Method for performing efficient site-directed gene knock-in in hepatocytes and application thereof

Abstract

The invention provides a method for efficient site-directed gene knock-in in hepatocytes and application of the method. The invention provides a technical scheme for carrying out gene knock-in operation by taking proliferated human hepatocytes (Pro HHs) as target cells, which is characterized in that the gene knock-in operation is carried out by taking the proliferated human hepatocytes (Pro HHs) as the target cells. Through the optimization strategy of the system, the Pro HHs subjected to gene editing can be effectively recolonized and mature, and the treatment of diseases is successfully realized. According to the invention, powerful concept verification is provided for autologous gene edited proliferative cell treatment of human hereditary liver diseases, and a new way is opened up for treatment of liver diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and more particularly, the present application relates to a method for efficient site-directed gene knock-in in hepatocytes and its application. BACKGROUND

[0002] The liver is a complex structure, function of a central metabolic organ, in which hepatocytes are the main function of the liver. Therefore, to establish a high-efficiency gene editing system in human hepatocytes has been an urgent goal for basic scientists. However, the existing main human hepatocyte system cannot fully meet such needs, for example, primary human hepatocytes (PHH) are the "gold standard" for measuring liver function, but PHHs rapidly differentiate in vitro, have functional loss, large donor differences, and extremely low gene manipulation efficiency; induced pluripotent stem cells (iPSCs) derived hepatocyte-like cells have good gene editing efficiency, but the differentiation efficiency and functional maturity of hiPSC-derived liver parenchymal cells are still much lower than PHHs, which may affect the accuracy and reliability of their application. Therefore, how to establish an amplifiable, easy to manipulate, simple and stable human hepatocyte system is still a basic problem in the field of liver, which has important significance and value for the research and treatment of liver diseases.

[0003] In vitro expansion of hepatocytes is a new source of human hepatocytes. Previously, the Ochiya team in Japan first established a small molecule inhibitor YAC culture medium of WNT, TGF-beta and Rock signaling pathway to induce the in vitro proliferation of rodent large and small mouse hepatocytes. The induced cells exhibit the characteristics of liver stem cells and liver precursor cells, and the in vitro expanded hepatocytes have the ability to differentiate into hepatocytes and bile duct epithelial cells. Although this breakthrough discovery is derived from rodents and has species limitations, because of the urgent need for human parenchymal hepatocytes, a large number of researchers have used similar techniques to challenge the in vitro expansion of human hepatocytes, and have reported the results of in vitro culture technology of human hepatocytes by different research groups in recent years. Although each research team has different combinations of chemical small molecules and cell growth factors, they all agree that EGF, HGF, TGF-beta inhibitor and WNT signaling pathway play a key role in the induction and expansion of human primary hepatocytes. With the rapid development of gene editing technology, precise gene editing in human primary cells has become possible, which has broadened the application field of basic research and clinical cell therapy.

[0004] However, one of the significant challenges in the field is the extremely low efficiency of exogenous gene knock-in in human primary hepatocytes. When attempting to edit hepatocytes using a homology directed repair (HDR) template, not only is the targeting efficiency less than 1%, but it is also accompanied by high cytotoxicity. Although the principle of not being able to easily knock in exogenous genes in primary hepatocytes is not clear, it greatly hinders the efficient integration of exogenous genes into the genome of the cells. SUMMARY

[0005] The purpose of the present application is to provide a method for efficient site-directed gene knock-in in hepatocytes and its application.

[0006] In a first aspect of the present application, a method for site-directed knock-in of a target gene in hepatocytes is provided, comprising: using proliferating human hepatocytes (ProliHHs) as target cells, introducing into the target cells: a Cas9-sgRNA complex, a DNA donor; wherein the DNA donor is a DNA donor for hitchhiking independent targeted integration (HITI), introduced into the target cells mediated by AAV2.7m8 virus; the target gene is included in the DNA donor.

[0007] In one or more embodiments, the Cas9-sgRNA complex is introduced into the target cells by electroporation; preferably, the electroporation is performed using a Lonza 4D 96-well electroporation system; more preferably, the electroporation is performed using an electroporation program DS198.

[0008] In one or more embodiments, the Cas9-sgRNA complex is generated by mixing Cas9 with sgRNA to form ribonucleoprotein (RNP).

[0009] In one or more embodiments, the Cas9 and sgRNA are mixed at a molar ratio of 1:1-10, preferably at a molar ratio of 1:1.5-8, more preferably at a molar ratio of 1:2-5, such as a molar ratio of 1:2.2, 1:2.5, 1:2.6, 1:3.

[0010] In one or more embodiments, the DNA donor is a linear donor; preferably, the DNA donor comprises the following operatively linked elements: a promoter, a target gene, a terminator; more preferably, upstream of the promoter and downstream of the terminator further comprise: Stuff, ITR.

[0011] In one or more embodiments, the proliferating human hepatocytes (ProliHHs) are cultured using HM or EHM medium.

[0012] In one or more embodiments, the proliferating human hepatocytes (ProliHHs) are derived from patients with genetic metabolic diseases. In one or more embodiments, the proliferating human hepatocytes (ProliHHs) are derived from patients with genetic metabolic diseases.

[0013] In one or more embodiments, for patient-derived ProliHHs, EHM or HM medium can be used. The EHM medium is HM medium with replacement of nicotinamide by recombinant human bFGF; preferably, the bFGF is used at 25 ± 10 ng / mL (preferably 25 ± 8 ng / mL, 25 ± 6 ng / mL, 25 ± 4 ng / mL, or 25 ± 2 ng / mL).

[0014] In one or more embodiments, the method for efficient site-directed gene knock-in in hepatocytes is a non-therapeutic, non-diagnostic method.

[0015] In one or more embodiments, the method for efficient site-directed gene knock-in in hepatocytes is an ex vivo method.

[0016] In one or more embodiments, under the guidance of sgRNA, the target gene achieves site-directed insertion.

[0017] In another aspect of the present application, a method for preparing therapeutic hepatocytes containing a target gene is provided, comprising: preparing proliferative human hepatocytes (ProliHHs) with the target gene introduced by any of the above-mentioned methods; the target gene is a gene for alleviating or treating a disease.

[0018] In one or more embodiments, the method for preparing therapeutic hepatocytes containing a target gene is a non-therapeutic, non-diagnostic method.

[0019] In one or more embodiments, the method for preparing therapeutic hepatocytes containing a target gene is an ex vivo method.

[0020] In one or more embodiments, the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 2.

[0021] In one or more embodiments, the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1.

[0022] In one or more embodiments, the target gene includes (but is not limited to): a genetic defect gene, a coding gene of a pharmaceutically active protein, a coding gene of a diagnostically active protein, a coding gene of an immunogenic protein, a coding gene of a structural protein, an enzyme coding gene, a reporter gene; preferably, the genetic defect gene includes (but is not limited to): tyrosinemia-related defect gene FAH; urea cycle disorder-related defect gene OTC, ASL, CPS1, ARG1, or ASS1; hypercholesterolemia-related defect gene LDLR; glycogen accumulation syndrome-related defect gene G6PC; Crigler-Najjar syndrome-related defect gene UGT1A1.

[0023] In one or more embodiments, the proliferated human hepatocytes (ProliHHs) are primary hepatocytes derived from a patient with a genetic deficiency disease, and have a genetic deficiency in their genome.

[0024] In another aspect of the present application, there is provided a use of a Cas9-sgRNA complex and a DNA donor for performing a targeted gene knock-in in a liver cell, or for preparing a reagent for performing a targeted gene knock-in in a liver cell, or for preparing a therapeutic liver cell comprising a target gene; wherein the liver cell is a proliferated human hepatocyte (ProliHH); the DNA donor is a DNA donor for homology independent targeted integration (HITI), introduced into the target cell mediated by AAV2.7m8 virus; the DNA donor comprises a target gene; preferably, the DNA donor is a linear donor.

[0025] In one or more embodiments, the target gene is a gene for alleviating or treating a disease; preferably, the target gene comprises (but not limited to) a genetic deficiency gene, a coding gene of a pharmaceutically active protein, a coding gene of a diagnostically active protein, a coding gene of an immunogenic protein, a coding gene of a structural protein, an enzyme coding gene, a reporter gene; preferably, the genetic deficiency gene comprises (but not limited to) a tyrosinemia related deficiency gene FAH, a tyrosinemia related deficiency gene FAH.

[0026] In one or more embodiments, the proliferated human hepatocytes are primary hepatocytes derived from a patient with a genetic deficiency disease, and have a genetic deficiency in their genome.

[0027] In another aspect of the present application, there is provided a liver cell comprising a target gene, wherein the liver cell is a recombinant proliferated human hepatocyte (ProliHH); and wherein the liver cell comprises exogenously: a Cas9-sgRNA complex, a DNA donor; wherein the DNA donor is a DNA donor for homology independent targeted integration (HITI), introduced into the proliferated human hepatocyte mediated by AAV2.7m8 virus; and wherein the DNA donor comprises a target gene.

[0028] In one or more embodiments, the liver cell comprising a target gene is prepared by any one of the methods described above; preferably, the Cas9-sgRNA complex is introduced into the target cell by electroporation; preferably, the electroporation is performed using a Lonza 4D 96-well electroporation system; more preferably, the electroporation is performed using an electroporation program DS198.

[0029] In another aspect of the present application, the use of the hepatocytes comprising the target gene in the preparation of a pharmaceutical composition for in vivo transplantation is provided; wherein the target gene is a gene for alleviating or treating a disease; preferably, the target gene includes (but not limited to): a genetic defect gene, a coding gene of a pharmaceutically active protein, a coding gene of a diagnostically active protein, a coding gene of an immunogenic protein, a coding gene of a structural protein, an enzyme coding gene, a reporter gene.

[0030] In one or more embodiments, the hepatocytes comprising the target gene maintain high efficiency of proliferation and passaging.

[0031] In one or more embodiments, the hepatocytes comprising the target gene stably secrete human albumin.

[0032] In one or more embodiments, after the inhibition, the edited and corrected ProliHHs hepatocytes transplanted can reestablish the zonal characteristics of the liver, and the integrated ProliHHs hepatocytes can develop into a mature hepatocyte state.

[0033] In one or more embodiments, the pharmaceutical composition for in vivo transplantation alleviates or treats liver diseases.

[0034] In one or more embodiments, the in vivo transplantation includes in vivo autologous transplantation.

[0035] In one or more embodiments, the method combined with the in vivo transplantation scheme includes: collecting primary hepatocytes from a patient with liver disease, preparing ProliHHs, and further preparing recombinant (knocking in an exogenous gene) ProliHHs using the aforementioned method to achieve treatment of the disease.

[0036] In one or more embodiments, the liver disease includes (but not limited to): liver injury.

[0037] In one or more embodiments, the liver disease includes (but not limited to): end-stage liver disease, liver cirrhosis, alcoholic liver disease, acute liver failure, hepatitis (including drug-induced hepatitis), liver fibrosis, liver cancer, liver metabolic disease, or liver injury caused by liver function failure.

[0038] In another aspect of the present application, a pharmaceutical composition or kit for in vivo transplantation is provided, which contains the hepatocytes comprising the target gene.

[0039] Other aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 : Optimization of exogenous gene integration strategy in ProliHHs.

[0041] (A) Schematic diagram depicts the process of genome targeting in ProliHHs using RNPs (Cas9-sgRNA) and DNA donor delivered by electroporation. The DNA donor contains EGFP gene as a reporter and knock-in efficiency in ProliHHs is checked by fluorescent imaging or flow cytometry.

[0042] (B) GFP fluorescent micrographs of ProliHHs, cells were electroporated with equal amount of plasmid expressing GFP to test the delivery ability of 14 low electric pulse electroporation programs.

[0043] (C) Array analysis of electroporation programs evaluated plasmid delivery efficiency and ProliHHs proliferation ability. DS198 was selected to optimize efficiency.

[0044] (D) Representative images of ProliHHs after electroporation program DS198 treatment.

[0045] (E) RNPs schematic for GAPDH 3’-UTR (GAPDH-sgRNA). RNPs were electroporated into ProliHHs and indel (%) of GAPDH-sgRNA was analyzed by tagmentation sequencing.

[0046] (F) Knock-in efficiency of circular HDR-mediated donor (IRES-EGFP) in ProliHHs was detected by fluorescent imaging and quantified by flow cytometry.

[0047] (G) Schematic diagram of targeting integration strategy for evaluating the combined effect of different integration strategies and donor template topology.

[0048] (H) Knock-in efficiency of different designed donors (IRES-EGFP) for GAPDH-sgRNA site was evaluated by flow cytometry.

[0049] (I) Representative fluorescent micrographs of linear donors with HDR, NHEJ or HITI integration strategy, respectively.

[0050] Figure 2 : High efficient infection efficiency of AAV2.7m8 in ProliHHs.

[0051] (A) AAV variant screening schematic. ProliHHs were infected with equal amount of AAV variants expressing nuclear localization signal (NLS)-mNeonGreen (green), infection of cells was analyzed by fluorescent imaging and flow cytometry 3 days post viral infection.

[0052] (B) Comparison of transduction efficiency of AAV variants in ProliHHs, representative fluorescence images were selected.

[0053] Figure 3 : Efficient CRISPR / Cas9 and AAV2.7m8-mediated targeted integration in ProliHHs.

[0054] (A) Quantification of AAV infection efficiency in ProliHHs by flow cytometry.

[0055] (B) Timeline of RNPs and AAV2.7m8-mediated targeted integration in ProliHHs. ProliHHs were infected with AAV2.7m8 donor (HITI-IRES-EGFP) 2 days before RNPs electroporation. Edited ProliHHs were further cultured and analyzed after electroporation.

[0056] (C) Schematic of targeted genome editing at GAPDH-sgRNA site using RNPs and AAV2.7m8 donor template. Cas9 (scissors) creates site-specific double-strand breaks (DSBs). DSBs stimulate the non-homologous end joining (NHEJ) pathway with AAV2.7m8 donor template as repair template. Blue boxes, GAPDH exons.

[0057] (D) ProliHHs were subjected to electroporation and analyzed for indels at GAPDH-sgRNA by deep sequencing. Mean values of n=5 independent donors are shown.

[0058] (E) Mean integration efficiency of donor template (HITI-IRES-EGFP) in ProliHHs (n=5 independent donors).

[0059] (F) ProliHHs electroporated as described above or ProliHHs transduced with AAV2.7m8 donor template were analyzed by flow cytometry 7 days after electroporation. Top, bright field images of edited ProliHHs. Middle, fluorescence images of edited ProliHHs. Bottom, representative FACS plot.

[0060] (G) Percentage of EGFP-expressing cells in three generations after electroporation was quantified using flow cytometry (n=3 independent donors).

[0061] Figure 4 : Correction of FAH gene deficiency by targeted integration in ProliHHs.

[0062] (A) Schematic of targeted editing using RNPs and AAV2.7m8 donor template at the AAVS1 site (AAVS1-sgRNA). Blue box, PPP1R12C exon.

[0063] (B) ProliHHs that were electroporated as described above were analyzed by flow cytometry two generations after electroporation. Top, brightfield images of edited ProliHHs. Middle, fluorescent images of edited ProliHHs. Bottom, representative FACS plot.

[0064] (C) Mean integration efficiency of AAV2.7m8 donors in ProliHHs. Mean values of n=5 independent donors are shown.

[0065] (D) Editing was performed on ProliHHs (P1) and cultured to obtain bulk edited cells (P2-P4) and sorted to generate pure edited cell populations (P5-P6). The percentage of EGFP+ cells was quantified using flow cytometry during cell passaging and sorting.

[0066] (E) Bulk edited ProliHHs cell populations were sorted to generate pure edited ProliHHs cell populations. Top, fluorescent images of ProliHHs. Bottom, representative FACS plot.

[0067] (F and G) FAH mRNA expression and protein expression in untreated, AAV only infected, edited or purified ProliHHs were quantified using RT-qPCR and Western blotting. All mRNA transcript levels and protein levels were referenced to fresh PHHs.

[0068] Figure 5 : Edited ProliHHs transplant therapy in tyrosinemia mouse model.

[0069] (A) Workflow of transplanting edited patient-derived ProliHHs into the FRG tyrosinemia mouse model. Left panel: FAH mutation information for this tyrosinemia patient. Negative immunohistochemical staining for FAH in the liver shows loss of FAH protein. Middle panel: PHHs were isolated from liver tissue of this patient and cultured in EHM. Patient ProliHHs were corrected by targeting the therapeutic donor to the AAVS1-sgRNA site and transplanted into FRG mice. Right panel: Schematic of targeted editing using RNPs and AAV2.7m8 donor at the AAVS1-sgRNA site.

[0070] (B) Kaplan-Meier survival curves of FRG mice with untransplanted cells and transplanted cells.

[0071] (C) Serum ALT, AST and TBIL levels of moribund control FRG mice (n=3) and unedited ProliHHs (n=3), surviving edited ProliHH-FRG mice (n=3) and surviving PHH-FRG mice (n=3).

[0072] (D) Dynamics of human ALB levels determined using ELISA in moribund unedited ProliHHs-FRG mice, surviving edited ProliHH-FRG mice and surviving PHH-FRG mice within 6 months after transplantation.

[0073] (E) Human ALB levels determined using ELISA in moribund unedited ProliHHs-FRG mice (n=7), surviving edited ProliHH-FRG mice (n=5) and surviving PHH-FRG mice (n=5) after transplantation.

[0074] (F) Immunohistochemical staining for FAH in the livers of mice after transplantation.

[0075] (G) Analysis of maturation of repopulated edited ProliHHs using co-immunofluorescence staining. Liver sections were analyzed using co-immunofluorescence staining. Data are shown as mean ± standard deviation. ns > 0.05, *p < 0.05; **p < 0.01; ***p < 0.001; (B) log-rank test, (C) and (E) Student's t-test. DETAILED DESCRIPTION

[0076] In the present application, the "target gene" can also be referred to as "gene of interest", which refers to a gene of interest useful for observing changes in hepatocytes, regulating hepatocyte performance or improving diseases after introduction into hepatocytes.

[0077] As used herein, the "element" refers to a series of functional nucleic acid / protein sequences useful for the expression of proteins, which are systematically constructed to form an expression construct. The sequence of the "element" can be those provided in the present application, and also includes variants thereof, as long as the variants substantially retain the function of the "element", which are obtained by inserting or deleting some bases (such as 1-50 bp; preferably 1-30 bp, more preferably 1-20 bp, more preferably 1-10 bp), or random or site-directed mutagenesis, etc.

[0078] As used herein, "operatively linked" or "operably linked" refers to the functional placement of two or more nucleic acid regions or nucleic acid sequences in a specific location relative to one another. For example, a promoter region is placed in a specific location relative to a nucleic acid sequence of interest such that transcription of the nucleic acid sequence is directed by the promoter region, and thus, the promoter region is "operably linked" to the nucleic acid sequence.

[0079] As used herein, "construct" refers to a single- or double-stranded DNA molecule that has been manipulated by human intervention to contain segments of DNA combined and arranged in a sequence not existing in nature. The "construct" includes "plasmid", "in vitro transcription product", or viral vector, etc.; or the "construct" is included in or as a part of an expression vector.

[0080] As used herein, "sgRNA" refers to "Single-guide RNA", which is designed based on the "target site on the target gene" and contains a sequence sufficient to cooperate with the endonuclease Cas9 to guide the Cas9-mediated DNA double-strand break at the target site. In the present application, the "sgRNA" includes sgRNA in the form of RNA (such as mRNA form), and also includes DNA form sequence corresponding to the sgRNA sequence or a construct containing the sequence, as long as they can be processed or converted into active "sgRNA" in cells.

[0081] Hepatocytes, as the core executors of liver functions, are naturally indispensable basic tools for basic research and treatment of liver diseases. However, so far, the exogenous gene site-directed knock-in of human hepatocytes has been maintained at a low level and is very inefficient, which greatly restricts the research progress and treatment effect in related fields. Therefore, constructing a human hepatocyte system that has both high gene knock-in efficiency and simple and stable production process has become a basic problem that needs to be solved in the field of liver research. Hepatocytes have different cell states at different stages of differentiation or development, and their expressed genes and presented functions and stability are not the same. Therefore, in addition to focusing on the success rate of cell knock-in, when considering the use of hepatocytes for transplantation / treatment, the selection of cells is also particularly important for the transplantation / treatment effect.

[0082] In recent years, genome editing technology has been more widely used. In the technology related to gene editing, it includes gene knock-in, gene knockout, gene mutation, etc. Among these different editing methods, the efficiency, accuracy or stability of gene knock-in is far lower than that of gene knockout. Therefore, the design difficulty of gene editing related to gene knock-in is much greater than that of other types of editing technology. In particular, the efficiency of gene knock-in and gene editing for hepatocytes is very low. Improvement is needed to meet the clinical application standards as much as possible, that is, to truly achieve the effect of clinical improvement, which has high requirements for the stability and function maintenance of cells.

[0083] In the present application, a technical solution of gene knock-in operation is proposed by using proliferated human hepatocytes (ProliHHs) as target cells. Through the research and analysis of hepatocytes, the inventors selected proliferated human hepatocytes (ProliHHs) as the target cells for editing and achieved the effect of in vivo treatment. Through systematic optimization strategy, AAV2.7m8 delivery and HITI strategy were successfully used to effectively realize CRISPR / Cas9-mediated targeted integration, aiming to correct the defective genes of patients in ProliHHs. Furthermore, it was found that the ProliHHs edited by gene editing could effectively reprogram and mature, and could successfully treat tyrosinemia and other genetic deficiency diseases. The present application provides a strong proof of concept for the autologous gene editing of proliferated cells for human genetic liver disease, has great clinical transformation potential, can open up a new way for the treatment of liver diseases, and further promote the development of the field of liver regeneration medicine.

[0084] In the present application, the ProliHHs are human hepatocytes that have regained proliferative capacity by using a strategy of WNT3a, HGF and other cell growth factors combined with chemical small molecules to induce the proliferation of non-dividing human hepatocytes. The ProliHHs are obtained by expansion in a limited number of passages. As a kind of hepatocytes, ProliHHs can better maintain the function of hepatocytes and the expression level of related genes such as ALB, TTR, CYP3A4, CYP1A2, CAR, C3, etc.

[0085] The method for expanding ProliHHs in vitro includes culturing hepatocytes in an in vitro expansion medium (for example, see ZL201811156216.1 or the HM medium listed in the present application), thereby obtaining intermediate cells between mature hepatocytes and liver precursor cells (with unique marker expression characteristics). The cells can further develop into mature hepatocyte state.

[0086] The method for in vitro expansion of hepatocytes comprises culturing hepatocytes in a cell culture medium containing a Wnt signaling pathway activator to expand the hepatocytes in vitro. The Wnt signaling pathway activator comprises an activator selected from the group consisting of Wnt3a protein, Wnt3a conditioned medium, CHIR, or a combination thereof. As a preferred embodiment, the cell culture medium further contains an ingredient selected from the group consisting of N-acetyl-cysteine, nicotinamide, FGF10, EGF, HGF, [Leu15]-gastrin I, A 83-01, Y-27632 cell culture medium, N2 supplement, B27 supplement, serum. The culture of ProliHHs can be carried out by a two-dimensional or three-dimensional culture system. The obtained hepatocytes can be frozen, recovered, subcultured, and maintained in culture for a long time.

[0087] To achieve efficient integration of exogenous genes, the inventors successfully established a culture system for in vitro expansion of hepatocytes (ProliHHs) through culture optimization and induction. The cells were in a suitable state, which was conducive to the implementation of the technical solutions of the present application. Further, the inventors systematically compared various gene knock-in strategies to seek the best solution that could match the liver cells with a specific state of the present application and had a high gene knock-in efficiency, including homology-directed repair (HDR)-mediated, non-homologous end joining (NHEJ)-mediated, and homology-independent targeted integration (HITI)-mediated targeted integration, and analyzed the donor template topology. The results showed that in ProliHHs, the knock-in efficiency using linear HITI donors was significantly better than other combination modes. To improve the efficiency of gene knock-in, the inventors screened AAV variant libraries and analyzed their ability to infect ProliHHs. The screening results showed that AAV2.7m8 performed best in terms of infection efficiency. The inventors also analyzed the electroporation technology and selected different low-voltage pulse electroporation programs of LONZA electroporator, and found that the DS198 program performed best in maintaining cell activity and providing high transfection capacity.

[0088] Thanks to the high transduction efficiency of HITI-linear donor mediated by AAV2.7m8, combined with appropriate electroporation technology, the CRISPR / Cas9-sgRNA protein complex achieved efficient delivery and successfully achieved integration efficiency as high as 23.6% to 66% at specific target sites in hepatocytes. This achievement not only proves the high efficiency of the method of the present application, but also provides the possibility for further expansion and transplantation application of gene knock-in cells.

[0089] Based on the new discovery of the inventor, a method for targeted gene knock-in in hepatocytes is provided, comprising: taking proliferating human hepatocytes (ProliHHs) as target cells, and introducing into the cells: a Cas9-sgRNA complex, and a DNA donor. In some embodiments, the method is in vitro (ex vivo), i.e., the operation is performed on ex vivo cells.

[0090] The present application also provides a method for preparing therapeutic hepatocytes containing a target gene, comprising: preparing proliferating human hepatocytes (ProliHHs) into which a target gene is introduced by the method described above; the target gene is a gene for alleviating or treating a disease. In some embodiments, the method is in vitro (ex vivo), i.e., the operation is performed on ex vivo cells.

[0091] In other aspects, based on the disclosure of the present application, cells or animals with genomic gene editing can also be prepared. In some embodiments, the method can also be for research purposes rather than for therapeutic purposes.

[0092] Genome targeted knock-in expression can be used as a general gene editing and correction strategy, which is independent of the patient's mutant gene, mutation type and site. The strategy of the present application is an ideal gene correction method for treating various genetic metabolic liver diseases caused by different gene mutations. Using the strategy of the present application, the therapeutic gene can be inserted into the target site in dividing and non-dividing cells.

[0093] Currently, there is an increasing demand for developing an efficient and stable gene editing method in clinical practice. The technical solution of the present application not only has important significance for promoting research in the field of liver, but also provides a new way for the treatment of liver diseases.

[0094] Based on the new discovery of the present application, a recombinant ProliHHs culture obtained by the method described in the present application or ProliHHs hepatocytes isolated and purified from the hepatocyte culture can be intermediate cells between mature hepatocytes and liver precursor cells, or further induced to obtain mature hepatocytes.

[0095] Methods for enriching or isolating and purifying cells from cell cultures are also well known to those skilled in the art, for example, cells can be enriched based on the epithelial-like morphological characteristics of hepatocytes; or collected based on special proteins (such as Albumin, etc.) or molecular markers expressed by hepatocytes (for example, using specific antibodies or ligands). In addition, it is also a feasible method to enrich hepatocytes by removing (such as digesting, lysing) other cells with non-epithelial-like morphology. As an alternative embodiment, flow cytometry sorting technology can be used to separate and purify cells by molecular markers on the surface of hepatocytes.

[0096] The cultured ProliHHs liver cells of the present application have a wide range of uses. These include, but are not limited to: use in the preparation of compositions (pharmaceutical compositions) for promoting liver regeneration or for liver transplantation; use in the preparation of compositions (pharmaceutical compositions) for treating liver damage (including, but not limited to: end-stage liver disease, liver cirrhosis, alcoholic liver disease, diabetes, obesity, acute liver failure, hepatitis, liver fibrosis, liver cancer, liver metabolic disease or liver damage caused by liver function failure); use as an in vitro model for the study of liver-related diseases or drug efficacy, for example, for the study of drug transport, drug metabolism, liver formation, liver regeneration, for liver toxicity testing, screening of hepatotoxic compounds, screening of compounds that modulate liver cell function, for the production of Albumin protein, etc.

[0097] In the technical solution for the preparation of compositions (pharmaceutical compositions) for promoting liver regeneration or for liver transplantation, liver cells (primary liver cells) can be obtained from the body of a patient with a genetic deficiency disease, cultured to obtain ProliHHs, and further prepared into recombinant ProliHHs with the target gene knocked in using the method of the present application. The recombinant ProliHHs can be used as a graft to inhibit the body, achieving the purpose of alleviating or treating liver disease.

[0098] The cultured liver cells of the present application can be used for liver toxicity research, and can also be applied to cell transplantation for the treatment of liver disease, the construction of a bioartificial liver, the detection of liver toxicity of new drugs, the evaluation of drug efficacy, and the identification of drug targets; can provide a sufficient source of liver cells or a liver cell model for basic research and clinical application in biology, medicine and pharmacy; the induction and differentiation process thereof can also provide an optimal research platform for the development and differentiation process of human liver cells, and has a very broad application prospect.

[0099] If necessary, the cultured liver cells of the present application can be further used for genetic engineering recombination to form further recombinant cells, for example, to impart further functions or characteristics to the cells, to introduce an exogenous gene expression cassette into the cells, or to perform gene knockout or gene editing on the genome of the cells, etc.

[0100] The present application also provides a composition (drug) containing: an effective amount of the ProliHHs liver cells (such as 1 x 10 4 -1 x 10 12 cells; preferably 1 x 10 5 -1 x 10 10 cells); and a pharmaceutically acceptable carrier. It contains an effective amount of the liver cells and a pharmaceutically acceptable carrier. The composition has no visible toxicity and side effects on animals.

[0101] The "effective amount" means an amount that can produce a function or activity to a human and / or animal and can be accepted by a human and / or animal. The "pharmaceutically acceptable carrier" means a carrier for administration of a therapeutic agent, including various excipients and diluents. The term means such a pharmaceutical agent carrier that is not an essential active ingredient itself and has no excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. The pharmaceutically acceptable carrier in the composition can contain a liquid such as water, saline, buffer. In addition, there can be present in these carriers auxiliary substances such as a filler, a lubricant, a glidant, a wetting agent or an emulsifying agent, a pH buffering substance, etc. The carrier can further contain a cell transfection reagent.

[0102] The present application also provides a method for liver cell transplantation / promoting liver regeneration, which comprises administering to a subject in need thereof an effective amount of the recombinant ProliHHs liver cell prepared according to the present application.

[0103] The composition, when used for administration, is usually 1 x 10 2 - 1 x 10 10 cells / kg body weight, preferably 1 x 10 3 - 1 x 10 8 cells / kg body weight, which is suitable depending on the diagnosis of a clinician and the severity of symptoms of a patient.

[0104] The present application also provides a kit containing the recombinant ProliHHs liver cell according to the present application or a composition containing the ProliHHs liver cell. Preferably, the kit further contains an instruction for use, thereby facilitating the use of the present application in research or in clinic by those skilled in the art.

[0105] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods in the following examples, for which no specific conditions are mentioned, are generally carried out according to the conventional conditions as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Science Press, or according to the conditions suggested by the manufacturer.

[0106] Materials and Methods

[0107] 1. Culture medium

[0108] The liver cell culture medium includes HM medium and EHM medium, the formulations of which are shown in Table 1 and Table 2.

[0109] Table 1

[0110]

[0111] Table 2

[0112]

[0113] This component can be replaced by 30% Wnt3a conditioned medium or 0.1 uM CHIR. The Wnt3a conditioned medium is taken from the supernatant of L-Wnt3a cell line (obtained from Hans Clevers lab) cultured for two days.

[0114] 2、Cell culture

[0115] Primary human hepatocytes were isolated from discarded liver tissues of liver transplantation of patients with genetic metabolic liver diseases in Renji Hospital. L-Wnt3a cell line was a gift from Hans Clevers lab. Snu-398 cell line was purchased from ATCC cell bank in USA.

[0116] General ProliHHs cell culture: HM medium was used;

[0117] ProliHHs culture based on primary hepatocytes from patients: EHM medium was used.

[0118] ProliHHs cell after electroporation: EHM medium was used preferentially.

[0119] 3、Human hepatocyte culture

[0120] Human hepatocytes were isolated from liver tissues of patients using collagenase. Isolated human hepatocytes were seeded in DMEM medium containing 10% serum, at a density of 2X 10 4 cells / cm2in collagen type I coated culture plates, which were placed in a 37°C, 5% CO 2 , 5% O 2 incubator. After 1 day of culture, the medium was changed to hepatocyte culture medium, and the cell culture medium was changed every 3 days. After 6 days of cell culture, the confluent cells were trypsinized and seeded in collagen type I coated culture plates at a density of 3X 10 4 cells / cm 2 , which were placed in a 37°C, 5% CO 2 , 5% O 2 incubator.

[0121] 4、Cell culture photography

[0122] Thawed human hepatocytes were recovered in DMEM medium containing 10% serum, and seeded in collagen type I coated six-well plates at a density of 2X 10 4 cells / cm 2 . After the cells adhered, the medium was changed to hepatocyte culture medium, and the cell plates were placed in a 37°C, 5% CO 2Cells were cultured in incubator, and pictures were taken every 30 minutes for three days. The video of cell proliferation was generated by the software.

[0123] 5. Gene expression detection

[0124] 1) Cell RNA was extracted by Trizol (Invitrogen) or RNeasy FFPE Kit (Qiagen).

[0125] 2) 1 ug of RNA extracted by Trizol or 1 ug or all of RNA extracted by RNeasy FFPE Kit was used to generate cDNA by M-MLV reverse transcriptase (Promega).

[0126] 3) SYBR Premix Ex Taq (TaKaRa) was used to perform real-time quantitative PCR, and ABIStepOnePlus real-time PCR system (Applied Biosystems) was used to detect gene expression. All Q-PCR data were repeated at least twice, and primer sequences were provided in Table 3.

[0127] Table 3

[0128]

[0129]

[0130] 6. Human albumin ELISA detection

[0131] 1) Capture antibody was diluted by coating buffer at 1:250, and 100 ul was added to each well of 96-well ELISA plate (Corning). The 96-well plate was sealed and incubated at 4°C overnight.

[0132] 2) After the capture antibody was removed, 400 ul of washing buffer was added to each well, soaked for 1 minute, then removed, and washed twice. The washing buffer was completely removed in the second time.

[0133] 3) 250 ul of blocking buffer was added, and incubated at room temperature for 2 hours.

[0134] 4) Washed twice according to step 2.

[0135] 5) The standard was diluted by two-fold, and 8 gradients were diluted. The sample was also diluted according to the standard curve to ensure that the final OD450 fell within the range of the standard curve.

[0136] 6) Take 100 ul of standard and sample and add to 96 well plate, after blocking, incubate at room temperature for 2 hours.

[0137] 7) Wash 4 times as per step 2.

[0138] 8) Add 100 ul of Detection antibody per well, incubate at room temperature for 1 hour.

[0139] 9) Wash 4 times as per step 2.

[0140] 10) Add 100 ul of Substrate Solution per well, incubate at room temperature for 15 minutes.

[0141] 11) Add 100 ul of Stop Solution per well, measure A450.

[0142] Note: The above mentioned solutions are taken from ALB ELISA kit (Bethyl Laboratories) which comes with the kit.

[0143] 7. Production of AAV vectors

[0144] To produce AAV vectors carrying a nuclear localization green fluorescent protein mNeonGreen (NLS-mNeonGreen) driven by a cytomegalovirus enhancer / chicken beta-actin (CAG) promoter and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in HEK 293T cells, a method of co-transfection with AAV vectors, rep / cap packaging plasmids, and adenoviral helper plasmids was used. Briefly, DMEM medium was collected twice every 48 hours after transfection. Cell lysates were treated with chloroform and supernatants were collected. The mixture of supernatants and medium was concentrated by 10% PEG 8000 and 1.0 M NaCl precipitation at 4°C overnight. The precipitate was then resuspended in PBS buffer containing Benzonase. Next, different gradient iodixanol solutions of 15%, 25%, 40%, and 60% were carefully overlaid, followed by the generated virus suspension, and centrifuged at 10°C, 350,000 x g for 90 minutes. The AAV-containing 40% iodixanol fraction obtained after ultracentrifugation was collected. The purified virus was further concentrated by buffer exchange into PBS containing 0.001% F68 (Gibico). The genome content titer of AAV was measured using SYBR (Roche) analysis with primers targeting the WPRE region. The RT-qPCR primers for WPRE were as follows:

[0145] AAV-forward, 5'-GTCAGGCAACGTGGCGTGGTGTG-3' (SEQ ID NO: 3);

[0146] AAV-reverse, 5'-GGCGATGAGTTCCGCCGTGGC-3' (SEQ ID NO: 4).

[0147] By this step, the quality and quantity of the produced AAV vectors can be accurately evaluated, providing a reliable material basis for subsequent experiments and applications.

[0148] 8. In vitro AAV library screening

[0149] First, 5 x 10^4 proliferating human hepatocytes (ProliHHs) were seeded in a 48-well plate and incubated overnight. Subsequently, an equal amount of AAV variants (1 x 10^12 genome copies of virions, GCs) was added to each well. After 3 days of incubation, the transduction of AAV in ProliHHs (i.e. mNeonGreen positive cells) was observed by inverted fluorescence microscopy (Olympus, BX71) and quantitatively analyzed using a flow cytometer (Invitrogen Attune NxT).

[0150] 9. Electroporation program screening

[0151] To transfect ribonucleoproteins (RNPs) into proliferating human hepatocytes (ProliHHs), the Lonza Nucleofector TM4D 96-well electroporation system. Given the lack of a suitable electroporation program for ProliHHs, a reporter plasmid expressing green fluorescent protein (pmaxGFP from Lonza P3 Primary Cell 4D-Nucleofector X Kit) was first used to screen programs according to the manufacturer's optimization guidelines. 2 x 10^5 ProliHHs were resuspended in 20 μΐ of Lonza electroporation buffer P3 (containing 18 μΐ of Nucleofector solution and 4 μΐ of Supplement Solution) with the addition of 500 ng of pmaxGFP DNA. Subsequently, cells were transferred to a cuvette and electroporated using the Lonza 4D 96-well electroporation system with a variety of different electroporation programs (CA137, CM138, CM150, DN100, DS120, DS137, DS150, DS156, DS198, EH100, EN138, EO100, EW113, and EX147). After 3 days of incubation, GFP fluorescence micrographs of cells were captured and quantified using an Operetta high-content imager (PerkinElmer). On day 8 after the electroporation program, cells were digested and counted using a cell counter to assess cell proliferation after electroporation. Through this series of screening steps, an electroporation condition suitable for ProliHHs was found, laying a solid foundation for subsequent experiments.

[0152] 10. Production of RNPs

[0153] Two single guide RNAs (sgRNAs, provided by GenScript) were chemically synthesized and resuspended using ddH2O. Subsequently, a recombinant Streptococcus pyogenes Cas9 (spCas9) protein was purchased from Integrated DNA Technologies (IDT). Next, spCas9 was mixed with sgRNAs at a 1 :2.6 molar ratio and incubated at room temperature for 10 minutes to generate ribonucleoproteins (RNPs). Once the RNP complex was formed, electroporation was performed immediately. The sgRNAs used were as follows:

[0154] GAPDH-sgRNA: 5'-AGCCCCAGCAAGAGCACAAG-3' (SEQ ID NO: 1); located at chr 12:91561377, the 3' untranslated region of GAPDH; and

[0155] AAVS1-sgRNA: 5'-CCGGAGAGGACCCAGACACG-3' (SEQ ID NO: 2); located at chr 19:55116314, the first intron of the PPP1R12C gene.

[0156] 11. ProliHHs electroporation procedure

[0157] The electroporation procedure was performed when ProliHHs reached 80-90% confluency in the culture dish. Two days before electroporation, AAV2.7m8-donor virus was added to the cells at a MOI of 100,000x (genome copies / cell) for infection and incubated overnight at 37°C. To prepare for targeted integration in ProliHHs, the procedure was performed using Lonza 4D electroporation system following the manufacturer’s instructions. First, a 6-well plate containing 3 ml of EHM medium per well (to avoid cell death due to the presence of penicillin-streptomycin after electroporation, penicillin-streptomycin was removed from the EHM medium) was pre-warmed. Two days after AAV infection, the cells were digested and counted. The digested cells were washed with PBS and centrifuged in an EP tube at 3 x 10^5 ProliHHs. Then, PBS was completely removed. After the RNPs were prepared according to the method mentioned earlier, the transfection solution for the cell suspension was prepared using Lonza P3 Primary Cell 4D-Nucleofector X Kit S (32RCT, product number: V4XP-3032). Before use, 18 μl of Nucleofector solution and 4 μl of supplement solution were mixed thoroughly. The mixed transfection solution was added to the cells, and 20 μl of pipette tip was used to add RNPs to the cell suspension. The cell suspension was gently blown to ensure thorough mixing. Then, the cell mixture was transferred to the electroporation dish plate, and the DS198 program was selected for electroporation. After electroporation, the cells were removed from the electroporation dish and gently added to the pre-warmed 37°C medium in the 6-well plate.

[0158] 12. Indel frequency analysis

[0159] First, genomic DNA was extracted from the sample using QIAGEN’s DNeasy Blood & Tissue Kit. Next, the DNA fragment containing the indel site was amplified by PCR technique using specific primers, and the PCR product was purified using Generay’s DNA purification kit. Subsequently, DNA sequencing was performed, and the frequency of indels was quantified by deep sequencing (100,000x coverage).

[0160] The primer sequences used are as follows:

[0161] AAVS1 -forward primer: 5’-AAAAGGGAACCCAGCGAGTG-3’ (SEQ ID NO: 5);

[0162] AAVS1 - reverse primer: 5'-CAGGTTCCGTCTTCCTCCAC-3' (SEQ ID NO: 6);

[0163] GAPDH - forward primer: 5'-TGGTGGCTGGCTCAGAAAAAG-3' (SEQ ID NO: 7);

[0164] GAPDH - reverse primer: 5'-TGTGAGGAGGGGAGATTCAGT-3' (SEQ ID NO: 8);

[0165] Deep sequencing work was done by China National Rice Research Institute.

[0166] 13. Cell transplantation Fah - / - Rag2 - / - IL2rg - / - Mouse

[0167] 1) 6 days before cell transplantation, liver injury was induced by removing NTBC from drinking water.

[0168] 2) The mouse's spleen was placed on its side, and the skin and muscle layers were cut 1-2 cm below the ribcage. The brown fat attached to the spleen was found and pulled out to pull out the spleen. The front end of the spleen was gently tied with a thread.

[0169] 3) 5X 10 5 proliHHs or human primary hepatocytes (about 100ul), the needle was inserted into the spleen, beyond the ligation position. After injection, stop for 30-60 seconds, then pull out the needle, tie the thread tightly to prevent backflow of liquid.

[0170] 4) The spleen was placed in the abdominal cavity, the muscle and skin were sutured, the wound was disinfected with alcohol cotton, and then placed in the cage. After transplantation, the body weight and death status were recorded every week. Mice with a 30% decrease in body weight without cell transplantation were used as negative controls.

[0171] 5) Mice surviving to 12 weeks were treated and liver, blood samples were collected for subsequent analysis.

[0172] 14. Statistical analysis

[0173] GraphPad Prism 5 software was used for statistical analysis throughout the text. Two-tailed Log-rank test was used to calculate the survival curve of rats after primary hepatocyte transplantation. Non-paired Student's t-test was used for statistical analysis of sample serological data. The data in the figure is shown as mean ± standard error. * indicates P<0.05, which is a significant difference.

[0174] Example

[0175] In order to optimize the efficient CRISPR-Cas9 gene editing strategy, the inventors previously cultured and expanded primary hepatocytes from various genetic metabolic disease patients in vitro, optimized the CRISPR-Cas9 gene editing strategy, and found that gene editing in the ProliHHs cell state was highly efficient and the edited cell state was good, and it was proved that the edited ProliHHs cells still had the ability of in vitro culture and transplantation, which could realize the intervention treatment of diseases.

[0176] Example 1, optimization and establishment of efficient gene editing system for proliferating hepatocytes

[0177] In order to be closer to the clinical application standard of gene editing cells, the CRISPR-Cas9 gene editing strategy for correcting the patient's in vitro expanded hepatocytes was referred to from the clinical treatment of autologous hematopoietic stem cells, so the Cas9-sgRNA protein complex (RNPs) and the exogenous gene fragment (Donor) were planned to be delivered into stem cells to realize gene editing-based disease intervention. It is planned to carry out EGFP gene site-directed knock-in expression and screening at an sgRNA site in the 3'UTR region of the GAPDH gene, which is a non-dependent promoter (A). The sgRNA used is GAPDH-sgRNA. Figure 1 A).

[0178] 1. Analysis of target cells to be transferred

[0179] Although it is commonly referred to as hepatocytes, hepatocytes include various cells in different differentiation states, such as liver stem cells, liver mature cells, etc. Through in-depth experimental research on different cell states, different transfer conditions and different gene editing systems, the inventors found that the cultured ProliHHs were in a specific "intermediate state" of cells, which had vigorous proliferation and division capacity, and had the possibility of greatly improving the efficiency of exogenous gene editing in human hepatocytes in ProliHHs.

[0180] 2. Targeted integration strategy and efficiency analysis

[0181] The homologous recombination (HDR) strategy still has low editing efficiency in ProliHHs. Various targeted integration strategies were analyzed, the combination of different integration strategies and donor template topological structures was evaluated, including homology-directed repair (HDR) pathway, non-homologous end joining (NHEJ) pathway and homology-independent targeted integration (HITI) strategy, and the donor template topological structure containing circular and linear DNA was also analyzed.

[0182] Through in-depth research experiments, the linear HITI-donor based on the HITI integration strategy significantly improved the knock-in efficiency of ProliHHs cells (Figure 1 FI).

[0183] 3. AAV-Donor

[0184] The inventors extensively analyzed delivery vectors and screened AAV2.7m8, which has high affinity for ProliHHs, from a library containing a large number of AAV subtypes. It possesses highly efficient delivery capabilities and is therefore preferred as a delivery vector for exogenous donors. Using it as a delivery vector for high-abundance cell donors significantly improves cell knock-in efficiency. Figure 2 A-2B and Figure 3 A).

[0185] Thanks to the high affinity of AAV2.7m8 for ProliHHs, combined with the HITI integration strategy-mediated Donor (HITI-IRES-GFP), and the DS198 program, RNPs were efficiently introduced into cells and cleaved at target sites. Figure 3 B-3C), compared to the AAV-infected control group, showed that up to 27% of the cells in the experimental group were GFP-positive. Figure 3 (D-3F). This demonstrates that, for the first time, a very high level of gene knock-in efficiency has been achieved in human hepatocytes through an optimized editing system.

[0186] The gene-edited cells were continuously passaged and cultured. Flow cytometry analysis during the cell culture process showed that the proportion of GFP in the cells remained stable, indicating that the successfully edited cells can proliferate stably. Figure 3 G).

[0187] In summary, by optimizing several key parameters in the editing system, a highly efficient human hepatocyte ProliHHs gene editing system was established, providing an efficient editing platform for targeted gene correction of ProliHHs in patients.

[0188] 4. Transformation Analysis

[0189] In primary hepatocyte in vitro gene editing, electroporation can cause significant cell damage. Therefore, after screening, it was found that when using various electroporation programs (CA137, CM138, CM150, DN100, DS120, DS137, DS150, DS156, DS198, EH100, EN138, EO100, EW113, and EX147) on the Lonza 4D 96-well electroporation system, DS198, EN138, and EX147 showed a certain electroporation rate, while other programs had very low efficiency. Among them, the DS198 electroporation program showed better results in maintaining cell proliferation and producing high-efficiency DSBs after ProliHHs electroporation. Figure 1 B-1D).

[0190] The deep sequencing results show that, under this procedure, RNPs achieved highly efficient cleavage at the target site, with indels reaching approximately 89.95%. Figure 1 E).

[0191] Example 2: Gene editing to correct patient-derived ProliHH defective gene

[0192] 1. System construction and efficiency and stability analysis

[0193] In this embodiment, the AAVS1 site within the human PP1R12C gene (chromosome 19), a "safe harbor" site for the transferred DNA fragment, was used as the test site. This site carries a low risk of insertional mutations in the host genome. The inventors selected a highly efficient cleavage sgRNA site within the AAVS1 intron region to express the patient's defective gene. Figure 4 A) to ensure the safety and expression stability of gene editing using non-homologous end joining strategies.

[0194] Here, a promoter-driven exogenous gene expression assembly (EF1α-EGFP-P2A-FAH) was constructed. Figure 4 A). After AAV with the expression component was used to infect cells alone, it was observed that cells expressing EGFP gradually lost EGFP expression during cell culture and proliferation within a short period of time, eventually decreasing to about 1%. Figure 4 B), however, following the aforementioned method for gene editing of ProliHHs, the defective gene that needs to be repaired is inserted into the AAVS1 site in patient-derived ProliHHs cells.

[0195] EGFP flow cytometry analysis of the edited cells showed that the site-specific insertion efficiency of FAH was approximately 30%, and ProliHHs hepatocytes from multiple different patients exhibited similar editing efficiencies (24%-60%). Figure 4 (B-4C). This indicates that the efficiency of the editing system is independent of the patient and has relatively high stability.

[0196] 2. Gene editing to correct patient-derived ProliHH passages and enrichment

[0197] To obtain more gene-edited patient cells for treatment, the gene-edited cells from Example 2 were continuously passaged and expanded, and the successfully gene-edited cells were further enriched by flow cytometry sorting. The EGFP flow cytometry results during cell culture showed that the edited cells maintained relatively stable EGFP expression during passage expansion. Furthermore, flow cytometry successfully enriched EGFP-positive gene-edited cells; these enriched edited cells expressed approximately 80% of EGFP and expanded stably.Figure 4 D-4E), these data demonstrate that the current culture and editing system can meet the stable gene correction and enrichment of edited hepatocytes.

[0198] To verify that the patient's defective gene is repaired after gene editing, the expression of RNA and protein levels of the FAH gene was detected, respectively, and it was found that the RNA and protein expression levels of the gene repaired cells and the enriched cells were close to those of PHHs (F-4G). These data show that the patient-derived ProliHHs can be expanded and enriched after gene repair and maintain normal gene expression. Figure 4

[0199] In summary, the repair and enrichment of the defective gene of patient-derived expandable ProliHHs are achieved, and the expression of functional proteins is verified.

[0200] Example 3, Gene editing patient-derived ProliHHs transplantation treatment of tyrosinemia mouse model

[0201] In this example, after verifying the gene repair of patient-derived gene-edited ProliHHs, the patient-derived ProliHHs edited to correct tyrosinemia were selected to treat the FRG mouse model to verify the treatment ability of the strategy. Here, the inventors knocked in EF1α-FAH at the AAVS1 site of the patient-derived ProliHHs of tyrosinemia to repair the patient's defective gene FAH, and transplanted the edited ProliHHs into the FRG mouse to detect the treatment effect of the cells (A). Figure 5

[0202] The results show that all the untransplanted FRG mice and the FRG mice transplanted with unedited ProliHHs died after repeated addition and removal of NTBC in mice, while only 4 of the 11 FRG mice transplanted with gene-edited ProliHHs died (survival rate of 64%), close to the survival rate of 72% of the PHH-transplanted FRG mice (B). Figure 5

[0203] Subsequently, the liver damage of the transplanted mice was further detected, and the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (T-BIL) of the mice were reduced, indicating that the transplantation of ProliHHs after gene editing can significantly reduce the damage of the liver, and there is no difference between the transplantation of ProliHHs and PHHs, further indicating the effect of ProliHHs transplantation in treating liver metabolic diseases (C). Figure 5

[0204] ​​​​​In addition, the secretion amount of human albumin in mouse serum can be used to roughly estimate the colonization of ProliHHs in the liver. The human ALB content in the blood of mice transplanted with unedited patient cells only slightly increased, while the secretion amount of human albumin significantly increased after the mice were transplanted with gene-edited ProliHHs. After 6 months of transplantation, the average secretion amount of human albumin reached 1.8 mg / mL, which was significantly higher than the secretion amount of 690 ng / mL of unedited patient cells transplanted Figure 5 D-5E).

[0205] In order to analyze the editing correction effect of ProliHHs in vivo, mouse liver tissues were collected after 6 months of transplantation of cells. Specific human FAH antibody was used for staining.

[0206] The staining results show that the integrated region of gene-edited ProliHHs is FAH-positive cells, which is close to the integrated region of PHH transplanted cells, while the unedited cells are FAH-negative cells Figure 5 F), immunofluorescence staining shows that the integrated mouse liver tissue of gene-edited ProliHHs is an FAH, ALB and HNF4A positive region, while CK19 and CK7 originally expressed by ProliHHs stop expressing. In some regions, the transplanted ProliHHs can express regional marker proteins such as CYP3A4 and ARG1, indicating that the edited and corrected ProliHHs transplanted can reconstruct the zonal characteristics of the liver, and the integrated ProliHHs develop into a mature hepatocyte state Figure 5 G).

[0207] In summary, based on the analysis of the inventors, the occurrence of high gene knock-in efficiency is the result of the comprehensive action of multiple factors including cell proliferation state, editing site DSB generation efficiency, targeted integration strategy, and the structure characteristics and abundance of the donor itself. The gene editing strategy optimized and improved by the inventors can achieve good therapeutic effect using ProliHHs derived from patients with tyrosinemia as treatment cells.

[0208] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the present application. For ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. At the same time, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference.

Claims

1. A method of performing site-directed knock-in of a gene of interest in a liver cell, comprising: The method comprises the following steps: introducing a Cas9-sgRNA complex and a DNA donor into proliferative human liver cells as target cells, wherein the DNA donor is a DNA donor for a target integration-independent strategy, and the DNA donor is introduced into the target cells by AAV2.7m8 virus mediation, and the DNA donor comprises a target gene. The Cas9-sgRNA complex is introduced into the target cells by electroporation, preferably, the electroporation is performed by using a Lonza 4D 96-well electroporation system, and more preferably, the electroporation is performed by using an electric conversion program DS198. The Cas9-sgRNA complex is generated by mixing Cas9 and sgRNA to form a ribonucleoprotein.

2. The method of claim 1, wherein, The DNA donor is a linear donor, and preferably, the DNA donor comprises the following operationally connected elements: a promoter, a target gene, and a terminator.

3. The method of claim 1, wherein, More preferably, upstream of the promoter and downstream of the terminator further comprise Stuff and ITR.

4. The method of claim 1, wherein, The proliferative human liver cells into which the target gene is introduced are prepared by the method according to any one of claims 1 to 4. The target gene is a gene for alleviating or treating a disease.

5. A method of making therapeutic hepatocytes comprising a gene of interest, comprising: The target gene comprises a genetic defect gene, a coding gene of a pharmaceutically active protein, a coding gene of a diagnostically active protein, a coding gene of an immunogenic protein, a coding gene of a structural protein, an enzyme coding gene, and a reporter gene. Preferably, the genetic defect gene comprises a tyrosinemia-related defect gene FAH, a urea cycle disorder-related defect gene OTC, ASL, CPS1, ARG1 or ASS1, a hypercholesterolemia-related defect gene LDLR, a glycogen accumulation syndrome-related defect gene G6PC, and a Crigler-Najjar syndrome-related defect gene UGT1A1.

6. The method according to any one of claims 1 to 5, characterized in that, The proliferative human liver cells are in vitro cultured primary liver cells derived from a patient with a genetic defect disease, and the genome of the proliferative human liver cells has a genetic defect.

7. The method according to any one of claims 1 to 5, characterized in that, 8. Application of a Cas9-sgRNA complex and a DNA donor to performing target gene site-directed knock-in in liver cells, or to preparing a reagent for performing target gene site-directed knock-in in liver cells, or to preparing therapeutic liver cells comprising a target gene. The liver cells are proliferative human liver cells, the DNA donor is a DNA donor for a target integration-independent strategy, and the DNA donor is introduced into the target cells by AAV2.7m8 virus mediation, and the DNA donor comprises a target gene, and preferably, the DNA donor is a linear donor. wherein ​ 9. Use according to claim 8, wherein the compound is ###0002### The target gene is a gene for alleviating or treating a disease; preferably, the target gene comprises a genetic defect gene, a coding gene of a pharmaceutically active protein, a coding gene of a diagnostically active protein, a coding gene of an immunogenic protein, a coding gene of a structural protein, an enzyme coding gene, a reporter gene; preferably, the genetic defect gene comprises a tyrosinemia-related defect gene FAH, a tyrosinemia-related defect gene FAH; a urea cycle disorder-related defect gene OTC, ASL, CPS1, ARG1 or ASS1; a hypercholesterolemia-related defect gene LDLR; a glycogen accumulation syndrome-related defect gene G6PC; a Crigler-Najjar syndrome-related defect gene UGT1A1. The proliferated human liver cells are primary liver cells derived from a patient with a genetic defect disease, and the genome of the proliferated human liver cells has a genetic defect.

10. A liver cell comprising a gene of interest, the liver cell being a recombinant proliferated human liver cell; wherein comprising exogenously: a Cas9-sgRNA complex, a DNA donor; wherein, The DNA donor is a DNA donor for a non-dependent targeted integration strategy, and is introduced into the proliferated human liver cells mediated by AAV2.7m8 virus; the DNA donor comprises a target gene.

11. The hepatocyte comprising a gene of interest of claim 10, wherein, The liver cells comprising the target gene are prepared by the method of any one of claims 1-5; preferably, the Cas9-sgRNA complex is introduced into the target cells by electroporation; preferably, the electroporation is performed by using a Lonza 4D 96-well electroporation system; more preferably, the electroporation is performed by using an electric conversion program DS198.

12. Use of the hepatocyte comprising a target gene according to claim 10 or 11 in the manufacture of a pharmaceutical composition for in vivo transplantation; wherein the target gene is a gene for alleviating or treating a disease; preferably, the target gene comprises: A genetic defect gene, a coding gene of a pharmaceutically active protein, a coding gene of a diagnostically active protein, a coding gene of an immunogenic protein, a coding gene of a structural protein, an enzyme coding gene, a reporter gene; Preferably, the genetic defect gene comprises a tyrosinemia-related defect gene FAH. A urea cycle disorder-related defect gene OTC, ASL, CPS1, ARG1 or ASS1; a hypercholesterolemia-related defect gene LDLR; a glycogen accumulation syndrome-related defect gene G6PC; a Crigler-Najjar syndrome-related defect gene UGT1A1.

13. A pharmaceutical composition or a kit for in vivo transplantation, comprising the liver cells comprising the target gene of claim 10 or 11.

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