WO2020187272A1 - Fusion protein for gene therapy and application thereof - Google Patents

Abstract

The present invention relates to a fusion protein for gene therapy and an application thereof. Specifically, provided by the present invention is an enhanced fusion protein. Compared with a wild-type gene editing protein, the enhanced fusion protein in the present invention can be used for gene therapy, and the gene therapy effect thereof is better than that of the wild-type gene editing protein.

Description

A fusion protein for gene therapy and its application Technical field

The present invention relates to the field of biotechnology, in particular, to a fusion protein for gene therapy and its application.

Background technique

Gene editing technology is a technology that artificially breaks double-stranded DNA and uses the repair mechanism of double-stranded DNA breaks to achieve gene manipulation. The existing gene editing technologies include ZFN, TALEN and CRISPR/Cas9 technologies, of which CRISPR/Cas9 technologies are the most widely used. CRISPR/Csa9 technology is an adaptive immune mechanism derived from bacteria or archaea. It uses a single-stranded guide RNA (sgRNA) and Csa9 protein to generate DNA double-strand breaks at specific locations in the genome, and then through endogenous non-identical Source end joining (NHEJ) or homologous recombination (HDR) repair mechanism to achieve target gene knockout or specific gene or fragment insertion.

Although CRISPR/Cas9 technology has extremely powerful functions, it also has its shortcomings, such as: 1. Off-target problem; 2. The limitation of PAM leads to the limitation of target selection; 3. The editing efficiency of some new tools is generally low ( XCas9 and SpCas9-NG), and the current optimization and modification of gene editing tools are mainly based on enhancing the accuracy of the tools and enhancing the modification of the target range. There is no extensive improvement of various gene editing tools for the gene editing tools themselves. Methods.

Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by defects and abnormal genes to achieve the purpose of treatment. Traditional gene therapy is based on overexpression of the correct gene to compensate for the missing genes in the patient's body to treat the disease. After the emergence of gene editing technology, gene therapy has developed into a precise treatment method that can be accurately corrected at the DNA level The disease-curing genes achieve the goal of fundamentally curing the disease. At present, gene therapy based on gene editing technology is developing in full swing. It has been reported that it has played a gratifying effect in the treatment of a variety of genetic diseases, but for most genetic diseases, the efficiency of gene editing-mediated gene therapy It is still low overall, which greatly limits its wide application. Therefore, the present invention is based on the newly invented enhanced gene editing tool, and delivers the enhanced gene editing tool to the disease model through a clinically feasible method, in an attempt to improve the efficiency of disease treatment.

Therefore, there is an urgent need to develop a new gene therapy method in this field.

Summary of the invention

The purpose of the present invention is to provide a new method of gene therapy.

The first aspect of the present invention provides the use of an active substance for the preparation of a drug or preparation for gene therapy, wherein the active substance is a fusion protein, or its coding gene, or its expression vector, wherein the fusion protein The structure of is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

Where

A is the gene editing protein,

B is the DNA double-strand binding domain,

C is an optional base editor element;

L is no or connecting peptide,

Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, the drug is used to treat diseases that can be treated by gene therapy.

In another preferred example, the disease includes genetic disease, nervous system disease, heart system disease, ophthalmological disease, autoimmune disease, hemophilia, blood disease, and/or cancer.

In another preferred embodiment, the genetic disease is selected from the group consisting of liver metabolic disease, β-thalassemia, cardiovascular disease, Duchenne muscular dystrophy, hyperoxalic acid, or a combination thereof.

In another preferred example, the metabolic disease of the liver is selected from the group consisting of familial hypercholesterolemia, hemophilia, phenylketonuria, tyrosineemia, hyperammonemia, or a combination thereof.

In another preferred embodiment, the disease includes a cell proliferative disorder.

In another preferred embodiment, the cell proliferative disorder is selected from the group consisting of lung cancer, colon cancer, rectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanin Tumor, stomach cancer, ovarian cancer, rheumatoid arthritis, or a combination thereof.

In another preferred example, the disease is a disease for an organ selected from the following group: liver, kidney, heart, pancreas, lung, bone, thymus, adrenal gland, oral cavity, pharynx, esophagus, stomach, brain, twelve Digital intestine, jejunum, ileum, large intestine, anus, or a combination thereof.

In another preferred embodiment, when the structure of the fusion protein is as shown in formula I', C is absent.

In another preferred embodiment, the non-peptide bond includes PEG.

In another preferred embodiment, the gene editing protein is selected from the following group: Cas9, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof.

In another preferred embodiment, the gene editing protein includes wild-type or mutant gene editing protein.

In another preferred embodiment, the gene editing protein is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, Or a combination.

In another preferred embodiment, the amino acid sequence of the wild-type gene editing protein is shown in SEQ ID NO.: 1, 14 or 15.

In another preferred embodiment, the amino acid sequence of the base editor element is shown in SEQ ID NO.: 2 or 12.

In another preferred embodiment, the DNA double-strand binding domain is a non-sequence-specific DNA double-strand binding domain.

In another preferred embodiment, the DNA double-strand binding domain is selected from the group consisting of HMG-D, Sac7d, or a combination thereof.

In another preferred example, the DNA double-stranded binding domain includes a wild-type DNA double-stranded binding domain and a mutant type of DNA double-stranded binding domain.

In another preferred embodiment, the DNA double-strand binding domain is derived from Drosophila or Archaea.

In another preferred embodiment, the amino acid sequence of the DNA double-strand binding domain is shown in SEQ ID NO.: 10 or 11.

In another preferred example, the length of the connecting peptide is 1-100 aa, preferably, 15-85 aa, more preferably, 25-70 aa.

In another preferred example, the connecting peptide is a sequence shown as Gly-Gly-Ser with n repeats, where n is 2-8, preferably n is 3-6.

In another preferred embodiment, the amino acid sequence of the connecting peptide is selected from the following group:

(1) The amino acid sequence of the polypeptide shown in any one of SEQ ID NO.: 3-7;

(2) Pass the amino acid sequence shown in any one of SEQ ID NO.: 3-7 through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8 It is formed by the substitution, deletion or addition of one, more preferably 1-3, most preferably one amino acid residue, and the amino acid shown in any one of SEQ ID NO.: 3-7 having the function of the polypeptide described in (1) A polypeptide derived from a sequence of polypeptides.

In another preferred embodiment, the base editor element includes cytosine deaminase and adenine deaminase.

In another preferred embodiment, the cytosine deaminase includes Apobec1 and Apobec3A.

In another preferred embodiment, the adenine deaminase includes TadA.

In another preferred embodiment, the fusion protein has an amino acid sequence shown in any one of SEQ ID NO.: 8, 9, and 13.

In another preferred embodiment, the medicine includes:

(a) Fusion protein, or its coding gene, or its expression vector (vector); the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

Where

A is the gene editing protein,

B is the DNA double-strand binding domain,

C is an optional base editor element;

L is no or connecting peptide,

Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond; and

(b) A pharmaceutically acceptable carrier.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the drug is an injection dosage form.

In another preferred embodiment, the medicine also includes other medicines for gene therapy.

In another preferred embodiment, the other drugs for gene therapy are selected from the group consisting of antisense nucleotide drugs, EDIT-101 drugs, CTX001, or a combination thereof.

In another preferred embodiment, the drug is a cell preparation.

In another preferred embodiment, the gene therapy is performed by gene editing.

In another preferred embodiment, the gene therapy is performed by gene editing reagents.

In another preferred example, the gene editing reagent comprises a fusion protein, and the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

Where

A is the gene editing protein,

B is the DNA double-strand binding domain,

C is an optional base editor element;

L is no or connecting peptide,

Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, the reagent further includes one or more reagents selected from the following group:

(a1) gRNA, crRNA, or a vector for producing the gRNA or crRNA;

(a2) Template for homologous targeted repair: single-stranded nucleotide sequence or plasmid vector.

In another preferred embodiment, the gene therapy is performed by the following method:

In the presence of gene editing reagents, cells are gene-edited.

In another preferred embodiment, the cells include human or non-human mammalian cells (such as primates or livestock).

In another preferred embodiment, the cells include cancer cells or normal cells.

In another preferred embodiment, the cells are selected from the group consisting of kidney cells, liver cells, nerve cells, heart cells, epithelial cells, muscle cells, somatic cells, bone marrow cells, endothelial cells, or combinations thereof.

In another preferred embodiment, the cell is selected from the group consisting of 293 cells, A549 cells, SW626 cells, HT-3 cells, PA-1 cells, or a combination thereof.

In another preferred embodiment, the cell includes HEK293T.

In another preferred embodiment, the gene editing is performed in an in vitro reaction system.

In another preferred embodiment, in the in vitro reaction system, the content of the gene editing reagent is 100ng-700ng, preferably, 200ng-600ng, more preferably, 300ng-500ng.

In another preferred embodiment, the cell is an in vitro cell.

The second aspect of the present invention provides a medicine kit including:

(a1) The first container, and the active substance in the first container, or the drug containing the active substance, wherein the active substance is a fusion protein, or its coding gene, or its expression vector, wherein The structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

Where

A is the gene editing protein,

B is the DNA double-strand binding domain,

C is an optional base editor element;

L is no or connecting peptide,

Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, the kit further includes:

(a2) The second container, and other medicines for gene therapy in the second container, or medicines containing other medicines for gene therapy.

In another preferred embodiment, the first container and the second container are the same or different containers.

In another preferred embodiment, the medicine in the first container is a unilateral preparation containing the active substance.

In another preferred embodiment, the medicine in the second container is a single preparation containing other medicines for gene therapy.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the drug is an injection dosage form.

The third aspect of the present invention provides a pharmaceutical composition comprising:

(a) Active substance, the active substance is a fusion protein, or its coding gene, or its expression vector (vector), wherein the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

Where

A is the gene editing protein,

B is the DNA double-strand binding domain,

C is an optional base editor element;

L is no or connecting peptide,

Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond; and

(b) A pharmaceutically acceptable carrier.

In another preferred embodiment, the expression vector includes a viral vector.

In another preferred example, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

In another preferred embodiment, the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof, preferably, the vector is adeno-associated virus (AAV).

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection dosage form.

In another preferred embodiment, the pharmaceutical composition also includes other drugs for gene therapy.

In another preferred embodiment, the other drugs for gene therapy are selected from the group consisting of antisense nucleotide drugs, EDIT-101 drugs, CTX001, or a combination thereof.

In another preferred embodiment, the pharmaceutical composition is a cell preparation.

The fourth aspect of the present invention provides a method for treating diseases, the method includes the steps of: administering an active substance or the kit according to the second aspect of the present invention to a subject in need, the active substance being a fusion protein, or The coding gene or its expression vector, wherein the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

Where

A is the gene editing protein,

B is the DNA double-strand binding domain,

C is an optional base editor element;

L is no or connecting peptide,

Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, before, simultaneously and/or after the administration of the active substance or the kit according to the second aspect of the present invention, other active substances for treating diseases are used in conjunction.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

Figure 1 shows the editing efficiency for two different endogenous targets, indicating that the different connection methods of the DNA double-strand binding domain and Cas9, as well as linkers of different lengths, have differences in the improvement of efficiency. Comprehensive selection of HMG-D The structural domain is best connected to the N-terminal of Cas9 through an L4 length linker. Namely: HMG-D-L4-Cas9, where H stands for HMG-D; S stands for Sac7d; L1-L5 stands for linkers of different lengths; mutH stands for mutant HMG-D (V32A and T33A mutations reduce the binding activity); C stands for Cas9.

Figure 2 shows that the editing efficiency of HMG-D-L4-Cas9 on other endogenous targets can also be improved. The efficiency is improved by> 20%, preferably,> 40%, and more preferably,> 60% (such as 80 %), up to 2 times, where H stands for HMG-D.

Figure 3 shows that the double-stranded binding domain HMG-D can increase the efficiency of Cas9 proteins from other sources (such as SaCas9), and the efficiency is increased by more than 20%.

Figure 4 shows that the double-stranded binding domain HMG-D can increase the efficiency of non-Cas9 proteins (such as AsCas12a) by 10-20%.

Figure 5 shows that the double-stranded binding domain HMG-D can increase the efficiency of apparent regulatory tools (such as CRISPR-VPR), and the efficiency can be increased by 2 times.

Figure 6 shows that the double-stranded binding domain HMG-D can improve the efficiency of the single-base editing tool ABE, where H stands for HMG-D.

Figure 7 shows the delivery of SpCas9 fused with HMG-D and sgRNA targeting the therapeutic target of hypercholesterolemia (PCSK9) to mouse liver by adeno-associated virus (AAV), successfully targeting the knockout of mouse PCSK9 gene , The knockout efficiency was significantly higher than the SpCas9 group without HMG-D. Through long-term monitoring of the blood cholesterol content of the treated mice, we found that the blood cholesterol content of the treated mice was significantly lower than that of the untreated group, and the HMG-D fusion treatment group was significantly lower than the treatment group without HMG-D fusion. It shows that SpCas9 fused with double-stranded binding protein can significantly help reduce blood cholesterol, which is beneficial to the treatment of cardiovascular diseases and familial hypercholesterolemia. Figure 7A shows the editing efficiency of PCSK9 targets in mice Figure 7B shows the decrease in blood cholesterol levels in the edited mice.

Figure 8 shows the expression of the fusion double-stranded binding protein (HMG-D) base editing tool (hA3A-CBE) protein, and sgRNA electrotransformation into hematopoietic stem cells to achieve -114 and -115 positions C to T mutation. The results showed that the editing efficiency of the hA3A-CBE group with HMG-D was significantly higher than that of the hA3A-CBE group without HMG-D; at the RNA level, the expression of gamma globin in the hA3A-CBE group with HMG-D was also obvious Increase; also at the protein level, the expression of globin in the hA3A-CBE group fused with HMG-D is also significantly higher than that in the hA3A-CBE group without HMG-D; Figure 8A shows the editing efficiency of hematopoietic stem cell targets, Figure 8B The expression of HBG gene mRNA after differentiation of edited hematopoietic stem cells is shown, and Fig. 8C shows the expression of gamma globin after differentiation of edited hematopoietic stem cells.

detailed description

After extensive and in-depth research, the inventors unexpectedly obtained an enhanced fusion protein. Compared with the wild-type gene editing protein, the enhanced fusion protein of the present invention can significantly improve the gene editing efficiency in vivo or in vitro, and the present invention also unexpectedly discovered that the gene editing protein and DNA double-strand binding domain, optional alkali The fusion protein formed by the base editor element and the optional connecting peptide can significantly improve gene editing efficiency (increased by ≥20%, such as 80%, or even up to 2 times), and the fusion protein of the present invention can be used in gene therapy. On this basis, the inventor completed the present invention.

the term

In order to make the present disclosure easier to understand, first define certain terms. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Other definitions are stated throughout the application.

The term "about" may refer to a value or composition within an acceptable error range of a particular value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "substantially consisting of" or "consisting of".

Sequence identity (or homology) is passed along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) ) Compare two aligned sequences and determine the number of positions where the same residue appears. Normally, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

As used herein, the term "EDIT-101 drug" belongs to gene therapy drugs, which is a type of cell. Specifically, EDIT-101 is a drug that uses CRISPR gene editing technology to treat hereditary retinal degeneration disease (LCA10 disease). 101 is administered by subretinal injection, and the gene editing system is directly delivered to the photoreceptor cells to achieve the therapeutic effect.

As used herein, the term "CTX001" belongs to gene therapy drugs, which is a type of cell. Specifically, CTX001 is based on CRISPR gene editing technology to achieve therapeutic purposes by cutting the BCL11A gene of patients with β-thalassemia.

Wild-type gene editing protein

As used herein, "wild-type gene editing protein" refers to a naturally occurring gene editing protein that has not been artificially modified. Its nucleotides can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR ) Etc. The amino acid sequence can be derived from the nucleotide sequence. The source of the wild-type gene editing protein includes (but is not limited to): Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium .

In a preferred embodiment of the present invention, the amino acid sequence of the wild-type gene editing protein is shown in SEQ ID NO.: 1 or 14 or 15.

In a preferred embodiment of the present invention, the gene editing protein includes, but is not limited to, Cas9, Cas12, Cas12a, Cas12b, Cas13, and Cas14.

DNA double-strand binding domain

As used herein, the term "DNA double-strand binding domain" is a DNA double-strand binding domain without sequence specificity. Compared with the sequence-specific DNA double-stranded binding domain, the non-sequence-specific DNA double-stranded binding domain of the present invention is not limited by the DNA sequence, and theoretically can bind to any DNA sequence, so it can be applied to any position The binding of DNA.

The sequence of a preferred DNA double-strand binding domain is shown in SEQ ID NO.: 10 or 11.

Base editor

Any base editor provided herein can modify specific nucleotide bases without producing a significant proportion of indels. As used herein, "insertion/deletion" refers to the insertion or deletion of nucleotide bases within a nucleic acid. Such insertions or deletions can lead to frameshift mutations in the coding region of the gene. In some embodiments, it is desirable to produce a base editor that effectively modifies (e.g., mutates or deamination) specific nucleotides within a nucleic acid without generating a large number of insertions or deletions (ie, indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of producing a larger proportion of the intended modification (eg, point mutation or deamination) relative to indels.

Any base editor of the present invention can effectively generate intended mutations, such as point mutations, in nucleic acids (for example, nucleic acids in the genome) without generating a large number of unintended mutations, such as unintended point mutations.

In the present invention, the base editor includes cytosine deaminase and adenine deaminase, and other types of base editors as long as they have the function of the base editor of the present invention are also within the protection scope of the present invention.

In the present invention, the structure after the gene editing protein is fused with the base editor is called ABE or CBE, where ABE is the structure after the gene editing protein is fused with adenine deaminase, and CBE is the gene editing protein and the cell The fusion structure of pyrimidine deaminase.

The sequence of a preferred base editor is shown in SEQ ID NO.: 2 or 12.

Fusion protein

As used herein, "fusion protein of the present invention" or "polypeptide" refers to the fusion protein described in the second aspect of the present invention. The structure of the fusion protein of the present invention is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

Where

A is the gene editing protein,

B is the DNA double-strand binding domain,

C is an optional base editor element;

L is no or connecting peptide,

Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.

In the present invention, the length of the connecting peptide has an effect on the activity of the fusion protein. The preferred length of the connecting peptide is 1-100 aa, preferably, 15-85 aa, and more preferably, 25-70 aa.

A preferred connecting peptide is shown in SEQ ID NO.: 3-7.

As used herein, the term "fusion protein" also includes variant forms shown in SEQ ID NO.: 8, 9, or 13 having the above-mentioned activity. These variants include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletion, insertion and/or substitution, and addition or addition at the C-terminal and/or N-terminal One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in this field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

The present invention also includes active fragments, derivatives and analogs of the above fusion protein. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogues of the present invention can be (i) one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in each amino acid residue, or (iii) a polypeptide formed by fusing an antigenic peptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused with a leader sequence, secretory sequence, or 6His tag sequence). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

A preferred type of active derivative means that compared with the amino acid sequence of Formula I, there are at most 3, preferably at most 2, and more preferably at most 1 amino acid replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by amino acid substitutions.

Table A

Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

The invention also provides analogs of the fusion protein of the invention. The difference between these analogues and the polypeptide shown in SEQ ID NO.: 8 or 9 or 13 may be the difference in the amino acid sequence, the difference in the modification form that does not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

Modified (usually without changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

In the present invention, in formula I, A is a gene editing protein, B is HMG-D or Sac7d, C is adenine deaminase or cytosine deaminase or none, L is L1 or L2 or L3 or L4 or L5 or none.

In a preferred embodiment, in Formula I, A is a gene editing protein, B is HMG-D, C is either adenine deaminase or cytosine deaminase or none, and L is L4 or L5.

In a preferred embodiment, the fusion protein of the present invention may also include two or more of the A, B, C, and L elements in Formula I.

In a preferred embodiment, in Formula I, A is a gene editing protein, B is HMG-9, C is none, and L is L4.

In a preferred embodiment, in Formula I, A is a gene editing protein, B is HMG-D, C is adenine deaminase, and L is L5.

In a preferred embodiment, in Formula I, A is a gene editing protein, B is HMG-D, C is cytosine deaminase, and L is L5.

In a preferred embodiment, the amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO.: 8, 9 or 13.

Adeno-associated virus

Because Adeno-associated virus (AAV) is smaller than other viral vectors, is non-pathogenic, and can transfect dividing and undivided cells, gene therapy methods for genetic diseases based on AAV vectors have been affected. Widespread concern.

Adeno-associated virus (adeno-associated virus, AAV), also known as adeno-associated virus, belongs to the Parvoviridae dependent virus genus. It is the simplest type of single-stranded DNA-deficient virus found so far and requires a helper virus (usually adenovirus). Viruses) participate in replication. It encodes the cap and rep genes in the inverted repeat (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.

Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo. Long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have accumulated a lot of data. In medical research, rAAV is used in the research of gene therapy for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice and other aspects.

In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large range of cells without any impact on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos. 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, they are all incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and/or cap genes are missing and replaced by the gene of interest, and these constructs are in vitro (into cultured cells) or in vivo (into the organism directly) ) The purpose of transporting the gene of interest. Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (such as adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeats (ITR) Region plasmids, and plasmids carrying AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

In some embodiments, the recombinant vector is capsidized to viral particles (e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV virus particles of AAV16). Therefore, the present disclosure includes recombinant virus particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in US Patent No. 6,596,535.

Expression vector and host cell

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention or the fusion protein coding sequence of the present invention, and methods for producing the polypeptides of the present invention through recombinant technology.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant fusion protein. Generally speaking, there are the following steps:

(1) Use the polynucleotide (or variant) of the present invention encoding the fusion protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;

(2). Host cells cultured in a suitable medium;

(3). Separate and purify protein from culture medium or cells.

In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors well known in the art. As long as it can replicate and stabilize in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

The methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

The host cell can be a prokaryotic cell (such as E. coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as yeast cells, plant cells or mammalian cells (including human and non-human mammals). Representative examples include: Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, yeast cells (such as Pichia pastoris, Kluyveromyces, or a combination thereof) are selected; preferably, the yeast cells include: Kluyveromyces, more preferably Maxsk Luwei and/or Kluyveromyces lactis) are host cells.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the replication initiation point, polyoma enhancers on the late side of the replication initiation point, and adenovirus enhancers.

Those of ordinary skill in the art know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture can be selected from various conventional mediums. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

Gene therapy

Gene Therapy for genetic diseases refers to the application of genetic engineering technology to introduce normal genes into patient cells to correct defective genes and cure the disease. The way of correction can be either to repair the defective gene in situ, or to transfer a functional normal gene into a certain part of the cell genome to replace the defective gene to play a role. Gene is the basic functional unit that carries biological genetic information and is a specific sequence located on the chromosome. Certain technical methods or vectors must be used to introduce foreign genes into biological cells. The methods of gene transfer are divided into biological methods, physical methods and chemical methods. Adenovirus vectors are currently one of the most commonly used viral vectors for gene therapy. Gene therapy is mainly used to treat diseases that pose a serious threat to human health, including, but not limited to: genetic diseases (such as hemophilia, cystic fibrosis, family hypercholesterolemia, etc.), malignant tumors, cardiovascular diseases, Infectious diseases (such as AIDS, rheumatoid, etc.). Gene therapy is a high-tech biomedical technology that introduces human normal genes or therapeutic genes into human target cells in a certain way to correct gene defects or exert therapeutic effects, so as to achieve the purpose of treating diseases. Gene therapy is different from conventional treatment methods: in general, the treatment of diseases is aimed at various symptoms caused by gene abnormalities, while gene therapy is aimed at the root cause of the disease-the abnormal gene itself. Target cells for gene therapy include, but are not limited to, somatic cells, bone marrow cells, liver cells, nerve cells, endothelial cells, and muscle cells.

In the present invention, the target gene is subjected to efficient gene editing (including gene insertion, replacement, etc.) through gene therapy, so as to restore normal gene expression or enhance gene expression, thereby treating related diseases.

Genetic disease

In the present invention, genetic disease refers to a type of disease caused by mutation of genomic DNA.

In a preferred embodiment, the genetic diseases of the present invention include, but are not limited to, liver metabolic diseases (such as familial hypercholesterolemia, hemophilia, phenylketonuria, tyrosinemia, hyperammonia Disease, etc.), β-thalassemia, cardiovascular disease, Duchenne muscular dystrophy, hyperoxalic acidosis.

Liver metabolic disease

The liver is the body's most important metabolic organ. Various substances are metabolized directly or indirectly through the liver. Therefore, liver cells express various metabolic enzymes to perform metabolic functions. Once a certain (or some) enzyme gene mutations affect the expression of the enzyme or its biological function, the upstream metabolites cannot be metabolized in time or important downstream metabolites cannot be supplemented, resulting in irreversible metabolic disorders , Leading to the occurrence of disease.

In addition, the level of cholesterol is often an important indicator for evaluating cardiovascular disease and familial hypercholesterolemia. The increase of cholesterol concentration in the blood or accumulation in blood vessels will lead to the occurrence of related cardiovascular diseases. The body's metabolism of cholesterol is carried out by the liver. Therefore, the lack of or reduction in the ability of the liver to metabolize cholesterol will lead to a high concentration of cholesterol in the blood, leading to the occurrence of diseases.

The research of the present invention shows that elevated blood cholesterol is an important factor in the occurrence of cardiovascular disease, and the present invention found that reducing cholesterol content through drug therapy or gene therapy is the main strategy for the treatment of cardiovascular disease and familial hypercholesterolemia .

beta thalassemia

Beta thalassemia is a blood genetic disease caused by mutations in the beta globin gene. When the beta globin gene is mutated and the beta strain protein cannot be expressed or function is lost, it will lead to premature apoptosis of red blood cell precursors and fail to form . At present, gene editing strategies can be used to reactivate the expression of gamma globin to compensate for the lack of beta globin. The production of gamma globin can also form a heterotetramer with alpha globin, that is, fetal hemoglobin HBF, which helps to treat beta. Thalassemia. However, gamma globin is mainly expressed in infancy, and then it is inhibited by various transcriptional regulators and cannot be expressed. At present, there are more and more discoveries that certain point mutations can reactivate gamma globin expression.

The main advantages of the present invention include:

(1) The present invention finds for the first time that the fusion protein of the present invention can significantly improve gene editing efficiency in vivo or in vitro.

(2) The present invention finds for the first time that the fusion protein of the present invention can significantly improve gene editing efficiency in vivo or in vitro by ≥20%, preferably,>40%, more preferably,>60% (such as 80%) , Up to 2 times.

(3) The present invention is the first to discover the mRNA of an in vitro transcription-enhanced gene editing tool, which improves the success of animal model construction.

(4) The present invention uses the AAV virus packaging the enhanced gene editing tool to express the protein of the enhanced gene editing tool to improve the effect of disease treatment.

(5) For the first time, the present invention utilizes fusion of double-stranded DNA binding domains to improve gene editing efficiency.

(6) The present invention screens for the first time to find a double-stranded DNA binding domain (such as HMG-D) that efficiently improves gene editing efficiency, and its excellent fusion method.

(7) The present invention finds for the first time that the double-stranded DNA binding domain of the present invention can broadly improve the gene editing efficiency of various gene editing tools.

(8) The enhanced gene editing tool of the present invention can improve the success rate of animal model construction and the efficiency of gene therapy.

(9) The enhanced gene editing tool of the present invention can be used for gene therapy.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

General method

Method 1: (Applicable to Example 1, Example 2, Example 3, Example 4 and Example 6)

1. The density of HEK293T cells in a 24-well plate grows to 60-70%, an equimolar amount of plasmid is transfected into the cells by the transfection reagent PEI, the medium is changed for 8-10 hours, and the cells are cultured for a period of time (Cas9 system culture for 72 hours, Culture the base editing system for 120 hours), harvest the cells and extract the genome.

2. Design suitable primers to amplify the sequence of 150-180bp around the target, carry out Hitom library construction, deep sequencing, analysis and calculation of editing efficiency.

Method 2: (Applicable to the construction of the animal model in Example 7)

1. In vitro transcription of mRNA of enhanced gene editing tools and sgRNA of corresponding target.

2. Mouse embryos were injected with mRNA and sgRNA and transplanted to surrogate mother mice to obtain F0 generation mice, identify genes, count mouse genotype mutation rates, and calculate model construction success rates.

Method 3: (Applicable to Example 5)

1. The density of HEK293T cells in a 24-well plate grows to 60-70%, an equimolar amount of plasmid is transfected into the cells by the transfection reagent PEI, the medium is changed for 8-10 hours, and the cells are cultured for a period of time (Cas9 system culture for 72 hours, Culture the base editing system for 120 hours), harvest cells, extract RNA, and reverse transcription to obtain cDNA.

2. Design appropriate primers to perform RT-qPCR for relative quantitative analysis, and compare the efficiency of target gene transcription activation.

Method 4: (Applicable to the in vivo gene therapy in Example 8)

1. Package the adeno-associated virus (AAV) of enhanced gene editing tools and sgRNA of therapeutic genes, or homologous repair templates.

2. Through intravenous injection (tail vein) or local injection (muscle, etc.) the packaged AAV to the disease animal model (mouse or rat), while injecting the control virus.

3. Regularly observe and test the phenotypes of the animal models of the treatment group and the control group to evaluate the treatment efficiency.

Method 5: (Applicable to the cell gene therapy of Example 9)

1. In vitro expression of enhanced gene editing tool proteins and synthesis of sgRNA for related targets.

2. Electrotransfer protein and RNA complex (RNP) into the cell.

3. Genotype testing and functional testing.

Example 1 Screening of enhanced gene editing tools

Synthesize different kinds of double-stranded DNA binding domains (HMG-D and Sac7d) respectively, by designing five different length linkers (L1, L2, L3, L4, and L5) (Table 1), and fused to the N-terminal of Cas9 Compare the two endogenous targets (VEGF and HBG1/2) with the C-terminal, and score the enhanced gene editing tools obtained by this series of optimizations based on the result statistics (the effect is very good. Based on the relative scores made) (bad: A; poor: AA; good: AAA; good: AAAA; very good: AAAAA), the results are as follows: (Note: C means Cas9; L1-L5 means different linkers ; H represents HMG-D domain; S represents sac7d.)

A: S-L1-C, H-L1-C

AA: S-L2-C, S-L3-C, S-L4-C, H-L2-C

AAA: H-L4-C-L4-S, H-L4-C-L5-S

AAAA: H-L3-C, C-L4-S, C-L5-S, C-L4-H, H-L4-C-L4-H, H-L4-C-L5-H, H-L4- H-L4-C, C-L5-H-L5-H

AAAAA: H-L4-C, C-L5-H

Comprehensive statistics show that the effect of HMG-D is very good, and the linker of L4 and L5 length (32 and 64 amino acids) is the optimal linker. By comparing the N-terminal and C-terminal connections, it is found that under the optimal linker conditions, The N-terminal and C-terminal connection effects are both very good. Therefore, the present inventors have obtained an enhanced gene editing tool, that is, HMG-D is fused to the N-terminal or C-terminal of Cas9 through L4 or L5 linker, which is a very good fusion method. (figure 1).

In the same way, this enhanced gene editing tool that enhances gene editing efficiency through the fusion of double-stranded DNA binding domains can also be compared to other types of double-stranded DNA binding domains, such as the widely used zinc finger protein (ZFP) and other DNA binding domains of transcription factors and HMG-D or Sac7d from other species, etc. The present invention can also improve gene editing efficiency through these double-stranded DNA binding domains. Therefore, the most important thing of the present invention is the discovery that a double-stranded DNA binding domain fusion gene editing tool can improve gene editing efficiency. DNA binding domain HMG-D.

Comparative example 1 does not work well after fusion with non-DNA binding domain

In order to further verify that the fusion of the DNA binding domain can improve the efficiency of gene editing, the DNA binding domain was changed to GFP protein (a non-DNA binding domain), an unrelated protein, and it was found that the fusion of GFP protein could not improve the efficiency of gene editing (figure 1). At the same time, the HMG-D domain was mutated to destroy its DNA binding ability, and a 3-amino acid mutant HMG-D domain (mutHMG-D, referred to as mutH) was constructed. Through experimental comparison, the mutHMG-D domain is also Can not improve gene editing efficiency (Figure 1).

Comparative Example 2 DNA binding domains other than HMG-D and Sac7d are not effective

In order to broaden the range of DNA binding domains, some single-stranded DNA binding domains (such as Rad51) were tested. The fusion of Rad51 also did not improve gene editing efficiency (Figure 1). Therefore, the results indicate that the double-stranded DNA binding domains of HMG-D and Sac7d screened in the present invention are very effective.

Example 2 Improve the gene editing efficiency of SpCas9

The obtained enhanced gene editing tool (ie the fusion protein of the present invention, such as HMG-D-L4-SpCas9) is further compared in terms of effects on more endogenous targets, and 293T cells are transfected at an equimolar ratio It was found that compared with SpCas9, the editing efficiency of the fusion protein of the present invention was improved by more than 20% (or 60%, or 80%) in the compared targets, up to 2 times (Figure 2).

Example 3 Improve the gene editing efficiency of Cas9 from other species (such as SaCas9)

By constructing the HMG-D-L4-SaCas9 expression vector, aiming at the endogenous target, transfecting 293T cells at an equimolar ratio, it was found that the editing efficiency of SaCas9 after HMG-D fusion also improved similarly. (image 3)

Example 4 Improve the gene editing efficiency of other non-Cas9 proteins (such as AsCas12a)

By constructing the HMG-D-L4-AsCas12a expression vector, aiming at the endogenous target, transfecting 293T cells at an equimolar ratio, it was found that the editing efficiency of AsCas12a after fusion with HMG-D has a similar effect. (Figure 4)

Example 5 Improve the transcriptional activation efficiency of transcriptional regulatory tools (CRISPR/Cas9)

By constructing the HMG-D-L4-dCas9-VPR expression vector, aiming at the endogenous target and transfecting 293T cells at an equimolar ratio, it was found that dCas9-VPR has similar transcriptional activation efficiency for endogenous genes after fusion of HMG-D The improvement of the effect. (Figure 5)

Example 6 Improving the editing efficiency of a base editor (base editor)

Since the base editor (base editor) fused cytosine deaminase (CBE) and adenine deaminase (ABE) at the N-terminal of Cas9, the C-terminal of Cas9 of the base editor passed L5linker To fuse HMG-D and develop an enhanced single-base editing tool. Through the comparison of endogenous targets, the editing efficiency of base editors (such as ABE) after fusion of HMG-D has been greatly improved, such as by >1.5-2 times (Figure 6).

The editing efficiency of the fusion protein fused with other types of base editors is similar or equivalent to that of the fusion protein fused with ABE.

Example 7 Improve the success rate of animal model construction

Through in vitro transcription of mRNA of enhanced gene editing tools, the success rate of animal model construction is increased.

Example 8 By delivering an enhanced gene editing tool fused with double-strand binding protein (HMG-D) to animals, the efficiency of Cas9 editing in vivo can be increased, and the efficiency of gene therapy can be improved

Take the gene therapy method of reducing blood cholesterol levels in a mouse model as an example.

method:

1. Package the adeno-associated virus of the AAV8 serotype of SpCas9 fused with HMG-D, and the sgRNA targeting the liver gene PCSK9, and use ultracentrifugation for virus purification, concentration, and qPCR titer determination; 2, 6-8 Mice of a suitable age were injected with a mixture of two viruses through the tail vein, and a control group was injected with a virus mixture of wild-type SpCas9 and sgRNA at the same time; 3. Surgery was performed 15 and 35 days after virus injection, and liver tissue was anesthetized. Methods Extract the genome and compare the gene editing efficiency by second-generation sequencing; 4. Start 7 days after injection, take blood every 7 days, and use enzyme labeling method to detect blood total cholesterol content; 5. Finally, compare wild-type gene editing tools uniformly And the efficiency of enhanced gene editing tools and disease treatment effects.

The inventors used adeno-associated virus (AAV) to deliver SpCas9 fused with HMG-D and sgRNA targeting the treatment target of hypercholesterolemia (PCSK9) to the liver of mice, and successfully targeted the mouse PCSK9 gene to be knocked out. The removal efficiency was significantly higher than that of the SpCas9 group without HMG-D (Figure 7A). Through long-term monitoring of the blood cholesterol content of the treated mice, it was found that the blood cholesterol content of the treated mice was significantly lower than that of the untreated group, and the HMG-D fusion treatment group was significantly lower than the treatment group without HMG-D fusion (Figure 7B).

The results show that SpCas9 fused with double-stranded binding protein can significantly help reduce blood cholesterol, which is beneficial to the treatment of cardiovascular diseases and familial hypercholesterolemia. At the same time, the delivery of enhanced gene editing tools through AAV significantly enhances editing efficiency in vivo, which also provides conditions for the treatment of more diseases.

Example 9 The enhanced gene editing tool of fusion double-stranded binding protein (HMG-D) by electroporation into therapeutic cells can improve the editing efficiency of therapeutic cells in vitro, which is beneficial to the treatment of diseases

Take the hematopoietic stem cell treatment of β thalassemia as an example.

The inventors used base editing tools to generate therapeutic point mutations on hematopoietic stem cells to activate the expression of gamma globin and treat beta thalassemia. By expressing the fusion double-stranded binding protein (HMG-D) base editing tool (hA3A-CBE) protein, and sgRNA electrotransformed into hematopoietic stem cells to achieve C to T mutations at positions -114 and -115.

method:

1. Prokaryotic expression and purification of hA3A-CBE protein fused with HMG-D; 2. Synthesis of target gene sgRNA; 3. Hematopoietic stem cells are isolated, cultured, and incubated with protein and RNA to form RNP and transferred to hematopoietic stem cells; 4. Electrotransfer 72 After hours, some cells were collected for genome extraction, PCR, sequencing, analysis and comparison of target gene mutation efficiency; 5. The remaining hematopoietic stem cells were differentiated into red blood cells by conventional methods, and the expression level of gamma globin in red blood cells was detected by RT-qPCR 6. Separate the protein in red blood cells and perform chromatography to qualitatively and quantify the expression of gamma globin; 7. Overall comparison of the therapeutic effects of enhanced editing tools on beta thalassemia.

The results showed that the editing efficiency of the hA3A-CBE group fused with HMG-D was significantly higher than that of the hA3A-CBE group without HMG-D (Figure 8A); at the RNA level, the gamma globin in the hA3A-CBE group fused with HMG-D The expression of hA3A-CBE group fused with HMG-D was also significantly higher than that of hA3A-CBE group without HMG-D (Fig. 8C). Therefore, the strategy of electroporation-enhanced gene editing tool proteins can greatly improve the editing efficiency of cells, which is of great significance for various cell therapies (HSC, CART, etc.) in the future.

discuss

In general, the present invention is based on the pre-clinical research and development of gene therapy for related diseases based on enhanced gene editing tools. It is implemented through two of the most important gene therapy strategies in the field of gene therapy, namely: in vivo therapy by delivering gene therapy tools in vivo and in vitro therapy in which patient cells are isolated for gene therapy and then transplanted into the patient.

First of all, most liver metabolic diseases can be treated by virus-targeted delivery of gene therapy drugs. The present invention takes cholesterolemia caused by cholesterol metabolism disorders as an example. The delivery of enhanced gene editing tools through AAV can greatly reduce blood levels in the body. Cholesterol content is not only beneficial for the treatment of cholesterolemia, but also has a great guiding effect on most liver metabolic diseases, and can be extended to general genetic diseases.

Secondly, the example of the use of hematopoietic stem cells in the treatment of β-thalassemia of the present invention proves that the enhanced gene editing tool of the present invention can greatly improve the gene editing efficiency of hematopoietic stem cells and improve the effect of β-thalassemia treatment. Of course, This can also be extended to more cell therapy, such as immune cells (T cells) for tumor therapy applications, mesenchymal stem cells, etc.

Finally, through the study of two representative cases of in vivo therapy and in vitro therapy, it can be seen that the enhanced gene editing tool of the present invention is superior to wild-type gene editing tools, and shows great prospects in gene therapy of diseases. In addition, the enhanced gene editing tool of the present invention is not limited to these two diseases, and includes all diseases that can be treated by wild-type gene editing tools.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

A use of an active substance, characterized in that it is used to prepare drugs or preparations for gene therapy, wherein the active substance is a fusion protein, or its coding gene, or its expression vector, wherein the structure of the fusion protein is as follows Formula I or I'as shown: C-A-L-B (I) B-L-A-C (I’) Where A is the gene editing protein, B is the DNA double-strand binding domain, C is an optional base editor element; L is no or connecting peptide, Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond. The use according to claim 1, wherein the medicine is used to treat diseases that can be treated by gene therapy. The use according to claim 1, wherein the gene editing protein is selected from the group consisting of Cas9, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof. The use according to claim 1, wherein the gene editing protein is selected from one or more sources of the following group: Streptococcus pyogenes, Staphylococcus aureus, Aminococcus ( Acidaminococcus sp), Lachnospiraceae bacterium (Lachnospiraceae bacterium). The use according to claim 1, wherein the DNA double-strand binding domain is a non-sequence-specific DNA double-strand binding domain. The use according to claim 1, wherein the DNA double-strand binding domain is derived from Drosophila or Archaea. The use according to claim 1, wherein the base editor element comprises cytosine deaminase and adenine deaminase. The use according to claim 1, wherein the gene therapy is performed by gene editing. The use according to claim 1, wherein the gene therapy is performed by gene editing reagents. The use according to claim 9, wherein the gene editing reagent comprises a fusion protein, and the structure of the fusion protein is shown in the following formula I or I': C-A-L-B (I) B-L-A-C (I’) Where A is the gene editing protein, B is the DNA double-strand binding domain, C is an optional base editor element; L is no or connecting peptide, Each "-" is independently a connecting peptide or a peptide bond or a non-peptide bond.

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