CN117959464A - Gene editor and anti-fibrosis inhibitor nucleic acid pharmaceutical compositions for the treatment of disease - Google Patents

Abstract

The invention provides a nucleic acid pharmaceutical composition of a gene editor and an anti-fibrosis inhibitor for use in the treatment of diseases. Specifically, the pharmaceutical composition comprises: (a) A first active ingredient which is a gene editor for base editing of a target gene; and (b) a second active ingredient, the second active ingredient being an anti-fibrotic inhibitor. The invention treats hereditary diseases by the nucleic acid sequence encoding the gene editor and the anti-fibrosis inhibitor through a virus or non-virus vector system, and can repair genes and inhibit fibrosis signals in microenvironment at the same time, thereby achieving the effect of cooperative treatment.

Description

Gene editor and anti-fibrosis inhibitor nucleic acid pharmaceutical compositions for the treatment of disease

Technical Field

The invention relates to the field of biotechnology, in particular to a nucleic acid pharmaceutical composition of a gene editor and an anti-fibrosis inhibitor for disease treatment.

Background

Duchenne muscular dystrophy (Duchene Muscular Dystrophy, DMD) is a group of X-linked recessive genetic rare diseases that result from mutations in dystrophin (dystrophin) and severe progressive muscle decline. It mainly involves skeletal and cardiac muscles, which decay in early childhood and progress rapidly. Typical clinical symptoms of DMD patients are muscle weakness with gastrocnemius pseudohypertrophy, abnormal gait after three years of age, loss of ability to jump, difficulty in going upstairs, difficulty in squatting, and obvious Gowers sign positive behavioral characteristics (supporting the thigh by hand while rising from the sitting position, shaking the thigh while walking, or only standing on the tip of the foot while walking, and stepping on the stairs while stepping on two steps). Symptoms of DMD patients generally remain relatively stable by age 7, with disease progression accelerated by age 12, losing mobility and requiring assistance from wheelchairs; following scoliosis, loss of upper limb function occurs with varying degrees of heart and respiratory failure in nearly all patients between 20 and 30 years of age, with an average life span of 26 years (Mercuri et al, 2019).

Currently, there is no cure for DMD disease, and corticosteroids are commonly used to delay DMD progression, but these hormonal drugs have insignificant therapeutic effects and large side effects. Existing marketed drugs are mainly those for DMD treatment by antisense oligonucleotide (ANTISENSE OLIGONUCLEOTIDES, ASO) mediated exon skipping, where about 69% of patients have large fragment deletions, 11% have large fragment duplications, 10% have nonsense mutations, 7% have missense mutations or small deletions, and 3% have mutations in introns or other regions. These mutations result in changes in the reading frame of the mRNA during translation, ultimately resulting in a Dystrophin protein deletion. ASO drugs can specifically bind to key sites on dystrophin gene pre-mRNA, preventing the exon from participating in the splicing process by inhibiting the transcription enhancer site, so that the mature mRNA transcript does not contain the segment of exon, and realizing exon skipping (Mann et al, 2001). While transcripts with exon skipping can encode a stable protein truncated to that of the normal Dystrophin protein due to the restoration of the reading frame, thus achieving functional restoration of Dystrophin protein (Aartsma-Rus et al 2003;Monaco et al, 1988).

ASO is a chemically modified single-stranded nucleotide polymer, usually 18-25 bases in length, that, after injection into a patient's body, it is required to pass through blood, biological barriers (e.g., blood brain barrier, vascular endothelial) and be endocytosed by cells before it enters target cells, and that, in cells, it is required to ensure that extracellular secretion is not achieved by lysosome degradation and secretion vesicles. Furthermore, the uptake capacity of ASO by different cell types is very different, so that the bioavailability of ASO in the target cell is low, greatly preventing the effect of ASO. From the data that have been published so far, the recovery level of Dystrophin protein is very low after clinical use of ASO drugs. Such as Viltolarsen (clinical three.gov Identifier: NCT 04337112), which recovered Dystrophin protein only 5.9% (CLEMENS ET al., 2020) in the high dose group (80 mg/kg/week) after 20 weeks of treatment, and in its clinical study 201, the side effects of elevated blood creatinine, elevated blood potassium, elevated blood urea nitrogen, etc. were reported in the high dose group of patients after receiving the drug.

The current gene editing treatment for DMD can achieve exon excision or jump and recover reading frame by editing the DMD gene, thereby expressing a truncated functional protein, reducing disease symptoms, improving life quality of patients and prolonging life of DMD patients. Thus, truncated, functional proteins are constantly produced by editing successful cells, with the potential for lifelong healing of one-needle therapy. The existing gene editing tools including TALEN, cas9, base editors and the like are widely used in the development of medicines related to monogenic genetic diseases.

However, whether ASO drugs are already on the market or the results of clinical trials to restore micro-dystrophin by alternative therapy strategies, it is not possible to achieve satisfactory clinical results for DMD patients with only restoration of Dystrophin protein expression. Although the base editor can restore dystrophin protein expression after editing in vitro and in vivo, there are different reports of functional restoration. This also suggests that gene mutation is merely responsible for DMD, and that progression of the disease is caused for a variety of reasons. In addition to the gene mutation itself being the initiating cause of the triggering disease, the development of the disease is often accompanied by a change in microenvironment, which also contributes to the disease progression. Therefore, when a disease is treated, simple gene editing treatment can repair gene mutation, and cannot well block the progress of the disease. Thus, while gene editing therapy, if it is possible to carry some therapeutic drugs, it is possible to provide a better treatment for diseases by controlling the microenvironment.

Disclosure of Invention

The object of the present invention is to provide a nucleic acid pharmaceutical composition for the treatment of a disease, and the use of said pharmaceutical composition in the treatment of a disease (e.g. DMD).

In a first aspect of the present invention there is provided a pharmaceutical composition for use in the treatment of a disease, the pharmaceutical composition comprising:

(a) A first active ingredient which is a gene editor for base editing of a target gene; and

(B) A second active ingredient which is an anti-fibrotic inhibitor.

In another preferred embodiment, the disease includes (but is not limited to): duchenne Muscular Dystrophy (DMD), idiopathic Pulmonary Fibrosis (IPF), amyotrophic Lateral Sclerosis (ALS).

In another preferred embodiment, the disease is Duchenne Muscular Dystrophy (DMD).

In another preferred embodiment, the gene editor is selected from the group consisting of: ABE, CBE, CRISPR/Cas9, or a combination thereof.

In another preferred embodiment, the gene editor is an ABE or CBE.

In another preferred embodiment, the target gene is a gene associated with the occurrence/progression of the disease.

In another preferred embodiment, the target gene is a gene encoding a dystrophin (dystrophin).

In another preferred embodiment, the anti-fibrosis inhibitor comprises an antibody, a cytokine, an RNA-based drug, or a combination thereof.

In another preferred embodiment, the anti-fibrosis inhibitor is selected from the group consisting of: anti-connective tissue growth factor (connective tissue growth factor, CTGF) antibodies, recombinant Interleukin 1receptor antagonist (Interleukin 1receptor antagonist,IL-1 ra), anti-IL-1 beta receptor antibodies, or combinations thereof.

In another preferred embodiment, the anti-fibrosis inhibitor is an anti-CTGF antibody.

In another preferred example, the heavy chain variable region of the anti-CTGF antibody X0304 comprises the following complementarity determining regions CDRs:

H-CDR1 as shown in SEQ ID NO. 1;

H-CDR2 as shown in SEQ ID NO. 2; and

H-CDR3 as shown in SEQ ID NO. 3.

In another preferred example, the light chain variable region of the anti-CTGF antibody comprises the following complementarity determining region CDRs:

L-CDR1 as shown in SEQ ID NO. 4;

L-CDR2 as shown in SEQ ID NO. 5 (AAS); and

L-CDR3 as shown in SEQ ID NO. 6.

In another preferred embodiment, the anti-CTGF antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 7 and a light chain variable region having the amino acid sequence shown in SEQ ID NO. 8.

In another preferred embodiment, the anti-CTGF antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9 and a light chain variable region having the amino acid sequence shown in SEQ ID NO. 10.

In another preferred embodiment, the anti-CTGF antibody comprises: single chain antibodies (scFv), double chain antibodies (bivalent antibody fragment variables, baFv), diabodies, monoclonal antibodies, chimeric antibodies (e.g., human murine chimeric antibodies), murine antibodies, or humanized antibodies.

In another preferred embodiment, the anti-CTGF antibody is a single chain antibody, the amino acid sequence of which is shown in SEQ ID NO. 11 or 12.

In another preferred embodiment, the anti-fibrosis inhibitor is a recombinant interleukin 1 receptor antagonist (IL-1 Rα) having the amino acid sequence shown in SEQ ID NO. 13.

In another preferred embodiment, the anti-fibrosis inhibition is an anti-IL-1. Beta. Receptor antibody (CANAKINUMAB) having the heavy chain variable region and light chain variable region amino acid sequences shown in SEQ ID NOS: 14-15.

In another preferred embodiment, the pharmaceutical composition comprises a first vector and a second vector for producing the first active ingredient and the second active ingredient, wherein the first vector comprises a first expression cassette and the second vector comprises a second expression cassette,

Wherein the first expression cassette has the structure of formula I of 5'-3' (5 'to 3'):

P1-X1-L1-X2-X3(I)

The second expression cassette has the structure of formula II 5'-3' (5 'to 3'):

P2-X4-L2-P3-X5-X3(II)

Wherein P1, P2 and P3 are a first promoter sequence, a second promoter sequence and a third promoter sequence respectively, the first promoter and the third promoter are the same type of promoter, and the second promoter is an RNApol III promoter;

X1 is the coding sequence of adenine deaminase or cytosine deaminase;

L1 and L2 are each independently absent or a linking sequence;

X2 is the coding sequence of a partially inactivated Cas9 nuclease (nCas), said Cas9 nuclease having nickase activity;

x3 is PolyA sequence;

X4 is the coding sequence of sgRNA;

X5 is the coding sequence for an anti-fibrotic inhibitor;

and each "-" is independently a bond or a nucleotide linking sequence.

In another preferred embodiment, the first vector and the second vector are both expression vectors selected from the group consisting of: adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, or retroviral vectors.

In another preferred embodiment, the first vector and the second vector are both adeno-associated viral vectors.

In another preferred embodiment, the first promoter and the third promoter are both tissue specific promoters or constitutive promoters.

In another preferred embodiment, the first promoter and the third promoter are both muscle-specific promoters, e.g., syn100, with the nucleotide sequence shown in SEQ ID NO. 16.

In another preferred embodiment, the anti-fibrosis inhibitor is an anti-CTGF antibody, and the anti-fibrosis inhibitor has a coding sequence as set forth in SEQ ID NO. 17, 18, 19 or 20.

In another preferred embodiment, the anti-fibrosis inhibition is a recombinant interleukin 1 receptor antagonist (IL-1α) having the amino acid sequence shown in SEQ ID NO. 13.

In another preferred embodiment, the anti-fibrosis inhibition is an anti-IL-1. Beta. Receptor antibody (CANAKINUMAB) having the heavy chain variable region and light chain variable region amino acid sequences shown in SEQ ID NOS: 14-15.

In another preferred embodiment, the coding sequence of the sgRNA is shown in SEQ ID NO. 21 or 22.

In another preferred embodiment, the first expression cassette comprises the sequence set forth in SEQ ID NO. 23.

In another preferred embodiment, the second expression cassette comprises a sequence selected from the group consisting of any one of SEQ ID NOs 24, 25, 26 or 27.

In another preferred embodiment, the pharmaceutical composition further comprises: (c) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation, preferably an injection.

In a second aspect of the invention, there is provided a kit comprising:

(c1) A first container, and a first vector in the first container, the first vector comprising a first expression cassette; and

(C2) A second container, and a second vector in the second container, the second vector comprising a second expression cassette;

wherein the first expression cassette has the structure of formula I of 5'-3' (5 'to 3'):

P1-X1-L1-X2-X3(I)

The second expression cassette has the structure of formula II 5'-3' (5 'to 3'):

P2-X4-L2-P3-X5-X3(II)

Wherein P1, P2 and P3 are a first promoter sequence, a second promoter sequence and a third promoter sequence respectively, the first promoter and the third promoter are the same type of promoter, and the second promoter is an RNApol III promoter;

X1 is the coding sequence of adenine deaminase or cytosine deaminase;

L1 and L2 are each independently absent or a linking sequence;

X2 is the coding sequence of a partially inactivated Cas9 nuclease (nCas), said Cas9 nuclease having nickase activity;

x3 is PolyA sequence;

X4 is the coding sequence of sgRNA;

X5 is the coding sequence for an anti-fibrotic inhibitor;

and each "-" is independently a bond or a nucleotide linking sequence.

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

In another preferred embodiment, the kit further comprises instructions describing: a method of simultaneously administering a first vector and a second vector to a subject in need thereof, thereby performing gene editing and anti-fibrotic therapy in said subject.

In a third aspect of the invention, there is provided an anti-CTGF antibody comprising a heavy chain variable region comprising the following complementarity determining region CDRs:

H-CDR1 as shown in SEQ ID NO. 1;

H-CDR2 as shown in SEQ ID NO. 2; and

H-CDR3 as shown in SEQ ID NO. 3.

In another preferred embodiment, the anti-CTGF antibody further comprises a light chain variable region comprising the following complementarity determining region CDRs:

L-CDR1 as shown in SEQ ID NO. 4;

L-CDR2 as shown in SEQ ID NO. 5 (AAS); and

L-CDR3 as shown in SEQ ID NO. 6.

In another preferred embodiment, the anti-CTGF antibody comprises a heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 7 and/or a light chain variable region having an amino acid sequence shown as SEQ ID NO. 8.

In another preferred embodiment, the anti-CTGF antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 7 and a light chain variable region having the amino acid sequence shown in SEQ ID NO. 8.

In another preferred embodiment, the anti-CTGF antibody comprises: single chain antibodies (scFv), double chain antibodies (bivalent antibody fragment variables, baFv), diabodies, monoclonal antibodies, chimeric antibodies (e.g., human murine chimeric antibodies), murine antibodies, or humanized antibodies.

In another preferred embodiment, the anti-CTGF antibody is a single chain antibody, the amino acid sequence of which is shown in SEQ ID NO. 11.

In another preferred embodiment, the anti-CTGF antibody is a double-chain antibody, the amino acid sequence of which is shown in SEQ ID NO. 19.

In a fourth aspect, the present invention provides the use of a pharmaceutical composition according to the first aspect of the invention in the manufacture of a medicament for the treatment of a disease.

In another preferred embodiment, the disease includes (but is not limited to): duchenne Muscular Dystrophy (DMD), idiopathic Pulmonary Fibrosis (IPF), amyotrophic Lateral Sclerosis (ALS).

In another preferred embodiment, the disease is Duchenne Muscular Dystrophy (DMD).

In a fifth aspect of the invention, there is provided a method of treating a disease comprising the steps of: administering to a subject in need thereof a pharmaceutical composition of the first aspect of the invention.

In another preferred embodiment, the disease includes (but is not limited to): duchenne Muscular Dystrophy (DMD), idiopathic Pulmonary Fibrosis (IPF), amyotrophic Lateral Sclerosis (ALS).

In another preferred embodiment, the disease is Duchenne Muscular Dystrophy (DMD).

In another preferred embodiment, the subject in need thereof comprises a human or non-human mammal.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows phage titers eluted per round of protein A four rounds of screening.

FIG. 2 shows the detection results of X3-4 monoclonal phage Monophage ELISA.

FIG. 3 shows the results of activity assays for anti-CTGF scFv.

Figure 4 shows the results of cross-reaction of X0304 and X3019 with human/mouse.

FIG. 5 shows mCTGF and mColla mRNA expression in a model of TGF- β induced in vitro fibrosis in mouse C2C12 cells; the abscissa in the figure represents whether TGF-beta and FG3019 are added or not and their concentrations, and the ordinate represents the relative expression amount of mRNA of mCTGF or mCOl a 1.

FIG. 6 shows that anti-CTGF scFv expression blocks transcription of the associated fibrosis factor mColla in a TGF-beta induced in vitro fibrosis model in mouse C2C12 cells; the abscissa indicates the presence or absence of TGF-. Beta.stimulation and the addition of the X0304 plasmid (Syn 100-X0304) or the X3019 plasmid (Syn 100-X3019), and the ordinate indicates the relative expression of mRNA of mCol a 1.

FIG. 7 shows the sequence alignment of X0304 and X3019.

FIG. 8 shows a schematic diagram of GOI sequence structure;

Figure 9 shows a 16 week tensile test following AAV administration; the abscissa in the figure represents the number of times corresponding to the 10-cycle tension, and the ordinate represents the percentage of the tension value with respect to the corresponding number of times to the first tension value.

FIG. 10 shows exon4 jump analysis of dystrophin genes in mouse hearts 16 weeks after AAV treatment; as shown in the left graph, the upper text represents the group represented by each lane, the leftmost lane represents the size of the DNA electrophoresis marker, the right arrow represents the positions of the E3-E4-E5 bands and the E3-E5 bands, and the lower number represents the ratio of each lane E3-E5 to E3-E4-E5; as shown in the right graph, the abscissa represents each group, and the ordinate represents the average value of the ratio of each group E3-E5 to E3-E4-E5.

Figure 11 shows WB plots of Dystrophin protein expression in mouse hearts 16 weeks after AAV treatment.

FIG. 12 shows immunofluorescence staining of Dystrophin protein expression in mouse hearts 16 weeks after AAV treatment; the left text in the figure represents the group to which each picture belongs, and the right picture represents the immunofluorescence picture of Dystrophin in the skeletal muscle to which each group corresponds.

FIG. 13 shows immunofluorescence staining of Col 1a protein expression in mouse hearts 16 weeks after AAV treatment; the left text in the figure represents the group to which each picture belongs, and the right picture represents the immunofluorescence picture of Col 1a in the skeletal muscle to which each group corresponds.

FIG. 14 shows a schematic diagram of the gene editor and anti-fibrosis co-therapy of the invention.

Detailed Description

The invention treats hereditary diseases through the nucleic acid sequences encoding the gene editing tool and the antibody activity by a virus or non-virus vector system, and can repair genes and inhibit fibrosis signals in microenvironment, thereby solving the problem that can not be solved by singly using gene editing products or antibody medicines for treatment. The present invention employs AAV vectors or mRNA forms, and combines gene editing tools and active molecule (including single chain antibody, baFv, or full length antibody) gene sequences against CTGF on the same nucleic acid vector, and then, the gene sequences are distributed to specific disease sites through the specificity of the vector system after intravenous or subcutaneous injection (as shown in FIG. 14). Under the drive of a tissue specific promoter, gene editing tools and antibody active molecules against CTGF were expressed. The gene editing tool edits the target genes, and meanwhile, active molecules of the antibody can be released into the microenvironment, so that the anti-fibrosis effect is achieved by neutralizing CTGF, and the muscle cells after being edited are more beneficial to survival because fibrosis is inhibited, and the editing effect of the gene editing tool is enhanced.

On this basis, the present invention has been completed.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

Connective Tissue Growth Factor (CTGF)

Connective tissue growth factor (connective tissue growth factor, CTGF) is widely recognized as a factor that promotes fibrosis, mainly produced by the secretion of fibroblasts. When the body is damaged locally, the fibroblasts secrete CTGF, which promotes local fibroblast proliferation, or muscle stem cells differentiate towards fibroblasts.

CTGF, a 38KDa size, extracellular matrix protein characterized by cysteine enrichment (22 cysteines at the N-terminus and 16 cysteines at the C-terminus), belongs to the CCN superfamily. It shares four domains, domain 1 being homologous to IGF1 binding protein, domain 2 being homologous to vWC (von Willebrand factor TYPE C REPEAT), domain 3 being homologous to thrombospondin type 1 repeats, domain 4 comprising a cysteine knot motif. Domains 1 and 2 at the N-terminus are linked to domains 3 and 4 at the C-terminus by a polypeptide homologous to any protein (z.chen et al 2020).

Expression of CTGF can be induced by the transforming growth factor (Transforming Growth Factor beta, tgfβ) superfamily, including tgfβ -1, -2,3 and vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) (Franklin, 1997), tgfβ induction with CTGF is achieved primarily by DNA regulatory elements on the CTGF promoter (Grotendorst et al, 1996).

CTGF expression stably increases the expression of collagen (collagen), integrin (integrin) and fibronectin (fibronectin) mRNA (Frazier et al, 1996; shi-wen et al, 2000). Thus, subcutaneous injection of CTGF in neonatal mice can result in the formation of localized granulomatous tissue, whereas combined or sequential injection of tgfβ and CTGF can result in the formation of stable granulomas (Mori et al, 1999). In addition, other molecules such as Insulin-like growth factor 1 (Insulin-like growth factor-1, IGF-1), bone morphogenic protein 4 (bone morphogenetic protein-4, BMP-4), bone morphogenic protein 4 (bone morphogenetic protein-7, BMP-7) can also interact with CTGF (Z.Chen et al, 2020).

Currently, CTGF is a protein that regulates cell-matrix interactions, as viewed in summary of its function, through five pathways: 1) CTGF inhibits downstream signaling (such as VEGF) by binding to cytokines and enhancing their downstream receptor response (increasing the downstream effects of TGF- β) or sequestering cytokines in mechanisms (Abreu et al, 2002; inoki et al, 2002); 2) CTGF can competitively bind to HSPG (heparan sulfate proteoglycan, heparan sulfate proteoglycans), reduce heparin-binding growth factors or antagonists thereof, and thereby alter downstream signaling pathways (Chen et al 2020); 3) CTGF can block or create new matrix binding sites, thereby altering the mechanisms signaling pathway, cell attachment, and motor function (Babic et al, 1999; chen et al, 2004; NISHIDA ET al, 2003); 4) CTGF can bind directly to cell surface receptors and stimulate downstream signal transduction (Babic et al, 1999; SEGARINI ET al, 2001; wahab et al, 2005); 5) CTGF can enter cells by endocytosis and regulate signaling to pathways in the cytoplasm or nucleus (WAHAB et al, 2001).

The muscle tissue of DMD patients had significant fibrosis compared to normal controls, high expression of CTGF mRNA could be detected (PESSINA ET al, 2014; sun et al, 2008), and the activity of CTGF in DMD model mice was proportional to the number of necrosis-regeneration nodules and fibrosis markers in the muscle tissue (Morales et al, 2018).

Idiopathic pulmonary fibrosis (Idiopathic pulmonary fibrosis, IPF) is a chronic, progressive pulmonary fibrotic disease characterized by extensive deposition and fibrosis of the extracellular matrix (Puglisi et al., 2016). In patients with IPF, the content of CTGF in plasma is significantly higher than in normal persons (Kono et al., 2011). In animal models of CTGF defects, deposition of extracellular matrix was significantly reduced (Liu et al, 2011). Whereas overexpression of CTGF and tgfβ significantly enhances collagen deposition and fibrosis in animal lung tissue (Sonnylal et al., 2010). High expression of mRNA and protein of CTGF was also found in spinal cord in both familial amyotrophic lateral sclerosis (famlial Amyotrophic lateral sclerosis, fALS) and sporadic amyotrophic lateral sclerosis patients (sporadic Amyotrophic lateral sclerosis, sALS) (Sun et al, 2008).

Monoclonal antibody FG-3019 to CTGF was effective in reducing muscle fibrosis and improving performance in exercise tolerance assays in MDX disease model mice (Maria Gabriela Morales et al., 2013), whereas inhibition of CTGF by FG-3019 was effective in promoting neuromuscular junction innervation and reducing myelin degeneration in SOD 1-deficient ALS disease model mice (Gonzalez et al., 2018). In clinical trials with IPF patients, patients receiving FG-3019 treatment were able to delay the progression of IPF by some amount, but at the same time 6% of patients were unable to continue to receive treatment due to serious side effects (RICHELDI ET al, 2020), while clinical trials with FG-3019 on DMD patients are also currently underway (clinical trims. Gov IDENTIFIER NCT 04371666).

However, the drugs can only change the microenvironment and can not eliminate the gene mutation which is the most initial cause of the final disease. In addition, antibodies are administered primarily by intravenous or subcutaneous injection, with only a portion of the drug reaching the fibrotic microenvironment. Thus, the dosage is high, and the high dosage also causes systemic side effects due to the wide expression of CTGF. If the CTGF-expressing antibodies can be controlled locally, the amount of the antibodies can be reduced, and the purpose of reducing toxicity can be achieved by regulating the local microenvironment.

Anti-fibrosis inhibitors

As used herein, the term "anti-fibrotic inhibitor" refers to a molecule that reduces the degree of fibrosis in a tissue and/or tissue cell microenvironment. The heavy chains of the "anti-fibrotic inhibitors" of the present invention include, but are not limited to, large molecules such as antibodies, cytokines, and small molecule compounds.

In one embodiment of the present invention, the anti-fibrosis inhibitor is an anti-CTGF antibody which binds to CTGF secreted by fibroblasts, thereby blocking the function of CTGF, reducing expression of molecules such as collagen, integrin, fibronectin, and the like, inhibiting local fibroblast proliferation, and thereby reducing the degree of fibrosis in the tissue microenvironment. In a preferred embodiment of the present invention, the anti-CTGF antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 7 and a light chain variable region shown in SEQ ID NO. 8. In another preferred embodiment of the present invention, the anti-CTGF antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9 and a light chain variable region shown in SEQ ID NO. 10.

In another embodiment of the present invention, the anti-fibrosis inhibitor is a recombinant Interleukin 1receptor antagonist (Intereukin 1receptor antagonist,IL-1Rα). In a preferred embodiment, the IL-1Rα has the amino acid sequence as set forth in SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

In yet another embodiment of the present invention, the anti-fibrosis inhibitor is an antibody to the IL-1. Beta. Receptor. In a preferred embodiment, the antibody has the amino acid sequence as set forth in SEQ ID NO:14, and a heavy chain variable region as set forth in SEQ ID NO:15, and a light chain variable region shown in seq id no.

Adeno-associated viral vectors

AAV is a common Gene drug delivery tool, since the load of AAV is typically 4.5Kb, and the size of GOI (Gene of Interest) is both too large and too small for viral production (Dong et al, 1996), the optimal packaging size is 4.2-4.5Kb. The genes of the enzymes of the gene editing tool are large, and the gene editing enzymes and sgrnas cannot be delivered in one vector. Thus, the gene editing enzyme will typically be loaded in one AAV vector, while the sgRNA is loaded in another AAV vector. Due to the size of the sgRNA gene in the range of 1-2Kb, efficient production is difficult (Dong et al, 1996)). Furthermore, when AAV is used to deliver some smaller-sized proteins, it is common to add non-expressed components as a filler to keep GOI size at 4.5kb for efficient production, as is common for bacterial or phage components, such as Lambda phage that is not expressed in eukaryotic cells by addition of stop codon mutations (Hirsch et al, 2009; parks et al, 1999), or genes that are used in conjunction with humans (Lee et al, 2019). Early studies found that stuffer non-specific transcription in AAV was potentially toxic (Keiser et al 2021), and the addition of a therapeutic gene was effective in reducing non-specific transcription to reduce toxicity. Therefore, selection of the appropriate stuffer to increase plasmid size while reducing stuffer toxicity is also a current concern for gene therapy.

Anti-CTGF antibodies

As used herein, the terms "anti-CTGF antibody of the present invention", "anti-connective tissue growth factor antibody of the present invention", "anti-CTGF antibody of the present invention" have the same meaning, and all refer to antibodies that specifically recognize and bind Connective Tissue Growth Factor (CTGF).

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are two types of light chains, λ (l) and κ (k). There are five major heavy chain species (or isotypes) that determine the functional activity of an antibody molecule: igM, igD, igG, igA and IgE. Each chain comprises a different sequence domain. The light chain comprises two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains, a heavy chain variable region (VH) and three constant regions (CH 1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity for an antigen. The constant domain of the light Chain (CL) and the constant region of the heavy Chain (CH) confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding and binding to Fc receptors (FcR). Fv fragments are the N-terminal part of immunoglobulin Fab fragments and consist of a variable part of one light chain and one heavy chain. The specificity of an antibody depends on the structural complementarity of the antibody binding site and the epitope. The antibody binding site consists of residues primarily from the highly variable region or Complementarity Determining Regions (CDRs). Occasionally, residues from non-highly variable or Framework Regions (FR) affect the overall domain structure and thereby the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that collectively define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, otherwise known as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR-H. Conventional antibody antigen binding sites thus comprise six CDRs, comprising a set of CDRs from each of the heavy and light chain v regions.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., refers to those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR-L and FR1-H, FR2-H, FR3-H, FR-H, respectively. Accordingly, the light chain variable domain may thus be referred to as (FR 1-L) - (CDR 1-L) - (FR 2-L) - (CDR 2-L) - (FR 3-L) - (CDR 3-L) - (FR 4-L) and the heavy chain variable domain may thus be denoted as (FR 1-H) - (CDR 1-H) - (FR 2-H) - (CDR 2-H) - (FR 3-H) - (CDR 3-H) - (FR 4-H). Preferably, the FR of the invention is a human antibody FR or a derivative thereof which is substantially identical to a naturally occurring human antibody FR, i.e. has a sequence identity of up to 85%, 90%, 95%, 96%, 97%, 98% or 99%.

Knowing the amino acid sequence of the CDRs, one skilled in the art can readily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR-H.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.

The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a host cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the antibodies of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the antibody sequences of the invention by chemical synthesis.

Pharmaceutical composition and kit

The present invention also provides a pharmaceutical composition which is a nucleic acid pharmaceutical composition of a gene editor and an anti-fibrosis inhibitor for use in the treatment of a disease, the pharmaceutical composition comprising: (a) A first active ingredient which is a gene editor for base editing of a target gene; and (b) a second active ingredient, the second active ingredient being an anti-fibrotic inhibitor. Wherein the disease includes (but is not limited to): duchenne Muscular Dystrophy (DMD), idiopathic Pulmonary Fibrosis (IPF), amyotrophic Lateral Sclerosis (ALS); the gene editor is selected from the group consisting of: ABE, CBE, CRISPR/Cas9, or a combination thereof; the target gene is a gene associated with the occurrence/development of the disease; the anti-fibrosis inhibitor includes an antibody, a cytokine, an RNA-based drug, or a combination thereof. In another preferred embodiment, the anti-fibrosis inhibitor is selected from the group consisting of: an anti-CTGF antibody, a recombinant Interleukin 1receptor antagonist (Interleukin 1receptor antagonist,IL-1rα), an anti-IL-1 beta receptor antibody, or a combination thereof.

In a preferred embodiment of the invention, the pharmaceutical composition is for the treatment of DMD comprising as a first active ingredient a gene editor for base editing of the gene encoding dystrophin (dystrophin), wherein the gene editor is ABE or CBE; anti-CTGF antibodies as a second active ingredient. Wherein, the anti-CTGF antibody comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 8; in another preferred embodiment, the anti-CTGF antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9 and a light chain variable region having the amino acid sequence shown in SEQ ID NO. 10.

Typically, these active ingredients are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the substance being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention can be used for gene editing therapy of a target gene related to occurrence/development of a disease, and anti-fibrosis therapy using an anti-fibrosis inhibitor, and thus can be used for treating diseases such as Duchenne Muscular Dystrophy (DMD), idiopathic Pulmonary Fibrosis (IPF), amyotrophic Lateral Sclerosis (ALS), and the like. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the above-described active ingredients of the present invention together with a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the first active ingredient and the second active ingredient is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

In general, the pharmaceutical compositions of the present invention may be prepared in a sterile container to form a kit comprising: (c1) A first container, and a first vector in the first container, the first vector comprising a first expression cassette for producing the first active ingredient described above; and (c 2) a second container, and a second vector in the second container, the second vector comprising a second expression cassette for producing the second active ingredient described above.

Typically, the kit contains one or more (e.g., at least two) unit dosage forms containing a first active ingredient and one or more (e.g., at least two) unit dosage forms containing a second active ingredient; preferably 4-10 each.

As used herein, the term "unit dosage form" refers to a dosage form that is intended for ease of use by preparing the composition into a single use, including but not limited to various solid (e.g., lyophilized), liquid, sustained release agents.

The description provided by the invention can be as follows: the method of use of the kit is to use a unit dosage form containing a first active ingredient and a unit dosage form containing a second active ingredient simultaneously.

The medicine box provided by the invention is prepared by the following steps: the formulation comprising the first active ingredient and the formulation comprising the second active ingredient are placed together with instructions to form a kit.

The main advantages of the invention include:

According to the invention, a gene editing tool and a nucleic acid molecule for encoding an antibody or an antibody active ingredient are delivered into specific tissues through a viral vector or a non-viral vector, and under the drive of a tissue specific promoter, only the gene editing tool and the antibody or an antibody active fragment with pharmaceutical activity are locally expressed, on one hand, a base editor repairs a pathogenic gene through gene editing, and meanwhile, after the antibody active ingredient of anti-CTGF is secreted from cells, a fibrosis signal in a microenvironment is reduced, so that the effects of treating both symptoms and root causes are achieved, and a synergistic therapeutic effect is generated. Because the tissue-specific promoter is adopted, the antibody component is only expressed in specific cells of the target organ, and the over-expression in blood or other tissues can not be caused, so that the off-target risk in non-target tissues is effectively reduced. In addition, by adding the antibody gene sequence to the plasmid of the gene editing tool, on the one hand, the plasmid size is increased as stuffer, and the production of AAV products is facilitated by adjusting the GOI size of AAV. The invention provides a new idea for selecting gene editing products stuffer.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are generally performed according to the conditions described in the conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents involved in the present invention are available from commercial sources unless otherwise specified.

Examples

Example 1 screening and functional identification of anti-CTGF antibodies

1.1 Screening of anti-CTGF phages

Screening was performed by a conventional phage display system from Shanghai university pharmaceutical college using the affinity panning technique from a full human antibody library, and 4 rounds of screening (FIG. 1) were performed to obtain monoclonal phage X3-4 having high affinity for CTGF.

The X3-4 monoclonal phage was tested Monophage ELISA using screening antigen human CTGF (CTF-H82E 6, beijing Baipessary, china) panel, using irrelevant antigen human phosphoribosyl pyrophosphate synthetase 1 (phosphoribosyl pyrophosphate synthetase, PRPS1) as a negative control, and Tris Buffer (TBS) as a blank control. Phage X3-4 was added, anti-Flag antibody (Abcam, ab1162, USA) was added and developed using TMB.

As shown in FIG. 2, monophage ELISA shows that X3-4 can specifically bind to human CTGF.

1.2 Activity detection of anti-CTGF antibodies

Sequencing the sequence of the X3-4 phage obtained in example 1.1 to obtain the variable region sequences of the heavy chain and the light chain aiming at CTGF, synthesizing a single-chain variable region antibody molecule according to the variable region sequences of the heavy chain and the light chain, namely X0304 scFv, wherein the nucleotide sequences are shown as SEQ ID NO. 28-29, and the amino acid sequence is shown as SEQ ID NO. 11. The variable region sequences of the control molecule X3019 scFv, heavy chain and light chain are derived from FG3019 of Fibrogen company, the nucleotide sequence is shown as SEQ ID NO. 18, and the amino acid sequence is shown as SEQ ID NO. 12. The sequence alignment of X0304 scFv and X3019 scFv is shown in FIG. 7.

Codon optimization is carried out on X0304 scFv and X3019 scFv respectively, flag-tag is added at the 5' end of the sequence, the sequence is constructed into pET28a plasmid (Novagen, cat No. 69864-3), the corresponding nucleotide sequence and amino acid sequence are shown as SEQ ID NO. 30-33, the E.coli is transformed and then expressed, his-tag in pET28a plasmid is utilized for purification, and the purified single-chain antibody is used for detecting CTGF binding activity.

CTGF binding activity of X0304 scFv and X3019 scFv was detected by ELISA. Human CTGF (hCTGF) coating was used and PRPS1 as an unrelated antigen coating was used as a control. The detection antibody adopts anti-Flag (Abcam, ab1162, USA) and TMB color development system. After termination of the reaction with 1M sulfuric acid (H2 SO 4), OD450 nm was detected.

The results of the detection are shown in the following table 1 and fig. 3:

TABLE 1 Single chain antibody Activity assay

ELISA results show that scFv of Flag-X0304 and Flag-X3019 can specifically bind to human CTGF, and the binding performance of the scFv is similar.

1.3X0304 and X3019 cross-reactivity with human, murine CTGF verification

Because of the drug development process, animal models are often required to verify the mechanism of the drug prior to entering clinical trials. Therefore, the molecule has the intersection of mice and human, the action mechanism of the molecule can be studied through a mouse model before clinic, and the concept verification of the drug mechanism can be accelerated. The cross-reactivity of X0304 scFv and X3019 scFv and hTGF and mCTGF was thus examined using ELISA. hCTGF and mCTGF coating were used in the same manner as in example 1.2.

As a result, as shown in FIG. 4, the horizontal axis shows whether or not each group has the added plate antigen, X0304, X3019, and the vertical axis represents the absorbance at OD 450. The left panel shows the detection values of the binding experiments at X0304 and X3019 and mCTGF, and the right panel shows the detection values of the binding experiments at X0304 and X3019 with hTGF. The results show that both X0304 scFv and X3019 scFv can well bind to CTGF of human and mouse, and have human-mouse cross-binding activity, and the difference between the two is not great.

1.4 Validation of in vitro anti-fibrotic function of X0304 and X3019 in a C2C12 cell TGF-beta induced in vitro fibrosis model in mice

Experiment one: the anti-fibrosis properties of the full length IgG FG3019 of X3019 were verified to determine whether the model was established.

The experimental method comprises the following steps: 1E+5C2C12 cells were seeded in 24-well plates and cultured in 10% FBS for 24 hours. The cells were switched to 2% HBS for 16 hours. FG3019 (synthesized from off-shore proteins) was then added to a final concentration of 30 μg/ml,60 μg/ml treatment, and TGF- β1 treatment (PEPROTECH, 100-21-10 μg, final concentration of 10 ng/ml) was added. After 8 hours, cells were harvested, RNA was extracted and usedThe HP Total RNA Kit (Omega Bio-tek, R6812, USA) extracts Total RNA from cells and cDNA was synthesized using PRIMESCRIPT ^TM RT Kit (Takara, RR047A, japan). The expression of mCol a1 and mCTGF was detected by qPCR using mCol a1-F/R (SEQ ID NO: 34-35) and mCTGF-F/R (SEQ ID NO: 36-37), respectively.

Results:

As shown in FIG. 5, FG3019 is capable of inhibiting the rising of the expression of mCTGF and the rising of the expression of mCOl a1 mediated by TGF-. Beta.on C2C12 cells.

The left panel of FIG. 5 shows that TGF-. Beta.can be induced in myotube cells of C2C12 with a significant increase in CTGF expression, consistent with the report. Whereas upon addition of FG3019, the mRNA expression of CTGF decreased, indicating that FG3019 is capable of inhibiting TGF-beta mediated upregulation of CTGF. In the right panel of FIG. 5, upon addition of TGF-beta stimulation, the fibrosis factor Cola1 was also significantly increased, indicating that TGF-beta activates the fibrotic signal. Whereas mRNA expression of mCola1 was down-regulated following FG3019 addition, indicating that fibrotic signals were inhibited.

Experiment II: constructing a Syn100-X0304 single-chain antibody and a Syn100-X3019 single-chain antibody vector, transducing C2C12 cells, stimulating the C2C12 cells by TGF-beta to generate fibrosis signals, and detecting whether anti-CTGF scFv expression can block transcription of related fibrosis factors.

The experimental method comprises the following steps: 1 x 10≡5c2c12 cells were seeded in 24 well plates and then cultured in 10% fbs for 24 hours. Each well was transfected with 500ng of plasmid comprising Syn100-X0304, the nucleotide sequence shown as SEQ ID NO:38, or Syn100-X3019, the nucleotide sequence shown as SEQ ID NO:39, respectively, by the transfection reagent lipofectamine 3000 (Invitrogen, L3000008). After 24 hours the medium was changed to 2% horse serum medium and incubated for 16 hours. TGF- β1 treatment (PEPROTECH, 100-21-10 μg, final concentration 10 ng/ml) was added. Cells were harvested after 8 hours, RNA and protein were extracted for qPCR detection, mCol a1 expression was detected.

As shown in FIG. 6, both the Syn100-X0304 and the Syn100-X3019 transduced C2C12 cells were effective in inhibiting TGF-beta induced ColIa1 transcription. Both X0304 and X3019 are shown to be effective in inhibiting TGF-beta induced fibrosis signaling.

Example 2 construction of anti-CTGF antibody AAV

2.1AAV GOI vector construction

The invention takes antibody genes or active fragments with therapeutic potential as the filling of AAV GOI, so that therapeutic genes can be introduced while gene editing. The anti-CTGF antibody fragment was made into a single-or bivalent single-stranded molecule (bivalent antibody fragment variable, abbreviated BaFv) and X0304 scFv and X3019 scFv (sequences as described in example 1.3) were designed into the sgRNA vector of AAV, respectively, in such a way that the final vector size was 4.2-4.5kb. The constructed GOI structure is shown in FIG. 8. The nucleotide sequences of mE4 sgRNA and hE50 sgRNA are shown as SEQ ID NO. 21 and 22, the nucleotide sequences of X0304 scFv and X3019 scFv and the GOI nucleotide sequences of mE4 sgRNA and X3019 scFv in AAV vectors are shown as SEQ ID NO. 24 and 25, and the nucleotide sequences of X0304 BaFv and X3019 BaFv and the GOI nucleotide sequences of hE50 sgRNA and AAV are shown as SEQ ID NO. 26 and 27. Wherein the promoter sequence adopts a muscle specific promoter, such as syn100, and the nucleotide sequence is SEQ ID NO. 16, so that the expression of the downstream coding gene in muscle cells can be controlled; other constitutive promoters are also possible, such as CMV promoter, etc.

2.2AAV viral packaging

The GOI components of AAV9-Syn100-eTAM include nCas, AIDx and UGI, construction references (Li et al, 2021) in which eTAM nucleotide and amino acid sequences are SEQ ID NOs 23 and 40.

The reference sgRNA GOI plasmid adopts a modified fragment of Lambda phage with gene mutation as the AAV9-mE4 sgRNA-L of stuffer, and the nucleotide is shown as a sequence SEQ ID NO. 41. GOI plasmids of the sgRNA are AAV9-sgRNA-Syn100-anti-CTGF scFv (the structure is shown as b in figure 8), and are named AAV9-mE4 sgRNA-Syn100-X0304 and AAV9-mE4 sgRNA-Syn100-X3019 respectively, and nucleotide sequences are shown as SEQ ID NO:24 and 25 respectively.

HEK293 cells were transduced by a three plasmid system (GOI, RC and helper plasmids) and subjected to lysis, clarification filtration, affinity chromatography, super-isolation concentration to obtain AAV virus. AAV titers were quantified by ddPCR. AAV viruses are used for functional verification in vivo assays.

EXAMPLE 3 in vivo demonstration of base editing and anti-fibrosis synergistic therapeutic effects

DMD E4 mice are typically amyotrophic by deletion of 4 bases in exon 4 of the mice by CRISPR/Cas9 technology, resulting in the occurrence of a stop codon in exon four, resulting in dystrophin deletion, and resulting in DMD E4 mice. By adopting a DMD E4 mouse model, the GT of the 5' SS of the 4 th intron of the mouse dystrophin gene can be used as AT by a base editor, so that the E4 of mRNA level is jumped in the splicing process, and a truncated functional protein with E4 deletion is formed, so that the muscle function of the mouse is recovered. In this example, the gene editing tool was delivered with anti-CTGF scFv via an sgRNA vector to obtain better therapeutic effect.

In vivo investigation of base editing and synergistic therapeutic action of anti-CTGF antibody fragments

DMD E4 mice (T003035, drug-collecting well) at 21 days of age were used and given i.v. according to 1: AAV9-Syn100-eTAM and AAV9-mE4-sgRNA-Syn100-X3019, mixed in a ratio of 1, were administered in accordance with 3E14 vg/kg. AAV9-mE4 sgRNA-L and AAV9-Syn100-eTAM were administered as 1E14vg/kg,3E14vg/kg as controls, mice were observed for 16 weeks, and muscle tone of mice was measured every two weeks after 8 weeks of treatment. And tissues of the mice were taken at 16 weeks, and the level of skipping of the E4 exon and the expression of Dystrophin protein in each muscle tissue of the mice were examined. The experimental design is as follows:

The experimental method comprises the following steps:

Western blot: to detect Dystrophin protein expression in the relevant tissues, the myocardium or skeletal muscle was homogenized and lysed with Western and IP cell lysates (Biyun Tian, P0013, china) for 45 min below 4℃and then centrifuged at 13,000g for 15 min at 4 ℃. The supernatant was then collected and the protein concentration was measured using the bicinchoninic acid (BCA) protein quantification kit. Protein extracts were separated on 8% surepage prep (M00662, gold, china) and transferred onto PVDF membranes. Membranes were blocked with 5% milk in TBST for 1 hour at room temperature and then incubated overnight at 4℃with primary antibodies (1:500-1,000 dilution for Dystrophin proteins; 1:10,000 dilution for Vinculin). Membranes were washed successively in TBST 3 times for 5 min each, then incubated with horseradish enzyme-labeled goat anti-rabbit IgG (h+l) and horseradish enzyme-labeled goat anti-mouse IgG (h+l) for 1 hour at room temperature. The membranes were then washed 3 more times in TBST for 10min, then chemiluminescent with ECL and finally scanned with tenability 4600SF (tenability 4600SF, tenability, china).

Muscle strength measurement:

Mice tensile force was measured beginning 4 or 6 weeks after AAV dosing and then measured with a grip dynamometer every 2 or 4 weeks until the end of the study (SA 417, jiangsu siren biosciences, china). The mouse's tail tip was held, the mouse was allowed to grasp the fence, and then the mouse was gently pulled off the grill. When the mice were no longer able to grasp the fence, the maximum force reading was recorded and repeated 10 more times at 10 second intervals. The calculation mode of the mouse tension reduction is as follows: the average of the number of pull-ups of the same group of mice was counted as the final pull-up value, as a percentage of the number of pull-ups of the individual mice compared to their baseline pull-ups (first grip strength readings).

The method for detecting the RNA skip condition comprises the following steps:

RNA and gDNA extraction, cDNA synthesis: using HP Total RNA Kit (Omega Bio-tek, R6812, USA) and Total RNA in tissues was extracted according to the protocol therein and cDNA was synthesized using PRIMESCRIPTTM RT Kit (Takara, RR047A, japan).

And (2) PCR: for detecting and analyzing exon 4 skipping, primers on exon 3 and exon 5 are designed to amplify cDNA, respectively, and the sequences of the primers are shown as SEQ ID NOs 42-43. The enzyme used was 2X HieffGold PCR MASTER Mix high fidelity enzyme premix (next holy, 10149ES03, china), PCR running program is: 98 deg.c for 3 min- & gt 98 deg.c for 10 s- & gt 58 deg.c for 20 s- & gt 72 deg.c for 20s ] & gt 40- & gt 72 deg.c for 5 min- & gt 4 deg.c for infinity.

Results:

As shown in fig. 9: the change in the muscle tension of the mice was examined under cyclic tension fatigue, the horizontal axis represents the number of times of tension, the vertical axis represents the ratio of each tension compared to the first tension, and the error bars represent the standard deviation of each group. After 16 weeks of drug injection, the tension changes were also examined in Control group, TAM L.D group, TAM H.D group, TAM-X3019 H.D group, and X3019 H.D group. DMD mice (TAM L.D and TAM H.D) in the base editor alone treatment group showed no significant improvement in muscle tone compared to Control mice (Control). While TAM-X3019 mice had a significant increase in muscle tone (P <0.05, two-way-ANOVA). Nor did the fourth group of X3019 delivered to the muscle with AAV9 alone improve muscle tone, suggesting that the effect of single chain antibodies against CTGF alone is insufficient to improve muscle function.

As shown in fig. 10: the result of skipping exon 4 (E4) of mRNA by dystrophin gene signature in the myocardium of each group of DMD mice was examined by agarose gel electrophoresis at 16 weeks after treatment. As shown in the left diagram of FIG. 10, the arrows E3-E4-E5 indicate wild-type bands, and the arrows E3-E5 indicate skipped bands. E4 exon skipping was observed in TAM L.D, TAM H.D, and TAM-X3019H.D groups, in which the base editor simultaneously expressed TAM-X3019H.D groups of anti-CTGF single-chain antibodies, with significantly higher efficiency than in other groups, whereas no exon skipping occurred in X3019H.D groups. The method shows that the editing effect can be enhanced by adding the anti-CTGF single-chain antibody into a base editor tool, and the base editor and the anti-CTGF single-chain antibody can be simultaneously administered to show the synergistic treatment effect.

As shown in fig. 11: immunoblotting staining detects the expression level of Dystrophin protein in heart tissue. It can be seen that the increase in protein expression was not evident in the TAM H.D and TAM L.D groups using only the base editor, whereas the increase in Dystrophin protein was very evident in the TAM-x3019h.d group, i.e., the anti-CTGF single chain antibody expression enhanced the base editor effect and restored Dystrophin protein expression. The reason may be due to inhibition of anti-fibrotic signals in the microenvironment, enhancing successful editing of muscle cell survival. The lanes correspond to the following groups: (1-3) WT hearts 100%,20%,4%; (4) Control; (5-6) TAM L.D; (7-8) TAM H.D (9-10) TAM+X3019 H.D; (11) X3019H.D

As shown in fig. 12: the Dystrophin protein in skeletal muscle was detected using immunofluorescent staining, the stronger the signal indicated better recovery of Dystrohpin protein. As shown in the left panel of FIG. 12, it can be seen that expression of Dystrophin protein was observed in TAM H.D, but protein expression was not restored after injection in X3019H.D alone, whereas stronger protein expression was observed in TAM-X3019H.D, consistent with immunoblotting results. Indicating that co-expression of anti-CTGF facilitates therapeutic action of the base editor. The right diagram of fig. 12 shows: the fluorescence intensity values of immunofluorescent staining of Dystrophin proteins from each group were significantly higher in the TAM-x3019h.d and TAM H.D groups than in the Control group, and the fluorescence intensity values were highest in the TAM-x3019h.d groups (11.43 times the Control group).

DMD mice develop more severe fibrosis symptoms at advanced stages, showing severe Col 1a deposition.

As shown in fig. 13: detection of Col 1a protein in skeletal muscle using immunofluorescent staining indicated that the stronger the signal, the more severe the deposition of Col 1 a. After base editor TAM treatment, a different degree of reduction in Col 1a deposition was observed in the treated mouse myocardium compared to Control groups, TAM L.D, TAM H.D, X3019H.D and TAM-X3019h.d, and TAM-X3019h.d almost completely eliminated Col 1a deposition. This demonstrates that concurrent base editor recovery dystrophin protein expression and anti-CTGF anti-fibrosis treatment can synergistically reduce fibrosis in DMD mouse muscle. The right diagram of fig. 13 shows: immunofluorescent staining of Col 1a proteins of each group showed significantly lower fluorescence intensity values than that of the Control group in TAM-X3019H.D and TAM H.D groups, and the fluorescence intensity values were the lowest in TAM-X3019H.D groups (corresponding to 0.56% of the Control group)

In summary, taking the combination of single-chain antibodies against Connective Tissue Growth Factor (CTGF) and a base editor as an example, the single-chain antibodies against Connective Tissue Growth Factor (CTGF) were first selected individually, and their affinities were similar to those of the single-chain antibodies corresponding to FibroGen full-length CTGF antibodies. In vitro, it was demonstrated that the single chain antibody was effective in inhibiting tgfβ -induced fibrosis of C2C12 in mouse muscle cells. The results of in vivo administration for treating duchenne muscular dystrophy show that the CTGF single chain antibody and the gene editing product are specifically expressed in muscle, and that administration together significantly reduces muscle fibrosis and muscle degeneration, improves recovery of dystrophin in muscle cells, and improves muscle function, as compared to administration alone or a control group.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. A pharmaceutical composition for use in the treatment of a disease, the pharmaceutical composition comprising:

(a) A first active ingredient which is a gene editor for base editing of a target gene; and

(B) A second active ingredient which is an anti-fibrotic inhibitor.

2. The pharmaceutical composition of claim 1, wherein the disease comprises (but is not limited to): duchenne Muscular Dystrophy (DMD), idiopathic Pulmonary Fibrosis (IPF), amyotrophic Lateral Sclerosis (ALS).

3. The pharmaceutical composition of claim 1, wherein the gene editor is selected from the group consisting of: ABE, CBE, CRISPR/Cas9, or a combination thereof.

4. The pharmaceutical composition of claim 1, wherein the anti-fibrosis inhibitor is selected from the group consisting of: an anti-CTGF antibody, a recombinant Interleukin 1receptor antagonist (Interleukin 1receptor antagonist,IL-1rα), an anti-IL-1 beta receptor antibody, or a combination thereof.

5. The pharmaceutical composition of claim 4, wherein the anti-CTGF antibody comprises a heavy chain variable region as shown in SEQ ID No. 7 and a light chain variable region as shown in SEQ ID No. 8.

6. The pharmaceutical composition of claim 4, wherein the anti-CTGF antibody comprises a heavy chain variable region as shown in SEQ ID No. 9 and a light chain variable region as shown in SEQ ID No. 10.

7. The pharmaceutical composition of claim 5 or 6, wherein the anti-CTGF antibody comprises: single chain antibodies (scFv), double chain antibodies (bivalent antibody fragment variables, baFv), diabodies, monoclonal antibodies, chimeric antibodies (e.g., human murine chimeric antibodies), murine antibodies, or humanized antibodies.

8. A kit, comprising:

(c1) A first container, and a first vector in the first container, the first vector comprising a first expression cassette; and

(C2) A second container, and a second vector in the second container, the second vector comprising a second expression cassette;

wherein the first expression cassette has the structure of formula I of 5'-3' (5 'to 3'):

P1-X1-L1-X2-X3 (I)

The second expression cassette has the structure of formula II 5'-3' (5 'to 3'):

P2-X4-L2-P3-X5-X3 (II)

Wherein P1, P2 and P3 are a first promoter sequence, a second promoter sequence and a third promoter sequence respectively, the first promoter and the third promoter are the same type of promoter, and the second promoter is an RNApol III promoter;

X1 is the coding sequence of adenine deaminase or cytosine deaminase;

L1 and L2 are each independently absent or a linking sequence;

X2 is the coding sequence of a partially inactivated Cas9 nuclease (nCas), said Cas9 nuclease having nickase activity;

x3 is PolyA sequence;

X4 is the coding sequence of sgRNA;

X5 is the coding sequence for an anti-fibrotic inhibitor;

and each "-" is independently a bond or a nucleotide linking sequence.

9. An anti-CTGF antibody comprising a heavy chain variable region comprising the following complementarity determining region CDRs:

H-CDR1 as shown in SEQ ID NO. 1;

H-CDR2 as shown in SEQ ID NO. 2; and

H-CDR3 as shown in SEQ ID NO. 3.

10. The anti-CTGF antibody of claim 9, further comprising a light chain variable region comprising the following complementarity determining region CDRs:

L-CDR1 as shown in SEQ ID NO. 4;

L-CDR2 as shown in SEQ ID NO. 5; and

L-CDR3 as shown in SEQ ID NO. 6.