WO2023046086A1 - Base editing system and application thereof - Google Patents

Abstract

Disclosed in the present invention are a base editing system and an application thereof. The base editing system of the present application comprises an ABE8e base editor and an sgRNA library targeting an HBG promoter, and the sgRNA library contains one or more of nucleotide sequences as shown in SEQ ID NO: 1, 2, and 3. According to the base editing system of the present invention, the HBG promoter is targeted in human cells to upregulate the expression of fetal hemoglobin, i.e., γ-globin, for the treatment of β hemoglobinopathies (such as β thalassemia, sickle cell anemia, and other diseases). The base editing system provided by the present invention has important clinical application prospects in the field of gene therapy for β hemoglobinopathies.

Description

A base editing system and its application

This application claims the priority of Chinese patent application 2021111239783 with a filing date of 2021/9/24. This application cites the full text of the above-mentioned Chinese patent application.

technical field

The invention belongs to the field of gene therapy, and in particular relates to an sgRNA library, a base editing system containing the same and its application, and also relates to an application of a highly specific ABE8e base editing system in the treatment of beta hemoglobinopathies.

Background technique

Thalassemia (thalassemia) is a monogenic genetic disease with the widest distribution and the largest number of affected people in the world, with more than 1,300,000 patients born each year with severe hemoglobin disorders. The globin in normal human hemoglobin (Hb) has 4 kinds of peptide chains, namely α, β, γ and δ. According to the different combinations of globin peptide chains, three kinds of hemoglobin are formed, namely HbA(α ₂ β ₂ ), HbA2 (α ₂ δ ₂ ) and HbF (α ₂ γ ₂ ). HbA is the main hemoglobin in adult erythrocytes, accounting for about 95% of Hb, HbA2 accounts for about 2% of adult Hb, HbF is the main hemoglobin in fetal and early neonatal period, in adults, HbF accounts for not less than 1% of total hemoglobin to 2%. The expression of human hemoglobin undergoes two transitions during development: embryo to fetus and fetus to adult, with the main form changing from HbF (α ₂ γ ₂ ) in fetal liver to HbA (α ₂ β _{2 )} in bone marrow after birth. ). γ-globin is a globin expressed in the fetus. The globin has a similar function to β-globin. The gene HBG (HBG1, HBG2) encoding the protein has a complete sequence in anemia patients, but it will be expressed in adulthood. be silenced. Allogeneic hematopoietic stem cell transplantation is the current method to cure "thalassemia", but it is expensive and lacks matching donors. Many patients cannot use hematopoietic stem cell transplantation. Therefore, gene therapy is becoming a new treatment method.

β-thalassemia (β-thalassemia, β-thalassemia) is a genetic disease in which the patient's own adult hemoglobin (HbA) is abnormal due to mutations in the β-globin subunit. One of the types of poverty. Studies have reported that the modification of improving the clinical symptoms of β-thalassemia can be achieved by reactivating the expression of HbF. The phenotypic diversity of β-thalassemia patients is due to the obvious individual differences in the content of HbF, which generally ranges from 0% to 98%. HbF The higher the content, the lighter the symptoms of anemia in patients. Innovative strategies for beta thalassemia and sickle cell disease (SCD) therapy facilitated by gene editing of hematopoietic stem cells, including correction of SCD-associated mutations using homology-directed DNA repair, BCL11A via forced promoter-LCR loop or NHEJ-mediated Encoding repressors of HBG1 and HBG2 transcription, however, these approaches have not been tested in patients.

Previous studies have found that the use of CRISPR/Cas9 editing technology can reactivate fetal hemoglobin HbF, enhance the expression of γ-globin, and replace defective β-globin, which is a useful disease in sickle cell disease (SCD) and β-thalassemia. hopeful treatment strategy. The essence of gene editing mediated by the CRISPR/Cas9 system is the generation of double-strand breaks, which are repaired by the host cell using its own nonhomologous end-joining (NHEJ) or based on homologous recombination repair (HDR) , but usually uncontrollable insertion or deletion mutations (Indels) are introduced at the target site, and non-targeted cleavage results in high off-target and low safety. Most genetic diseases in humans (Homo sapiens) are caused by base mutations, and the repair efficiency of point mutations by HDR is very low, and HDR basically does not occur in non-dividing cells. Base editing technology (base editing) is a new type of target gene modification technology developed on the basis of the CRISPR/Cas9 system. Base editing directly mutates a single nucleotide on the genome, with almost no double-strand breaks (DSB), It also does not require a template and is a precise gene editing technique.

There are more than 300 kinds of β-thalassemias caused by mutations. The current method of gene editing to modify the β-globin gene has two main problems: one is to introduce the normally expressed β-globin gene by means of homologous recombination in situ repair. The globin gene is usually limited due to its low efficiency; the second is to delete the mutation site or base edit to repair a certain site through gene editing, which may only target one or several types of β-thalassemia, Universality is very low.

Therefore, based on this demand, in order to develop β-thalassemia that can treat different gene mutation types at the same time, scientists have further developed base editing technology. In 2017, the research team of Chinese-American scientist David R. Liu developed an adenine single base editor (ABE) that efficiently induces point mutations. The adenine base editor (ABE) can convert A T into G·C, which can achieve high-efficiency single-base substitutions without introducing double-strand breaks. Base editing technology is produced by fusion of deaminase, sgRNA mainly guides the base editor to the target position, Cas9 nickase (Cas9nickase, nCas9) binds DNA double strands and forms a single-stranded gap, deaminase makes non-gap Adenine or cytosine near the target site is deaminated, and then the base is introduced through DNA replication to achieve the purpose of base replacement. The base editing method rarely causes indels, does not need to rely on homologous recombination, and has significant advantages in safety and efficiency. Among the known pathogenic single-base mutations in humans, 48% (about 15,000) of the mutations are G to A mutations, and 14% (about 4,500) of the disease-causing mutations are A to G mutations. Single base editing has been widely used in gene knockout, knockin, base repair and other fields, and its continuous development has corrected a certain proportion of pathogenic SNP (single nucleotide polymorphism, SNP) to a certain extent.

Since the emergence of single base editors, they have been continuously developed and optimized due to their advantages of being safe and efficient, not relying on DNA double-strand breaks, and not requiring the participation of donor DNA. They have been used to treat genetic diseases such as thalassemia and hemophilia, including in Gene editing of the inner ear of mice will be an effective tool for gene therapy of deafness. With the development of single-base editing tools, gene therapy may be realized through arbitrary conversion of bases in the future.

On March 16, 2020, David Liu's team found that ABE8e can effectively generate natural mutations and upregulate the expression of fetal hemoglobin by editing the BCL11A enhancer or HBG promoter in human cells, which enhances the effectiveness of adenine base editing and applicability. No strategy for high-throughput saturation screening of HBG non-coding and coding sequences by ABE8e base editors has been reported so far.

Contents of the invention

In order to solve the above technical problems, the present invention provides a sgRNA library, a base editing system containing it, a base editing method, a method for increasing the expression of gamma globin RNA, and a method for increasing the expression of HbF; and the base editing system application. The base editing system of the present invention targets the HBG promoter in human cells to up-regulate the expression of fetal hemoglobin, that is, γ-globin, to treat β-hemoglobinopathy (such as β-thalassemia, sickle cell anemia, etc.), the present invention provides The base editing system has important clinical application prospects in the field of gene therapy for beta hemoglobinopathies.

Elevating fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with β-thalassemia and remains a key goal in the treatment of patients with sickle cell disease and thalassemia. In order to identify potential targets that can effectively increase HbF levels in red blood cells, the newly optimized deoxyadenosine deaminase TadA-8e was used in the experiment to conduct high-throughput screening for targets at the HBG promoter through the ABE8e system.

In order to effectively identify targets that may induce fetal hemoglobin (HbF) at the HBG1 and HBG2 promoters, the present invention uses adult-type (low HbF) HUDEP-2 cells stably expressing the human γ-globin gene promoter region design 870 sgRNAs were used for point mutation saturation screening of ABE8e. After HbF staining, 10% cells with high expression of HbF and 10% cells with low expression of HbF were sorted by flow cytometry, and the expression of each sgRNA was analyzed by deep sequencing (that is, the score of each sgRNA, which scored higher The sgRNAs are listed in Table 1). The results showed that the control sgRNAs were evenly distributed between the HbF high and low populations.

The sgRNAs are ranked according to the deep sequencing results. The invention selects the top-ranked sgRNAs from the saturation screening library. These sgRNAs are enriched in high-expressing HbF cells, and then a single sgRNA is introduced into the cells for verification. First, it was verified in HUDEP-2 cells, using 1620 as a positive control and AAVS1 as a negative control. The results showed that the γ-globin gene level in most cells edited by sgRNAs was significantly increased, confirming the screening results.

In addition, further validated in CD34 cells by RNP electroporation, sgRNA-edited cells showed similar levels of γ-globin reactivation. The research results of the present invention show that a potential target for new therapy for treating hemoglobin can be screened out through ABE8e high-throughput screening of the HBG promoter. Compared with other editors, ABE8e has a lower off-target rate and higher editing efficiency. And importantly, screening against the HBG promoter region does not adversely affect cell differentiation and maturation, which is the optimal location. Therefore, this patent designs a saturated library targeting the PAM site (NGG) on the HBG (HBG1, HBG2) promoter, and uses the ABE8e editing tool to screen new sites to increase the expression level of HbF.

One aspect of the technical solution of the present invention provides a sgRNA library targeting the HBG promoter, which comprises one or more of the nucleotide sequences shown in SEQ ID NO:1, 2 and 3.

When the sgRNA library comprises one of the nucleotide sequences shown in SEQ ID NO: 1, 2 and 3, the sgRNA library is essentially a sgRNA, and the sgRNA library of the present invention is nucleoside The acid sequence is the sgRNA of SEQ ID NO: 1, 2 or 3.

In some embodiments of the present invention, the sequence of sgRNA405 is shown in SEQ ID NO:1; the sequence of sgRNA408 is shown in SEQ ID NO:2; the sequence of sgRNA411 is shown in SEQ ID NO:3.

The HBG in the present invention is the genes HBG1 and HBG2 encoding γ-globin protein.

In the sgRNA library of the present invention, the sgRNA is preferably located on a viral vector, more preferably the viral vector is a lentiviral vector.

When the sgRNA is located in a viral vector, the present invention is intended to protect one or more recombinant viral vectors, such as recombinant lentiviral vectors, containing one or more of the nucleotide sequences of SEQ ID NO: 1-SEQ ID NO: 870.

In some embodiments of the present invention, the sgRNA library further comprises one or more of the nucleotide sequences shown in SEQ ID NO:4-SEQ ID NO:59.

In some embodiments of the present invention, the sgRNA library further comprises one or more of the nucleotide sequences shown in SEQ ID NO:59-SEQ ID NO:870.

Another aspect of the technical solution of the present invention provides a base editing system, which comprises an ABE8e base editor and an sgRNA library according to the present invention.

The ABE8e base editor of the present invention contains deoxyadenosine deaminase TadA-8e.

In some embodiments of the present invention, the sequence of the ABE8e base editor is shown in SEQ ID NO:871.

Another aspect of the technical solution of the present invention provides a pharmaceutical composition, which comprises the sgRNA library and the base editing system as described in the present invention.

In some embodiments of the present invention, the pharmaceutical composition further includes a drug delivery system, for example, the drug delivery system is liposome, exosome, phage or bacterial minicell.

Another aspect of the technical solution of the present invention provides a genetically engineered cell comprising the sgRNA library and the base editing system of the present invention.

The starting cells of the genetically engineered cells of the present invention are preferably hematopoietic stem cells, hematopoietic progenitor cells or erythroid progenitor cells; and/or, the source of the starting cells is mammals.

In some embodiments of the present invention, the starting cells are CD34 ⁺ hematopoietic stem cells or CD34 ⁺ hematopoietic progenitor cells; and/or, the source of the starting cells is human or mouse; for example, human HUDEP-2 cells.

Another aspect of the technical solution of the present invention provides a base editing method, which uses the base editing system, the pharmaceutical composition or the genetically engineered cells of the present invention to base edit the HBG promoter.

The base editing method of the present invention can be conventional in the art, such as using lentivirus infection or RNP electroporation, wherein, when using RNP electroporation, the sgRNA can be chemically modified, such as at the 3' end and 5' end of the sgRNA Each end is modified by adding 3 thio and methoxy groups. Another aspect of the technical solution of the present invention provides a method for increasing the expression of γ-globin RNA, which includes the base editing method according to the present invention.

Another aspect of the technical solution of the present invention provides a method for increasing the expression of HbF, which includes the base editing method described in the present invention.

In the present invention, the HbF refers to HbF (α ₂ γ ₂ ) hemoglobin.

In the present invention, the base editing method, the method for increasing the expression of γ-globin RNA, and the method for increasing the expression of HbF may be non-therapeutic methods. Another aspect of the technical solution of the present invention provides a sgRNA library as described in the present invention, a base editing system as described in the present invention, a pharmaceutical composition as described in the present invention or a genetically engineered cell as described in the present invention Application in the preparation of base-edited drugs.

Preferably, the base editing drug is a drug for treating genetic diseases.

More preferably, the genetic disease is thalassemia.

In a certain preferred embodiment, the genetic disease is β-thalassemia.

Another aspect of the technical solution of the present invention provides a sgRNA library as described in the present invention, a base editing system as described in the present invention, a pharmaceutical composition as described in the present invention or a genetically engineered cell as described in the present invention Application in preparing base editing tools or base editing models.

The reagents and raw materials used in the present invention are all commercially available.

The drug delivery system described in the present invention is a delivery system in a narrow sense, such as liposome.

The positive progress effect of the present invention is:

Through the latest high-efficiency ABE8e base editing system and the latest optimized deoxyadenosine deaminase TadA-8e high-throughput screening saturation library of new targets in the promoter regions of HBG1 and HBG2, the library includes 870 specific HBG genes Highly specific sgRNA for the promoter region. The base editing system of the present invention targets the HBG promoter in human cells to up-regulate the expression of fetal hemoglobin, namely γ-globin, to treat β-hemoglobinopathy (such as β-thalassemia, sickle cell anemia and other diseases). The base editing system provided by the present invention has important clinical application prospects in the field of gene therapy for beta hemoglobinopathies.

Description of drawings

Figure 1 is a schematic diagram of LentiABE8e-Blast lentiviral vector.

Figure 2 shows that the HUDEP-2 cell line that has successfully and stably expressed ABE8e has been constructed by Sanger sequencing. Compared with unedited cells, the genome editing efficiency of edited cells can be as high as 70%.

Fig. 3 is the detection of the effect of the ABE8e system on the erythroid differentiation ability of HUDEP-2 cells by flow cytometric analysis.

Figure 4 shows that after HUDEP-2 cells were infected with lentivirus erythroid differentiation by RT-qPCR, compared with unedited cells, the levels of γ-globin mRNA in edited cells were significantly increased relative to β-globin mRNA (numbers Representative sequence numbers representing different sgRNAs).

Figure 5 shows that RNP-delivered ABE8e/sgRNA was highly edited in CD34 ⁺ HSPCs by RT-qPCR, and the expression levels of γ-globin relative to β-globin increased after erythroid differentiation.

Figure 6 shows the level of enucleation rate after erythroid differentiation of CD34 ⁺ hematopoietic stem cells detected by flow cytometric analysis.

Figure 7 shows that the expression level of γ-globin relative to β-globin is increased after RNP delivery of ABE8e/sgRNA in CD34 ⁺ hematopoietic stem cells of β-thalassemia patients by RT-qPCR.

Figure 8 shows the proportion of base-edited and non-edited humanized cells in mouse bone marrow detected by flow cytometry.

Figure 9 shows the proportions of human myeloid cells (CD33+), B cells (CD19+) and other blood cells detected by flow cytometry.

Figure 10 shows the editing efficiencies of cells in edited and unedited groups before and after transplantation analyzed by CRISPResso2, and the editing efficiencies of cells of various lines sorted by flow cytometry.

Figure 11 shows the increased expression level of gamma globin mRNA in cells after transplantation as studied by RT-qPCR.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Example 1

1. Construction of sgRNA screening library.

Design sgRNA for the HBG1 and HBG2 promoters on the γ-globin genome, and simultaneously design a control sgRNA without a target site on the genome, and include a previously known site that can significantly increase the expression of γ-globin sgRNA (provided by Jin Synthesized by Sri Company). The synthetic sgRNA library was cloned into the BsmBI-digested phko9 lentiviral vector (addgeng official website), and viral particles were obtained through viral packaging for infecting HUDEP-2 cells. After the construction of the vector is completed, the vector library is used in a competent state, amplified and cultured, and colonies are collected, and expanded and cultivated, and finally the plasmid is extracted to obtain the amplified vector library.

sgRNA library lentiviral packaging: the amplified gRNA library was transfected into 293T cells (Shanghai Bangyao Biological Co., Ltd.) with the assistance of lentiviral packaging vectors, and the lentiviruses were packaged and concentrated.

2. Construct the HUDEP-2 cell line stably expressing ABE8e.

HUDEP-2 cells are an immortal human hematopoietic stem and progenitor cell line, which can undergo erythroid differentiation after induction in vitro, and obtain erythrocytes with high expression of HbA and low expression of HbF. It is an ideal cell model for studying hemoglobin conversion.

Construct the lentiABE8e-Blast lentiviral expression vector (its map is shown in Figure 1), and infect HUDEP-2 cells with the lentivirus expressing ABE8e (refer to Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, et al.( 2013)Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells.PLoS ONE8(3):e59890.doi:10.1371/journal.pone.0059890), screened with drug blast for 5 days, and obtained pyogenic chains with stable expression HUDEP-2 cell line of Coccus ABE8e (TadA-8e).

Before screening, first verify the targeting activity of the ABE8e protein (its sequence is shown in SEQ ID NO: 871), by using the article from David Liu (refer to Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity ), such as -198 (ie sgRNA418 in the sgRNA library), -175 (ie sgRNA424 in the sgRNA library), Site1 mutagenizes the target site in the ABE8e-HUDEP-2 cell line, and also sgRNAs from different positions in the library were selected, and the selected sgRNAs were constructed into the phko9 lentiviral vector for verification. After packaging the virus and infecting the ABE8e-HUDEP-2 cell line, an editing efficiency of more than 70% can be produced 5 days after the infection of the cells (as shown in Figure 2). In addition, this application also tested the system for its erythroid differentiation The results showed that the introduction of ABE8e protein into the cells did not affect the erythroid differentiation ability of the cells (as shown in Figure 3). This application includes two ways to introduce sgRNA and ABE8e into cells, one is through lentivirus infection, and the other is through RNP electroporation. For cells edited in two ways, the genome is generally extracted 5 days later, and primers about 200 bp away from the editing site are designed to detect the editing efficiency.

3. The sgRNA library was screened in HUDEP-2 cells.

Cloning the sgRNA library of HBG into the phko9 lentiviral vector generated a lentiviral vector library encoding 870 gRNAs and a puromycin resistance, infected HUDEP-2-ABE8e cells at a multiplicity of infection of 0.3, and expressed in Puromycin Infected cells were selected for 7 days in prime and then induced to differentiate into erythrocytes. After red blood cells are matured and cultured, the sites involved in hemoglobin conversion are edited, which will affect the expression of HbF in the cells. Stain with HbF antibody and sort the 10% cells with the highest and lowest HbF expression by FACS, and perform PCR amplification of the lentiviral vector on the enriched and expressed cells in the screening, the amplified fragment contains the gRNA sequence carried by the vector, The 870 sgRNAs in these cells were analyzed by deep sequencing, 59 of which scored higher (refer to Table 1), and the enrichment of sgRNAs in enriched cells can be obtained, so as to find the corresponding sgRNA sequences. It is very likely to be involved in the HbF raising process, so it can be used as a target for further research. Since the sgRNA library is a mixture of multiple different sgRNA vectors, the lentivirus obtained in this application is a mixed virus library, and each virus carries a single gRNA vector. Perform virus infection pre-experiments on the screened target cells to obtain the infection percentage of the cells at a lower MOI (30%) (to ensure that each cell enters at most one lentivirus) to confirm the amount of virus in the sgRNA library during infection (detection of virus titers When the degree is 30%, the amount of cells is 30,000, and the amount of infected virus is (10 μL).

As shown in Table 1, according to the results of deep sequencing, the enrichment of the significantly enriched sgRNA in the cells was analyzed compared with the group with high expression of 10% HbF and the group with low expression of 10% HbF. When using the robust rank aggregation (RRA) method of MAGeCK and the default parameter bioinformatics analysis, a certain threshold is set, and filters of FDR<0.05 and log ₂ Fold-Change>log ₂ (1.5) are used to define up-regulated Candidate sgRNA. Then rank the sgRNA, which represents the relative enrichment of the sgRNA, that is, if the ranking exceeds a certain threshold, it has the effect of activating the expression of HbF.

Table 1: sgRNAs with higher scores

sgRNA ID Score SEQ ID NO: sgRNA ID Score SEQ ID NO: sgRNA 541 34.395 4 sgRNA 142 3.9528 32

sgRNA 1066 23.539 5 sgRNA 418 (control) 3.5567 33 sgRNA 689 21.34 6 sgRNA 65 3.3742 34 sgRNA 562 21.187 7 sgRNA 225 3.232 35 sgRNA 32 15.82 8 sgRNA 351 3.1693 36 sgRNA 62 14.906 9 sgRNA 31 2.7039 37 sgRNA 944 12.671 10 sgRNA 342 2.5178 38 sgRNA 445 12.449 11 sgRNA 661 2.4697 39 sgRNA 234 11.072 12 sgRNA 974 2.4628 40 sgRNA 543 10.548 13 sgRNA 470 2.373 41 sgRNA 616 9.6405 14 sgRNA 931 2.3715 42 sgRNA 621 9.0611 15 sgRNA 309 2.271 43 sgRNA 1058 8.4345 16 sgRNA 479 2.0708 44 sgRNA 90 8.4343 17 sgRNA 1021 2.0362 45 sgRNA 409 8.0433 18 sgRNA 986 2.0039 46 sgRNA 118 7.882 19 sgRNA 312 1.5273 47 sgRNA 347 7.6729 20 sgRNA 14 1.495 48 sgRNA 242 7.6281 twenty one sgRNA 513 1.2857 49 sgRNA 1061 7.2964 twenty two sgRNA 706 1.1925 50 sgRNA 658 7.2245 twenty three sgRNA 405 1.1237 1 sgRNA 1065 7.2136 twenty four sgRNA 223 1.0884 51 sgRNA 955 6.8254 25 sgRNA 496 1.039 52 sgRNA 626 6.7586 26 sgRNA 245 1.0353 53 sgRNA 653 6.5252 27 sgRNA 424 (control) 0.7537 54 sgRNA 27 6.4956 28 sgRNA 120 0.70886 55 sgRNA 520 6.45 29 sgRNA 657 0.68753 56 sgRNA 665 6.3462 30 sgRNA 370 0.68623 57 sgRNA 987 5.9911 31 sgRNA 558 0.57447 58 sgRNA 408 4.303 2 sgRNA 491 4.004 59

In Vitro Verification of Example 2 Candidate sgRNA

1. Validate the candidate sgRNA in HUDEP2-ABE8e cells.

Introduce the top-ranked sgRNA into HUDEP-2 cells highly expressing ABE8e protein, and use the sgRNA transfection group of sgRNA1620 (sgRNA1620 refers to Therapeutic base editing of human hematopoietic stem cells) (its sequence is shown in SEQ ID NO:872) as Positive control, the AAVS1sgRNA (as shown in SEQ ID NO:873) transfection group with no target on the genome is negative control. sgRNA1620 was used as a positive control because it has been reported in the literature that editing this site by a single base editing tool has high editing efficiency and significantly increases the level of γ-globin. In this application, the selected sgRNA was constructed on the phko9 lentiviral vector, and the virus was produced by 293T packaging, and the HUDEP-2 cells were infected with the same virus amount (200,000 cells were cultured in a 24-well plate, and 10 μL virus amount was infected). After the lines were induced to differentiate, RT-qPCR was used to detect the changes in the expression of γ-globin RNA.

The results of RT-PCR showed that the mRNA expression level of γ-globin in edited cells was significantly increased compared with the average mRNA expression level of unedited cells. After erythroid differentiation, the expression of intracellular HbF was detected by FACS, and the average expression of HbF in edited cells was significantly higher than that in unedited cells (as shown in A and B of Figure 4 ). Based on this result, it shows that the editing of normal human HUDEP-2 cells by lentivirus infection can reactivate the expression of γ-globin gene in the cells, so that the γ-globin and HbF expression level increased.

2. The next step is to verify the candidate sgRNA in CD34 ⁺ cells.

Compared with lentiviral vectors, electroporation (ribonucleoprotein, RNP) is a more commonly used physical delivery method, which directly delivers ABE8e protein and sgRNA, and has higher transduction efficiency in human CD34 ⁺ HSPCs cells, which can maximize The off-target effect can be greatly reduced, and RNP can be rapidly degraded in cells, which improves the safety. The experiment used electroporation to deliver ABE8e protein and chemically modified (3' end and 5' end each with 3 sulfo and methoxy groups) sgRNA to CD34 ⁺ HSPCs in the form of RNP (Shanghai Bangyao Biological Co., Ltd. company), targeting the γ-globin gene promoter region for the treatment of β-thalassemia. In the experiment, 1620 was used as a positive control, and AAVS1sgRNA was used as a negative control. After erythroid differentiation, the expression of γ-globin gene was detected by RT-qPCR, and the enucleation rate of cells was detected by flow cytometry after Hochest staining on the 18th day.

The present application found that by synthesizing sgRNAs targeting the promoter region of the γ-globin gene and chemically modifying both ends, ABE8e protein and sgRNA were delivered to CD34 ⁺ hematopoietic stem/ In the absence of sorting, the average editing efficiency in CD34 ⁺ cells can reach more than 80%. After erythroid differentiation, the average expression levels of edited intracellular γ-globin mRNA relative to β-like globin mRNA were generally elevated (as shown in Figure 5), especially at the sgRNA408, sgRNA405, and sgRNA411 loci , the expression of the gamma-globin gene was significantly increased relative to the average content of the beta-globin (as shown in Figure 5), and the denucleation rate was also about 40% (as shown in Figure 6).

Based on the above results, it is shown that the ribonucleoprotein delivery method can efficiently edit CD34 ⁺ hematopoietic stem/progenitor cells while avoiding the safety hazards brought by lentiviral vectors, providing new methods and experiments for gene editing treatment of β-thalassemia Base.

3. Select CD34 ⁺ cells derived from patients with β-thalassemia to verify the sgRNA of the regulatory target.

Studies have shown that increasing HbF can improve β-thalassemia patients to a certain extent. Therefore, in order to further prove that ABE8e/sgRNA editing of human CD34 ⁺ HSPCs can induce the expression of γ-globin, we chose to electrotransfer the sgRNA of the candidate target into CD34 ⁺ cells derived from β-thalassemia patients, and then differentiate them in vitro. Expression of gamma globin was detected after differentiation. The results showed that after 18 days of erythroid differentiation of edited patient CD34+HSPCs cells, the average expression level of γ-globin mRNA relative to β-like-globin mRNA was significantly increased (as shown in Figure 7).

These results indicated that gene editing of ABE8e/sgRNA RNP in CD34 ⁺ HSPCs of β-thalassemia patients increased the expression of γ-globin in the edited cells. Therefore, ABE8(RNP)-mediated disruption of the binding sites in the promoters of HBG1 and HBG2 is a potentially feasible and safe therapeutic strategy for the treatment of β-thalassemia.

Example 3 In vivo verification of candidate sgRNA

NCG-X immunodeficiency mouse model: the recipient mouse is immunodeficiency mouse NOD-Prkdc ^em26Cd52 Il2rg ^em26Cd22 kit ^{em1Cin(V831M)} /Gpt, this strain can accept human hematopoietic stem cell transplantation without radiation, has T/B/ Defective NK immune cells and inhibited homing function of murine hematopoietic stem cells are a good model for human hematopoietic stem cell transplantation.

ABE8e protein and sgRNA (sgRNA1620 and sgRNA408) were used to efficiently edit healthy human CD34+ cells by RNP electroporation, and 1 million cells were injected into the tail vein of NCG-X immunodeficient mice (4-5 weeks old). Four months later, the mice were euthanized, and the bone marrow was separated to prepare a mononuclear cell suspension. The humanized ratio and the ratio of various blood cells in the bone marrow of NCG-X mice were detected. The proportion of humanized cells in the base-edited and non-edited groups in the mouse bone marrow was analyzed by flow cytometry hCD45+ cells/(hCD45+ cells+mCD45+ cells)×100 (as shown in Figure 8), and the types of cells differentiated into The proportion of blood cells, including the proportion of human myeloid cells (CD33+), B cells (CD19+) and other blood cells (as shown in Figure 9). CRISPResso2 was used to analyze the editing efficiency of cells in edited and unedited groups before and after transplantation, as well as the editing efficiency of cells of various lines sorted by flow cytometry, and analyze the insertion, deletion and mutation of bases in cells ( As shown in A and B of Figure 10). RT-qPCR was used to study the expression level of γ-globin mRNA of the transplanted cells (as shown in FIG. 11 ).

The results showed that we transplanted the edited hematopoietic stem cells into immunodeficient mice and successfully homing four months later. The proportion of human cells in the mouse bone marrow was analyzed by FACS, which showed that the edited group had a homing efficiency similar to that of unedited cells. Then, the differentiation of different lineage cells was detected by FACS, and the results showed that the edited cells could successfully differentiate into various blood cells including B cells and myeloid cells in the mouse bone marrow. The base editing efficiency of human cells in mouse bone marrow was analyzed by deep sequencing, and the results showed that the target genes of most cells remained edited after bone marrow transplantation. The above results indicate that hematopoietic stem cells edited with ABE8e base can homing and rebuild the whole blood system for a long time.

Although the specific implementations of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these implementations without departing from the principle and essence of the present invention. Revise. Accordingly, the protection scope of the present invention is defined by the appended claims.

Claims (10)

A sgRNA library targeting the HBG promoter, comprising one or more of the nucleotide sequences shown in SEQ ID NO:1, 2 and 3. The sgRNA library according to claim 1, wherein the sgRNA library also comprises one or more of the nucleotide sequences shown in SEQ ID NO:4-SEQ ID NO:59; Preferably, the sgRNA library further comprises one or more of the nucleotide sequences shown in SEQ ID NO:59-SEQ ID NO:870; More preferably, the sgRNA is located on a viral vector, and the viral vector is preferably a lentiviral vector. A base editing system comprising an ABE8e base editor and the sgRNA library according to claim 1 or 2; preferably, the sequence of the ABE8e base editor is as shown in SEQ ID NO:871. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the sgRNA library as claimed in claim 1 or 2 or the base editing system as claimed in claim 3; preferably, the pharmaceutical composition also Including drug delivery system; more preferably, the drug delivery system is liposome, exosome, phage or bacterial microcell. A genetically engineered cell, characterized in that the genetically engineered cell comprises the sgRNA library according to claim 1 or 2 or the base editing system according to claim 3. The genetically engineered cell according to claim 5, wherein the starting cell of the genetically engineered cell is hematopoietic stem cell, hematopoietic progenitor cell or erythroid progenitor cell; and/or, the source of the starting cell is a mammal; Preferably, the starting cells are CD34 ⁺ hematopoietic stem cells or CD34 ⁺ hematopoietic progenitor cells; and/or, the source of the starting cells is human or mouse; for example, human HUDEP-2 cells. A base editing method, characterized in that, the base editing method uses the base editing system according to claim 3, the pharmaceutical composition according to claim 4, or the base editing method according to claim 5 or 6. Genetically engineered cells to base edit the HBG promoter. A method for increasing the expression of HbF, which comprises using the base editing method as claimed in claim 7; preferably, said increasing the expression of HbF is achieved by increasing the expression of γ-globin RNA. A sgRNA library as claimed in claim 1 or 2, the base editing system as claimed in claim 3, the pharmaceutical composition as claimed in claim 4, or the genetically engineered cell as claimed in claim 5 or 6 in preparation Application of base editing in medicine; Preferably, the base editing drug is a drug for treating genetic diseases; More preferably, the genetic disease is thalassemia; Further more preferably, the genetic disease is β-thalassemia. A sgRNA library as claimed in claim 1 or 2, the base editing system as claimed in claim 3, the pharmaceutical composition as claimed in claim 4, or the genetically engineered cell as claimed in claim 5 or 6 in preparation Applications in base editing tools or base editing models.

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