Method for efficient site-specific gene knock-in in hepatocytes and use thereof

Abstract

Provided in the present invention are a method for efficient site-specific gene knock-in in hepatocytes and the use thereof. Provided in the present invention is a technical solution for performing gene knock-in operation using proliferated human hepatocytes (ProliHHs) as target cells. By means of a systematic optimization strategy, gene-edited ProliHHs can effectively repopulate and mature to realize successful disease treatment. The present invention provides a powerful concept verification for autologous gene-edited proliferative cell therapy for human hereditary liver diseases, and opens up a new way for the treatment of liver diseases.

Description

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

[0001] The present invention claims priority to the patent application with the application number CN 202410622578.4, filed on May 17, 2024; the entire content of which is incorporated herein. TECHNICAL FIELD

[0002] The present invention belongs to the field of biotechnology, and more specifically, the present invention relates to a method for efficient site-directed gene knock-in in hepatocytes and application thereof. BACKGROUND

[0003] The liver is a complex structure and a central metabolic organ with multiple functions, in which hepatocytes are the main bearers of liver functions. Therefore, establishing an efficient gene editing system in human hepatocytes has always been an urgent goal for basic scientists. However, the existing main human hepatocyte systems cannot fully meet such needs. For example, although primary human hepatocytes (PHH) are the "gold standard" for measuring liver function, they have the disadvantages of rapid differentiation in vitro, loss of function, large differences between donors, and extremely low efficiency of gene manipulation; induced pluripotent stem cells (iPSCs) derived hepatocyte-like cells have a good gene editing efficiency, but the differentiation efficiency and functional maturity of hiPSC-derived hepatocytes are still much lower than that of PHHs, which may affect the accuracy and reliability of their application. Therefore, how to establish an amplifiable, easy-to-gene-manipulate, simple-to-produce, and stable human hepatocyte system is still a basic problem in the field of liver, which has important significance and value for the research of liver field and the treatment of liver diseases.

[0004] Hepatocyte expansion in vitro is a new cell source for human hepatocytes. Previously, the Ochiya team in Japan first established a small molecule inhibitor YAC medium of WNT, TGF-beta and Rock signaling pathway to induce the in vitro proliferation of rodent 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 the research groups have reported the results of the in vitro culture technology of human hepatocytes 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.

[0005] However, a significant challenge 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 easily knocking in exogenous genes in primary hepatocytes is not clear, it greatly hinders the efficient integration of exogenous genes into the cell genome. SUMMARY

[0006] The purpose of the present application is to provide a method for efficient site-directed gene knock-in in hepatocytes and its application.

[0007] In the first aspect of the present application, a method for site-directed knock-in of a target gene in hepatocytes is provided, comprising: using proliferative 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 homology-independent targeted integration (HITI), introduced into the target cells mediated by AAV2.7m8 virus; the DNA donor includes a target gene.

[0008] 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.

[0009] In one or more embodiments, the Cas9 is mixed with the sgRNA to generate ribonucleoproteins (RNPs) as Cas9-sgRNA complexes.

[0010] In one or more embodiments, the Cas9 is mixed with the sgRNA 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.

[0011] 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 gene of interest, a terminator; more preferably, upstream of the promoter and downstream of the terminator further comprises: Stuff, ITR.

[0012] In one or more embodiments, the proliferated human hepatocytes (ProliHHs) are cultured in HM or EHM medium.

[0013] In one or more embodiments, the proliferated human hepatocytes (ProliHHs) are derived from a patient with a genetic metabolic disease.

[0014] In one or more embodiments, for the patient-derived ProliHHs, EHM or HM medium can be used. The EHM medium is a HM medium with replacement of nicotinamide by recombinant human bFGF; preferably, the amount of bFGF is 25±10 ng / mL (preferably 25±8 ng / mL, 25±6 ng / mL, 25±4 ng / mL, or 25±2 ng / mL).

[0015] In one or more embodiments, the method for efficient site-directed gene knock-in in hepatocytes is a non-therapeutic, non-diagnostic method.

[0016] In one or more embodiments, the method for efficient site-directed gene knock-in in hepatocytes is an ex vivo method.

[0017] In one or more embodiments, under the guidance of the sgRNA, the gene of interest is inserted at a specific site.

[0018] In another aspect of the present application, a method for preparing therapeutic hepatocytes comprising a gene of interest is provided, comprising: preparing proliferated human hepatocytes (ProliHHs) with the gene of interest introduced by the method described in any one of the preceding aspects; the gene of interest is a gene for alleviating or treating a disease.

[0019] In one or more embodiments, the method for preparing therapeutic hepatocytes comprising a gene of interest is a non-therapeutic, non-diagnostic method.

[0020] In one or more embodiments, the method for preparing therapeutic hepatocytes comprising a target gene is an ex vivo method.

[0021] In one or more embodiments, the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 2.

[0022] In one or more embodiments, the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1.

[0023] In one or more embodiments, 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 urea cycle disorder related deficiency gene OTC, ASL, CPS1, ARG1 or ASS1, a hypercholesterolemia related deficiency gene LDLR, a glycogen storage syndrome related deficiency gene G6PC, a Crigler-Najjar syndrome related deficiency gene UGT1A1.

[0024] In one or more embodiments, the proliferated human hepatocytes (ProliHHs) are primary hepatocytes derived from a patient with a genetic deficiency disease, and the genome of the hepatocytes has a genetic deficiency.

[0025] In another aspect of the present application, there is provided a use of a Cas9-sgRNA complex and a DNA donor for performing a site-directed knock-in of a target gene in a hepatocyte, or for preparing a reagent for performing a site-directed knock-in of a target gene in a hepatocyte, or for preparing a therapeutic hepatocyte comprising a target gene; wherein the hepatocyte is a proliferated human hepatocyte (ProliHHs); the DNA donor is a DNA donor for homology independent targeted integration (HITI), and is introduced into the target cell mediated by an AAV2.7m8 virus; the DNA donor comprises a target gene; preferably, the DNA donor is a linear donor.

[0026] 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.

[0027] In one or more embodiments, the proliferated human hepatocytes are primary hepatocytes derived from a patient with a genetic deficiency, and the genome of the primary hepatocytes has the genetic deficiency.

[0028] In another aspect of the present application, there is provided a hepatocyte comprising a target gene, wherein the hepatocyte is a recombinant proliferated human hepatocyte (ProliHHs); and wherein the hepatocyte comprises exogenously: a Cas9-sgRNA complex, a DNA donor; wherein the DNA donor is a HITI-compatible DNA donor, and wherein the DNA donor is introduced into the ProliHHs via AAV2.7m8 virus mediation; and wherein the DNA donor comprises the target gene.

[0029] In one or more embodiments, the hepatocyte 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; and more preferably, the electroporation is performed using an electroporation program DS198.

[0030] In another aspect of the present application, there is provided a use of the hepatocyte comprising a target gene in the preparation of a pharmaceutical composition for in vivo transplantation; wherein the target gene is a gene that alleviates or treats a disease; preferably, the target gene comprises (but is not limited to): a genetic deficiency gene, a gene encoding a pharmaceutically active protein, a gene encoding a diagnostically active protein, a gene encoding an immunogenic protein, a gene encoding a structural protein, a gene encoding an enzyme, a reporter gene.

[0031] In one or more embodiments, the hepatocyte comprising a target gene maintains efficient proliferation and passaging.

[0032] In one or more embodiments, the hepatocyte comprising a target gene stably secretes human albumin.

[0033] In one or more embodiments, after the inhibition, the edited and corrected ProliHHs hepatocytes transplanted can reconstitute the zonal features of the liver, and the integrated ProliHHs hepatocytes can develop into a mature hepatocyte state.

[0034] In one or more embodiments, the pharmaceutical composition for in vivo transplantation alleviates or treats a liver disease.

[0035] In one or more embodiments, the in vivo transplantation comprises in vivo autologous transplantation.

[0036] In one or more embodiments, the method in conjunction with the in vivo transplantation protocol comprises: collecting primary hepatocytes from a patient with liver disease, preparing ProliHHs, further preparing recombinant (knock-in exogenous gene) ProliHHs using the aforementioned method, and achieving treatment of the disease.

[0037] In one or more embodiments, the liver disease includes, but is not limited to, liver injury.

[0038] In one or more embodiments, the liver disease includes, but is 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.

[0039] In another aspect of the present application, a pharmaceutical composition or kit for in vivo transplantation is provided, which contains the liver cells containing the target gene.

[0040] 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

[0041] Figure 1: Optimization of exogenous gene integration strategy in ProliHHs.

[0042] (A) Schematic diagram describes the process of genome targeting in ProliHHs using RNPs (Cas9-sgRNA) and DNA donor delivered by electroporation. The DNA donor contains the EGFP gene as a reporter gene, and the knock-in efficiency in ProliHHs is checked by fluorescence imaging or flow cytometry.

[0043] (B) GFP fluorescence micrographs of ProliHHs, cells were electroporated with equal amount of GFP expressing plasmid to test the delivery ability of 14 low electric pulse electroporation programs.

[0044] (C) Array analysis of electroporation programs evaluated plasmid delivery efficiency and ProliHHs proliferation ability. DS198 was selected to optimize efficiency.

[0045] (D) Representative images of ProliHHs after electroporation program DS198 treatment.

[0046] (E) RNPs schematic for GAPDH 3'-UTR (GAPDH-sgRNA). RNPs were electroporated into ProliHHs and analyzed for indel (%) of GAPDH-sgRNA by tagmentation sequencing.

[0047] (F) Knock-in efficiency of the circular HDR-mediated donor (IRES-EGFP) in ProliHHs was detected by fluorescent imaging and quantified by flow cytometry.

[0048] (G) Schematic of the targeted integration strategy for evaluating the combined effect of different integration strategies and donor template topology.

[0049] (H) Knock-in efficiency of different designed donors (IRES-EGFP) against the GAPDH-sgRNA site was evaluated by flow cytometry.

[0050] (I) Representative fluorescent micrographs of linear donors using HDR, NHEJ or HITI integration strategies, respectively.

[0051] Figure 2: High efficiency of infection of AAV2.7m8 in ProliHHs.

[0052] (A) Schematic of AAV variant screening. ProliHHs were infected with equal amounts of AAV variants expressing nuclear localization signal (NLS)-mNeonGreen (green), and cells were analyzed for infection by fluorescent imaging and flow cytometry 3 days post-viral infection.

[0053] (B) Comparison of transduction efficiency of AAV variants in ProliHHs, representative fluorescent images were selected.

[0054] Figure 3: Efficient CRISPR / Cas9 and AAV2.7m8-mediated targeted integration in ProliHHs.

[0055] (A) Quantification of AAV infection efficiency in ProliHHs by flow cytometry.

[0056] (B) Timeline schematic of RNPs and AAV2.7m8-mediated targeted integration in ProliHHs. ProliHHs were infected with AAV2.7m8 donor (HITI-IRES-EGFP) 2 days prior to RNP electroporation. Edited ProliHHs were continued to be cultured and analyzed post-electroporation.

[0057] (C) Schematic of targeted genome editing at the 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 the AAV2.7m8 donor template as a repair template. Blue boxes, GAPDH exons.

[0058] (D) ProliHHs were subjected to electroporation and indels of GAPDH-sgRNA were analyzed by deep sequencing. Mean values of n=5 independent donors are shown.

[0059] (E) Mean integration efficiency of the donor template (HITI-IRES-EGFP) in ProliHHs (n=5 independent donors).

[0060] (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.

[0061] (G) Percentage of cells expressing EGFP in three generations after electroporation was quantified using flow cytometry (n=3 independent donors).

[0062] Figure 4: Correction of FAH gene deficiency by targeted integration in ProliHHs.

[0063] (A) Schematic of targeted editing using RNPs and AAV2.7m8 donor template at the AAVS1 site (AAVS1-sgRNA). Blue box, PPP1R12C exon.

[0064] (B) ProliHHs electroporated as described above were analyzed by flow cytometry two generations after electroporation. Top, bright field images of edited ProliHHs. Middle, fluorescence images of edited ProliHHs. Bottom, representative FACS plot.

[0065] (C) Mean integration efficiency of AAV2.7m8 donor in ProliHHs. Mean values of n=5 independent donors are shown.

[0066] (D) ProliHHs (P1) were subjected to editing 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.

[0067] (E) Bulk edited ProliHHs cell populations were sorted to generate pure edited ProliHHs cell populations. Top, fluorescence images of ProliHHs. Bottom, representative FACS plot.

[0068] (F and G) Quantification of FAH mRNA expression and protein expression in untreated, AAV only infected, edited or purified ProliHHs using RT-qPCR and Western blotting. All mRNA transcript levels and protein levels were referenced to fresh PHHs.

[0069] Figure 5: Edited ProliHHs transplantation therapy in tyrosinemia mouse model.

[0070] (A) Workflow of transplanting edited patient-derived ProliHHs into 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 AAVS1-sgRNA site with a therapeutic donor and transplanted into FRG mice. Right panel: Schematic of targeted editing at the AAVS1-sgRNA site using RNPs and AAV2.7m8 donor.

[0071] (B) Kaplan-Meier survival curves of FRG mice with untransplanted cells and transplanted cells.

[0072] (C) Serum ALT, AST, and TBIL levels of dying control FRG mice (n=3) and unedited ProliHHs (n=3), surviving edited ProliHH-FRG mice (n=3), and surviving PHH-FRG mice (n=3).

[0073] (D) Dynamics of human ALB levels determined using ELISA in dying unedited ProliHHs-FRG mice, surviving edited ProliHH-FRG mice, and surviving PHH-FRG mice within 6 months post-transplantation.

[0074] (E) Human ALB levels determined using ELISA in dying unedited ProliHHs-FRG mice (n=7), surviving edited ProliHH-FRG mice (n=5), and surviving PHH-FRG mice (n=5) post-transplantation.

[0075] (F) Immunohistochemical staining for FAH in mouse livers post-transplantation.

[0076] (G) Analysis of maturation of refilled edited ProliHHs using co-immunofluorescent staining. Liver sections were analyzed using co-immunofluorescent 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

[0077] 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.

[0078] As used herein, the "element" refers to a series of functional nucleic acid / protein sequences useful for the expression of a protein, which is 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 is 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.

[0079] As used herein, the "operatively linked" or "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest, so that the transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operatively linked" to the nucleic acid sequence.

[0080] As used herein, the "construct" refers to a single-stranded or double-stranded DNA molecule that has been artificially intervened to contain DNA fragments combined and arranged in sequences that do not exist in nature. The "construct" includes "plasmids", "in vitro transcription products", or viral vectors, etc.; or the "construct" is included in an expression vector, as part of an expression vector.

[0081] As used herein, the "sgRNA" or "Single-guide RNA" is designed based on the "target site on the target gene", which 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 sequences corresponding to the sgRNA sequence or constructs containing the sequence, as long as they can be processed or converted into active "sgRNA" in cells.

[0082] 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 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 effectiveness of transplantation / treatment.

[0083] In addition, genome editing technology has been used more in recent years. In gene editing related technologies, including gene knock-in, gene knockout, gene mutation, etc., the efficiency, accuracy or stability of gene knock-in is much 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; and needs to be improved 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.

[0084] In the present application, a technical solution of gene knock-in operation is proposed by taking proliferated human hepatocytes (ProliHHs) as target cells. Through research and analysis of hepatocytes, the inventors select proliferated human hepatocytes (ProliHHs) as the target cells for editing and can achieve in vivo therapeutic effect. Through systematic optimization strategy, AAV2.7m8 delivery and HITI strategy are successfully used to effectively realize CRISPR / Cas9-mediated targeted integration, aiming to correct the defective genes of patients in ProliHHs. Furthermore, it is found that the ProliHHs edited by gene editing can be effectively reprogrammed and matured, and can successfully treat tyrosinemia and other genetic deficiency diseases. The present application provides a strong proof of concept for 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.

[0085] In the present application, the ProliHHs are proliferated human hepatocytes induced to regain proliferative capacity by using WNT3a, HGF and other cell growth factors combined with chemical small molecules strategy, and are obtained by expansion in a limited number of passages. As a kind of hepatocyte, ProliHHs can better maintain the function and related gene expression level of hepatocytes, such as ALB, TTR, CYP3A4, CYP1A2, CAR, C3, etc.

[0086] 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), so as to obtain intermediate cells (having unique marker expression characteristics) between mature hepatocytes and liver precursor cells. The cells can further develop into mature hepatocyte state.

[0087] The method for expanding hepatocytes in vitro includes culturing hepatocytes in a cell culture medium containing a Wnt signaling pathway activator to expand the hepatocytes in vitro. The Wnt signaling pathway activator includes 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, and serum. The culture of ProliHHs can be carried out by a two-dimensional or three-dimensional culture system. The obtained hepatocytes can be frozen, thawed, subcultured, and maintained in culture for a long time.

[0088] To achieve efficient integration of exogenous genes, the inventors successfully established a culture system of in vitro proliferated hepatocytes (ProliHHs) through culture optimization and induction, and analyzed that the cells are in a suitable state, which is 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 can be particularly matched with the liver cells of the present application and has 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 show that in ProliHHs, the knock-in efficiency of linear HITI donor is significantly better than other combination modes. In order to improve the efficiency of gene knock-in, the inventors screened AAV variant library and analyzed its ability to infect ProliHHs. The screening results show that AAV2.7m8 performs best in terms of infection efficiency. The inventors also analyzed the electroporation technology and screened different low electric pulse electroporation programs of LONZA electroporator, and found that the DS198 program performs best in maintaining cell activity and providing high transfection capacity.

[0089] 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 achieves efficient delivery and successfully achieves an integration efficiency of up to 23.6% to 66% at a specific target site 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.

[0090] Based on the new findings of the inventors, a method for targeted gene knock-in in hepatocytes is provided, comprising: introducing a Cas9-sgRNA complex and a DNA donor into proliferated human hepatocytes (ProliHHs) as target cells. In some specific embodiments, the method is in vitro (ex vivo), that is, the operation is performed on ex vivo cells.

[0091] The present application also provides a method for preparing therapeutic hepatocytes containing a target gene, comprising: preparing proliferated human hepatocytes (ProliHHs) with the target gene introduced by the above method; and the target gene is a gene for alleviating or treating diseases. In some specific embodiments, the method is in vitro (ex vivo), that is, the operation is performed on ex vivo cells.

[0092] In other aspects, based on the disclosure of the present application, genome editing cells or animals can also be prepared. In some specific embodiments, the method can also be for research purposes rather than for therapeutic purposes.

[0093] Genome site knock-in expression can be used as a general gene editing and correction treatment strategy, which does not depend on the patient's mutant gene, mutation type and site. The strategy of the present application is an ideal gene correction method to treat 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 the dividing and non-dividing cells.

[0094] Currently, there is an increasing demand for developing an efficient and stable gene editing method in the clinic. The technical scheme of the present application not only has important significance for promoting the research in the field of liver, but also provides a new way for the treatment of liver diseases.

[0095] Based on the new findings of the present application, a recombinant ProliHHs culture obtained by the method of the present application or ProliHHs hepatocytes isolated and purified from the hepatocyte culture can be an intermediate cell between mature hepatocytes and liver precursor cells, or further induced to obtain mature hepatocytes.

[0096] The method for enriching or isolating and purifying cells from cell culture is also well known to those skilled in the art, for example, the enrichment can be based on the epithelial-like morphological characteristics of hepatocytes; or based on the special proteins (such as Albumin, etc.) or molecular markers expressed by hepatocytes to select and collect (for example, using specific antibodies or ligands). In addition, it is also a feasible method to enrich hepatocytes by removing (such as digestion, lysis) 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.

[0097] The ProliHHs hepatocytes cultured by the present application have various uses. Including but not limited to: for preparing a composition (pharmaceutical composition) for promoting liver regeneration or liver transplantation; for preparing a composition (pharmaceutical composition) for treating liver damage (including but not limited to: end-stage liver disease, liver cirrhosis, alcoholic liver, diabetes, obesity, acute liver failure, hepatitis, liver fibrosis, liver cancer, liver metabolic disease or liver damage caused by liver function failure); for use as an in vitro model to study liver-related diseases or drug efficacy, such as for studying drug transport, drug metabolism, liver formation, liver regeneration, for liver toxicity testing, screening of hepatotoxic compounds, screening of compounds that regulate hepatocyte function, for producing Albumin protein, etc.

[0098] In the technical solution for preparing the liver regeneration or liver transplantation, preferably, the liver cells (primary liver cells) can be obtained from the body of the patient with genetic defect disease, the ProliHHs are obtained by culture, and the recombinant ProliHHs with the target gene knocked in are obtained by further using the method of the application, which can be used as a transplant to inhibit the in-vivo, so as to achieve the purpose of relieving or treating the liver disease.

[0099] The cultured liver cells of the application can be used for hepatotoxicity research, and can also be applied to cell transplantation treatment of liver diseases, construction of bioartificial liver, detection of hepatotoxicity of new drugs, evaluation of drug efficacy, identification of drug targets; can provide sufficient liver cell sources or liver cell models for basic research and clinical application of biology, medicine and pharmacy; the induction and differentiation process can also provide an optimal research platform for the development and differentiation process of human liver cells, and has a very broad application prospect.

[0100] In the case of need, the cultured liver cells of the application can be further used for genetic engineering recombination to form further recombined cells, for example, to impart further functions or characteristics to the cells, to transfer an exogenous gene expression cassette into the cells, or to perform gene knockout or gene editing on the genome of the cells, etc.

[0101] The application further provides a composition (drug) containing: an effective amount of the ProliHHs liver cells (such as 1×10 4 -1×10 12 , preferably 1×10 5 -1×10 10 ); 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.

[0102] The "effective amount" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals. The "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to some pharmaceutical carriers that are not essential active ingredients and have 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 auxiliary substances in these carriers, such as fillers, lubricants, flow aids, wetting agents or emulsifying agents, pH buffering substances, etc. The carrier can also contain a cell transfection reagent.

[0103] The application further provides a method for liver cell transplantation / liver regeneration, which comprises: administering an effective amount of the recombinant ProliHHs liver cells prepared by the application to a subject in need of treatment.

[0104] When used for administration, the composition is typically 1×10 2 -1×10 10 Cells / kg body weight, optimal 1×10 3 -1×10 8 One cell / kg body weight is appropriate, but this also depends on the clinician's diagnosis and the severity of the patient's symptoms.

[0105] The present invention also provides a kit containing the recombinant ProliHHs hepatocytes of the present invention or a composition containing the ProliHHs hepatocytes. Preferably, the kit also includes instructions for use, thereby facilitating use by those skilled in the art in research or clinical application.

[0106] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0107] Materials and Methods

[0108] 1. Culture medium

[0109] Hepatocyte culture media include HM medium and EHM medium, and the formulations are shown in Table 1 and Table 2.

[0110] Table 1

[0111] Table 2

[0112] *This component can be replaced with 30% Wnt3a conditioned medium or 0.1 uM CHIR. The Wnt3a conditioned medium is obtained from the cell supernatant produced after two days of culture of the L-Wnt3a cell line (obtained from Hans Clevers' laboratory).

[0113] 2. Cells

[0114] Primary human hepatocytes were isolated from discarded liver tissue from liver transplants of patients with hereditary metabolic liver disease at Renji Hospital. The L-Wnt3a cell line was donated by Hans Clevers' laboratory. The Snu-398 cell line was purchased from the American Type Culture Collection (ATCC).

[0115] Standard ProliHHs cell culture: using HM medium;

[0116] ProliHHs were obtained by culturing primary hepatocytes derived from patients using EHM medium.

[0117] ProliHHs cells after electroporation: EHM medium is used preferentially.

[0118] 3. Human hepatocyte culture

[0119] Human hepatocytes are isolated from patient liver tissue using collagenase. Isolated human hepatocytes are seeded at a density of 2 X 10 4 cells / cm2 in collagen type I coated plates in a 37°C, 5% CO 2 , 5% O 2 incubator. After 1 day of culture, the medium is changed to hepatocyte medium and the medium is changed every 3 days. After 6 days of culture, the confluent cells are trypsinized and re-seeded at a density of 3 X 10 4 cells / cm 2 in collagen type I coated plates in a 37°C, 5% CO 2 , 5% O 2 incubator.

[0120] 4. Time-lapse imaging of cell culture

[0121] Thawed human hepatocytes are recovered in DMEM medium with 10% serum and seeded at a density of 2 X 10 4 cells / cm 2 in a six-well plate coated with collagen type I. After cell attachment, the medium is changed to hepatocyte medium and the cell plate is placed in a 37°C, 5% CO 2 incubator in an incuCyte FLR instrument. Cells are continuously cultured for 3 days and a set of images is taken every 30 minutes in the same field of view. A video of cell proliferation is generated by the software.

[0122] 5. Gene expression detection

[0123] 1) Cell RNA is extracted using Trizol (Invitrogen) or RNeasy FFPE Kit (Qiagen) reagents.

[0124] 2) 1 ug of RNA extracted with Trizol or according to the specific value with RNeasy FFPE Kit is taken and cDNA is obtained using M-MLV reverse transcriptase (Promega) kit.

[0125] 3) Real-time quantitative PCR was performed using SYBR Premix Ex Taq (TaKaRa) kit and the ABI StepOnePlus real-time PCR system (Applied Biosystems) to detect gene expression. All Q-PCR data were repeated at least twice. Primer sequences are provided in Table 3.

[0126] Table 3

[0127] 6. Human albumin ELISA assay

[0128] 1) Dilute capture antibody 1:250 with coating buffer, add 100ul per well to 96 well ELISA plate (Corning). Seal the 96 well plate and incubate at 4°C overnight.

[0129] 2) Remove capture antibody and add 400ul per well wash buffer, soak for 1 minute, then remove, wash 2 times. The second time, make sure to remove all wash buffer.

[0130] 3) Add 250ul blocking buffer, incubate at room temperature for 2 hours.

[0131] 4) Wash 2 times as in step 2.

[0132] 5) Dilute standards 2-fold using Assay buffer A, 8 dilutions. Dilute samples according to the standard curve to ensure the final OD450 falls within the range of the standard curve.

[0133] 6) Add 100ul of standards and samples to the 96 well plate, seal and incubate at room temperature for 2 hours.

[0134] 7) Wash 4 times as in step 2.

[0135] 8) Add 100ul per well detection antibody, incubate at room temperature for 1 hour.

[0136] 9) Wash 4 times as in step 2.

[0137] 10) Add 100ul per well substrate solution, incubate at room temperature for 15 minutes.

[0138] 11) Add 100ul per well stop solution, measure A450.

[0139] Note: The solutions mentioned above are from the ALB ELISA kit (Bethyl Laboratories).

[0140] 7. Production of AAV vectors

[0141] For the production of AAV vectors carrying the nuclear localization green fluorescent protein mNeonGreen (NLS-mNeonGreen) driven by the cytomegalovirus enhancer / chicken beta-actin (CAG) promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) in HEK 293T cells, a co-transfection approach with AAV vector, rep / cap packaging plasmid and adenoviral helper plasmid was employed. 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. Subsequently, 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 after ultracentrifugation was collected. The purified virus was further concentrated by buffer exchange into PBS containing 0.001% F68 (Gibico). The genomic content titer of AAV was measured using SYBR (Roche) analysis with primers targeting the WPRE region. The RT-qPCR primers for WPRE are as follows:

[0142] AAV-forward, 5'-GTCAGGCAACGTGGCGTGGTGTG-3' (SEQ ID NO: 3);

[0143] AAV-reverse, 5'-GGCGATGAGTTCCGCCGTGGC-3' (SEQ ID NO: 4).

[0144] By this step, the quality and quantity of the produced AAV vectors can be accurately assessed, providing a reliable material basis for subsequent experiments and applications.

[0145] 8. In vitro AAV library screening

[0146] First, 5 x 10^4 of proliferating human hepatocytes (ProliHHs) were seeded in a 48-well plate and cultured 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 culture, 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).

[0147] 9. Electroporation program screening

[0148] To transfect ribonucleoproteins (RNPs) into proliferating human hepatocytes (ProliHHs), the Lonza Nucleofector TM 4D 96-well electroporation system. Given the lack of a suitable electroporation program for ProliHHs, the program was first screened using a reporter plasmid expressing green fluorescent protein (pmaxGFP from Lonza P3 Primary Cell 4D-Nucleofector X Kit) according to the manufacturer's optimization guidelines. 2 x 10^5 of ProliHHs were resuspended in 20 pL of Lonza electroporation buffer P3 (containing 18 pL of Nucleofector solution and 4 pL of Supplement Solution) with the addition of 500 ng of pmaxGFP DNA. Subsequently, the 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 culture, GFP fluorescence micrographs of the cells were captured and counted using an Operetta high-content imager (PerkinElmer). On day 8 after the electroporation program, the cells were digested and counted using a cell counter to assess cell proliferation after electroporation. Through this series of screening steps, the electroporation conditions suitable for ProliHHs were found, laying a solid foundation for subsequent experiments.

[0149] 10. Production of RNPs

[0150] 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:

[0151] GAPDH-sgRNA: 5'-AGCCCCAGCAAGAGCACAAG-3' (SEQ ID NO: 1); located at chr 12:91561377, the 3' untranslated region of GAPDH; and

[0152] AAVS1-sgRNA: 5'-CCGGAGAGGACCCAGACACG-3' (SEQ ID NO: 2); located at chr 19:55116314, the first intron of the PPP1R12C gene.

[0153] 11. ProliHHs electroporation

[0154] Electroporation 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) and incubated overnight at 37°C. To prepare for targeted integration in ProliHHs, the Lonza 4D electroporation system was used 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 preheated. Two days after AAV infection, the cells were digested and counted. The digested cells were washed with PBS and centrifuged at 3x10^5 ProliHHs in an EP tube. Then, the PBS was completely removed. After the RNPs were prepared according to the method mentioned earlier, the transfection solution for the suspended cells was prepared using the 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 preheated 37°C medium in a 6-well plate.

[0155] 12. Indel frequency analysis

[0156] First, genomic DNA was extracted from the samples using the DNeasy Blood & Tissue Kit from QIAGEN. Next, the DNA fragments containing the indel sites were amplified by PCR using specific primers, and the PCR products were purified using the DNA purification kit from Generay. Subsequently, DNA sequencing was performed, and the frequency of indels was quantified by deep sequencing (100,000x coverage).

[0157] The primer sequences used are as follows:

[0158] AAVS1 -forward primer: 5'-AAAAGGGAACCCAGCGAGTG-3' (SEQ ID NO: 5);

[0159] AAVS1 -reverse primer: 5'-CAGGTTCCGTCTTCCTCCAC-3' (SEQ ID NO: 6);

[0160] GAPDH - forward primer: 5'-TGGTGGCTGGCTCAGAAAAAG-3' (SEQ ID NO: 7);

[0161] GAPDH - reverse primer: 5'-TGTGAGGAGGGGAGATTCAGT-3' (SEQ ID NO: 8);

[0162] Deep sequencing work was done by China National Rice Research Institute.

[0163] 13. Cell transplantation Fah - / - Rag2 - / - IL2rg - / - Mouse

[0164] 1) 6 days before cell transplantation, liver injury was induced by removing NTBC from drinking water.

[0165] 2) The mouse's spleen was placed on its side, and the skin and muscle layers were cut 1-2 cm below the rib. 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.

[0166] 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 liquid backflow.

[0167] 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 condition were recorded every week. Mice with a 30% decrease in body weight without cell transplantation were used as negative controls.

[0168] 5) Mice surviving to 12 weeks were treated and liver, blood samples were collected for subsequent analysis.

[0169] 14. Statistical analysis

[0170] The whole text was statistically analyzed by using GraphPad Prism 5 software. When calculating the survival curve of rats after primary hepatocyte transplantation, a two-tailed Log-rank test was used. The sample serological data was statistically analyzed by using a non-paired Student's t-test. The data in the figure was shown as mean ± standard error. * indicates P<0.05, which is a significant difference.

[0171] Example

[0172] In order to optimize the efficient CRISPR-Cas9 gene editing strategy, the inventors previously cultured and expanded a variety of primary hepatocytes from patients with genetic metabolic diseases 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, and could realize the intervention treatment of diseases.

[0173] Example 1, optimization and establishment of efficient gene editing system for proliferating hepatocytes

[0174] In order to be closer to the clinical application standard of gene edited 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 the disease intervention based on gene editing. 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 (Figure 1A). The sgRNA used is GAPDH-sgRNA.

[0175] 1. Analysis of target cells to be transferred

[0176] Although commonly known as hepatocytes, hepatocytes include a variety of 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.

[0177] 2. Targeted integration strategy and efficiency analysis

[0178] The homologous recombination (HDR) strategy still has low editing efficiency in ProliHHs. A variety of targeted integration strategies were analyzed, and the combined effect 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 including circular and linear DNA was also analyzed.

[0179] Through in-depth research experiments, linear HITI-donor based on HITI integration strategy significantly improved the knock-in efficiency of ProliHHs cells (Figures 1F-I).

[0180] 3. AAV-Donor

[0181] 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. AAV2.7m8, with its efficient delivery capability, was selected as the preferred delivery vector for exogenous donors. Using it as a delivery vector for high-abundance cell donors significantly improved cell knock-in efficiency (Figures 2A-2B and 3A).

[0182] Thanks to the high affinity of AAV2.7m8 delivery to ProliHHs combined with the HITI integration strategy-mediated Donor (HITI-IRES-GFP), and the DS198 program's efficient introduction of RNPs into cells and cleavage of target sites (Figures 3B-3C), compared to the AAV-infected control group, up to 27% of the cells in the experimental group were GFP-positive (Figures 3D-3F). This demonstrates that a high level of gene knock-in efficiency has been significantly achieved in human hepatocytes for the first time through an optimized editing system.

[0183] 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 could proliferate and be cultured stably (Figure 3G).

[0184] 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.

[0185] 4. Transformation Analysis

[0186] In in vitro gene editing of primary hepatocytes, 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 was more effective in maintaining the cell proliferation state and generating efficient DSBs after ProliHHs electroporation (Figures 1B-1D).

[0187] The deep sequencing results show that RNPs produced a highly efficient cleavage effect at the target site under this procedure, with indels reaching approximately 89.95% (Figure 1E).

[0188] Example 2, Gene editing corrects patient-derived ProliHH defective gene

[0189] 1. Editing system construction and efficiency, stability analysis

[0190] In this example, AAVS1 in human PP1R12C gene (chromosome 19) was used as the test site for the "safe harbor" site of the introduced DNA fragment, which has a low risk of insertion mutation in the host genome. The inventors selected a high-efficiency sgRNA site in the intron region of AAVS1 to express the patient's defective gene (Figure 4A), to ensure the safety and stability of gene editing using the non-homologous end joining strategy.

[0191] Here, a promoter EF1a-driven exogenous gene expression module (EF1a-EGFP-P2A-FAH) was constructed (Figure 4A). After AAV infection with the expression module alone, it was observed that the EGFP-expressing cells gradually lost EGFP expression over time as the cell culture proliferated, and finally decreased to about 1% (Figure 4B). However, according to the previously established method of ProliHHs gene editing, the AAVS1 site in the patient-derived ProliHHs cells was inserted with the defective gene that needed to be repaired.

[0192] The results of EGFP flow cytometric analysis of edited cells showed that the efficiency of site-specific insertion of FAH was about 30%, and the ProliHHs hepatocytes of multiple different patients had similar editing efficiency (24%-60%) (Figures 4B-4C). This indicates that the efficiency of the editing system is independent of the patient and has relatively high stability.

[0193] 2. Gene editing corrects patient-derived ProliHH passage and enrichment

[0194] In order to obtain more gene-edited patient cells for treatment, the cells edited in Example 2 were continuously passaged and expanded, and further enriched for successfully edited cells by flow sorting. From the results of EGFP flow cytometric detection during cell culture, it can be seen that the edited cells still maintain relatively stable EGFP expression during passage expansion, and in addition, flow cytometry can successfully enrich EGFP-positive successfully edited cells. The proportion of these enriched edited cells expressing EGFP is as high as about 80% and is stable in expansion (Figures 4D-4E), which proves that the current culture and editing system can meet the stable gene correction and enrichment of successfully edited hepatocytes.

[0195] To verify the patient's defective gene was repaired after gene editing, the expression of RNA and protein levels of FAH gene were 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 PHH (Fig. 4F-4G). These data showed that the patient-derived ProliHHs could be amplified and enriched after gene repair and maintain normal gene expression.

[0196] In summary, the repair and enrichment of the defective gene of patient-derived amplifiable ProliHHs were achieved and verified in the expression of functional protein.

[0197] Example 3, gene editing patient-derived ProliHHs transplantation treatment of tyrosinemia mouse model

[0198] In this example, after verifying the patient-derived gene-edited ProliHHs with gene repair, the inventors selected the edited tyrosinemia patient-derived ProliHHs to correct the FRG mouse model to verify the treatment ability of the strategy. Here, the inventors knocked in EF1a-FAH at the AAVS1 site of the tyrosinemia patient-derived ProliHHs 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 (Fig. 5A).

[0199] The results showed 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 was 64%), close to the survival rate of 72% of the PHH transplanted FRG mice (Fig. 5B).

[0200] 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 could significantly reduce the damage of the liver, and there was no difference between the transplantation of PHH, further indicating the effect of ProliHHs transplantation in the treatment of liver metabolic diseases (Fig. 5C).

[0201] In addition, the secretion amount of human albumin in the serum of the mice can be used to estimate the repopulation of ProliHHs in the liver, and the human ALB content in the blood of the mice transplanted with unedited patient cells only increased slightly, while the secretion amount of human albumin significantly increased after the mice were transplanted with gene-edited ProliHHs, and the average secretion amount of human albumin reached 1.8 mg / mL after 6 months of transplantation, which was significantly higher than the secretion amount of 690 ng / mL of the unedited patient cells (Fig. 5D-5E).

[0202] To analyze the editing correction effect of ProliHHs in vivo, the mouse liver tissues were collected 6 months after the transplantation of cells. The human FAH antibody was used for staining.

[0203] The staining results showed that the integrated region of the gene-edited ProliHHs was FAH-positive cells, close to the integrated region of the PHH, while the unedited cells were FAH-negative cells (Figure 5F). The immunofluorescence staining showed that the integrated mouse liver tissues of the gene-edited ProliHHs were FAH, ALB and HNF4A-positive regions, while the CK19 and CK7 originally expressed by the ProliHHs stopped expressing. In some regions, the transplanted ProliHHs could express regional marker proteins such as CYP3A4 and ARG1, indicating that the edited and corrected ProliHHs could reconstruct the zonal characteristics of the liver, and the integrated ProliHHs developed into a mature hepatocyte state (Figure 5G).

[0204] 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 donor structure characteristics, abundance, etc. The gene editing strategy optimized and improved by the inventors can achieve good therapeutic effect by using the ProliHHs derived from patients with tyrosinemia as therapeutic cells.

[0205] 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 all belong to 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. Meanwhile, 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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