CN110172442A

CN110172442A - A kind of people's induced pluripotent stem cells, construction method and application thereof

Info

Publication number: CN110172442A
Application number: CN201910408744.XA
Authority: CN
Inventors: 梁德生; 胡志青; 周妙金; 邬玲仟
Original assignee: Central South University
Current assignee: Shanghai Pingpu Medical Technology Co ltd
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2019-08-27
Anticipated expiration: 2039-05-16
Also published as: CN114703142B; CN110172442B; CN114703142A

Abstract

The present invention relates to a kind of pluripotent stem cell (iPSCs), energy directed differentiation is endothelial progenitor cells.The invention further relates to the construction methods of the pluripotent stem cell, and its purposes in pharmacy.Claimed basic technical scheme is: a kind of people's induced pluripotent stem cells, it is characterised in thatF8Mutational site is nearby by in-frame deletion in the area gene B coded sequence, or the entire area B coded sequence is targeted missing.The present invention overcomesF8The method of abnormal gene expression is to provide a kind of people's induced pluripotent stem cells of modified, wherein improper expressionF8Sequence is targeted missing by in-frame deletion or the entire area B coded sequence near mutational site in the area gene B coded sequence.In the preparation method of the people's induced pluripotent stem cells, building donor vehicle is not needed, it is only necessary to which directly synthesis ssODN technically realizes relatively easily, will not add any screening-gene, and efficiency is higher.

Description

A kind of people's induced pluripotent stem cells, construction method and application thereof

Technical field

The present invention relates to a kind of pluripotent stem cell (iPSCs), energy directed differentiation is endothelial progenitor cells.The invention further relates to The construction method of the pluripotent stem cell, and its purposes in pharmacy.

Background technique

Hemophilia A (Hemophilia A, HA) is a kind of due to lacking X linkage inheritance caused by functional FVIII Hemorrhagic disease, the disease incidence in male are about 1/5000^[1,2].It is mainly shown as different degrees of coagulation disorders, Light-duty, osculant and heavy type are clinically classified as according to the blood coagulation activity of plasma F VIII.(FVIII blood coagulation is living by light-duty patient Property be 5%-30%) bleeding after general experience wound or major operation, account for about the 40% of HA patient；Osculant patient (FVIII blood coagulation Activity is 1%-5%) occasionally there is spontaneous bleeding, experience postoperative hemorrhage is serious, accounts for about the 10% of HA patient；Heavy patient (FVIII Blood coagulation activity < 1%) account for about the 50% of HA patient, it often will appear joint Repeated Hemorrhage and disable, in some instances it may even be possible to intracranial hemorrhage And threat to life.

HA there is no radical cure method at present, clinically mainly takes alternative medicine for the disease, that is, passes through infusion blood plasma separation FVIII or the FVIII albumen of recombination prevent to be transfused in time after bleeding or bleeding to stop blooding.But the half of FVIII Declining, the phase is extremely short, and normal to need be transfused repeatedly all the life, there is also the risks of latent viral infection, it is often more important that prolonged and repeated infusion can produce Raw neutrality antibody is invalid so as to cause treatment^[3,4]。

HA be caused due to F8 gene mutation its generate FVIII molecular structure defect or FVIII content reduce and cannot Caused by the coagulation function brought into normal play.F8 reaches 186kb as one of the maximum gene cloned, overall length^[5], outer comprising 26 Aobvious son, wherein 14 exons are its maximum exons (3.1kb).There are many mutation type of F8, so far human mutant data The mutation type of library (The Human Gene Mutation Database, HGMD) record is 3231 kinds existing, including missense is prominent Change, nonsense mutation, small fragment missing and insertion etc., cause FVIII function to reduce or lose^[6]。

F8 coding generates the Precursor Peptide for containing 2351 amino acid, is formed by a series of processing modification and contains 2332 amino The FVIII maturation protein of acid.According to sequence similarity, FVIII structure can be divided into several different functional areas, including 3 The area A, 1 area B and 2 areas C, structure composition are N-A1-A2-B-A3-C1-C2-C (as shown in Figure 1)^[7,8].Wherein exist in the area A The binding site of calcium ion, plays a role in intrinsic coagulation pathway.

In secretion process, the albumen of single stranded form can be cut by not woods proteolytic enzyme to be generated by cuprous ion FVIII The heterodimer of one heavy chain (A1-A2-B) of connection and a light chain (A3-C1-C2) composition.By blood coagulation enzyme hydrolysis, The area B has been cut off, A1, A2, the A3-C1-C2 connected by cuprous ion has been formed, becomes active FVIII, i.e. FVIIIa, thus It plays blood coagulation activity (as shown in Fig. 2)^[7,9]。

In addition, the area B high glycosylation, it is little with FVIII activity relationship that some researches show that it, and the missing major part area B is not The activity of FVIII can be had an impact^[10].Although it is living that the area B is not present in final performance blood coagulation in protein function level Property FVIII in, but in all F8 gene mutations included, there are nearly 500 kinds of mutation to occur in the area B coded sequence, In 90% or more mutation cause FVIII protein translation to terminate in advance, lose FVIII coagulation function, lead to HA^[6]。

HA has always been considered as being most possible one of the single gene inheritance disease for realizing gene therapy.One of ideal side Formula is the in-situ immobilization of mutated gene or carries out functional correction by gene editing in situ appropriate, i.e., in the original of dcc gene Site carries out accurate gene repair by the approach of homologous recombination or eliminates the pathogenic effect of mutation.This mode is not only extensive The function of gene has been answered, while having also retained the controlling element in situ of gene, mutated gene has been transformed into just to greatest extent The consistent physiological status of ordinary person.Have at present for No. 22 introne inversion types of F8 gene and the mutation of No. 1 introne inversion type HA gene in-situ immobilization strategy^[11,12].Another gene therapy mode is gene substitution, is exactly by the treatment base with promoter Because random or site-directed integration is into deficient cells, to achieve the purpose that treatment.Due to the limitation of technology, what is carried out at present is exhausted Most gene Therapy study is all to use gene substitution strategy, such as have through virus-mediated random integration strategy, non-disease The gene that poisonous carrier mediates adds strategy.

Virus-mediated random integration strategy, exactly using the plasmid of the coded sequence and promoter sequence that include F8 The cell of F8 defect is transfected, F8 expression cassette has certain probability to be integrated into genome, to express FVIII protein exhibits work With.Its disadvantage has: (1) F8 is very big, and coded sequence is 8kb or so, even the version of the area B missing is added also greater than 4kb Promoter sequence, this plasmid is very big, and in general plasmid is bigger, and building is also more troublesome, and integration efficiency is also very low；(2) if Gene random integration is into genome, and the expression of gene may be subjected to the influence of position effect, it is possible to which expression efficiency is low, very It cannot extremely express；(3) it is possible to destroy endogenous gene or activation if plasmid integration to other endogenous gene sites The deleterious gene that do not express originally, so as to cause bad consequence.

And the gene addition strategy that non-virus carrier mediates has following defect: (1) since F8 very big, coded sequence 8kb Left and right, even the version of the area B missing adds promoter sequence also greater than 4kb, plasmid is very big, and in general plasmid is bigger, Construct also more troublesome, integration efficiency is also very low, and can generally introduce external source screening-gene, the residual of exogenous genetic fragment for The remote effect of cell cannot temporarily define；(2) exogenous origin gene integrator passes through exogenous promoter to the safe site of genome Regulate and control with controlling element, it is possible to influence expression efficiency.

Early-stage study shows that F8 (B domain deletion F8, BDD-F8) gene of the area external source Ba Ru B deletion form can With effectively expressing functionality BDD-FVIII^[13-15], and using the BDD-FVIII of recombination equally there is certain clinic to control Therapeutic effect^[10,16].Therefore be directed to the pathogenic mutation of the area F8 gene B coded sequence, the present invention propose one it is completely new based on original position Pathogenic mutation situ converting is that the area B coded sequence is small that is, by target gene editing technique by the therapeutic strategy of gene editing In-frame deletion eliminates these mutation and FVIII is caused to translate the pathogenic effect terminated in advance to restore the translation of FVIII albumen It answers, restores the blood coagulation activity of endogenous FVIII.And on this basis, in-frame deletion is further extended to the entire area B code sequence Lieque is lost, this novel original position HA strategies in gene therapy is applied to all area B pathogenic mutation patients.

Summary of the invention

An object of the present invention is to provide a kind of people's induced pluripotent stem cells (iPSCs), the pluripotent stem cell energy Directed differentiation is endothelial progenitor cells, and the normal expression F8 gene in human body.

It is another object of the invention to provide a kind of methods for obtaining above-mentioned iPSCs.

Another object of the present invention is, according to iPSCs obtained, to provide its a kind of purposes for being used for HA treatment.

Claimed basic technical scheme is: a kind of people's induced pluripotent stem cells, it is characterised in that F8 base Because by in-frame deletion, or entirely, the area B coded sequence is targeted missing near mutational site in the area B coded sequence.

The applicant's deposit number obtained are as follows: two cell strains of C201968 and C201990, the two cell strains are only It is a specific embodiment of the claimed technical solution of the present invention.Wherein one plant is that patient iPSCs has lacked 54 bases Cell, deposit number C201968, another strain is the cell that patient iPSCs has lacked the entire area B, deposit number C201990.

Because of mutational site in the different area in-frame deletion F8 gene B coded sequences or the targeting missing entire area B coded sequence Method, the particular sequence details of related gene in people's induced pluripotent stem cells obtained can be made to have a certain difference, But this species diversity does not impact the expression of related gene, then belongs to the scope of technical solution of the present invention.

According to an embodiment of the invention, its mutational site being directed to is c.3167delCTGA to make a variation.

In order to efficiently obtain the iPSCs of the area B coded sequence in-frame deletion in situ, with reference to the prior art, feasible method packet It includes but is not limited to: can be by means of artificial nuclease.Artificial nuclease mainly has the short palindrome in Regularity interval to repeat sequence at present Arrange (clustered regularly interspaced short palindrome repeats, CRISPRs) gene editing System, zinc finger enzyme (Zinc-finger nucleases, ZFNs) and TALE nuclease (transcription activator- like effector nucleases,TALENs)^[17,18].These gene editing tools are all to cut target DNA by identification, are made (double-strand breaks, DSBs) is broken at DNA double chain^[19].DSB is mainly with inaccurate non-homologous end joining (non-homologous end joining, NHEJ) approach is repaired^[20], it can be achieved that efficient gene knockout^[21,22].Together When DSB the homologous recombination (homologous recombination, HR) near broken site can be activated active, repaired homologous Gene targeting efficiency can be significantly improved in the presence of multiple template^[23], realize the addition, replacement, accurate gene piece of gene Section missing or point mutation.

The present invention needs the method especially set out to be: being mediated using ssODN (single-stranded oligodeoxynucleotide) homologous The advantage of recombination^[24], genetic fragment is carried out using ssODN as homologous recovery template accurately to be lacked.Specifically, pass through CRISPR/Cas9 and ssODN carries out consideration convey to the HA-iPSCs from patient, obtains in the small frame of the original position area B coded sequence The iPSCs of missing or the entire area B coded sequence targeting missing.

If it is the position near only deletion mutation sequence, be according to mutational site near sequence design it is nearest The sgRNA of CRISPR/Cas9 (design of Cas9 has the limitation of PAM sequence), missing comprising including the base of mutational site in total 3 bases several again, that is, realize in-frame deletion.On the other hand, the mutation type in the area B has several hundred kinds to cause HA, we The strategy of the entire area the B missing of design can be directed to the variation in any one area B, and in order to exclude possible patient's heredity back The influence of scape, we have also carried out the experiment of the missing in the entire area B in normal person iPSCs simultaneously, referring to the in embodiment the 2nd (7).This is the experiment proves that the missing in the entire area B will not have an impact the expression of F8 gene.

It is that relatively common two sides stay the SQ type of 14 amino acid to connect employed in specific embodiment in the present invention.Such as If fruit lack just for some specific mutation site the bases several again of a part 3 near it, then basis is needed Specific mutation location proximate constructs the purpose of in-frame deletion, different ssODN is designed, specifically, being exactly in in-frame deletion alkali The upstream and downstream of base takes about 40 bases to synthesize the ssODN of 80nt or so to mediate accurate genetic fragment to lack respectively^[25].Often One case-specific situation is different；It dashes forward if it is directly realizing to cause a disease in all areas B by the missing entire area B coded sequence If the treatment of change, most conventional is exactly to obtain a kind of this SQ connection (Ser743 of the area B N-terminal and the Gln1638 of C-terminal melt Close) the area B missing type, such words ssODN can be using the F8-BDD-ssODN sequence in following table, i.e. SEQ ID NO.20.The ssODN can be applied to all mutation in the area B, if the area the B deletion form F8 of other types connection is constructed, such as RH connection (Arg747 of the area B N-terminal is merged with the His1646 of C-terminal), then will accordingly adjust the sequence of ssODN.

According to an embodiment of the invention, HA-iPSCs is to collect Urine in Patients cell, and being induced is that patient HA is special Property iPSCs (HA-iPSCs).Used abductive approach bibliography^[11]。

After obtaining iPSCs, directs it and be induced to differentiate into endothelial progenitor cells for cell transplantation, play therapeutic effect.It closes In how Induction of committed differentiation be endothelial progenitor cells, can be with bibliography^[26]。

It usually should be to acquire the body cell of patient, and being induced is HA-iPSCs for clinical practice application, Then mutational site in the area B coded sequence in F8 gene therein lack in small frame using method provided by the invention It loses or the entire area B coded sequence carries out targeting missing, so that people's induced pluripotent stem cells of gene correction are obtained, it is this dry thin Born of the same parents can be reduced rejection for transplanting after directed differentiation is endothelial progenitor cells.

The method that the present invention overcomes F8 abnormal gene expression is to provide a kind of people's induced pluripotent stem cells of modified, Wherein nearby sequence is encoded in the mutational site in the area the F8 gene B coded sequence of improper expression by in-frame deletion or the entire area B Sequence is targeted missing.In the preparation method of the people's induced pluripotent stem cells, building donor vehicle is not needed, it is only necessary to directly SsODN is synthesized, technically realizes relatively easily, any screening-gene will not be added, and efficiency is higher.It is provided by the invention The method for obtaining iPSCs is properly termed as a kind of gene correction in situ, in this way can be in the original promoter of gene and regulation member Under part, gene normal expression is realized, it is comparatively safe.

Detailed description of the invention

Fig. 1 is FVIII tactic pattern figure；

Fig. 2 is the structure change ideograph of FVIII molecular structure and activation process；Wherein red arrow indicates not woods albumen The cleavage site of hydrolase, yellow arrows indicate the site of fibrin ferment cutting, and purple M expression connects heavy chain and light chain Metal ion.Full length FVIII indicates that overall length FVIII, FVIIIa indicate the FVIII of activation；

Fig. 3 is method process schematic provided by the invention；

Fig. 4 is the induction and qualification figure of mutation iPSCs in the area F8 gene B coded sequence；Wherein A. collects the urine of culture Cell expresses β-catenin, KRT7 and ZO-1 etc., prompts its renal cells source；B. iPSC grams of acquisition is reprogrammed Grand form；C. the iPSCs caryogram detection no abnormality seen obtained；D. the iPSCs obtained still retains the deletion mutation in the area B；E. it is immunized Fluorescence display iPSCs expresses a variety of stem-cell marker albumen；F. teratoma experiment confirms that iPSCs can break up in vivo in body Form the tissue in three germinal layer sources.

Fig. 5 is the small in-frame deletion result of CRISPR gene editing System-mediated F8 gene B area's coded sequence；Wherein Schematic diagram is knocked out in the area A.CRISPR gene editing System-mediated F8 gene B frame；B. constructed sgRNA Efficiency testing is respectively 41.3% and 18.92%, green base is sgRNA sequence, and blue base is PAM, and dotted line indicates missing, and black arrow is slotting Entering, red base is the base sequence of insertion ,+indicating insertion, △ indicates missing, × indicate number.C. after gene targeting experiments, Preliminary Identification is carried out by clone of the PCR to acquisition.D.PCR positive band sequence verification；

Fig. 6 is that N-iPSCs accurately lacks 54bp PCR and sequencing qualification result；Wherein figure A is N-del 54-15-iPSCs The electrophoretogram of PCR amplification is carried out through F8-E14-F/R with N-del 54-42-iPSCs, N-iPSCs is control cell；Figure B is N- The PCR product sequencing result of del 54-15-iPSCs and N-del 54-42-iPSCs, black vertical line represents deletion fragment in figure Place, N-del 54-15-iPSCs and N-del 54-42-iPSCs are that two sides sequence is directly connected to after lacking 54bp base；

Fig. 7 is that HA-iPSC s stem cell surface marker after gene targeting is identified；Wherein Nanog, Oct4 are labeled as green Color fluorescence, SSEA-1, SSEA-4 are labeled as red fluorescence；DAPI is used to contaminate core；

Fig. 8 is that N-iPSCs stem cell surface marker after gene targeting is identified；Wherein Nanog, Oct4 are labeled as green Fluorescence, SSEA-1, SSEA-4 are labeled as red fluorescence；DAPI is used to contaminate core；

Fig. 9 is that HA-iPSCs in-frame deletion 54bp clones caryogram detection；Wherein figure A is 2-6-iPSCs caryogram, and figure B is 2- 46-iPSCs caryogram, karyotyping show 46, XY, no abnormality seen；

Figure 10 is that N-iPSCs in-frame deletion 54bp clones caryogram detection；Wherein figure A is N-del 54-42-iPSCs caryogram, Figure B is N-del 54-15-iPSCs caryogram, and karyotyping shows 46, XY, no abnormality seen.

Figure 11 is that RT-PCR detects the iPSCs F8 transcription after HA-iPSCs in-frame deletion 54bp；Wherein the iPSCs stage is logical Cross the detection F8 expression of RT-PCR transcriptional level, H₂O is blank control, and HA-iPSCs is patient group, 2-6-iPSCs and 2-46- IPSCs is in-frame deletion group, and N-iPSCs is Normal group, and GAPDH is internal reference, and F8 (E14) is the expansion of accurate gene delection area Increase, F8 (E23-26) is across 23-26 exon primer amplification；

Figure 12 is that RT-PCR detects the iPSCs F8 transcription after N-iPSCs in-frame deletion 54bp；Wherein the iPSCs stage passes through RT-PCR transcriptional level detects F8 expression, and H2O is blank control, and N-iPSCs is Normal group, N-del 54-42-iPSCs It is accurate missing 54bp clone group with N-del 54-42-iPSCs, GAPDH is internal reference, and F8 (E14) is the expansion of accurate gene delection area Increase, F8 (E23-26) is across 23-26 exon primer amplification；

Figure 13 is iPSCs stage FVIII secretion detection；Wherein ELISA detects iPSCs phase cell lysate and cell is trained Support FVIII expression in supernatant；

Figure 14 is iPSCs stage LMAN1 detection of expression；Wherein detected in iPSCs phase cell by Western blot The expression of LMAN1, β-actin are used as internal reference.

Figure 15 is that endothelial progenitor cells break up the 5th day cell progress flow cytometer detection；Wherein upper row schemes from left to right successively are as follows: 1. Cell scatter plot to be analyzed, the target complex cell that the interior a group cell relatively concentrated of door is analyzed as us carry out subsequent analysis； 2.HA-iEPCs group analysis is 23.24% as a result, the positive ratios of the bis- marks of right upper quadrant display CD31/CD34；3.2-6-iEPCs Group analysis is as a result, the double positive ratios of right upper quadrant are 20.85%；4.2-46-iEPCs group analysis is as a result, right upper quadrant is double positive Ratio is 11.27%；Lower row schemes from left to right successively are as follows: 1.N-iEPCs group analysis is as a result, the double positive ratios of right upper quadrant are 10.09%；2.N-del 54-42-iEPCs group analysis is as a result, the double positive ratios of right upper quadrant are 27.37%；3.N-del 54- 15-iEPCs group analysis is as a result, the double positive ratios of right upper quadrant are 24.54%；

Figure 16 is flow cytometer detection after endothelial progenitor cells sorting；Wherein upper row schemes from left to right successively are as follows: after 1. sortings Endothelial progenitor cells form, growth rate is fast, be epithelioid cell；2. cell flow cytometer detection scatter plot after sorting, door inner cell Group is cell population of interest to be analyzed；3. single mark CD34-PE group, left upper quadrant is positive cell group；Lower row figure from left to right according to It is secondary are as follows: 1. single mark CD31-FITC groups, right lower quadrant is positive cell group；2.2-6 group sorts cell analysis as a result, double positive thin Born of the same parents' ratio is 93.24%；3.N-iPSCs group sorts cell analysis as a result, double positive cells ratio is 92.36%.

Figure 17 is the endothelial cell immunotoxin Fluorescence Identification after sorting；Wherein CD34 is labeled as red fluorescence；CD31 is labeled as Red fluorescence；CD144 is labeled as green fluorescence, and DAPI is used to contaminate core；

Figure 18 is mature endothelial cell identified by immunofluorescence；Wherein CD31 is labeled as red fluorescence, and vWF is glimmering labeled as green Light, DAPI are used to contaminate core；

Figure 19 is mature endothelial cell N-terminal FVIII Immunofluorescence test；Wherein FVIII-N is labeled as red fluorescence, vWF Labeled as green fluorescence, DAPI contaminates core；

Figure 20 is mature endothelial cell C-terminal FVIII Immunofluorescence test；Wherein FVIII-C is labeled as red fluorescence, vWF Labeled as green fluorescence, DAPI contaminates core；

Figure 21 is mature endothelial cell stage FVIII ELISA detection；

Figure 22 is endothelial cell stage LMAN1 detection of expression；Endothelial cell phase cell is detected by Western blot The expression of interior LMAN1, β-actin are used as internal reference；

Figure 23 is that endothelial progenitor cells carry out FVIII blood coagulation activity detection after transplanting in Mice Body；

Figure 24 is the mouse survival curve after docking experiment；Wherein HA mice (n=9)；HA-iEPCs (n=9)；2-6- IEPCs (n=12)；2-46-iEPCs (n=10)；N-iEPCs (n=9)；N-del 54-42-iEPCs (n=9).Ns, with HA Mice compares no difference of science of statistics；* *, p < 0.001, * *, p < 0.01, (log-rank compared with HA-iEPCs test)；

Figure 25 is docking experiment mice mean survival time；Wherein experience docking experiment, in the experimental record time, (48 is small When) after survive the mouse survival time do not counted.Ns, the no difference of science of statistics compared with HA mice.* *, p < 0.001, * *, p < 0.01, *, p < 0.05, compared with HA-iEPCs group；

Figure 26 is human archeocyte detection in mouse liver；Wherein CD31 is labeled as red fluorescence, and vWF is glimmering labeled as green Light, DAPI contaminate core；

Figure 27 is human archeocyte detection in mouse major organs；Wherein CD31 is labeled as red fluorescence, and vWF is labeled as green Fluorescence, DAPI contaminate core；

Figure 28 is that the targeting area B coded sequence lacks target practice schematic diagram；Wherein black base is the area F8 gene B internal sequence, Green represents the identification sequence of the CRISPR/Cas9 of design, and blue is PAM sequence, and red small arrow is cutting position, yellow yin Shadow part is the upstream homologous sequence of ssODN, and gray shade part is the downstream homologous sequence of ssODN, and orange-yellow base is same Two positions of justice mutation, the area B Reframed F8 (BDD F8) are the F8 structural schematic diagram in the missing area B in situ after sequence；

Figure 29 is the CRISPR/Cas9 cutting efficiency detection for the area B coded sequence targeting missing；Wherein figure A is to utilize T Carrier connect PCR product again sequencing approach identification F8-BDU-sg1 efficiency, indels generate ratio be 13.33%；Scheming B is benefit With carrier T connection PCR product again sequencing approach identification F8-BDD-sg4 efficiency, indels generate ratio be 35.71%.WT shows Original series, blue identification division are PAM sequence, and dotted line shows the part of missing, and red mark base is to be mutated and be inserted into base, △ is deletion mutation base number ,+it is insertion mutation base number；

Figure 30 is the clone in the sequencing identification HA-iPSCs missing area B；Wherein figure A is that BD21-iPSCs and BD25-iPSCs is passed through F8-E14-F/R carries out electrophoresis result after PCR amplification, and Marker is DL 2000, and HA-iPSCs is the cell that do not practice shooting, amplification 498bp band out, BD21-iPSCs and BD25-iPSCs do not amplify band, and N-iPSCs is normal control, amplify 502bp Band.Scheming B is that BD21-iPSCs and BD25-iPSCs electrophoresis result, Marker after F8-BUF/BDR progress PCR amplification are DL 2000, HA-iPSCs are the cell that do not practice shooting, amplify 3019bp band, and BD21-iPSCs and BD25-iPSCs are amplified 341bp band, N-iPSCs are normal control, amplify 3023bp band.Scheming C is that BD21-iPSCs and BD25-iPSCs is passed through F8-BUF/BDR amplification PCR product carry out Sanger sequencing as a result, Predicted be HA-iPSCs lack the area B (retain SQ Sequence) theoretical sequence, black vertical line represents at deletion fragment in figure, and peak figure is that BD21-iPSCs and BD25-iPSCs lacks B Two sides sequence is directly connected to after base in area, the same sense mutation at blue arrow meaning to introduce in SQ sequence；

Figure 31 is that N-iPSCs lacks the area B PCR and sequencing qualification result；Figure A is N-BD9-iPSCs and N-BD14-iPSCs The electrophoresis result after F8-E14-F/R progress PCR amplification, Marker is DL 2000, and N-iPSCs is normal control, is amplified 502bp band, N-BD9-iPSCs and N-BD14-iPSCs do not amplify band.Figure B is N-BD9-iPSCs and N-BD14- IPSCs electrophoresis result after F8-BUF/BDR progress PCR amplification, Marker is DL 2000, and N-iPSCs is normal control, 3023bp band is amplified, N-BD9-iPSCs and N-BD14-iPSCs amplify 341bp band.Scheme C be N-BD9-iPSCs and PCR product that N-BD14-iPSCs is expanded through F8-BUF/BDR carry out Sanger sequencing as a result, Predicted is N-iPSCs Lack the theoretical sequence in the area B (retain SQ sequence), black vertical line represents at deletion fragment in figure, peak figure be BD21-iPSCs and Two sides sequence is directly connected to after base in the BD25-iPSCs missing area B, synonymous for what is introduced in SQ sequence at blue arrow meaning Mutation.

Figure 32 is the identification of the area HA-iPSCs B deletion clone stem cell surface marker；Nanog, Oct4 are glimmering labeled as green Light, SSEA-1, SSEA-4 are labeled as red fluorescence；DAPI is used to contaminate core.

Figure 33 is the identification of the area N-iPSCs B deletion clone stem cell surface marker；Nanog, Oct4 are glimmering labeled as green Light, SSEA-1, SSEA-4 are labeled as red fluorescence；DAPI is used to contaminate core.

Figure 34 is the detection of the area HA-iPSCs B deletion clone caryogram；Figure A is BD21-iPSCs caryogram, and figure B is BD25- IPSCs caryogram, karyotyping show 46, XY, no abnormality seen.

Figure 35 is the detection of the area N-iPSCsB deletion clone caryogram；Figure A is N-BD9-iPSCs caryogram, and figure B is N-BD14- IPSCs caryogram, karyotyping show 46, XY, no abnormality seen.

Figure 36 is that RT-PCR detects iPSCs stage F8 transcription；The iPSCs stage detects F8 table by RT-PCR transcriptional level It reaches, H₂O is blank control, and HA-iPSCs is that patient group, BD21-iPSCs and BD25-iPSCs are the area B missing group, N-iPSCs For Normal group, GAPDH is internal reference, and F8 (BD) is across 14 exon primer amplifications, and F8 (E23-26) is across 23-26 extra Show sub- primer amplification.

Figure 37 is that RT-PCR detects iPSCs stage F8 transcription；The iPSCs stage detects F8 table by RT-PCR transcriptional level It reaches, H₂O is blank control, and N-iPSCs is Normal group, and N-BD9-iPSCs and N-BD14-iPSCs are the area B deletion clone Group, GAPDH are internal reference, and F8 (BD) is across 14 exon primer amplifications, and F8 (E23-26) is across 23-26 exon primer Amplification.

Figure 38 is iPSCs stage FVIII secretion detection；ELISA is detected in iPSCs phase cell lysate and cell culture FVIII expression in clear.

Figure 39 is iPSCs stage LMAN1 detection of expression；LMAN1 in iPSCs phase cell is detected by Western blot Expression, β-actin be used as internal reference.

Figure 40 is that the 5th day cell of differentiation carries out flow cytometer detection；From left to right successively are as follows: 1. cell scatter plots to be analyzed, door The interior a group cell relatively concentrated carries out subsequent analysis as the target complex cell that we analyze；2.BD21-iEPCs group analysis knot Fruit, right upper quadrant show the positive ratio of the bis- marks of CD31/CD34, are 20.59%；3.BD25-iEPCs group analysis as a result, upper right as The double positive ratios of limit are 16.84%；4.N-BD9-iEPCs group analysis is as a result, the double positive ratios of right upper quadrant are 19.55%； 5.N-BD14-iEPCs group analysis is as a result, the double positive ratios of right upper quadrant are 21.23%.

Figure 41 is the endothelial progenitor cells identified by immunofluorescence after sorting；CD31 is labeled as red fluorescence；CD144 is labeled as green Color fluorescence, DAPI are used to contaminate core.

Figure 42 is mature endothelial cell identified by immunofluorescence；CD31 is labeled as red fluorescence, and vWF is labeled as green fluorescence, DAPI is used to contaminate core.

Figure 43 is that RT-PCR detects iECs stage F8 transcription；The iECs stage detects F8 expression by RT-PCR transcriptional level, H₂O is blank control, and HA-iECs is that patient group, BD21-iECs and BD25-iECs are the area B missing group, and N-iECs is normal right According to group, GAPDH is internal reference, and F8 (BD) is across 14 exon primer amplifications, and F8 (E23-26) is that across 23-26 exon draws Object amplification.

Figure 44 is that RT-PCR detects iECs stage F8 transcription；The iECs stage detects F8 expression by RT-PCR transcriptional level, H₂O is blank control, and N-iECs is Normal group, and N-BD9-iECs and N-BD14-iECs are the area B deletion clone group, GAPDH For internal reference, F8 (BD) is across 14 exon primer amplifications, and F8 (E23-26) is across 23-26 exon primer amplification.

Figure 45 is mature endothelial cell N-terminal FVIII Immunofluorescence test；FVIII-N is labeled as red fluorescence, vWF label For green fluorescence, DAPI contaminates core.

Figure 46 is mature endothelial cell C-terminal FVIII Immunofluorescence test；FVIII-C is labeled as red fluorescence, vWF label For green fluorescence, DAPI contaminates core.

Figure 47 is mature endothelial cell stage FVIII ELISA detection；

Figure 48 is endothelial cell stage LMAN1 detection of expression；Endothelial cell phase cell is detected by Western blot The expression of interior LMAN1, β-actin are used as internal reference.

Figure 49 is that endothelial progenitor cells carry out FVIII blood coagulation activity detection after transplanting in Mice Body；* *, p < 0.001, with HA- IEPCs group compares, n=6.

Figure 50 is the mouse survival curve after docking experiment；Ns, the no difference of science of statistics compared with HA mice；* *, p < 0.001, (log-rank test), (n=9) compared with HA-iEPCs.

Figure 51 is docking experiment mice mean survival time；Experience docking experiment, the experimental record time (48 hours) it The mouse survival time survived afterwards is not counted.Ns, the no difference of science of statistics compared with HA mice.* *, p < 0.001, * *, P < 0.01, compared with HA-iEPCs group.

Figure 52 is the PCR amplification thermal cycle item of F8-E14-sg1, F8-E14-sg2, F8-BDU-sg1, F8-BDD-sg4 group Part figure.

Figure 53 is that CRISPR/Cas9 target practice fixed point deletion detects PCR system thermal cycle conditions figure in embodiment step 4.2.

Specific embodiment

Below with reference to embodiment, the present invention will be described in detail.It should be noted that in the absence of conflict, the present invention In embodiment and embodiment in feature can be combined with each other.

Such as the following table 1 of Primer and sequence involved in the embodiment of the present invention.

1. Primer of table, sequence and its corresponding purposes

The experimental method and steps are as follows that the present invention is implemented:

The building of CRISPR/Cas9 expression plasmid

It is suitable to design in the way of the Opimized CRISPR Design that FengZhang Lab is provided The short chain guide RNA (sgRNA) (http://crispr.mit.edu/) [27] of CRISPR/Cas9.Order Feng Zhang The CRISPR/Cas9 skeleton pX330 for the Cas9 codon optimization that Lab is provided.

(1) sgRNA (20nt or so) is designed according to experiment purpose, sequence is shown in Table sequence SEQ ID NO.1, SEQ in 1 ID NO. 2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15 is added in the identification sequence C ACC of the end sequence 5' sgRNA addition Bbs I at the end 5' of sgRNA complementary series AAAC；

(2) BbsI digestion pX330 skeleton is used, digestion system is as follows:

37 DEG C digestion 3 hours, digestion products carry out agarose gel electrophoresis, electroresis appraisal digestion is complete, i.e., progress glue recycling Product adds 12 μ L ddH2O lysates after recycling, for connecting.

(3) oligonucleotides by the raw work biology Co., Ltd synthesis in Shanghai carries out respectively according to following four groups of difference systems Annealing forms the double-strand containing cohesive end；

F8-E14-sg1 annealing system is as follows:

After 95 DEG C, 5 minutes, it is naturally cooling to room temperature, for connecting；

F8-E14-sg2 annealing system is as follows:

F8-BDU-sg1 annealing system is as follows:

F8-BDD-sg4 annealing system is as follows:

(4) it connects, system is as follows:

16 DEG C of connections overnight；

(5) conversion and monoclonal colonies picking

DH5 α competence is taken out from -75 DEG C of ultra low temperature freezers, 10 μ L connection products are added into 50 μ L competence, is used Middle pipette tips gently blow and beat mixing；

30 minutes are stood on ice, while recirculated water bath cabinet is preheated to 42 DEG C；

1.5mL centrifuge tube after ice bath is completed is put in 42 DEG C of circulator baths after heat shock 45 seconds, immediately on ice Stand 2 minutes；

It is operated in superclean bench, the LB liquid medium of 500 μ L non-resistants is added；

37 DEG C of air table 180r/min shake 45 minutes；

Bacterium solution is take out of the shaker, with 2500g centrifugation 5 minutes, part supernatant is discarded in superclean bench, about leaves and takes 100 μ L bacterium solutions mix；

Bacterium solution is added in the LB solid plate of ammonia benzyl resistance good in advance, is uniformly coated on bacterium solution with spreading rod On solid LB culture dish；It is inverted in 37 DEG C of constant incubator cultures 12~16 hours.

Picking single bacterium colony is into the LB liquid medium of the 500 μ L resistance of benzyl containing ammonia, 37 DEG C of 220 reincarnations of air table long 4 Bacterium solution is sent to go to be sequenced after~6 hours, sequencing primer is U6 universal primer.

Sequencing result and the consistent bacterium colony of notional result expand culture again, and bacterium solution is seeded to about 60mL added with ammonia benzyl It is 14~16 hours long with 280 reincarnations on 37 DEG C of air tables in LB liquid medium.Utilize OMEGA Endofree plasmid Midi Kit extracts plasmid, can be obtained the successful CRISPR/Cas9 expression vector of building according to specification operation.

2. Hemophilia A patients' specificity iPSCs induces (HA-iPSCs)

Collecting an example saltant type is the heavy HA Urine in Patients that c.3167delCTGA F8 gene leads to p.D1055fsX5, point From its urine cell is cultivated, Urine in Patients cell is reprogrammed using four factor methods of classics and clones and reflects for iPSC sample It is fixed.

The acquisition of 2.1 urine specimens

Under the premise of patient signs informed consent form, non-invasive collection Urine in Patients, the specific steps are as follows:

(1) urethral orifice is sterilized, dips Iodophor using clean cotton swab, gently wipes urethral orifice and surrounding, the cotton swab more renewed It is repeated 1 times, then dips 70% alcohol wipe twice；

(2) it waits several seconds and collects urine after drying alcohol.The lid of receiving flask is opened, the 20mL urine most started of urinating Liquid is discharged into urinal, collects mud-stream urine into receiving flask, when urine is drained fastly, last part is discharged into urinal, carefully covers receipts Collect the lid of bottle.100-200mL urine is collected under normal circumstances.

The culture of 2.2 urine cells

(1) urine in collecting bottle is dispensed into 50mL centrifuge tube in desinfection chamber, is centrifuged 10 minutes with 400g room temperature, Careful reject supernatant stays less than 1mL raffinate, urine raffinate after centrifugation of all packing into 50mL centrifuge tube is incorporated into In one 50mL centrifuge tube, 10mL DPBS is added, with 200g room temperature centrifugation 10 minutes, careful reject supernatant stayed 200 μ L residual Liquid adds 1mL initial medium that cell is resuspended, is inoculated in a hole of 12 porocyte culture plates for being coated with 0.1%gelatin In；

(2) second days directly 500 μ L of addition fresh initial mediums；

(3) third day directly adds the fresh initial medium of 500 μ L；

Start within (4) the 4th days, is measured with REBM complete medium half change liquid daily, it under normal circumstances, 5 days or so after inoculation can To observe a few cell colony, observe after cell colony can full dose replacement REBM complete medium, continue to cultivate, directly When to cell confluency degree up to 50% or so, is passed on, be denoted as P1, cultivated with REBM complete medium.

(5) it takes P1-P3 for cell, carries out counting and be seeded in six well culture plate holes with 60,000 cells/wells inoculation, two six holes Plate hole.Remaining cell cryopreservation.

2.3 induction iPS cells

It needs to carry out retroviral infection twice in Induction Process, is Day by the timing definition of first time virus infection 0.

(1) Day-3 6 pm counts inoculation HEK293T cell in six orifice plates, with the inoculation of 800,000 cells/wells；

(2) Day-2 packaging virus, at 4 points in afternoon, to yesterday inoculation HEK293T cell replace 1.5mL complete medium, 2 Start to transfect after hour, cell density should be 80% or so when transfection.

Prepare calcium phosphate transfection liquid:

First plus Milli-Q water is into EP pipe, adds plasmid mixing, adds 2M CaCl2 mixing, it is eventually adding 2 × HBS is firmly blown and beaten, and generates bubble.Transfection liquid is added dropwise on cell, transfection liquid is enable to cover all areas in hole as far as possible；

(3) HEK293T of transfection is carefully replaced the fresh DMEM complete medium of 2mL (12 is small after transfection by the Day-1 morning When)；Afternoon, every hole inoculation 60,000 was inoculated with two holes to inducing cell in six porocyte culture plates.Day 0, in six orifice plates to Inducing cell replaces 2mL REGM fresh culture.The HEK293T cells and supernatant comprising retrovirus is collected (about to transfect 36 hours afterwards), it is added in the hole to inducing cell after being filtered with 0.45 μm of syringe filter.Hole to be induced add Oct4, Tetra- kinds of viral supernatants of Sox, Klf4 and c-Myc in the hole of control only plus the viral supernatants containing GFP, and are added in system dense eventually Degree is the polybrene of 8 μ g/mL to increase efficiency of infection.HEK293T cell per well adds the fresh DMEM of 2mL and trains completely Support base；

(4) viral supernatants will be changed to REGM after infection overnight to promote cell to restore by 1 Day.Receive in the afternoon for the second time Collection viral supernatants infect once again, operate as before, the HEK293T cell after does not continue to cultivate.2 morning of Day is changed to REGM culture medium.Day 3 replaces REGM culture medium, and virus packaging and infection can be judged by observing the GFP efficiency of control wells Efficiency, normally should be close to 100%.Day 4 replaces REGM culture medium, part cell can be observed, metamorphosis occurs, cell becomes Small, form becomes polygon and flat, and nucleocytoplasmic ratio increases while having agglomerating aggregation growth tendency, and growth rate becomes faster, with training Feeding continuation metamorphosis can gradually increase, and form sharp contrast with the density and form of GFP control cell；

(5) it generally can be transferred on MEF in Day 5, prejudging second day cell can be close to preparing MEF when converging, and second day With Accutase vitellophag and count.For inoculating cell to 10 cm cell culture dishes, each ware inoculation 100,000 or 200,000 is thin Born of the same parents are cultivated with ES/DFBS 1:1 culture medium, change liquid daily；

(6) VPA to final concentration of 1mM is added every other day, it is continuous to add 7 days.Can be seen in culture dish since Day 6 has greatly The small clone of amount occurs, and clone shows smooth clear border, and diopter is high and cell nucleocytoplasmic ratio is high.But with the continuation of culture The gradually differentiation or dead these small clones since Day 9；

(7) clone that some ES forms can be seen since Day 16 gradually grows up, what some were a separately formed, some are It grows, is grown to wait clone when 10 times of object lens visual field after i.e. available Pasteur pipe picking among the clone broken up before For expanding and identifying.

2.4 iPSCs recovery

(1) recovery day before cell, culture cell orifice plate have been coated with Matrigel；

(2) water bath is first opened, temperature is set to 37 DEG C, when water bath temperature is stablized at 37 DEG C, is taken from liquid nitrogen container The cell frozen out unscrews cryopreservation tube lid, make wherein remaining liquid nitrogen vapor away, then tighten cryopreservation tube lid rapidly, quickly set In 37 DEG C of water baths, melts when pipe medium floe to taking-up when only remaining a mung bean size, quickly recover into desinfection chamber；

(3) desinfection chamber operation room completes station, takes a clean 15mL centrifuge tube, and addition freezes 9 times of liquid in pipe The good mTeSR1 of pre-temperature in advance of volume, then cryopreservation tube inner cell is drawn in the centrifuge tube for adding good culture medium, with 170g in room Temperature centrifugation 5 minutes；

(4) supernatant is gently sucked out to discard, adds appropriate mTeSR1 culture medium that cell precipitation is resuspended, Y27632 is added to final concentration It is 10 μM, after mixing, is seeded in the orifice plate for being coated with Matrigel in advance, cross shakes up in 37 DEG C of cell incubators of postposition and trains It supports.

2.5 iPSCs culture is passed on and is frozen

(1) after iPSCs recovery, maintain culture that need to change liquid daily, with mTeSR1 culture medium culture；

(2) when cell confluency degree reaches 70%, it is coated with Matrigel, needs to be coated with suitable orifice plate according to experiment.Coating It passes within second day after good Matrigel, is digested with 0.5mM EDTA when passage: culture medium is first sucked out and discards, addition can not have The 0.5mM EDTA rinsing of culture hole bottom surface twice, raffinate is blotted only, slightly volume 0.5mM EDTA is added and is stored at room temperature and disappear Change 3 minutes, inhale clean liquid, the desired amount of cell of appropriate mTeSR1 piping and druming is added and gets off, is seeded to and has been coated with Matrigel's In orifice plate, cross is shaken up, and is set in 37 DEG C of cell incubators and is cultivated；

(3) when cell confluency degree reaches 80%-90%, freeze-stored cell, digestion process and passage one be can according to need Sample, final step directly use frozen stock solution (90%mTeSR1+10%DMSO, in advance 4 DEG C of pre-coolings) that cell is resuspended, directly go to and freeze Guan Zhong is placed in -80 DEG C of ultra low temperature freezers in freezing storing box and stays overnight, is then transferred in liquid nitrogen container, makes a record.

The identification of 2.6 stem cell surface markers

(1) the HA-iPSCs passage by culture on Matrigel is seeded to the 24 of the cell climbing sheet for being coated with Matrigel In orifice plate, when continuing culture 2-3 days, identified by immunofluorescence is carried out；

(2) culture medium is discarded, DPBS is rinsed 2 times, exhausts raffinate, and 4% paraformaldehyde room temperature is added and fixes 15 minutes；

(3) raffinate is exhausted, PBST (DPBS+1/1000Triton-100) is added and is permeabilized 15 minutes；

(4) PBST is discarded, DPBS is rinsed 2 times -3 times, and 5%BSA is closed 30 minutes；

(5) Oct4/Nanog/SSEA-1/SSEA-4 primary antibody is diluted with 1:200 with confining liquid, primary antibody is incubated at room temperature 1 hour；

(6) primary antibody is discarded, after adding DPBS to rinse 2 times -3 times, DPBS is added and washes 3 times, every time 10 minutes；Again with 5%BSA Closing 30 minutes；

(7) secondary antibody is diluted with 1:300 with confining liquid, corresponding primary antibody kind selects suitable secondary antibody, and it is small that room temperature is protected from light incubation 1 When；

(8) liquid is discarded, after DPBS is rinsed 2 times -3 times, DPBS is added and washes 5 times, every time 5 minutes；

(9) plus 1 μ g/mL DAPI room temperature is protected from light and is incubated for 6min, after DPBS is rinsed 2 times -3 times, DPBS is added and washes 5 times, often Secondary 5 minutes；

(10) ultrapure water rinsing is primary, adds 90% glycerol mounting, nail polish edge sealing is taken pictures under laser confocal microscope It saves.

The detection of 2.7 caryogram

When HA-iPSCs convergence degree is to 80% in (1) six orifice plate, into culture medium plus colchicine is to final concentration 40ng/ ML is cultivated 5 hours in 37 DEG C of cell incubators；

(2) exhaust culture medium, and physiological saline rinsing is added twice, is digested 3 minutes, is added with 37 DEG C of 0.05%EDTA- pancreatin Enter culture medium and terminates digestion；

(3) cell was collected into 15mL centrifuge tube, with 1200g centrifugation 5 minutes；

(4) supernatant is abandoned, it is hypotonic that addition 8mL 0.07%KCl hypotonic medium is incubated for progress in 10 minutes in 37 DEG C of water baths；

(5) (glacial acetic acid and methanol are mixed according to the ratio of 3:1 for the hypotonic fixers that 37 DEG C of 1.5mL preheatings are added after the completion Close) gently piping and druming mixing, it sets in 37 DEG C of water baths 5 minutes, is pre-fixed；

(6) it pre-fixes after completing with 1200g room temperature centrifugation 10 minutes；

(7) it discards supernatant, the fixer of 37 DEG C of 8mL preheatings is added, piping and druming mixes；

(8) with 1200g room temperature centrifugation 10 minutes；

(9) it discards supernatant, repeats to fix once, gently inhale and abandon supernatant, proper amount of fresh fixer resuspension cell is added (to hair Hyaloid is muddy)；

(10) in drop piece separating apparatus in drip piece, number, 75 DEG C roasting piece 3 hours, carry out Giemsa staining, under the microscope, adopt Collect image.

Triploblastica breaks up in 2.8 bodies

(1) due to the presence of cell immunogenicity, heterograft will appear immunological rejection, therefore the iPSC of source of people is subcutaneous It is injected in nude mouse and carries out internal triploblastica Analytical Chemical Experiment.Prepare convergence degree in 2 6cm dish and reaches 80% or more HA-iPSCs；

(2) 37 DEG C of vitellophags of 0.05%EDTA- pancreatin about 5 minutes, after mTeSR1 culture medium terminates digestion, with 1200g Centrifugation 5 minutes exhausts supernatant, and cell is resuspended with 140 μ L mTeSR1 culture mediums, adds 70 μ L Matrigel mixing, sets ice On；

(3) nude mice for using 6-8 week old fixes after nude mice to its lower limb groin position Iodophor partly sterilised, uses Cell suspension is slowly at the uniform velocity subcutaneously injected to nude mice groin 1mL syringe, local compression after the completion of injection；

(4) nude mice is cut into toe label, and recorded；

(5) locally there is kick, visible apparent knurl after 6 weeks in about two weeks visible injection sites.At about 8 weeks, Teratoma it is long to diameter 1cm or so when, can perform the operation and harvest teratoma；

(6) it completely takes out, measure Tumor diameter and takes pictures together together with teratoma coating, 4% paraformaldehyde is fixed overnight After do paraffin tissue sections parallel HE dyeing.Dyeing can be observed and be taken pictures under an optical microscope after completing.

The detection of 3.CRISPR/Cas9 cleavage activity

In order to detect CRISPR/Cas9 cleavage activity, we turn CRISPR/Cas9 expression plasmid by nuclear transfection mode Enter into HA-iPSCs cell, express CRISPR/Cas9, when it plays dissection and generates DSB, cell passes through The mechanism reparation of NHEJ generates indels, can reflect this by ratio shared by target gene of the detection containing indels The cleavage activity of sgRNA.

3.1 nuclear transfection

1. HA-iPSCs is seeded in six orifice plates for being coated with Matrigel, cultivated using mTeSR1, about six orifice bores converge It can be used to practice shooting when right 90%；

2. changing within 2.5 hours before gene targeting liquid, the fresh cultured that final concentration of 10 μM of Y27632 are added is based on 37 DEG C of cultures Case continues to cultivate；

3. according to the consideration convey reagent Human Stem Cell of Lonza company2 specification of Kit carries out Operation first takes 82 μ L Solution II and 18 μ L Supplement I in kit to mix and is placed on equilibrium at room temperature temperature Degree；It is stored at room temperature 15 minutes, every pipe is separately added into the CRISPR/Cas9 expression plasmid of the corresponding sgRNA of an expression, is stored at room temperature 5 minutes；

4. utilizing TrypLE SELECT vitellophag: after DPBS is washed 3-4 times, TrypLE SELECT is added, sets 37 DEG C of trainings It supports and digest 3-5 minute in case, when microscopic observation cell clone edge curl, the termination of ES culture medium is added and digests, by cell pressure-vaccum Get off, be transferred in centrifuge tube, counted using blood cell counting plate, takes out the room cell suspension 170g for containing 1,000,000 cells Temperature centrifugation 5 minutes；

5. abandoning supernatant after centrifugation, exhaust culture medium raffinate, by step 3. in plus the liquid of good plasmid be added in centrifuge tube, weight Outstanding cell, is transferred in pole cup, carries out consideration convey using LONZA consideration convey instrument B-016 program, adds 400 μ L after completing immediately MTeSR1 is stored at room temperature 5 minutes into pole cup；

6. 1.5mL mTeSR1 is added in six orifice plates for completing Matrigel in advance, the cell in 5. middle pole cup is turned It moves in six orifice plates for adding good mTeSR1, Y27632 is added, after cross shakes up, puts 37 DEG C of incubator cultures；

7. consideration convey 12 hours, changing fresh culture, add Y27632, cultivate 3 days, extracts cell DNA.

3.2 gDNA extracting

(1) after rinsing the cell of DNA to be extracted 3 times using DPBS, 37 DEG C of pancreatin digest 5 minutes, and ES culture medium is added Digestion is terminated, cell is collected into 1.5mL centrifuge tube, with 5000g room temperature centrifugation 5 minutes；

(2) supernatant is abandoned after centrifugation, and 500 μ L nucleus lysates are added, add 1/200 Proteinase K and 1/1000RNase, After piping and druming mixes, 70 μ L 10%SDS are added, after being mixed by inversion, set in shaking table 37 DEG C, 100 turn over night cracking；

(3) cell of overnight cracking is taken out, isometric Tris- phenol is added, is mixed by inversion repeatedly, until occurring fine and closely woven equal It is centrifuged when even small oily particle, with 17000g room temperature centrifugation 10 minutes；

(4) after the completion of being centrifuged, supernatant liquid is sucked out into a new 1.5mL centrifuge tube for liquid in pipe layering, be added etc. The 1:1 phenol/chloroform mixed liquor of volume, is mixed by inversion repeatedly, is centrifuged when until forming uniform chyle shape particle, with the room 17000g Temperature centrifugation 10 minutes；

(5) after the completion of being centrifuged, liquid in pipe layering draws supernatant liquid into a new 1.5mL centrifuge tube, is added 2 times The dehydrated alcohol of volume pre-cooling, is mixed by inversion repeatedly, occurs being centrifuged when white filiform DNA using refrigerated centrifuge, with 17000g is centrifuged 10 minutes at 4 DEG C；

(6) after the completion of being centrifuged, supernatant is outwelled, 75% dehydrated alcohol that 500 μ L are pre-chilled in advance is added, is overturned 10 times, with 17000g is centrifuged 10 minutes at 4 DEG C；

(7) supernatant is outwelled, is put from exhaustion raffinate dries in superclean bench, and appropriate ddH20 dissolution is added.

Acquired gDNA is as template, sequence near PCR amplification cleavage site, and system is as follows: (F8-E14-sg1, It is expanded with F8-E14-sg2 group with F8-E14-F/R；F8-BDU-sg1 group is expanded with F8-BU-F/R；F8-BDD- Sg4 group is expanded with F8-BD-F/R)

Thermal cycle conditions are as shown in Figure 52.

3.3 are detected using sequencing approach

PCR product carries out electrophoresis, and glue recycles target fragment, and recovery product carries out carrier T connection, and linked system is as follows:

It takes 10 μ L to convert after 16 DEG C of water-bath connections overnight, and carries out blue and white screening, about 14 hours left sides of bacterial growth after conversion The right side, the bacterium colony of picking white shake or so 3 hours of bacterium and send sequencing to identify the bacterium solution, with general into the liquid LB added with ammonia benzyl Primer T7 is sequenced, and carries out sequence alignment using DNA star sequence alignment program, indels person nearby occurs in cleavage site It is calculated as dissector, calculates corresponding CRISPR/Cas9 cutting efficiency.

4.CRISPR/Cas9 combines ssODN consideration convey HA-iPSCs and N-iPSCs (with 3.1)

For small in-frame deletion in the missing area B, transfected plasmids are divided to two groups, wherein one group is 4 μ g F8-E14-sg1 With 4 μ g F8-E14-sg2, another group is 4 μ g F8-E14-sg1,4 μ g F8-E14-sg2 and 50pmol F8-E14-del54- SsODN is stored at room temperature 5 minutes；

For lacking entire B district's groups, transfected plasmids are 1 group, be 4 μ g F8-BDU-sg1,4 μ g F8-BDD-sg4 with 50pmol F8-BDD-ssODN is stored at room temperature 5 minutes；

After consideration convey, cultivates 3-5 days, when cell concentration is more, take 10000 cell counts to be seeded to MEF feeder layer thin On born of the same parents, to obtain monoclonal, remaining cell mixing freezes in case subsequent applications；

It is unicellular be seeded on MEF after, need to replace daily the culture of ES culture medium about 12 days or so, clonal growth to 10 times of mirrors When the visual field 1/2 to 2/3, by monoclonal picking into 48 orifice plates for being covered with Matrigel, clonal expansion is carried out；DNA is extracted to carry out PCR is identified to obtain positive colony.

4.1 gDNA extracting (with 3.2)

4.2 PCR Preliminary detection fixed point deletions

Small in-frame deletion in the area B, which is carried out, using F8-E14-F/R primer clones Preliminary Identification

It is as follows that CRISPR/Cas9 target practice fixed point deletion detects PCR system:

Thermal cycle conditions are shown in Figure 52.

PCR product carries out electrophoresis through 1% Ago-Gel, and the HA-iPSCs not practiced shooting or non-integrator cell of practicing shooting amplify 498bp band；The PCR product of positive fixed point deletion clone is 448bp size strip.The PCR for amplifying about 448bp band is produced Object send sequencing, using F8-E14-F as sequencing primer, sequence alignment is carried out using SeqMan software, to the clone accurately lacked completely It expanded, frozen, while carrying out next step experiment.For N-iPSCs, non-target practice person or non-integrator can be expanded The PCR product of 502bp band out, positive fixed point deletion clone is 448bp size strip.

The entire area B, which is carried out, using F8-BU-F/F8-BD-R primer targets deletion clone Preliminary Identification

Thermal cycle conditions are shown in Figure 53.

PCR product carries out electrophoresis through 1% Ago-Gel, and the HA-iPSCs not practiced shooting or non-integrator cell of practicing shooting amplify 3019bp band；The PCR product of positive fixed point deletion clone is 341bp size strip.The PCR of about 341bp band will be amplified Product send sequencing, using F8-BU-F as sequencing primer, sequence alignment is carried out using SeqMan software, to gram accurately lacked completely It is grand to be expanded, frozen, while carrying out next step experiment.

The accurate deletion clone stem cells surface marker identification of 4.3 fixed points, step is the same as 2.6.

The expression of 5.RT-PCR detection F8

1. by after HA-iPSCs and gene targeting iPSCs and N-iPSCs cultivated in 12 orifice plates, reached to convergence degree Cell total rna is extracted with Trizol method when to 80% or more；By (the N-del 54- of clone N-del 54 of N-iPSCs missing 54bp 15, N-del 54-42) it is cultivated in 12 orifice plates, cell total rna is extracted with Trizol method；Gram that the entire area B is lacked simultaneously Grand (BD21, BD25) is cultivated in 12 orifice plates, extracts cell total rna when convergence degree reaches 80% or more with Trizol method； The N-iPSCs clone N-BD (N-BD9, N-BD14) for lacking the entire area B is cultivated in 12 orifice plates, cell is extracted with Trizol method Total serum IgE；

2. utilizing the possible existing DNA of the digestion removal of DNase I of Thermo ScientificTM company；

3. taking 1 μ g total serum IgE to PCR pipe, is handled 10 minutes for 70 DEG C in PCR instrument, be placed in 5 minutes on ice, use Promega The Reverse Transcription System of company prepares reverse transcription system:

42 DEG C, 3Omin → 95 DEG C, the 5min of 5min → on ice

Reverse transcription is carried out in PCR instrument, response procedures:

4. it is as follows to prepare PCR system using the cDNA of previous step reverse transcription as template:

Reference gene GAPDH is GAPDH-F and GAPDH-R using primer (theoretical size is 453bp).For lacking 54bp For clone, F8 transcription detection passes through two pairs of RT primers: F8-E14-F/R, F8-RT-E23F and F8-RT-E26R carry out PCR.It is right For lacking the entire area B clone, F8 transcription detection passes through two pairs of RT primers: F8-RT-BDF/R, F8-RT-E23F and F8-RT- E26R carries out PCR.Thermal cycle conditions are shown in Figure 53.

5 μ L product agarose gel electrophoresis are taken to analyze.

6.ELISA detects FVIII expression

1. collecting HA-iPSCs of the culture in 12 orifice plates respectively, identification pinpoints accurate deletion clone 2-6,2-46 and just The culture supernatant of often control people iPSCs (N-iPSCs) 24 hours culture supernatant of cell, collection is centrifuged after five minutes with 5000g Take supernatant, 1/100 protease inhibitors be added, be stored in -80 DEG C it is spare.It is collected simultaneously the clone N- of N-iPSCs missing 54bp The supernatant of del 54 (N-del 54-15, N-del 54-42) collects the cell cultivated in orifice plate after supernatant and corresponds to counting；It is corresponding For the clone of the entire area B missing, and equally collects cell conditioned medium and count cell concentration；

2. the Paired Antibodies for ELISA-Factor VIII using CEDARLANE company is detected.

7.iPSCs orients endothelial cell differentiation

In order to which whether the gene repair strategy for detecting this research use is effective, need to carry out cylinder therapeutic effect evaluation, and Endothelial cell is the major cell types of synthesis and secretion FVIII, therefore is in proliferative capacity by iPSCs directed differentiation Skin progenitor cells provide advantage for research and clinical application before follow-up clinical.

Referring to existing document report [26], realize that iPSCs directed differentiation is that endothelium ancestral is thin by micromolecular inhibitor Born of the same parents are broken up using 6 μM of CHIR99021 in experiment, and specific differentiation step is as follows:

(1) iPSCs by culture in six orifice plates for being coated with Matrigel is first rinsed 3 times with DPBS, adds 500 μ L Accutase stands digestion 5 minutes in 37 DEG C of incubators, and mTeSR1 culture medium is added and terminates digestion, cell is collected to sterile It in 15mL centrifuge tube, is counted using blood cell counting plate, with 170g room temperature centrifugation 5 minutes, inhales and abandon supernatant, add appropriate culture Base weight hangs cell, is seeded in the culture orifice plate for being coated with Matrigel with 50,000 cell/cm2, and Y27632 is added to final concentration It is 10 μM, is cultivated with mTeSR1, cross shakes up in 37 DEG C of incubators of postposition and cultivates, and is denoted as Day-3 at this time；

(2) Day-2 and Day-1 only needs to replace culture medium mTeSR1 daily；

(3) when Day 0 replace culture medium LaSR basal medium add 6 μM of CHIR99021 (because CHIR99021 be using What DMSO was prepared, therefore fresh addition stand-by LaSR basal medium when use, carry out changing liquid after mixing)；

(4) Day 1 replaces culture medium as Day 0, and microscope can find that dead cell increases under the microscope；

(5) Day 2 starts, and LaSR basal medium is replaced daily, until Day 5；

(6) Flow cytometry progenitor endothelial cell surface marker CD31 and CD34 are carried out when Day 5, to detect differentiation Efficiency；

Day 5 can also carry out CD31 magnetic bead sorting simultaneously, obtain the higher endothelial progenitor cells of purity；

The sorting of 7.1 endothelial progenitor cells

The cell of differentiation Day 5 can carry out CD31 magnetic bead sorting:

(1) differential medium is discarded, DPBS is added and rinses 3-4 times, sucks raffinate；

(2) Accutase is added, 37 DEG C digest 5 minutes, and LaSR basal medium terminates digestion；

(3) cell is collected into a clean and sterile 50mL centrifuge tube, using cell sieve filtration cell agglomerate, with 170g room temperature Centrifugation 5 minutes；

(4) supernatant is abandoned, the MACS buffer 10mL of pre-cooling is added, cell is resuspended, with 170g room temperature centrifugation 5 minutes；

(5) it gently inhales and abandons supernatant, when remaining a small amount of raffinate, put from blotting only raffinate；

(6) 60 μ L MACS buffers are added, piping and druming mixes and then is added 20 μ L FcR Blocking Reagent, 20 μ L CD31MicroBeads piping and druming is added after mixing to mix；

(7) 4 DEG C of refrigerator accurate incubations 15 minutes are set；

(8) add the MACS buffer of 1mL pre-cooling after the completion of being incubated for, with 170g room temperature centrifugation 5 minutes；

(9) it inhales and abandons supernatant, cell is resuspended in the MACS buffer that 1mL pre-cooling is added；

(10) prepare sorting column, LS sorting column is fixed on MACS sorting adapter rack；

(11) first MACS buffer is added in LS sorting column with the MACS buffer balance sorting column of 3mL pre-cooling It is flowed through naturally to it, prepares 50mL centrifuge tube and collect filtrate；

(12) it in the LS sorting column after cell suspension to be added to balance, is flowed freely over to it, the MACS of pre-cooling is added Buffer is washed column 3mL and is washed 3 times, and the generation of bubble must be avoided in operating process；

(13) after having been flowed completely to liquid, LS sorting column is removed and is detached from magnetic field, prepare a sterile 15mL centrifugation Pipe；

(14) the MACS buffer of 5mL pre-cooling is added in LS sorting column, sorts the sterile work in column kit using LS Buffer is disposably quickly pushed through sorting column by plug, and is collected into 15mL centrifuge tube；

(15) 170g room temperature is centrifuged 5 minutes after counting cell suspension, is inoculated in the orifice plate for being coated with Collagen IV In, it is cultivated with EGM-2.

7.2 Flow cytometry endothelial progenitor cells differentiation efficiencies

(1) endothelial progenitor cells break up Day 5, carry out flow cytometer detection endothelial progenitor cells differentiation efficiency, are rinsed and are broken up with DPBS Cell 3 times, be added the Accutase of pre-temperature, 37 DEG C digest 5 minutes, and LaSR basal medium terminates digestion, by cell collect to In the 15mL centrifuge tube of sterile clean, with 170g room temperature centrifugation 5 minutes；

(2) after the completion of being centrifuged, liquid is discarded supernatant, 5mL DPBS is added, cell is resuspended, be centrifuged 5 points again with 170g room temperature Clock；

(3) it inhales and abandons supernatant, 400 μ L DPBS are added, cell is resuspended, and dispense the 1.5mL centrifuge tube completely to sterilize to 4 In, 100 μ L are dispensed in each 1.5 mL centrifuge tube；

(4) wherein a pipe is as blank control, and a pipe 20 μ L CD34-PE Antibody of addition, gently piping and druming mixes, and sets It is protected from light incubation 30 minutes on ice, 20 μ L CD31-FITC Antibody are added in a pipe, and gently piping and druming mixes, and set to be protected from light on ice and incubate It educates 30 minutes, there are also a pipes, and 20 μ L CD34-PE Antibody and 20 μ L CD31-FITC Antibody are added, and gently blows and beats After mixing, sets and be protected from light incubation 30 minutes on ice；

(5) after antibody incubation is completed, 1mL DPBS is added in every pipe, after gentle inversion mixes, with 5000g room temperature Centrifugation 5 minutes；

(6) it after centrifugation is completed, gently inhales and abandons supernatant, 300 μ L DPBS are added, cell is resuspended, sample is set and is protected from light on ice Hematology, Qu Xiangya hospital fluidic cell room is detected, using in BD FACSCaliburTM Flow Cytometer instrument CellQuest Pro software is detected, statistical data analysis.

The 7.3 Flow cytometry efficiencies of separation (step is with 7.2)

Endothelial progenitor cells break up Day 5 after the sorting of CD31 marked by magnetic bead antibody, and it is thin to carry out flow cytometer detection endothelium ancestral Born of the same parents' efficiency of separation.

7.4 endothelial cell immunotoxin Fluorescence Identification surface markers

(1) endothelial progenitor cells of the culture after the sorting on Collagen IV room temperature is seeded to be coated with In 24 orifice plates of the cell climbing sheet of Matrigel, when continuing culture 1 day and 7 days, identified by immunofluorescence is carried out respectively, step is same 2.6, the marker detected when cultivating 1 day is the diluted CD31/CD144/CD34 primary antibody of 1:100；The mark detected when cultivating 7 days Remember that object is the diluted vWF of 1:100 and N-terminal FVIII antibody and C-terminal FVIII antibody.

8. the detection of endothelial cell stage F8 expression

The expression of 8.1 RT-PCR detection F8

(1) endothelial cell differentiated is cultivated in 12 orifice plates, when convergence degree reaches 80% or more with Trizol method extracts total serum IgE；Remaining step is the same as step 5.

8.2 ELISA detect FVIII expression

Culture is collected respectively in 12 orifice plates by 24 hours culture supernatants of the iPSCs endothelial cell differentiated, is collected Culture supernatant supernatant is taken after five minutes with 5000g centrifugation, 1/100 protease inhibitors is added, be stored in -80 DEG C it is spare.It receives Collect the corresponding counting of cell in supernatant metapore；Utilize the Paired Antibodies for ELISA- of CEDARLANE company Factor VIII is detected.

9.Western blot detects intracellular LMAN1 expression

9.1 extract total protein of cell

(1) cell culture medium in net 12 orifice plate is blotted, DPBS is added and rinses 2-3 times；

(2) 200 μ L RIPA lysates (1/100 protease inhibitors is added) is added, is digested 5 minutes in standing on ice, it Postdigestive cell lysate is scraped and is drawn in 1.5mL centrifuge tube by pipette tips in afterwards；

(3) ultrasound is carried out to cell lysate using Ultrasound Instrument to interrupt, ultrasonic probe is inserted under liquid level, is persistently surpassed Sound 5 seconds, stand 5 seconds, repeatedly 5-6 times；

(4) 95 DEG C are denaturalized for heating 10 minutes；

(5) it is centrifuged 10 minutes with 14000g at 4 DEG C, supernatant is sucked out into a clean 1.5mL centrifuge tube, mark is performed Note, is stored in -80 DEG C.

9.2 BCA protein quantifications

(1) albumen for taking 9 μ L to extract is added RIPA lysate according to 1:4 and is diluted to albumen；

(2) simultaneously, take BSA standard items (protein concentration 2mg/mL) according to proportional diluted, totally 6 gradients, 2mg/mL, 1mg/ ML, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0mg/mL are diluted with RIPA lysate；

(3) all samples and standard items detection are respectively provided with two multiple holes, and required BCA is calculated according to hole count to be detected The amount of liquid is detected, every hole needs to be added 200 μ L BCA detection liquid.BCA detection liquid is divided into A liquid and B liquid, and working solution is A liquid and B liquid It is mixed with 1:50, after being mixed by inversion, each hole of ELISA Plate is added 200 μ L BCA and detects liquid, and 20 μ L measuring samples or standard is added Product.It should be noted that avoiding bubble from generating during sample-adding；

(4) ELISA Plate of sample to be tested and BCA detection liquid will have been added to be placed in 37 DEG C of water isolation type constant incubators and has been incubated for 30 Minute；

(5) it is incubated for after completing, the corresponding absorbance of test sample under optical wavelength is absorbed with 570nm in microplate reader, lead to It crosses standard items and makes standard curve, the concentration of sample is conversed in conjunction with dilution.The R2 of standard curve fit curve must be greater than 0.99, it is accurate for can prompting this time to detect；

(6) different samples are uniformly diluted to same concentration using RIPA lysate, then with 5 × loading buffer It is mixed according to 4:1 and protein sample；

(7) 100 DEG C are boiled 10 minutes, and albumen can carry out electrophoresis.

9.3 Western blot

(1) prepare polyacrylamide gel (10% separation gel): wash clean glass plastic plate is fixed in Bio-Rad It on gum-making rack, according to preparing separation gel in following table, and mixes, soft to be added between offset plate, about 7mL, upper layer is added and goes Ionized water sealing；

(2) upper layer concentration glue (5%) is prepared after gelling to be separated is solid: according to preparing concentration glue in following table, and It mixes, the deionized water that sealing is used in (1) step is outwelled, concentration glue is added, is inserted into comb teeth；

(3) electrophoresis: 1 × electrophoresis liquid is added in Vertial electrophorestic tank, the glue connection prepared is put into togerther electrophoresis tank with offset plate Interior, short glass plate pulls out comb teeth towards inside, and the albumen after taking 20 μ g to add loading buffer is added in loading wells, 5 μ L pre-stained protein ladder are added in one of loading wells, with 80V, about 30 minutes or so electrophoresis, to When protein sample electrophoresis is into separation gel, voltage is adjusted to 120V, (electrophoresis time was according to stripe size and glue in about 60 minutes for electrophoresis Concentration determine)；

(4) transferring film: electrophoresis terminates preceding about 20 minutes preparation 1 × transferring film liquid, is placed in -20 DEG C of pre-coolings.It, will after electrophoresis Glue takes out, and chooses destination region and cuts away the glue of surrounding, is cut according to the size of the PAGE glue to transferring film of the same size with it PVDF film is dipped in methanol and activates 5 minutes, later, glue and pvdf membrane is dipped to 1 × transferring film of pre-cooling by pvdf membrane In liquid, transferring film folder black is placed in the following, be sequentially placed foam-rubber cushion, double-layer filter paper, glue, pvdf membrane, double-layer filter paper, foam-rubber cushion, Transferring film folder white is covered into clamping, is placed in transferring film slot, transferring film folder black side is corresponding with black side in slot, in white face and slot Red is corresponding, and the Bioice ice chest that -20 DEG C are pre-chilled is put into transferring film slot, electrode is plugged, with 252mA constant current transferring film 90 Minute；

(5) primary antibody is incubated for: it is after transferring film finishes, film is face-up, it is put into and washes in bellows, with 0.1%TBST rinsing one Secondary, with 5% skim milk, room temperature is closed 1 hour on decolorization swinging table, after having closed, what addition was prepared with 5% skim milk Primary antibody gently shakes overnight (β-actin is diluted with 1:10000, and LMAN1 is diluted with 1:1000) for 4 DEG C on decolorization swinging table；

(6) secondary antibody is incubated for: recycling primary antibody is shaken washing 3 times on decolorization swinging table with 0.1%TBST, 10 minutes every time, added Enter the secondary antibody prepared with 5% skim milk to shake incubation on decolorization swinging table (anti-mouse secondary antibody was diluted with 1:10000, anti-rabbit in 1 hour Secondary antibody is diluted with 1:10000)；

(7) develop: after secondary antibody has been incubated for, shaken on decolorization swinging table with 0.1%TBST wash 3 times, 10 minutes every time, ECL developer solution is prepared, A liquid and B liquid are mixed with 1:1, are added dropwise on film, and room temperature is protected from light incubation 3-5 minutes, and film is put into Develop in Biorad visualizer, according to the band brightness adjustment time for exposure, saves picture.

10.EPCs carries out transplanting in HA model mouse body

HA model mice is the F8tm1Kaz strain bought from the laboratory Jackson^[28].The strain Mice homozygous will appear Joint or soft tissue hematostaxis, but when the pregnancy production of pregnant mouse generally will not bleeding, no production difficulty.Strain HA model Mouse shows the key feature of HA, for HA research and probe into strategies in gene therapy and provide a good model.

It takes 6-8 week old female mice hero mouse fifty-fifty, divides the correspondence of mouse needed for each experimental group to cage before being tested, same gender is small Mouse is distributed into a cage, carries out cell transplantation experiment after mouse adapts to 2-3 days.

Cell transplantation experiment: by the endothelial progenitor cells in culture 1-3 days after the sorting of CD31 magnetic bead antibody with DPBS rinsing 3 After secondary, Accutase is added, is digested 5 minutes in 37 DEG C of incubators, 5%FBS/DPBS is added and terminates digestion, collects cell extremely It in sterile 15mL centrifuge tube, is counted using blood cell counting plate, with 170g room temperature centrifugation 5 minutes, inhales and abandon supernatant, be added Cell is resuspended in 5mL DPBS, with 170g room temperature centrifugation 5 minutes, inhales and abandons supernatant, puts from blotting net supernatant liquid, every 2,000,000 cell Add 100 μ L DPBS that cell is resuspended, is very much a pipe by 200, is transplanted for HA mouse cell.

It is anaesthetized, is injected with 10 μ L avertins (25mg/mL)/g (mouse weight) intraperitoneal injection hemophilia A model mice After about 5 minutes later fall in a swoon to mouse anesthesia, 100 i.e. 2,000,000 endothelial progenitor cells of μ L cell suspension, note are inhaled using insulin needle Bubble need to be discharged in meaning, be injected in Mice Body by vena orbitalis posterior, mouse is put back to cage, be placed on IVC frame to its revival It is raised on son, every mouse peritoneal injects 40 μ L FK506 and carries out immunosupress after 24 hours, carries out every two days later primary FK506 injection.

The correction of the area B excalation type gene is divided into 8 groups in this experiment:

HA model mice group, does not inject cell, only injects FK506；

100 μ L DPBS vena orbitalis posteriors are injected in HA model mice by DPBS group, other processing are all the same；

HA-iEPCs group, i.e. 2,000,000 HA-iEPCs groups of injection；

2-6-iEPCs group injects the 2000000 endothelial progenitor cells groups differentiated by 2-6-iPSCs；

2-46-iEPCs group injects the 2000000 endothelial progenitor cells groups differentiated by 2-46-iPSCs；

The endothelial progenitor cells group that N-iEPCs group, i.e. 2,000,000 normal control N-iPSCs of injection differentiate；

The endothelial progenitor cells that N-del 54-42-iEPCs group, i.e. 2,000,000 N-del 54-42-iPSCs of injection differentiate Group；

C57BL/6 group, i.e. wild-type mice group, do not inject cell, only inject FK506.

The complete gene deletion type correction in the area B is divided into 8 groups in this experiment:

HA model mice group, does not inject cell, only injects FK506；

HA-iEPCs group, i.e. 2,000,000 HA-iEPCs groups of injection；

BD21-iEPCs group injects the 2000000 endothelial progenitor cells groups differentiated by 2-6-iPSCs；

BD25-iEPCs group injects the 2000000 endothelial progenitor cells groups differentiated by 2-46-iPSCs；

The endothelial progenitor cells group that N-BD14-iEPCs group, i.e. 2,000,000 N-del 54-42-iPSCs of injection differentiate；

11. blood coagulation activity detection and docking experiment after cell transplantation

At cell transplantation 14 days, after avertin anesthesia mouse is injected intraperitoneally, blood is taken by eyeball, sodium citrate is anticoagulant, point From blood plasma, FVIII blood coagulation activity in blood plasma is detected.The mouse after transplanted cells 14 days is taken simultaneously, after avertin intraperitoneal anesthesia Using pin fixing limbs, 75% alcohol disinfecting abdomen cuts off skin and peritonaeum, will cut off at auricle, quickly from the apex of the heart About 25mL DPBS is injected, DPBS is changed to 4% paraformaldehyde when the liquid wait flow out substantially is presented transparent, is infused from the apex of the heart It injects in Mice Body, stops perfusion when the protein-denatured performances such as tail tilting, four limbs spasm occurs in mouse, by Mouse Liver The major organs such as dirty, heart, spleen, lungs and kidney are taken out, and are placed in 4% paraformaldehyde and fix, and change into after 24 hours 15% sucrose is dehydrated 24 hours, is changed 30% sucrose into later and is dehydrated 24 hours progress frozen sections, thickness is cut to 15-20 μm, cuts 55 DEG C roasting piece 30 minutes after complete, piece can be used for immunofluorescence dyeing.

Docking experiment: by the mouse after cell transplantation 14 days, after avertin intraperitoneal anesthesia, apart from mouse mouse Rat-tail is cut at the position of tail end mousetail diameter about 1.5mm, its vertical natural is allowed to bleed 5 minutes, and it is close to press the broken ends of fractured bone later Stop blooding within 1 minute at the heart, then puts back in cage and observe and record the mouse survival time.

12. mouse tissue frozen section immunofluorescence dyeing

(1) it will be taken out by the frozen tissue section of roasting piece and carry out immunofluorescence dyeing, to confirm that it is small that human archeocyte enters Mouse liver, due to we inject be endothelial progenitor cells, main surface marker has CD31/CD144/vWF, pre- by early period Experimental identification vWF antibody can distinguish source of people and source of mouse, therefore immunofluorescence carries out the dyeing of anti-human vWF antibody in this research；

(2) it fixes, thus can be directly permeabilized because tissue has already passed through paraformaldehyde, in the organized block of slide Position add PBST (DPBS containing 1/1000Triton-100) to be permeabilized 17 minutes；Remaining subsequent step is the same as 2.6.

Embodiment

1. it is total to miss out four bases comprising missing for the HA patient mutations' location proximate c.3167delCTGA to make a variation The segment of 54 bases, so that frameshift mutation be made to restore normal reading code:

(1) Hemophilia A patients' specificity iPSCs identifies (HA-iPSCs)

Have collected patient's (c.3167delCTGA leading to p.D1055fsX5) urine that an example contains the area B frameshift mutation HA early period Liquid sample, and epithelial cell therein (expression ZO-1, KRT7, β-catenin) (Fig. 4 A) is cultivated, and then reprogrammed and be IPSCs, form is cloning growth, and Edge divider, nucleocytoplasmic ratio are high (Fig. 4 B)；Caryogram detection has no that Chromosome level makes a variation (Fig. 4 C), the iPSCs come to induction are sequenced, it was demonstrated that it still retains patient's special c.3167delCTGA variation (figure It 4D), is good disease cells model.By identified by immunofluorescence stem cell surface marker, can express NANOG, OCT4, SSEA-4 does not express SSEA-1, meets stem cell surface marker expression characteristic (Fig. 4 E)；Break up by internal triploblastica, hair The iPSCs now induced can be in nude mouse at teratoma, the interior alimentary canal containing entoderm of HE dyeing display teratoma Epithelial cell, mesoderm cartilaginous tissue and ectodermic neural epithelium sample tissue (Fig. 4 F).

(2) building, Efficiency testing and the core of the CRISPR/Cas9 expression plasmid of 54bp are accurately lacked for mutational site Turn HA-iPSCs and obtains the accurate deletion clone of fixed point

The applicant has introduced CRISPR/Cas9 gene editing system from the laboratory Zhang Feng, for patient spy Anisotropic variant sites design sgRNA, and combine ssODN and carry out gene therapy in situ in B region mutation type HA-iPSCs (as schemed 5A)；Cutting efficiency detection (Fig. 5 B) is carried out to the CRISPR/Cas9 of building in the way of carrier T connection sequencing；Joint mapping Good CRISPR/Cas9 and ssODN carries out gene targeting to HA-iPSCs, is tentatively reflected by PCR to the clone after target practice It is fixed, it randomly selects two 2-6 and 2-46 and carries out subsequent experimental, PCR qualification result is as shown in Figure 5 C；PCR product is carried out Sanger sequencing, sequencing confirm that the two clones have accurately lacked 54 bases (figure including making a variation 4 bases comprising patient 5D)。

Joint CRISPR/Cas9 and ssODN carries out gene targeting to HA-iPSCs, while CRISPR/Cas9 is used alone To HA-iPSCs carry out consideration convey, it is unicellular be seeded on MEF feeder layer after, picking monoclonal, amplification after extract DNA, lead to PCR, sequencing identification are crossed, the positive colony number of acquisition is counted, as shown in following table (table 2), joint ssODN group target practice efficiency is 14.71%, and it is 2.08% that CRISPR/Cas9 person's target practice efficiency, which is used alone,.This indicate that joint CRISPR/Cas9 with SsODN mediates the more efficient of accurate genetic fragment missing.

The joint of table 2 CRISPR/Cas9 is with ssODN compared with independent CRISPR/Cas9 consideration convey HA-iPSCs efficiency

(3) joint CRISPR/Cas9 and ssODN consideration convey N-iPSCs accurately lack 54bp

In order to avoid the genetic background of HA-iPSCs influences, while it is accurate to carry out 54bp segment to normal control N-iPSCs Missing.Since joint CRISPR/Cas9 and ssODN mediates the more efficient of accurate genetic fragment missing, N-iPSCs is carried out Accurate missing 54bp is carried out by the way of combining CRISPR/Cas9 and ssODN.After consideration convey, connect cell mixing is unicellular In kind to MEF, when cell clone grows to ten times of mirror visual field 1/2-2/3 or so, picking monoclonal, amplification, extracting DNA, PCR Identification, PCR amplification go out 448bp band person and send sequencing, and sequencing is accredited as the clone that positive is accurate genetic fragment missing, N-del 54-15-iPSCs and N-del 54-42-iPSCs are selected at random carries out subsequent experimental (Fig. 6).

(4) the positive colony identified by immunofluorescence that target practice obtains

Stem cell surface marker identified by immunofluorescence, the positive colony obtained after target practice are carried out to the positive colony of acquisition The surface marker consistent with embryonic stem cell can be expressed, Oct4, Nanog, SSEA-4 expression are positive, SSEA-1 expression yin Property (Fig. 7 and Fig. 8), it was demonstrated that target practice process does not have an impact iPSCs characteristic.

(5) the positive colony caryogram obtained of practicing shooting detects

Gene repair clone passage 10 instead of carries out caryogram detections afterwards, with practice shooting before consistent, caryogram 46, XY, (Fig. 9 and It Figure 10) is shown in Chromosome level and has no obvious variation.

(6) iPSCs stage F8 detection of expression

In the iPSCs stage, the inspection of F8 transcriptional level is carried out by positive colony of the RT-PCR to the missing 54bp obtained that practices shooting It surveys (Figure 11 and Figure 12), discovery lacks at the iPSCs stage, 14 exon of precise fragments deletion clone transcriptional level to be accurate Mistake type, patient and reparation, which are cloned, and 23 exon of normal person is to the transcription between 26 exons band and stripe size Unanimously, illustrate that the RNA degradation of nonsense mediation does not occur for the patient, and it is reported in the literature consistent^[29]。

Secretory volume of the FVIII in cytoplasm in expression quantity and culture supernatant is detected by ELISA, is found in iPSCs rank Section FVIII secretory volume is lower (Figure 13).Have in document report [30] FVIII secretion process and needs among endoplasmic reticulum-Golgi The participation of body, this intermediate are the complex that LMAN1 and MCFD2 is formed.Clinically any one albumen homozygous mutation can all be led The joint of FV and FVIII is caused to lack, we have detected the expression (Figure 14) of LMAN1 by Western blot, find in iPSCs LMAN1 protein expression is had no in phase cell, this can explain the lower phenomenon of iPSCs stage FVIII secretory volume.

(7) iPSCs orients endothelial progenitor cells differentiation and the F8 detection of expression in endothelial cell stage

The cell broken up the 5th day carries out flow cytometer detection using CD31-FITC/CD34-PE, to determine that endothelial progenitor cells break up Efficiency.Homophyletic cell differentiation efficiency does not have difference as seen from Figure 15, and CD31/CD34 double positive cells proportion is about For 10%-30%, the endothelial progenitor cells differentiated have proliferative capacity, this is obtained for us largely can be used in transplanting Skin progenitor cells provide possibility.

Using CD31 magnetic bead by after endothelial progenitor cells sorting purifying, streaming inspection is carried out using CD31-FITC/CD34-PE Survey, with determine we used in sort system separating effect, as shown in figure 16, the cell after sorting is seen from form It examines, is epithelioid cell's representative configuration, cell is uniform, and growth rate is fast, CD31/CD34 double positive cells institute after sorting Accounting example is greater than 90%, and the sorting system for illustrating that we use is feasible, and the endothelial progenitor cells purity obtained after sorting is preferable, has Conducive to the subsequent experiment of progress.

Endothelial progenitor cells after sorting carry out Immunofluorescence test relevant surfaces marker, as shown in figure 17, CD31/ CD144/CD34 expresses the positive, further confirms that the cell after sorting is endothelial progenitor cells.

When endothelial progenitor cells after sorting were with EGM-2 culture medium culture 6-7 days, the growth rate of endothelial cell slows down, And mature endothelial cell marker vWF expression (Figure 18) can be detected.

Endothelial cell can synthesize FVIII, and can synthesize vWF, be stored in Weibel-Palade corpusculum, work as blood vessel When damaging, vWF and FVIII discharges jointly and in conjunction with stable vWF/FVIII are formed, and avoid FVIII by fast degradation, To more effectively participate in hemostasis.Therefore we are detected by dyeing vWF with N-terminal FVIII antibody and C-terminal FVIII antibody The expression and positioning of FVIII in the cell.Such as Figure 19, terminated in advance because Patient cells express FVIII, thus in HA-iPSCs It can detecte N-terminal FVIII positive signal, the maturation of gene repair clone and normal person source in the endothelial cell of induction Endothelial cell co-expresses FVIII and vWF.But patient iPSCs induces the mature endothelial cell C-terminal FVIII differentiated Dyeing has no obvious positive signal (Figure 20), and the endothelial cell that gene repair clone and normal iPSCs are differentiated not only may be used To detect N-terminal FVIII, while it may also detect that C-terminal FVIII, this is also confirmed used by this research from protein level The validity of correcting strategy.

Secretory volume of the FVIII in mature endothelial cell endochylema in expression quantity and culture supernatant is detected by ELISA, is such as schemed 21, compared with the iPSCs stage, mature endothelial cell stage FVIII secretory volume increases, and detects LMAN1 albumen in endothelial cell Expression, discovery LMAN1 albumen expresses (Figure 22) in endothelial cell, thus endothelial cell stage FVIII secretory volume is higher than The iPSCs stage.

(8) endothelial progenitor cells carry out transplanting in HA model mouse body

The each group endothelial progenitor cells differentiated are passed through into vena orbitalis posterior according to grouping and dosage described in method and step Carry out mouse cell transplantation in vivo, transplant HA-iEPCs, C-iPSCsization and EPCs (2-6-iEPCs and 2-46-iEPCs), The EPCs (N-iEPCs) that normal control N-iPSCs is differentiated, N-iPSCs lack 54bp Immune Clone Selection wherein N-del 54- N-del 54-42-iEPCs that 42-iPSCs is differentiated carries out transplanting in Mice Body, totally five groups of cell transplantation groups and one group It injects DPBS as a control group, when transplanting 14 days, takes each group mice plasma, detect FVIII blood coagulation activity, we are small with each group The practical blood coagulation activity of mouse accounts for the ratio of wild-type mice blood coagulation activity to show every group of blood coagulation activity after transplanting.Such as Figure 23 institute Show, 2-6-iEPCs and 2-46-iEPCs transplantation group blood coagulation activity is apparently higher than DPBS group and HA-iEPCs transplantation group, with N- IEPCs and N-del 54-42-iEPCs group no significant difference, experiment in vivo confirm that the correcting strategy that we use is effective.

Judge the therapeutic effect of the strategy by docking experiment, to its time-to-live after mouse docking, by Figure 24 with 25 as can be seen that 2-6-iEPCs, 2-46-iEPCs, N-iEPCs and N-del 54-42-iEPCs group mouse survival time it is obvious It is longer than HA-iEPCs group, it is notable that survivorship curve can be seen that at the end of docking Germicidal efficacy (48 hours), 2- It is survived after thering is mouse to experienced docking experiment in 6-iEPCs, 2-46-iEPCs and N-del 54-42-iEPCs group, this Treatment for disease is significantly.There are 4 survivals in the mouse of 12 experience docking experiments in 2-6-iEPCs group Get off, there are 4 to survive in the mouse of 10 experience docking experiments in 2-46-iEPCs group, N-del 54-42-iEPCs group In 9 experience docking experiment mouse in there is 1 to survive.This just further demonstrates the validity of gene repair strategy.

In the human archeocyte of mouse vivo detection transplanting

By endothelial progenitor cells after vena orbitalis posterior is injected in Mice Body, the cell of these source of people needs to enter and be colonized In in Mice Body, more lasting effect can be just played.The liver of transplanted cells group mouse is taken out after perfusion, into Row frozen section, using CD31 mark endothelial cell, then by anti-human vWF antibody carry out immunofluorescence dyeing, by with DPBS Negative control group compares, and observes that we have injected in the mouse liver slice of cell and is observed that vWF stained positive Cell, and positive signal (Figure 26) is had no in DPBS group.Other major organs of mouse, heart, spleen, lungs, kidney are taken simultaneously Dirty and brain tissue, by frozen section and immunofluorescence dyeing, discovery has also discovered human archeocyte (figure in the lungs of mouse 27), this provides experiment basis for the therapeutic strategy long-term effectiveness.

2. for the HA patient iPSCs targeting missing entire area the B coded sequence c.3167delCTGA to make a variation, to make to move Code mutation restores normal reading code:

(1) building of the CRISPR/Cas9 expression plasmid of the targeting missing area B coded sequence, Efficiency testing

According to sgRNA primer sequence is synthesized shown in target practice schematic diagram (Figure 28), pX330 skeleton is connected to by annealing In, two CRISPR/Cas9-sgRNA are constructed, combines a ssODN using two CRISPR/Cas9-sgRNA and accurately lacks B Structural domain, while retaining SQ sequence, SQ sequence is 741-743 amino acids and 1638-1648 amino acids, 1638 amino acids For glutamine, 1639 amino acids stay 744 and 745 ammonia due to the limitation of PAM sequence for asparagine in the research Base acid, it is glutamine and asparagus fern that gene order CAGAAT is consistent with 1638 and 1639 CAAAAC coding amino acid Therefore amide is equivalent to and introduces two same sense mutations.I.e. accurate missing 2678bp for iPSCs patient-specific for HA The segment of size makes reading frame reading code restore normal to correct frameshift mutation.It is i.e. accurate for normal control iPSCs Lack the segment of 2682bp size, the iPSCs of the building original position area B missing.

CRISPR/Cas9 Efficiency testing

The NHEJ ratio generated using the method detection CRISPR/Cas9 that carrier T connection PCR product is sequenced again, with This efficiency to prompt CRISPR/Cas9.The cutting efficiency of F8-BDU-sg1 is 13.33% (Figure 29 A), and F8-BDD-sg4's cuts Cutting efficiency is 35.71% (Figure 29 B).

(2) joint CRISPR/Cas9 and ssODN carries out gene targeting to iPSCs

Joint CRISPR/Cas9 and ssODN carries out HA-iPSCs consideration convey to obtain the thin of the accurate genetic fragment missing in the area B Born of the same parents.Consideration convey is carried out to HA-iPSCs using Lonza consideration convey instrument, after consideration convey, by after cell count with unicellular inoculation, picking Monoclonal simultaneously expands, and extracts cell genomic dna, PCR identification, while identifying with two pairs of primers, is expanded with F8-E14-F/R Increase, the clonal expansion not shaping band of the area B missing is expanded with F8-BUF/BDR, and the clone of the area B missing can amplify The band of 341bp size.PCR amplification goes out 341bp band person and PCR product is sequenced, and carries out by primer of F8-BD-R Sanger sequencing, sequencing are accredited as the cell that positive is the accurate missing area B.Selecting two is accredited as accurate gene piece at random The clone BD21-iPSCs and BD25-iPSCs (Figure 30) of section missing carry out next step experiment.

In order to avoid the genetic background of HA-iPSCs influences, the area B piece is carried out to normal control N-iPSCs simultaneously in an experiment Section missing.It is identified simultaneously with two pairs of primers, is expanded with F8-E14-F/R, the clonal expansion not shaping band of the area B missing, with F8-BUF/BDR is expanded, and the clone of the area B missing can amplify the band of 341bp size.PCR amplification goes out 341bp band PCR product is sequenced in person, carries out Sanger sequencing by primer of F8-BD-R, it is accurate lack that sequencing, which is accredited as positive, Lose the cell in the area B.N-BD9-iPSCs and N-BD14-iPSCs is selected at random carries out subsequent experimental (Figure 31).

(3) area the B deletion clone identified by immunofluorescence obtained of practicing shooting and caryogram detection

Stem cell surface marker identified by immunofluorescence is carried out to the positive colony of the area the B missing of acquisition, is obtained after target practice Positive colony can express the surface marker consistent with embryonic stem cell, Oct4, Nanog, SSEA-4 expression are positive, SSEA-1 expresses negative (Figure 32 and Figure 33), it was demonstrated that target practice process does not have an impact iPSCs stem cell properties.

The area B deletion clone passage 10 instead of carries out caryogram detection afterwards, preceding consistent with practicing shooting, caryogram 46, XY (Figure 34 and figure 35) it is shown in Chromosome level and has no obvious variation.

(4) iPSCs stage F8 detection of expression

In the iPSCs stage, the detection of F8 transcriptional level is carried out by positive colony of the RT-PCR to the area the B missing obtained of practicing shooting (Figure 36 and Figure 37) has found that the area B deletion clone transcriptional level goes out 552bp across 14 exon primer amplifications in the iPSCs stage Band, and patient and normal control do not amplify band；Patient and the area B deletion clone and 23 exon of normal control are to 26 Transcription between exon has band and stripe size is consistent, illustrates that the RNA degradation of nonsense mediation does not occur for the patient, has Frameshift mutation causes protein translation to terminate in advance in the area document report B, but do not occur nonsense mediation RNA degradation, this research and Result reported in the literature is consistent^[29]。

Secretory volume of the FVIII in cytoplasm in expression quantity and culture supernatant is detected by ELISA, is found in iPSCs rank Section FVIII secretory volume is lower (Figure 38).The participation of endoplasmic reticulum-Golgi intermediate is needed in FVIII secretion process, among this Body is the complex that LMAN1 and MCFD2 is formed^[30].The expression (Figure 39) that LMAN1 is had detected by Western blot, LMAN1 protein expression is not detected in iPSCs phase cell, thus, it may be possible to due to needed for iPSCs stage FVIII secretion GAP-associated protein GAP does not express to keep iPSCs stage FVIII secretory volume lower.

(5) iPSCs directed differentiation is endothelial progenitor cells

The iPSCs that the area B is lacked passes through addition little molecules in inhibiting agent method^[26]Directed differentiation is endothelial progenitor cells, is being divided Pass through flow cytometer detection endothelial progenitor cells differentiation efficiency when changing the 5th day.As shown in Figure 40, homophyletic cell differentiation efficiency is not slightly poor Not, CD31/CD34 double positive cells proportion is each about 15%-30%.Since endothelial progenitor cells have proliferative capacity, thus Enough endothelial progenitor cells can be obtained by culture amplification.

The endothelial progenitor cells differentiated are sorted using CD31 magnetic bead, the endothelial progenitor cells after sorting are by exempting from Epidemic disease fluorescence detection cell surface marker, as shown in figure 41, CD31/CD144 expresses the positive.

When endothelial progenitor cells after sorting were with EGM-2 culture medium culture 6-7 days, the growth rate of endothelial cell slows down, And mature endothelial cell marker vWF expression (Figure 42) can be detected.

(6) endothelial cell stage F8 detection of expression

In the endothelial cell stage, F8 transcriptional level is carried out by positive colony of the RT-PCR to the area the B missing obtained of practicing shooting It detects (Figure 43 and Figure 44), it can be observed that the area B deletion clone transcriptional level draws across 14 exons in the endothelial cell stage Object amplifies 552bp band, and patient and normal control do not amplify band；Patient and the area B deletion clone and normal control 23 exons have band to the transcription between 26 exons and stripe size is consistent.

Endothelial cell is not only the major cell types of synthesis FVIII^[31,32], and vWF can be synthesized, it is stored in In Weibel-Palade corpusculum, when angiogenesis damage, vWF and FVIII discharges jointly, can be to avoid FVIII by fast prompt drop Solution, to more effectively participate in hemostasis.In this project by dyeing vWF and N-terminal FVIII antibody and C-terminal FVIII antibody come Detect the expression and positioning of FVIII in the cell.Because frameshift mutation occurs in patient's F8 gene, translation is caused to terminate in advance, such as Chapter 1, it can detecte N-terminal FVIII positive signal in HA-iECs shown in detection, but be the failure to detect C-terminal FVIII sun Property signal.Regardless of the endothelial cell that the area the B deletion clone for being derived from patient differentiates or the B for being derived from normal person The mature endothelial cell that area's deletion clone differentiates while can detecte N-terminal FVIII (Figure 45) and C-terminal FVIII (figure 46) positive signal.This also confirms the validity of the used area the B missing correcting strategy of this research from protein level.

Secretory volume of the FVIII in mature endothelial cell culture supernatant, such as Figure 47, with the iPSCs stage are detected by ELISA It compares, mature endothelial cell stage FVIII secretory volume increases, and detects expression of the LMAN1 albumen in endothelial cell, discovery LMAN1 albumen has expression (Figure 48) in endothelial cell, this may be the original that endothelial cell stage FVIII secretory volume increases Cause.

(7) endothelial progenitor cells carry out transplanting in HA model mouse body

The each group endothelial progenitor cells differentiated are subjected to mouse cell transplantation in vivo by vena orbitalis posterior, transplant HA- The EPCs (BD21-iEPCs and BD25-iEPCs) that the iPSCs of the area iEPCs, B missing is differentiated, normal control N-iPSCs points Change EPCs (N-iEPCs), N-iPSCs carry out the area B missing obtain Immune Clone Selection wherein N-BD14-iPSCs break up and The N-BD14-iEPCs come carries out transplanting in Mice Body, and totally five groups of cell transplantation groups and one group of injection DPBS are as control Group.When transplanting 14 days, each group mice plasma is taken, detects FVIII blood coagulation activity, every group of blood coagulation activity is small with each group after transplanting The practical blood coagulation activity of mouse accounts for the ratio of wild-type mice blood coagulation activity to indicate.Such as Figure 49, BD21-iEPCs and BD25-iEPCs Transplantation group blood coagulation activity is apparently higher than DPBS group and HA-iEPCs transplantation group, with N-iEPCs and N-BD14-iEPCs group without obvious Difference, the area the B in situ missing strategy that experiment in vivo confirms that we use is effective for hemophilia A treatment.

The therapeutic effect that the original position area B missing strategy is judged by docking experiment is deposited to it is recorded after mouse docking Live time, BD21-iEPCs, BD25-iEPCs, N-iEPCs and N-BD14-iEPCs group mouse it can be seen from Figure 50 and 51 Time-to-live is considerably longer than HA-iEPCs group, it is notable that survivorship curve can be seen that be terminated in docking Germicidal efficacy When (48 hours), there is mouse to experienced docking in BD21-iEPCs, BD25-iEPCs, N-iEPCs and N-BD14-iEPCs group It is survived after experiment, this is significantly for the treatment of disease.9 experience dockings are tested in BD21-iEPCs group There is 1 to survive in mouse, there are 2 to survive in the mouse of 9 experience docking experiments in BD25-iEPCs group, N- There is 1 to survive in the mouse of 9 experience docking experiments in iEPCs group, 9 experience docking experiments in N-BD14-iEPCs group Mouse in there are 2 to survive.This just further demonstrates missing strategy in the area B in situ for the validity of hemophilia A treatment.

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The content that above-described embodiment illustrates should be understood as that these embodiments are only used for being illustrated more clearly that the present invention, without For limiting the scope of the invention, after the present invention has been read, those skilled in the art are to various equivalent forms of the invention Modification each fall within the application range as defined in the appended claims.

SEQUENCE LISTING

<110>Central South University

<120>a kind of people's induced pluripotent stem cells, construction method and application thereof

<130> 1

<160> 22

<170> PatentIn version 3.5

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Claims

1. a kind of people's induced pluripotent stem cells, it is characterised in that be mutated position in the area human blood coagulation factor VII I gene B coded sequence Point is by in-frame deletion, or the entire area B coded sequence is targeted missing, so that it is normal for correcting the reading frame of mutation in situ Reading frame.

2. a kind of people's induced pluripotent stem cells have deposit number C201968.

3. a kind of people's induced pluripotent stem cells have deposit number C201990.

4. a kind of method for constructing people's induced pluripotent stem cells, it is characterised in that by CRISPR/Cas9 and ssODN to source The HA-iPSCs of patient Yu carries out consideration convey, obtains the small in-frame deletion mutational site of the original position area B coded sequence or the entire area B coding The iPSCs of sequence targeting missing.

5. the method for building people's induced pluripotent stem cells according to claim 4, it is characterised in that the ssODN With sequence SEQ ID NO 9 and SEQ ID NO 20.

6. people's induced pluripotent stem cells described in claims 1 or 2 or 3 are applied in preparation treatment HA disease medicament.

7. a kind of drug for treating HA contains people's induced pluripotent stem cells described in claims 1 or 2 or 3.

8. the method for the cell strain being mutated in a kind of building area in-frame deletion human blood coagulation factor VII I gene B, including the use of molecule Biological means will have the gene in-frame deletion in situ being mutated in the area human blood coagulation factor VII I gene B or the entire area B in cell Missing.

9. the cell strain being mutated in a kind of building area in-frame deletion human blood coagulation factor VII I gene B according to claim 8 Method, it is characterised in that the molecular biology method refers to using CRISPR/Cas9, carries out to the gene in situ of mutation Shearing.

10. the cell strain being mutated in a kind of building area in-frame deletion human blood coagulation factor VII I gene B according to claim 9 Method, it is characterised in that in the area human blood coagulation factor VII I gene B be mutated gene in situ be c.3167delCTGA.