CN108239645B - Liver specific transcription regulation sequence and application thereof - Google Patents
Liver specific transcription regulation sequence and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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- C12N15/09—Recombinant DNA-technology
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/09—Recombinant DNA-technology
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Abstract
The invention relates to a liver specific transcription regulation and control sequence. Specifically, the invention provides a regulatory sequence capable of driving and enhancing the specific expression of downstream genes in liver cells, the regulatory sequence comprises regulatory sequences of albumin and transthyretin, can be used for specifically expressing therapeutic genes in the liver cells, such as coagulation factor IX genes, and has very important significance for gene therapy strategies expressed by the liver cells.
Description
Technical field
The present invention relates to genetic engineering field, relates more specifically to a kind of liver specificity transcription regulating nucleotide sequence and its answer
With.
Background technique
Hemophilia B (Hemophilia B, HB) belongs to monogenic inheritance disease, is people's blood coagulation on chromosome x q27.1
Sex chromosome linkage inheritance hemorrhagic disease caused by factors IX encoding gene (hF9) defect, the disease incidence in global male
About 1/25000.Normal people F9 full length gene about 34kb, wherein coded sequence cDNA length is 1383bp, and it is long to be located at X chromosome
The arm end region Xq27.1-q27.2 includes 8 exons and 7 intrones.People's F9 gene defect leads to FIX albumen in blood plasma
Missing or dysfunction are to lead to the basic cause of disease of hemophilia B.Currently, the primary treatment measure of haemophiliac is to substitute
It treats (protein replacement therapy, PRT), including preventative or therapeutic be transfused recombinant blood coagulation factor repeatedly
IX or blood plasma product etc..Although these remedy measures improve the survival rate and quality of life of patient to a certain extent,
Do not have curative.At the same time, the expensive expense ($ 100-300,000/) of long-term treatment and blood transfusion or intravenous injection are drawn
The complication such as the infection of hair all bring many drawbacks to patient.
Gene therapy is one of current most potential haemophiliachemophiliac strategy of healing.It, can by gene cloning and transfer techniques
Therapeutic gene is effectively transferred to gene defect cell with the normal coagulation factor of expressive function steadily in the long term, restore to suffer from
The normal coagulation function of person, to achieve the purpose that cure all the life.In conjunction with current hemophilia gene treat basic research and face
Bed test progress, the relatively mature technology of potentiality of most curing of development is with gland relevant viral vector (adeno-
Associated virus, AAV) carry out vivo gene therapy (In vivo gene therapy).
World's the first Gene Therapy for Hemophilia B clinical test of 2011 and consecutive publications in 2014 reports AAV carrier
Mediate the significant curative effect that obtains in 10 severe HB patients of gene therapy, some patientss clinical symptoms are relieved, interrupt or
Reduce the use of hFIX factor substitute.Nevertheless, the intracorporal hFIX level of Most patients can only achieve the 1- of normal person
6%, in high, medium and low three groups, mainly the patient of high dose group and part middle dose group achieves preferable clinical effect
It answers, but there are also certain distances relative to > 30% thorough criterion of cure.The main reason for effect is limited in this clinical test
Are as follows:
(1) transcriptional efficiency that AAV carries intracorporal expression frame can also further increase；
(2) production technology of clinical test preparation needs to be further increased, and to improve the ratio of entity virion, reduces
The pollution of gutless；
(3) patient's body produces the immunological effect for carrier involucrum albumen.Although this immunological effect can pass through
Immunosuppressive therapy is controlled, but is resulted in AAV carrier to a certain extent and removed by host body.
In summary, it is further improved the bio-carrier of optimization gene treatment hemophilia B and carries out clinical conversion, for filling out
Mending the domestic blank in this field has very important meaning.
Summary of the invention
The purpose of the present invention is to provide a kind of liver specificity transcription regulating nucleotide sequence and its applications.
Another object of the present invention is to provide a kind of, and the adeno-associated virus comprising liver specificity transcription regulating nucleotide sequence carries
Body, the gland relevant viral vector can be used for haemophiliachemophiliac gene therapy.
In the first aspect of the present invention, a kind of isolated liver specificity transcription regulating nucleotide sequence is provided, which is characterized in that
The regulating and controlling sequence includes albumin regulating and controlling sequence and transthyretin regulating and controlling sequence.
In another preferred example, the albumin regulating and controlling sequence includes the bound site of transcription factor HNF-1 and C/EBP
Point.
In another preferred example, the transthyretin regulating and controlling sequence include transcription factor HNF-1, HNF-3,
The binding site of HNF-4 and C/EBP.
In another preferred example, the transcription factor is liver specific transcription factor.
In another preferred example, the liver specificity transcription regulating nucleotide sequence is to appoint polynucleotides selected from the group below:
(a) nucleotide sequence polynucleotides as shown in SEQ ID NO.:1；
(b) homology >=90% of sequence shown in nucleotide sequence and SEQ ID NO.:1 is (preferably >=95%, more preferably
>=98%, most preferably >=99%), and the polynucleotides with liver specificity transcriptional activity；
(c) 5 ' ends and/or 3 ' ends of the polynucleotides as shown in SEQ ID NO.:1 increase and/or reduce 1-50 (preferably
Ground 1-20, more preferably 1-10, more preferably 1-5) nucleotide, and the polynucleotides with liver specificity transcriptional activity.
In another preferred example, the liver specificity transcriptional activity refers to driving downstream DNA segment in liver cell
The activity of middle specific transcriptional.
In another preferred example, " specific transcriptional in liver cell " refers to that the transcription in the liver cell is living
Property the ratio between (or transcription amount) Z1 and transcriptional activity (or transcription amount) Z2 in non-liver cell (such as fibroblast) (Z1/Z2)
>=2, preferably >=5, more preferably >=10.
In another preferred example, the polynucleotides selected from (b) or (c) remain polynucleotides shown in SEQ ID NO.:1
Liver specificity transcriptional activity at least >=50%, >=60%, >=70%, >=80%, >=90%, >=100%.
In another preferred example, the regulating and controlling sequence is artificial recombination regulating and controlling sequence.
In another preferred example, the regulating and controlling sequence derives from people or non-human mammal.
In the second aspect of the present invention, a kind of nucleic acid constructs is provided, which is characterized in that the construction contains outer
Source gene or exogenous dna fragment, and the first aspect present invention institute being operatively connected with the foreign gene or DNA fragmentation
The regulating and controlling sequence stated.
In another preferred example, the foreign gene is selected from the group: therapeutic gene, riddled basins, resistant gene,
Antigenic protein gene, RNAi gene, microRNA gene, or combinations thereof.
In another preferred example, the therapeutic gene is selected from the group: human coagulation factor IX gene, human blood coagulation
VIII, people's alpha1-antitrypsin, people's aryl sulfatase B (ARSB) gene, human ldl receptor (LDLR) gene,
Or combinations thereof.
In another preferred example, the clotting factor IX gene includes that wildtype factor IX gene and saltant type are solidifying
Blood factor IX gene.
In another preferred example, the saltant type clotting factor IX gene includes the coagulation factor of (i) codon optimization
IX gene, and (ii) point mutation clotting factor IX gene, wherein the polypeptide of the point mutation clotting factor IX gene coding exists
The 338th arginine corresponding to wildtype factor IX sports leucine.
In another preferred example, the sequence of the clotting factor IX gene of the codon optimization such as SEQ ID NO.:2 institute
Show.
In another preferred example, the sequence of the point mutation clotting factor IX gene is as shown in SEQ ID NO.:3.
In the third aspect of the present invention, a kind of carrier is provided, which is characterized in that the carrier contains the present invention second
Construction described in aspect.
In another preferred example, the carrier is plasmid, viral vectors.
In another preferred example, the carrier is slow virus carrier, gland relevant viral vector.
In another preferred example, the carrier is AAV carrier, it is therefore preferable to AAV8 carrier.
In another preferred example, the carrier is AAV8 carrier, and the carrier (side) has the present invention the
The ITR sequence of mutation described in eight aspects.
In the fourth aspect of the present invention, a kind of host cell is provided, contains third of the present invention in the host cell
Regulating and controlling sequence described in the first aspect present invention of external source or the present invention second are integrated in carrier described in aspect or chromosome
Nucleic acid constructs described in aspect.
In another preferred example, the cell is isolated cell and/or the cell is genetically engineered cell.
In another preferred example, the cell behaviour or nonhuman mammalian cells.
In another preferred example, the cell is liver cell.
In the fifth aspect of the invention, regulating and controlling sequence described in first aspect present invention, second aspect of the present invention are provided
The purposes of carrier described in the nucleic acid constructs or third aspect present invention, which is characterized in that be used to prepare a preparation or
Composition, the preparation or composition are for regulating and controlling foreign gene or exogenous dna fragment in liver cell or liver organization
It carries out specific expressed.
In the sixth aspect of the present invention, the purposes of regulating and controlling sequence described in first aspect present invention is provided, feature exists
In for constructing an expression vector, the expression vector is used for the specific expressed external source in liver cell or liver organization
Gene.
In another preferred example, the foreign gene include structural gene, nonstructural gene (such as encoding antisense RNA,
The nonstructural gene of miRNA or siRNA).
In the seventh aspect of the present invention, a kind of pharmaceutical composition is provided, which is characterized in that the pharmaceutical composition contains
There are carrier described in third aspect present invention and pharmaceutically acceptable excipient.
In another preferred example, the dosage form of the pharmaceutical composition is peroral dosage form or injection.
In the eighth aspect of the present invention, a kind of ITR sequence (inverted terminal repeat sequence) of mutation, the mutation are provided
ITR sequence contain 106 bases, between 3 ' ends the 25th to the 130th of wild type ITR sequence (SEQ ID NO.:5)
Base sequence it is identical or complementary.
In another preferred example, the ITR sequence of the mutation delete wild type ITR sequence correspond to SEQ ID
1-24 (5 ' ends) of NO.:5 and 131-145 (3 ' end).
In another preferred example, the ITR sequence of the mutation is as shown in SEQ ID NO.:4.
In another preferred example, the ITR sequence of the mutation is the base sequence complementary with SEQ ID NO.:4.
In the ninth aspect of the present invention, a kind of carrier is provided, which is characterized in that the carrier contains the present invention the 8th
ITR sequence described in aspect.
In another preferred example, the carrier is plasmid, viral vectors.
In another preferred example, the carrier is slow virus carrier, gland relevant viral vector.
In another preferred example, the carrier is AAV carrier, it is therefore preferable to AAV8 carrier.
In another preferred example, 5 ' ITR sequences of the AAV carrier are mutation described in eighth aspect present invention
ITR sequence.
In another preferred example, 3 ' ITR sequences of the AAV carrier are mutation described in eighth aspect present invention
ITR sequence.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 show through population analysis in hemophilia B patient 1113 kinds of gene mutation types of antidiastole and this
Distribution of a little mutation in human blood coagulation 9 (hF9) gene extron, introne and UTR region.Wherein, each code is containing as follows: E,
Exon；Int, introne；UTR, upstream regulatory region.
Fig. 2 shows wild type AAV virus and AAV carrier schematic diagram.
Fig. 2A show wild type AAV carrier be diameter be 20-25nm without coating DNA virus, include and be about 4.7kb's
Single stranded DNA genome.Albumen needed for AAV genome has two groups of encoding genes, rep gene to encode virus replication and assemble, cap base
The virus encapsidation of 60-mer is formed because of 3 kinds of albumen of coding.The coat protein of AAV carrier and the consistent of wild type AAV virus or
It is very similar, include the treatment correlated expression gene with transcription driving element.Have in the molecular wt of AAV carrier 74% by egg
White composition, the carrying amount of maximum DNA are 5kb.
Fig. 2 B shows that wild type AAV virus is engineered to recombinate AAV carrier, wherein itself viral encoding gene is treated
Property gene replace.
Fig. 3 shows that the carrier mediated selectively targeted liver cell of AAV generates coagulation factor gene treatment hemophilia B signal
Figure.
Fig. 4 shows that AAV carrier structure figure and hFIX coded sequence optimization front and back compare.
Fig. 4 A shows AAV carrier structure schematic diagram.
Fig. 4 B show people's FIX coded sequence optimization front and back compares, including Optimal Parameters (left side), G/C content (in) and
CDNA secondary structure (right side).
Fig. 5 shows that sequence optimisation people FIX cDNA helps to improve hFIX in the intracorporal activity of HB mouse and albumen water
It is flat.
Fig. 5 A shows the result of ELISA method measurement hFIX antigen levels.Carry wild type hFIX, Optimization-type hFIX
(cohFIX) or the DNA plasmid of Optimization-type hFIX positive control (+ve ctrl) expression frame enters blood through high-pressure injection mode
Friendly disease B mouse (HB) is internal.Mice plasma acquires after 48 hours and measures hFIX antigen levels by ELISA method.As a result
It is indicated with mean ± standard deviation.
Fig. 5 B and Fig. 5 C respectively illustrate the result of ELISA method measurement hFIX activity and antigen levels.GFP is carried, it is excellent
The AAV8 carrier of change type hFIX (cohFIX) or Optimization-type hFIX positive control (+ve ctrl) expression frame is infused by tail vein
The mode of penetrating enters HB mouse.Since first week, mice plasma was primary every surrounding acquisition, wherein hFIX activity and antigen levels
It is measured respectively by Chromogenic assay and ELISA method, is as a result expressed as mean ± standard deviation.
Fig. 6 shows that carrying high blood coagulation activity mutation hFIX cDNA facilitates the biological dose that AAV carrier is effectively reduced.
The AAV8 carrier of expression hFIXR338L mutant enters HB mouse by tail vein injection mode.Since first week, mouse blood
Slurry is primary every surrounding acquisition, and wherein hFIX activity and antigen levels pass through Chromogenic assay respectively and ELISA method is measured,
As a result it is expressed as mean ± standard deviation.
Fig. 7 shows gel electrophoresis analysis double-strand AAV carrier and single-stranded AAV carrier result.Wherein ITRm1It is to contain
ITR structure, ITR in the carrier that Nathwani etc. is deliveredm2The ITR mutation structure being transformed in this project is included, shape is both mediated
At the carrier (scAAV) containing double-stranded DNA core.ITRControlControl type ITR structure is included, core containing single stranded DNA is packaged to form
Carrier (ssAAV).AAV carrier can retain its mono-/bis-chain structure under alkaline gel electrophoresis, show as single-stranded or double-stranded molecule
Amount；And the duplex structure of scAAV is destroyed in non-deformed glue, to all show as the molecular weight of single stranded DNA.
Fig. 8 shows that liver specificity controlling element activity compares in HB Mice Body.Carry different liver specificity tune
The AAV carrier of the cohFIX expression frame of control sequence enters factor Ⅸ deficient mouse (HB) in vivo through tail vein injection.It is latter from injecting
In week, mice plasma is primary every surrounding acquisition, wherein hFIX activity and antigen levels pass through respectively Chromogenic assay and
ELISA method is measured, and is as a result expressed as mean ± standard deviation.
Fig. 9 shows that the AAV8 carrier for carrying pSyn.cohFIX expression frame carries out dosage in HB Mice Body and increases examination
It tests.AAV carrier is respectively according to height (2 × 1012/ kg/ only), in (4 × 1011/ kg/ is only) and low (1 × 1011/ kg/ is only) three kinds not
Same dosage is in tail vein input HB Mice Body (n=3~6).AAV.GFP carrier (2 × 1012/ kg/ is only) it is used as yin
Property control.Mouse first week and, plasma activities and antigen levels difference of hFIX primary every surrounding acquisition blood plasma after injection
It is measured by Chromogenic assay and ELISA method, is as a result expressed as mean ± standard deviation.
Figure 10 shows the RNA table that AAV carrier dosage increases in test, in each group HB mouse liver, spleen and thymic tissue
Relatively up to level.The 12nd week after having injected various dose AAV carrier, each group mouse has 2-3 only to acquire liver, spleen and thymus gland group
It knits and extracts RNA, and reverse transcription generates cDNA.Pass through the cDNA water of real-time PCR semiquantitative determination hFIX and internal reference GAPDH
It is flat, the level difference of RNA in relatively more each tissue.The rna level of Human factor IX is expressed as opposite RNA copy number (2-Δ(hFIX-GAPDH)).It is as the result is shown individual mouse data and mean ± standard deviation.
Figure 11 shows that AAV carrier increases the biological distribution in test in each group mouse tissue internal organs in dosage.Dosage
Increasing each group mouse 2-3 in test, only 12 weeks acquisition livers, spleen and thymic tissue after injection of AAV vectors extract RNA liver, spleen, and
DNA in thymic tissue passes through concentration of the real-time PCR semiquantitative determination AAV carrier DNA in each histocyte.Knot
Fruit is shown as mean ± standard deviation.
Figure 12 shows that dosage increases each group mouse liver function index measurement in test.Each group mouse 2-3 is only in injection of AAV
Before carrier, and injection after the 4th and 12 week acquisition blood plasma by Chromogenic assay measure AST and ALT concentration, as the result is shown for
Mean ± standard deviation.
Figure 13 shows that dosage increases the high blood coagulation activity marker measurement of each group mouse in test.Each group mouse 2-3 is only being infused
The blood plasma of acquisition in the 4th and 12 week detects the concentration of D dimer by ELISA method before penetrating AAV carrier, and after injection, as a result shows
It is shown as mean ± standard deviation.
Specific embodiment
The present inventor is surprised to find that a kind of liver specificity transcriptional control sequence by depth studying extensively for the first time
Column can drive enhancing downstream gene specific expressed in liver cell.Experiment shows that regulating and controlling sequence of the invention can
Specific expressed in liver cell to enhance foreign gene (such as Human factor IX), transcriptional control is high-efficient (
Through being verified in factor Ⅸ deficient mouse model).Regulating and controlling sequence of the invention can also be used in the specific earth's surface in liver cell
Up to other therapeutic gene, all have very important significance for the strategies in gene therapy through liver cell expression.
PSyn regulating and controlling sequence
As used herein, term " regulating and controlling sequence ", " transcription regulating nucleotide sequence ", " pSyn regulating and controlling sequence " are used interchangeably, and are
Refer to a kind of nucleic acid sequence of accurate and effective initial gene functional transcription.The regulating and controlling sequence of the invention includes deriving from white egg
White, transthyretin transcription regulating nucleotide sequence.Preferably, the regulating and controlling sequence is to appoint polynucleotides selected from the group below:
(a) nucleotide sequence polynucleotides as shown in SEQ ID NO.:1；
(b) homology >=90% of sequence shown in nucleotide sequence and SEQ ID NO.:1 is (preferably >=95%, more preferably
>=98%, most preferably >=99%), and the polynucleotides with liver specificity transcriptional activity；
(c) 5 ' ends and/or 3 ' ends of the polynucleotides as shown in SEQ ID NO.:1 increase and/or reduce 1-50 (preferably
Ground 1-20, more preferably 1-10, more preferably 1-5) nucleotide, and the polynucleotides with liver specificity transcriptional activity.
PSyn regulatory nucleotide sequence can drive enhancing downstream gene specific expressed in liver cell.In this hair
In bright, it is specific expressed in liver cell to be mainly used for enhancing Human factor IX, transcriptional control efficiency and other livers
Dirty transcription regulating nucleotide sequence compares in factor Ⅸ deficient mouse model.Gene therapy in addition to being applied to hemophilia B
Outside, pSyn regulating and controlling sequence can also be used in the other therapeutic gene of specific expression in liver cell.Therefore, this section of sequence pair
It all has very important significance in the strategies in gene therapy through liver cell expression.
Herein, the regulating and controlling sequence includes the variant of regulating and controlling sequence shown in SEQ ID NO.:1, and the variant can
To carry out random or rite-directed mutagenesis etc. to obtain by being inserted into or deleting regulatory region.
The invention also includes with regulating and controlling sequence of the invention have 50% or more (preferably 60% or more, 70% or more,
80% or more, more preferable 90% or more, more preferable 95% or more, most preferably 98% or more, such as nucleic acid of 99%) homology, institute
Stating nucleic acid also has the function of that specific regulatory control starts Plant aboveground tissue expression." homology " refers to according to position identical hundred
Divide ratio, the similar level (i.e. sequence similarity or identity) between two or more pieces nucleic acid.
As used herein, term " specific expressed " refers to what target gene was organized in the specific time and/or specifically
Expression.
As used herein, " external source " or " heterologous " refers to the two or more pieces nucleic acid or protein sequence of separate sources
Between relationship.For example, if the combination of promoter and objective gene sequence is not usually naturally occurring, promoter for
It is external source for the target gene.It is " external source " for cell that particular sequence is inserted into it or organism.
Promoter of the invention can operationally be connect with foreign gene, and the foreign gene is for promoter
It can be external source (heterologous).Foreign gene (also referred to as target gene) of the present invention is not particularly limited, preferably
Foreign gene is the foreign gene for gene therapy through liver cell expression, such as human coagulation factor IX gene.
The present invention also provides nucleic acid constructs and carrier comprising the regulating and controlling sequence.It is used to prepare the side of recombinant vector
Method is well known to those of ordinary skill in the art.It is thin that expression vector can be bacterial plasmid, bacteriophage, yeast plasmid, plant
Cellular virus, mammalian cell virus or other carriers.A kind of particularly preferred carrier is viral for AAV8.
Those of ordinary skill in the art can be used well known method building and contain promoter of the present invention and/or mesh
Gene order expression vector.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..
The building of pSyn regulating and controlling sequence
In order to improve the curative effect of AAV vector-mediated gene treatment, screening and optimizing has been carried out to transcriptional regulatory element.It is thin in liver
In born of the same parents' directed gene therapy, although the viral vectors such as AAV8 are affinity to liver organization and the infected tissue that can succeed is thin
Born of the same parents, but also have some other non-liver cell of certainly possible infection, structure miss the target generation expression and corresponding adverse reaction.Cause
The controlling element of this design screening primarily requires to be to have the effect of liver cell specific regulatory control.In addition, being based on double-strand complementary type AAV
The bale capacity of carrier only has the half of wild type, and in order to successfully be assembled into viral entities particle, controlling element is also required to wrap
Containing base number as few as possible, the bale capacity for being more than virion is otherwise generated into invalid product.Transcription regulating nucleotide sequence master
It to include promoter and enhancer.Promoter is responsible for the identification and combination of RNA polymerase, and enhancer includes to promote regulatory transcription
The binding site of the factor can increase the transcription effect of downstream gene.
In the clinical test of the progress such as Nathwani, the controlling element of hFIX sequence is special by human apolipoprotein E liver
Anisotropic control zone (Human apolipoprotein E-Hepatocyte Control Region, ApoE-HCR) group is enhancing
Son, the composite construction sequence formed with the starting sub-portfolio of people's alpha-1 antitrypsin, and in order to adapt to the bale capacity tune of AAV
Whole size.The carrier for carrying this controlling element belongs to one of strongest carrier of the expression efficiency in liver organization, and successfully
Ground embodies certain curative effect in clinical test.However, this expression has only obtained the phenotype of patient to a certain degree
Alleviate, and not fully achieved the standard of healing.
In order to obtain the liver specific genes expression of higher clinical efficacy level, start in liver specific genes
Retrieved in subdata base (The Liver Specific Gene Promoter Database) (http: //
rulai.cshl.org/LSPD/index.html).In the efficiently specifically expressed albumen of liver cell, albumin (Albumin)
It is wherein one of main member.It can be seen that the enhancer and promoter of albumin have more liver specificity regulation
Element, thus close with the transcriptional control interconnection in liver cell.The promoter of albumin include transcription initiation site extremely
Region between the 217bp of upstream includes the binding site (table 5) of different transcription factors in sequence, such as liver specificity turns
Record factor HNF-1 (Hepatocyte nuclear factor 1) (LF-B1, PAF, AFP1), C/EBP, CTF/NF1, C/EBP
With DBP etc..And at least there are three enhancers in the upstream region at human albumin gene 5 ' end, including promoter upstream about-
265bp, -1.7kb, and the region -6kb.These belong to transcription factor, such as the main knot of HNF-1 rich in the region AT and GC
Coincidence point, this protein-dna interaction have the function of being obviously promoted for the transcription of downstream gene.
The region of AT and GT sequence is rich in the promoter and enhancer of 5. albumin of table and a-fetoprotein gene
Transthyretin (transthyretin, TTR), also known as prealbumin (prealbumin), and mainly exist
One of albumen being synthetically produced in liver cell.The promoter length of mouse TTR gene includes from transcription initiation site to 5 ' upstreams
The region of 202 base-pairs, wherein from -108 to -202 regions, which belong to TTR specific transcriptional in liver cell, expresses required area
Domain.In addition, thering is the DNA fragmentation of about 100bp to make with very strong enhancer between -1.86kb to -1.96kb in 5 ' upstreams
With the expression efficiency under proximal promoter can be promoted to drive in hepatoma cell strain enhances 5~10 times.It is identified, TTR's
Promoter and enhancer region include the binding site of at least 4 kinds different transcription factors, as HNF-1, HNF-3, HNF-4, with
And C/EBP etc..These factors largely belong to liver specific transcription factor, for promoting table of the downstream gene in liver cell
Up to stronger facilitation.
The main synthesising part of Human factor IX (hFIX) also is located at liver cell, the promoter of encoding gene hF9
It is positioned between -219 to+29 base-pairs, belongs to the promoter that TATA sequence lacks, transcriptional efficiency is weaker.However, identified hair
It include 5 cis-regulating elements in present hF9 initiating sequence.In these elements can with the transcription abundant of content in liver because
Son, such as C/EBP α, NF1, AR, DBP or HNF-4, in conjunction with to promote hF9 to transcribe.In all elements, with No. 5 sites
The transcription facilitation of regulating and controlling sequence (- 219 to -199) is most strong.Although only carrying the reporter plasmid of hF9 proximal promoter
Expression is not high, but after it joined No. 5 site sequences of several copies, transcriptional efficiency can with contain HCR tune
Control the plasmid roughly the same 32 of sequence.These data are to have obtained conclusion in cell line test in vitro at present, are not yet carried out
In vivo studies verifying.Therefore, in order to obtain the plasma thromboplastin component of physiological expression, it is worth attempting to apply these physiological tune
It controls sequence construct and expresses frame, in the gene therapy vector of liver cell internal specific expression hFIX.
The above through the encoding gene of liver cell specificity synthetic proteins have the enhancer sequence that can be used for reference with
The high controlling element of transcriptional efficiency is screened or synthesized to mediate liver cell specificity for the expression frame in gene therapy
Transcriptional expression.
The ITR sequence of mutation
The present invention provides a kind of ITR sequence (inverted terminal repeat sequence) of mutation, the ITR sequence of the mutation contains
Base sequence complementary between 106 bases, and the 25th to the 130th of 3 ' end wild type ITR sequence SEQ ID NO.:5
Or it is identical.
Specifically, wild type ITR structure contains 145 base-pairs, plays a role in virus replication and mediates to form list
The AAV virus of chain DNA core.The present invention adjusts wild type ITR structure, at the both ends of 3 ' end wild type ITR sequences
After each removal number of base sequence, end termination site therein (terminal resolution site, TRS) is deleted,
And the complementary series of this ITR structure is implemented in 5 ' ends of expression vector, to promote to be formed in AAV reproduction process complementary double
Chain structure promotes transcriptional expression.The ITR structure of the transformations such as this structure ratio Nathwani further reduces several base-pairs, makes
It still can form complementary double-stranded viruses DNA.In summary structure, adjustment and construct AAV carrier bioactivity and peace
Full property has carried out system evaluation in HB Mice Body.
Human coagulation factor IX gene
Hemophilia B (Hemophilia B, HB) belongs to monogenic inheritance disease, is people's blood coagulation on chromosome x q27.1
Sex chromosome linkage inheritance hemorrhagic disease caused by factors IX encoding gene (hF9) defect, the disease incidence in global male
About 1/25000.Normal people F9 full length gene about 34kb, wherein coded sequence cDNA length is 1383bp, and it is long to be located at X chromosome
The arm end region Xq27.1-q27.2 includes 8 exons and 7 intrones.People's F9 gene defect leads to FIX albumen in blood plasma
Missing or dysfunction are to lead to the basic cause of disease of hemophilia B.Through 3721 HB patients of population analysis and its family member, in total
Identification diagnosis goes out 1113 species specificity mutation (Fig. 1).In these mutation, about 73% is point mutation, in single or multiple extensive
Ground is distributed in exon (700/812).About 50% mutation causes hFIX protein expression obstacle or functional defect, to induce
Serious clinical phenotypes (table 1).
The correlation analysis of table 1. hemophilia B patient mutations type and disease severity
The haemophiliachemophiliac disease incidence of China is 5-10/10 ten thousand at present, and about 15% belongs to hemophilia B patient, and patient with severe symptoms accounts for it
Middle half or so.With current medical economics situation, most severe HB patients can not be undertaken needed for preventative replacement therapy
Expensive expense.
Strategies in gene therapy imports normal gene by gland relevant viral vector AAV to be had in expression of liver cell coagulation factor
There is the potential cured for a long time, tentatively achieves certain Clinical efficacy and safety result in external clinical test at present.So
And there is also certain Optimal improvements spaces for bio-carrier used in gene therapy.Most of all, the country there is no tool at present
There is the gene therapy product of similar intellectual property.Therefore, the present invention combines international forward position progress, has carried out gene therapy hemophilia
The research of B, exploitation optimizes the AAV carrier for Gene Therapy for Hemophilia B, and confirms in the animal model of preclinical phase
Its biological activity and safety, to provide solid foundation for its clinic conversion.The exploitation of this carrier is certain
China is filled up in degree at present in the blank in gene therapy hemophilia B field, clinical application will include blood for gene therapy
Genetic disease including friendly disease establishes solid theory and practice basis.
In addition, being used to mediate the AAV carrier for expressing Disease-causing gene in liver cell and liver therein special in the present invention
Specific regulatory sequences have versatility.For other kinds of single-gene defective genetic disease, if dcc gene size
Suitable for AAV carrier package capacity, liver specific expression can be carried out by this carrier.Therefore, the AAV involved in the present invention
Carrier and controlling element theoretically have very extensive applicable prospect in field of gene, are the more genes of future development
Therapeutic strategy provides advantageous research tool.
Gland relevant viral vector
AAV Carrier diameters about 20-25nm, wild type AAV virus are made of albumen involucrum and nucleic acid core, may include big
The DNA fragmentation (Fig. 2A) of small about 4.7kb.Engineered restrovirus itself encoding gene can replaced being treated property gene, thus
Constitute recombination AAV viral vectors (Fig. 2 B).The AAV carrier involucrum albumen that natural separation is identified from humans and animals tissue so far
(capsid protein) has more than 120.Tissue affinity testing result shows that different AAV carriers can be a variety of groups to human body
Knit it is affinity, at the same time it is same tissue can also by the AAV carrier of different serotypes infect (table 2).For blood friend
Ospc gene treats main target organs liver (Fig. 3), and AAV2, AAV7, AAV8, AAV9 and AAVrh10 have higher parent
And property, wherein the efficiency of infection highest of AAV8 and AAVrh10, therefore it is applied to the clinical examination of hemophilia gene treatment
It tests.
The thermophilic parent's property of tissue of 2. different serotypes AAV of table
Carrier of the invention
The present invention also relates to the carriers comprising pSyn regulating and controlling sequence of the invention, and utilize carrier or pSyn of the invention
The genetically engineered host cell of regulating and controlling sequence.
Term " carrier " refers to that bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, lactation are dynamic
Object cell virus or other carriers.As long as any plasmid and carrier can be used in short, can replicate and stablize in host.
For carrier, replication orgin, promoter, marker gene and translation control element are usually contained.Preferably, carrier of the present invention
For AAV carrier.
It can use method well known to those skilled in the art and construct carrier of the invention.These methods include vitro recombination
DNA technique, DNA synthetic technology, In vivo recombination technology etc..The DNA sequence dna can be effectively connected to suitable in carrier of the present invention
In promoter, to instruct mRNA to synthesize.Carrier of the present invention further includes the ribosome bind site of translation initiation and transcribes eventually
It is only sub.
In addition, carrier of the present invention preferably includes one or more selected markers, to provide for selecting conversion
Host cell phenotypic character, such as the dihyrofolate reductase, neomycin resistance and green fluorescence of eukaryotic culture
Albumen (GFP), or tetracycline or amicillin resistance for Escherichia coli.
Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable
When host cell, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, such as plant cell (cells of such as crops and forestry plant).Representative example has: Escherichia coli, streptomycete
Belong to, Agrobacterium；Fungal cell's such as yeast；Plant cell, zooblast etc..Preferably, host cell of the invention behaviour or non-
People's mammalian cell, preferably liver cell
Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.
AAV carrier of the invention
In the strategy of gene therapy hemophilia B, AAV carrier has critical transmitting translocation.Express base of curing the disease
The sequence of cause needs to transport in body blood circulation by AAV carrier and specific localization is in specific target organ.
The AAV carrier of gene therapy hemophilia B mainly includes to consist of two parts, and: a. cures the disease gene expression construct；B. there is mutation ITR
The AAV structure sequence (Fig. 4) of structure.The present invention, which reaches to improve to have in gene therapy by two component parts of optimization, to be controlled
The expression of the normal coagulation function hFIX of the property treated effect.Specifically, the liver of gene therapy hemophilia B of the invention is special
Property expression gland relevant viral vector (AAV carrier) include:
1. liver specificity transcription regulating nucleotide sequence, pSynthetic (abbreviation pSyn)；
2. the Human factor IX coded sequence of sequence optimisation；
3. optimizing the ITR structure (the ITR sequence of mutation) of improvement.
Main advantages of the present invention include:
(a) in liver organization specific efficient driving downstream gene transcriptional expression；
(b) there is the activity that downstream gene expression is driven in liver organization steady in a long-term；
(c) there is preferable safety, do not expressed substantially in non-liver organization, do not interfere the table of other non-genes of curing the disease
Up to level.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
Embodiment 1
Sequence optimisation people's F9 encoding gene
The expression frame of people FIX includes the regulating and controlling sequences such as hFIX cDNA coded sequence and enhancers/promoters (figure
4A).Firstly, having carried out codon optimization to hFIX coded sequence.The principle of codon optimization is by assessing different codons
The difference of frequency of use, by the higher codon of expression frequency is replaced relatively in eukaryocyte corresponding to the same amino acid
The lower codon of frequency of use, to enhance the transcriptional expression efficiency of gene.
In addition, carrying out sequence optimisation to hFIX coded sequence reduces its quilt to improve the degree of stability of mRNA molecular structure
The risk of endogenous RNase degradation.Sequence optimisation mainly utilizes OptimumGeneTM analysis to complete.The parameter packet wherein optimized
It includes: (1) genetic transcription efficiency regulation parameter, including increase Preference amino acid codes, reduce or delete utilization rate and is lowly close
The use of numeral；(2) G/C content is adjusted, being applicable in section is 30%~70%；(3) the mRNA secondary structure of hFIX is modified, including
Reduce or delete unstable structures, repetitive sequence and the applicable terminator codons such as implicit shearing site, ring-type or branch
TGA etc. (Fig. 4 B).
In order to verify whether optimisation strategy of the invention can effectively improve the expression efficiency of hF9 gene, same
Optimized hFIX sequence is constructed under the driving of transcriptional regulatory element, by Hydrodynamic injection hemophilia B (HB) mouse,
The plasmid for carrying expression frame is imported in mouse liver cell using physical pressure.
As a result as shown in Figure 5A, compared with wild type cDNA, codon optimizing strategy of the invention makes the hFIX in blood plasma
(cohFIX) antigenic expression improves about 3 times, it is this it is horizontal with obvious clinical efficacy is achieved in clinical test
HFIX optimization (hFIX.ctrl) is almost the same.
Then, liver organization of the cohFIX expression frame by AAV8 carrier transport to HB mouse will be carried, blood plasma is regular
Detection as a result, it has been found that, cohFIX optimization (AAV.cohFIX) of the invention can in mouse liver cell stability and high efficiency
Expression antigen and blood coagulation activity are carried out, transcriptional efficiency and positive control (AAV.hFIXctrl) are almost the same, and result is effective
The bleeding phenotype (Fig. 5 B) for alleviating gene therapy mouse.These results advantageously demonstrate sequence optimisation strategy of the invention
The transcriptional efficiency of hF9 gene, therefore the expression vector suitable for gene therapy can effectively be improved.
In addition to effectively optimizing cDNA sequence, the present invention also introduces a point mutation in encoding gene, to make
The 338th arginine replaces with leucine (hFIX.SIHR338L) in hFIX albumen.This mutation is originally found anticipates in one
Big benefit family, when the 338th in hFIX arginine is replaced by leucine, although as a result leading to FIX albumen in patient's blood circulation
It is horizontal suitable with normal person, but blood coagulation activity reaches 5-8 times of normal person.It has been investigated that the site R338 be Stuart factor with
The significant points that FIX is combined, can be enhanced joint efficiency between the two, to be remarkably reinforced FIX's after being replaced by leucine
Blood coagulation activity.In addition, carrying the anti-fibrinolytic effect of the FIX albumen of this mutation also enhances.Therefore, this mutational site is introduced
CohFIX sequence is expressed in HB mouse liver cell through AAV carrier, and the expression of hFIXR338L mutant can have as the result is shown
The dosage of the reduction AAV preparation of effect, in low dosage 1 × 1011It is normal that~100% can be generated on the basis of vg/kg in Mice Body
The coagulation factor activity and antigen of human plasma level reduce several times (Fig. 6) compared with wild type AAV carrier.
Embodiment 2
It constructs and screens liver specificity regulating and controlling sequence
In order to improve the curative effect of AAV vector-mediated gene treatment, other than being optimized to hF9 gene, also transcription is adjusted
Control element has carried out screening and optimizing.In order to obtain the transcriptional control of full blast, different regulating and controlling sequences is combined, and with
The pLP1 regulating and controlling sequence that AAV carrier carries in the clinical tests such as Nathwani has carried out parallel comparison.
1. artificial synthesized pSynthetic (pSyn) regulating and controlling sequence, combines the regulating and controlling sequence of TTR and Alb, includes
The binding site of multiple liver cell idiosyncratic transcription factors such as HNF-1, HNF-3, HNF-4 and C/EBP.
2. Human factor IX regulating and controlling sequence (pF9) has the two different versions of length.Wherein pF9.v1 contains F9 base
Because of the sequence between -219 to+29 base-pairs, and pF9.v2 also introduces the enhancer sequence of 5 copies, and the sequence is in vitro
The transcriptional efficiency of pF9 proximal promoter is effectively raised in cell line test.
In addition to this, also the ITR structure of carrier is adjusted.Control type ITR structure (ITRControl) mediate formed it is single-stranded
The AAV carrier (Fig. 7, sample F) of DNA core, Nathwani etc. delete the ITR (ITR of AAV carrier sidem1) in nearly more than 20
Base-pair promotes transcriptional efficiency (Fig. 7, sample to form complementary double-stranded DNA structure during AAV is packed and produced
A).Further several base-pair (ITR have been deleted in transformation on this basism2), but retain it and form complementary double-stranded viruses DNA's
Ability (Fig. 7, sample B-E).Also it joined one section of artificial synthesized intron sequences in the expression frame of hFIX, and two
End adds splicing donor and splicing acceptor, to not influence coded sequence.In summary structural adjustment and
The bioactivity of the AAV carrier of building and safety have carried out system evaluation in HB Mice Body.
In order to relatively determine whether the liver specificity controlling element of design construction of the present invention has the transcription of efficient stable
Drive efficiency constructs the hFIX expression frame comprising different controlling elements and is packaged into AAV8 carrier.These carriers pass through
Enter in HB Mice Body after tail vein injection, and mediates liver specific expression's hFIX albumen.
As a result it as shown in figure 8, by determination of activity and elisa assay, is shown in nearly tracing study in four months, injects
HFIX antigen and activity level highest in the HB mouse group blood plasma for the AAV carrier for including pSyn controlling element.Regulate and control with LP1
Element group compares, and the albumen of hFIX and activity level are relatively weak in the HB mice plasma of pTTR and pF9 group, prompts its transcription
Regulate and control relatively inefficient.
Embodiment 3
The internal dosage of pSyn regulating and controlling sequence increases test
According to the above results show that pSyn regulating and controlling sequence has relatively stronger liver specificity transcriptional activity, therefore utilization packet
The AAV.pSyn.cohFIX carrier of the regulating and controlling sequence containing pSyn has carried out internal dosage and has increased test, wherein high, medium and low concentration group
The carrier dosage of injection is thought with the dosage applied in the clinical test of the designs such as Nathwani corresponding respectively.
The results show that the hFIX antigen and activity in blood plasma begin to increase from injection carrier latter week, reached to 4th week
Metastable level is maintained to peak, and in 4 months observation periods.In addition, the coagulation factor activity in blood plasma
With antigen levels with the directly proportional rising of increasing of dosage, the activity and concentration of hFIX can reach in the mice plasma of middle dose group
To normal person it is horizontal 100% or so, and high dose group normal person it is horizontal 600~900% between (Fig. 9).These
As a result demonstrating AAV.pSyn.cohFIX carrier effectively in the stable expression of HB vivo long-term and can play its blood coagulation
Effect.
Embodiment 4
The tissue specificity of pSyn regulating and controlling sequence is tested
For the tissue specificity of the carrier mediated expression hFIX of clear AAV, part HB mouse is at three, injecting virus carrier
It is extracted histoorgan after month to be analyzed, the RNA in liver, spleen and thymic tissue is extracted and analyzes hFIX phase
It is horizontal to the rna expression of reference gene GAPDH.
The results are shown in Figure 10, and the RNA of Human factor IX is mainly expressed in liver organization, and expression with
The raising of carrier dosage and increase.Compared with liver organization, hFIX rna expression level is in baseline level in spleen and thymus gland,
Rna level in respectively organizing with the HB mouse for having injected AAV.GFP carrier is consistent.These results illustrate that AAV.pSyn.hFIX is carried
The rna expression that body mediates has liver organization specificity.
Embodiment 5
The biological distribution of AAV.pSyn.cohFIX carrier
Then, AAV carrier is analyzed in the intracorporal biological distribution of HB mouse.According to previously reported as a result, AAV8 is carried
Body should be mainly affinity to mouse liver tissue.
As a result as shown in figure 11, liver, spleen, thymus gland and myeloid tissue through analysis detection gene therapy mouse, viral DNA
It is mainly distributed in liver organization, and distributed density and carrier dose proportional.Furthermore also there is minimal amount of disease in spleen
Malicious DNA distribution, in mean concentration about 0.23 copy/cell of high dose group, in other dosage groups and its hetero-organization with
Baseline level in AAV.GFP group HB mouse is almost consistent.In conjunction with these data and rna expression level analysis as a result,
Illustrate that the gene delivery strategies of AAV8 combination liver specificity regulating and controlling sequence can effectively guarantee that purpose cures the disease gene in liver
Specific expressed in tissue, there is the AAV.pSyn.cohFIX carrier in the application control to express in liver internal specific
HFIX is to treat the purpose of hemophilia B.
Embodiment 6
Liver function index measurement
Show that the carrier mediated gene therapy of AAV can induce organism immune response and slight in the clinical test of the past
Liver damage, along with the rising of liver function index.It compares before AAV vector injection and the 4th, 12 week blood plasma after injection
The level of glutamic-oxalacetic transaminease (AST) and glutamic-pyruvic transaminase (ALT).
As a result as shown in figure 12, (Week 0) is compared with the level in the HB mice plasma of non-receptor gene treatment, infused
AST the or ALT level after viral vectors in 4 weeks or 12 weeks mice plasmas is penetrated all to increase without conspicuousness.This is also and in blood plasma
The hFIX level for stablizing expression is corresponding, illustrates that AAV carrier and its expression product do not induce apparent liver under these dosage
Functional lesion.
Embodiment 7
The high blood coagulation activity marker measurement of mouse
The excessive extrinsic coagulation factor can induce the high coagulation of body.In order to detect various dose AAV carrier
Whether the hFIX of expression can generate high blood coagulation risk, have detected the D dimer concentration in blood plasma.
As a result it is compared as shown in figure 13 with horizontal and AAV.GFP control group level before gene therapy, under various dose
AAV carrier expression coagulation factor does not cause excessive Coagulation test, it was demonstrated that is not present in HB Mice Body under these dosage
High blood coagulation risk.
In summary data, the AAV.pSyn carrier that the present invention constructs sufficiently have the specific expressed mesh in liver organization
Gene of curing the disease function.In hemophilia gene treatment, plasma thromboplastin component can be expressed steadily in the long term under pSyn driving,
To restore the normal coagulation function of mouse, achievees the purpose that long-term or even permanently cure.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Ruijin Hospital, Shanghai Jiao Tong University School of Medicine
<120>liver specificity transcription regulating nucleotide sequence and its application
<130> P2017-1550
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 447
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agctttctga acagccaaac agagattcca aagttcaggc accaaagttc agaccctaac 60
agttatttac aagggtcagt tcctgaattt gtcaattagt aacaattgta ttcaacagta 120
aggattttat gtttgggtag gttagtttgg ttagtaatta ctaaacaccc tgaatttgtc 180
aattagtaac aattgtattc aacagtaagg attttatgtt tgggtaggat cctattaaga 240
atatttcata gaacgaatgt tccgatgctc taatctctct aggcaaggtt catatttgta 300
tgggttactt attctctctt tgttgactaa gtcaataata caaagatgag tctagttaat 360
aatctacaat tattggttaa agaagtatat tagtgctaat ttccctccgt ttgtcctagc 420
ttttctcttc tgtcaacccc acacgcc 447
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ggctacctgc tgtccgccga gtgtaccgtg ttcctggacc atgagaacgc aaataagatc 120
ctgaaccggc ccaaaagata taatagtgga aagctggagg aatttgtgca gggcaacctg 180
gagagagaat gcatggagga aaagtgtagc ttcgaggaag cccgcgaggt gtttgaaaat 240
accgagcgaa ccacagagtt ctggaagcag tatgtggacg gcgatcagtg cgagagcaac 300
ccctgtctga atggcgggag ttgcaaagac gatatcaact catacgaatg ctggtgtcct 360
ttcggatttg aaggcaaaaa ttgcgagctg gacgtgacct gtaacattaa gaatgggagg 420
tgcgagcagt tttgtaaaaa ctctgccgat aataaggtgg tctgcagttg tacagaaggg 480
tatcgcctgg ccgagaacca gaagtcctgc gaaccagctg tgcccttccc ttgtggacgg 540
gtgagcgtct cccagacttc aaaactgacc agagctgagg cagtgtttcc cgacgtggat 600
tacgtcaaca gcacagaggc tgaaactatc ctggacaaca ttactcagtc tacccagagt 660
ttcaatgact ttacccgggt ggtcggaggc gaggatgcta aaccaggcca gttcccctgg 720
caggtggtcc tgaacgggaa ggtggatgca ttttgcgggg gatctatcgt gaatgagaaa 780
tggattgtca ccgccgctca ctgcgtggaa actggggtca agatcaccgt ggtcgccgga 840
gagcacaaca ttgaggaaac agaacatact gagcagaaga ggaatgtgat ccgcatcatt 900
cctcaccata actacaatgc agccatcaac aaatataatc atgacattgc cctgctggaa 960
ctggatgagc ctctggtgct gaacagctac gtcacaccaa tctgcattgc tgacaaggag 1020
tacactaaca tcttcctgaa gttcgggtca ggatacgtga gcggctgggg gagagtcttc 1080
cacaagggca ggtccgcact ggtgctgcag tatctgcgag tgcctctggt cgatcgagcc 1140
acttgtctgc ggtctaccaa gttcaccatc tacaacaata tgttctgcgc tggatttcac 1200
gagggaggac gagactcctg tcagggcgat tctggaggcc cacatgtgac agaggtcgaa 1260
ggcaccagct tcctgacagg catcatttcc tggggggagg aatgcgcaat gaaggggaaa 1320
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acctaa 1386
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ctgaaccggc ccaaaagata taatagtgga aagctggagg aatttgtgca gggcaacctg 180
gagagagaat gcatggagga aaagtgtagc ttcgaggaag cccgcgaggt gtttgaaaat 240
accgagcgaa ccacagagtt ctggaagcag tatgtggacg gcgatcagtg cgagagcaac 300
ccctgtctga atggcgggag ttgcaaagac gatatcaact catacgaatg ctggtgtcct 360
ttcggatttg aaggcaaaaa ttgcgagctg gacgtgacct gtaacattaa gaatgggagg 420
tgcgagcagt tttgtaaaaa ctctgccgat aataaggtgg tctgcagttg tacagaaggg 480
tatcgcctgg ccgagaacca gaagtcctgc gaaccagctg tgcccttccc ttgtggacgg 540
gtgagcgtct cccagacttc aaaactgacc agagctgagg cagtgtttcc cgacgtggat 600
tacgtcaaca gcacagaggc tgaaactatc ctggacaaca ttactcagtc tacccagagt 660
ttcaatgact ttacccgggt ggtcggaggc gaggatgcta aaccaggcca gttcccctgg 720
caggtggtcc tgaacgggaa ggtggatgca ttttgcgggg gatctatcgt gaatgagaaa 780
tggattgtca ccgccgctca ctgcgtggaa actggggtca agatcaccgt ggtcgccgga 840
gagcacaaca ttgaggaaac agaacatact gagcagaaga ggaatgtgat ccgcatcatt 900
cctcaccata actacaatgc agccatcaac aaatataatc atgacattgc cctgctggaa 960
ctggatgagc ctctggtgct gaacagctac gtcacaccaa tctgcattgc tgacaaggag 1020
tacactaaca tcttcctgaa gttcgggtca ggatacgtga gcggctgggg gagagtcttc 1080
cacaagggca ggtccgcact ggtgctgcag tatctgcgag tgcctctggt cgatcgagcc 1140
acttgtctgc tatctaccaa gttcaccatc tacaacaata tgttctgcgc tggatttcac 1200
gagggaggac gagactcctg tcagggcgat tctggaggcc cacatgtgac agaggtcgaa 1260
ggcaccagct tcctgacagg catcatttcc tggggggagg aatgcgcaat gaaggggaaa 1320
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acctaa 1386
<210> 4
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ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgg 106
<210> 5
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<213>artificial sequence (Artificial Sequence)
<400> 5
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60
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gagcgcgcag agagggagtg gccaa 145
Claims (16)
1. a kind of isolated liver specificity transcription regulating nucleotide sequence, which is characterized in that the regulating and controlling sequence includes albumin tune
It controls sequence and transthyretin regulating and controlling sequence, the albumin regulating and controlling sequence includes transcription factor HNF-1 and C/EBP
Binding site, the transthyretin regulating and controlling sequence include transcription factor HNF-1, HNF-3, HNF-4 and C/EBP
Binding site；
The liver specificity transcription regulating nucleotide sequence is the polynucleotides as shown in SEQ ID NO.:1.
2. a kind of nucleic acid constructs, which is characterized in that the construction contains foreign gene or exogenous dna fragment, Yi Jiyu
The regulating and controlling sequence described in claim 1 that the foreign gene or DNA fragmentation is operatively connected.
3. construction as claimed in claim 2, which is characterized in that the foreign gene is selected from the group: therapeutic gene, screening
Marker gene, resistant gene, antigenic protein gene, RNAi gene, microRNA gene, or combinations thereof.
4. construction as claimed in claim 3, which is characterized in that the therapeutic gene is selected from the group: Human factor IX
Gene, human blood coagulation factor VII I, people's alpha1-antitrypsin, people's aryl sulfatase B (ARSB) gene, human ldl
Receptor (LDLR) gene, or combinations thereof.
5. construction as claimed in claim 4, which is characterized in that the clotting factor IX gene include wild type blood coagulation because
Sub- IX gene and saltant type clotting factor IX gene.
6. construction as claimed in claim 5, which is characterized in that the saltant type clotting factor IX gene includes (i) close
The clotting factor IX gene of numeral optimization, and (ii) point mutation clotting factor IX gene, wherein the point mutation coagulation factor
The polypeptide of IX gene coding sports leucine in the 338th arginine corresponding to wildtype factor IX.
7. construction as claimed in claim 6, which is characterized in that the sequence of the clotting factor IX gene of the codon optimization
As shown in SEQ ID NO.:2.
8. construction as claimed in claim 6, which is characterized in that the sequence of the point mutation clotting factor IX gene is such as
Shown in SEQ ID NO.:3.
9. a kind of carrier, which is characterized in that the carrier contains construction as claimed in claim 2.
10. carrier as claimed in claim 9, which is characterized in that the carrier is slow virus carrier, adeno-associated virus load
Body.
11. carrier as claimed in claim 10, which is characterized in that the carrier is AAV8 carrier.
12. a kind of host cell, which is characterized in that contain carrier as claimed in claim 9 or dyeing in the host cell
The regulating and controlling sequence described in claim 1 or construction as claimed in claim 2 of external source are integrated in body.
13. host cell as claimed in claim 12, which is characterized in that the cell is behaved or nonhuman mammalian cells.
14. regulating and controlling sequence described in claim 1, construction as claimed in claim 2 or carrier described in any one of claim 10
Purposes, which is characterized in that be used to prepare a preparation or composition, the preparation or composition are used to foreign gene or outer
Source DNA segment carries out specific expressed in liver cell or liver organization.
15. the purposes of regulating and controlling sequence described in claim 1, which is characterized in that for constructing an expression vector, the expression
Carrier is used for the specific expressed foreign gene in liver cell or liver organization.
16. a kind of pharmaceutical composition, which is characterized in that the pharmaceutical composition contains carrier and medicine described in any one of claim 10
Acceptable excipient on.
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| CN102628041B (en) * | 2008-02-14 | 2013-09-11 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expressing coded sequence used for gene therapy |
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