CN112741906B - A product for treating hemophilia B - Google Patents

Abstract

The application discloses a product for treating hemophilia B. The product comprises: an agent capable of inserting a gene of interest into Alb (Albumin ) intron 13 in hepatocytes and co-expressing the gene of interest with Alb; the target gene comprises a gene encoding F9 factor. The method selects the intron 13 of the Alb gene in the hepatocyte as the exogenous gene insertion site for the first time, and can ensure the complete expression of Alb and co-expression with the exogenous gene according to the integration donor sequence designed by the site, thereby realizing the purpose of realizing the high expression of the exogenous gene on the premise of not destroying the expression of the endogenous gene. The product provided by the application can improve the treatment and prevention effects of hemophilia B, has high efficiency and high safety, and has good application prospect in the aspect of clinical treatment.

Description

A product for treating hemophilia B

Technical Field

The invention relates to the field of gene therapy, in particular to a product for treating hemophilia B.

Background

Many genetic diseases occur as a result of genetic mutations in certain metabolic-related proteins expressed by the liver. These mutated proteins often lose function, leading to failure of the relevant metabolic processes or normal physiological and biochemical processes, and diseases such as hemophilia A, hemophilia B, tyrosinemia, phenylketonuria, and the like. Most of the existing treatment mechanisms are protein replacement treatment, liver transplantation, gene therapy in a gene supplementation mode and the like, and the site-specific integrated gene therapy provides a new choice for treating the diseases.

Protein replacement therapy and small molecule drug therapy tend to be expensive and require long-term treatment, which is a heavy burden on both the patient's body and the economy. For liver transplantation, donor deficiency, operation risk and immune rejection are also faced with several problems. Gene supplementation by viral delivery solves the problem of delivery of therapeutic genes in vivo, but therapeutic efficacy decreases with cell division and insertional mutagenesis by random integration by viruses limits the clinical utility of this approach. In gene therapy attempts, for example hemophilia B deficient in coagulation factor F9, selection of highly expressed protein Alb in the liver as an insertion site is a new attempt. However, studies have been performed or directed to homologous recombination, which cannot be inserted in non-dividing cells and have a low repair efficiency, and furthermore, due to the presence of the longer homology arms, no larger segment of the CDS can be delivered by AAV vectors to treat more diseases; or the currently performed clinical test for repairing liver metabolic diseases based on ZFN targeted integration of exogenous genes at the Alb site usually needs to introduce three virus systems, which are more complex than CRISPR.

The first prior art is as follows: "US 20190225991A1 METHOD AND COMPOSITIONS FOR GENEME EDITING IN NON-DIVIDING CELLS" discloses a HITI (homology-independent targeted integration) technique:

the sgRNA target present on the genome is typically 20nt in length, whereas Cas9 cleavage occurs 3nt upstream of the PAM sequence (NGG), thus dividing the target sequence into two segments, 17nt and 6 nt. If no donor (donor) sequence is present, the cell can be repaired by means of non-homologous end joining (NHEJ); however, if we provide an exogenous donor and the corresponding target sequence is also present on the donor, cleavage will occur on the cell genome and donor, respectively, after introduction of the CRISPR/Cas9 system and donor into the cell, resulting in blunt-ended DSBs. Because the homologous template does not exist, the NHEJ repair can be carried out between the two blunt-end interfaces at the moment, the connection mode can be a forward connection mode and a reverse connection mode, and if the repair is in the forward connection mode, the Cas9 recognition site can be damaged to stop the cutting and realize the integration of the exogenous gene; if the repair is reverse ligation, the Cas9 recognition site is recombined to continue cleavage at the target site until finally a forward ligation repair occurs. This strategy, called HITI, is based on the repair mechanism of NHEJ, and therefore allows for efficient targeted knock-in of foreign genes in both dividing and non-dividing cells without the need for homology arms, and experiments have demonstrated HITI-mediated in vitro gene integration efficiencies as high as 90%, much higher than traditional homologous recombination.

The HITI technique in the first prior art provides only the means (or strategy) for integration, and the problems of selection of specific insertion sites, optimization of inserts, and production of Indels at binding sites have not been discussed or studied in detail.

The second prior art is: CN 108977464A-composition, medicine and sgRNA for improving blood coagulation activity of patients with hemophilia B disease are integrated into a No. 2-8 exon fragment of cDNA of human F9(human F9, hF9) in a way of homologous recombination (HDR) in a No. one intron of an Alb gene through CRISPR/Cas9, and long-term stable expression of the hF9 gene is realized by utilizing a strong promoter of the Alb, so that hemophilia B phenotype is repaired.

This technique has the following disadvantages:

1. selecting the intron one of the Alb gene as an insertion site, and destroying the expression of Alb while inserting the gene segment, namely destroying the expression of endogenous protein as a direct result of knocking-in;

2. selecting hF9 cDNA No. 2-8 exon fused with No.1 exon of Alb gene, wherein the expressed F9 factor is not complete hF9 factor;

3. homologous recombination is selected for integration of the exogenous gene fragment, knockin (integration) cannot be achieved in non-dividing cells, there are limitations on the type of cell treated, and only suitable for treatment of neonatal mice, and the presence of the long homology arm limits the loading capacity of AAV virus for exogenous CDS.

Disclosure of Invention

In view of the drawbacks of the prior art, it is an object of the present invention to provide a product for the treatment of haemophilia B.

The invention aims to establish a safer and more effective hemophilia B treatment platform by an efficient nonhomologous integration strategy like HITI on the premise of not destroying the expression of endogenous proteins (most other schemes reported in the literature destroy the expression of the endogenous proteins while knocking in genes). The product of the invention has the following advantages: the complete hF9 factor can be expressed without destroying the expression of endogenous protein, and large fragment gene integration can be realized in both dividing cells and non-dividing cells.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:

in one aspect, the invention provides a product for the treatment and/or prevention of haemophilia B, which product comprises: an agent capable of inserting a gene of interest into the intron 13 of Alb in hepatocytes and co-expressing the gene of interest with Alb;

the target gene comprises a gene encoding F9 factor, and the F9 factor can be a wild type with normal activity or a mutant with improved activity;

preferably, the sequence of the gene coding for the F9 factor is shown in SEQ ID No. 6.

The gene coding for factor F9 shown in SEQ ID No.6 is the gene coding for an gain-of-function mutant FIX Padua (R338L).

In a preferred embodiment, the reagent comprises: a nuclease and a sgRNA, or a delivery vector for the nuclease and a delivery vector for the sgRNA;

the sgRNA can guide the nuclease to cut the No.13 intron of Alb in the liver cell and form a fracture site;

preferably, the target sequence of the sgRNA comprises a sequence as set forth in any one of SEQ ID nos. 1 to 5 (i.e., sg6, sg7, sg10, sg11, sg 15);

more preferably, the target sequence of the sgRNA comprises the sequence shown in SEQ ID No.2 (i.e., sg 7).

In a preferred embodiment, the nuclease is selected from one or any of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1 and Cpf 1; preferably, the nuclease is Cas9, preferably, the nuclease is Cas9, more preferably, the Cas9 is selected from Cas9 derived from streptococcus pyogenes, streptococcus pneumoniae or streptococcus thermophilus, more preferably, the Cas9 is selected from Cas9 derived from streptococcus pyogenes, i.e. spCas9

(streptococcus pyogenes Cas9)。

In a preferred embodiment, the reagent further comprises: a donor repair template comprising the exon 14 of Alb and the gene of interest connected in sequence, or a delivery vehicle for the donor repair template,

preferably, the sequence of the 14 exon of Alb is shown as SEQ ID No. 7.

In a preferred embodiment, the donor repair template has an SA sequence upstream of exon 14 of Alb, preferably, the SA sequence is shown in SEQ ID No. 8;

preferably, a self-splicing polypeptide encoding gene is arranged between the exon 14 of Alb and the target gene in the donor repair template, more preferably, the self-splicing polypeptide is a 2A polypeptide, more preferably, the 2A polypeptide is a P2A polypeptide (porcine teschovirus) 2A-like polypeptide), a T2A polypeptide (Chorista punctata beta-tetrad virus (Thosea asigna virus) 2A-like polypeptide), an E2A polypeptide and/or an F2A polypeptide, more preferably, the 2A polypeptide is a T2A polypeptide, more preferably, the encoding gene sequence of the T2A polypeptide is shown as SEQ ID No. 9;

preferably, a polyA sequence is arranged at the downstream of the target gene in the donor repair template, preferably, the polyA is bGH polyA, and more preferably, the bGH polyA sequence is shown as SEQ ID No. 10.

In a preferred embodiment, the target sequence of the sgRNA with PAM connected to the 3' end thereof or homology arms identical to the sequences on both sides of the cleavage site are arranged at both ends of the donor repair template,

preferably, the target sequences of the sgRNAs with 3' ends connected with the PAM sequences are arranged at two ends of the donor repair template,

more preferably, PAM sequences are connected to the 3' ends of the target sequences of the sgRNAs at the two ends of the donor repair template,

more preferably, the target sequence of the sgRNA with PAM sequence linked to the 3 'end of both ends of the donor repair template is reverse complementary to the target sequence of the sgRNA with PAM sequence linked to the 3' end of intron 13 of Alb in the hepatocyte for gene insertion for HITI strategy.

In addition to the HITI strategy, any method which can perform gene insertion on a genome by adopting homologous recombination, non-homologous end integration, homologous arm mediated end joining, micro-homologous end connection and the like such as HDR, NHEJ, HMEJ, MMEJ and the like can be used as a substitute integration strategy to achieve the insertion purpose.

In a preferred embodiment, the vector comprises a viral vector, and/or a non-viral vector; the viral vector comprises an adeno-associated viral vector (i.e., an AAV vector), an adenoviral vector, a lentiviral vector, a retroviral vector, and/or an oncolytic viral vector, and the non-viral vector comprises a cationic high molecular polymer, and/or a liposome;

preferably, the vector comprises a viral vector;

more preferably, the vector comprises an adeno-associated viral vector.

In a preferred embodiment, the nuclease delivery vector is a first AAV vector (i.e., the first AAV vector contains a coding sequence thereon for expression of the nuclease), preferably, the first AAV vector is pAAV-LP1-spCas 9;

the delivery vector of the sgRNA and the delivery vector of the donor repair template are both second AAV vectors (i.e. the second AAV vector contains the donor repair template and a coding sequence for expressing the sgRNA), preferably, the second AAV vector is PAAV-HITI-donor.

In a preferred embodiment, the serotype of the first AAV vector and the second AAV vector is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh64, or AAVrh 74; preferably, the serotype of the delivery vector is AAV8 (specific infection of liver), and different AAV types can be selected according to the type of infected tissue;

more preferably, the first AAV vector is pAAV-LP1-spCas 9; the second AAV vector is pAAV-HITI-donor.

In a preferred embodiment, the product may further comprise sgrnas for use in constructing an animal model of hemophilia B, the target sequences of which comprise the sequences shown in SEQ ID nos. 12 and 13, preferably the animal is a mammal, more preferably the mammal is a rat, more preferably the rat is a Sprague-dawley (sd) rat.

Suitable species for any of the products provided herein include, but are not limited to, rats.

In another aspect, the invention provides a sgRNA for use in a CRISPR/Cas9 gene editing system, the target sequence of the sgRNA comprising a sequence set forth in any one of SEQ ID nos. 1-5;

preferably, the target sequence of the sgRNA comprises the sequence shown in SEQ ID No. 2.

In another aspect, the invention also protects the use of the sgRNA in editing the Alb gene of a cell, preferably, the cell is a hepatocyte.

In another aspect, the invention provides a sgRNA for use in constructing an animal model of hemophilia B, the target sequence of which comprises the sequences shown in SEQ ID nos. 12 and 13;

preferably, the animal is a mammal, more preferably, the mammal is a rat, more preferably, the rat is a Sprague-dawley (sd) rat.

The invention has the following beneficial effects:

1. the intron 13 of the Alb gene in hepatocytes was selected for the first time as the insertion site for the foreign gene. The integration donor sequence designed according to the site can ensure the complete expression of Alb and the normal expression of the inserted gene (the Alb and the inserted gene are fused and co-expressed), thereby realizing the purpose of completing the gene knock-in under the premise of not damaging the expression of the endogenous gene.

2. The comprehensive advantages of the treatment strategy provided by the invention are that: the method has the advantages of no need of a promoter (strong promoter of endogenous Alb), no need of a homologous arm (HITI strategy), no damage to endogenous protein expression (complement of Alb 14 exon while inserting exogenous gene), high-efficiency integration in dividing cells and non-dividing cells (HITI strategy), great clinical treatment potential (AAV packaging can be performed, and the homologous arm is not needed, so that the insertable capacity of an exogenous fragment can be improved to the maximum extent, the method is particularly advantageous in integration of long-fragment genes), and the F9 mutant (R338L) with the highest activity at present is used, so that the virus titer is reduced, the immune response and the generation of F9 antibodies are reduced, and the treatment effect of hemophilia B can be improved.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a schematic diagram of an integration strategy. Wherein ITR represents the inverted terminal repeat of AAV, SA represents the cleavage sequence, 2A represents the self-cleaving peptide, PA represents bGH-polyA, the fragment between 2A and PA represents the target gene hF9P to be integrated into Alb, P-F1, P-R1, P-F2 and P-R2 represent the positions of PCR primers for detecting 5 'integration and 3' integration.

FIG. 2 shows the Alb target screening results. Wherein, fig. 2A is the primary screening result of the target by the luciferase reporting system, fig. 2B is the T7E1 result graph of the primary screening target, and fig. 2C is the primary sequencing result analysis of sg7 target.

FIG. 3 shows the results of in vitro integration and identification of hF9 p. In which FIG. 3A shows the results of electrophoresis of the integrated band, "blank" shows the results of the untransformed plasmid, "HITI" shows the results of the transformed plasmids pAAV-HITI-donor and px458-sg7, and FIG. 3B shows the analysis of the second generation sequencing results of the integrated band.

FIG. 4 is a schematic diagram of the main structure of the viral plasmid. Wherein LP1 represents a liver-specific promoter, spCas9 represents Cas9 from Streptococcus pyogenes (Streptococcus pyogene), PA represents bGH-polyA, hF9p represents a human-derived highly active F9 mutant with gain-of-function mutation (R338L), U6 represents a U6 promoter, and sg7 represents a coding sequence of sgRNA 7.

FIG. 5 is a schematic structural diagram of plasmid vector pAAV-LP1-spCas 9.

FIG. 6 is a schematic structural view of plasmid vector pAAV-HITI-donor.

FIG. 7 is a hemophilia B rat (F9)^-/-Rat). Wherein, FIG. 7A is F9^-/-A schematic diagram of a rat construction method; FIG. 7B is a sequence comparison of rat F9 and human F9 in the knockout region; FIG. 7C is F9^-/-mRNA expression level in rats; FIG. 7D is F9^-/-APTT detection of rats; FIG. 7E is F9^-/-Detecting the bleeding amount of the rat; FIG. 7F is F9^-/-Survival experiments in rats; WT in FIG. 7 represents a wild-type rat, KO represents F9^-/-Rats.

FIG. 8 shows the in vivo integration assay of hF9 p. Wherein, fig. 8A is a schematic diagram of treatment of hemophilia B rats, fig. 8B is a port PCR amplification electrophoresis result of the integrated band, and fig. 8C is a second generation sequencing result analysis of the integrated port PCR amplification band.

FIG. 9 shows the identification of transcripts co-expressed with Alb and hF9 p. Wherein, fig. 9A is a schematic diagram of the identification of the co-expressed transcript, fig. 9B is an electrophoresis result of the PCR amplification band of the co-expressed transcript, fig. 9C is a qPCR relative quantification result of the co-expressed transcript, and fig. 9D is a first generation sequencing result analysis of the PCR amplification band of the co-expressed transcript.

FIG. 10 shows the result of hF9p protein expression assay. Among them, FIG. 10A shows the result of Western Blot detection of hF9p expression in liver, FIG. 10B shows the result of RT-PCR detection of hF9p expression in liver, FIG. 10C shows the result of immunohistochemical detection of hF9p expression in liver, and FIG. 10D shows the result of immunofluorescence detection of hF9p expression in liver.

FIG. 11 is a result analysis for evaluating the effect of treatment. Wherein, FIG. 11A is ELISA quantitative analysis of hF9p expression in rat plasma, FIG. 11B is APTT result detection of rat plasma, FIG. 11C is blood loss detection of rat within 6 minutes after tail-off experiment, and FIG. 11D is survival analysis of rat 48 hours after tail-off experiment.

Figure 12 is a safety analysis of treatment strategies. Fig. 12A shows HE staining results of rat liver, and fig. 12B shows Alb expression and liver function index (AST and ALT) analysis results in rat serum.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited. Such as described in Sambrook et al, molecular cloning, A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

As shown in FIG. 1, the present invention is based on HITI integration strategy, and selects high activity target as CRISPR/Cas9 cleavage site in the 13 th intron of Alb gene, wherein the integration donor comprises SA (splicing acceptor) sequence for completing splicing, the last Exon (14 th Exon, Exon 14) for completing Alb expression cassette, 2A sequence (T2A) for completing protein co-expression separation, and target gene insertion for completing gene therapy. In the examples of the present invention, we exemplified the treatment of hemophilia B, and verified the proposed treatment regimen by selecting a human F9 mutant (R338L, indicated by human F9Padua, which is a mutant F9 protein, abbreviated as hF9p, the sequence of the coding gene of which is shown in SEQ ID No.6) with high activity.

(one) determination of knock-in site of foreign gene

We first screened 18 potential targets with higher CRISPR/Cas9 cleavage efficiency in the 3' region near intron 13 of rat Alb gene by means of Benchling website (table 1), and each target had the same number as its corresponding sgRNA (e.g., sg1 corresponds to sgRNA 1). We first screened the cleavage efficiency of these 18 targets using the tLuc-SSA in vitro target screening system. Six targets with higher CRISPR cleavage activity, namely sg4, sg6, sg7, sg10, sg11 and sg15, are obtained after screening (figure 2A). In order to further detect the cutting efficiency of the 6 high-efficiency targets screened by the tLuc-SSA system in rat cells, the 6 targets are transfected into PC12 cells and then subjected to T7E1 detection, and the result shows that strips with corresponding sizes are cut out of the other targets except the sgRNA 4. The results further demonstrate that these targets are efficiently cleaved within the cell (FIG. 2B). In conclusion, sg6, sg7, sg10, sg11 and sg15 (corresponding to sgRNAs 6, 7, 10, 11 and 15; target sequences are respectively shown in SEQ ID Nos. 1-5) are selected for further target screening, 5 target-cleaved sequences are subjected to PCR amplification, and the mutation condition of each target is subjected to first-generation sequencing analysis. Based on the sequencing results, analysis of the cleavage efficiency of each target site using the synthiego website (https:// ice. synthieo. com) showed that sg7 had the highest cleavage efficiency (89.3%) (fig. 2C). In conclusion, among the 18 target libraries, sg7 (corresponding to sgRNA7, the target sequence is 5'-GAATCATTTCACATTCCCTC-3' (SEQ ID No.2)) has higher cleavage efficiency and is used as the target for subsequent cell and animal experiments.

TABLE 1 target screening information

Target numbering	Score of	Position of	Target sequence	PAM motif
					sg1	37.14	chr14：+19177937	5′CCATGTAATATGCAGCAGTG3′	TGG
sg2	46.74	chr14：-19177880	5′ATTTGTGACGCATACGAACA 3′	TGG
					sg3	33.23	chr14：+19177826	5′AAAAGTAGCATTTCAGGGCA 3′	AGG
sg4	28.47	dhr14：+19177821	5′GTTCAAAAAGTAGCATTTCA 3′	GGG
					sg5	34.70	chr14：-19177787	5′TGAGTTATTTTATACACTAA 3′	TGG
sg6	24.55	chr14：+19177765	5′GAAATGATTCAGACATTGAA 3′	TGG
					sg7	36.69	chr14：-19177741	5′GAATCATTTCACATTCCCTC 3′	CGG
sg8	46.40	chr14：+19177737	5′ACTGTTGTTAGGGCACCGGA 3′	GGG
					sg9	45.28	chr14：+19177736	5′AACTGTTGTTAGGGCACCGG 3′	AGG
sg10	41.79	chr14：+19177733	5′ATAAACTGTTGTTAGGGCAC 3′	CGG
					sg11	34.68	chr14：+19177727	5′AAAAGGATAAACTGTTGTTA 3′	GGG
sg12	35.13	chr14：+19177726	5′TAAAAGGATAAACTGTTGTT3′	AGG
					sg13	32.81	chr14：+19177710	5′TCTGTTGTGTCCAAAATAAA 3′	AGG
sg14	35.48	chr14：+19177677	5′TCCATTGGAATTCTTAAAAC 3′	AGG
					sg15	32.48	chr14：-19177667	5′GCCTGTTTTAAGAATTCCAA 3′	TGG
sg16	36.94	chr14：+19177662	5′TTGCTTAATTACAGTTCCAT 3′	TGG
					sg17	34.80	chr14：-19177649	5′AATGGAACTGTAATTAAGCA 3′	AGG
sg18	41.07	chr14：-19177644	5′AACTGTAATTAAGCAAGGCC 3′	AGG

For therapeutic purposes in this protocol, we constructed the plasmid on the pAAV plasmid vector backbone. According to the design principle of HITI, a homodromous double-cutting donor plasmid vector pAAV-HITI-donor is constructed: pAAV-HITI-SA-EX14-T2A-hF9p-U6-sg7 (shown in FIGS. 4 and 6). This co-cut donor was used in packaging AAV virus with another plasmid vector pAAV-LP1-spCas9 (shown in FIGS. 4 and 5).

The plasmid vector pAAV-HITI-donor is a DNA fragment formed by sequentially connecting the following sequences between two ITRs of the vector pAAV: a sequence which is reversely complementary to the sg7 sequence connected with the PAM sequence at the 3 'end, an SA sequence (SEQ ID No.8), an exon 14 sequence of Alb (SEQ ID No.7), a T2A sequence (SEQ ID No.9), a coding gene sequence of hF9p (SEQ ID No.6), a bGH polyA coding sequence (SEQ ID No.10), a sequence which is reversely complementary to the sg7 sequence connected with the PAM sequence at the 3' end, a U6 promoter sequence (SEQ ID No.11) and a coding sequence of sgRNA7 (SEQ ID No. 2).

The plasmid vector pAAV-LP1-spCas9 is a DNA fragment in which the following sequences are sequentially connected between two ITRs of the vector pAAV: the LP1 promoter sequence, the spCas9 coding sequence, and the polyA sequence.

(II) in vitro validation of protocol

We transfected rat PC12 cells with successfully constructed donor plasmid pAAV-HITI-donor and px458-sg7 plasmid (for insertion of sg7 (coding sequence for sgRNA 7) at the multiple cloning site of px458 (from Addgene: # 48138)), and flow-sorted 72 hours later to obtain positive cells. Extraction of sorted GFP⁺The genome of the cell was subjected to PCR (primers and their positions are shown in FIG. 1) to determine whether hF9p gene was successfully knocked in, and the result of agarose gel electrophoresis showed that a band indicating the integration of the target gene was detected (FIG. 3A). The first round PCR product was subjected to secondary PCR to verify the correctness of integration by second-generation sequencing, andthe integration at this target site was analyzed (FIG. 3B), and based on the port type of integration we found that HITI was integrated mostly with precise integration, but partial Indels were generated, and the sequence alignment showed that these Indels did not affect the expression of ALB and hF9p according to the 3n principle.

The PCR primer sequences are as follows:

one-round PCR primer

P-F1：5’-GGCTGCCGACAAGGATAACT-3’(SEQ ID No.14)；

P-R1：5’-TCGACGTCACCGCATGTTAG-3’(SEQ ID No.15)；

P-F2：5’-CACTTGTTGACCGAGCCA-3’(SEQ ID No.16)；

P-R2：5’-TCCCGAGTATAGTTACCTGAGATG-3’(SEQ ID No.17)。

Two-round PCR primer

Hitom-P-F1(SEQ ID No.18)：

5’-GGAGTGAGTACGGTGTGCTGCCCTGAAATGCTACTTTTTGAA-3’；

Hitom-P-R1(SEQ ID No.19)：

5’-GAGTTGGATGCTGGATGGCAAGGTTTGGCCCTCCGC-3’；

Hitom-P-F2(SEQ ID No.20)：

5’-GGAGTGAGTACGGTGTGCCATTGTCTGAGTAGGTGTCATTCT-3’；

Hitom-P-R2(SEQ ID No.21)：

5’-GAGTTGGATGCTGGATGGTCCATTGGAATTCTTAAAACAGGCT-3’。

Wherein, the amplification product of P-F1 and P-R1 is 569bp and is used for judging 5' integration; the amplification product of P-F2 and P-R2 is 799bp, which is used to judge 3' integration. The amplification product of Hitom-P-F1 and Hitom-P-R1 is 188bp, and is used for the second-generation sequencing of 5' integration; the amplification product of Hitom-P-F2 and Hitom-P-R2 is 190bp and is used for 3' integrated second-generation sequencing.

(III) in vivo validation of protocols

(1) Hemophilia B rat (F9)^-/-Rat) construction

We collected embryos from wild type rats (Sprague-dawley (sd) rats), injected the CRISPR/Cas9 complex into the cytoplasm at the unicellular stage, and established a hemophilia B rat model (ref: Shao, y., Guan, y., Wang, l., Qiu, z., Liu, m., Chen, y., Wu, l., Li, y., Ma, x., Liu, m., and Li, D. (2014) CRISPR/Cas-mediated genome editing in the rate vision direction of one-cell embryo myys. nat protocol.9, 2493-2512) in which the target sequences of two sgrnas were used as follows:

sg1’：5’-CGTCCAAAGAGATATAACTC-3’(SEQ ID No.12)；

sg2’：5’-ATATTAATCAGGTATGATGT-3’(SEQ ID No.13)。

the CRISPR/Cas9 complex targets rat F9 gene exon 2, and a 130bp long fragment is deleted between neonatal rat F9 genome exon 2 exon and intron 3, so that the F9 gene has a frame shift mutation (figure 7A), and the deletion sequence has homology with human F9 factor (figure 7B).

RT-PCR results showed F9^-/-Rat liver did not express F9 mRNA (fig. 7C). The Activated Partial Thromboplastin Time (APTT) assay showed F9^-/-The blood coagulation time of the plasma of the rat is obviously prolonged, wherein the blood coagulation time of the wild rat is 20s, F9^-/-The clotting time of rats was extended to about 65s (fig. 7D). Through tail-breaking experiments, the blood loss within 6 minutes and the survival rate within 48 hours are detected, and the results show that compared with wild rats, F9 is increased^-/-The blood loss of the rats was significantly increased from 800ul to 2800ul in wild type rats (FIG. 7E). F9^-/-Rats (n-5) all died within 48 hours, whereas no death was seen in wild-type rats (fig. 7F).

The above results show that F9^-/-The F9 gene of rat is completely knocked out, resulting in severe bleeding phenotype, and can be used as an ideal animal model for hemophilia B.

(2) AAV delivery of therapeutic systems

We packaged pAAV-LP1-spCas9 and pAAV-HITI-donor 2 AAV8 viruses, and performed tail vein injection of AAV vector to 1 week old rats at the following dose for each group:

WT(n＝3)：PBS，

F9^-/-(Untreated, Untreated group 1, n ═ 3): the amount of the PBS is reduced to a value of PBS,

F9^-/-+2×10¹²GC pAAV-HITI-Donor (Donor, untreated group 2, n-3),

HITI low dose: f9^-/-+2×10¹¹GC pAAV-LP1-spCas9+2×10¹²GC pAAV-HITI-donor (HITI or HITI-Low, treatment group 1, n-3),

HITI high dose: f9^-/-+2×10¹²GC pAAV-LP1-spCas9+4×10¹²GC pAAV-HITI-donor (HITI-High, treatment group 2, n-3);

the experimental rats were monitored for long periods of time (fig. 8A).

(2) Gene level analysis of treatment regimens

We performed Partial biopsy of the livers (Partial hepatomyc, PH) from rats at 8 weeks after AAV injection, extracted liver tissue genomes for PCR, and examined whether the hF9p gene was successfully knocked in, which indicated that integration of the gene of interest was detected (FIG. 8B). Second generation sequencing of the integration region ports by secondary PCR verified the accuracy of integration and analyzed the integration at the target site (fig. 8C). The result is consistent with the in vitro verification result, and the port integration types have similarity. The above results demonstrate that the HITI integration strategy of the present treatment regimen can achieve efficient site-specific integration of the hF9p gene fragment in vivo.

(3) Co-expression analysis of treatment regimens

To verify the co-expression of hF9p gene and Alb gene in vivo, we extracted liver mRNA simultaneously and performed RT-PCR. Reverse transcription of mRNA into cDNA, designing primers Q-C-F (sequence: 5'-AGGCTGCCGACAAGGATAAC-3' (SEQ ID No.22)) and Q-C-Q (sequence: 5'-GGTGATGAGGCCTGGTGATT-3' (SEQ ID No.23)) to perform RT-PCR (FIG. 9A), detecting a band with a size corresponding to the ALB-T2A-hF9p co-expression transcript by agarose gel electrophoresis (FIG. 9B), detecting the relative expression condition of the co-expression transcript by RT-PCR, and displaying that the HITI integration strategy has high integration efficiency (FIG. 9C). Sanger sequencing of the bands of interest indicated that integration of the HITI resulted in the correct transcript (FIG. 9D). The above results demonstrate that the present treatment regimen allows the co-expression of Alb and hF9p genes without disrupting the expression of the endogenous gene Alb.

(4) mRNA and protein level analysis for treatment regimens

We extracted liver protein and carried out Western Blot with beta-actin as internal reference, and the result shows that the expression of hF9p protein is detected (FIG. 10A), and then the expression of hF9p factor in rat by RT-PCR is analyzed, and the high expression of hF9p is also shown (FIG. 10B). To further verify the expression profile of hF9p in liver, we performed paraffin and frozen sections of liver tissue samples and observed the expression profile of hF9p protein in liver tissue by immunohistochemistry (FIG. 10C) and immunofluorescence (FIG. 10D). The results all show that the treatment scheme can realize the expression of the hF9p gene in the liver, and successfully edited cells are uniformly distributed in the liver.

(5) Functional testing of treatment protocols

To verify the functional activity of hF9p protein expressed by HITI knock-in rat hepatocytes, we performed orbital bleeds of rats four weeks after AAV injection, collected plasma samples every 4 weeks for ELISA experiments, and quantitatively monitored the hF9p content in plasma. The results showed that the hF9p content in the plasma of the treated group was up to 3000ng/ml, accounting for 60% of normal plasma in humans (FIG. 11A). At the same time, we also monitored the Activated Partial Thromboplastin Time (APTT) in plasma simultaneously. The results show that the APTT value of the rats in the treatment group is obviously reduced and is close to the background level, and the APTT value of the rats in the high titer group is basically equal to that of the WT (figure 11B), and the results all show that the blood coagulation function of the rats in the treatment group is obviously improved. At month 8 of AAV injection, rats were anesthetized and tested for blood loss at 4cm of the tail (fig. 11C) and for survival within 48 hours after tail breakage (fig. 11D). The results showed that the blood loss of the treated rats was close to that of the WT rats and no death occurred after tail-breaking. Whereas the bleeding volume of the untreated group increased by as much as three-fold compared to the WT group with the donor-only injection group and almost all died within 48 hours after tail-break (fig. 11D). The results further show that the blood coagulation function of the rats in the treatment group is obviously improved.

(6) Safety assessment of treatment regimens

To evaluate the safety of AAV injection, we performed paraffin sectioning of liver tissue samples from rats after liver biopsy, and stained with Hematoxylin-Eosin (HE), which showed that AAV injection did not cause accumulation of inflammatory cells in the body and there was no histological difference in the overall morphology of the liver (fig. 12A). The serum markers of liver damage aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) did not differ between untreated and treated groups. In addition, the serum Alb content of rats in the treated group was not different from that in the untreated group (fig. 12B).

To identify whether the liver-specifically expressed CRISPR/Cas9 system had off-target effects in the treated rats, we selected 11 Alb potential highly active off-target sites via the benchmark website (table 2). The deep sequencing results showed that no significant indels production (less than 0.1%) was seen at these 11 off-target sites in the treated rats compared to the untreated rats (table 3). These data all indicate that a strategy for AAV-delivered HITI components to repair hemophilia B is safe and effective.

TABLE 2 off-target site information

TABLE 3 off-target analysis of treatment strategies

Numbering	Post-off-target sequence	Coordinates of the object	Miss ratio information
				rAlb	GAATCATTTCACATTCCCTC	chr14∶19084511
OT1	aAgTCATTTCACATTCCCTC	chr13：99525284	0.05％±0.01％
				OT2	ccAcCATTTtACATTCCCTC	chr17：16290752	0.07％±0.02％
OT3	GAAaCATTTtACATTCaCTC	chr15：81461028	0.02％±0.01％
				OT4	GAATCATcTgtCATTCCCTC	chr9：101189192	0.06％±0.02％
OT5	cAATCATTTgACcTTCCCTC	chr4：242284766	0.03％±0.01％
				OT6	ctATgAaTTCACATTCCCTC	chr9：97607931	0.04％±0.01％
OT7	cAAcCcTcTCACATTCCCTC	chr11：36751925	0.03％±0.01％
				OT8	ctAaCATTTCcCATTCCCTC	chr18：58108152	0.05％±0.01％
OT9	GAtTCATTTCAgATTCCCTa	chr1：167588939	0.09％±0.01％
				OT10	GtgTaATTTCtCATTCCCTC	chr20：36749557	0.06％±0.02％
OT11	GtAcCtTTTaACATTCCCTC	chr3：101013665	0.03％±0.01％

Note: the second lower case letter in Table 3 represents the nucleotide site of the mutation after off-target.

In conclusion, the therapeutic system delivered by AAV does not obviously activate the innate immune response (inflammatory response), does not change the Alb content in rat serum, and has no abnormal liver function or obvious off-target effect. These results fully demonstrate the superior safety of our treatment regimen.

Those not described in detail in this specification are within the skill of the art. The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement or the like made within the spirit and principle of the present application shall be included in the scope of the claims of the present application.

Sequence listing

<110> Shanghai Bodhisae Biotech Co., Ltd, university of east China

<120> a product for treating hemophilia B

<130> JH-CNP191232

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Claims (32)

1. A product for the treatment and/or prevention of haemophilia B, comprising: an agent capable of inserting a gene of interest into the intron 13 of Alb in hepatocytes and co-expressing the gene of interest with Alb;

the target gene is an encoding gene of the F9 factor and is shown as SEQ ID No. 6;

the reagent comprises: a nuclease and a sgRNA, or a delivery vector for the nuclease and a delivery vector for the sgRNA;

the sgRNA can guide the nuclease to cut the No.13 intron of Alb in the liver cell and form a fracture site;

the target sequence of the sgRNA is a sequence shown in any one of SEQ ID Nos. 1-5;

the reagent comprises: a donor repair template, or a delivery vehicle for the donor repair template;

the donor repair template comprises a No. 14 exon of Alb and the target gene which are connected in sequence;

the sequence of the 14 exon of Alb is shown as SEQ ID No. 7.

2. The product of claim 1, wherein: the target sequence of the sgRNA is a sequence shown in SEQ ID No. 2.

3. The product of claim 1, wherein: the nuclease is selected from one or more of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1 and Cpf 1.

4. The product of claim 3, wherein: the nuclease is Cas 9.

5. The product of claim 4, wherein: the Cas9 is selected from Cas9 derived from streptococcus pyogenes, streptococcus pneumoniae, or streptococcus thermophilus.

6. The product of claim 5, wherein: the Cas9 is selected from Cas9 derived from streptococcus pyogenes.

7. The product of claim 1, wherein: and an SA sequence is arranged upstream of the 14 exon of the Alb in the donor repair template.

8. The product of claim 7, wherein: the SA sequence is shown as SEQ ID No. 8.

9. The product of claim 1, wherein: a splicing polypeptide coding gene is arranged between the No. 14 exon of the Alb and the target gene in the donor repair template.

10. The product of claim 9, wherein: the self-cleaving polypeptide is a 2A polypeptide.

11. The product of claim 10, wherein: the 2A polypeptide is P2A, T2A, E2A, and/or F2A.

12. The product of claim 11, wherein: the 2A polypeptide is a T2A polypeptide.

13. The product of claim 12, wherein: the coding gene sequence of the T2A polypeptide is shown in SEQ ID No. 9.

14. The product of claim 1, wherein: and a polyA sequence is arranged at the downstream of the target gene in the donor repair template.

15. The product of claim 14, wherein: the polyA is bGH polyA.

16. The product of claim 15, wherein: the sequence of the bGH polyA is shown as SEQ ID No. 10.

17. The product of claim 1, wherein: and the two ends of the donor repair template are provided with target sequences of the sgRNA or homologous arms which are the same as the sequences on the two sides of the fracture site.

18. The product of claim 17, wherein: and target sequences of the sgRNAs are arranged at two ends of the donor repair template.

19. The product of claim 18, wherein: and PAM sequences are connected to the 3' ends of the target sequences of the sgRNAs at the two ends of the donor repair template.

20. The product of claim 19, wherein: the target sequence of the sgRNA of which the 3 'ends at the two ends of the donor repair template are connected with the PAM sequence is reversely complementary with the target sequence of the sgRNA of which the 3' ends on the 13 # intron of the Alb in the liver cell are connected with the PAM sequence.

21. The product of claim 1, wherein: the delivery vector includes a viral vector, and/or a non-viral vector.

22. The product of claim 21, wherein: the viral vector includes an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector, and/or an oncolytic viral vector.

23. The product of claim 21, wherein: the non-viral vector comprises a cationic high molecular polymer, and/or a liposome.

24. The product of claim 21, wherein: the delivery vector includes a viral vector.

25. The product of claim 24, wherein: the delivery vector includes an adeno-associated viral vector.

26. The product of claim 1, wherein: the delivery vector of the nuclease is a first AAV vector, and both the delivery vector of the sgRNA and the delivery vector of the donor repair template are second AAV vectors.

27. The product of claim 26, wherein: the serotype of the first AAV vector and the second AAV vector is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh64, or AAVrh 74.

28. The product of claim 27, wherein: the serotype of the delivery vector is AAV 8.

29. An sgRNA used in a CRISPR/Cas9 gene editing system, wherein the target sequence of the sgRNA is a sequence shown in any one of SEQ ID Nos. 1-5.

30. The sgRNA of claim 29, wherein: the target sequence of the sgRNA is a sequence shown in SEQ ID No. 2.

31. Use of the sgRNA of claim 30 for non-disease diagnostic and therapeutic purposes in editing cellular Alb genes.

32. The use of claim 31, wherein: the cells are hepatocytes.

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