CN108239645A - Liver specificity transcription regulating nucleotide sequence and its application - Google Patents
Liver specificity transcription regulating nucleotide sequence and its application Download PDFInfo
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Abstract
The present invention relates to a kind of liver specificity transcription regulating nucleotide sequences.Specifically, the present invention provides a kind of specific expressed regulating and controlling sequences that can drive enhancing downstream gene in liver cell, the regulating and controlling sequence includes the regulating and controlling sequence of albumin and transthyretin, it can be used for specific express therapeutic gene in liver cell, such as clotting factor IX gene, all have very important significance for the strategies in gene therapy through liver cell expression.
Description
Technical field
The present invention relates to genetic engineering fields, relate more specifically to a kind of liver specificity transcription regulating nucleotide sequence and its answer
With.
Background technology
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 incidence in global male
About 1/25000.Normal people F9 full length gene about 34kb, wherein coded sequence cDNA length are 1383bp, long positioned at X chromosome
Arm end Xq27.1-q27.2 regions include 8 extrons and 7 intrones.People's F9 gene defects lead to FIX albumen in blood plasma
Missing or dysfunction are to lead to the basic cause of disease of hemophilia B.At present, the primary treatment measure of haemophiliac is to be substituted
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, so as to achieve the purpose that cure all the life.With reference to the basic research that current hemophilia gene is treated and face
Bed experiment progress, the relatively ripe technology for most curing potentiality 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 carriers
The significant curative effect that is obtained in 10 severe HB patients of mediation gene therapy, some patientss clinical symptoms are relieved, interrupt or
Reduce the use of hFIX factor substitutes.Nevertheless, the hFIX levels in Most patients body 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
Should, but relative to>30% thorough criterion of cure also has certain distance.The main reason for effect is limited in this clinical test
For：
(1) transcriptional efficiency of the expression frame in AAV carriers can also further improve；
(2) production technology of clinical test preparation needs to be further improved, 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 carriers 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.
Invention content
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 adeno-associated virus load comprising liver specificity transcription regulating nucleotide sequence
Body, the gland relevant viral vector can be used for haemophiliachemophiliac gene therapy.
In the first aspect of the present invention, a kind of liver specificity transcription regulating nucleotide sequence of separation 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 appoints polynucleotides selected from the group below：
(a) nucleotide sequence such as SEQ ID NO.:Polynucleotides shown in 1；
(b) nucleotide sequence and SEQ ID NO.:Homology >=90% of sequence shown in 1 is (preferably >=95%, more preferably
>=98%, most preferably >=99%), and the polynucleotides with liver specificity transcriptional activity；
(c) such as SEQ ID NO.:5 ' the ends and/or 3 ' ends of polynucleotides shown in 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 drive downstream DNA segment in liver cell
The activity of middle specific transcriptional.
In another preferred example, the transcription that " specific transcriptional in liver cell " refers in the liver cell is lived
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 SEQ ID NO.:Polynucleotides shown in 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 genes, microRNA genes, or combination.
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 combination.
In another preferred example, the clotting factor IX gene includes wildtype factor IX genes and saltant type is coagulated
Blood factor IX genes.
In another preferred example, the saltant type clotting factor IX gene includes the coagulation factor of (i) codon optimization
IX genes and (ii) point mutation clotting factor IX gene, wherein the polypeptide of the point mutation clotting factor IX gene coding exists
Leucine is sported corresponding to the 338th arginine of wildtype factor IX.
In another preferred example, the sequence of the clotting factor IX gene of the codon optimization such as SEQ ID NO.:2 institutes
Show.
In another preferred example, the sequence of the point mutation clotting factor IX gene such as SEQ ID NO.:Shown in 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 carriers, it is therefore preferable to AAV8 carriers.
In another preferred example, the carrier is AAV8 carriers, and the carrier (side) has the present invention the
The ITR sequences 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
The regulating and controlling sequence or the present invention second being integrated in carrier or chromosome described in aspect described in the first aspect present invention of external source
Nucleic acid constructs described in aspect.
In another preferred example, the cell is the cell of separation 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 present 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 used to 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 the regulating and controlling sequence described in first aspect present invention is provided, feature exists
In for building 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 the 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 sequences (inverted terminal repeat sequence) of mutation, the mutation are provided
ITR sequences contain 106 bases, with 3 ' end wild type ITR sequences (SEQ ID NO.:5) between the 25th to the 130th
Base sequence it is identical or complementary.
In another preferred example, the ITR sequences of the mutation delete wild type ITR sequences correspond to SEQ ID
NO.:5 1-24 (5 ' ends) and 131-145 (3 ' ends).
In another preferred example, the ITR sequences of the mutation such as SEQ ID NO.:Shown in 4.
In another preferred example, the ITR sequences of the mutation are and SEQ ID NO.:4 complementary base sequences.
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 sequences 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 carriers, it is therefore preferable to AAV8 carriers.
In another preferred example, 5 ' ITR sequences of the AAV carriers are the mutation described in eighth aspect present invention
ITR sequences.
In another preferred example, 3 ' ITR sequences of the AAV carriers are the mutation described in eighth aspect present invention
ITR sequences.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to form new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Fig. 1 show through population analysis in hemophilia B patient 1113 kinds of gene mutation types of antidiastole and this
A little mutation are in the distribution of human blood coagulation 9 (hF9) gene extron, introne and UTR region.Wherein, each code is containing as follows：E,
Extron；Int, introne；UTR, upstream regulatory region.
Fig. 2 shows wild type AAV virus and AAV carrier schematic diagrames.
Fig. 2A show wild type AAV carriers be a diameter of 20-25nm without coating DNA virus, include and be about 4.7kb's
Single stranded DNA genome.AAV genomes have two groups of encoding genes, albumen needed for rep gene codes virus replication and assembling, cap bases
Because 3 kinds of albumen of coding are to form the virus encapsidation of 60-mer.The coat protein of AAV carriers and the consistent of wild type AAV virus or
It is very similar, include the treatment correlated expression gene with transcription driving element.Have 74% in the molecular wt of AAV carriers by egg
White composition, the carrying amount of maximum DNA is 5kb.
Fig. 2 B show that wild type AAV virus is engineered to recombinate AAV carriers, wherein itself viral encoding gene is treated
Property gene substitution.
Fig. 3 shows the carrier mediated selectively targeted liver cell generation coagulation factor gene treatment hemophilia B signals of AAV
Figure.
Fig. 4 shows that AAV carrier structures figure and the optimization of hFIX coded sequences are front and rear relatively.
Fig. 4 A show AAV carrier structure schematic diagrames.
Fig. 4 B show that the optimization of people FIX coded sequences is front and rear relatively, including Optimal Parameters (left side), G/C content (in) and
CDNA secondary structures (right side).
Fig. 5 shows that sequence optimisation people FIX cDNA help to improve activity and albumen water of the hFIX in HB Mice Bodies
It is flat.
Fig. 5 A show that ELISA method measures the result of hFIX antigen levels.Carry wild type hFIX, Optimization-type hFIX
(cohFIX) or the DNA plasmid of Optimization-type hFIX positive controls (+ve ctrl) expression frame enters blood through high-pressure injection mode
Friendly disease B mouse (HB) are internal.Mice plasma acquired after 48 hours and measures hFIX antigen levels by ELISA method.As a result
It is represented with mean ± standard deviation.
Fig. 5 B and Fig. 5 C respectively illustrate ELISA method and measure hFIX activity and the result of antigen levels.GFP is carried, it is excellent
The AAV8 carriers of change type hFIX (cohFIX) or Optimization-type hFIX positive controls (+ve ctrl) expression frame are noted 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 helps effectively to reduce the biological dose of AAV carriers.
The AAV8 carriers of expression hFIXR338L mutant enter 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 are measured respectively by Chromogenic assay and ELISA method,
As a result it is expressed as mean ± standard deviation.
Fig. 7 shows gel electrophoresis analysis double-strand AAV carriers and single-stranded AAV carriers result.Wherein ITRm1It is to contain
ITR structures, ITR in the carrier that Nathwani etc. is deliveredm2The ITR mutation structures being transformed in this project are included, both mediate shape
Into the carrier (scAAV) containing double-stranded DNA core.ITRControlControl type ITR structures are included, are packaged to form core containing single stranded DNA
Carrier (ssAAV).AAV carriers 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, so as to all show as the molecular weight of single stranded DNA.
Fig. 8 shows that liver specificity controlling element activity compares in HB Mice Bodies.Carry different liver specificity tune
The AAV carriers of the cohFIX expression frames of control sequence enter 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 respectively by Chromogenic assay and
ELISA method is measured, and is as a result expressed as mean ± standard deviation.
Fig. 9 shows that the AAV8 carriers for carrying pSyn.cohFIX expression frames carry out dosage in HB Mice Bodies and increase examination
It tests.AAV carriers are 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 Bodies (n=3~6).AAV.GFP carriers (2 × 1012/ kg/ is only) it is used as the moon
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 that AAV carrier dosage increases in experiment, the RNA tables in each group HB mouse livers, spleen and thymic tissue
Up to level relatively.The 12nd week after various dose AAV carriers have been injected, each group mouse has 2-3 only to acquire liver, spleen and thymus gland group
Knit extraction RNA, and reverse transcription generation cDNA.Pass through the cDNA water of real-time PCR semiquantitative determinations 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 numbers (2-Δ(hFIX-GAPDH)).As a result individual mouse data and mean ± standard deviation are shown as.
Figure 11 shows that AAV carriers increase the biological distribution in experiment in each group mouse tissue internal organs in dosage.Dosage
Increase in experiment each group mouse 2-3 only 12 weeks acquisition livers, spleen and thymic tissue after injection of AAV vectors extract RNA livers, spleen and
DNA in thymic tissue passes through concentration of the real-time PCR semiquantitative determination AAV carrier DNAs in each histocyte.Knot
Fruit is shown as mean ± standard deviation.
Figure 12 shows that dosage increases each group mouse liver function index in experiment and measures.Each group mouse 2-3 is only in injection of AAV
The blood plasma of acquisition in the 4th and 12 week measures the concentration of AST and ALT by Chromogenic assay before carrier and after injection, is as a result shown as
Mean ± standard deviation.
Figure 13 shows that dosage increases the high blood coagulation activity marker of each group mouse in experiment and measures.Each group mouse 2-3 is only being noted
It penetrates before AAV carriers and the blood plasma of acquisition in the 4th and 12 week detects the concentration of D dimer by ELISA method after injection, as a result show
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 for the first time by depth studying extensively
Row 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 efficient (
Through being verified in factor Ⅸ deficient mouse model).The regulating and controlling sequence of the present invention can be additionally 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 sequences
As used herein, term " regulating and controlling sequence ", " transcription regulating nucleotide sequence ", " pSyn regulating and controlling sequences " 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 present invention is included from white egg
In vain, the transcription regulating nucleotide sequence of transthyretin.Preferably, the regulating and controlling sequence is appoints polynucleotides selected from the group below：
(a) nucleotide sequence such as SEQ ID NO.:Polynucleotides shown in 1；
(b) nucleotide sequence and SEQ ID NO.:Homology >=90% of sequence shown in 1 is (preferably >=95%, more preferably
>=98%, most preferably >=99%), and the polynucleotides with liver specificity transcriptional activity；
(c) such as SEQ ID NO.:5 ' the ends and/or 3 ' ends of polynucleotides shown in 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 sequences 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 sequences can be additionally 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 SEQ ID NO.:The variant of regulating and controlling sequence shown in 1, the variant can
By being inserted into or deleting regulatory region, to carry out random or rite-directed mutagenesis etc. to obtain.
The invention also includes with the present invention regulating and controlling sequence have 50% or more (preferably more than 60%, more than 70%,
More than 80%, more preferable more than 90%, the nucleic acid of more preferable more than 95%, most preferably more than 98%, such as 99%) homology, institute
Stating nucleic acid also has the function of that specific regulatory control starts Plant aboveground tissue expression." homology " refer to according to position it is identical hundred
Divide and compare, the similar level (i.e. sequence similarity or homogeneity) between two or more pieces nucleic acid.
As used herein, term " specific expressed " refers to target gene in the specific time and/or specifically organizes
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.Particular sequence is " external source " for cell that it is inserted into or organism.
The promoter of the present invention can operationally be connect with foreign gene, and the foreign gene is for promoter
Can be external source (heterologous).Foreign gene (also referred to as target gene) of the present invention is not particularly limited, preferably
Foreign gene be 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.Expression vector can be that bacterial plasmid, bacteriophage, yeast plasmid, plant are thin
Cellular virus, mammalian cell virus or other carriers.A kind of particularly preferred carrier is AAV8 viruses.
Those of ordinary skill in the art can use well known method structure to contain promoter of the present invention and/or mesh
Gene order expression vector.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..
The structure of pSyn regulating and controlling sequences
The effect of in order to improve the treatment of AAV vector-mediated genes, screening and optimizing is 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
Primarily requirement is that have the effect of liver cell specific regulatory control to the controlling element of this design screening.In addition, 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, otherwise invalid product will be generated more than the bale capacity of virion.Transcription regulating nucleotide sequence master
To include promoter and enhancer.Promoter is responsible for the identification and combination of RNA polymerase, and enhancer includes and promotes 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 sequences is by human apolipoprotein E livers spy
Different in nature control zone (Human apolipoprotein E-Hepatocyte Control Region, ApoE-HCR) group is enhancing
Son, the composite construction sequence formed with the startup sub-portfolio of people's alpha-1 antitrypsin, and for the bale capacity tune for adapting to AAV
Whole size.The carrier for carrying this controlling element belongs to one of expression efficiency is most strong in liver organization carrier, and successfully
Ground embodies certain curative effect in clinical test.However, this expression only makes the phenotype of patient obtain to a certain degree
Alleviate, and not fully achieved the standard of healing.
In order to obtain the expression of the liver specific genes of higher clinical efficacy level, start in liver specific genes
(http is retrieved in subdata base (The Liver Specific Gene Promoter Database)://
rulai.cshl.org/LSPD/index.html).In the efficiently specifically expressed albumen of liver cell, albumin (Albumin)
It is one of wherein main member.It can be seen that the enhancer and promoter of albumin have more liver specificity regulation and control
Element, so as to close with the transcriptional control interconnection in liver cell.The promoter of albumin includes 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 the factor HNF-1 (Hepatocyte nuclear factor 1) (LF-B1, PAF, AFP1), C/EBP, CTF/NF1, C/EBP
With DBP etc..And human albumin gene 5 ' end upstream region at least there are three enhancers, including promoter upstream about-
265bp, -1.7kb and -6kb regions.These belong to transcription factor, such as the main knot of HNF-1 rich in AT and GC regions
Site is closed, this protein-dna interaction has the function of to be obviously promoted for the transcription of downstream gene.
The region of AT and GT sequences 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 exists
One of albumen being synthetically produced in liver cell.The promoter length of mouse TTR genes is included from transcription initiation site to 5 ' upstreams
The region of 202 base-pairs, wherein from -108 to -202 regions, which belong to TTR specific transcriptionals in liver cell, expresses required area
Domain.In addition, there is the DNA fragmentation for having about 100bp between -1.86kb to -1.96kb in 5 ' upstreams very strong enhancer to make
With, can promote in hepatoma cell strain proximal promoter drive under expression efficiency enhance 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 sequences lack, transcriptional efficiency is weaker.However, identified hair
Include 5 cis-regulating elements in present hF9 initiating sequences.In these elements can with the transcription of rich content in liver because
Son, such as C/EBP α, NF1, AR, DBP or HNF-4, with reference to so as to which hF9 be promoted 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 carry the reporter plasmid of hF9 proximal promoters
Expression is not high, but after No. 5 site sequences of several copies are added, and transcriptional efficiency can be with containing HCR tune
Control the plasmid roughly the same 32 of sequence.Only cell line has obtained conclusion to these data in testing in vitro at present, not yet carries out
In vivo studies is verified.Therefore, in order to obtain the plasma thromboplastin component of physiological expression, it is worth attempting using these physiological tune
Sequence construct expression frame is controlled, in the gene therapy vector of liver cell internal specific expression hFIX.
Encoding gene of the above through 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 for the expression frame in gene therapy, mediation liver cell specificity
Transcriptional expression.
The ITR sequences of mutation
The present invention provides a kind of ITR sequences (inverted terminal repeat sequence) of mutation, the ITR sequences of the mutation contain
106 bases, with 3 ' end wild type ITR sequence SEQ ID NO.:Base sequence complementary between the 25th to the 130th of 5
It is or identical.
Specifically, wild type ITR structures contain 145 base-pairs, play a role in virus replication and mediate to form list
The AAV viruses of chain DNA core.The present invention adjusts wild type ITR structures, 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, it is complementary double so as to promote to be formed in AAV reproduction processes
Chain structure promotes transcriptional expression.This structure further reduces several base-pairs than the ITR structures of the transformations such as Nathwani, makes
It still can form complementary double-stranded viruses DNA.In summary structure, the bioactivity and peace of AAV carriers for adjusting and building
Full property has carried out system evaluation in HB Mice Bodies.
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 incidence in global male
About 1/25000.Normal people F9 full length gene about 34kb, wherein coded sequence cDNA length are 1383bp, long positioned at X chromosome
Arm end Xq27.1-q27.2 regions include 8 extrons and 7 intrones.People's F9 gene defects lead 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 extron (700/812).About 50% mutation causes hFIX protein expressions obstacle or functional defect, so as to induce
Serious clinical phenotypes (table 1).
The correlation analysis of 1. hemophilia B patient mutations type of table and disease severity
The haemophiliachemophiliac 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 achieve certain Clinical efficacy and safety result in external clinical test at present.So
And also there are certain Optimal improvements spaces for the 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 and is in progress, and has carried out gene therapy hemophilia
The research of B, exploitation optimizes the AAV carriers for Gene Therapy for Hemophilia B, and is confirmed in the animal model of preclinical phase
Its biological activity and safety, so as to provide solid foundation for its clinical converts.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 practice will be that gene therapy includes blood
Genetic disease including friendly disease establishes solid theory and practice basis.
In addition, the AAV carriers of Disease-causing gene and liver therein spy are expressed in liver cell for mediating 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, can liver specific expression 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, can be included big
The DNA fragmentation (Fig. 2A) of small about 4.7kb.Engineered restrovirus itself encoding gene being treated property gene replaced, so as to
Form 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 to plant.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 same tissue can also by the AAV carriers 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, the efficiency of infection highest of wherein 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
The carrier of the present invention
Carrier the present invention also relates to the pSyn regulating and controlling sequences comprising the present invention and the carrier or pSyn using the present invention
The genetically engineered host cell of regulating and controlling sequence.
Term " carrier " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, lactation are moved
Object cell virus or other carriers.As long as in short, can replicate and stablize in host, any plasmid and carrier can be used.
For carrier, replication orgin, promoter, marker gene and translation control element are usually contained.Preferably, carrier of the present invention
For AAV carriers.
The carrier of the method well known to those skilled in the art structure present invention can be utilized.These methods include vitro recombination
DNA technique, DNA synthetic technologys, In vivo recombination technology etc..The DNA sequence dna can be effectively connected to suitable in carrier of the present invention
When in promoter, mRNA to be instructed 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, conversion is selected to provide
Host cell phenotypic character, such as the dihyrofolate reductase, neomycin resistance and green fluorescence of eukaryotic culture
Albumen (GFP) or the tetracycline or amicillin resistance for Escherichia coli.
Comprising above-mentioned appropriate DNA sequence dna and the carrier of appropriate promoter or control sequence, it is suitable to can be used for conversion
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；It is or high
Eukaryocytes are waited, 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 appropriate carrier, promoter, enhancer and host cell.
The AAV carriers of the present invention
In the strategy of gene therapy hemophilia B, AAV carriers have critical transmission translocation.Express base of curing the disease
The sequence of cause needs to transport in body blood cycle by AAV carriers and specific localization is in specific target organ.
The AAV carriers of gene therapy hemophilia B are mainly included and are made of two parts：A. it cures the disease gene expression construct；B. there is mutation ITR
The AAV structure sequences (Fig. 4) of structure.The present invention is controlled by optimizing two component parts so as to reach to improve to have in gene therapy
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 carriers) include：
1. liver specificity transcription regulating nucleotide sequence, pSynthetic (abbreviation pSyn)；
2. the Human factor IX coded sequence of sequence optimisation；
3. optimize the ITR structures (the ITR sequences 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.
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Test method without specific conditions in the following example, 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 genes
The expression frame of people FIX includes the regulating and controlling sequences such as hFIX cDNA coded sequences and enhancers/promoters (figure
4A).First, codon optimization has been carried out to hFIX coded sequences.The principle of codon optimization is by assessing different codons
The difference of frequency of use replaces the higher codon of the expression frequency in eukaryocyte corresponding to same amino acid opposite
The relatively low codon of frequency of use, so as to enhance the transcriptional expression efficiency of gene.
In addition, carrying out sequence optimisation to hFIX coded sequences, to improve the degree of stability of mRNA molecular structures, its quilt is reduced
The risk of endogenous RNase degradations.Sequence optimisation is mainly completed using OptimumGeneTM analyses.The parameter packet wherein optimized
It includes：(1) genetic transcription efficiency regulation and control parameter, it is lowly close including increasing Preference amino acid codes, reducing or deleting utilization rate
The use of numeral；(2) G/C content is adjusted, it is 30%~70% to be applicable in section；(3) the mRNA secondary structures of hFIX are changed, 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 which whether the optimisation strategy for verifying the present invention can effectively improve the expression efficiency of hF9 genes, same
Optimized hFIX sequences are 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, and this level is with achieving apparent clinical efficacy in clinical test
HFIX optimizations (hFIX.ctrl) are basically identical.
Then, cohFIX expression frames will be carried by AAV8 carrier transports to the liver organization of HB mouse, blood plasma is regular
Detection as a result, it has been found that, cohFIX optimizations (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 basically identical, and result is effective
The bleeding phenotype (Fig. 5 B) for alleviating gene therapy mouse.These results advantageously demonstrate the sequence optimisation strategy of the present invention
The transcriptional efficiency of hF9 genes, 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, so as to make
The arginine of the 338th replaces with leucine (hFIX.SIHR338L) in hFIX albumen.This mutation is originally found in a meaning
Big profit family, when the arginine of the 338th in hFIX 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 R338 sites be Stuart factor with
The significant points that FIX is combined, can enhance joint efficiency between the two, so as 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 sequences are expressed through AAV carriers in HB mouse liver cells, and as a result showing the expression of hFIXR338L mutant can have
The dosage of the reduction AAV preparations 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 carriers.
Embodiment 2
It builds and screens liver specificity regulating and controlling sequence
The effect of in order to improve the treatment of AAV vector-mediated genes, other than being optimized to hF9 genes, also adjusts transcription
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 sequences that AAV carriers carry in the clinical tests such as Nathwani have carried out parallel comparison.
1. artificial synthesized pSynthetic (pSyn) regulating and controlling sequence combines the regulating and controlling sequence of TTR and Alb, comprising
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 bases
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 promoters is effectively raised in cell line experiment.
In addition to this, also the ITR structures of carrier are adjusted.Control type ITR structures (ITRControl) mediation formed it is single-stranded
The AAV carriers (Fig. 7, sample F) of DNA cores, Nathwani etc. delete the ITR (ITR of AAV carriers sidem1) in nearly more than 20
Base-pair so as to form complementary double-stranded DNA structure during AAV packaging productions, promotes transcriptional efficiency (Fig. 7, sample
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 one section of artificial synthesized intron sequences is added, and two in the expression frame of hFIX
End is plus splicing donor and splicing acceptor, so as to not influence coded sequence.In summary structural adjustment and
The bioactivity of the AAV carriers of structure and safety have carried out system evaluation in HB Mice Bodies.
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 expression frames of the hFIX comprising different controlling elements and is packaged into AAV8 carriers.These carriers pass through
Enter in HB Mice Bodies after tail vein injection, and mediate liver specific expression's hFIX albumen.
The results are shown in Figure 8, by determination of activity and elisa assay, is shown in the tracing study of nearly four months, injection
HFIX antigens and activity level highest in the HB mouse group blood plasma of AAV carriers that includes pSyn controlling elements.Regulate and control with LP1
Element group compares, and the albumen of hFIX and activity level are relatively weak in the HB mice plasmas of pTTR and pF9 groups, prompt its transcription
Regulate and control relatively inefficient.
Embodiment 3
The internal dosage of pSyn regulating and controlling sequences increases experiment
According to the above results show that pSyn regulating and controlling sequences have relatively stronger liver specificity transcriptional activity, therefore utilize packet
The AAV.pSyn.cohFIX carriers of the regulating and controlling sequence containing pSyn have carried out internal dosage and have increased experiment, wherein high, medium and low concentration group
The carrier dosage of injection is thought corresponding with the dosage applied in the clinical test of the designs such as Nathwani respectively.
The results show that the hFIX antigens and activity in blood plasma begin to increase from injection carrier latter week, reached to 4th week
To peak, and metastable level is maintained in the observation period of 4 months.In addition, the coagulation factor activity in blood plasma
With antigen levels with the directly proportional rising of increasing of dosage, the activity of hFIX and concentration 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 it demonstrates the expression that AAV.pSyn.cohFIX carriers can effectively be stablized in HB vivo long-terms and plays its blood coagulation
Effect.
Embodiment 4
The tissue specificity experiment of pSyn regulating and controlling sequences
For the tissue specificity of the carrier mediated expression hFIX of clear and definite AAV, part HB mouse are in three, injecting virus carrier
Histoorgan is extracted after month to be analyzed, the RNA in liver, spleen and thymic tissue is extracted and analyzes hFIX phases
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 expressions level is in baseline level in spleen and thymus gland,
Rna level in respectively being organized with the HB mouse for having injected AAV.GFP carriers is consistent.These results illustrate that AAV.pSyn.hFIX is carried
The rna expression of body mediation has liver organization specificity.
Embodiment 5
The biological distribution of AAV.pSyn.cohFIX carriers
Then, biological distribution of the AAV carriers in HB Mice Bodies is analyzed.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.In addition 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.With reference to these data and the analysis result of rna expression level,
Illustrating the gene delivery strategies of AAV8 combination liver specificity regulating and controlling sequences can effectively ensure that purpose cures the disease gene in liver
Specific expressed in tissue, there is the AAV.pSyn.cohFIX carriers in the application control to be expressed in liver internal specific
HFIX is to treat the purpose of hemophilia B.
Embodiment 6
Liver function index measures
Show that the carrier mediated gene therapies 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 injections and the blood plasma of the 4th, 12 week after injection
The level of glutamic-oxalacetic transaminease (AST) and glutamic-pyruvic transaminase (ALT).
As a result (Week 0) is compared with the level in the HB mice plasmas of non-receptor gene treatment as shown in figure 12, noted
AST the or ALT levels after viral vectors in the mice plasma of 4 weeks or 12 weeks are penetrated all to increase without conspicuousness.This also in blood plasma
The hFIX levels for stablizing expression are corresponding, illustrate that AAV carriers and its expression product do not induce apparent liver under these dosage
Functional lesion.
Embodiment 7
The high blood coagulation activity marker of mouse measures
The excessive extrinsic coagulation factor can induce the high coagulation of body.In order to detect various dose AAV carriers
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 levels before gene therapy, under various dose
AAV carriers expression coagulation factor does not cause excessive Coagulation test, it was demonstrated that is not present in HB Mice Bodies under these dosage
High blood coagulation risk.
In summary data, the AAV.pSyn carriers that the present invention is built fully 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 drivings,
So as to restore the normal coagulation function of mouse, achieve 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 be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited
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
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Claims (10)
1. the liver specificity transcription regulating nucleotide sequence of a kind of separation, which is characterized in that the regulating and controlling sequence includes albumin tune
Control sequence and transthyretin regulating and controlling sequence.
2. liver specificity transcription regulating nucleotide sequence as described in claim 1, which is characterized in that the albumin regulating and controlling sequence
Include the binding site of transcription factor HNF-1 and C/EBP.
3. liver specificity transcription regulating nucleotide sequence as described in claim 1, which is characterized in that the transthyretin
Regulating and controlling sequence includes the binding site of transcription factor HNF-1, HNF-3, HNF-4 and C/EBP.
4. liver specificity transcription regulating nucleotide sequence as described in claim 1, which is characterized in that the liver specificity transcription
Regulating and controlling sequence is appoints polynucleotides selected from the group below：
(a) nucleotide sequence such as SEQ ID NO.:Polynucleotides shown in 1；
(b) nucleotide sequence and SEQ ID NO.:Sequence shown in 1 homology >=90% (preferably >=95%, more preferably >=
98%, most preferably >=99%), and the polynucleotides with liver specificity transcriptional activity；
(c) such as SEQ ID NO.:5 ' the ends and/or 3 ' ends of polynucleotides shown in 1 increase and/or reduce 1-50 (preferably 1-
20, more preferably 1-10, more preferably 1-5) nucleotide, and the polynucleotides with liver specificity transcriptional activity.
5. liver specificity transcription regulating nucleotide sequence as claimed in claim 4, which is characterized in that the liver specificity transcription
Activity refers to drive the activity of downstream DNA segment specific transcriptional in liver cell.
6. liver specificity transcription regulating nucleotide sequence as claimed in claim 5, which is characterized in that described " in liver cell
Specific transcriptional " refer to transcriptional activity (or transcription amount) Z1 in the liver cell with non-liver cell (such as fibroblast)
In the ratio between transcriptional activity (or transcription amount) Z2 (Z1/Z2) >=2, preferably >=5, more preferably >=10.
7. 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.
8. construction as claimed in claim 7, which is characterized in that the foreign gene is selected from the group：Therapeutic gene, screening
Marker gene, resistant gene, antigenic protein gene, RNAi genes, microRNA genes, or combination, preferably, described controls
Gene is treated to be selected from the group：Human coagulation factor IX gene, human blood coagulation VI II, people's alpha1-antitrypsin, people's aromatic yl acid ester
Enzyme B (ARSB) gene, human ldl receptor (LDLR) gene, or combination.
9. a kind of carrier, which is characterized in that the carrier contains the construction described in claim 7.
10. the use of the carrier described in construction or claim 9 described in regulating and controlling sequence described in claim 1, claim 7
On the way, which is characterized in that be used to prepare a preparation or composition, the preparation or composition are used to foreign gene or external source
DNA fragmentation carries out specific expressed in liver cell or liver organization.
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| CN201711098538.0A CN108239645B (en) | 2017-11-09 | 2017-11-09 | Liver specificity transcription regulating nucleotide sequence and its application |
| PCT/CN2018/114844 WO2019091457A1 (en) | 2017-11-09 | 2018-11-09 | Liver-specific transcriptional regulatory sequence and applications thereof |
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| CN111218446A (en) * | 2019-12-25 | 2020-06-02 | 武汉枢密脑科学技术有限公司 | Liver specific promoter and application thereof |
| CN114846141A (en) * | 2019-12-23 | 2022-08-02 | 苏州华毅乐健生物科技有限公司 | Isolated nucleic acid molecule and application thereof |
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| WO2019091457A1 (en) * | 2017-11-09 | 2019-05-16 | 上海交通大学医学院附属瑞金医院 | Liver-specific transcriptional regulatory sequence and applications thereof |
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| CN111218446A (en) * | 2019-12-25 | 2020-06-02 | 武汉枢密脑科学技术有限公司 | Liver specific promoter and application thereof |
| CN111218446B (en) * | 2019-12-25 | 2023-03-28 | 劲帆生物医药科技（武汉）有限公司 | Liver specificity promoter and application thereof |
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| WO2019091457A1 (en) | 2019-05-16 |
| CN108239645B (en) | 2019-08-09 |
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