5 or 7.

42. recombinant expression carrier according to claim 1, wherein the nucleic acid encode V1 capsid protein.

43. recombinant expression carrier according to claim 1, wherein the nucleic acid encode V2 capsid protein.

44. recombinant expression carrier according to claim 1, wherein the nucleic acid encode V3 capsid protein.

45. a kind of nucleic acid, it includes the sequences for being selected from SEQ ID NO:410-434,876-1718 and 1989-2251.

46. nucleic acid according to claim 45, wherein by the engineered nucleic acid to express AAV capsid protein or its variant And/or AAV assembles activator protein (AAP) or its variant.

47. nucleic acid according to claim 45 is opened wherein the AAP is located at the nucleic acid different from the AAV capsid protein It puts in reading frame.

48. nucleic acid according to claim 46, wherein the AAP is AAV2 AAP (AAP-2) or its variant.

49. a kind of isolated albumen, the nucleic acid encode as described in claim 45.

50. a kind of recombination AAV, it includes the isolated albumen described in claim 49.

Invention field

In some respects, this disclosure relates to be used for isolated nucleic acid, composition and the examination of adeno-associated virus in identification of cell Agent box.In some respects, present disclose provides new AAV and its application method and related kits.

Background of invention

Recombination AAV adeno-associated virus (rAAV) can drive stable and lasting transgene expression in target tissue, and not have There are significant toxicity and host immune originality.Therefore, rAAV is the promising delivering load for long-tenn therapeutic gene expression Body.However, be currently available that the low transduction efficiency of rAAV carrier and limited tissue tropism can limit they as feasible and The application effectively treated.In addition, the clinical translation of the loyalty of the main therapeutic AAV serotype from non-human tissue is one and asks Topic.Therefore, there is still a need for being used for the new AAV carrier of gene delivery.

Summary of the invention

In some respects, this disclosure relates to the new AAV applied for gene therapy.In some embodiments, this paper institute The AAV stated includes the amino acid variation of one or more capsid proteins, assigns new or enhancing tissue tropism's property.According to Some embodiments, change identified and disclosed herein is AAV2, AAV2/3 (for example, AAV2/3 heterozygote) and AAV8 Body, with useful tissue-targeting matter.For example, provide the variant of AAV8, it can be used for transducer cell, such as people liver is thin Born of the same parents' (for example, being present in hepatic tissue), central nervous system cell (CNS cell) etc..Provide AAV2, AAV2/3 (for example, AAV2/3 heterozygote) and the variant of AAV8 can be used for targeting ocular tissue's (for example, eyes), stomach and intestine in some embodiments Road, respiratory system, breast tissue, pancreatic tissue, the urinary tract tissue, uterine tissue and certain cancers are (for example, breast cancer, forefront Gland cancer etc.) it is relevant tissue and its hetero-organization cell.In some embodiments, modification A AV as described herein targeting in addition to Tissue other than the tissue of its corresponding wild type AAV targeting.

The disclosure provides the nucleic acid of separation in some respects, and it includes the sequences for encoding polypeptide selected from the following: SEQ ID NO:1-409,435-868 and 1726-1988 encode AAV capsid protein.In some embodiments, separation is provided Nucleic acid segment.In certain embodiments, the segment of isolated nucleic acid does not encode and SEQ ID NO:869,870 or 871 Any of the identical peptide of sequence.

In some respects, the disclosure provides nucleic acid, and it includes be selected from SEQ ID NO:410-434,876-1718 and 1989- 2251 sequence.In some embodiments, the nucleic acid encode expression AAV capsid protein or its variant and/or AAV assembling swash Living protein (AAP) or its variant.In some embodiments, the AAP and the AAV capsid protein are located at the nucleic acid not With in open reading frame.In some embodiments, the AAP is AAV2AAP (AAP-2) or its variant.

In some respects, the disclosure provides isolated AAV capsid protein, and it includes be selected from SEQ ID NO:1-409,435- The amino acid sequence of 868 and 1726-1988.In some embodiments, isolated AAV capsid protein includes ammonia selected from the following Base acid sequence: SEQ ID NO:1-409,837-852 or 1726-1814, wherein by the sequence with SEQ ID NO:869 institute Show that the different amino acid substitution of the corresponding amino acid of sequence is conservative substitution.

In some respects, the disclosure provides AAV2/3 heterozygote capsid protein.In some embodiments, isolated AAV Capsid protein includes amino acid sequence selected from the following: SEQ ID NO:435-628 and 1815-1988, wherein by the sequence In to sequence shown in SEQ ID NO:869 or 870 the different amino acid substitution of corresponding amino acid be conservative substitution.

In some embodiments, isolated AAV capsid protein includes sequence selected from the following: SEQ ID NO:629- 836 or 853-868, wherein by amino acid different to the corresponding amino acid of sequence shown in SEQ ID NO:871 in the sequence Replace with conservative substitution.

In the disclosure in some terms, providing the composition comprising any aforementioned isolated AAV capsid protein.Some In embodiment, the composition also includes pharmaceutically acceptable carrier.In some embodiments, the disclosure is provided The composition of one or more isolated AAV capsid proteins and physiologically compatible carrier.

In the disclosure in some terms, providing the recombination AAV comprising any aforementioned isolated AAV capsid protein (rAAV).In some embodiments, the composition comprising rAAV is provided.It in certain embodiments, include the people AAV The composition also include pharmaceutically acceptable carrier.Recombination AAV is additionally provided, wherein recombination AAV includes a kind of or more The isolated AAV capsid protein of the kind disclosure.

In some aspects of the disclosure, host cell is provided, contains the nucleic acid comprising coded sequence selected from the following: SEQ ID NO:410-434,876-1718 and 1989-2251, are operably connected with promoter.In some embodiments In, provide the composition comprising host cell and sterile cell maintenance medium.In some embodiments, it provides comprising host The composition of cell and cryoprotector.

According to some aspects of the disclosure, provide for by the method for transgene delivery to subject.In some implementations In scheme, this method includes applying aforementioned any rAAV to subject, wherein the rAAV includes at least one transgenosis, and And wherein the rAAV infects the cell of the target tissue of the subject.In some embodiments, subject is selected from mouse, big Mouse, rabbit, dog, cat, sheep, pig and non-human primate.In one embodiment, the subject is people.

In some embodiments, at least one transgenosis is protein coding gene.In certain embodiments, institute State at least one transgenes encoding small RNA.In certain embodiments, small RNA is miRNA.In certain implementations In scheme, the small RNA is miRNA sponge or TuD RNA, inhibits the work of at least one of subject miRNA Property.In certain embodiments, the miRNA is expressed in the cell of the target tissue.In certain embodiments, the target Tissue is liver, central nervous system (CNS), eye, stomach and intestine, breathing, mammary gland, pancreas, the urinary tract or uterine tissue.

In some embodiments, the transgene expression includes the transcript of at least one miRNA binding site, wherein The miRNA is by hybridizing the activity for inhibiting the transgenosis in the tissue other than target tissue with the binding site.

In certain embodiments, rAAV by intravenous, transdermal, intraocular, intrathecal, intracerebral, oral, intramuscular, it is subcutaneous, Intranasally or by sucking it is applied to subject.

According to some aspects of the disclosure, the method for generating body cell transgenic animal model is provided.Some In embodiment, this method includes applying aforementioned any rAAV to non-human animal, wherein the rAAV includes at least one Transgenosis, and wherein the rAAV infects the cell of the target tissue of the non-human animal.

In some embodiments, the transgenosis is at least one protein coding gene.In certain embodiments, institute State transgenes encoding at least one small RNA.In some embodiments, the transgenes encoding at least one report point Son.In certain embodiments, small RNA is miRNA.In certain embodiments, the small RNA is miRNA Sponge or TuD RNA, inhibit the activity of at least one of animal miRNA.In certain embodiments, in the target group The miRNA is expressed in the cell knitted.In certain embodiments, the target tissue be liver, central nervous system (CNS), Eye, stomach and intestine, breathing, mammary gland, pancreas, the urinary tract or uterine tissue.

In some embodiments, the transgene expression includes the transcript of at least one miRNA binding site, wherein The miRNA is by hybridizing the activity for inhibiting the transgenosis in the tissue other than target tissue with the binding site.

According to some aspects of the disclosure, the method for generating body cell animal model is provided comprising move to inhuman Object applies aforementioned any rAAV, wherein the rAAV includes at least one transgenosis, wherein the transgene expression includes The transcript of at least one binding site of miRNA, wherein the miRNA is by hybridizing suppression with the binding site of the transcript Make activity of the transgenosis in the tissue other than target tissue.

In some embodiments, the transgenosis includes tissue-specific promoter or inducible promoter.Certain In embodiment, the tissue-specific promoter is liver specificity thyroxine-binding globulin (TBG) promoter, pancreas islet Plain promoter, growth hormone release inhibiting hormone promoter, -2 promoter of mucoprotein, pancreatic polypeptide (PPY) promoter, is dashed forward at glucagon promoter Touch albumen -1 (Syn) promoter, retinoschisis element promoter, K12 promoter, CC10 promoter, surfactant protein C (SP- C) promoter, PRC1 promoter, RRM2 promoter, (UPII) promoter of uroplakin 2 or lactoferrin promoter.

In certain embodiments, rAAV passes through intravenous, transdermal, intraocular, intrathecal, oral, intramuscular, subcutaneous, intranasal Or animal is applied to by sucking.According to some aspects of the disclosure, the body cell generated by any preceding method is provided Transgenic animal model.

In terms of other of the disclosure, the kit for generating rAAV is provided.In some embodiments, the examination Agent box includes the container for accommodating isolated nucleic acid, the isolated nucleic acid with SEQ ID NO:410-434,876-1718 and The sequence of any of 1989-2251.In some embodiments, the kit includes the container for accommodating isolated nucleic acid, The isolated nucleic acid encode has the polypeptide of the sequence of any of SEQ ID NO:1-409,435-868 or 1726-1988. In some embodiments, the kit also includes the specification for producing the rAAV.In some embodiments, institute Stating kit also includes that at least one accommodates the container for recombinating AAV carrier, wherein the recombination AAV carrier includes transgenosis.

In terms of other of the disclosure, kit is provided, it includes the container for accommodating recombination AAV, the recombination AAV tool There is any aforementioned isolated AAV capsid protein.In some embodiments, the container of kit is syringe.

In other respects, this disclosure relates to based on the carrier of AAV as carrier, gene delivery, treatment, prevention and research mesh And body cell transgenic animal model exploitation purposes.

In some respects, this disclosure relates to which AAV serotype, has been proven that different tissue/cell type tropisms simultaneously And it can be steady with horizontal realization similar with adenovirus vector in animal tissue in the case where no carrier correlation toxicology Fixed somatic cell gene transfer (for example, up to 100% in-vivo tissue is transduceed, depending on target tissue and carrier dosage).At it In terms of him, this disclosure relates to have liver, central nervous system (CNS), eye, stomach and intestine, breathing, mammary gland, pancreas, the urinary tract or son The AAV serotype of palace tissue targeting ability.These tissues are related to extensive human diseases, including the nervous system disease, metabolism Disease, diabetes system, eye disease, respiratory disease, enterogastric diseases, disease of urinary tract and reproductive disease and certain cancers Disease.

In some embodiments, the rAAV includes at least one transgenosis.Transgenosis, which can be, causes pathological state Transgenosis.In some embodiments, the albumen of transgenes encoding treatment of pathological conditions.

On the other hand, can be used for will be in the method for transgene delivery to subject by the new AAV of the disclosure.This method It is carried out by applying the rAAV of the disclosure to subject, wherein rAAV includes at least one transgenosis.In some embodiments In, rAAV targets the predetermined tissue of subject.

On the other hand, the AAV of the disclosure can be used for generating in the method for body cell transgenic animal model.The party Method is carried out by applying the rAAV of the disclosure to animal, and wherein rAAV includes at least one transgenosis, and transgenic causes Pathological state, and wherein rAAV targets the predetermined tissue of animal.

Transgenosis can express many genes, including cancer related gene, rush apoptogene and apoptosis-related genes.One In a little embodiments, transgene expression is able to suppress the small RNA of cancer related gene expression.In other embodiments, Transgene expression is able to suppress the small RNA of the expression of apoptosis-associated genes.Small RNA in other embodiments is MiRNA or shRNA.According to other embodiments, transgene expression toxin, optionally wherein toxin is DTA.In other embodiments In, transgene expression reporter gene is optionally reporter enzyme, such as beta galactosidase or fluorescin, such as GFP or firefly Light element enzyme.

Transgenosis can express miRNA.In other embodiments, transgene expression miRNA sponge, wherein miRNA is extra large Silk floss inhibits the activity of one or more miRNA in animal.In some embodiments, miRNA can be interior miRNAs or it can To be expressed in the cell of liver, central nervous system (CNS), eye, stomach and intestine, breathing, mammary gland, pancreas, the urinary tract or uterine tissue.

RAAV can transduce many different types of tissues, such as neuron, squamous cell, kidney proximal end or distal end are returned Renal tubular cell, mucous membrane gland cell, vascular endothelial cell, endometrial cell, retina cell or the certain cancer cell (examples of rotation Such as, breast cancer cell, prostate gland cancer cell etc.).

In some embodiments, with 10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵The agent of a genome copies/subject Amount application rAAV.In some embodiments, with 10¹⁰、10¹¹、10¹²、10¹³Or 10¹⁴The dosage of a genome copies/subject Apply rAAV.RAAV can be applied by any approach.For example, in some embodiments, it can intravenously apply (example Such as, pass through introportal infusion).

In some embodiments, transgenosis includes tissue-specific promoter, such as liver specificity thyroxine knot Globulin (TBG) promoter, insulin promoter, glucagon promoter, growth hormone release inhibiting hormone promoter, mucoprotein -2 is closed to start Son, pancreatic polypeptide (PPY) promoter, synapsin -1 (Syn) promoter, retinoschisis element promoter, K12 promoter, CC10 Promoter, surfactant protein C (SP-C) promoter, PRC1 promoter, RRM2 promoter, uroplakin 2 (UPII) starting Son or lactoferrin promoter.

Body cell transgenic animal model can be mammal, such as mouse, rat, rabbit, dog, cat, sheep, pig, non- People primate.

In some embodiments, the therapeutic agent of presumption can be applied to body cell transgenic animal model, with determination Influence of the therapeutic agent of presumption to the pathological state of animal.

On the other hand, the disclosure is the body cell transgenic animals generated by methods described herein.

According to another aspect of the present disclosure, the kit for generating rAAV is provided, generation has in predetermined tissue There are the body cell transgenic animals of pathological state.The kit include at least one accommodate recombination AAV carrier container, at least one A container for accommodating rAAV packing composition, and the specification of building and packaging recombination AAV.

RAAV packing composition may include the host cell for expressing at least one rep gene and/or at least one cap gene. In some embodiments, host cell is 293 cells.In other embodiments, host cell expression at least one assists Viral gene products influence the generation of the rAAV containing recombination AAV carrier.At least one described cap gene, which can encode, to be come From the capsid protein for the AAV serotype for targeting predetermined tissue.

In other embodiments, rAAV packing composition includes helper virus, and optionally wherein helper virus is adenovirus Or herpesviral.

RAAV carrier and component therein may include any element as described herein.For example, in some embodiments, RAAV carrier includes transgenosis, such as any transgenosis as described herein.In some embodiments, transgene expression miRNA Inhibitor (for example, miRNA sponge or TuD RNA), wherein miRNA inhibitor inhibits a kind of or more in body cell transgenic animals The activity of kind miRNA.

Each limitation of the disclosure can include the various embodiments of the disclosure.Therefore, it is contemplated that be related to any one The each limitation for the disclosure that component or component group is closed can include in each aspect of the disclosure.The present disclosure is not limited to it The application of structure detail and component layout illustrating in the following description or shown in the accompanying drawings.The disclosure can have other It embodiment and can be practiced or carried out in various ways.

Brief Description Of Drawings

Figure 1A -1B shows the workflow schematic diagram for identifying AAV variant.Figure 1A is depicted in chosen tissue The high-throughput detection of new AAV variant.Using high circulation PCR amplification provirus capsid sequence, low circulation PCR is then carried out to expansion It is bar coded to carry out (SMRT) sequencing in real time of multiple unimolecule to increase sublibrary progress.Figure 1B shows the life for sequencing data The general introduction of the pipeline (pipeline) of object bioinformatics analysis.

Fig. 2A -2D shows number related with the selected AAV8 variant vivo detection FFLuc transgene activity of different administration According to.Fig. 2A shows the firefly of the different AAV8 variants of assessment in the 6th week after (intranasal) injection of IV (intravenous), IM (intramuscular) or IN Light element enzymatic activity.The data of Fig. 2 B-2D are related to every kind of variant B2 (Fig. 2 B), B3 (Fig. 2 C) and B61 (Fig. 2 D) compared with AAV8 The active assessment of FFLuc (average value ± SD, n=3, t are examined).

The FFLuc that Fig. 3 A-3B shows that the 21st day AAV8 variant B61 is delivered compared with AAV9 after new born animal injection turns The relevant data of the assessment of gene activity.Have detected the luciferase activity and genome copies of brain (Fig. 3 A) and spinal cord (Fig. 3 B) (average value ± SD, n=5, t are examined).

Fig. 4 A-4B shows vivo detection FFLuc after right hind of the AAV8 variant B44 compared with AAV8 intramuscular (IM) injection The data of transgene activity.Fig. 4 A shows the entire animal luciferase expression of the 6th week assessment variant B44 after IM injection. Fig. 4 B shows muscle (RTA, right tibialis anterior；LTA, flesh before left tibia), the assessment of liver and heart.B44 is compared with AAV8 Luciferase activity (left side bar chart) and relative ratios' (right bars figure) (average value ± SD, n=3).

Compared with Fig. 5 shows that AAV8 variant (B2, B3, B61) occurs with the system of other AAV serotypes.

Fig. 6 A shows the schematic diagram of the workflow by characterizing new AAV variant in high-throughput tropism screen body.

Fig. 6 B, which is shown, screens the schematic diagram that NHP characterizes the workflow of new AAV variant by high-throughput tropism.

Fig. 7 shows scatter plot, shows that the different AAV2 capsid variants with one or more monamino acid variants are (total Totally 409) and AAV2/3 variant (in total 194) distribution.

Fig. 8 shows the figure of the vector construct used in the multiplex screening of the capsid variants of discovery.By unique 6- Bp bar code is cloned into transgenosis and is packaged into candidate capsid variants.

Fig. 9 shows index transgenosis and high-throughput sequencing library design for assessing capsid variants tropism profile analysis Schematic diagram.Being able to use the site flank BsrGI and SacI will limit containing 6-bp bar code (the first bar code) and BstEII The index and adapter box in property site are cloned into vector construct.It is cut to come from BstEII digestion and contains host genome and load The complete thick DNA of the rAAV processing tissue of body genome.Obtained 5'- jag is connected to for specificity containing the second bar code Adapter, this allow further it is multiple sequencing and streamlining (streamlining)；With 5'- biotin modification, can be used The segment containing adapter is selected in use enrichment with magnetic bead.Then use the primer special to adapter and transgenic sequence can PCR amplification is carried out to the material of enrichment, to generate the library for being used for high-flux sequence.SEQ ID NO is shown from top to bottom: 1719-1725。

It is described in detail

Adeno-associated virus (AAV) is a kind of small (about 26nm) replication defect type nonenveloped virus, often relies on second Such as presence of adenovirus or herpesviral of kind virus, it could grow in cell.Disease can be caused and induce non-by being unaware of AAV Often slight immune response.AAV can infect division and non-dividing cell, and can be by its genome conformity to host cell Genome in.These features make AAV become the very attractive candidate for creating gene therapy viral vector.Based on serum The prototype AAV carrier of type 2 provides Proof of Concept for the nontoxic and stable gene transfer in mouse and larger animal model, but The gene transfering efficiency gone on business is showed in many main target tissues.In some respects, the disclosure attempts to be used for by providing to have The different tissues of gene therapy and research application target the new AAV of ability to overcome the disadvantage.

In some aspects of the disclosure, the new AAV capsid protein with different tissues targeting ability is provided.In some realities It applies in scheme, the tissue targeted from the AAV comprising capsid protein separates AAV capsid protein.In some respects, it provides for inciting somebody to action The method of target tissue of the transgene delivery into subject.Transgene delivery method can be used for gene therapy (for example, treatment disease Disease) or research (for example, creation body cell transgenic animal model) application.

It was found that the method for AAV

Most of biology of AAV is all influenced by its capsid.Thus, it is found that the method for new AAV is concentrated mainly on separation On the DNA sequence dna of AAV capsid.The central feature of the latent life cycle of adeno-associated virus (AAV) is in host cell with integration And/or the form of episome group persistently exists.Method for separating new AAV includes the latent AAV DNA gene of based on PCR The molecule rescue of group, the infectious virus of the latent provirus genome of vitro tissue DNA in the presence of adenovirus miscellaneous function Rescue, and it is polymerase-mediated by isothermal bacteriophage Phi-29, save from tissue DNA by rolling ring linear amplification it is cyclic annular former sick Virus gene group.The incubation period of AAV proviral DNA genome is all utilized in all these separation methods, and it is lasting to be absorbed in rescue Property virus genom DNA.

In some respects, this disclosure relates to find to identify that there is desired tissue tropism from the in-vivo tissue of subject New AAV variant.Be not intended to be any particular theory, the use of in-vivo tissue be utilized from the normal tissue of subject and The natural reservoir for the genome diversity observed in the virus genome sequence of tumor tissues separation.Therefore, in some implementations In scheme, it is multifarious that in-vivo tissue by selection pressure and/or immune evasion serves as viral (for example, viral capsid proteins) Natural incubator.

In some respects, this disclosure relates to find the PCR product generated by the amplification of AAV DNA (for example, from host cell In the in-vivo tissue of subject separate or extract AAV DNA) can be subjected to high-throughput unimolecule, in real time (SMRT) sequencing with Identify new capsid protein variant.As used herein, " unimolecule, in real time (SMRT) sequencing " refers to the single-molecule sequencing of parallelization Method, such as Roberts et al. (2013) Genome Biology 14:405, doi:10.1186/gb-2013-14-7-405 It is described.It is not intended to be any particular theory, the use of SMRT sequencing is eliminated to from other routine high-throughput genomes The short reading segment for the alignment that sequencing approach obtains carries out the needs that viral genome is rebuild and chimera is predicted.

The latent AAV genome of endogenous is in mammalian cell (for example, non-human primate tissue such as liver, spleen The cell of dirty and lymph node) in there is transcriptional activity.It is not wishing to be bound by theory, it is assumed that in order to maintain AAV holding in host Long property, it may be necessary to the low-level transcription from AAV gene, and resulting cap RNA can be used as it is more suitable and abundant Substrate come retrieve for carrier exploitation functional cap sequence.By RNA detection method (for example, RT-PCR) can be changed abundance Detect rep and cap genetic transcription object.The presence of cap genetic transcription object and cap RNA is generated by external reverse transcription (RT) The ability of cDNA significantly increases the abundance for the rescue template of the new cap sequence of based on PCR from tissue, and enhances new The sensitivity of AAV discovery.

New cap sequence can also be total thin by what is separated from the tissue with extremely low-abundance provirus AAV genome Born of the same parents DNA transfects cell to identify.Cell can further be turned with the gene for providing helper viral function (for example, adenovirus) Dye in the cell of transfection to trigger and/or enhance AAV genetic transcription.It in some embodiments, can be by from transfection Cap mRNA is separated in cell, and cDNA (such as passing through RT-PCR) is generated from mRNA and the new of the disclosure is identified to cDNA sequencing Cap sequence.

Isolated capsid protein and the nucleic acid for encoding it

The AAV separated from mammal, particularly non-human primate can be used for generating for clinical development and the mankind The gene transfer vector of gene therapy application.In some respects, present disclose provides use method disclosed herein in various bodies What is found in inner tissue's (for example, liver, brain, stomach, breathing, mammary gland, pancreas, rectum, prostate, uropoiesis and cervical tissue) is new AAV.In some embodiments, wherein finding that the tissue of new AAV variant is cancerous tissue (for example, tumour or cancer cell).One In a little embodiments, organizing to have found the capsid protein for encoding these new AAV in isolated virus genom DNA from people Nucleic acid.The example it has been found that tissue of new AAV capsid protein is described in table 1.About AAV nucleic acid and protein sequence and Other information is listed in table 3-5 and 8 and sequence table.

The isolated nucleic acid for encoding the disclosure of AAV capsid protein includes having SEQ ID NO:410-435,876-1718 Or any nucleic acid of sequence shown in any of 1989-2251, and with any nucleic acid with its substantially homologous sequence.In In some embodiments, the isolated nucleic acid of the disclosure includes any nucleic acid of the sequence with coding polypeptide, the polypeptide tool There are the sequence of any of SEQ ID NO:1-409,435-868 or 1726-1988.In some embodiments, the disclosure mentions Isolated nucleic acid is supplied, and with sequence shown in any of SEQ ID NO:410-435,876-1718 and 1989-2251 Nucleic acid there is basic homology, but do not encode the egg with amino acid sequence shown in SEQ ID NO:869,870 or 871 It is white.

In some embodiments, the isolated AAV capsid protein of the disclosure include with SEQ ID NO:1-409, Any albumen of amino acid sequence shown in any of 837-852 or 1726-1814 and with it with basic homology Any albumen.In some embodiments, present disclose provides isolated capsid protein, with SEQ ID NO:1-409, The albumen of sequence shown in any of 837-852 and 1726-1814 has basic homology, but does not have SEQ ID NO: Amino acid sequence shown in 869.

In some embodiments, the isolated AAV capsid protein of the disclosure include with SEQ ID NO:435-628 or Any albumen of amino acid sequence shown in any of 1815-1988 and with its any albumen with basic homology. In some embodiments, present disclose provides isolated capsid protein, and with SEQ ID NO:435-628 and 1815- The albumen of sequence shown in any of 1988 has basic homology, but does not have amino shown in SEQ ID NO:869 or 870 Acid sequence.

In some embodiments, the isolated AAV capsid protein of the disclosure include with SEQ ID NO:629-836 or Any albumen of amino acid sequence shown in any of 853-868 and with its any albumen with basic homology.In In some embodiments, present disclose provides isolated capsid protein, and with SEQ ID NO:629-836 or 853-868 Any of shown in sequence albumen have basic homology, but do not have SEQ ID NO:871 shown in amino acid sequence.

" homology " refers to the homogeneity percentage between two polynucleotides or two polypeptide portions.When refer to nucleic acid or When its segment, term " basic homology " is indicated, when with nucleotides inserted appropriate or missing, (or its is complementary with another nucleic acid Chain) optimal comparison when, there are the nucleotide sequence homologies of about 90 to about 100% of aligned sequences.When referring to polypeptide or its piece Duan Shi, term " basic homology " indicate that there are ratios when with notch appropriate, insertion or missing with another polypeptide optimal comparison To about 90 to 100% nucleotide sequence homology of sequence.Term " highly conserved " refers at least 80% identity, preferably extremely Few 90% identity, more preferably above 97% identity.In some cases, it is highly conserved may refer to 100% it is same Property.Those skilled in the art are readily determined together by using algorithm well known by persons skilled in the art and computer program One property.

As described herein, the comparison between nucleic acid or polypeptide sequence uses a variety of disclosures or commercially available multisequencing Any one of alignment programs carry out, such as " Clustal W ", can be accessed by the Web server on internet.Alternatively, Also Vector NTI utility program can be used.It is same that many algorithms known in the art can be used in measurement nucleotide sequence Property, including in above procedure comprising those of.As another example, polynucleotide sequence can be compared using BLASTN, BLASTN provides the comparison of best overlapping region and Percent sequence identity between inquiry and search sequence.Similar program can For comparing amino acid sequence, such as " Clustal X " program, BLASTP.In general, any program in these programs is all silent Recognize the lower use of setting, but those skilled in the art can according to need these settings of change.Alternatively, those skilled in the art's energy It is enough using another algorithm or computer program, the algorithm or computer program are at least provided to be provided by reference algorithm and program Identity compares horizontal.Comparison can be used for identifying two kinds of corresponding amino acid between albumen or peptide." corresponding amino acid " is The amino acid of albumen or peptide sequence, with the amino acid alignment of another albumen or peptide sequence.Corresponding amino acid can be with It is identical or not identical.Corresponding amino acid as not same amino acid can be described as variant amino acids.Table 6 provides variant amino acids Example.

Or for nucleic acid, hybrid polynucleotide under conditions of stablizing duplex by being formed between homology region, so Homology is determined with the size measurement of single-stranded specific nucleic acid enzymic digestion and digestion fragment afterwards.Substantially homologous DNA sequence dna It can be identified under such as stringent condition in Southern hybrid experiment, as defined in the particular system.It determines and closes Suitable hybridization conditions are within the skill of the art.

" nucleic acid " sequence refers to DNA or RNA sequence.In some embodiments, term trapping nucleic acids include any known DNA and RNA base analogue sequence, such as, but not limited to 4- acetyl group cytimidine, 8- hydroxy-n 6- methyladenosine, nitrogen third Piperidinyl cytimidine, false iso-cytosine, 5- (carboxy hydroxy-methyl) uracil, 5 FU 5 fluorouracil, 5-bromouracil, 5- carboxyl first Base amino methyl -2- thiouracil, 5- carboxymethyl-amino methyl uracil, dihydrouracil, inosine, N6- iso-amylene gland are fast Purine, 1- methyl adenine, 1- methyl pseudouracil, 1- methyl guanine, 1-methylinosine, 2,2- dimethylguanine, 2- first Base adenine, 2- methyl guanine, 3- methylcystein, 5-methylcytosine, N6- methyl adenine, 7- methyl guanine, 5- Methylaminomethyl uracil, 5- methoxyl group-amino methyl -2- thiouracil, β-d- mannose distinguish glycosides, 5'- methoxycarbonyl Methyluracil, 5- methoxyuracil, 2- methyl mercapto-N6- isopentennyladenine, uracil -5- oxy acetic acid methyl ester, urine are phonetic Pyridine -5- fluoroacetic acid, pseudouracil, distinguishes that glycosides, 2- sulphur cytimidine, 5-methyl-2-thiouracil, 2- sulphur urine are phonetic at oxybutoxosine Pyridine, 4- thiouracil, methyl uracil,-uracil -5- ethoxyacetic acid methyl esters, uracil -5- ethoxyacetic acid, pseudouracil, Distinguish glycosides, 2- sulphur cytimidine and 2,6-diaminopurine.

In some embodiments, the albumen and nucleic acid of the disclosure are separated.As used herein, term " separation " refers to people What work was obtained or generated by.As herein in regard to used in nucleic acid, term " separation " generally means: (i) is anti-for example, by polymerase chain Answer (PCR) amplification in vitro；(ii) it is generated by clone's recombination；(iii) it is isolated and purified by cracking and gel；Or (iv) passes through example As chemical synthesis synthesizes.Isolated nucleic acid is the nucleic acid for being easy to operate by recombinant DNA technology well known in the art.Therefore, It is disclosed 5' and 3' restriction site or is had been disclosed in the carrier of polymerase chain reaction (PCR) primer sequence and include Nucleotide sequence is considered as separation, but is not considered with the nucleic acid sequence that its native state is present in its natural host It is separation.Isolated nucleic acid, which can be, substantially to be purified, but is not required.For example, dividing in clone or expression vector From nucleic acid be not pure because it can substance only comprising the small percentage in cell where it.However, this nucleic acid It is separation, because the term is used herein, because it is easy to through standard skill known to persons of ordinary skill in the art Art operation.As herein in regard to used in albumen or peptide, term " separation " generally refers to the albumen being manually obtained or generated by or peptide (example Such as, by chemical synthesis, pass through recombinant DNA technology etc.).

Replace it should be understood that conserved amino acid can be carried out to provide the function equivalent modifications of capsid protein or homologous Object.In some respects, the disclosure includes that the sequence for causing conserved amino acid to replace changes.As used herein, conserved amino acid takes Generation, which refers to, does not change the relative charge for the albumen for wherein carrying out amino acid substitution or the amino acid substitution of size characteristic.Variant can be with According to the method preparation for changing polypeptide sequence known to persons of ordinary skill in the art, such as in the reference text for editing these methods Offer middle discovery, such as Molecular Cloning:A Laboratory Manual, J.Sambrook, et al. compile, second Version, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M.Ausubel et al. volume, John Wiley&Sons, Inc.,New York.The conservative substitution of amino acid includes in the substitution to carry out between the amino acid in the following group: (a) M, I, L, V；(b)F,Y,W；(c)K,R,H；(d)A,G；(e)S,T；(f)Q,N；(g) E, D.Therefore, can to albumen disclosed herein and The amino acid sequence of polypeptide carries out conserved amino acid substitution.

The example of the isolated nucleic acid of polypeptide of the coding comprising AAV capsid protein is the core with sequence selected from the following Acid: SEQ ID NO:410-434,876-1718 and 1989-2251.The isolated nucleic acid fragment for encoding AAV capsid sequence is available The nucleic acid of the capsid sequence needed for building encodes.Segment can have any suitable length.In some embodiments, it encodes The isolated nucleic acid fragment (part) of AAV capsid sequence can be used for constructing the nucleic acid for encoding required capsid sequence.Segment can have Have any suitable length (for example, length is at least 6, at least 9, at least 18, at least 36, at least 72, at least 144, at least 288, At least 576, at least 1152 or more nucleotide).For example, the nucleic acid sequence of the polypeptide of the first AAV capsid protein of coding Segment can be used for constructing or can introducing in the nucleic acid sequence of the 2nd AAV capsid sequence of coding, to change the property of AAV capsid.In In some embodiments, the AAV capsid protein comprising the capsid sequence segment from a variety of AAV serotypes is referred to as chimeric AAV Capsid.Segment can be the segment for not encoding peptide identical with the sequence of any of SEQ ID NO:869,870 or 871.Example Such as, the segment for encoding the nucleic acid sequence of variant amino acids (compared with known AAV serotype) can be used for constructing or can introducing volume In the nucleic acid sequence of code AAV capsid sequence, to change the property of AAV capsid.In some embodiments, with known AAV blood Clear type (for example, AAV serotype 2, AAV2/3 (for example, AAV2/3 heterozygote) or AAV8) comparing, encodes the nucleic acid sequence of AAV variant Column can include about 1 to about 100 amino acid variant.In some embodiments, with known AAV serotype (for example, AAV blood Clear type 2, AAV2/3 (for example, AAV2/3 heterozygote) AAV8) are compared, and the nucleic acid sequence of coding AAV variant can include about 5 to about 50 amino acid variants.In some embodiments, with known AAV serotype (for example, AAV serotype 2, AAV2/3 (example Such as, AAV2/3 heterozygote) or AAV8) compare, the nucleic acid sequence of coding AAV variant can include about 10 to about 30 amino acid and become Body.In some embodiments, with known AAV serotype (for example, AAV serotype 2, AAV2/3 are (for example, AAV2/3 heterozygosis Body) or AAV8) compare, the nucleic acid sequence of coding AAV variant may include 1 or 2 or 3 or 4,5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 amino acid variants.For example, coding AAV The nucleic acid sequence (for example, SEQ ID NO:861) of variant may include 3 compared with known AAV serotype (for example, AAV8) A amino acid variant.By that will include that the segment for the nucleic acid sequence in region for encoding variant amino acids introduces the known AAV of coding In the nucleic acid sequence of serotype, the recombination cap sequence with one or more of 3 amino acid variants can be constructed.Segment It can be introduced by any suitable method, including use direct mutagenesis.Therefore, the new AAV with new attribute can be created to become Body.

In some respects, present disclose provides coding AAV assembling activator protein (AAP) or the isolated nucleic acid of its variant. As used herein, " assembling activator protein " or " AAP " are chaperones, and function is by newly synthesized capsid protein (for example, VP Albumen, such as AAV VP1, VP2 and VP3) the cytotropic kernel of target, to promote virus genomic involucrum.In general, AAP is compiled Code is in the cap gene of adeno-associated virus.For example, AAP-2 coding is in the cap gene of AAV2.Other examples of AAP include but It is not limited to AAP-1, AAP-3, AAP-4, AAP-5, AAP-8, AAP-9, AAP-11 and AAP-12, such as Sonntag et al., J.Virol.2011 .85 in December (23): described in 12686-12697.It in some embodiments, is never capsid protein (example Such as, VP1, VP2, VP3) cap gene different open reading frame (ORF) translate AAP.For example, in some embodiments, from The ORF1 of cap gene translates capsid protein (for example, AAV2VP1, VP2, VP3), and translates AAP (example from the ORF2 of cap gene Such as, AAP-2).In some embodiments, the isolated nucleic acid for encoding AAP includes selected from SEQ ID NO:410-434 and 876- 1718 sequence is made from it.

Recombinate AAV

In some respects, present disclose provides isolated AAV.As herein in regard to used in AAV, term " separation " refers to people The AAV that work is obtained or generated by.Recombination method can be used and generate isolated AAV.Such AAV is referred to herein as " recombination AAV ". Recombinating AAV (rAAV) preferably there is tissue specificity to target ability so that the transgenosis of rAAV by specific delivery to a kind of or A variety of predetermined tissues.An important factor for AAV capsid is determining these tissue specificities targeting ability.It is thereby possible to select having It is suitable for being targeted the rAAV of the capsid of tissue.In some embodiments, rAAV include with SEQ ID NO:1-409, The capsid protein of amino acid sequence shown in any of 435-852,859-874 or 1726-1988, or have substantially together with it The albumen of source property.

It is well known in the art that obtaining, which has the method for the recombination AAV of required capsid protein,.(see, e.g., US 2003/ 0138772), content is incorporated herein by reference in their entirety).In general, this method includes culture host cell, contain coding The nucleic acid sequence of AAV capsid protein is (for example, coding has any institute in SEQ ID NO:1-409,435-868 or 1726-1988 Show the nucleic acid of the polypeptide of sequence) or its segment；Functional rep gene；By AAV inverted terminal repeat (ITR) and transgenosis The recombination AAV carrier of composition；With enough miscellaneous functions, to allow to recombinate AAV carrier package into AAV capsid protein.In In some embodiments, capsid protein is the structural proteins encoded by the cap gene of AAV.In some embodiments, AAV packet Containing three kinds of capsid proteins, virion protein 1 to 3 (is named as VP1, VP2 and VP3), it is all these all can be from single cap gene table It reaches.Therefore, in some embodiments, VP1, VP2 and VP3 albumen share common core sequence.In some embodiments, The molecular weight of VP1, VP2 and VP3 are respectively about 87kDa, about 72kDa and about 62kDa.In some embodiments, after translation, clothing Glutelin forms spherical 60 glycoprotein polyprotein precursor shells around viral genome.In some embodiments, protein shell is mainly by VP3 clothing Glutelin composition.In some embodiments, the function of capsid protein is protection viral genome, delivery of gene group and and host Interaction.In some respects, viral genome is delivered to host with tissue specific way by capsid protein.In some implementations In scheme, VP1 and/or VP2 capsid protein can contribute to the tissue tropism of the AAV of packaging.In some embodiments, it packs The tissue tropism of AAV determined by VP3 capsid protein.In some embodiments, increased by the mutation occurred in capsid protein Tissue tropism that is strong or changing AAV.

In some respects, the present disclosure describes the variants of wild type AAV serotype.In some embodiments, variant has There is the tissue tropism of change.In some embodiments, AAV variant as described herein includes the intragenic amino acid variation of cap (for example, replacing, missing, insertion).As described above, all three capsid proteins are all from single cap genetic transcription.Cause This, in some embodiments, the intragenic amino acid variation of cap is present in all three clothing encoded by the cap gene In glutelin.Alternatively, in some embodiments, amino acid variation may be not present in all three capsid proteins.One In a little embodiments, amino acid variation is occurred over just in VP1 capsid protein.In some embodiments, amino acid variation is only sent out Life is in VP2 capsid protein.In some embodiments, amino acid variation occurs over just in VP3 capsid protein.In some implementations In scheme, AAV variant includes the variation of one or more of cap gene.In some embodiments, more than one variation occurs In identical capsid protein (for example, in VP3).In some embodiments, more than one variation occurs in different clothing In glutelin (for example, at least one variation is in VP2 and at least one variation is in VP3).

In some embodiments, AAV variant described herein be AAV2, AAV2/3 (for example, AAV2/3 heterozygote) or The variant of AAV8.Known AAV2 effectively transduction human central nervous system (CNS) tissue, renal tissue, ocular tissue (for example, sense Photo-cell and retinal pigment epithelium (RPE)) and its hetero-organization.Therefore, in some embodiments, AAV3 as described herein becomes Body can be used for delivering gene therapy to CNS tissue, renal tissue or ocular tissue.It it is known that AAV3 effectively transduces carcinous people liver Cell.Therefore, in some embodiments, AAV3 variant as described herein can be used for delivering gene therapy to carcinous and normal Human liver cell.The tissue of known AAV8 targeting liver organization, respiratory tissue and eye.Therefore, in some embodiments, this paper institute The AAV8 variant stated can be used for delivering gene therapy to the tissue of liver organization, respiratory tissue and eye.

It should be understood that AAV2, AAV2/3 as described herein are (for example, AAV2/3 compared with corresponding wild type AAV Heterozygote) and AAV8 variant may include the intragenic one or more variations of cap.Therefore, in some embodiments, herein Described AAV2, AAV2/3 (for example, AAV2/3 heterozygote) and AAV8 variant can have tissue tropism, can be used for treating gene It is delivered to wild type AAV2, AAV2/3 (for example, AAV2/3 hybrid) or other organization types that AAV8 is not targeted.For example, one In a little embodiments, AAV8 variant described herein is (for example, B61；SEQ ID NO:865) it can be used for treating gene and be delivered to Pivot nervous system (CNS).In some embodiments, AAV2, AAV2/3 (for example, AAV2/3 hybrid) as described herein or AAV8 Variant can be used for targeting nephrocyte or liver cell.In some embodiments, AAV2, AAV2/3 as described herein (for example, AAV2/3 hybrid) or AAV8 variant can be used for by gene treat be delivered to liver, spleen, heart or brain.

In some respects, AAV variant described herein can be used for treating CNS associated disease.As used herein, " CNS is related Illness " is the disease or the patient's condition of central nervous system.CNS associated disease may influence spinal cord (such as myelopathy), brain (such as brain Disease) or brain and spinal cord around tissue.CNS associated disease can be genetic origin, by somatic mutation heredity or obtain. CNS associated disease can be the psychological patient's condition or illness, such as attention deficit hyperactivity disorder, autism spectrum disorder, mood barrier Hinder, schizophrenia, depression, rett's syndrome etc..CNS associated disease can be autoimmunity disease.CNS associated disease can also To be the cancer of CNS, such as the cancer of the brain.It can be the preinvasive cancer of CNS as the CNS associated disease of cancer, such as star is thin Born of the same parents' tumor, spongioblastoma etc., or it can be transferred into the cancer of CNS tissue, such as be transferred to the lung cancer of brain.CNS Other non-limiting examples of associated disease include Parkinson's disease, lysosomal storage disease, ischemic, neuropathic pain, amyotrophia Lateral schlerosis (ALS), multiple sclerosis (MS) and Canavan are sick (CD).

In some embodiments, AAV variant as described herein can be used for delivering gene therapy to heart cell (example Such as, heart tissue).Therefore, in some embodiments, AAV variant described herein can be used for treating cardiovascular disorder.Such as this Used in text, " cardiovascular disorder " is the disease or the patient's condition of cardiovascular system.Cardiovascular disease may influence heart, the circulatory system, Artery, vein, blood vessel and/or capillary.Cardiovascular disorder can be genetic origin, by somatic mutation heredity or obtain .The non-limiting example of cardiovascular disease include rheumatic heart disease, valvulopathy, hypertensive cardiopathy, aneurysm, Atherosclerosis, hypertension (hypertension) (for example, hypertension (high blood pressure)), peripheral arterial Disease (PAD), ischemic heart disease, angina pectoris, coronary heart disease, coronary artery disease, myocardial infarction, cranial vascular disease, transience Cerebral ischemia attack, inflammatory heart disease, cardiomyopathy, pericardial disease, congenital heart disease, heart failure, apoplexy and due to just plus this Myocarditis caused by disease.

In some embodiments, AAV variant described herein can target the tissue of lung and/or pulmonary system (for example, exhaling Desorption system).Therefore, in some embodiments, AAV variant described herein can be used for treating tuberculosis.As used herein, " lung Disease " is the disease or the patient's condition of pulmonary system.Tuberculosis may influence to breathe related lung or muscle.Tuberculosis can be genetic origin , by somatic mutation heredity or obtain.Tuberculosis can be lung cancer, including but not limited to non-small cell lung cancer, cellule lung Cancer and lung class cancer.The further non-limiting example of tuberculosis include acute bronchitis, acute respiratory distress syndrome (ARDS), Asbestosis, asthma, bronchiectasis, capillary bronchitis, bronchiolitis obliterans (BOOP), broncho-pulmonary development are not Good, byssinosis, chronic bronchitis, coccidioidomycosis (coccus), chronic obstructive pulmonary disease (COPD), hidden source property sense of organization pneumonia (COP), cystic fibrosis, pulmonary emphysema, Hantavirus pulmonary syndrome, histoplasmosis, mankind's pneumonitis virus, anaphylaxis lung Inflammation, influenza, Lymphangiomatosis, celiothelioma, Middle East respiration syndrome, non-tuberculous mycobacteria, pertussis, pneumoconiosis (black lung), pneumonia, primary ciliary dyskinesia, primary pulmonary hypertension, pulmonary hypertension, pulmonary fibrosis, Pulmonary Vascular Disease, Respiratory Syncytial Virus(RSV) (RSV), sarcoidosis, SARS (Severe Acute Respiratory Syndrome) (SARS), silicosis, sleep-respiratory are temporary Stop, unexpected infant death syndrome (SIDS) and pulmonary tuberculosis.

In some embodiments, AAV variant described herein can target hepatic tissue.Therefore, in some embodiments, AAV variant described herein can be used for treating hepatopathy.As used herein, " hepatopathy " is the disease or the patient's condition of liver.Hepatopathy can be Genetic origin, by somatic mutation heredity or obtain.Hepatopathy can be liver cancer, including but not limited to hepatocellular carcinoma (HCC), Fibrolamellar cancer, cholangiocarcinoma, angiosarcoma and hepatoblastoma.The further non-limiting example of tuberculosis includes Alagille comprehensive Simulator sickness, 1 antitrypsin deficiency disease of Alpha, oneself immunity hepatitis, Biliary atresia, cirrhosis, Hepatic cystiform diseases, fat Hepatopathy, galactosemia, gall stone, Gilbert syndrome, hemochromatosis, pregnant hepatopathy, neonatal hepatitis, primary Biliary cirrhosis, primary sclerotic cholangitis, porpharia, thunder Cotard, sarcoidosis, toxic hepatitis, the storage of 1 type glycogen Product disease, tyrosinemia, A type-, it is B-mode-, the third type-virus hepatitis, hepatolenticular degeneration and snail fever.

In some embodiments, AAV variant described herein can target renal tissue.Therefore, in some embodiments In, AAV variant described herein can be used for treating nephrosis.As used herein, " nephrosis " is the disease or the patient's condition of liver.Nephrosis can be with It is genetic origin, by somatic mutation heredity or obtains.Nephrosis can be kidney, including but not limited to clear-cell carcinoma, thoroughly Clear cell carcinoma, 1 type papillary carcinoma, 2 type papillary carcinomas, chromophobe cell tumor, acidophil carcinoma, Collecting duct carcinoma, renal plevis migrate it is thin Born of the same parents' cancer and Wilm'stumor.Other non-limiting examples of nephrosis include A Bude Halden-Kaufman-Li Niyake comprehensive Levy (nephropathic cystinosis), acute renal failure/acute kidney injury, acute great Ye ephritis, acute phosphate nephrosis, acute kidney Tubular necrosis, adenine phosphoribosiltransferase deficiency, adenovirus ephritis, Alport syndrome, amyloidosis, blood Manage smooth myolipoma, analgesia nephrosis, angiotensins antibody and focal segmental glomerulosclerosis, antiphospholipid syndrome, The treatment-related glomerulonephritis of anti-tnf-alpha, APOL1 mutation, the excessive syndrome of apparent cortin, Aristolochic acid nephropathy, Balkan region nephrosis, Bartter syndrome, beet urinate disease, β-thalassemia nephrosis, biliar nephrosis, BK polyoma, C1q kidney Disease, Cardiorenal syndrome, CFHR5 nephrosis, cholesterol embolism, allergic granuloma syndrome, chyluria, collapse resistance glomerulus Disease, disintegration glomerulopathy relevant to CMV, congenital nephrotic syndrome, Conorenal syndrome (Mainzer-Saldino Syndrome or Saldino-Mainzer disease), contrast induced nephropathy, sulfuric acid copper poisoning, cortical necrosis, cryoglobulinemia, crystal lure The acute kidney injury led, acquired cystic kidney disease, cystinuria, compactness storage disorders (MPGN2 type), (X is chain hidden for Dent disease Property nephrosis), dialyse uneven syndrome, diabetic nephropathy, diabetes insipidus, EAST syndrome, ectopic ureter, oedema, Ai Dehai Nurse-chester's disease, Fabry disease, familial hypocalcemia, fanconi syndrome, Fraser syndrome, fibronectin glomerulus Disease, fibroid glomerulonephritis and immunization type glomerulopathy, not Rayleigh syndrome, focal segmental glomerulosclerosis, Focal sclerosis disease, focal glomerulosclerosis, Galloway Mowat syndrome, lucky special Mann syndrome, renal glomerular disease, Glomerulus renal tubular backflow, glycosuria, Goodpasture's syndrome, hemolytic uremic syndrome (HUS), atypia hemolytic urine The related urine pool of toxication syndrome (aHUS), hemophagocytic syndrome, hemorrhagic cystitis, blood stasis and Paroxysmal Nocturnal hemoglobin Stay with hemolytic anemia, hepatic veno-occulusive disease, sinus hepaticus Chiari syndrome, hepatitis C associated kidney disease, hepatorenal syndrome, HIV associated kidney disease (HIVAN), horseshoe kidney (kidney fusion), elusive ulcer, aldosteronism, hypercalcinemia, potassemia, Renal function caused by hypermagnesemia, hypernatremia, hyperuricemia, hyperphosphatemia, hypocalcemia, hypopotassaemia, hypopotassaemia Incomplete, hypomagnesemia, hyponatremia, hypophosphatemia, IgA nephrosis, IgG4 nephrosis, interstitial cystitis, painful bladder syndrome, Interstitial nephritis, Ivemark syndrome, kidney stone (Kidney Stones), kidney stone (Nephrolithiasis), hook end spiral shell Revolve body disease nephrosis, light chain deposition disease, monoclonal immunoglobulin storage disorders, Liddle syndrome, Lightwood-Albright Syndrome, lipoprotein glomerulopathy, lithium renal toxicity, LMX1B mutation lead to heredity FSGS, pain in the loins blood urine, lupus, systemic red The solitary ephritis of the relevant glomerulus of yabbi sore, Lupus nephritis, lupus nephritis, Lyme disease-, malarial nephropathy, pernicious high blood Pressure, soft pinta, meatal stenosis, medullary cystic disease of kidney, sponge kidney,medullary, megaureter, melamine toxicity and kidney, film hyperplasia Property glomerulonephritis, membranous nephropathy, Central America nephrosis, metabolic acidosis, metabolic alkalosis, microscopic polyangitis, Milk alkalinity syndrome, Minimal change, polycystic Kidney depauperation, Huppert's disease, myeloproliferative tumour and glomerulus Disease, nailpatella syndrome, nephrocalcinosis, renal systemic fibrosing, nephrosis (wandering kidney, nephroptosis), nephrosis are comprehensive Sign, neurogenic bladder, nodular glomerulosclerosis, nongonococcal, cracker syndrome, orodigitofacial dysostosis, orthostatic (kidney-cataract is comprehensive for low blood pressure, orthostatic albuminuria, osmotic diuresis, Page kidney, papillary necrosis, nipple kidney syndrome Sign, isolatism renal aplasia), peritonaeum-kidney syndrome, post-urethral valve, kidney after glomerulonephritis, streptococcal infection after infection Bead ephritis, nodular polyarteritis, polycystic kidney disease, post-urethral valve, pre-eclampsia, hyperplasia glomerulonephritis are with monoclonal IgG deposition (Nasr disease), albuminuria (protein in urine), false aldosteronism, Albright's syndrome to type Disease, pyelonephritis (renal infection), pyonephrosis, radiation nephropathy, feeds syndrome again, is reflux nephropathy, fast at pneumorenal syndrome Fast progressivity glomerulonephritis, nephrapostasis, perinephric abscess, renal aplasia, aneurysm of renal artery, renal artery stenosis, clear-cell carcinoma, kidney Tumour, renal Hypouricemia and exercise induced acute renal failure, infarction of kidney, renal osteodystrophy, renal tubule Acid poisoning, Retrocaval ureter, retroperitoneal fibrosis, rhabdomyolysis, has with bariatric surgery Reset Osmostat The rhabdomyolysis of pass, rheumatoid arthritis related renal disease, sarcoidosis nephrosis, to lose salt, kidney and brain, Schimke immune Osteodysplasty, scleroderma renal crisis, serpentine fibula-polycystic kindey syndrome, Exner syndrome, Sickle kidney It is nephrosis after disease, silicon exposure and chronic kidney disease, hematopoietic cell transplantation, nephrosis related with stem cell transplantation, thin basement membrane disease, good Property familial hematuria, trigonitis, tuberous sclerosis, renal tubule hypoplasia, tumor lysis syndrome, uremia, urine Toxication optic neuropathy, ureteral orifice cyst, urethral sarcoma, ankylurethria, the urinary incontinence, urinary tract infections, urinary obstruction, bladder intestines Fistula, ureteral vesicoureteral reflux, Xi Peier-lindau's syndrome, warfarin associated kidney disease, Wegner's granulomatosis, Wegener Syndrome is wished in swollen Polyangiitis and warm Delhi.

In some embodiments, AAV variant as described herein can be used for delivering gene therapy to ocular tissue (for example, The tissue or cell of eye).Therefore, in some embodiments, AAV variant described herein can be used for treating eye disorders.Such as Used herein, " eye disorders " are the disease or the patient's condition of eye.Eye disease may influence eye, sclera, cornea, anterior chamber, back room, rainbow Film, pupil, crystalline lens, vitreous humor, retina or optic nerve.Eye disorders can be genetic origin, prominent by body cell Become heredity or obtains.The non-limiting example of eye disease and illness includes but is not limited to: age-related macular degeneration, view Film disease, diabetic retinopathy, macular edema, glaucoma, retinitis pigmentosa and cancer eye.

In some embodiments, AAV variant as described herein can be used for delivering gene therapy to gastrointestinal tissue (such as Stomach intestinal tissue).Therefore, in some embodiments, AAV variant described herein can be used for treating disorder of gastrointestinal tract.Such as this Used in text, " disorder of gastrointestinal tract " is the disease or the patient's condition of gastrointestinal tract.Gastrointestinal disease may influence mucous membrane (for example, epithelium, intrinsic Layer, muscular layer of mucosa etc.), submucosa (for example, submucous plexus, enteric plexus etc.), the muscle layer of gastrointestinal tract, serous coat and/or outer membrane, Oral cavity, esophagus, pylorus, gastroduodenal, small intestine, caecum, appendix, colon, anal canal or rectum.Disorder of gastrointestinal tract can be heredity Origin, by somatic mutation heredity or obtain.The non-limiting example of enterogastric diseases and illness includes but is not limited to: scorching Property enteropathy (IBD), Crohn disease, ulcerative colitis, irritable bowel syndrome, celiaca, gastroesophageal reflux disease (GERD), lose It relaxes disease (achakasua), diverticulitis (diverticulitus), diarrhea and certain cancers are (for example, intestinal cancer, gastric cancer, colon Cancer, carcinoma of the rectum etc.).

In some embodiments, AAV variant as described herein can be used for delivering gene therapy to breast tissue (example Such as, the tissue of breast).Therefore, in some embodiments, AAV variant described herein can be used for treating disorder of breast.Such as this Used in text, " disorder of breast " is the disease or the patient's condition of breast.Breast disease may influence the fibr tissue of breast, adipose tissue, Leaflet or conduit.Disorder of breast can be genetic origin, by somatic mutation heredity or obtain.Breast disease and illness Non-limiting example includes but is not limited to: mazoitis, Breast Calcifications, adiponecrosis, adenofibroma, fibrosis and simple cyst, Excessive cream, hyperplasia and breast cancer.

In some embodiments, AAV variant as described herein can be used for delivering gene therapy to pancreatic tissue (example Such as, the tissue of pancreas).Therefore, in some embodiments, AAV variant described herein can be used for treating pancreas illness.Such as this Used in text, " pancreas illness " is the disease or the patient's condition of pancreas.Pancreatic disease may influence pancreas head, pancreas neck, pancreas gland Body, pancreas tail portion, pancreas islet (such as langerhans islands), acinus or columnar epithelium.Pancreas illness can be genetic origin, pass through Somatic mutation heredity obtains.The non-limiting example of pancreatic disease and illness includes but is not limited to: diabetes are (for example, 1 type Diabetes and diabetes B), pancreatitis (such as acute pancreatitis, chronic pancreatitis) and cancer of pancreas.

In some embodiments, AAV variant as described herein can be used for delivering gene therapy to the urinary tract tissue (example Such as the urinary tract tissue, such as bladder body).Therefore, in some embodiments, AAV variant described herein can be used for treating and secrete Disorder of urethra.As used herein, " disorder of urinary tract " is the disease or the patient's condition of the urinary tract.Disease of urinary tract may will affect wing Guang, ureter, urethra or prostate.Disorder of urinary tract can be genetic origin, by somatic mutation heredity or obtain.It secretes The non-limiting example of urethral disease and illness includes but is not limited to: urinary tract infections, kidney stone, bladder control problem are (for example, urine Retention, urinary incontinence etc.), cystitis and bladder cancer.

In some embodiments, AAV variant as described herein can be used for delivering gene therapy to uterine tissue (such as The tissue in uterus).Therefore, in some embodiments, AAV variant described herein can be used for treating uterine disorder.As herein Used, " uterine disorder " is the disease or the patient's condition in uterus.Uterine disease may influence cervix, canal of uterine cervix, corpus uteri (uterus Bottom), endometrium, mesometrium or perimetrium.Uterine disorder can be genetic origin, by somatic mutation heredity or It obtains.The non-limiting example of uterine disease and illness includes but is not limited to: adenomyosis, endometriosis, uterus Endometrial hyperplasia, Asherman syndrome and carcinoma of endometrium.

It is cultivated in host cell thin component of the rAAV carrier package in AAV capsid trans- can be supplied to host Born of the same parents.Alternatively, component needed for any one or more of (for example, recombination AAV carrier, rep sequence, cap sequence and/or miscellaneous function) It can be provided by stable host cell, the host cell is engineered to contain using method known to those skilled in the art There are one or more required components.Most suitable to be, this stable host cell contains under inducible promoter control Required component.But required component is likely to be under the control of constitutive promoter.It is being suitble to be used together with transgenosis Regulating element discussion in, there is provided herein the examples of suitable induction type and constitutive promoter.In another alternative In case, selected stabilization host cell can be lured containing the selected component under constitutive promoter control and one or more Other selected components under the control of conductivity type promoter.For example, can produce stable host cell, it is derived from 293 cell (its Under the control of constitutive promoter contain E1 miscellaneous function), but its contain inducible promoter control under rep and/or Cap albumen.Those skilled in the art can produce other stable host cells.

Can be used any suitable genetic elements (carrier) by recombination AAV carrier needed for the rAAV for generating the disclosure, Rep sequence, cap sequence and miscellaneous function are delivered to packaging host cell.In some embodiments, all three capsids are encoded The single nucleic acid of albumen (such as VP1, VP2 and VP3) is delivered in packaging host cell in single carrier.In some embodiment party In case, the nucleic acid of encoding capsid protein is by two kinds of vehicle deliveries into packaging host cell；Comprising encoding two kinds of capsid proteins The first vector of first nucleic acid of (such as VP1 and VP2) and the second nucleic acid comprising encoding single capsid protein (such as VP3) Second support.In some embodiments, by three kinds of carriers (every kind of carrier includes the nucleic acid for encoding different capsid proteins) delivering To packaging host cell.Selected genetic elements can include that those described herein is passed by any suitable method It send.The method of any embodiment for constructing the disclosure is known to the technical staff of nucleic-acid manipulation field, including gene work Journey, recombined engineering and synthetic technology.See, e.g., Sambrook et al., Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Press,Cold Spring Harbor,N.Y..Similarly, rAAV virion is generated Method be well-known, and the selection of appropriate method is not the limitation to the disclosure.See, e.g., K.Fisher etc. People, J.Virol., 70:520-532 (1993) and U.S. Patent number 5,478,745.

In some embodiments, can be used triple transfection methods generate recombination AAV (in U.S. Patent number 6,001, It is described in detail in 650).In general, by thin with recombination AAV carrier (including transgenosis) transfecting host to be packaged at AAV particle Born of the same parents, AAV helper function vector and helper function vector recombinate AAV to generate." AAV assists function to AAV helper function vector coding Can " sequence (such as rep and cap), it is trans- to work to carry out effective AAV duplication and capsidation.Preferably, AAV is assisted Function carrier supports effective AAV carrier to generate, without generating any detectable wild type AAV virus body (such as containing active The AAV virion of energy property rep and cap gene).The non-limiting example for being suitable for the invention carrier is special including being described in the U.S. PHLP19 in benefit number 6,001,650 and the pRep6cap6 carrier being described in U.S. Patent number 6,156,303, in whole Appearance is incorporated herein by reference.Helper function vector encodes virus derived from non-AAV and/or AAV depends on the cell of its duplication The nucleotide sequence of function (such as " miscellaneous function ").Miscellaneous function includes those required to AAV duplication function, including but unlimited In participate in AAV gene transcriptional activation, phase specificity AAV mRNA montage, AAV DNA replication dna, cap expression product synthesis and Those of AAV Mouth Disease Virus Proteins part.Miscellaneous function based on virus can be derived from any of helper virus, such as adenopathy Poison, herpesviral (in addition to herpes simplex virus type 1) and vaccinia virus.

In some respects, present disclose provides the host cells of transfection.Term " transfection " is for referring to cell to exogenous DNA Intake, when exogenous DNA is introduced into for example intracellular (cross-cell membrane), cell is by " transfection ".Many rotaring dyeing technologies are usually It is known in the art.See, e.g., Graham et al. (1973) Virology, 52:456；Sambrook et al. (1989) Molecular Cloning,a laboratory manual,Cold Spring Harbor Laboratories,New York；Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier；With Chu et al. (1981)Gene13:197.Such technology can be used for by one or more exogenous nucleic acids (such as nucleotide integration vector and other Nucleic acid molecules) it is introduced into suitable host cell.

" host cell " refer to it is any containing or can carry the cell of substances of interest.Host cell is usually that lactation is dynamic Object cell.Host cell may be used as AAV helper construct, AAV mini gene plasmid, helper function vector or with recombination AAV Generate the recipient of other relevant transfer DNAs.The term includes the offspring for the initial cell being transfected.Therefore, such as this " host cell " used in text can refer to the cell transfected with exogenous DNA array.It should be appreciated that due to natural, accidental Or intentional mutation, the offspring of single parent cell is on morphology or in genome or total DNA complement It (complement) may be not necessarily identical with original parent in.

As used herein, term " cell line " is to refer to cell mass that is continuous in vitro or extending growth and division.It is logical Often, cell line is derived from the clonal population of single progenitor cells.This field it is further known that, this clonal population storage or turn It may occur from the variation sent out or induced during shifting, in nuclear staining.Therefore, the cell from mentioned cell line may be with ancestors Cell or culture are not exactly the same, and signified cell line includes these variants.

As used herein, term " recombinant cell " refers to that having had been incorporated into exogenous DNA section (such as causes bioactivity more Peptide transcription or generate biologically active nucleic acid such as RNA DNA section) cell.

Cell can also be transfected with the carrier (for example, assistant carrier) for providing miscellaneous function to AAV.Miscellaneous function is provided Carrier can provide adenovirus function, including such as E1a, E1b, E2a and E4ORF6.The sequence of the adenoviral gene of these functions is provided Column can be obtained from any of adenoviral serotype, such as serotype 2,3,4,7,12 and 40, and further include this field Known any mankind's type identified at present.Therefore, in some embodiments, the method be related to expression AAV duplication, The carrier of one or more genes necessary to AAV genetic transcription and/or AAV are packed transfects cell.

As used herein, term " carrier " includes any genetic elements, such as plasmid, bacteriophage, transposons, clay, dye Colour solid, artificial chromosome, virus, virion etc., can replicate when in conjunction with control element appropriate and it can be thin Metastatic gene sequence between born of the same parents.Therefore, which includes clone and expression vector and viral vectors.In some embodiments In, it is contemplated that useful carrier be nucleic acid segment wherein to be transcribed (such as nucleic acid sequence) be located at promoter transcription control under that A little carriers." promoter " refers to the cell synthesis mechanism as needed for promotor gene specific transcriptional or the identification of the synthesis mechanism of introducing DNA sequence dna.Phrase " being operatively positioned ", " under control " or " transcription control under " mean promoter relative to nucleic acid at In correct position and direction, to control the starting of RNA polymerase and the expression of gene.Term " expression vector or construct " meaning Refer to that any kind of genetic constructs containing nucleic acid, part of or whole nucleic acid coding sequences can be transcribed.Some In embodiment, expression includes the transcription of nucleic acid, for example, to generate biologically active polypeptide product or inhibition from the gene of transcription RNA (such as shRNA, miRNA, miRNA inhibitor).

In some cases, using method well known in the art, isolated capsid gene can be used in constructing and packing weight Group AAV, to determine functional character relevant to the capsid protein that the gene encodes.For example, isolated capsid gene can be used in Building and packaging include the recombination AAV (rAAV) of reporter gene (for example, beta-galactosidase, GFP, luciferase etc.).Then RAAV can be delivered to animal (for example, mouse), and by audit report gene various animal tissues (for example, heart, Liver, kidney) in expression can determine the tissue-targeting matter of the capsid gene newly separated.Disclosed herein is the new separation of characterization Capsid gene other methods, be well known in the art there are also other methods.

It is not meant that for recombinant vector to be packaged in required AAV capsid with the preceding method for generating the rAAV of the disclosure Limitation, and other suitable methods are apparent to a skilled reader.

Recombinate AAV carrier

" recombination AAV (rAAV) carrier " of the disclosure is typically at least anti-by transgenosis and its adjusting sequence and 5' and 3'AAV Terminad repetitive sequence (ITR) composition.By this recombination AAV carrier package into capsid protein and to be delivered to selected target thin Born of the same parents.In some embodiments, transgenosis is the nucleic acid sequence heterologous with carrier sequence, encodes desired polypeptides, albumen, function Property RNA molecule (for example, miRNA, miRNA inhibitor) or other gene products.Nucleic acid coding sequence is to allow transgenosis in target The mode transcribed, translate and/or expressed in the cell of tissue is operably connected with component is adjusted.

The AAV sequence of carrier generally comprises 5' the and 3' inverted terminal repeat of cis acting (see, e.g., for example B.J.Carter,in"Handbook of Parvoviruses",ed.,P.Tijsser,CRC Press,pp.155 168 (1990)).The length of ITR sequence is about 145bp.Preferably, the entire sequence for substantially encoding ITR is used in molecule, although The small modification to a certain degree of these sequences is allowed.Modify the ability of these ITR sequences within the skill of the art. (see, for example, " Molecular Cloning.A Laboratory Manual ", the second edition, Cold Spring Harbor Laboratory,New York(1989)；With K.Fisher et al., the textbook of J Virol., 70:520 532 (1996)). The example of this molecule for the disclosure is containing transgenosis " cis acting " plasmid, wherein selected transgenic sequence 5' and 3'AAV ITR sequence is flanked with relevant regulating element.AAV ITR sequence can be obtained from any of AAV, including The mammal AAV type identified at present.

In some embodiments, present disclose provides the AAV carriers of self-complementary.As used herein, " itself is mutual for term The AAV carrier of benefit " (scAAV) refers to that the carrier containing double-stranded vector genome, the carrier are by the way that there is no from AAV The end of one ITR splits site (resolutionn site, TR) and generates.There is no TR to prevent in the load that TR is not present Body end starts to replicate.In general, scAAV carrier generates single-stranded inverted repeat gene group, wherein every end has wild type (wt) AAV TR and intermediate there is mutation T R (mTR).

In some embodiments, the rAAV of the disclosure is the rAAV of false type parting.False type parting is and exogenous virus packet Memebrane protein combination generates the process of virus or viral vectors.The result is that the virion of false type parting.Use this method, external source disease Malicious envelope protein can be used in changing increase/reduction stability of host's tropism or virion.In some respects, false type point The rAAV of type includes the nucleic acid from two or more differences AAV, wherein the nucleic acid encode capsid protein from a kind of AAV, And other virus proteins of the nucleic acid encode of other at least one AAV and/or viral genome.In some embodiments, false The rAAV of type parting refers to the capsid of the inverted terminal repeat (ITR) comprising a kind of AAV serotype and different AAV serotypes The AAV of albumen.For example, the AAV carrier of the false type parting of the ITR of the serotype X containing useful Y protein encapsulation will be named as AAVX/Y (for example, capsid of ITR and AAV1 of the AAV2/1 with AAV2).In some embodiments, the rAAV of false type parting can be used for The tissue specificity targeting ability and the disease from another AAV serotype for combining the capsid protein from a kind of AAV serotype Malicious DNA, to allow transgenosis targeted delivery to target tissue.

Other than the main element for the recombination AAV carrier identified above, carrier further includes conventional necessary control element, To allow its transcription, translation and/or expression in the cell of the virus generated with plamid vector transfection or the infection disclosure Mode is operably connected with transgenosis.As used herein, the sequence " being operably connected " includes adjacent with target gene Expression control sequence and trans- or work at a distance to control the expression control sequence of target gene.

Expression control sequence includes suitable transcription initiation, termination, promoter and enhancer sequence；Effective RNA processing Signal, such as montage and polyadenylation (poly A) signal；Stablize the sequence of cytoplasm mRNA；Improve the sequence (example of translation efficiency Such as Kozak consensus sequence)；Enhance the sequence of protein stability；And when needed, the sequence of enhancing coded product secretion.Greatly Measure expression control sequence, including natural, composing type, derivable and/or tissue specificity promoter, be this field It is knowing and can be used.

As used herein, when nucleic acid sequence (for example, coded sequence) and adjust sequence so that nucleic acid sequence expression or When the mode that transcription is placed under being influenced or controlled by adjusting sequence is covalently attached, it is claimed to be operably connected.If it is desired to will Nucleic acid sequence translates into functional protein, then if 5' adjust sequence in promoter cause the transcription of coded sequence with And if connection property between two DNA sequence dnas will not (1) lead to the introducing of frameshift mutation, (2) interference promoter region refers to The ability of coded sequence transcription is led, or corresponding RNA transcript is translated into the ability of albumen by (3) interference, then it is assumed that two DNA Sequence is operably connected.Therefore, if promoter region can influence the transcription of the DNA sequence dna, so that resulting transcript can To translate into required albumen or polypeptide, then promoter region will be operably connected with nucleic acid sequence.Similarly, two or more Multiple code areas are operably connected when they are connected in this way, i.e., they lead to two from the transcription of common promoter The expression of a or more albumen is translated in frame.In some embodiments, the coded sequence being operably connected generates Fusion protein.In some embodiments, the coded sequence that is operably connected generate functional r NA (for example, shRNA, MiRNA, miRNA inhibitor).

For encoding the nucleic acid of albumen, polyadenous glycosides is inserted into usually after transgenic sequence and before 3'AAV ITR sequence Polyadenylation sequence.RAAV construct for use in the present invention can also contain introne, be preferably located on promoter/enhancer sequence and Between transgenosis.A kind of possible intron sequences are originated from SV-40, and referred to as SV-40T intron sequences.It can be used Another carrier element be internal ribosome entry site (IRES).IRES sequence is used to generate one from individual gene transcript Kind or more polypeptide.IRES sequence will be used to generate the albumen containing more than one polypeptide chain.These and other common vectors member The selection of part is conventional, and many such sequences are available [see, for example, Sambrook etc. and cited therein Bibliography, for example, page 3.18 3.26 and 16.17 16.27 and Ausubel et al., Current Protocols in Molecular Biology,John Wiley&Sons,New York,1989].In some embodiments, foot and mouth disease virus 2A sequence is included in polyprotein；This is a kind of small peptide (length is about 18 amino acid), has been demonstrated that polyprotein can be mediated Cracking (Ryan, M D et al., EMBO, 1994；4:928-933；Mattion, N M et al., J Virology, November 1996；p.8124-8127；Furler, S et al., Gene Therapy, 2001；8:864-873；And Halpin, C et al., The Plant Journal,1999；4:453-459).It has included previously plasmid and gene therapy vector (AAV and retrovirus) Manual system in demonstrate 2A sequence cleavage activity (Ryan, M D et al., EMBO, 1994；4:928-933；Mattion,N M et al., J Virology, November 1996；p.8124-8127；Furler, S et al., Gene Therapy, 2001；8: 864-873；And Halpin, C et al., The Plant Journal, 1999；4:453-459；De Felipe, P et al., Gene Therapy,1999；6:198-208；De Felipe, P et al., Human Gene Therapy, 2000；11:1921-1931.； And Klump, H et al., Gene Therapy, 2001；8:811-817).

The definite property of regulating and controlling sequence needed for gene expression may be due to species, tissue or cell type in host cell It is different, but in general (as required) should include the 5' non-transcribed sequences for the starting for relating separately to transcription and translation and 5' is non-turns over Translate sequence, such as TATA frame, capped sequence, CAAT sequence, enhancer element etc..Particularly, this 5' non-transcribed adjusts sequence It will include promoter region comprising for being operatively connected the promoter sequence of the transcription control of gene.Regulating and controlling sequence can be with It as needed include enhancer sequence or upstream activat subsequence.The carrier of the disclosure optionally includes 5' leader sequence or letter Number sequence.The selection and design of suitable carrier are within the scope of the ability and discretion of those of ordinary skill in the art.

The example of constitutive promoter includes but is not limited to that retrovirus Rous sarcoma virus (RSV) LTR promoter (is appointed Selection of land is with RSV enhancer), cytomegalovirus (CMV) promoter (optionally have cmv enhancer) [see, e.g. Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter, dihyrofolate reductase promoter, beta-actin Promoter, phosphoglycerokinase (PGK) promoter and EF1 α promoter [Invitrogen].

Inducible promoter allow adjust gene expression, and can by external source provide compound, environmental factor example Adjusted such as the presence of temperature or specific physiological status, for example, acute stage, cell specific differentiation state or only in replicating cell In.Inducible promoter and inducible type systems can be obtained from various commercial sources, including but not limited to Invitrogen, Clontech and Ariad.Have been described many other systems, and those skilled in the art can be readily selected these and be System.The example for the inducible promoter that the promoter provided by external source is adjusted includes that zinc induction type sheep metallothionine (MT) opens Mover, dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system (WO 98/10088)；Moulting hormone insect promoter (No et al., Proc.Natl.Acad.Sci.USA, 93:3346-3351 (1996)), tetracycline suppressor system (Gossen et al., Proc.Natl.Acad.Sci.USA, 89:5547-5551 (1992)), (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey etc. to tetracycline-inducible People, Curr.Opin.Chem.Biol., 2:512-518 (1998)), RU486 inducible system (Wang et al., Nat.Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and rapamycin inducible system (Magari et al., J.Clin.Invest., 100:2865-2872 (1997)).What is come in handy herein is other kinds of Inducible promoter is the promoter that those are adjusted by specific physiological status, such as the specific differentiation shape of temperature, acute stage, cell State or only in replicating cell.

In another embodiment, the natural promoter of transgenosis will be used.When the expression for wishing transgenosis should mould When quasi- natural expression, natural promoter may be preferred.When the expression of transgenosis must in time or developmentally, or with group When knitting specificity pattern, or responding specific transcription stimulation, natural promoter can be used.In another embodiment, other Natural expression control element, such as enhancer element, polyadenylation site or Kozak consensus sequence can also be used for simulation naturally Expression.

In some embodiments, it adjusts sequence and assigns tissue-specific gene ability to express.In some cases, it organizes The specific regulator sequences combination tissue-specific transcription factor, with tissue specific way inducible transcription.Such organizing specific Property adjust sequence (for example, promoter, enhancer etc.) be well known in the art.Example organization specific regulatory sequence include but It is not limited to following tissue-specific promoter: liver specificity thyroxine-binding globulin (TBG) promoter, insulin promoter Son, glucagon promoter, growth hormone release inhibiting hormone promoter, pancreatic polypeptide (PPY) promoter, synapsin -1 (Syn) promoter, flesh Acid kinase (MCK) promoter, mammal desmin (DES) promoter, α-myoglobulin heavy chain (a-MHC) promoter, stomach and intestine - 2 promoter of Specific mucin, eye specificity retina hormone promoter, eye specificity K12 promoter, respiratory tissue are special Property CC10 promoter, respiratory tissue specific surfaces activated protein C (SP-C) promoter, breast tissue specificity PRC1 starting Son, breast tissue specificity RRM2 promoter, the urinary tract tissue specificity uroplakin 2 (UPII) promoter, uterine tissue Specific lactoferrin promoter or serum cardiac troponin T (cTnT) promoter.Other Exemplary promoters include that β-flesh moves egg White promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996)；Alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum.Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol.Biol.Rep., 24:185-96 (1997))；Bone sialoprotein promoter (Chen et al., J.Bone Miner.Res., (1996) 11:654-64), CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998)； Heavy chain immunoglobulin promoter；Cell receptor α-chain promoter, neuron such as neuronspecific enolase (NSE) starting Sub (Andersen et al., Cell.Mol.Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc.Natl.Acad.Sci.USA, 88:5611-5 (1991)) and neuronal specificity vgf gene open Mover (Piccioli et al., Neuron, 15:373-84 (1995)), etc., these are aobvious to those skilled in the art And it is clear to.

In some embodiments, one or more binding sites of one or more miRNA are introduced into rAAV carrier In transgenosis, to inhibit expression of the transgenosis in one or more tissues of the subject of carry genetic modification.Art technology Personnel will be understood that, can choose binding site with the expression of tissue specific way control transgenosis.For example, can be special by liver The binding site of property miR-122 is introduced into transgenosis to inhibit expression of the transgenosis in liver.Target site in mRNA can In 5'UTR, 3'UTR or code area.In general, target site is located in the 3'UTR of mRNA.Make in addition, transgenosis can be designed Multiple miRNA are by identifying identical or multiple sites come regulating mRNA.The presence of multiple miRNA binding sites can lead to multiple The synergistic effect of RISC simultaneously provides efficient expression inhibiting.Target site sequence may include 5-100,10-60 or more in total Nucleotide.Target site sequence may include at least five nucleotide of target gene binding site sequence.

Recombinate AAV carrier: transgene coding sequence

The composition of the transgenic sequence of rAAV carrier will depend on the purposes of resulting vehicle.For example, a type of turn base Because sequence includes report sequence, detectable signal is generated in expression.In another example, transgenes encoding is therapeutic Albumen or therapeutic functional r NA.In another example, transgenes encoding is intended for the albumen or functionality of research purpose RNA, for example, to generate the body cell transgenic animal model of carry genetic modification, for example, to study the function of transgene product. In another example, transgene-encoded protein or functional r NA, are intended for generating the animal model of disease.It is appropriate Transgene coding sequence is apparent to those skilled in the art.

The report sequence that can be provided in transgenosis includes but is not limited to encoding beta-lactamase, beta galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase and Be well known in the art other those.When associated with the controlling element of its expression is driven, report sequence offer can The signal detected by conventional means, including enzyme, radiography, colorimetric, fluorescence or other spectroscopic assays, fluorescence activated cell point Choosing measurement and immunoassays, including enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA) and immunohistochemistry.Example Such as, in the case where flag sequence is LacZ gene, the load for carrying signal is detected by the measurement of betagalactosidase activity The presence of body.In the case where transgenosis is green fluorescent protein or luciferase, the carrier for carrying signal can be by shining Color or light in meter generate to estimate measurement.For example, such report molecule can be used for verifying the tissue specificity targeting of rAAV Ability and tissue-specific promoter's regulation activity.

In some respects, present disclose provides rAAV carrier, it is used to prevent or treat the one or more of mammal The method of genetic defect or dysfunction, the polypeptide such as in mammal lacks or polypeptide is excessive, especially for controlling The severity or degree for treating or reducing deficiency disease in human body show to lack with such polypeptide in cell and tissue related One or more diseases.This method include amount to be enough to treat disease or illness in the subject with this illness and when Between to subject apply one of pharmaceutically acceptable carrier or a variety of therapeutic peptides, polypeptide, siRNA, Microrna, anti- The rAAV carrier of adopted nucleotide etc..

Therefore, the disclosure include encode one or more peptides, more peptide or proteins rAAV carrier delivering, can be used for controlling Treat or prevent the morbid state of mammalian subject.Exemplary treatment albumen include it is one or more selected from growth factor, Interleukins, interferon, anti-apoptosis factor, cell factor, diabetes resisting factor, anti-apoptotic agent, coagulation factor, it is antitumor because The polypeptide of son.Other non-limiting examples of human cytokines include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, Promoting sexual gland hormone, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10 (187A), virus IL- 10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17 and IL-18.

RAAV carrier may include the gene to be transferred to subject, with treatment and gene expression reduction, expression deletion or function It can the relevant disease of obstacle.Exemplary gene and associatcd disease state include but is not limited to: G-6-Pase, with glycogen It is related to store up shortage type 1A；Phosphoenolpyruvate-carboxylic kinases, it is related with Pepck shortage；The transfer of -1 phosphoric acid uridine acyl of galactolipin Enzyme, it is related with galactosemia；Phenylalanine hydroxylase, it is related with phenylketonuria；Branched-chain alpha-keto acid dehydrogenase is urinated with maple sugar Disease is related；Fumaroyl acetoacetate hydrolase, it is related with 1 type tyrosinemia；Methylmalonyl-CoA isomerase, with methyl Malonic acid mass formed by blood stasis is related；Medium chain acyl coa dehydrogenase, it is related with middle chain acetyl coenzyme A shortage；Positive ornithine carbamoyl transfer Enzyme, it is related with positive ornithine carbamyltransferase defect；Argininosuccinate synthetase, it is related with citrullinemia；Low-density Lipoprotein receptor protein, it is related with familial hypercholesterolemia；UDP-glucoronosyl/transferase (glucouronosyltransferase), related with Crigler-Na Jiaer disease；Adenosine deaminase, with Severe Combined Immune Defect disease is related；Hypoxanthine guanine phosphoribosyltransferase, it is related with gout and Lai Shi-Nai En syndrome；Biotin enzyme, It is related with biotinidase deficiency；β-glucocerebrosidase, it is related with Gaucher disease；GRD beta-glucuronidase has with Sly syndrome It closes；Peroxisomal membrane protein 70kDa, it is related with cerebrohepatorenal syndrome；Pancreatin deaminase, with Accute porphyrin Disease is related；α -1 antitrypsin treats α -1 antitrypsin deficiency disease (pulmonary emphysema)；Hematopoietin is for treating ground Anaemia caused by middle sea anaemia or kidney failure；Treat the vascular endothelial growth factor of ischemic disease, Ang-1 and at Fibroblast growth factor；For treating the occluded blood vessel such as thrombomodulin of atherosclerosis, thrombosis or embolism White and tissue factor approach restrainer；For treating the aromatic amino acid decarboxylase (AADC) and tyrosine hydroxylase of Parkinson's disease Enzyme (TH)；For treating phospholamban, sarco (endo) the blood plasma net adenosine triphosphatase -2 of congestive heart failure (SERCA2) Beta-3 adrenergic receptor, antisense or mutant form and heart adenyl cyclase；For treating various cancers The tumor suppressors gene such as p53 of disease；Cell factor, such as the various leucocytes for treating inflammation and immunologic derangement and cancer One of interleukin；For treating the dystrophin or dystrophin and dystrophin of muscular dystrophy Or small nutrient；With for treating the insulin of diabetes.

In some embodiments, this disclosure relates to which AAV, it includes codings can be used for treatment and central nervous system (CNS) nucleic acid of the albumen of the relevant patient's condition, disease or illness or functional r NA.It is gene relevant to CNS disease below Non-limiting list: DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SYP, SYT1, CHRNA7,3Rtau/4rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1, AGER, Ahl tribulus sea silent sickness are related；UCH-L1,SKP1,EGLN1,Nurr-1, BDNF, TrkB, gstm1, S106 β, it is related with Parkinson's disease；IT15,PRNP,JPH3,TBP,ATXN1,ATXN2,ATXN3, Atrophin 1, FTL, TITF-1 is related to Huntington disease；FXN, it is related with Friedreich ataxia；ASPA, with Canavan disease is related；DMD, it is related with muscular dystrophy；And SMN1, UBE1, DYNC1H1, it is related with Duchenne-Arandisease.In In some embodiments, this disclosure relates to recombinate AAV, it includes the nucleic acid for expressing one or more forementioned genes or its segment. In some embodiments, this disclosure relates to comprising expressing one or more functions of inhibiting one or more forementioned gene expression The recombination AAV of the nucleic acid of property RNA.

In some embodiments, this disclosure relates to which encoding can be used for treating the patient's condition relevant to cardiovascular system, disease Illness albumen or functional r NA nucleic acid.It is the non-limiting list of gene relevant to cardiovascular disease below: VEGF, FGF, SDF-1, linker protein 40, linker protein 43, SCN4a, HIF1 α, SERCa2a, ADCY1 and ADCY6.In In some embodiments, this disclosure relates to recombinate AAV, it includes the nucleic acid for expressing one or more forementioned genes or its segment. In some embodiments, this disclosure relates to comprising expressing one or more functions of inhibiting one or more forementioned gene expression The recombination AAV of the nucleic acid of property RNA.

In some embodiments, this disclosure relates to which AAV, can be used for treating disease relevant to pulmonary system it includes coding The nucleic acid of condition, the albumen of disease or illness or functional r NA.It is the non-limiting list of gene relevant to tuberculosis: TNF below α、TGFβ1、SFTPA1、SFTPA2、SFTPB、SFTPC、HPS1、HPS3、HPS4、ADTB3A、IL1A、IL1B、LTA、IL6、 CXCR1 and CXCR2.In some embodiments, this disclosure relates to recombinate AAV, it includes express one or more forementioned genes Or the nucleic acid of its segment.In some embodiments, this disclosure relates to comprising express it is one or more inhibit it is one or more before State the recombination AAV of the nucleic acid of the functional r NA of gene expression.

In some embodiments, this disclosure relates to AAV, it includes coding can be used for treating the patient's condition relevant to liver, The nucleic acid of the albumen or functional r NA of disease or illness.It is the non-limiting list of gene relevant to hepatopathy below: α 1-AT, HFE, ATP7B, fumaroyl acetoacetate hydrolase (FAH), G-6-Pase, NCAN, GCKR, LYPLAL1 and PNPLA3.In some embodiments, this disclosure relates to recombinate AAV, it includes express one or more forementioned genes or its piece The nucleic acid of section.In some embodiments, this disclosure relates to comprising expressing one or more one or more forementioned genes of inhibition The recombination AAV of the nucleic acid of the functional r NA of expression.

In some embodiments, this disclosure relates to AAV, it includes coding can be used for treating the patient's condition relevant to kidney, The nucleic acid of the albumen or functional r NA of disease or illness.It is the non-limiting list of gene relevant to nephrosis below: PKD1, PKD2、PKHD1、NPHS1、NPHS2、PLCE1、CD2AP、LAMB2、TRPC6、WT1、LMX1B、SMARCAL1、COQ2、PDSS2、 SCARB3、FN1、COL4A5、COL4A6、COL4A3、COL4A4、FOX1C、RET、UPK3A、BMP4、SIX2、CDC5L、USF2、 ROBO2, SLIT2, EYA1, MYOG, SIX1, SIX5, FRAS1, FREM2, GATA3, KAL1, PAX2, TCF2 and SALL1.One In a little embodiments, this disclosure relates to recombinate AAV, it includes the nucleic acid for expressing one or more forementioned genes or its segment.In In some embodiments, this disclosure relates to comprising expressing one or more functionality for inhibiting one or more forementioned gene expression The recombination AAV of the nucleic acid of RNA.

In some embodiments, this disclosure relates to which AAV, can be used for treating the patient's condition relevant to eye, disease it includes coding The nucleic acid of the albumen or functional r NA of disease or illness.It is the non-limiting list of gene relevant to eye disease below: CFH, C3, MT-ND2、ARMS2、TIMP3、CAMK4、FMN1、RHO、USH2A、RPGR、RP2、TMCO、SIX1、SIX6、LRP12、ZFPM2、 TBK1, GALC, actin, CYP1B1, CAV1, CAV2, optic nerve albumen and CDKN2B.In some embodiments, this public affairs It opens and is related to recombinating AAV, it includes the nucleic acid for expressing one or more forementioned genes or its segment.In some embodiments, originally It is open to be related to the recombination of the nucleic acid comprising expressing one or more functional r NA for inhibiting one or more forementioned gene expression AAV。

In some embodiments, this disclosure relates to AAV, it includes coding can be used for treating the patient's condition relevant to breast, The nucleic acid of the albumen or functional r NA of disease or illness.It is the non-limiting list of gene relevant to breast disease below: BRCA1, BRCA2, Tp53, PTEN, HER2, BRAF and PARP1.In some embodiments, this disclosure relates to recombinate AAV, Nucleic acid comprising expressing one or more forementioned genes or its segment.In some embodiments, this disclosure relates to include expression The recombination AAV of the nucleic acid of one or more functional r NA for inhibiting one or more forementioned gene expression.

In some embodiments, this disclosure relates to which AAV, can be used for treating disease relevant to gastrointestinal tract it includes coding The nucleic acid of condition, the albumen of disease or illness or functional r NA.It is the non-limiting column of gene relevant to gastrointestinal disease below Table: CYP2C19, CCL26, APC, IL12, IL10 and IL-18.In some embodiments, this disclosure relates to recombinate AAV, packet Containing the nucleic acid for expressing one or more forementioned genes or its segment.In some embodiments, this disclosure relates to include expression one The recombination AAV of the nucleic acid of kind or a variety of functional r NA for inhibiting one or more forementioned genes expression.

In some embodiments, this disclosure relates to AAV, it includes coding can be used for treating the patient's condition relevant to pancreas, The nucleic acid of the albumen or functional r NA of disease or illness.It is the non-limiting list of gene relevant to pancreatic disease below: PRSS1, SPINK1, STK11, MLH1, KRAS2, p16, p53 and BRAF.In some embodiments, this disclosure relates to recombinate AAV, it includes the nucleic acid for expressing one or more forementioned genes or its segment.In some embodiments, this disclosure relates to wrap Recombination AAV containing the nucleic acid for expressing one or more functional r NA for inhibiting one or more forementioned gene expression.

In some embodiments, this disclosure relates to AAV, it includes coding can be used for treating the patient's condition relevant to urinary tract, The nucleic acid of the albumen or functional r NA of disease or illness.It is the non-limiting list of gene relevant to urinary disease below: HSPA1B, CXCR1&2, TLR2, TLR4, TGF-1, FGFR3, RB1, HRAS, TP53 and TSC1.In some embodiments, originally Open to be related to recombinating AAV, it includes the nucleic acid for expressing one or more forementioned genes or its segment.In some embodiments, This disclosure relates to include the recombination for the nucleic acid for expressing one or more functional r NA for inhibiting one or more forementioned gene expression AAV。

In some embodiments, this disclosure relates to AAV, it includes coding can be used for treating the patient's condition relevant to uterus, The nucleic acid of the albumen or functional r NA of disease or illness.It is the non-of gene relevant to eye disease (ocular disease) below Restricted list: DN-ER, MLH1, MSH2, MSH6, PMS1 and PMS2.In some embodiments, this disclosure relates to recombinate AAV, it includes the nucleic acid for expressing one or more forementioned genes or its segment.In some embodiments, this disclosure relates to wrap Recombination AAV containing the nucleic acid for expressing one or more functional r NA for inhibiting one or more forementioned gene expression.

The rAAV of the disclosure can be used in restoring expression in subject reduce, silencing or the otherwise base of functional disturbance Cause expression (for example, with cancer subject in silencing tumor suppressor).The rAAV of the disclosure can also be enough In the expression (for example, the oncogene expressed in the subject with cancer) for the gene for striking the low unconventionality expression in subject. In some embodiments, the rAAV that rAAV carrier can be carried by applying to the subject with cancer, using comprising compiling The rAAV carrier of the nucleic acid (for example, tumor suppressor) of code gene product relevant to cancer carrys out treating cancer.In some implementations It, can be by the way that the rAAV for carrying rAAV carrier be administered to the subject with cancer, with comprising encoding small RNA in scheme (it can inhibit gene product (such as oncogene) relevant to cancer to the rAAV carrier of the nucleic acid of (for example, shRNA, miRNA) Expression) carry out treating cancer.In some embodiments, comprising encode relevant to cancer gene product nucleic acid (or inhibit and The functional r NA of the relevant gene expression of cancer) rAAV carrier can be used for research purpose, for example, for studying cancer or mirror Determine the therapy for the treatment of cancer.It is non-limiting list (such as the cancer base of known Exemplary gene relevant to cancer development below Cause and tumor suppressor): AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2、AKT1、ALB、ANPEP、ANXA5、ANXA7、AP2M1、APC、ARHGAP5、ARHGEF5、ARID4A、ASNS、ATF4、 ATM、ATP5B、ATP5O、AXL、BARD1、BAX、BCL2、BHLHB2、BLMH、BRAF、BRCA1、BRCA2、BTK、CANX、 CAP1、CAPN1、CAPNS1、CAV1、CBFB、CBLB、CCL2、CCND1、CCND2、CCND3、CCNE1、CCT5、CCYR61、 CD24、CD44、CD59、CDC20、CDC25、CDC25A、CDC25B、CDC2L5、CDK10、CDK4、CDK5、CDK9、CDKL1、 CDKN1A、CDKN1B、CDKN1C、CDKN2A、CDKN2B、CDKN2D、CEBPG、CENPC1、CGRRF1、CHAF1A、CIB1、 CKMT1、CLK1、CLK2、CLK3、CLNS1A、CLTC、COL1A1、COL6A3、COX6C、COX7A2、CRAT、CRHR1、CSF1R、 CSK、CSNK1G2、CTNNA1、CTNNB1、CTPS、CTSC、CTSD、CUL1、CYR61、DCC、DCN、DDX10、DEK、DHCR7、 DHRS2、DHX8、DLG3、DVL1、DVL3、E2F1、E2F3、E2F5、EGFR、EGR1、EIF5、EPHA2、ERBB2、ERBB3、 ERBB4、ERCC3、ETV1、ETV3、ETV6、F2R、FASTK、FBN1、FBN2、FES、FGFR1、FGR、FKBP8、FN1、FOS、 FOSL1、FOSL2、FOXG1A、FOXO1A、FRAP1、FRZB、FTL、FZD2、FZD5、FZD9、G22P1、GAS6、GCN5L2、 GDF15、GNA13、GNAS、GNB2、GNB2L1、GPR39、GRB2、GSK3A、GSPT1、GTF2I、HDAC1、HDGF、HMMR、 HPRT1、HRB、HSPA4、HSPA5、HSPA8、HSPB1、HSPH1、HYAL1、HYOU1、ICAM1、ID1、ID2、IDUA、IER3、 IFITM1、IGF1R、IGF2R、IGFBP3、IGFBP4、IGFBP5、IL1B、ILK、ING1、IRF3、ITGA3、ITGA6、ITGB4、 JAK1、JARID1A、JUN、JUNB、JUND、K-ALPHA-1、KIT、KITLG、KLK10、KPNA2、KRAS2、KRT18、KRT2A、 KRT9、LAMB1、LAMP2、LCK、LCN2、LEP、LITAF、LRPAP1、LTF、LYN、LZTR1、MADH1、MAP2K2、MAP3K8、 MAPK12、MAPK13、MAPKAPK3、MAPRE1、MARS、MAS1、MCC、MCM2、MCM4、MDM2、MDM4、MET、MGST1、 MICB、MLLT3、MME、MMP1、MMP14、MMP17、MMP2、MNDA、MSH2、MSH6、MT3、MYB、MYBL1、MYBL2、MYC、 MYCL1、MYCN、MYD88、MYL9、MYLK、NEO1、NF1、NF2、NFKB1、NFKB2、NFSF7、NID、NINJ1、NMBR、 NME1、NME2、NME3、NOTCH1、NOTCH2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR2F6、NRAS、NRG1、 NSEP1、OSM、PA2G4、PABPC1、PCNA、PCTK1、PCTK2、PCTK3、PDGFA、PDGFB、PDGFRA、PDPK1、PEA15、 PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3CB、PIK3CG、PIM1、PKM2、PKMYT1、PLK2、PPARD、PPARG、 PPIH、PPP1CA、PPP2R5A、PRDX2、PRDX4、PRKAR1A、PRKCBP1、PRNP、PRSS15、PSMA1、PTCH、PTEN、 PTGS1、PTMA、PTN、PTPRN、RAB5A、RAC1、RAD50、RAF1、RALBP1、RAP1A、RARA、RARB、RASGRF1、 RB1、RBBP4、RBL2、REA、REL、RELA、RELB、RET、RFC2、RGS19、RHOA、RHOB、RHOC、RHOD、RIPK1、 RPN2、RPS6KB1、RRM1、SARS、SELENBP1、SEMA3C、SEMA4D、SEPP1、SERPINH1、SFN、SFPQ、SFRS7、 SHB、SHH、SIAH2、SIVA、SIVA TP53、SKI、SKIL、SLC16A1、SLC1A4、SLC20A1、SMO、SMPD1、SNAI2、 SND1、SNRPB2、SOCS1、SOCS3、SOD1、SORT1、SPINT2、SPRY2、SRC、SRPX、STAT1、STAT2、STAT3、 STAT5B、STC1、TAF1、TBL3、TBRG4、TCF1、TCF7L2、TFAP2C、TFDP1、TFDP2、TGFA、TGFB1、TGFBI、 TGFBR2、TGFBR3、THBS1、TIE、TIMP1、TIMP3、TJP1、TK1、TLE1、TNF、TNFRSF10A、TNFRSF10B、 TNFRSF1A、TNFRSF1B、TNFRSF6、TNFSF7、TNK1、TOB1、TP53、TP53BP2、TP53I3、TP73、TPBG、 TPT1、TRADD、TRAM1、TRRAP、TSG101、TUFM、TXNRD1、TYRO3、UBC、UBE2L6、UCHL1、USP7、VDAC1、 VEGF、VHL、VIL2、WEE1、WNT1、WNT2、WNT2B、WNT3、WNT5A、WT1、XRCC1、YES1、YWHAB、YWHAZ、 ZAP70 and ZNF9.

RAAV carrier, which may include, adjusts the albumen of Apoptosis or the nucleic acid of functional r NA as the coding of transgenosis. It is that the nucleic acid of the product of gene relevant to Apoptosis and these genes of coding and its homologue and coding inhibit these below Gene and its be used as in certain embodiments of the disclosure transgenosis homologue expression small RNA (for example, ShRNA, miRNA) nucleic acid non-limiting list: RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAK1、BAX、BCL10、BCL2、BCL2A1、BCL2L1、BCL2L10、BCL2L11、BCL2L12、BCL2L13、BCL2L2、 BCLAF1、BFAR、BID、BIK、NAIP、BIRC2、BIRC3、XIAP、BIRC5、BIRC6、BIRC7、BIRC8、BNIP1、 BNIP2、BNIP3、BNIP3L、BOK、BRAF、CARD10、CARD11、NLRC4、CARD14、NOD2、NOD1、CARD6、CARD8、 CARD9、CASP1、CASP10、CASP14、CASP2、CASP3、CASP4、CASP5、CASP6、CASP7、CASP8、CASP9、 CFLAR、CIDEA、CIDEB、CRADD、DAPK1、DAPK2、DFFA、DFFB、FADD、GADD45A、GDNF、HRK、IGF1R、 LTA、LTBR、MCL1、NOL3、PYCARD、RIPK1、RIPK2、TNF、TNFRSF10A、TNFRSF10B、TNFRSF10C、 TNFRSF10D、TNFRSF11B、TNFRSF12A、TNFRSF14、TNFRSF19、TNFRSF1A、TNFRSF1B、TNFRSF21、 TNFRSF25、CD40、FAS、TNFRSF6B、CD27、TNFRSF9、TNFSF10、TNFSF14、TNFSF18、CD40LG、FASLG、 CD70、TNFSF8、TNFSF9、TP53、TP53BP2、TP73、TP63、TRADD、TRAF1、TRAF2、TRAF3、TRAF4、 TRAF5DRD2、GRIA1、GRIA2,GRIN1、SLC1A1、SYP、SYT1、CHRNA7、3Rtau/4rTUS、APP、BAX、BCL-2、 GRIK1、GFAP、IL-1、AGER、UCH-L1、SKP1、EGLN1、Nurr-1、BDNF、TrkB、gstm1、S106β、IT15、 PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, Atrophin 1, FTL, TITF-1, FXN, ASPA, DMD and SMN1, UBE1、DYNC1H1。

It will also be appreciated by the skilled artisan that in the case where encoding the transgenosis of albumen or polypeptide base can be being turned The mutation for causing conserved amino acid to replace is generated, because in provide the homologue of the equivalent variant of function or albumen or polypeptide.In Some aspects, the disclosure include that the sequence for causing the conserved amino acid of transgenosis to replace changes.In some embodiments, turn base Because including the gene with dominant negative mutation.It interacts for example, transgenosis can be expressed with wild-type protein similar elements Mutain, to block some aspects of wild-type protein function.

Useful transgene product further includes miRNA.MiRNA and other small RNAs by the cutting of target RNA transcript/ The Translational repression of degradation or target mRNA (mRNA) adjusts gene expression.In general, miRNA is naturally expressed as final 19-25 A untranslatable rna product.MiRNA shows it by the sequence-specific interaction of the 3' non-translational region (UTR) with said target mrna Activity.The miRNA of these endogenous expression forms hairpin precursor, is subsequently processed into miRNA duplex, and be further processed into " mature " single-stranded miRNA molecule.Maturation miRNA instructs multiprotein complex miRISC, based on them and maturation miRNA Complementary identification said target mrna target site, such as in the area 3'UTR.

Unrestricted miR-96 gene list and its homologue can be used as in certain embodiments of this method below The target of transgenosis or the small RNA as transgenes encoding is (for example, miRNA sponge, antisense oligonucleotides, TuD RNA): hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c, hsa-let-7c*, hsa-let-7d、hsa-let-7d*、hsa-let-7e、hsa-let-7e*、hsa-let-7f、hsa-let-7f-1*、hsa- let-7f-2*、hsa-let-7g、hsa-let-7g*、hsa-let-7i、hsa-let-7i*、hsa-miR-1、hsa-miR- 100、hsa-miR-100*、hsa-miR-101、hsa-miR-101*、hsa-miR-103、hsa-miR-105、hsa-miR- 105*、hsa-miR-106a、hsa-miR-106a*、hsa-miR-106b、hsa-miR-106b*、hsa-miR-107、hsa- miR-10a、hsa-miR-10a*、hsa-miR-10b、hsa-miR-10b*、hsa-miR-1178、hsa-miR-1179、hsa- miR-1180、hsa-miR-1181、hsa-miR-1182、hsa-miR-1183、hsa-miR-1184、hsa-miR-1185、 hsa-miR-1197、hsa-miR-1200、hsa-miR-1201、hsa-miR-1202、hsa-miR-1203、hsa-miR- 1204、hsa-miR-1205、hsa-miR-1206、hsa-miR-1207-3p、hsa-miR-1207-5p、hsa-miR-1208、 hsa-miR-122、hsa-miR-122*、hsa-miR-1224-3p、hsa-miR-1224-5p、hsa-miR-1225-3p、hsa- miR-1225-5p、hsa-miR-1226、hsa-miR-1226*、hsa-miR-1227、hsa-miR-1228、hsa-miR- 1228*、hsa-miR-1229、hsa-miR-1231、hsa-miR-1233、hsa-miR-1234、hsa-miR-1236、hsa- miR-1237、hsa-miR-1238、hsa-miR-124、hsa-miR-124*、hsa-miR-1243、hsa-miR-1244、hsa- miR-1245、hsa-miR-1246、hsa-miR-1247、hsa-miR-1248、hsa-miR-1249、hsa-miR-1250、 hsa-miR-1251、hsa-miR-1252、hsa-miR-1253、hsa-miR-1254、hsa-miR-1255a、hsa-miR- 1255b、hsa-miR-1256、hsa-miR-1257、hsa-miR-1258、hsa-miR-1259、hsa-miR-125a-3p、 hsa-miR-125a-5p、hsa-miR-125b、hsa-miR-125b-1*、hsa-miR-125b-2*、hsa-miR-126、hsa- miR-126*、hsa-miR-1260、hsa-miR-1261、hsa-miR-1262、hsa-miR-1263、hsa-miR-1264、 hsa-miR-1265、hsa-miR-1266、hsa-miR-1267、hsa-miR-1268、hsa-miR-1269、hsa-miR- 1270、hsa-miR-1271、hsa-miR-1272、hsa-miR-1273、hsa-miR-127-3p、hsa-miR-1274a、hsa- miR-1274b、hsa-miR-1275、hsa-miR-127-5p、hsa-miR-1276、hsa-miR-1277、hsa-miR-1278、 hsa-miR-1279、hsa-miR-128、hsa-miR-1280、hsa-miR-1281、hsa-miR-1282、hsa-miR-1283、 hsa-miR-1284、hsa-miR-1285、hsa-miR-1286、hsa-miR-1287、hsa-miR-1288、hsa-miR- 1289、hsa-miR-129*、hsa-miR-1290、hsa-miR-1291、hsa-miR-1292、hsa-miR-1293、hsa- miR-129-3p、hsa-miR-1294、hsa-miR-1295、hsa-miR-129-5p、hsa-miR-1296、hsa-miR- 1297、hsa-miR-1298、hsa-miR-1299、hsa-miR-1300、hsa-miR-1301、hsa-miR-1302、hsa- miR-1303、hsa-miR-1304、hsa-miR-1305、hsa-miR-1306、hsa-miR-1307、hsa-miR-1308、 hsa-miR-130a、hsa-miR-130a*、hsa-miR-130b、hsa-miR-130b*、hsa-miR-132、hsa-miR- 132*、hsa-miR-1321、hsa-miR-1322、hsa-miR-1323、hsa-miR-1324、hsa-miR-133a、hsa- miR-133b、hsa-miR-134、hsa-miR-135a、hsa-miR-135a*、hsa-miR-135b、hsa-miR-135b*、 hsa-miR-136、hsa-miR-136*、hsa-miR-137、hsa-miR-138、hsa-miR-138-1*、hsa-miR-138- 2*、hsa-miR-139-3p、hsa-miR-139-5p、hsa-miR-140-3p、hsa-miR-140-5p、hsa-miR-141、 hsa-miR-141*、hsa-miR-142-3p、hsa-miR-142-5p、hsa-miR-143、hsa-miR-143*、hsa-miR- 144、hsa-miR-144*、hsa-miR-145、hsa-miR-145*、hsa-miR-146a、hsa-miR-146a*、hsa-miR- 146b-3p、hsa-miR-146b-5p、hsa-miR-147、hsa-miR-147b、hsa-miR-148a、hsa-miR-148a*、 hsa-miR-148b、hsa-miR-148b*、hsa-miR-149、hsa-miR-149*、hsa-miR-150、hsa-miR-150*、 hsa-miR-151-3p、hsa-miR-151-5p、hsa-miR-152、hsa-miR-153、hsa-miR-154、hsa-miR- 154*、hsa-miR-155、hsa-miR-155*、hsa-miR-15a、hsa-miR-15a*、hsa-miR-15b、hsa-miR- 15b*、hsa-miR-16、hsa-miR-16-1*、hsa-miR-16-2*、hsa-miR-17、hsa-miR-17*、hsa-miR- 181a、hsa-miR-181a*、hsa-miR-181a-2*、hsa-miR-181b、hsa-miR-181c、hsa-miR-181c*、 hsa-miR-181d、hsa-miR-182、hsa-miR-182*、hsa-miR-1825、hsa-miR-1826、hsa-miR-1827、 hsa-miR-183、hsa-miR-183*、hsa-miR-184、hsa-miR-185、hsa-miR-185*、hsa-miR-186、 hsa-miR-186*、hsa-miR-187、hsa-miR-187*、hsa-miR-188-3p、hsa-miR-188-5p、hsa-miR- 18a、hsa-miR-18a*、hsa-miR-18b、hsa-miR-18b*、hsa-miR-190、hsa-miR-190b、hsa-miR- 191、hsa-miR-191*、hsa-miR-192、hsa-miR-192*、hsa-miR-193a-3p、hsa-miR-193a-5p、 hsa-miR-193b、hsa-miR-193b*、hsa-miR-194、hsa-miR-194*、hsa-miR-195、hsa-miR-195*、 hsa-miR-196a、hsa-miR-196a*、hsa-miR-196b、hsa-miR-197、hsa-miR-198、hsa-miR-199a- 3p、hsa-miR-199a-5p、hsa-miR-199b-5p、hsa-miR-19a、hsa-miR-19a*、hsa-miR-19b、hsa- miR-19b-1*、hsa-miR-19b-2*、hsa-miR-200a、hsa-miR-200a*、hsa-miR-200b、hsa-miR- 200b*、hsa-miR-200c、hsa-miR-200c*、hsa-miR-202、hsa-miR-202*、hsa-miR-203、hsa- miR-204、hsa-miR-205、hsa-miR-206、hsa-miR-208a、hsa-miR-208b、hsa-miR-20a、hsa- miR-20a*、hsa-miR-20b、hsa-miR-20b*、hsa-miR-21、hsa-miR-21*、hsa-miR-210、hsa-miR- 211、hsa-miR-212、hsa-miR-214、hsa-miR-214*、hsa-miR-215、hsa-miR-216a、hsa-miR- 216b、hsa-miR-217、hsa-miR-218、hsa-miR-218-1*、hsa-miR-218-2*、hsa-miR-219-1-3p、 hsa-miR-219-2-3p、hsa-miR-219-5p、hsa-miR-22、hsa-miR-22*、hsa-miR-220a、hsa-miR- 220b、hsa-miR-220c、hsa-miR-221、hsa-miR-221*、hsa-miR-222、hsa-miR-222*、hsa-miR- 223、hsa-miR-223*、hsa-miR-224、hsa-miR-23a、hsa-miR-23a*、hsa-miR-23b、hsa-miR- 23b*、hsa-miR-24、hsa-miR-24-1*、hsa-miR-24-2*、hsa-miR-25、hsa-miR-25*、hsa-miR- 26a、hsa-miR-26a-1*、hsa-miR-26a-2*、hsa-miR-26b、hsa-miR-26b*、hsa-miR-27a、hsa- miR-27a*、hsa-miR-27b、hsa-miR-27b*、hsa-miR-28-3p、hsa-miR-28-5p、hsa-miR-296-3p、 hsa-miR-296-5p、hsa-miR-297、hsa-miR-298、hsa-miR-299-3p、hsa-miR-299-5p、hsa-miR- 29a、hsa-miR-29a*、hsa-miR-29b、hsa-miR-29b-1*、hsa-miR-29b-2*、hsa-miR-29c、hsa- miR-29c*、hsa-miR-300、hsa-miR-301a、hsa-miR-301b、hsa-miR-302a、hsa-miR-302a*、 hsa-miR-302b、hsa-miR-302b*、hsa-miR-302c、hsa-miR-302c*、hsa-miR-302d、hsa-miR- 302d*、hsa-miR-302e、hsa-miR-302f、hsa-miR-30a、hsa-miR-30a*、hsa-miR-30b、hsa-miR- 30b*、hsa-miR-30c、hsa-miR-30c-1*、hsa-miR-30c-2*、hsa-miR-30d、hsa-miR-30d*、hsa- miR-30e、hsa-miR-30e*、hsa-miR-31、hsa-miR-31*、hsa-miR-32、hsa-miR-32*、hsa-miR- 320a、hsa-miR-320b、hsa-miR-320c、hsa-miR-320d、hsa-miR-323-3p、hsa-miR-323-5p、 hsa-miR-324-3p、hsa-miR-324-5p、hsa-miR-325、hsa-miR-326、hsa-miR-328、hsa-miR- 329、hsa-miR-330-3p、hsa-miR-330-5p、hsa-miR-331-3p、hsa-miR-331-5p、hsa-miR-335、 hsa-miR-335*、hsa-miR-337-3p、hsa-miR-337-5p、hsa-miR-338-3p、hsa-miR-338-5p、hsa- miR-339-3p、hsa-miR-339-5p、hsa-miR-33a、hsa-miR-33a*、hsa-miR-33b、hsa-miR-33b*、 hsa-miR-340、hsa-miR-340*、hsa-miR-342-3p、hsa-miR-342-5p、hsa-miR-345、hsa-miR- 346、hsa-miR-34a、hsa-miR-34a*、hsa-miR-34b、hsa-miR-34b*、hsa-miR-34c-3p、hsa-miR- 34c-5p、hsa-miR-361-3p、hsa-miR-361-5p、hsa-miR-362-3p、hsa-miR-362-5p、hsa-miR- 363、hsa-miR-363*、hsa-miR-365、hsa-miR-367、hsa-miR-367*、hsa-miR-369-3p、hsa-miR- 369-5p、hsa-miR-370、hsa-miR-371-3p、hsa-miR-371-5p、hsa-miR-372、hsa-miR-373、hsa- miR-373*、hsa-miR-374a、hsa-miR-374a*、hsa-miR-374b、hsa-miR-374b*、hsa-miR-375、 hsa-miR-376a、hsa-miR-376a*、hsa-miR-376b、hsa-miR-376c、hsa-miR-377、hsa-miR- 377*、hsa-miR-378、hsa-miR-378*、hsa-miR-379、hsa-miR-379*、hsa-miR-380、hsa-miR- 380*、hsa-miR-381、hsa-miR-382、hsa-miR-383、hsa-miR-384、hsa-miR-409-3p、hsa-miR- 409-5p、hsa-miR-410、hsa-miR-411、hsa-miR-411*、hsa-miR-412、hsa-miR-421、hsa-miR- 422a、hsa-miR-423-3p、hsa-miR-423-5p、hsa-miR-424、hsa-miR-424*、hsa-miR-425、hsa- miR-425*、hsa-miR-429、hsa-miR-431、hsa-miR-431*、hsa-miR-432、hsa-miR-432*、hsa- miR-433、hsa-miR-448、hsa-miR-449a、hsa-miR-449b、hsa-miR-450a、hsa-miR-450b-3p、 hsa-miR-450b-5p、hsa-miR-451、hsa-miR-452、hsa-miR-452*、hsa-miR-453、hsa-miR-454、 hsa-miR-454*、hsa-miR-455-3p、hsa-miR-455-5p、hsa-miR-483-3p、hsa-miR-483-5p、hsa- miR-484、hsa-miR-485-3p、hsa-miR-485-5p、hsa-miR-486-3p、hsa-miR-486-5p、hsa-miR- 487a、hsa-miR-487b、hsa-miR-488、hsa-miR-488*、hsa-miR-489、hsa-miR-490-3p、hsa- miR-490-5p、hsa-miR-491-3p、hsa-miR-491-5p、hsa-miR-492、hsa-miR-493、hsa-miR- 493*、hsa-miR-494、hsa-miR-495、hsa-miR-496、hsa-miR-497、hsa-miR-497*、hsa-miR- 498、hsa-miR-499-3p、hsa-miR-499-5p、hsa-miR-500、hsa-miR-500*、hsa-miR-501-3p、 hsa-miR-501-5p、hsa-miR-502-3p、hsa-miR-502-5p、hsa-miR-503、hsa-miR-504、hsa-miR- 505、hsa-miR-505*、hsa-miR-506、hsa-miR-507、hsa-miR-508-3p、hsa-miR-508-5p、hsa- miR-509-3-5p、hsa-miR-509-3p、hsa-miR-509-5p、hsa-miR-510、hsa-miR-511、hsa-miR- 512-3p、hsa-miR-512-5p、hsa-miR-513a-3p、hsa-miR-513a-5p、hsa-miR-513b、hsa-miR- 513c、hsa-miR-514、hsa-miR-515-3p、hsa-miR-515-5p、hsa-miR-516a-3p、hsa-miR-516a- 5p、hsa-miR-516b、hsa-miR-517*、hsa-miR-517a、hsa-miR-517b、hsa-miR-517c、hsa-miR- 518a-3p、hsa-miR-518a-5p、hsa-miR-518b、hsa-miR-518c、hsa-miR-518c*、hsa-miR-518d- 3p、hsa-miR-518d-5p、hsa-miR-518e、hsa-miR-518e*、hsa-miR-518f、hsa-miR-518f*、hsa- miR-519a、hsa-miR-519b-3p、hsa-miR-519c-3p、hsa-miR-519d、hsa-miR-519e、hsa-miR- 519e*、hsa-miR-520a-3p、hsa-miR-520a-5p、hsa-miR-520b、hsa-miR-520c-3p、hsa-miR- 520d-3p、hsa-miR-520d-5p、hsa-miR-520e、hsa-miR-520f、hsa-miR-520g、hsa-miR-520h、 hsa-miR-521、hsa-miR-522、hsa-miR-523、hsa-miR-524-3p、hsa-miR-524-5p、hsa-miR- 525-3p、hsa-miR-525-5p、hsa-miR-526b、hsa-miR-526b*、hsa-miR-532-3p、hsa-miR-532- 5p、hsa-miR-539、hsa-miR-541、hsa-miR-541*、hsa-miR-542-3p、hsa-miR-542-5p、hsa- miR-543、hsa-miR-544、hsa-miR-545、hsa-miR-545*、hsa-miR-548a-3p、hsa-miR-548a-5p、 hsa-miR-548b-3p、hsa-miR-548b-5p、hsa-miR-548c-3p、hsa-miR-548c-5p、hsa-miR-548d- 3p、hsa-miR-548d-5p、hsa-miR-548e、hsa-miR-548f、hsa-miR-548g、hsa-miR-548h、hsa- miR-548i、hsa-miR-548j、hsa-miR-548k、hsa-miR-548l、hsa-miR-548m、hsa-miR-548n、 hsa-miR-548o、hsa-miR-548p、hsa-miR-549、hsa-miR-550、hsa-miR-550*、hsa-miR-551a、 hsa-miR-551b、hsa-miR-551b*、hsa-miR-552、hsa-miR-553、hsa-miR-554、hsa-miR-555、 hsa-miR-556-3p、hsa-miR-556-5p、hsa-miR-557、hsa-miR-558、hsa-miR-559、hsa-miR- 561、hsa-miR-562、hsa-miR-563、hsa-miR-564、hsa-miR-566、hsa-miR-567、hsa-miR-568、 hsa-miR-569、hsa-miR-570、hsa-miR-571、hsa-miR-572、hsa-miR-573、hsa-miR-574-3p、 hsa-miR-574-5p、hsa-miR-575、hsa-miR-576-3p、hsa-miR-576-5p、hsa-miR-577、hsa-miR- 578、hsa-miR-579、hsa-miR-580、hsa-miR-581、hsa-miR-582-3p、hsa-miR-582-5p、hsa- miR-583、hsa-miR-584、hsa-miR-585、hsa-miR-586、hsa-miR-587、hsa-miR-588、hsa-miR- 589、hsa-miR-589*、hsa-miR-590-3p、hsa-miR-590-5p、hsa-miR-591、hsa-miR-592、hsa- miR-593、hsa-miR-593*、hsa-miR-595、hsa-miR-596、hsa-miR-597、hsa-miR-598、hsa-miR- 599、hsa-miR-600、hsa-miR-601、hsa-miR-602、hsa-miR-603、hsa-miR-604、hsa-miR-605、 hsa-miR-606、hsa-miR-607、hsa-miR-608、hsa-miR-609、hsa-miR-610、hsa-miR-611、hsa- miR-612、hsa-miR-613、hsa-miR-614、hsa-miR-615-3p、hsa-miR-615-5p、hsa-miR-616、 hsa-miR-616*、hsa-miR-617、hsa-miR-618、hsa-miR-619、hsa-miR-620、hsa-miR-621、hsa- miR-622、hsa-miR-623、hsa-miR-624、hsa-miR-624*、hsa-miR-625、hsa-miR-625*、hsa- miR-626、hsa-miR-627、hsa-miR-628-3p、hsa-miR-628-5p、hsa-miR-629、hsa-miR-629*、 hsa-miR-630、hsa-miR-631、hsa-miR-632、hsa-miR-633、hsa-miR-634、hsa-miR-635、hsa- miR-636、hsa-miR-637、hsa-miR-638、hsa-miR-639、hsa-miR-640、hsa-miR-641、hsa-miR- 642、hsa-miR-643、hsa-miR-644、hsa-miR-645、hsa-miR-646、hsa-miR-647、hsa-miR-648、 hsa-miR-649、hsa-miR-650、hsa-miR-651、hsa-miR-652、hsa-miR-653、hsa-miR-654-3p、 hsa-miR-654-5p、hsa-miR-655、hsa-miR-656、hsa-miR-657、hsa-miR-658、hsa-miR-659、 hsa-miR-660、hsa-miR-661、hsa-miR-662、hsa-miR-663、hsa-miR-663b、hsa-miR-664、hsa- miR-664*、hsa-miR-665、hsa-miR-668、hsa-miR-671-3p、hsa-miR-671-5p、hsa-miR-675、 hsa-miR-7、hsa-miR-708、hsa-miR-708*、hsa-miR-7-1*、hsa-miR-7-2*、hsa-miR-720、hsa- miR-744、hsa-miR-744*、hsa-miR-758、hsa-miR-760、hsa-miR-765、hsa-miR-766、hsa-miR- 767-3p、hsa-miR-767-5p、hsa-miR-768-3p、hsa-miR-768-5p、hsa-miR-769-3p、hsa-miR- 769-5p、hsa-miR-770-5p、hsa-miR-802、hsa-miR-873、hsa-miR-874、hsa-miR-875-3p、hsa- miR-875-5p、hsa-miR-876-3p、hsa-miR-876-5p、hsa-miR-877、hsa-miR-877*、hsa-miR- 885-3p、hsa-miR-885-5p、hsa-miR-886-3p、hsa-miR-886-5p、hsa-miR-887、hsa-miR-888、 hsa-miR-888*、hsa-miR-889、hsa-miR-890、hsa-miR-891a、hsa-miR-891b、hsa-miR-892a、 hsa-miR-892b、hsa-miR-9、hsa-miR-9*、hsa-miR-920、hsa-miR-921、hsa-miR-922、hsa- miR-923、hsa-miR-924、hsa-miR-92a、hsa-miR-92a-1*、hsa-miR-92a-2*、hsa-miR-92b、 hsa-miR-92b*、hsa-miR-93、hsa-miR-93*、hsa-miR-933、hsa-miR-934、hsa-miR-935、hsa- miR-936、hsa-miR-937、hsa-miR-938、hsa-miR-939、hsa-miR-940、hsa-miR-941、hsa-miR- 942、hsa-miR-943、hsa-miR-944、hsa-miR-95、hsa-miR-96、hsa-miR-96*、hsa-miR-98、hsa- MiR-99a, hsa-miR-99a*, hsa-miR-99b and hsa-miR-99b*.

The function for the mRNA that miRNA inhibits it to target, and therefore inhibit by the expression of the mRNA polypeptide encoded.Therefore, it hinders The activity (for example, silencing miRNA) of disconnected (partially or completely) miRNA can effectively induce or restore the repressed polypeptide of expression Expression (derepressing to polypeptide).In one embodiment, it is by the derepressing for polypeptide that the mRNA target of miRNA encodes The miRNA activity in cell is inhibited to realize by any one of a variety of methods.For example, blocking the activity of miRNA can With by small RNA that is complementary with miRNA or being substantially complementary (for example, antisense oligonucleotides, miRNA sponge, TuD RNA) hybridization is to realize, to block the interaction of miRNA Yu its said target mrna.As used herein, it is substantially complementary with miRNA Small RNA be that can hybridize with miRNA and block the active nucleic acid of miRNA.In some embodiments, with miRNA base Complementary small RNA is in addition to 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18 base in sheet Except small RNA with miRNA complete complementary.In some embodiments, small RNA sequence and miRNA be substantially Complementation, or the small RNA sequence complementary with having the miRNA of at least one base.

" miRNA inhibitor " is the medicament for blocking miRNA function, expression and/or processing.For example, these molecules include but It is not limited to inhibit Microrna specific antisense, the Microrna sponge, tough and tensile bait of miRNA and the interaction of Drosha compound RNA (TuD RNA) and Microrna oligonucleotides (double-strand, hair clip, short oligonucleotide).As described above, microrna inhibitor energy It is enough to be expressed in the cell of the transgenosis from rAAV carrier.Microrna sponge is special by complementary heptamer seed sequence Inhibit miRNA (Ebert, M.S.Nature Methods, Epub August, 12,2007) to property.In some embodiments, may be used To use the entire miRNA family of single sponge sequences silences.TuD RNA realization has miRNA specific in mammalian cell Effect and it is long-term inhibit (see, e.g., Takeshi Haraguchi, et al., Nucleic Acids Research, 2009, Vol.37, No.6e43, the content for being related to TuD RNA are incorporated herein by reference).For miRNA function in silenced cell The other methods of (derepressing for miRNA target) will be apparent to practitioners skilled in the art.

In some embodiments, the cloning capacity (capacity) of recombinant RNA carrier may the required code sequence of limitation Column, and may need to replace 4.8 kilobase genomes of virus completely.Therefore, in some cases, big gene may be uncomfortable It shares and recombinates AAV carrier in standard.It will be understood by those skilled in the art that being can get in this field for overcoming Limited-Coding ability Option.The jugate to form head to tail for example, the AAV ITR of two genomes can anneal, almost adds the ability of carrier Times.The insertion permission of splice site removes ITR from transcript.Overcome other selections of limited cloning capacity for technical staff For be obvious.

The body cell transgenic animal model generated using the gene transfer based on rAAV

Present disclosure also relates to use the body cell transgenosis for being based on the method production disease of recombinant adeno-associated virus (rAAV) Animal model.This method is at least partially based on AAV serotype and its variant is mediated in adults with tissue specific way The observation result of effective and stable gene transfer.By rAAV element (capsid, promoter, transgene product) combine with obtain with Time and tissue specific way express the body cell transgenic animal model for stablizing transgenosis.It is generated by disclosed method Body cell transgenic animals may be used as human diseases, pathological state and/or characterize gene effect useful model, function It can (for example, tissue specificity, disease act on) unknown or incomplete understanding.For example, animal (for example, mouse) can be in different hairs (for example, age) is educated the stage to be infected with rAAV, the rAAV include have specific organization targeting ability (for example, liver, heart, Pancreas) capsid and with tissue-specific promoter drive participate in disease gene expression transgenosis.After infection, rAAV It infects the different cells of target tissue and generates transgene product.

In some embodiments, the sequence of the code area of transgenosis is modified.Modification can change by transgenes encoding The function of product.Then it can be studied and be repaired in vivo by using method disclosed herein generation body cell transgenic animal model The effect of decorations.In some embodiments, the modification of coding region sequence is nonsense mutation, generates segment (for example, truncating shape Formula).In other cases, modification is missense mutation, leads to amino acid substitution.Other modifications are possible and for ability It is obvious for field technique personnel.

In some embodiments, transgenosis causes pathological state.The transgenosis for leading to pathological state is its product in disease It works in disease or illness and (for example, causing disease or illness, makes animal susceptible disease or illness) and/or inducible Animal diseases Or the gene of illness.Then animal can be observed to assess any amount of aspect of disease (for example, being in progress, to the anti-of treatment It should wait).These embodiments are not intended to limit, other aspects are disclosed herein with example and are described in greater detail below.

In some respects, present disclose provides destroy particular cell types by targeting to generate body cell transgenic animals The method of model.For example, the model of type 1 diabetes can be destroyed by the targeting of pancreas β pancreas islet to generate.In other examples In, the targeting destruction of particular cell types can be used for assessing effect of the particular cell types to human diseases.In this respect, it encodes Cytotoxin (for example, diphtheria toxin A (DTA)) or the transgenosis for promoting apoptogene (NTR, Box etc.) can be used as specific cells The transgenosis of the functional ablation of type.Other exemplary transgenosis that its product kills cell are included in method disclosed herein In, and will be apparent to practitioners skilled in the art.

In some respects, present disclose provides for generate body cell transgenic animal model with study gene overexpression or The method for striking low long-term effect.In specific target tissue gene for a long time be overexpressed or strike it is low (for example, by shRNA, miRNA, MiRNA inhibitor etc.) it can interfere with normal metabolic balance and establish pathological state, to generate the animal of disease such as cancer Model.In some respects, present disclose provides the method for generating body cell transgenic animal model, the method is latent for studying Oncogene and other genes overexpression or strike the long term of low gene, and for studying the hair of the tumour in target tissue Raw and gene function.Useful transgene product includes known albumen relevant to cancer and the small of these protein expressions is inhibited to do Disturb nucleic acid.

Those skilled in the art can be readily selected other suitable transgenosis, and condition is that they can be used for generating tissue The animal model of specific pathologies state and/or disease.

Recombinate AAV method of administration

RAAV can be delivered to subject with composition forms according to any proper method known in the art.It is preferred that outstanding The rAAV for floating in physiologically compatible carrier (such as in the composition) can be applied to subject, such as host animal, Such as people, mouse, rat, cat, dog, sheep, rabbit, horse, ox, goat, pig, cavy, hamster, chicken, turkey or non-human primates are dynamic Object (such as macaque).In some embodiments, host animal does not include people.

By rAAV be delivered to mammalian subject can for example, by intramuscular injection or by be applied to mammal by The blood flow of examination person.It can be administered in blood flow by being injected into vein, artery or any other blood vessel.In some embodiment party In case, rAAV is administered in blood flow by isolated limb perfusion, the isolated limb perfusion is known in surgical field Technology, this method substantially enables a technician to separate limbs with body circulation before applying rAAV virion.Ability Field technique personnel can also use the deformation for the isolated limb perfusion technology being described in United States Patent (USP) No.6,177,403 will Virion is administered in the vascular system of the limbs of separation, potentially to enhance the transduction to muscle cell or tissue.In addition, In In some cases, it may be necessary to which virion is delivered to the CNS of subject." CNS " refers to the brain of vertebrate and the institute of spinal cord There are cell and tissue.Therefore, which includes but is not limited to neuronal cell, Deiter's cells, astroglia, brain ridge Marrow liquid (CSF), interstitial space, bone, cartilage etc..Recombinating AAV can be by being injected into such as ventricular area and corpus straitum (example Such as, the caudate nucleus of corpus straitum or shell core), spinal cord and neuromuscular junction or subfolium, be directly delivered to CNS or brain, it is described Injection uses needle, conduit or relevant apparatus, using neurosurgery technology known in the art, such as passes through stereotactic injection (see, e.g. Stein et al., J Virol 73:3424-3429,1999；Davidson et al., PNAS 97:3428-3432, 2000；Davidson et al., Nat.Genet.3:219-223,1993；With Alisky and Davidson, Hum.Gene Ther.11:2315-2329,2000)。

The composition of the disclosure can individually include rAAV, or with other one or more viruses (for example, having one kind Or second of rAAV coding of a variety of different transgenosis) combination.In some embodiments, composition include 1,2,3,4,5,6, 7,8,9,10 or more different rAAV, every kind of rAAV have one or more different transgenosis.

In view of the targeted indication of rAAV, those skilled in the art can be readily selected suitable carrier.For example, A kind of suitable carrier includes salt water, can be prepared with various buffer solutions (such as phosphate buffered saline (PBS)).Other examples Property carrier includes Sterile Saline, lactose, sucrose, calcium phosphate, gelatin, glucan, agar, pectin, peanut oil, sesame oil and water. The selection of carrier is not the limitation to the disclosure.

Optionally, other than rAAV and carrier, the composition of the disclosure can contain other conventional medicine ingredients, such as Preservative or chemical stabilizer.Suitable exemplary preservative includes methaform, potassium sorbate, sorbic acid, sulfur dioxide, does not eat Sub- propyl propionate, p-hydroxybenzoate, ethyl vanillin, glycerol, phenol and parachlorophenol.Suitable chemical stabilizer includes bright Glue and albumin.

RAAV is applied to transfect the required cell organized with enough amounts, and the gene transfer and table of enough levels are provided It reaches, without excessive adverse effect.Conventional and pharmaceutically acceptable administration method is including but not limited to directly delivered to institute Select organ (for example, portal vein is delivered to liver), oral, sucking (including intranasal and intratracheal delivering), intraocular, intravenous, flesh Meat is interior, in subcutaneous, intradermal, tumor and other parenteral administration method.If desired, approach can be administered in combination.

The dosage of rAAV virion needed for realizing specific " therapeutic effect ", such as genome copies/every kg body weight (GC/kg) dosage unit in, will based on several factors which and change, including but not limited to: rAAV virion administration method reaches Gene needed for therapeutic effect or rna expression are horizontal, the specified disease or illness and gene treated or RNA product it is steady It is qualitative.It can readily determine that based on above-mentioned factor and other factors well-known in the art, those skilled in the art RAAV virion dose range suffers from the patient of specified disease or illness to treat.

A effective amount of rAAV is the amount for being enough to target infection animal, targeting expectation tissue.In some embodiments, effectively The rAAV of amount is the amount for being enough to generate stable body cell transgenic animal model.Effective quantity depends primarily on such as species, year The factors such as age, weight, subject's health and tissue to be targeted, therefore can change between animal or tissue.For example, a effective amount of RAAV is usually containing about 10⁹To 10¹⁶In the range of the solution of the about 1ml to about 100ml of a genome copies.In some implementations In scheme, with 10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵The dosage of a genome copies/subject applies rAAV.In some realities It applies in scheme, with 10¹⁰、10¹¹、10¹²、10¹³Or 10¹⁴The dosage of a genome copies/subject applies rAAV.In some cases Under, about 10¹¹To 10¹²The dosage of a rAAV genome copies is suitable.In certain embodiments, 10¹²A rAAV genome Copy is effective to targeting heart, liver and pancreatic tissue.In some cases, it is generated by the rAAV of multi-dose stable Transgenic animals.

In some embodiments, rAAV composition is prepared to reduce the aggregation of AAV particle in composition, is especially being deposited (for example, about 10 in the case where high rAAV concentration¹³GC/ml or higher).Method for reducing rAAV aggregation is this field crowd Well known, including such as adding surfactant, pH is adjusted, salinity is adjusted.(see, e.g., Wright FR, etc. People, Molecular Therapy (2005) 12,171-178, content is incorporated herein by reference.)

The preparation of pharmaceutically acceptable excipient and carrier solution is well known to the skilled artisan, because To develop suitable dosage and therapeutic scheme to use particular composition as described herein in various therapeutic schemes.

In general, these preparations contain at least about 0.1% or more reactive compound, although the percentage of active constituent Can of course change, and can be convenient about the 1 of total formulation weight or volume or 2% to about 70% or 80% or more it Between change.Of course, it is possible to the amount of reactive compound in the upper useful composition of every kind for the treatment of be prepared, so that in any given unit Suitable dosage is obtained in the compound of dosage.The technical staff for preparing the field of such pharmaceutical preparation such as dissolves consideration The factors such as degree, bioavilability, biological half-life, administration method, shelf life of products and other pharmacological considerations, and Therefore, various dosage and therapeutic scheme may be desirable.

In some cases, it is desirable to by the way that subcutaneous, pancreas is interior, intranasal, parenteral, intravenous, intramuscular, intrathecal or mouth It is constructed in clothes, peritonaeum or by the treatment based on rAAV in the inhalation delivery pharmaceutical composition disclosed herein suitably prepared Body.In some embodiments, such as U.S. Patent number 5,543,158；5,641,515 and 5,399,363 is (whole each by quoting Body is incorporated herein) described in administration mode can be used for delivering rAAV.In some embodiments it is preferred that method of application be Pass through introportal infusion.

It include aseptic aqueous solution or dispersion liquid and for extemporaneous preparation of sterile injectable suitable for injecting the medicament forms that use The aseptic powdery of solution or dispersion liquid.Dispersion can also be prepared in glycerol, liquid macrogol and its mixture and oil.In Under common storage and use condition, these preparations contain preservative to prevent the growth of microorganism.In many cases, the shape Formula is sterile and flow to the degree for being easy to inject.It must be stable under conditions of manufacture and storage, and must be prevented from The contamination of microorganism such as bacterium and fungi.Carrier can be solvent or decentralized medium, contain such as water, ethyl alcohol, polynary Alcohol (such as glycerol, propylene glycol and liquid macrogol etc.), suitable mixture and/or vegetable oil.For example, by using packet Clothing such as lecithin, by the granularity needed for maintaining and by using surfactant in the case where dispersion, it is appropriate to keep Mobility.Various antibacterial agents and antifungal agent, such as p-hydroxybenzoate, methaform, phenol, sorb can be passed through Acid, thimerosal etc. prevent the effect of microorganism.In many cases it is preferred to include isotonic agent, such as sugar or sodium chloride.Pass through The reagent absorbed in the composition using delay, such as aluminum monostearate and gelatin, can be realized the extension of Injectable composition It absorbs.

Administration for injectable aqueous solutions, such as, if it is desired, can suitably buffer solution, and first with foot Enough salt water or glucose keeps liquid diluent isotonic.These specific aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous It is applied in peritonaeum.In this regard, the sterile aqueous media being able to use will be known to the skilled in the art.For example, can A dosage to be dissolved in the isotonic NaCl solution of 1ml, and be added in the subcutaneous injection liquid of 1000ml or It is recommended that infusion site injection (see, for example, " Remington's Pharmaceutical Sciences " the 15th edition, the 1035-1038 pages and 1570-1580).Depending on the situation of host, some variations of dosage necessarily occur.Under any circumstance, The personnel for being responsible for application will determine the suitable dosage of individual host.

By the way that the desired amount of activity rAAV is mixed in solvent appropriate, as needed with it is enumerated herein it is various other at Divide and prepare sterile injectable solution together, then filtration sterilization.In general, by the way that the active constituent of various sterilizings is mixed sterile load Prepare dispersion in body, the sterile carrier contain basic dispersion medium and needed for those exemplified above other at Point.In the case where being used to prepare the aseptic powdery of sterile injectable solution, preferred preparation method is vacuum drying and freezing Dry technology, from the powder for generating active constituent and ingredient needed for any other in the solution being previously sterile filtered.

RAAV composition disclosed herein can also be configured to neutral or salt form.Pharmaceutically acceptable salt includes that acid adds At salt (being formed with the free amine group of albumen) and with inorganic acid such as hydrochloric acid or phosphoric acid or organic acid such as acetic acid, oxalic acid, winestone The formation such as acid, tussol.The salt formed with free carboxy can also be derived from inorganic base, such as sodium hydroxide, potassium hydroxide, hydrogen Amine-oxides, calcium hydroxide or iron hydroxide and organic base, such as isopropylamine, trimethylamine, histidine, procaine.It is preparing When, solution will be in the mode compatible with dosage formulation and to treat effective amount application.Preparation is easy to the application of a variety of dosage forms, example Such as Injectable solution, drug release capsules.

As used herein, " carrier " includes any and all solvents, decentralized medium, carrier, coating, diluent, antibacterium With antifungal agent, isotonic and absorption delaying agent, buffer, carrier solution, suspension, colloid etc..These media and medicament are used for The purposes of pharmaceutically active substance is well known in the art.The active constituent of supplement can also mix in composition.Phrase is " pharmaceutically It is acceptable " refer to the molecular entity and composition that allergy or similar adverse reaction are not generated when giving host.

Delivery vector such as liposome, Nano capsule, particle, microballoon, lipid granule, vesica etc. can be used for the group of the disclosure Object is closed to be introduced into suitable host cell.Particularly, the rAAV carrier of delivering transgenosis can be prepared for being encapsulated in lipid Delivering in grain, liposome, vesica, nanosphere or nano particle etc..

Such preparation is preferably used to be incorporated herein the pharmaceutically acceptable preparation of disclosed nucleic acid or rAAV construct. The formation and use of liposome are generally known to those skilled in the art.Recently, developing has improved serum stability With the liposome (U.S. Patent number 5,741,516) of circulating half-life.Further, it is described that liposome and liposome-like preparations Various methods (U.S. Patent number 5,567,434 as potential drug carrier；5,552,157；5,565,213；5,738,868 With 5,795,587).

Liposome is successfully used together with many cell types, these cell types usually turn other programs It contaminates resistant.In addition, liposome does not have DNA length limitation, this is the delivery system typically based on virus.Liposome by It is efficiently used for introducing gene, drug, radiotherapy dose, virus, transcription factor and allosteric effector into the cell of a variety of cultures In system and animal.In addition, have been completed several successful clinical tests, the liposome-mediated drug of these experimental examinations is passed The validity sent.

Liposome is formed by phosphatide, and phosphatide disperses in an aqueous medium and spontaneously forms multilayer concentric bilayer vesicle (also referred to as For multi-layer vesicles (MLV)).MLV usually has 25nm to 4 μm of diameter.The ultrasonic treatment of MLV result in diameter 200 to The small monolayer vesicle (SUV) of 500.ANG., contains aqueous solution in core.

Alternatively, the nanocapsule formulations of rAAV can be used.Nano capsule usually can be in a manner of stablizing and be repeatable Capture substance.Side effect caused by order to avoid being overloaded by intracellular polymeric, it should use the polymerization for capableing of degradation in vivo Object designs this ultra-fine grain (about 0.1 μm of size).Consider using the biodegradable poly- alkyl-cyano for meeting these requirements Acrylate nano particle.

Other than above-mentioned delivering method, it is also contemplated that following technology is as the alternative that rAAV composition is delivered to host Method.It uses and describes in United States Patent (USP) No.5,656,016 ultrasonic delivery (that is, ultrasonic wave), as raising medicine Object penetrates into and by the rate of the circulatory system and the device of effect.The other drugs delivering substitute of consideration is intraosseous injection (United States Patent (USP) No.5,779,708), microchip device (United States Patent (USP) No.5,797,898), ophthalmic preparation (Bourlais etc. People, 1998), the delivering (U.S. Patent number of transdermal matrix (U.S. Patent number 5,770,219 and 5,783,208) and feedback control 5,697,899)。

Kit and compositions related

In some embodiments, medicament as described herein can be assembled into drug or diagnosis or research kit, to promote Into their purposes in treatment, diagnosis or research application.Kit may include the one or more for accommodating the component of the disclosure Container and operation instruction.Specifically, such kit may include one or more reagents as described herein, and description expection is answered With the proper use of specification with these reagents.In certain embodiments, the medicament in kit can be suitable for spy Apply and apply calmly the pharmaceutical preparation and dosage of the method for the medicament.Kit for research purposes may include each for carrying out The debita spissitudo of kind experiment or the component of amount.

Kit can be designed so as to researcher using method described herein, and can take various forms. Under applicable circumstances, every kind of composition of kit can in liquid form (for example, in the solution) or in solid form (example Such as dry powder) it provides.In some cases, some compositions can be (for example, in an active) may make up or machinable, For example, by adding suitable solvent or other substances (for example, water or cell culture medium), may be provided in kit or It may not provide.As it is used herein, " specification " can define the component of explanation and/or promotion, and be usually directed to The packaging of the disclosure is associated or associated printed instructions.Specification can also be any including providing in any way Oral or electronic description, so that user will readily recognize that specification is associated with the kit, for example, audiovisual (for example, Video-tape, DVD etc.), internet and/or it is network-based communication etc..Printed instructions can be by management drug or biology system Form as defined in the government organs of the manufacture, use or sale of product, the specification also can reflect manufacture, use or sale and be used for The approval of the mechanism of the care of animal.

Kit can include any one or more of component as described herein in one or more containers.As reality Example, in one embodiment, kit may include for one or more components of mix reagent box and/or separation and mixing Sample and the specification for being applied to subject.The kit may include the container for accommodating medicament as described herein.Medicament can be The form of liquid, gel or solid (powder).Medicament sterile can be prepared, is packaged in syringe and Refrigerated Transport.Alternatively, it It can be contained in bottle or other containers for storing.Second container can have other medicaments of sterile preparation.Alternatively, examination Agent box may include the activating agent of premixing, and transport in syringe, bottle, pipe or other containers.Kit can have and will try Agent is applied to one or more needed for animal or all components, such as syringe, local application device or intravenous injection needle tube And bag, especially for generate particular volume multicellular animal model kit in the case where.

The kit can have a diversified forms, for example, the pack of blister pouch, shrink pack, vacuum seal bag, sealable heat at Type pallet or similar bag or tray form, wherein accessory be loosely packaged in bag (pouch), one or more pipe, container, In box or bag (bag).It after adding accessory, can sterilize to kit, to allow each accessory in container Otherwise it is unfolded.It is able to use any sterilization technology appropriate to sterilize to kit, such as radiation sterilization, heating are gone out Bacterium or other sterilizing methods known in the art.Depending on concrete application, which can also include other components, for example, Container, cell culture medium, salt, buffer, reagent, syringe, needle, such as gauze of the fabric for applying or removing disinfectant, Disposable glove, before administration to support of medicament etc..

The method that the specification for including in kit can be related to latent AAV in detection cell.In addition, the kit of the disclosure It may include specification, feminine gender and/or positive control, container, diluent and the buffer for sample, sample preparation cartridge and be used for The table of printing or electronic reference AAV sequence that sequence compares.

Embodiment