WO2021083073A1 - 一种基于肝细胞Alb基因的疾病治疗的产品 - Google Patents

一种基于肝细胞Alb基因的疾病治疗的产品 Download PDF

Info

Publication number
WO2021083073A1
WO2021083073A1 PCT/CN2020/123621 CN2020123621W WO2021083073A1 WO 2021083073 A1 WO2021083073 A1 WO 2021083073A1 CN 2020123621 W CN2020123621 W CN 2020123621W WO 2021083073 A1 WO2021083073 A1 WO 2021083073A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
sequence
vector
liver
seq
Prior art date
Application number
PCT/CN2020/123621
Other languages
English (en)
French (fr)
Inventor
李大力
陈曦
牛煦然
席在喜
刘明耀
Original Assignee
华东师范大学
上海邦耀生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201911055309.XA external-priority patent/CN112741906B/zh
Priority claimed from CN202010879736.6A external-priority patent/CN113058041B/zh
Application filed by 华东师范大学, 上海邦耀生物科技有限公司 filed Critical 华东师范大学
Publication of WO2021083073A1 publication Critical patent/WO2021083073A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the invention relates to the field of gene therapy, in particular to a disease treatment product based on the Alb gene of liver cells.
  • liver cancer Many genetic diseases are caused by genetic mutations in certain metabolic-related proteins expressed by the liver. These mutated proteins often lose their functions, leading to the inability of related metabolic processes or normal physiological and biochemical processes, such as hemophilia A, hemophilia B, tyrosinemia, phenylketonuria and other diseases.
  • the existing treatment mechanisms are mostly protein replacement therapy (or small molecule drug therapy), liver transplantation, and gene therapy in the form of gene supplementation.
  • Protein replacement therapy and small molecule drug therapy are often expensive and require long-term treatment, which is a heavy burden for the patient's body and economy.
  • liver transplantation it also faces problems such as insufficient donors, surgical risks, and immune rejection.
  • non-integrated viruses such as Adeno-associated virus, AAV
  • AAV Adeno-associated virus
  • the delivery based on non-integrated viruses will reduce the efficacy of the cell division.
  • the delivery of Lentivirus (LV) also has the risk of random integration leading to insertion mutation, which also limits the clinical application of this method. Targeted integration of gene therapy provides new options for the treatment of such diseases.
  • factor 9 Factor 9, F9
  • Alb albumin
  • Pompe disease is a severe metabolic myopathy caused by mutations in the acid alpha-glucosidase (GAA) gene, named after the Dutch pathologist Johannes Cassianus Pompe.
  • GAA exists in the lysosome during normal metabolism, decomposes glycogen in the acidic environment of the lysosome, and provides energy for the body. The lack of GAA will cause glycogen accumulation in lysosomes in many tissues, with the heart and skeletal muscle being the most severely affected. Due to the accumulation of glycogen in the myocardium, skeletal muscle and nervous system, the muscle tissue and nervous system will have serious pathological changes.
  • the patient’s myocardium often results in myocardial hypertrophy due to compensatory hyperplasia, and the tongue often becomes hypertrophy; the muscle function is obviously degraded, manifested as muscle weakness and weakened respiratory function; in the nervous system, nerve cells (including neurons and glial The number of cells) has been significantly reduced; the weight of patients is often lighter than normal, causing a serious burden of survival for patients. Therefore, for patients with severe Pompe disease, there is an urgent need for an effective cure.
  • enzyme replacement therapy is the only effective treatment in clinic, but it has certain side effects, is expensive and has limited effects.
  • the targeted integration of gene therapy also provides new options for the treatment of such diseases.
  • HDR mainly occurs in the S/G2 phase of cell division
  • the repair efficiency is low in non-dividing adult hepatocytes.
  • rAAV recombinant AAV
  • ZFN zinc finger nuclease
  • the target sequence of sgRNA on the genome is usually 20bp in length, and the cleavage of Cas9 occurs 3bp upstream of the PAM (protospacer-adjacent motifs) sequence (NGG), so the target sequence is divided into two segments, 17bp and 6bp.
  • NGS protospacer-adjacent motifs sequence
  • the cell can be repaired by non-homologous end joining (NHEJ); if an exogenous donor is provided, and the target fragment on the donor has the same genome at both ends
  • NHEJ non-homologous end joining
  • the reverse CRISPR/Cas9 recognition sequence after the CRISPR/Cas9 system and donor are introduced into the cell, both the cell genome and donor will be cleaved, resulting in blunt-ended DNA double-strand breaks (DSBs).
  • NHEJ repair will occur between the two blunt-ended interfaces at this time. There will be two connection methods, forward and reverse. If the repair is forwardly connected, it will destroy the CRISPR/Cas9 recognition sequence and Terminate the cutting and realize the integration of the foreign gene; if the repair is reversed connection, it will repair the CRISPR/Cas9 recognition sequence and perform two rounds of cutting, resulting in the removal of the inserted fragment. This method is called HITI.
  • HITI is based on the NHEJ pathway, it can achieve high-efficiency targeted knock-in of foreign genes in non-dividing cells, without the need for homology arms, and experiments have shown that the efficiency of HITI-mediated in vitro gene integration is as high as 90%, which is much higher than HDR .
  • the existing HITI technology only provides an integration method (or strategy), but there are no issues regarding the selection of specific insertion sites, the optimization of insert fragments, and the insertions and deletions (indels) generated by the binding site. Conduct detailed discussions and research.
  • Prior art 2 "CN108977464A-composition, drug and sgRNA for improving the coagulation activity of patients with hemophilia B” through CRISPR/Cas9 in the first intron of the Alb gene through homologous recombination (homology- Directed repair, HDR) is integrated into part of the human F9 (human F9, hF9) cDNAs sequence (exons 2-8), using the strong promoter of Alb to achieve long-term stable expression of the hF9 gene, thereby repairing type B blood Friendly phenotype.
  • This technique has the following disadvantages:
  • the purpose of the present invention is to provide a disease treatment product based on the Alb gene of liver cells.
  • the purpose of the present invention is to use HITI, an efficient non-homologous integration strategy, based on the premise of not disrupting the expression of endogenous proteins (other solutions previously reported in the literature all destroy part of the endogenous protein coding frame while knocking in genes). Establish a safer and more effective disease treatment platform.
  • the product of the present invention has the following advantages: it does not substantially damage the expression of endogenous proteins; it can achieve high-efficiency integration of exogenous genes in dividing cells and non-dividing cells; specifically, it can express complete hF9 factor or hGAA protein, and both To achieve long-term protein expression and secretion.
  • the present invention provides a product for treating and/or preventing diseases, comprising: a reagent capable of inserting a target gene into intron 13 of Alb gene in liver cells and co-expressing the target gene and Alb;
  • the diseases include inherited liver metabolic diseases or diseases that can be treated with metabolic cross correction by the liver,
  • the target gene includes a gene that is mutated or deleted in the disease.
  • the hereditary liver metabolic disease refers to a metabolic disease caused by mutation or deletion of genes in liver cells or liver, and the disease can be achieved by synthesizing or secreting corresponding normal proteins in liver cells or liver.
  • the effect of treatment such as hemophilia B, hemophilia A, phenylketonuria, tyrosinemia, etc.;
  • hemophilia B the lack of F9 factor is synthesized by hepatocytes or liver and secreted from hepatocytes or liver, but the F9 gene of the patient's hepatocytes is mutated or deleted, so a safe site for F9 insertion is found in hepatocytes In this way, the inactivating protein synthesized by the liver cell itself is inserted into the liver cell, so as to achieve the effect of treating hemophilia B.
  • the diseases that can be treated by the liver for metabolic cross-correction treatment refer to metabolic diseases caused by gene mutations or deletions in cells or tissues (such as muscle) other than liver cells or liver, and the diseases can be synthesized by hepatocytes or liver Or secrete the corresponding normal protein and transport it through the circulatory system to other cells or tissues where the gene has been mutated or deleted, and be taken up by diseased cells or tissues to achieve the effect of disease treatment, that is, metabolic cross-correction treatment of disease, such as Pompeii S disease, mucopolysaccharidosis, Gaucher disease (glucocerebrosidosis), Sanhoff disease, Niemann-Pick disease, Fabry disease, or Farber disease;
  • Pompe's disease the mutant or lacking protein GAA is located in peripheral tissues (such as muscle), but the secretory activity of hepatocytes or liver can be used to select hepatocytes or liver as target cells or target organs, after inserting the normal genes of GAA .
  • the synthesized and secreted protein GAA will follow the circulatory system to reach the peripheral tissues or their cells, and the peripheral tissues or their cells will re-uptake the protein GAA for disease treatment-that is, metabolic cross-correction.
  • This crossover means that the diseased cells or tissues (protein mutation or lack) are different from the normal protein-producing cells or tissues, but the resulting protein can achieve the ultimate goal of correcting and treating the diseased cells or tissues.
  • the inherited liver metabolic disease includes hemophilia B, hemophilia A, phenylketonuria, or tyrosinemia, preferably, hemophilia B,
  • the target gene includes the coding gene of factor F9, and the F9 factor can be a wild-type with normal activity or a mutant with increased activity. More preferably, the sequence of the coding gene of the F9 factor is as SEQ ID No As shown in .28, the F9 factor encoding gene shown in SEQ ID No. 28 is the encoding gene of a gain-of-function mutant FIX Padua (R338L);
  • the product can improve the symptoms of reduced coagulation activity and/or prolonged coagulation time of the hemophilia B.
  • the diseases that can be treated with metabolic cross correction by the liver include Pompe disease, mucopolysaccharidosis, Gaucher disease (glucocerebrosidosis), Sanhoff disease, Niemann-Pick disease, Fabry disease , Or Farber disease, preferably Pompe disease;
  • the target gene includes a GAA coding gene, and more preferably, the GAA coding gene sequence is shown in SEQ ID No.6.
  • the reagent includes: a nuclease and sgRNA, and/or a delivery vector for the nuclease and a delivery vector for the sgRNA;
  • the sgRNA can guide the nuclease to cut the intron 13 of the Alb gene in the liver cell and form a break site;
  • the target sequence of the sgRNA includes the sequence shown in any one of SEQ ID No. 1-5 (ie sg6, sg7, sg10, sg11, sg15);
  • the target sequence of the sgRNA includes the sequence shown in SEQ ID No. 2 (ie, sg7).
  • the nuclease is selected from one or more of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, Cpf1;
  • the nuclease is Cas9, more preferably, the Cas9 is selected from Cas9 derived from Streptococcus pyogenes, and/or Streptococcus pneumoniae, and/or Streptococcus thermophilus, more preferably, the Cas9 It is selected from Cas9 derived from Streptococcus pyogenes.
  • the reagent includes: a donor repair template, and/or a delivery vehicle for the donor repair template,
  • the donor repair template includes the coding sequence of exon 14 of the Alb gene and the coding sequence of the target gene, which are sequentially connected,
  • the coding sequence of exon 14 of the Alb gene is shown in SEQ ID No. 7;
  • a splicing acceptor (SA) sequence is provided upstream of exon 14 of the Alb gene in the donor repair template, and more preferably, the SA sequence is shown in SEQ ID No. 8;
  • a self-spliced polypeptide encoding gene is set between exon 14 of the Alb gene in the donor repair template and the target gene.
  • the self-spliced polypeptide is a 2A polypeptide, more preferably
  • the 2A polypeptides are P2A (porcine teschovirus 2A-like polypeptide), T2A (thosea asigna virus 2A-like polypeptide), E2A (equine rhinitis virus ( equine rhinitis A virus) 2A-like polypeptide) and/or F2A (foot-and-mouth disease virus 2A-like polypeptide), more preferably, the 2A polypeptide is a T2A polypeptide, more preferably, the T2A
  • the polypeptide coding gene sequence is shown in SEQ ID No. 9,
  • the 3'end of the self-splicing polypeptide encoding gene and the 5'end of the target gene are connected to a secreted peptide encoding gene, and the secretion
  • the peptide is used to secrete the protein of the target gene to achieve therapeutic purposes.
  • the coding gene sequence of the secreted peptide is shown in SEQ ID No. 26,
  • the 3'end of the secreted peptide coding gene and the 5'end of the target gene are connected to the coding gene of the protein tag. More preferably, the coding gene sequence of the protein tag is shown in SEQ ID No. 27;
  • a polyA sequence is provided downstream of the target gene in the donor repair template. More preferably, the polyA is bGH (bovine growth hormone)-polyA, and more preferably, the bGH-polyA sequence is as SEQ ID No Shown in .10.
  • the donor repair template includes the target sequence of the sgRNA provided at both ends of the sgRNA, or homology arms with the same sequence on both sides of the fracture site are provided,
  • the donor repair template includes the target sequence of the sgRNA provided at both ends thereof,
  • the 3'ends of the target sequence of the sgRNA provided at both ends of the donor repair template are connected with a PAM sequence
  • the target sequence of the sgRNA with the PAM sequence connected to the 3'ends of the donor repair template is connected to the 3'end of the PAM sequence connected to the 3'ends of the intron 13 of the Alb gene in the liver cell.
  • the target sequence of the sgRNA is reverse-complemented for gene insertion of the HITI strategy.
  • HDR homologous recombination
  • NHEJ non-homologous end integration
  • HMEJ homology arm-mediated end joining
  • MMEJ micro-homologous end joining
  • the delivery vector includes a viral vector and/or a non-viral vector;
  • the viral vector includes an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector, and/or an oncolytic viral vector ,
  • the non-viral vector includes cationic high molecular polymer and/or liposome;
  • the delivery vector includes a viral vector; more preferably, the delivery vector includes an adeno-associated virus vector.
  • the reagent includes: a delivery vector for the donor repair template, and a delivery vector for the nuclease is a first rAAV vector (for example, the first rAAV vector contains a coding sequence for expressing the nuclease) ), preferably, the first rAAV vector is pAAV-LP1-spCas9;
  • the delivery vector of the sgRNA and the delivery vector of the donor repair template are both a second rAAV vector; (that is, the second rAAV vector contains the donor repair template and the coding sequence for expressing the sgRNA), preferably Yes, the second rAAV vector is pAAV-HITI-donor.
  • the serotypes of the first rAAV vector and the second rAAV vector are rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVrh10, rAAVrh64, or rAAVrh74;
  • the serotype of the delivery vector is rAAV8 (specifically infects the liver), and specifically, different rAAV types can be selected according to the type of infected tissue;
  • the reagent may also include at least one of the following detection combinations A)-C) (including any one, any two or all three):
  • a droplet digital PCR probe set for detecting the insertion of the target gene into the break site including SEQ ID No. 20-23;
  • any of the above products provided by the present invention include but are not limited to rats (specifically Sprague-Dawley rats, SD rats), all higher organisms expressing Alb pass the intron 13 of the Alb gene All gene therapy methods that perform gene insertion in order to achieve the purpose of treatment should fall within the protection scope of the present invention.
  • the present invention also provides any one of the following 1)-3) biological materials:
  • a nucleic acid molecule comprising a DNA molecule and/or an RNA molecule, and the DNA sequence corresponding to the nucleic acid molecule is any of the donor repair templates described above;
  • the recombinant vector includes a viral vector and/or a non-viral vector; the viral vector includes an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, and a retroviral vector , And/or oncolytic viral vectors, the non-viral vectors include cationic high molecular polymers, and/or liposomes, and/or plasmids;
  • the recombinant vector includes a viral vector; more preferably, the delivery vector includes an adeno-associated virus vector, and the serotype of the adeno-associated virus vector is rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVrh10 , RAAVrh64, or rAAVrh74;
  • the serotype of the delivery vehicle is rAAV8;
  • the "r” in “rAAV” stands for recombinant type.
  • the recombinant cell is a hepatocyte.
  • the present invention protects the application of the above-mentioned biological materials in the preparation of products for the treatment and/or prevention of diseases,
  • the hereditary liver metabolic disease includes hemophilia B, hemophilia A, phenylketonuria, or tyrosinemia, preferably, hemophilia B, more preferably, the product can Improving the symptoms of reduced blood clotting activity and/or prolonged clotting time of the hemophilia B;
  • the diseases that can be treated by liver for metabolic cross correction include Pompe disease, mucopolysaccharidosis, Gaucher disease, Sanhoff disease, Niemann-Pick disease, Fabry disease, or Farber disease.
  • Pompe disease More preferably, the product can ameliorate at least one of the following af symptoms of Pompe disease:
  • liver myocardium, diaphragm, quadriceps, tongue, brain (including olfactory bulb, cerebral cortex, midbrain, cerebellum and hindbrain), and/or spinal cord (including spinal dorsal horn and ventral horn) are elevated in glycogen,
  • At least one of the a-f symptoms is specifically any one, any two, any three, any four, any five or all six of the a-f symptoms,
  • any one of the a-f symptoms is the a, b, c, d, e, or f symptoms
  • any two of the af symptoms are the a and b, a and c, a and d, a and e, a and f, b and c, b and d, b and e, b and f, c and d, c and e, c and f, d and e, d and f, or e and f symptoms,
  • any three of the af symptoms are the a, b and c, a, b and d, a, b and e, a, b and f, a, c and d, a, c and e, a, c and f, a, d and e, a, d and f, a, e and f, b, c and d, b, c and e, b, c and f, b, d and e, b, d and f, b, e and f, c, d and e, c, e and f, or d, e and f symptoms,
  • any four of the af symptoms are the a, b, c and d, a, b, c and e, a, b, c and f, a, b, d and e, a, b, d and f, a, b, e and f, a, c, d and e, a, c, d and f, a, c, e and f, a, d, e and f, b, c, d and e, b, c, d and f, b, c, e and f, b, d, e and f, or c, d, e and f symptoms,
  • any five of the af symptoms are the a, b, c, d and e, a, b, c, d and f, a, b, c, e and f, a, b, d, e and f, a, c, d, e, and f, or b, c, d, e, and f symptoms,
  • All six of the a-f symptoms are a, b, c, d, e, and f symptoms.
  • the present invention provides a sgRNA used in a CRISPR/Cas9 gene editing system, the sgRNA can guide a nuclease to cut intron 13 of the Alb gene in a cell and form a double-strand break site;
  • the target sequence of the sgRNA comprises a sequence shown in any one of SEQ ID No. 1-5;
  • the target sequence of the sgRNA includes the sequence shown in SEQ ID No. 2;
  • the cells are hepatocytes.
  • the present invention also protects the application of the sgRNA in editing cell Alb genes.
  • the present invention provides an sgRNA for constructing an animal model of hemophilia B, the target sequence of which includes the sequences shown in SEQ ID No. 29 and SEQ ID No. 30;
  • the animal is a mammal, more preferably, the mammal is a rat, and more preferably, the rat is an SD rat.
  • the comprehensive advantages of the treatment strategy provided by the present invention are: no promoter (using endogenous Alb strong promoter), no homology arm (using HITI strategy), and no damage to endogenous protein expression (in Alb gene 13
  • the exogenous gene is inserted into the intron number and exon 14 of the Alb gene is added at the same time)
  • the efficient integration of the target gene can be achieved in both dividing cells and non-splitting cells (using HITI strategy), which has great clinical Therapeutic potential (rAAV packaging can be carried out, and since the homology arm is not required, the insertable capacity of foreign fragments can be maximized, and the advantages in the integration of long fragment genes are particularly obvious. Inserted genes are mainly inserted in the forward direction. One of the main advantages of this strategy).
  • the present invention provides a new universal product for the treatment of inherited liver metabolic diseases, especially hemophilia B, and a product for diseases that can be cross-corrected by the liver for metabolic cross-correction treatment, especially Pompe disease.
  • hemophilia B treatment products using the currently most active F9 mutant (R338L), while reducing the virus titer, reduce the immune response and the production of F9 antibodies, and can improve the treatment of hemophilia B effect).
  • the product has higher treatment efficiency and better safety, and has great clinical treatment potential.
  • Figure 1 is a schematic diagram of the integration strategy.
  • ITR in the upper and lower figures represents the inverted terminal repeat sequence of rAAV
  • SA represents the splice acceptor sequence
  • 2A represents the self-cleaving peptide
  • PA represents bGH-polyA
  • MD represents the target gene to be integrated into Alb, such as hGAA or hF9p CDS sequence
  • P-F1, P-R1, P-F2 and P-R2 represent the PCR primer positions for detecting 5'integration and 3'integration.
  • Figure 2 shows the screening results of sgRNA in intron 13 of the Alb gene.
  • Figure 2A is the preliminary screening result of the sgRNA target through the luciferase reporter system
  • Figure 2B is the T7E1 digestion result of the preliminary screening target
  • Figure 2C is the Sanger sequencing result of the sg7 target.
  • FIG. 3 is a schematic diagram of the main structure of the viral plasmid.
  • LP1 in the above figure represents a liver-specific promoter
  • spCas9 represents Cas9 from Streptococcus pyogene
  • PA represents bGH-polyA
  • hF9p represents a human-derived highly active F9 with gain-of-function mutations.
  • Mutant (R338L) U6 stands for U6 promoter
  • sg7 stands for the coding sequence of sgRNA7
  • hGAA stands for human GAA CDS.
  • Figure 4 is a schematic diagram of the structure of the plasmid vector pAAV-LP1-spCas9.
  • Figure 5 is a schematic diagram of the structure of the plasmid vector pAAV-HITI-donor-1.
  • Figure 6 is a schematic diagram of the structure of the plasmid vector pAAV-HITI-donor-2.
  • Figure 7 shows the in vitro integration and identification results of hF9p.
  • Figure 7A is the result of the electrophoresis of the integrated band
  • blank is the result of the untransformed plasmid
  • HITI is the result of the transformed plasmid pAAV-HITI-donor-1 and px458-sg7
  • Figure 7B is the result of the integrated band Analysis of second-generation sequencing results.
  • Figure 8 shows the construction of hemophilia B rats (F9 -/- rats).
  • Figure 8A is a schematic diagram of the F9 -/- rat construction method, where the position indicated by the arrow is the CRISPR/Cas9 cleavage position
  • Figure 8B is the sequence comparison of rat F9 and human F9 in the knockout region
  • the WT in Figure 8 represents For wild-type rats, KO stands for F9 -/- rats.
  • Figure 9 shows the detection of hemophilia B rats (F9 -/- rats).
  • Figure 9A is a schematic diagram of knocking out the F9 gene using the CRISPR/Cas9 system
  • Figure 9B is the rat genotype sequencing peak map
  • Figure 9C is the determination of the F9mRNA expression level in the rat liver
  • Figure 9D is the rat plasma PT and APTT Detection
  • Figure 9E is the blood flow detection within 6 minutes of the tail of the rat
  • Figure 9F is the survival rate of the rat within 48 hours of the tail
  • Figure 9G is the spontaneous blood of F9 -/- rats causing hemorrhage in the joints
  • WT Wild-type
  • F9 -130/-130 represents F9 -/- rats
  • F9KO represents F9 -/- rats.
  • Figure 10 shows HITI-mediated identification of hF9p targeted integration.
  • Figure 10A is a schematic diagram of treatment of hemophilia B rats
  • Figure 10B is the result of PCR amplification and electrophoresis of the targeted integration binding site port
  • Figure 10C is the result of next-generation sequencing of the PCR amplified band of the binding site port analysis.
  • Figure 11 shows the identification results of co-expression transcripts of rAlb and hF9p.
  • Figure 11A is a schematic diagram of the identification of co-expressed transcripts
  • Figure 11B is the electrophoresis results of the PCR amplified bands of co-expressed transcripts
  • Figure 11C is the relative quantitative results of qPCR of co-expressed transcripts
  • Figure 11D is the PCR amplification of co-expressed transcripts. Analysis of the first-generation sequencing results of increased bands.
  • Figure 12 shows the detection results of HITI-mediated liver hF9p protein expression.
  • Figure 12A is the Western Blot detection result of hF9p expression in the liver
  • Figure 12B is the gray value result of the band in Figure 12A
  • Figure 12C is the immunohistochemical detection result of the hF9p expression in the liver
  • Figure 12D is the hF9p expression in the liver. The immunofluorescence test results.
  • Figure 13 is the identification result of HITI-mediated high expression of plasma hF9p protein to restore the coagulation function of F9 -/- rats.
  • Figure 13A is the ELISA quantitative analysis of hF9p protein in rat plasma
  • Figure 13B is the APTT result of rat plasma
  • Figure 13C is the blood flow measurement of rats within 6 minutes after the tail docking experiment
  • Figure 13D is the tail docking experiment Rat survival rate after 48 hours.
  • Figure 14 shows the safety analysis of the treatment strategy.
  • Figure 14A is the HE staining result of rat liver
  • Figure 14B is the analysis result of Alb expression and liver function indexes (AST and ALT) in rat serum.
  • Figure 15 shows the result of electrophoresis of in vitro integrated PCR products of target genes.
  • WT is the result of untreated PC12 cells (rat adrenal pheochromocytoma cells)
  • HITI is the result of PC12 cells transfected with plasmid pAAV-HITI-donor-1 and px458-sg7
  • the left arrow represents 972bp
  • the right The side arrow represents 789bp
  • the rightmost lane is the DNA molecular weight standard.
  • Figure 16 shows the analysis of Sanger sequencing results of in vitro integrated bands.
  • Figure 17 shows the analysis of the second-generation sequencing results of in vitro integrated bands.
  • Figure 18 is an analysis of Sanger sequencing results of in vivo integrated bands.
  • Figure 19 is an analysis of the results of next-generation sequencing of integrated bands in vivo.
  • Figure 20 shows the electrophoresis results of the PCR products of hGAA cDNA integration in vivo, where PC represents a positive control using the PC12 genome that has been correctly integrated in vitro as a template.
  • Figure 21 shows the analysis results of the in vivo integration efficiency of next-generation sequencing and ddPCR.
  • Figure 22 shows the identification of rAlb-T2A-hGAA fusion transcripts, in which Figure 22A is the PCR electrophoresis result, and Figure 22B is the qPCR relative quantification result.
  • Figure 23 shows the Sanger sequencing result analysis of rAlb-T2A-hGAA fusion transcript.
  • Figure 24 shows the weight change of rats for 9 consecutive months after virus injection.
  • Figure 25 shows the comparison of muscle strength of rats 9 months after virus injection.
  • Figure 26 shows the comparison of hGAA protein activity in rat plasma samples 9 months after virus injection.
  • Figure 27 shows the detection results of hGAA protein activity in main tissues of rats 9 months after virus injection.
  • Figure 28 is an immunohistochemical analysis of protein expression in the liver, where the second row is an enlarged view of the dotted box in the first row.
  • Figure 29 shows the PAS (Periodic Acid-Schiff) staining results of main muscle tissues, where the larger box is an enlarged view of the smaller box.
  • PAS Periodic Acid-Schiff
  • Figure 30 shows the results of PAS staining of brain tissue.
  • Figure 31 shows the results of PAS staining of the spinal cord.
  • Figure 32 shows the comparison result of the weight index of the left ventricle.
  • Figure 33 shows the off-target analysis results of the CRISPR/Cas9 system.
  • Figure 34 shows the results of qPCR detection of the relative expression of RNA of major immune inflammatory factors.
  • Figure 35 shows the hematoxylin-eosin staining (HE) staining of liver tissue.
  • Figure 36 shows the results of serological analysis of major liver function indicators.
  • the present invention is based on the HITI integration strategy.
  • intron 13 of the Alb gene a highly active target is screened as a CRISPR/Cas9 cleavage site, and its integration donor contains SA (splice acceptor, The splicing acceptor sequence is used to complete the splicing of mRNA, the last exon (exon 14, Exon 14) of the Alb gene is used to complete the Alb expression box, and the 2A sequence (T2A) is used to complete the isolation after translation of the co-expressed protein , And the therapeutic gene insertion is used to complete the purpose of protein expression and gene therapy, and the bGH-polyA (PA) sequence is used to maintain the stability of therapeutic gene mRNA.
  • SA splice acceptor
  • the splicing acceptor sequence is used to complete the splicing of mRNA
  • the last exon (exon 14, Exon 14) of the Alb gene is used to complete the Alb expression box
  • the 2A sequence (T2A) is used to complete the
  • the plasmid vector pAAV-HITI-donor-1 is a DNA fragment with the following sequence connected in sequence between the two ITRs of the vector pAAV: the sequence opposite to the CRISPR/Cas9 recognition site sequence (sg7+PAM) in the Alb gene, the SA sequence (SEQ ID No. 8), Alb gene exon 14 sequence (SEQ ID No. 7), T2A sequence (SEQ ID No. 9), hF9p gene coding sequence (SEQ ID No.
  • bGH polyA sequence (SEQ ID No.10), the reverse sequence of the CRISPR/Cas9 recognition site sequence (sg7+PAM) in the Alb gene, the U6 promoter sequence (SEQ ID No.11), the gene coding sequence of sgRNA7 (its target sequence) It is SEQ ID No. 2).
  • the plasmid vector pAAV-HITI-donor-2 is a DNA fragment with the following sequence connected in sequence between the two ITRs of the vector pAAV: the sequence opposite to the CRISPR/Cas9 recognition site sequence (sg7+PAM) in the Alb gene, the SA sequence (SEQ ID No. 8), Alb gene exon 14 sequence (SEQ ID No. 7), T2A sequence (SEQ ID No. 9), secretion peptide chymotrypsinogen B2 (to ensure that GAA can be secreted) gene coding sequence ( SEQ ID No. 26), the gene coding sequence (SEQ ID No. 27) of the Flag tag (easy to detect protein), the gene coding sequence of hGAA (SEQ ID No.
  • the target sequence is SEQ ID No. 2;
  • the plasmid vector pAAV-LP1-spCas9 is a DNA fragment in which the following sequences are sequentially connected between the two ITRs of the vector pAAV: LP1 promoter sequence, spCas9 coding sequence, and polyA sequence.
  • P-F1 5'-GGCTGCCGACAAGGATAACT-3' (SEQ ID No. 31);
  • P-R1 5’-TCGACGTCACCGCATGTTAG-3’ (SEQ ID No. 32);
  • P-F2 5’-CACTTGTTGACCGAGCCA-3’ (SEQ ID No. 33);
  • P-R2 5'-TCCCGAGTATAGTTACCTGAGATG-3' (SEQ ID No. 34).
  • the amplified product of P-F1 and P-R1 is 569 bp, which is used to identify the integration of the 5'port; the amplified product of P-F2 and P-R2 is 799 bp, which is used to identify the integration of the 3'port.
  • the amplified product of Hitom-P-F1 and Hitom-P-R1 is 188bp, used for 5'integrated second-generation sequencing; the amplified product of Hitom-P-F2 and Hitom-P-R2 is 190bp, used for 3' Integrated next-generation sequencing.
  • sgrF9-1 5’-CGTCCAAAGAGATATAACTC-3’ (SEQ ID No. 29);
  • sgrF9-2 5'-ATATTAATCAGGTATGATGT-3' (SEQ ID No. 30).
  • the CRISPR/Cas9 complex targets exon 2 of the rat F9 gene, and deletes a 130bp fragment between exon 2 and intron 3 of the F9 genome of newborn rats, leading to premature termination of the F9 gene Codon ( Figure 8A, Figure 9B), the deleted sequence has homology with human F9 factor ( Figure 8B).
  • mice We packaged two AAV8 viruses, pAAV-LP1-spCas9 and pAAV-HITI-donor-1, and injected the AAV vector into the tail vein of 4-week-old rats.
  • the injection doses of rats in each group were as follows:
  • HITI's integration methods are mostly based on precise integration.
  • 5-P1-F 5’-GCAGTAACTCGAATGCACCG-3’ (SEQ ID No. 12);
  • 5-P1-R 5’-ACGCATGTCCCCAAGCTATC-3’ (SEQ ID No. 13);
  • the amplified product of 5-P1-F and 5-P1-R is 972bp, which is used to identify the integration at the 5'binding site;
  • the amplified product of 3-P1-F and 3-P1-R is 789bp, Used to identify integration at the 3'binding site.
  • the Pompe disease rat used in this study comes from an animal model constructed in the published literature of this research group :Yang L,Zhang X,Wang L,Yin S,Zhu B,Xie L,Duan Q,Hu H,Zheng R,Wei Y,Peng L,Han H,Zhang J,Qiu W,Geng H,Siwko S,Zhang X,Liu M,Li D*.
  • Gaa DG/DG rat can be used as an ideal animal model for Pompe disease.
  • the plasmids pAAV-LP1-spCas9 and pAAV-HITI-donor-2 were co-transfected with AAV8 and AAV helper helper vectors to HEK293T cells, and the AAV virus was purified to obtain pAAV-LP1-spCas9 virus and pAAV-HITI-donor virus.
  • the integration area port was amplified by nested PCR, and the results of Sanger sequencing showed that there was integration of the target gene at the binding site ( Figure 18). At the same time, next-generation sequencing was used to analyze the integration of the target site, and the results were the same as the in vitro results. Consistent, the splicing sequence type of the port is mainly based on precise integration ( Figure 19).
  • the probes used by ddPCR are as follows:
  • Ref3 cy5-ACACAACAGATGTCAGAGAGCC-BHQ3 (SEQ ID No. 21);
  • Ref52 Fam-AGCTTTATCCTCTCTC-MGB (SEQ ID No. 22);
  • Ins32 AF594-AGCTCCGGGTCATTCTAACTAGT-BHQ (SEQ ID No. 23).
  • HITI strategy can generate the correct rAlb-T2A-hGAA fusion transcript
  • liver mRNA for RT-PCR. Reverse transcription of mRNA into cDNA, and design primers QcF (sequence: 5'-CGTGGACAAGTGTTGCAAGG-3' (SEQ ID No. 24)) and QcR (sequence: 5'-AGCATGAAAGGAGCCACAAGA-3' (SEQ ID No. 25)).
  • the above-mentioned muscle strength testing method is as follows:
  • the maximum number of grasping indications can reflect the rat Muscle strength.
  • GAA protein activity detection method is as follows:
  • PAS staining was used to detect glycogen in different tissues of rats in each group. The results are as follows:
  • the purple-red particles indicated by the arrows in the cells are glycogen.
  • the glycogen of the rats in the treatment group was effectively eliminated in the myocardium, tongue, diaphragm, and quadriceps muscle;
  • the purple-red flakes surrounding neuronal cells and glial cells are glycogen.
  • the glycogen of the rats in the treatment group is in the brain (including the olfactory bulb, cerebral cortex, The midbrain, cerebellum and hindbrain) are effectively eliminated;
  • the purple-red flakes surrounding neuronal cells and glial cells are glycogen.
  • glycogen in the treated group is located in the spinal cord (including the dorsal horn and ventral horn of the spinal cord). ) Has been effectively cleared;
  • the left ventricular mass index (LVMI) of the rats in each group was measured. As shown in Figure 32, the symptoms of elevated left ventricular mass index of the rats in the treatment group were significantly improved.
  • the results are shown in Figure 34A-C. Compared with the untreated group, there was no significant difference in the expression levels of the above-mentioned typical immune inflammatory factors in the liver of the treated group of rats, which indicates that the treatment strategy based on rAAV delivery did not produce a significant immune response.
  • the therapeutic system delivered by rAAV8 in the present application does not significantly activate the body's innate immune response (inflammation response), does not cause liver damage, and does not appear to have obvious off-target effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Hematology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

本发明提供一种基于肝细胞Alb基因的疾病治疗的产品，包含能够将目的基因插入到肝细胞中Alb的13号内含子上并使所述目的基因与Alb共表达的试剂，所述疾病包括遗传性肝脏代谢疾病或可用肝脏进行代谢交叉校正治疗的疾病，所述目的基因包括所述疾病中突变或缺失的基因。

Description

一种基于肝细胞Alb基因的疾病治疗的产品 技术领域
本发明涉及基因治疗领域，具体涉及一种基于肝细胞Alb基因的疾病治疗产品。
背景技术
很多遗传疾病的发生是肝脏表达的某些代谢相关蛋白的基因突变导致的。这些突变后的蛋白往往失去功能，导致相关代谢过程或正常生理生化过程无法进行，如A型血友病、B型血友病、酪氨酸血症、苯丙酮尿症等疾病。现有的治疗机制多为蛋白替代治疗(或小分子药物治疗)、肝脏移植以及以基因增补方式的基因治疗等。
蛋白替代治疗和小分子药物治疗的价格往往较为昂贵，且需要长期接受治疗，对于患者的身体和经济而言都是沉重的负担。而对于肝脏移植而言，也面临着供体不足，手术风险和免疫排斥等问题。通过病毒递送的基因增补方式虽然解决了治疗基因在体内的递送问题，但基于非整合型病毒(如Adeno-associated virus,AAV)的递送会伴随细胞的分裂而疗效下降，基于整合型的病毒(如Lentivirus,LV)的递送也存在随机整合导致插入突变的风险，故而也限制了该方法的临床应用。定点整合的基因治疗为这类疾病的治疗提供了新的选择。在使用定点整合策略的基因治疗研究中，以凝血因子9(Factor 9,F9)缺乏的B型血友病为例，选择肝脏中高表达蛋白Alb(白蛋白)基因作为插入位点是一个新的尝试。F9蛋白由肝实质细胞合成并分泌，是凝血级联反应过程中的重要组成蛋白。该蛋白的缺乏会导致患者出现凝血功能障碍，严重者会因为无法正常凝血而死亡。该疾病为一种典型的遗传性肝脏代谢疾病。
庞贝氏病(Pompe disease)是一种由酸性α葡萄糖苷酶(acid alpha-glucosidase,GAA)基因发生突变引起的严重代谢性肌病，以荷兰病理学家Johannes Cassianus Pompe的名字命名。GAA在正常代谢中存在于溶酶体内，在溶酶体的酸性环境中分解糖原，为机体提供能量。GAA的缺乏将在多个组织中导致溶酶体内出现糖原积累，其中心脏和骨骼肌受到的影响最为严重。由于心肌，骨骼肌和神经系统中出现糖原积累，故而肌肉组织和神经系统会出现严重的病变现象。患者心肌往往会由于代偿性增生导致心肌肥大，舌头也往往变得肥大；肌肉功能出现明显退化，表现为肌无力，呼吸功能减弱；在神经系统中，神经细胞(包括神经元和神经胶质细胞)的数量出现明显减少；患者体重也往往轻于正常人，给患者造成严重的生存负担。所以对于重症庞贝氏病患者来说，亟需一种能够有效治愈的方法。目前，酶替代治疗是临床上唯一有效的治疗手段，但是存在一定的副作用，价格昂贵且效果有限。而定点整合的基因治疗同样为这类疾病的治疗提供了新的选择。
在基因治疗的尝试上，先前研究已经发现采用肝脏作为分泌器官过表达人GAA(human GAA,hGAA)蛋白可以实现庞贝氏病的体内代谢交叉校正。而针对定点整合的治疗，选择肝脏中高表达蛋白Alb(白蛋白)基因作为插入位点同样是一个新的尝试。然而不论是针对以B型血友病为代表的遗传性肝脏代谢疾病还是以庞贝氏病为代表的可通过肝脏进行代谢交叉校正治疗的疾病，之前的相 关基因治疗研究大都采用同源重组修复(homology-directed repair,HDR)的方式。由于HDR主要发生在细胞分裂的S/G2期，因而在非分裂的成体肝细胞中修复效率较低。此外，由于较长同源臂的存在，无法通过临床较为安全的重组AAV(rAAV)载体递送更大片段的cDNAs来治疗更多的疾病；此外，目前进行的基于锌指核酸酶(zinc finger nuclease,ZFN)系统修复肝代谢类疾病的临床前研究通常需要导入三个病毒系统，相比新型的CRISPR(clustered regularly interspaced short palindromic repeats)系统更为复杂，且靶向整合效率不到0.5％，不适用于编辑细胞需求更高的遗传疾病。
现有技术一：“US20190225991A1 METHODS AND COMPOSITIONS FOR GENOME EDITING IN NON-DIVIDING CELLS.”公开了一种HITI(homology-independent targeted integration，非同源依赖的靶向整合)技术：
在基因组上sgRNA的靶序列通常长度为20bp，而Cas9的切割是发生在PAM(protospacer-adjacent motifs)序列(NGG)上游3bp处，因此就将靶点序列分为了17bp与6bp两段。如果不存在供体(donor)序列，细胞可以通过非同源末端连接(non-homologous end joining,NHEJ)的方式进行修复；如果提供外源的donor，且donor上的目的片段两端存在同基因组反向的CRISPR/Cas9识别序列，则在将CRISPR/Cas9系统及donor导入细胞后，细胞基因组和donor均会被切割，产生平末端的DNA双链断裂(double-strand breaks,DSBs)。由于不存在同源模板，此时两个平端接口之间会发生NHEJ的修复，连接方式会有正向与反向两种，如果修复是正向连接的，那么就会破坏CRISPR/Cas9识别序列并终止切割并实现外源基因的整合；如果修复是反向连接的，则会修复CRISPR/Cas9识别序列并进行二轮切割，导致插入片段的移除，这种方式被称为HITI。由于HITI是基于NHEJ途径，因而可以在非分裂细胞中实现高效的外源基因靶向敲入，不需要同源臂，且实验证明HITI介导的体外基因整合效率高达90％，远高于HDR。
现有的HITI技术只提供了整合的方式(或策略)，但是对于具体的插入位点的选择，插入片段的优化以及结合位点产生的插入或缺失(insertions and deletions,indels)等问题上没有进行详细的讨论与研究。
现有技术二：“CN108977464A-用于提高B型血友病患者的凝血活性的组合物、药物和sgRNA”中通过CRISPR/Cas9在Alb基因的一号内含子中通过同源重组(homology-directed repair,HDR)的方式整合入部分人F9(human F9，hF9)cDNAs序列(2-8号外显子)，利用Alb的强启动子实现了hF9基因的长期稳定表达，从而修复了B型血友病表型。这一技术存在以下缺点：
1.选择Alb基因的一号内含子作为插入位点，在插入基因片段的同时破坏了Alb的表达，即敲入的一个直接结果是破坏了内源蛋白的表达；
2.选择hF9 cDNA 2-8号外显子融合Alb基因的1号外显子，表达的F9因子不是完整的hF9因子；
3.选择同源重组进行外源基因片段整合，在非分裂细胞中无法实现敲入(整合)，存在治疗细胞类型的限制，仅适合用于新生小鼠的治疗，且长同源臂的存在限制了AAV病毒对外源基因的装载容量。
发明内容
针对现有技术中存在的缺陷，本发明的目的在于提供一种基于肝细胞Alb基因的疾病治疗产品。
本发明旨在基于不破坏内源蛋白表达的前提下(之前文献报道的其它方案都在敲入基因的同时破坏了部分内源蛋白编码框)，通过HITI这一高效的非同源整合策略，建立一个更为安全、有效的疾病治疗平台。本发明产品具有如下优势：基本不破坏内源蛋白表达；在分裂细胞和非分裂细胞中都能实现外源基因的高效整合；具体地，可以表达完整的hF9因子，或hGAA蛋白，且均可实现蛋白的长期表达和分泌。
为达到以上目的，本发明采取的技术方案是：
一方面，本发明提供一种治疗和/或预防疾病的产品，包含：能够将目的基因插入到肝细胞中Alb基因的13号内含子上并使所述目的基因与Alb共表达的试剂；
所述疾病包括遗传性肝脏代谢疾病或可用肝脏进行代谢交叉校正治疗的疾病，
所述目的基因包括在所述疾病中发生突变或缺失的基因。
在上述产品中，所述遗传性肝脏代谢疾病是指肝细胞或肝脏中基因发生突变或缺失导致的代谢疾病，且该疾病可以通过在肝细胞或肝脏中合成或分泌对应的正常蛋白以达到疾病治疗的效果，如B型血友病、A型血友病、苯丙酮尿症、酪氨酸血症等；
例如B型血友病，其缺乏的F9因子为肝细胞或肝脏进行合成并从肝细胞或肝脏处分泌，但是患者的肝细胞F9基因突变或缺失，因此在肝细胞中找到安全位点插入F9基因，这样在肝细胞中就插入了肝细胞本身合成的失活蛋白，从而达到治疗B型血友病的效果，同理，A型血友病、苯丙酮尿症、酪氨酸血症等也可以用同样的方式治疗，只需把F9换成其它疾病突变的基因，那么插入的正常基因就会在肝脏中表达正常的蛋白，从而达到治疗效果；
所述可用肝脏进行代谢交叉校正治疗的疾病是指除肝细胞或肝脏之外的其它细胞或组织(如肌肉)中基因发生突变或缺失导致的代谢疾病，且该疾病可以通过肝细胞或肝脏合成或分泌对应的正常蛋白并通过循环系统输送到所述基因发生突变或缺失的其它细胞或组织中并被病变细胞或组织摄取以达到疾病治疗的效果，即疾病的代谢交叉校正治疗，如庞贝氏病、黏多糖贮积症、戈谢病(葡糖脑苷脂病)、Sanhoff病、Niemann-Pick病、Fabry病、或Farber病；
例如庞贝氏病，其突变或缺乏的蛋白GAA位于外周组织(比如肌肉)，但是可以利用肝细胞或肝脏的分泌活性，选择肝细胞或肝脏作为靶细胞或靶器官，插入GAA的正常基因后，合成并分泌的蛋白GAA会顺着循环系统到达外周组织或其细胞，外周组织或其细胞再摄取蛋白GAA，进行疾病治疗——即进行代谢交叉校正。这个交叉是指发病(蛋白突变或缺乏)的细胞或组织与产生正常蛋白的细胞或组织不同，但是所产生的蛋白可以达到最终校正治疗发病的细胞或组织的目的。
在上述产品中，所述遗传性肝脏代谢疾病包括B型血友病、A型血友病、苯丙酮尿症、或酪氨酸血症，优选，B型血友病，
更优选的，所述目的基因包括F9因子的编码基因，所述F9因子可为活性正常的野生型或活性提高的突变体，更优选的，所述F9因子的编码基因的序列如SEQ ID No.28所示，SEQ ID No.28所示的F9因子的编码基因为一种功能获得性突变体FIX Padua(R338L)的编码基因；
更优选的，所述产品能够改善所述B型血友病的凝血活性降低症状和/或凝血时间延长症状。
在上述产品中，所述可用肝脏进行代谢交叉校正治疗的疾病包括庞贝氏病、黏多糖贮积症、戈谢病(葡糖脑苷脂病)、Sanhoff病、Niemann-Pick病、Fabry病、或Farber病，优选，庞贝氏病；
更优选的，所述目的基因包括GAA的编码基因，更优选的，所述GAA的编码基因序列如SEQ ID No.6所示。
在上述产品中，所述试剂包括：核酸酶和sgRNA，和/或所述核酸酶的递送载体和所述sgRNA的递送载体；
所述sgRNA能够引导所述核酸酶对肝细胞中Alb基因的13号内含子进行切割并形成断裂位点；
优选的，所述sgRNA的靶序列包含SEQ ID No.1-5(即sg6，sg7，sg10，sg11，sg15)中任一所示的序列；
更优选的，所述sgRNA的靶序列包含SEQ ID No.2(即sg7)所示的序列。
在上述产品中，所述核酸酶选自Cas9、Cas3、Cas8a、Cas8b、Cas10d、Cse1、Csy1、Csn2、Cas4、Cas10、Csm2、Cmr5、Fok1、Cpf1中的一种或任意几种；
优选的，所述核酸酶为Cas9，更优选的，所述Cas9选自来源于化脓性链球菌、和/或肺炎链球菌、和/或嗜热链球菌的Cas9，更优选的，所述Cas9选自来源于化脓性链球菌的Cas9。
在上述产品中，所述试剂包括：供体修复模板、和/或所述供体修复模板的递送载体，
所述供体修复模板包括依次连接的Alb基因的14号外显子编码序列和所述目的基因的编码序列，
优选的，所述Alb基因的14号外显子编码序列如SEQ ID No.7所示；
优选的，所述供体修复模板中所述Alb基因的14号外显子的上游设剪接受体(splicing acceptor,SA)序列，更优选的，所述SA序列如SEQ ID No.8所示；
优选的，所述供体修复模板中所述Alb基因的14号外显子与所述目的基因之间设自剪切多肽编码基因，更优选的，所述自剪切多肽为2A多肽，更优选的，所述2A多肽为P2A(猪捷申病毒(porcine teschovirus)2A样多肽)、T2A(明脉扁刺蛾β四体病毒(thosea asigna virus)2A样多肽)、E2A(马甲型鼻炎病毒(equine rhinitis A virus)2A样多肽)和/或F2A(手足口病毒(foot-and-mouth disease virus)2A样多肽)，更优选的，所述2A多肽为T2A多肽，更优选的，所述T2A多肽的编码基因序列如SEQ ID No.9所示，
更优选的，在针对所述可用肝脏进行代谢交叉校正治疗的疾病产品中，所述自剪切多肽编码基因3’端和所述目的基因5’端之间连接分泌肽编码基因，所述分泌肽用于将所述目的基因的蛋白分泌，以达到治疗目的，优选的，所述分泌肽 的编码基因序列如SEQ ID No.26所示，
更优选的，所述分泌肽编码基因3’端、所述目的基因5’端连接蛋白标签的编码基因，更优选的，所述蛋白标签的编码基因序列如SEQ ID No.27所示；
优选的，所述供体修复模板中所述目的基因下游设polyA序列，更优选的，所述polyA为bGH(bovine growth hormone)-polyA，更优选的，所述bGH-polyA序列如SEQ ID No.10所示。
在上述产品中，所述供体修复模板包括设于其两端的所述sgRNA的靶序列、或设与所述断裂位点两侧序列相同的同源臂，
优选的，所述供体修复模板包括设于其两端的所述sgRNA的靶序列，
更优选的，设于所述供体修复模板两端的所述sgRNA的靶序列的3’端连接PAM序列，
更优选的，设于所述供体修复模板两端的3’端连接PAM序列的所述sgRNA的靶序列与所述肝细胞中Alb基因的13号内含子上的3’端连接PAM序列的所述sgRNA的靶序列反向互补，以用于HITI策略的基因插入。
除HITI策略之外，采用同源重组(HDR)，非同源末端整合(NHEJ)，同源臂介导的末端连接(HMEJ)，微同源末端连接(MMEJ)等任一可以在基因组上进行基因插入的方法都可以作为替代整合策略达到插入目的。
在上述产品中，所述递送载体包括病毒载体、和/或非病毒载体；所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、和/或溶瘤病毒载体，所述非病毒载体包括阳离子高分子聚合物、和/或脂质体；
优选的，所述递送载体包括病毒载体；更优选的，所述递送载体包括腺相关病毒载体。
在上述产品中，所述试剂包括：所述供体修复模板的递送载体，所述核酸酶的递送载体为第一rAAV载体(如所述第一rAAV载体上含有表达所述核酸酶的编码序列)，优选的，所述第一rAAV载体为pAAV-LP1-spCas9；
所述sgRNA的递送载体和所述供体修复模板的递送载体均为第二rAAV载体；(即所述第二rAAV载体上含有所述供体修复模板和表达所述sgRNA的编码序列)，优选的，所述第二rAAV载体为pAAV-HITI-donor。
在上述产品中，所述第一rAAV载体和所述第二rAAV载体的血清型为rAAV1、rAAV2、rAAV5、rAAV6、rAAV7、rAAV8、rAAV9、rAAVrh10、rAAVrh64、或rAAVrh74；
优选的，所述递送载体的血清型为rAAV8(特异性感染肝脏)，具体可以根据感染组织的类型选择不同的rAAV类型；
和/或，所述试剂还可包括如下检测用组合A)-C)中的至少一种(包括任一种、任两种或全部三种)：
A)用于检测所述目的基因插入所述断裂位点的PCR引物组：包括SEQ ID No.12-19；
B)用于检测所述目的基因插入所述断裂位点的微滴式数字PCR探针组：包括SEQ ID No.20-23；
C)用于检测所述目的基因与所述Alb基因形成的融合转录本的PCR引物组： 包括SEQ ID No.24-25。
本发明所提供以上任一所述产品的适用种属包含但不限于大鼠(具体可为Sprague-Dawley rats，SD大鼠)，一切表达Alb的高等生物通过在Alb基因的13号内含子中进行基因插入以达到治疗目的的基因治疗方式均应在本发明的保护范围内。
另一方面，本发明还提供了如下1)-3)生物材料中的任一种：
1)一种核酸分子，包括DNA分子和/或RNA分子，所述核酸分子对应的DNA序列如以上任一所述供体修复模板；
2)含有1)所述核酸分子的重组载体，所述重组载体包括病毒载体、和/或非病毒载体；所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、和/或溶瘤病毒载体，所述非病毒载体包括阳离子高分子聚合物、和/或脂质体、和/或质粒；
优选的，所述重组载体包括病毒载体；更优选的，所述递送载体包括腺相关病毒载体，所述腺相关病毒载体的血清型为rAAV1、rAAV2、rAAV5、rAAV6、rAAV7、rAAV8、rAAV9、rAAVrh10、rAAVrh64、或rAAVrh74；
更优选的，所述递送载体的血清型为rAAV8；
其中，“rAAV”中的“r”代表重组型。
3)含有1)所述核酸分子的重组细胞或重组菌，优选的，所述重组细胞为肝细胞。
另一方面，本发明保护以上所述生物材料在制备治疗和/或预防疾病产品中的应用，
优选的，所述遗传性肝脏代谢疾病包括B型血友病、A型血友病、苯丙酮尿症、或酪氨酸血症，优选，B型血友病，更优选，所述产品能够改善所述B型血友病的凝血活性降低症状和/或凝血时间延长症状；
优选的，所述可用肝脏进行代谢交叉校正治疗的疾病包括庞贝氏病、黏多糖贮积症、戈谢病、Sanhoff病、Niemann-Pick病、Fabry病、或Farber病，优选，庞贝氏病，更优选的，所述产品能够改善所述庞贝氏病的包括如下a-f症状中的至少一种：
a体重减轻，
b肌肉力量丧失，
c血浆中GAA蛋白活性降低，
d肝脏、心肌、膈肌、股四头肌、舌、脑、和/或脊髓中GAA蛋白活性降低，
e肝脏、心肌、膈肌、股四头肌、舌、脑(包括嗅球，大脑皮层，中脑，小脑和后脑)、和/或脊髓(包括脊髓背角和腹角)中糖原升高，
f左心室重量指数升高。
所述a-f症状中的至少一种具体为所述a-f症状中的任一种、任两种、任三种、任四种、任五种或全部六种，
所述a-f症状中的任一种为所述a、b、c、d、e或f症状，
所述a-f症状中的任两种为所述a和b、a和c、a和d、a和e、a和f、b和c、b和d、b和e、b和f、c和d、c和e、c和f、d和e、d和f、或e和f症状，
所述a-f症状中的任三种为所述a、b和c，a、b和d，a、b和e，a、b和f，a、c和d，a、c和e，a、c和f，a、d和e，a、d和f，a、e和f，b、c和d，b、c和e，b、c和f，b、d和e，b、d和f，b、e和f，c、d和e，c、d和f，c、e和f，或d、e和f症状，
所述a-f症状中的任四种为所述a、b、c和d，a、b、c和e，a、b、c和f，a、b、d和e，a、b、d和f，a、b、e和f，a、c、d和e，a、c、d和f，a、c、e和f，a、d、e和f，b、c、d和e，b、c、d和f，b、c、e和f，b、d、e和f，或c、d、e和f症状，
所述a-f症状中的任五种为所述a、b、c、d和e，a、b、c、d和f，a、b、c、e和f，a、b、d、e和f，a、c、d、e和f，或b、c、d、e和f症状，
所述a-f症状中的全部六种为a、b、c、d、e和f症状。
另一方面，本发明提供了一种用于CRISPR/Cas9基因编辑系统中的sgRNA，所述sgRNA能够引导核酸酶对细胞中Alb基因的13号内含子进行切割并形成双链断裂位点；
优选的，所述sgRNA的靶序列包含SEQ ID No.1-5中任一所示的序列；
更优选的，所述sgRNA的靶序列包含SEQ ID No.2所示的序列；
优选的，所述细胞为肝细胞。
另一方面，本发明还保护所述sgRNA在编辑细胞Alb基因中的应用。
另一方面，本发明提供了一种用于构建B型血友病动物模型的sgRNA，其靶序列包含SEQ ID No.29和SEQ ID No.30所示的序列；
优选的，所述动物为哺乳动物，更优选的，所述哺乳动物为大鼠，更优选的，所述大鼠为SD大鼠。
本发明的有益效果如下：
1、选择肝细胞中Alb基因的13号内含子作为外源目的基因的插入位点。根据该位点设计的整合供体序列可以保证Alb同插入基因的正常表达(二者进行融合共表达)，从而实现在不破坏内源基因表达的前提下完成基因敲入的目的并实现治疗基因的高效表达。
2、本发明所提供的治疗策略的综合优势在于：无需启动子(利用内源性Alb的强启动子)，无需同源臂(利用HITI策略)，不破坏内源蛋白表达(在Alb基因13号内含子中插入外源基因的同时补齐了Alb基因的14号外显子)，在分裂细胞和非裂细胞中都能实现目的基因的高效整合(利用HITI策略)，有极大的临床治疗潜力(可以进行rAAV包装，且由于不需要同源臂，因而可以最大限度提高外源片段的可插入容量，在长片段基因的整合上优势尤其明显，插入基因主要以正向插入为主也是该策略的主要优势之一)。
3、本发明提供了一种新的可通用型治疗遗传性肝脏代谢疾病尤其是B型血友病的产品以及可用肝脏进行代谢交叉校正治疗的疾病尤其是庞贝氏病的产品。其中，B型血友病治疗产品(使用了目前最高活性的F9突变体(R338L)，在降低病毒滴度的同时，减少免疫反应以及F9抗体的产生，且能改善B型血友病的治疗效果)。该产品具有较高的治疗效率和较好的安全性，有极大的临床治疗潜 力。
附图说明
图1为整合策略示意图。其中，上图和下图中ITR代表rAAV的反向末端重复序列，SA代表剪接受体序列，2A代表自裂解肽，PA代表bGH-polyA，MD代表欲整合入Alb的目的基因，如hGAA或hF9p CDS序列，P-F1、P-R1、P-F2和P-R2代表检测5’整合和3’整合的PCR引物位置。
图2为Alb基因的13号内含子中sgRNA的筛选结果。其中，图2A为sgRNA靶点通过荧光素酶报告系统初筛结果，图2B为初筛靶点的T7E1酶切结果，图2C为sg7靶点的Sanger测序结果。
图3为病毒质粒主要结构示意图。其中，上图中LP1代表肝脏特异性启动子，spCas9代表来自化脓性链球菌(Streptococcus pyogene)的Cas9，PA代表bGH-polyA；中图中hF9p代表一种功能获得性突变的人源高活性F9突变体(R338L)，U6代表U6启动子，sg7代表sgRNA7的编码序列；下图中hGAA代表人GAA CDS。
图4为质粒载体pAAV-LP1-spCas9的结构示意图。
图5为质粒载体pAAV-HITI-donor-1的结构示意图。
图6为质粒载体pAAV-HITI-donor-2的结构示意图。
图7为hF9p体外整合鉴定结果。其中，图7A为整合目的条带的电泳结果，“空白”为未转化质粒的结果，“HITI”为转化质粒pAAV-HITI-donor-1和px458-sg7的结果，图7B为整合条带的二代测序结果分析。
图8为B型血友病大鼠(F9 -/-大鼠)的构建。其中，图8A为F9 -/-大鼠构建方法示意图，其中箭头所示位置为CRISPR/Cas9切割位置；图8B为大鼠F9和人F9在敲除区域的序列比较，图8中的WT代表野生型大鼠，KO代表F9 -/-大鼠。
图9为B型血友病大鼠(F9 -/-大鼠)的检测。其中，图9A为利用CRISPR/Cas9系统敲除F9基因设计示意图，图9B为大鼠基因型测序峰图，图9C为大鼠肝脏中F9mRNA表达水平的测定；图9D为大鼠血浆PT和APTT检测；图9E为大鼠断尾6分钟内的血流量检测；图9F为大鼠断尾48小时内存活率；图9G为F9 -/-大鼠自发出血引发关节积血，图9中的WT(Wild-type)代表野生型大鼠，F9 -130/-130代表F9 -/-大鼠，F9KO代表F9 -/-大鼠。
图10为HITI介导的hF9p靶向整合鉴定。其中，图10A为B型血友病大鼠治疗示意图，图10B为靶向整合的结合位点端口的PCR扩增电泳结果，图10C为结合位点端口PCR扩增条带的二代测序结果分析。
图11为rAlb与hF9p共表达转录本鉴定结果。其中，图11A为共表达转录本鉴定示意图，图11B为共表达转录本PCR扩增条带的电泳结果，图11C为共表达转录本的qPCR相对定量结果，图11D为共表达转录本PCR扩增条带的一代测序结果分析。
图12为HITI介导肝脏hF9p蛋白表达的检测结果。其中，图12A为肝脏中hF9p表达的Western Blot检测结果，图12B为图12A中条带的灰度值结果，图12C为肝脏中hF9p表达的免疫组化检测结果，图12D为肝脏中hF9p表达的免 疫荧光检测结果。
图13为HITI介导的血浆hF9p蛋白的高效表达恢复F9 -/-大鼠的凝血功能的鉴定结果。其中，图13A为大鼠血浆中hF9p蛋白的ELISA定量分析，图13B为大鼠血浆的APTT结果检测，图13C为断尾实验后6分钟内大鼠的血流量检测，图13D为断尾实验后48小时大鼠存活率。
图14为治疗策略的安全性分析。其中，图14A为大鼠肝脏的HE染色结果，图14B为大鼠血清中Alb表达及肝功能指标(AST和ALT)分析结果。
图15为目的基因体外整合PCR产物电泳结果。其中，WT为未处理组PC12细胞(大鼠肾上腺嗜铬细胞瘤细胞)的结果，HITI为转染质粒pAAV-HITI-donor-1和px458-sg7的PC12细胞结果，左侧箭头代表972bp，右侧箭头代表789bp，最右列泳道为DNA分子量标准。
图16为体外整合条带的Sanger测序结果分析。
图17为体外整合条带的二代测序结果分析。
图18为体内整合条带的Sanger测序结果分析。
图19为体内整合条带的二代测序结果分析。
图20为hGAA cDNA体内整合PCR产物电泳结果，其中，PC代表以体外已正确整合的PC12基因组为模板的阳性对照。
图21为二代测序和ddPCR进行体内整合效率的分析结果。
图22为rAlb-T2A-hGAA融合转录本鉴定，其中图22A为PCR电泳结果，图22B为qPCR相对定量结果。
图23为rAlb-T2A-hGAA融合转录本的Sanger测序结果分析。
图24为注射病毒后连续9个月大鼠体重变化。
图25为注射病毒9个月后的大鼠肌肉力量对比。
图26为注射病毒9个月后的大鼠血浆样品hGAA蛋白活性对比。
图27为注射病毒9个月后的大鼠主要组织hGAA蛋白活性检测结果。
图28为免疫组化分析肝脏中蛋白表达情况，其中，第二行为第一行虚线框处的放大图。
图29为主要肌肉组织的PAS(Periodic Acid-Schiff)染色结果，其中，较大方框为较小方框位置处的放大图。
图30为脑组织的PAS染色结果。
图31为脊髓的PAS染色结果。
图32为左心室的重量指数对比结果。
图33为CRISPR/Cas9系统的脱靶分析结果。
图34为主要免疫炎症因子RNA相对表达量的qPCR检测结果。
图35为肝脏组织的苏木精-伊红染色(hematoxylin-eosin staining,HE)染色。
图36为主要肝功能指标的血清学分析结果。
具体实施方式
如图1所示，本发明以HITI整合策略为基础，在Alb基因的13号内含子中筛选高活性靶点作为CRISPR/Cas9切割位点，其整合供体上包含SA(剪接受体，splicing acceptor)序列用以完成mRNA的剪接，Alb基因最后一个外显子(14号 外显子，Exon 14)用以补全Alb表达框，2A序列(T2A)用以完成共表达蛋白转译后的分离，以及治疗基因插入用以完成蛋白表达和基因治疗的目的，bGH-polyA(PA)序列用以维持治疗基因mRNA的稳定性。
在本发明一个实施例中，我们以B型血友病的治疗为例，且选择了一个具有高活性的人F9突变体(R338L，用human F9 Padua表示，是一个突变型的F9蛋白，简称hF9p，其编码基因的序列如SEQ ID No.6所示)对提出的治疗方案进行了验证。
在本发明另一个实施例中，我们进一步以庞贝氏病的治疗为例，且选择了hGAA cDNA作为插入的目的基因对提出的治疗方案进行了验证。
(一)筛选目的基因的整合位点
我们首先借助Benchling网站在大鼠Alb基因的13号内含子靠近3’区域筛选了18个潜在的切割效率较高的sgRNAs(表1)，每个靶点与其对应的sgRNA的数字相同(如靶点sg1对应sgRNA1)。我们首先使用tLuc-SSA体外靶点筛选系统在HEK293T细胞中对这18个靶点的切割效率进行初步的筛选。筛选后得到6个CRISPR切割活性较高的靶点(sg4，sg6，sg7，sg10，sg11和sg15)(图2A)。为了进一步检测tLuc-SSA系统筛选出的6个高效靶点在大鼠细胞内的切割效率，我们将这6个靶点转染PC12细胞后进行T7E1检测，结果显示除sg4外，其它靶点均切出了相应大小的条带。该结果进一步证明这些靶点在内源基因位点可进行高效的切割(图2B)。综上，我们选择靶点sg6，sg7，sg10，sg11，sg15(对应sgRNA6，7，10，11，15，靶序列分别如SEQ ID No.1-5所示)进行进一步的靶点筛选，将5个靶点切割后的序列进行PCR扩增，对各靶点突变情况进行Sanger测序分析。根据测序结果，利用Synthego网站(https：//ice.synthego.com)对各靶点的切割效率进行分析，结果显示sg7具有最高的切割效率(～89.3％)(图2C)。综上分析，在18个候选sgRNA中，sg7具有较高的切割效率，可作为后续细胞和动物实验的靶点。
表1、靶点筛选信息
靶点编号 得分 位置 靶序列 PAM基序
sg1 37.14 ch r14：+19177937 5′CCATGTAATATGCAGCAGTG 3′ TGG
sg2 46.74 ch r14：-19177880 5′ATTTGTGACGCATACGAACA 3′ TGG
sg3 33.23 ch r14：+19177826 5′AAAAGTAGCATTTCAGGGCA 3′ AGG
sg4 28.47 ch r14：+19177821 5′GTTCAAAAAGTAGCATTTCA 3′ GGG
sg5 34.70 ch r14：-19177787 5′TGAGTTATTTTATACACTAA 3′ TGG
sg6 24.55 Ch r14：+19177765 5′GAAATGATTCAGACATTGAA 3′ TGG
sg7 36.69 ch r14：-19177741 5′GAATCATTTCACATTCCCTC 3′ CGG
sg8 46.40 ch r14：+19177737 5′ACTGTTGTTAGGGCACCGGA 3′ GGG
sg9 45.28 ch r14：+19177736 5′AACTGTTGTTAGGGCACCGG 3′ AGG
sg10 41.79 ch r14：+19177733 5′ATAAACTGTTGTTAGGGCAC 3′ CGG
sg11 34.68 ch r14：+19177727 5′AAAAGGATAAACTGTTGTTA 3′ GGG
sg12 35.13 ch r14：+19177726 5′TAAAAGGATAAACTGTTGTT 3′ AGG
sg13 32.81 ch r14：+19177710 5′TCTGTTGTGTCCAAAATAAA 3′ AGG
sg14 35.48 ch r14：+19177677 5′TCCATTGGAATTCTTAAAAC 3′ AGG
sg15 32.48 ch r14：-19177667 5′GCCTGTTTTAAGAATTCCAA 3′ TGG
sg16 36.94 ch r14：+19177662 5′TTGCTTAATTACAGTTCCAT 3′ TGG
sg17 34.80 ch r14：-19177649 5′AATGGAACTGTAATTAAGCA 3′ AGG
sg18 41.07 ch r14：-19177644 5′AACTGTAATTAAGCAAGGCC 3′ AGG
(二)质粒载体构建
考虑到本方案的治疗目的，我们将供体片段构建在pAAV质粒载体骨架上。根据HITI的设计原则，我们构建了同向双切供体质粒载体pAAV-HITI-donor-1(图3中，图5)和pAAV-HITI-donor-2(图3下，图6)，每个双切供体质粒载体与另一质粒载体pAAV-LP1-spCas9(图3上，图4)被用于AAV病毒的包装。
质粒载体pAAV-HITI-donor-1为在载体pAAV的两个ITR之间依次连接如下序列的DNA片段：与Alb基因中CRISPR/Cas9识别位点序列(sg7+PAM)反向的序列，SA序列(SEQ ID No.8)，Alb基因的14号外显子序列(SEQ ID No.7)，T2A序列(SEQ ID No.9)，hF9p的基因编码序列(SEQ ID No.28)，bGH polyA序列(SEQ ID No.10)，与Alb基因中CRISPR/Cas9识别位点序列(sg7+PAM)反向的序列，U6启动子序列(SEQ ID No.11)，sgRNA7的基因编码序列(其靶序列为SEQ ID No.2)。
质粒载体pAAV-HITI-donor-2为在载体pAAV的两个ITR之间依次连接如下序列的DNA片段：与Alb基因中CRISPR/Cas9识别位点序列(sg7+PAM)反向的序列，SA序列(SEQ ID No.8)，Alb基因的14号外显子序列(SEQ ID No.7)，T2A序列(SEQ ID No.9)，分泌肽chymotrypsinogen B2(保证GAA可以实现分泌)的基因编码序列(SEQ ID No.26)，Flag标签(便于检测蛋白)的基因编码序列(SEQ ID No.27)，hGAA的基因编码序列(SEQ ID No.6)，bGH polyA序列(SEQ ID No.10)，与Alb基因中CRISPR/Cas9识别位点序列(sg7+PAM)反向的序列，U6启动子序列(SEQ ID No.11)，sgRNA7的基因编码序列(其靶序列为SEQ ID No.2)；
其中，由于rAAV装载容量的限制，我们将sgRNA的表达框安置在靠近3’ITR的位置。
质粒载体pAAV-LP1-spCas9为在载体pAAV的两个ITR之间依次连接如下序列的DNA片段：LP1启动子序列，spCas9编码序列，和polyA序列。
(三)方案1的体外验证
我们将构建成功的供体质粒pAAV-HITI-donor-1与px458-sg7质粒(在px458(来自Addgene:#48138)BbsI克隆位点插入sgRNA7的编码序列)转染大鼠PC12细胞，72小时后进行流式分选，获得阳性细胞。提取分选后的GFP +细胞基因组进行PCR(引物及其位置如图1所示)，检测hF9p基因是否成功敲入，琼脂糖凝胶电泳结果显示了目的基因整合的条带(图7A)。通过二代测序进一步验证了整合的正确性，并分析了该靶点处的整合情况(图7B)，根据端口的整合结果我们发现HITI的整合方式大都以精准整合为主，但也有部分indels的产生，且不会导致rAlb和hF9p基因的移码。
一轮PCR引物
P-F1：5’-GGCTGCCGACAAGGATAACT-3’(SEQ ID No.31)；
P-R1：5’-TCGACGTCACCGCATGTTAG-3’(SEQ ID No.32)；
P-F2：5’-CACTTGTTGACCGAGCCA-3’(SEQ ID No.33)；
P-R2：5’-TCCCGAGTATAGTTACCTGAGATG-3’(SEQ ID No.34)。
二轮PCR引物
Hitom-P-F1(SEQ ID No.35)：
5’-GGAGTGAGTACGGTGTGCTGCCCTGAAATGCTACTTTTTGAA-3’；
Hitom-P-R1(SEQ ID No.36)：
5’-GAGTTGGATGCTGGATGGCAAGGTTTGGCCCTCCGC-3’；
Hitom-P-F2(SEQ ID No.37)：
5’-GGAGTGAGTACGGTGTGCCATTGTCTGAGTAGGTGTCATTCT-3’；
Hitom-P-R2(SEQ ID No.38)：
5’-GAGTTGGATGCTGGATGGTCCATTGGAATTCTTAAAACAGGCT-3’。
其中，P-F1与P-R1的扩增产物为569bp，用于鉴定5’端口的整合；P-F2与P-R2的扩增产物为799bp，用于鉴定3’端口的整合。Hitom-P-F1与Hitom-P-R1的扩增产物为188bp，用于5’整合的二代测序；Hitom-P-F2与Hitom-P-R2的扩增产物为190bp，用于3’整合的二代测序。
(四)方案1的体内验证
1、B型血友病大鼠(F9 -/-大鼠)构建
我们收集来自野生型大鼠(Sprague-Dawley(SD)大鼠)的胚胎，在单细胞期将CRISPR/Cas9复合物注射到细胞质中，建立B型血友病大鼠模型(具体步骤参见文献：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 rat via direct injection of one-cell embryos.Nat Protoc.9,2493-2512)，其中采用的两个sgRNA的靶序列如下(图9A)：
sgrF9-1：5’-CGTCCAAAGAGATATAACTC-3’(SEQ ID No.29)；
sgrF9-2：5’-ATATTAATCAGGTATGATGT-3’(SEQ ID No.30)。
CRISPR/Cas9复合物靶向大鼠F9基因2号外显子，在新生大鼠F9基因组2号外显子和3号内含子之间删除了一个长为130bp的片段，导致F9基因产生提前的终止密码子(图8A，图9B)，该删除序列与人F9因子存在同源性(图8B)。
RT-PCR结果显示F9 -/-大鼠肝脏中不表达F9mRNA(图9C)。凝血酶原时间(PT)测定结果相同，但活化部分凝血活酶时间(APTT)测定显示F9 -/-大鼠血浆凝血时间明显延长，但野生型大鼠凝血时间约为20s，F9 -/-大鼠凝血时间延长至约65s(图9D)。通过断尾实验，我们检测了6分钟内的失血量和48小时内的存活率，结果表明，与野生型大鼠相比，F9 -/-大鼠失血量明显增加，由野生型大鼠的约800μl增至约2800μl(图9E)。F9 -/-大鼠(n＝5)均于20小时内死亡，而野生型大鼠未见死亡(图9F)，且F9 -/-大鼠自发出血引发关节积血。
以上结果表明，F9 -/-大鼠的F9基因被完全敲除，导致严重的出血表型，可作为B型血友病的理想动物模型。
2、治疗系统的AAV递送
我们包装了pAAV-LP1-spCas9和pAAV-HITI-donor-1两个AAV8病毒，对4周龄的大鼠进行了AAV载体的尾静脉注射，各组大鼠注射剂量情况为：
野生型组(WT)：注射等体积PBS的野生型大鼠，n＝3；
未处理组(Untreated)：注射等体积PBS的F9 -/-大鼠，n＝3；
Donor组(Only donor)：仅注射pAAV-HITI-donor-1病毒(2×10 12GC/只)的F9 -/-大鼠，n＝3；
低剂量HITI组(HITI或Low-HITI)：注射pAAV-LP1-spCas9病毒(2×10 11GC/只)和pAAV-HITI-donor-1病毒(2×10 12GC/只)的F9 -/-大鼠，n＝3；
高剂量HITI组(High-HITI)：注射pAAV-LP1-spCas9病毒(2×10 12GC/只)和pAAV-HITI-donor-1病毒(4×10 12GC/只)的F9 -/-大鼠，n＝3；
对以上大鼠按对应时间进行定期采样和长期监测(图10A)。
3、方案1的基因型鉴定
我们在注射rAAV后第8周对大鼠进行部分活体取肝，提取肝脏基因组。如图1所示，在结合位点左右各设计一对引物进行PCR，检测hF9p基因是否成功敲入，凝胶电泳结果显示了目的基因的整合(图10B)。二代测序确认了整合的精准性，并进一步分析了结合位点处的整合类型和比例(图10C)。同体外结果一致，端口的整合类型具有相似性。以上结果说明本治疗方案可以实现hF9p基因片段在体内有效的位点特异性整合。
4、方案1的共表达分析
为了验证体内hF9p基因同rAlb基因的共表达，我们提取了肝脏mRNA。反转录成cDNA，如图11A，设计引物Q-c-F(序列：5’-AGGCTGCCGACAAGGATAAC-3’(SEQ ID No.39))和Q-c-R(序列：5’-GGTGATGAGGCCTGGTGATT-3’(SEQ ID No.40))进行RT-PCR(图11A)，琼脂糖凝胶电泳结果显示了rAlb-T2A-hF9p共表达转录本对应大小的条带(图11B)，通过RT-PCR定量检测了共表达转录本的相对表达情况，结果显示HITI整合策略具有较高的整合效率(图11C)。对目的条带进行Sanger测序，结果表明HITI的整合产生了正确的转录本(图11D)。以上结果说明本治疗方案在不破坏内源基因Alb表达的前提下可实现rAlb和hF9p基因的共表达。
5、方案1的mRNA与蛋白水平分析
我们提取肝脏蛋白进行Western Blot，以β-actin为内参，结果显示检测到了hF9p蛋白的表达(图12A)，随后对大鼠hF9p蛋白的表达量进行定量分析也显示hF9p的高表达(图12B)。为了进一步验证hF9p在肝脏中的表达分布情况，我们将肝脏组织样品进行石蜡切片和冰冻切片，通过免疫组化(图12C)与免疫荧光(图12D)对hF9p蛋白在肝组织中的表达分布进行观察。结果均表明本治疗方案可以实现肝脏中hF9p基因的表达，编辑成功的细胞在肝脏中均匀分布。
6、方案1的功能检测
为了验证HITI敲入大鼠肝细胞表达hF9p蛋白的功能活性，我们在注射rAAV四周后，对大鼠进行眼眶取血，每隔4周收集血浆样品进行ELISA实验，对血浆中hF9p的含量进行定量监测。结果显示，治疗组血浆中的hF9p含量最高可达3000ng/ml，占人正常血浆的60％(图13A)。同时，我们也同步监测了血浆中的活化部分凝血活酶时间(APTT)。结果显示，治疗组大鼠的APTT值明显下降，并接近于本底水平，且高滴度组的APTT值基本与WT持平(图13B)，以上结果均表明治疗组的凝血功能得到了明显改善。在注射AAV的第8个月，我们对大鼠麻醉处理后于尾部4cm处进行断尾，检测其6分钟内的失血量(图13C)以及断尾 后48小时内的存活情况(图13D)。结果显示治疗组大鼠的失血量接近于WT大鼠，且在断尾后未发生任何死亡。而未治疗组与仅注射donor组的流血量相较WT组增加三倍之多，且在断尾后48小时内全部死亡(图13D)。以上结果进一步表明HITI介导的hF9p敲入可明显恢复F9 -/-大鼠的凝血功能。
7、方案1的安全性评估
为了评价rAAV注射的安全性，我们对大鼠活体取肝后的肝脏组织样品进行石蜡切片，通过苏木精-伊红(Hematoxylin-Eosin staining，HE)染色，结果显示rAAV注射并未引起机体炎症细胞的聚集，肝脏整体形态没有组织学差异(图14A)。肝脏损伤的血清标志物天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)在未治疗组和治疗组间没有差异。另外，治疗组大鼠血清中的Alb含量同未治疗组相比也没有区别(图14B)。
为了鉴定肝脏特异性表达的CRISPR/Cas9系统是否在治疗组大鼠中发生脱靶效应，我们通过Benchling网站选择了11个rAlb潜在的高活性脱靶位点(表2)。较未治疗组大鼠相比，二代测序结果显示治疗组大鼠在这11个脱靶位点未见明显的indels产生(低于0.1％)(表3)。这些数据均表明，通过rAAV递送的HITI策略对血友病B大鼠具有治疗效果，不会诱导严重的脱靶效应和肝脏毒性。
表2、脱靶位点信息
Figure PCTCN2020123621-appb-000001
表3、治疗策略的脱靶分析
Figure PCTCN2020123621-appb-000002
注：表3中第二列小写字母代表脱靶后突变的核苷酸位点。
综上说明，我们通过rAAV递送的治疗体系没有明显激活机体的固有免疫反应(产生炎性反应)，未引起大鼠血清中Alb含量的改变，肝功能未见异常，也未产生明显的脱靶效应。这些结果充分地说明我们的治疗方案具有较好的安全性。
(五)方案2的体外验证
我们将构建成功的供体质粒pAAV-HITI-donor-2与px458-sg7质粒(在px458(来自Addgene:#48138)BbsI克隆位点插入sgRNA7的编码序列)转染大鼠PC12细胞，72小时后进行流式分选，获得GFP +细胞。提取分选后的GFP +细胞基因组进行巢式PCR，检测hGAA基因是否成功敲入，琼脂糖凝胶电泳实验的结果显示检测到了正确整合的条带(图15)。将PCR产物进行通过Sanger测序(图16)和二代测序(图17)验证了整合的正确性，并分析了该靶点处的整合情况。根据端口的整合类型，我们发现HITI的整合方式大都以精准整合为主。
上述巢式PCR引物序列如下：
一轮PCR引物：
引物对5-P1：
5-P1-F：5’-GCAGTAACTCGAATGCACCG-3’(SEQ ID No.12)；
5-P1-R：5’-ACGCATGTCCCCAAGCTATC-3’(SEQ ID No.13)；
引物对3-P1：
3-P1-F：5’-CCCTGGACACCATCAACGTCC-3’(SEQ ID No.14)；
3-P1-R：5’-CCTGGCCTTGCTTAATTACAGTTC-3’(SEQ ID No.15)；
二轮PCR引物：
引物对5-P2：
Hitom-5-P2-F(SEQ ID No.16)：
5’-GGAGTGAGTACGGTGTGCTGCCCTGAAATGCTACTTTTTGAA-3’；
Hitom-5-P2-R(SEQ ID No.17)：
5’-GAGTTGGATGCTGGATGGCAAGGTTTGGCCCTCCGC-3’；
引物对3-P2：
Hitom-3-P2-F(SEQ ID No.18)：
5’-GGAGTGAGTACGGTGTGCCATTGTCTGAGTAGGTGTCATTCT-3’；
Hitom-3-P2-R(SEQ ID No.19)：
5’-GAGTTGGATGCTGGATGGTCCATTGGAATTCTTAAAACAGGCT-3’；
其中，5-P1-F与5-P1-R的扩增产物为972bp，用于鉴定5’结合位点处的整合；3-P1-F与3-P1-R的扩增产物为789bp，用于鉴定3’结合位点处的整合。
(六)方案2的体内验证
1、庞贝氏病大鼠(Gaa KO大鼠)构建
本研究所采用的庞贝氏病大鼠(点突变位点为鼠Gaa蛋白第645位氨基酸，突变情况为D645G，简写为Gaa DG/DG)来自本课题组已发表的文献所构建的动物模型：Yang L,Zhang X,Wang L,Yin S,Zhu B,Xie L,Duan Q,Hu H,Zheng R,Wei Y,Peng L,Han H,Zhang J,Qiu W,Geng H,Siwko S,Zhang X,Liu M,Li D*.Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants.Protein Cell.2018Sep；9(9):814-819.
根据文献报道，该Gaa DG/DG大鼠可作为庞贝氏病的理想动物模型。
2、治疗系统的rAAV递送
将质粒pAAV-LP1-spCas9和pAAV-HITI-donor-2分别同AAV8和AAV helper辅助载体共转染HEK293T细胞，AAV病毒纯化后获得pAAV-LP1-spCas9病毒和pAAV-HITI-donor病毒。我们将4周龄的SD大鼠分为四组，进行尾静脉注射，各组大鼠注射情况如下：
野生型组(WT)：注射等体积PBS的野生型大鼠，n＝3；
未处理组(Untreated)：注射等体积PBS的KO大鼠，n＝3；
Donor组(Only donor)：仅注射pAAV-HITI-donor-2病毒(1.2×10 12GC/只)的KO大鼠，n＝3；
治疗组：注射pAAV-LP1-spCas9病毒(1.2×10 12GC/只)和pAAV-HITI-donor-2病毒(2×10 12GC/只)的KO大鼠，n＝3(组织取样检测时有两个重复：HITI-1及HITI-2)；
对以上注射后的实验大鼠按对应时间进行后续监测。
3、方案2的基因水平分析
注射8周后，对步骤2中各组大鼠进行部分活体取肝(Partial hepatectomy,PH)，提取肝脏基因组进行PCR(方法同图15)，检测目的基因是否成功敲入，结果如图20所示，凝胶电泳显示了整合的目的基因对应的条带。
通过巢式PCR对整合区域端口处进行扩增，通过Sanger测序结果显示结合位点处存在目的基因的整合(图18)，同时利用二代测序分析该靶点处的整合情况，结果同体外结果一致，端口的拼接序列类型以精准整合为主(图19)。
以上结果说明，本治疗方案可以实现体内外源基因片段的有效整合。通过ddPCR(Droplet Digital PCR，微滴式数字PCR)进一步定量靶向整合效率，结果显示效率最高可达11％(图21和表4)。
ddPCR使用的探针如下：
Ins5：Vic-TGTCTGAATCATTTC-MGB(SEQ ID No.20)；
Ref3：cy5-ACACAACAGATGTCAGAGAGCC-BHQ3(SEQ ID No.21)；
Ref52：Fam-AGCTTTATCCTCTCTC-MGB(SEQ ID No.22)；
Ins32：AF594-AGCTCCGGGTCATTCTAACTAGT-BHQ(SEQ ID No.23)。
表4
Figure PCTCN2020123621-appb-000003
4、HITI策略可产生正确的rAlb-T2A-hGAA融合转录本
为了验证体内hGAA基因同Alb基因的共表达，我们提取了肝脏mRNA进行RT-PCR。将mRNA反转录成cDNA，设计引物Q-c-F(序列：5’-CGTGGACAAGTGTTGCAAGG-3’(SEQ ID No.24))和Q-c-R(序列：5’-AGCATGAAAGGAGCCACAAGA-3’(SEQ ID No.25))进行RT-PCR，琼脂糖凝胶电泳结果显示了共表达转录本(rAlb-T2A-hGAA)对应大小为634bp的条带(图22A)，通过qPCR(引物对为Q-c-F和Q-c-R，内参基因为β-actin，)进而对共表达转录本进行了相对定量分析(图22B)，Sanger测序结果进一步验证了共表达转录本的精准剪接(图23)。以上结果说明本治疗方案在不破坏内源基因rAlb表达的前提下可实现rAlb和hGAA基因的共表达。
5、方案2的治疗效果分析
1)rAAV注射后即开始进行大鼠的治疗效果评估。首先，我们在注射时，注射后1个月，3个月和9个月进行了各组大鼠体重的监测。结果显示，治疗组大鼠随着时间的推移显示出同野生型大鼠(WT)相似的体重增加，而未处理组(Untreated)和仅donor组大鼠体重增加并不明显，且在一段时间后出现了下降的趋势(图24)。以上说明本申请的治疗策略能够有效阻止庞贝氏病大鼠体重的减轻。
2)在注射病毒的9个月后，对各组大鼠的肌肉力量进行测量。结果发现，治疗组大鼠(HITI)的平均握力有了显著的提升，而仅donor组和未处理组(Untreated)接近，均低于野生型大鼠(WT)和治疗组大鼠(HITI)。这说明本申请的治疗方法能够有效恢复庞贝氏病大鼠肌肉的力量(图25)。
上述肌肉力量检测方法如下：
一手抓尾，一手托腹，将大鼠两只前爪放置在测力计的金属杆上，让大鼠前爪在金属杆上进行抓握，抓握示数的最大值即可以反映大鼠的肌肉力量。
3)在注射病毒9个月后，分别通过眼眶采集各组大鼠的外周血，将获得的血浆样品进行hGAA蛋白活性检测，结果显示在治疗组大鼠(HITI)的血浆中hGAA蛋白活性均出现明显上调(图26)。
上述GAA蛋白活性检测方法如下：
预先使用稀释至10mM的对硝基苯酚标准品按0、2、4、6、8、10μl加入到96孔板中，使用α-葡萄糖苷酶检测缓冲液将体积调整到100μl/孔，并在410nm处 读取吸光度，绘制标准曲线。待测样品调整为50μl/孔，并加入50μl的反应混合液(α-葡萄糖苷酶检测缓冲液47μl与α-葡萄糖苷酶检测底物3μl)，反应30分钟后在410nm处读取OD值，根据标准曲线测定酶活性。
4)在注射病毒9个月后，分别采集各组大鼠的主要组织(肝脏，心肌，膈肌，股四头肌，舌，脑，脊髓)，制备成匀浆后进行hGAA蛋白活性检测(方法同步骤3)，结果，与未处理组和仅donor组相比，治疗组大鼠(HITI)的肝脏、心肌、膈肌和舌中hGAA蛋白活性明显更高，股四头肌、脊髓和脑中略有升高(图27)。
免疫组化检测各组大鼠肝脏中hGAA蛋白的表达情况，结果显示，在治疗组大鼠肝脏中检测到了hGAA蛋白的表达，如图28所示，其中，hGAA阳性信号为黄色颗粒状，未处理组和donor组的没有黄色点状分布，因为GAA是定位在溶酶体上的蛋白，不完全是成片分布，因而呈不规则的点状。
通过PAS染色检测各组大鼠不同组织中的糖原，结果如下：
如图29所示，细胞中箭头所示处的紫红色颗粒为糖原，与未处理组相比，治疗组大鼠的糖原在心肌、舌、膈肌、股四头肌得到了有效清除；
如图30所示，神经元细胞与神经胶质细胞周围的紫红色片状物即为糖原，与未处理组相比，治疗组大鼠的糖原在脑部(包括嗅球，大脑皮层，中脑，小脑和后脑)得到了有效清除；
如图31所示，神经元细胞与神经胶质细胞周围的紫红色片状物即为糖原，与未处理组相比，治疗组大鼠的糖原在脊髓(包括脊髓背角和腹角)得到了有效的清除；
5)在注射病毒9个月后，测量各组大鼠左心室的重量指数(LVMI)，结果如图32所示，治疗组大鼠的左心室重量指数升高的症状得到了明显的改善。
6、方案2的安全性评估
为了验证CRISPR系统体内的脱靶效应，基于Benchling网站针对sg7靶点在基因组范围内选取12个分值较高的脱靶位点，对这12个脱靶位点进行了二代测序分析，结果显示均未发生明显的脱靶情况(低于0.1％)(图33)。
将各组大鼠活体取肝得到的肝脏样品提取总RNA并反转录得到cDNA，通过qPCR对IL6、IL10、IFN-β1等炎症因子的表达进行了检测，结果如图34A-C所示，与未处理组相比，治疗组大鼠的肝脏中上述典型的免疫炎症因子的表达水平未见显著差异，这说明基于rAAV递送的治疗策略没有产生明显的免疫反应。
对各组大鼠活体取肝后的肝脏组织样品进行HE染色，在组织细胞周围未观察到存在明显的免疫细胞浸润(图35)。
对各组大鼠通过眼眶采集的外周血的血清样品进行肝功能分析，与未处理组相比，治疗组大鼠血清中的ALB蛋白含量并未出现明显的差异，且血清中谷丙转氨酶(ALT)与谷草转氨酶(AST)的含量也保持正常水平(图36A-C)。
综上说明，本申请通过rAAV8递送的治疗体系没有明显激活机体的固有免疫反应(产生炎性反应)，未引起肝功能损伤，且未出现明显的脱靶效应。这些结果充分地说明本申请的治疗方案具有较好的安全性。

Claims (13)

  1. 一种治疗和/或预防疾病的产品，其特征在于，所述产品中包含：能够将目的基因插入到肝细胞中Alb基因的13号内含子上并使所述目的基因与Alb共表达的试剂；
    所述疾病包括遗传性肝脏代谢疾病或可用肝脏进行代谢交叉校正治疗的疾病，
    所述目的基因包括在所述疾病中发生突变或缺失的基因。
  2. 如权利要求1所述的产品，其特征在于：所述遗传性肝脏代谢疾病包括B型血友病、A型血友病、苯丙酮尿症、或酪氨酸血症，优选，B型血友病，
    更优选的，所述目的基因包括F9因子的编码基因，更优选的，所述F9因子的编码基因如SEQ ID No.28所示；
    更优选的，所述产品具有改善所述B型血友病的凝血活性降低症状和/或凝血时间延长症状；
    所述可用肝脏进行代谢交叉校正治疗的疾病包括庞贝氏病、黏多糖贮积症、、戈谢病、Sanhoff病、Niemann-Pick病、Fabry病、或Farber病，优选，庞贝氏病，
    更优选的，所述目的基因包括GAA的编码基因，更优选的，所述GAA的编码基因如SEQ ID No.6所示；
    更优选的，所述产品具有改善所述庞贝氏病包括如下a-f症状中的至少一种：
    a体重减轻，
    b肌肉力量丧失，
    c血浆中GAA蛋白活性降低，
    d肝脏、心肌、膈肌、股四头肌、舌、脑、和/或脊髓中GAA蛋白活性降低，e肝脏、心肌、膈肌、股四头肌、舌、脑、和/或脊髓中糖原升高，
    f左心室重量指数升高。
  3. 如权利要求2所述的产品，其特征在于，所述试剂包括：核酸酶和sgRNA，和/或所述核酸酶的递送载体和所述sgRNA的递送载体；
    所述sgRNA能够引导所述核酸酶对肝细胞中Alb基因的13号内含子进行切割并形成双链断裂位点；
    优选的，所述sgRNA的靶序列包含SEQ ID No.1-5中任一所示的序列；
    更优选的，所述sgRNA的靶序列包含SEQ ID No.2所示的序列。
  4. 如权利要求3所述的产品，其特征在于：所述核酸酶选自Cas9、Cas3、Cas8a、Cas8b、Cas10d、Cse1、Csy1、Csn2、Cas4、Cas10、Csm2、Cmr5、Fok1、Cpf1中的一种或任意几种；
    优选的，所述核酸酶为Cas9，更优选的，所述Cas9选自来源于化脓性链球菌、和/或肺炎链球菌、和/或嗜热链球菌的Cas9，更优选的，所述Cas9选自来源于化脓性链球菌的Cas9。
  5. 如权利要求3或4所述的产品，其特征在于：所述试剂包括：供体修复模板、和/或所述供体修复模板的递送载体，
    所述供体修复模板包括依次连接的Alb基因的14号外显子编码序列和所述目的基因的编码序列；
    优选的，所述Alb基因的14号外显子编码序列如SEQ ID No.7所示；
    优选的，所述供体修复模板中所述Alb基因的14号外显子的上游设SA序列，更优选的，所述SA序列如SEQ ID No.8所示；
    优选的，所述供体修复模板中所述Alb基因的14号外显子与所述目的基因之间设自剪切多肽编码基因，更优选的，所述自剪切多肽为2A多肽，更优选的，所述2A多肽为P2A、T2A、E2A和/或F2A，更优选的，所述2A多肽为T2A多肽，更优选的，所述T2A多肽的编码基因序列如SEQ ID No.9所示，
    更优选的，所述自剪切多肽编码基因3’端与所述目的基因5’端之间连接分泌肽编码基因，更优选的，所述分泌肽的编码基因序列如SEQ ID No.26所示；
    优选的，所述供体修复模板中所述目的基因下游设polyA序列，更优选的，所述polyA为bGH-polyA，更优选的，所述bGH-polyA序列如SEQ ID No.10所示。
  6. 如权利要求5所述的产品，其特征在于：所述供体修复模板包括设于其两端的所述sgRNA的靶序列、或设与所述断裂位点两侧序列相同的同源臂，
    优选的，所述供体修复模板包括设于其两端的所述sgRNA的靶序列，
    更优选的，设于所述供体修复模板两端的所述sgRNA的靶序列的3’端连接PAM序列，
    更优选的，所述供体修复模板两端的3’端连接PAM序列的所述sgRNA的靶序列与所述肝细胞中Alb的13号内含子上的3’端连接PAM序列的所述sgRNA的靶序列反向或反向互补。
  7. 如权利要求3-6中任一所述的产品，其特征在于：所述递送载体包括病毒载体、和/或非病毒载体；所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、和/或溶瘤病毒载体，所述非病毒载体包括阳离子高分子聚合物、和/或脂质体；
    优选的，所述递送载体包括病毒载体；更优选的，所述递送载体包括腺相关病毒载体。
  8. 如权利要求7所述的产品，其特征在于：所述试剂包括：所述供体修复模板的递送载体，所述核酸酶的递送载体为第一rAAV载体，所述sgRNA的递送载体和所述供体修复模板的递送载体均为第二rAAV载体；
    优选的，所述第一rAAV载体和所述第二rAAV载体的血清型为rAAV1、rAAV2、rAAV5、rAAV6、rAAV7、rAAV8、rAAV9、rAAVrh10、rAAVrh64、或rAAVrh74；
    更优选的，所述递送载体的血清型为rAAV8；
    和/或，所述目的基因包括GAA的编码基因，所述试剂包括如下检测用组合A)-C)中的至少一种：
    A)用于检测所述目的基因插入所述断裂位点的PCR引物组：包括SEQ ID No.12-19；
    B)用于检测所述目的基因插入所述断裂位点的微滴式数字PCR探针组：包括SEQ ID No.20-23；
    C)用于检测所述目的基因与所述Alb基因形成的融合转录本的PCR引物组： 包括SEQ ID No.24-25。
  9. 如下1)-3)生物材料中的任一种：
    1)一种核酸分子，包括DNA分子和/或RNA分子，所述核酸分子对应的DNA序列如权利要求5或6中所述供体修复模板；
    2)含有1)所述核酸分子的重组载体，所述重组载体包括病毒载体、和/或非病毒载体；所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、和/或溶瘤病毒载体，所述非病毒载体包括阳离子高分子聚合物、和/或脂质体、和/或质粒；
    优选的，所述重组载体包括病毒载体；更优选的，所述递送载体包括腺相关病毒载体，所述腺相关病毒载体的血清型为rAAV1、rAAV2、rAAV5、rAAV6、rAAV7、rAAV8、rAAV9、rAAVrh10、rAAVrh64、或rAAVrh74；更优选的，所述递送载体的血清型为rAAV8；
    3)含有1)所述核酸分子的重组细胞或重组菌，优选的，所述重组细胞为肝细胞。
  10. 权利要求9所述生物材料在制备治疗和/或预防疾病产品中的应用，所述疾病包括遗传性肝脏代谢疾病或可用肝脏进行代谢交叉校正治疗的疾病，
    优选的，所述遗传性肝脏代谢疾病包括B型血友病、A型血友病、苯丙酮尿症、或酪氨酸血症，优选，B型血友病，更优选，所述产品能够改善所述B型血友病的凝血活性降低症状和/或凝血时间延长症状；
    优选的，所述可用肝脏进行代谢交叉校正治疗的疾病包括庞贝氏病、黏多糖贮积症、戈谢病、Sanhoff病、Niemann-Pick病、Fabry病、或Farber病，优选，庞贝氏病，更优选的，所述产品能够改善所述庞贝氏病的包括如下a-f症状中的至少一种：
    a体重减轻，
    b肌肉力量丧失，
    c血浆中GAA蛋白活性降低，
    d肝脏、心肌、膈肌、股四头肌、舌、脑、和/或脊髓中GAA蛋白活性降低，
    e肝脏、心肌、膈肌、股四头肌、舌、脑、和/或脊髓中糖原升高，
    f左心室重量指数升高。
  11. 一种用于CRISPR/Cas9基因编辑系统中的sgRNA，其特征在于，所述sgRNA能够引导核酸酶对细胞中Alb基因的13号内含子进行切割并形成双链断裂位点；
    优选的，所述sgRNA的靶序列包含SEQ ID No.1-5中任一所示的序列；
    更优选的，所述sgRNA的靶序列包含SEQ ID No.2所示的序列；
    优选的，所述细胞为肝细胞。
  12. 权利要求11所述sgRNA在编辑细胞Alb基因中的应用。
  13. 一种用于构建B型血友病动物模型的sgRNA，其特征在于，所述sgRNA的靶序列包含SEQ ID No.29和SEQ ID No.30所示的序列；
    优选的，所述动物为哺乳动物，更优选的，所述哺乳动物为大鼠，更优选的，所述大鼠为SD大鼠。
PCT/CN2020/123621 2019-10-31 2020-10-26 一种基于肝细胞Alb基因的疾病治疗的产品 WO2021083073A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201911055309.XA CN112741906B (zh) 2019-10-31 2019-10-31 一种用于治疗b型血友病的产品
CN201911055309.X 2019-10-31
CN202010879736.6A CN113058041B (zh) 2020-08-27 2020-08-27 一种用于治疗庞贝氏病的产品
CN202010879736.6 2020-08-27

Publications (1)

Publication Number Publication Date
WO2021083073A1 true WO2021083073A1 (zh) 2021-05-06

Family

ID=75715754

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/123621 WO2021083073A1 (zh) 2019-10-31 2020-10-26 一种基于肝细胞Alb基因的疾病治疗的产品

Country Status (1)

Country Link
WO (1) WO2021083073A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114015708A (zh) * 2021-11-16 2022-02-08 中南大学 一种深海细菌来源的α-葡萄糖苷酶QsGH13及其编码基因与应用
US11622547B2 (en) 2019-06-07 2023-04-11 Regeneran Pharmaceuticals, Inc. Genetically modified mouse that expresses human albumin

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105940013A (zh) * 2013-12-09 2016-09-14 桑格摩生物科学股份有限公司 用于治疗血友病的方法和组合物
WO2019079527A1 (en) * 2017-10-17 2019-04-25 Casebia Therapeutics Limited Liability Partnership COMPOSITIONS AND METHODS FOR GENETIC EDITION FOR HEMOPHILIA A
CN111492060A (zh) * 2017-09-28 2020-08-04 株式会社绿十字 因子viii或因子ix基因敲除兔、其制备方法及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105940013A (zh) * 2013-12-09 2016-09-14 桑格摩生物科学股份有限公司 用于治疗血友病的方法和组合物
CN111492060A (zh) * 2017-09-28 2020-08-04 株式会社绿十字 因子viii或因子ix基因敲除兔、其制备方法及其用途
WO2019079527A1 (en) * 2017-10-17 2019-04-25 Casebia Therapeutics Limited Liability Partnership COMPOSITIONS AND METHODS FOR GENETIC EDITION FOR HEMOPHILIA A

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN HAINAN, SHI MI, GILAM AVITAL, ZHENG QI, ZHANG YIN, AFRIKANOVA IVKA, LI JINLING, GLUZMAN ZOYA, JIANG RUHONG, KONG LING-JIE, CH: "Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII.", SCIENTIFIC REPORTS, vol. 9, no. 1, 14 November 2019 (2019-11-14), pages 1 - 15, XP055807516, ISSN: 2045-2322, DOI: 10.1038/s41598-019-53198-y *
WANG QIHAN, HUAI CONG, SUN RUILIN, ZHUANG HUA, CHEN HONGYAN, FEI JIAN, LU DARU: "A Quick and Efficient Method to Generate Hemophilia B Mouse Models by the CRISPR/Cas System", HEREDITAS, vol. 37, no. 11, 30 November 2015 (2015-11-30), pages 1143 - 1148, XP055789498, ISSN: 0253-9772, DOI: 10.16288/j.yczz.15-117 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11622547B2 (en) 2019-06-07 2023-04-11 Regeneran Pharmaceuticals, Inc. Genetically modified mouse that expresses human albumin
CN114015708A (zh) * 2021-11-16 2022-02-08 中南大学 一种深海细菌来源的α-葡萄糖苷酶QsGH13及其编码基因与应用
CN114015708B (zh) * 2021-11-16 2023-07-21 中南大学 一种深海细菌来源的α-葡萄糖苷酶QsGH13及其编码基因与应用

Similar Documents

Publication Publication Date Title
US11654201B2 (en) Liver-specific nucleic acid regulatory elements and methods and use thereof
EP4186921A1 (en) Gene editing for autosomal dominant diseases
TWI811219B (zh) 高活性調控元件
CN105567735A (zh) 一种凝血因子基因突变的定点修复载体系统及方法
JP2017512466A (ja) オルニチントランスカルバミラーゼ（ｏｔｃ）欠損症の処置において有用な組成物
US20230287419A1 (en) DUCHENNE MUSCULAR DYSTROPHY-RELATED EXONIC SPLICING ENHANCER, sgRNA AND GENE EDITING TOOL, AND APPLICATIONS
US20200270618A1 (en) Compositions and methods for treating alpha-1 antitrypsin deficiency
WO2021083073A1 (zh) 一种基于肝细胞Alb基因的疾病治疗的产品
JP2022530457A (ja) 遺伝子操作ａａｖ
US20150045416A1 (en) Methods and Compositions for Gene Delivery
CN111718420B (zh) 一种用于基因治疗的融合蛋白及其应用
WO2023202469A1 (zh) 一种用于遗传性凝血因子缺乏病治疗的核酸构建体及其用途
CN113058041B (zh) 一种用于治疗庞贝氏病的产品
CN109337928B (zh) 通过过表达腺相关病毒受体提高基因治疗效率的方法
WO2019036484A1 (en) COMPOSITIONS AND METHODS FOR THE TREATMENT OF ARGININOSUCCINIC ACIDURIA
CN112741906B (zh) 一种用于治疗b型血友病的产品
WO2020187272A1 (zh) 一种用于基因治疗的融合蛋白及其应用
EP3929294A1 (en) Pyruvate kinase deficiency (pkd) gene editing treatment method
WO2023173072A1 (en) Systems and methods for genetic modulation to treat liver disease
CN117187215A (zh) 受化学小分子诱导重新组装的CRISPR/CasRx系统及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20882209

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20882209

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC