Expression vector of targeted transcriptional activation Lama1 gene and application of expression vector

Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to an expression vector of a targeted transcriptional activation Lama1 gene and application of the expression vector to preparation of drugs for treating LAMA2-related muscular dystrophy. According to the present invention, the expression vector of the target transcription activation Lama1 gene is constructed through the construction of AAV9-mini CMV-SpdCas9-VP64 and AAV9-U6-gRNA2-U6-gRNA3-CAG-MPH-GFP of the AAV9 mediated transcription activation substitution gene Lama1, and the improvement effect of the AAV9 mediated transcription activation Lama1 on the LAMA2-CMD mouse disease phenotype is proved;

Description

Technical Field

[0001] The present invention belongs to the technical field of biological gene engineering, and specifically relates to an expression vector for targeted transcriptional activation of Lama1 gene and use thereof in preparing a drug for treating LAMA2-related muscular dystrophy. Background Art

[0002] LAMA2-related muscular dystrophy (LAMA2-MD) is an autosomal recessive myopathy caused by pathogenic variants in the LAMA2 gene. According to the age of onset and the severity of clinical manifestations, it is usually divided into two subtypes, namely LAMA2-related congenital muscular dystrophy (LAMA2-CMD) and autosomal recessive limb-girdle muscular dystrophy type 23 (LGMDR23). Among them, LAMA2-CMD is the most important subtype of LAMA2-MD and the most common subtype of congenital muscular dystrophy (CMD), accounting for about one-third of CMD patients worldwide. LAMA2-MD is a rare disease that is severely disabling and fatal. Its clinical manifestations include widespread symmetrical muscle weakness, hypotonia, abnormal white matter signals, joint and spinal deformities, etc. Currently, there are still many challenges in the diagnosis and treatment of LAMA2-MD, and there is still a lack of effective treatments in clinical practice.

[0003] Studies have shown that the human LAMA2 gene is located at 6q22.23, contains 65 exons, and the cDNA length is about 9.4kb. This gene encodes laminin-α2, which is composed of 3122 amino acids and is an important extracellular matrix protein. Laminin-α2 is mainly expressed in muscle, skin, trophoblasts, peripheral nerves and cerebrovascular endothelial cells. Laminin-α2, β1 and γ1 proteins are assembled together to form a heterotrimeric protein laminin-211, which participates in the formation of the skeletal muscle basement membrane and plays an important role in muscle cell contraction, regeneration, differentiation and migration. LAMA2 pathogenic variants lead to complete or partial deletion of laminin-α2, which can cause insufficient assembly of laminin-211, unstable extracellular matrix structure, destruction of the connection between cell membrane and extracellular matrix, separation of basement membrane and sarcoplasmic membrane, and subsequent damage to muscle fiber contraction load and abnormal cell signal transduction pathways. Muscle biopsy of patients showed muscular dystrophy-like pathological changes, muscle fiber dysplasia, degeneration, necrosis and regeneration, and interstitial connective tissue hyperplasia, leading to LAMA2-MD.

[0004] According to the possible pathogenesis and intervention of LAMA2-MD muscle disease, the treatment research of LAMA2-MD is mainly divided into three categories: one is to use various technical means to intervene in the LAMA2 gene, such as knockout mice dy W / dy W The first is to overexpress LAMA2 transgenes, or to read through nonsense mutations in LAMA2 at the cellular level. Antisense oligonucleotides can also be used to mediate LAMA2 exon 4 skipping and CRISPR / Cas9 to correct LAMA2 splicing mutations. The second is to replace connexins or homologous proteins, such as mini-agrin, Integrin-α7 and other connexins transgenic overexpression, homologous proteins laminin-α1 and αLNNd targeting laminin-α4 transgenic overexpression, or exogenous supplementation of laminin-111. The third is to improve downstream events, such as Bcl-2 overexpression, omigapil and other inhibition of apoptosis, N-acetylcysteine ​​and vitamin E and other antioxidants, prednisone anti-inflammatory, losartan to improve muscle fibrosis, insulin-like growth factor-1 overexpression to promote dysregulation. W / dy W Mouse muscle regeneration, etc.

[0005] At present, although the treatment research for LAMA2-MD has received increasing attention, there are still many problems that have not been solved. On the one hand, although correcting the LAMA2 gene mutation seems to be the most fundamental treatment option, due to the full length of LAMA2 cDNA of about 9.4kb, there is no suitable vector to achieve the exogenous introduction of full-length LAMA2 cDNA, and no functional truncated cDNA sequence has been found, making LAMA2 gene replacement therapy difficult to carry out in basic research and clinical trials; on the other hand, studies have shown that nonsense read-through failed to improve laminin-α2 expression at the cellular level, and exon 4 skipping did not improve LAMA2-MD mice. Although CRISPR / Cas9 has a certain effect in correcting splicing mutations, there are many types of LAMA2 mutations and no hotspot mutations. Gene modification for a certain LAMA2 mutation has great limitations in application. In addition, only some of the treatment studies on increasing the expression of connexins and homologous proteins and improving secondary cellular events have achieved a certain degree of improvement in motor function and muscle pathology in animal models, while there are relatively few studies on the evaluation methods for motor function improvement and the extension of survival. Therefore, how to choose a suitable carrier, route of administration, and when to provide therapeutic intervention are all unknown, and the treatment of LAMA2-MD is still full of difficulties and challenges.

[0006] At present, among the five known homologous laminin chains, only laminin-α1 and laminin-α2 have the most similar structures and functions. Among them, laminin-α1 is encoded by the LAMA1 gene and is also an important component of the extracellular matrix. It plays an important regulatory role in cell adhesion, cell migration, tissue and organ development, and nerve axon growth during embryonic development. Laminin-α1, β1, and γ1 form the laminin-111 trimer. Laminin-γ1 connects to the nidogen-1 protein, which indirectly connects laminin-111 to type IV collagen and perlecan protein, stabilizing the loose laminin network and forming a stable basement membrane. LAMA1 is a homologous gene of LAMA2, and its encoded product laminin-α1 is gradually being recognized for its role in the treatment of LAMA2-MD. Studies have shown that laminin-111 protein extracted from Engelbreth-Holm-Sarm (EHS) mouse tumors or recombinant human laminin-111 protein can improve dy W / dy W Muscular dystrophy phenotype in mice. However, this approach requires multiple consecutive dosings rather than a single lifelong dosing. Since laminin-α1 is mainly expressed during embryonic development, its expression decreases rapidly after birth and is almost not expressed in the adult neuromuscular system. The two homologous genes LAMA1 and LAMA2 are similar in size and full-length replacement is also impossible. Therefore, how to increase the expression of endogenous full-length laminin-α1 has become an important therapeutic target.

[0007] With the development and application of AAV vectors and CRISPR / Cas9 technology, gene-related treatments for monogenic genetic diseases have ushered in a new dawn, especially in gene-related treatments for hereditary neuromuscular diseases. There are precedents for achieving therapeutic goals by increasing the expression of homologous genes. For example, in the gene modification therapy for spinal muscular atrophy (SMA) caused by pathogenic variants in the SMN1 gene, splicing regulation is used to promote the transcription of exon 7 of the homologous gene SMN2, thereby increasing the expression of full-length SMN2 and obtaining full-length SMN protein. Two drugs have been approved for marketing and are used worldwide. Therefore, it is possible to achieve a replacement therapy effect by transcriptionally activating LAMA1 expression in the early postnatal period. Previous studies have demonstrated the CRISPR / Cas9 transcriptional activation system for dy 2J / dy 2J The efficacy in mice, however, was due to 2J / dy 2J The mice have a normal lifespan, so it was not possible to study the effect of this approach on the survival of Lama2-deficient mice.

[0008] Therefore, the evaluation of LAMA1 transcriptional activation therapy requires an appropriate disease model. The mouse models commonly used to study LAMA2-MD abroad are the dy / dy and dy 2J / dy 2J ,dy 3K / dy 3K anddy W / dy W Among them, the Lama2 mutation of dy / dy is unknown; dy 2J / dy 2J It is caused by a splicing mutation that results in the deletion of Lama2 exon 2, which has a mild phenotype and hardly affects survival; 3K / dy 3K anddy W / dy W Although both have severe muscular dystrophy phenotypes, these two mouse models are constructed according to the old animal model method, caused by mutations at the 3' end of Lama2 exon 4 and the 5' end of exon 1. However, there are no relevant animal models in China.

[0009] For example, the Lama2 gene knockout mouse model disclosed in Chinese patent CN116814687A is based on the clinical discovery of a high-frequency mutation region of LAMA2 in China. CRISPR / Cas9 technology has been used to successfully construct a model mouse with a homozygous exon 3 deletion and a 5'-end frameshift mutation of exon 4 that causes Lama2 out-of-frame mutations, and the model mouse is named dy H / dy H . Previous studies have shown that this is a new LAMA2-CMD mouse model that is suitable for studying mouse survival, body weight, exercise, muscle pathology, imaging, and molecular mechanisms before and after treatment. Based on this model, the next step is to simultaneously introduce the transcriptional activation domain and accurately reach the mouse Lama1 gene, which requires the design of gRNA (guideRNA) targeting the Lama1 promoter, which is also an urgent problem to be solved in this field. Summary of the invention

[0010] To this end, the technical problem to be solved by the present invention is to provide an expression vector for targeted transcriptional activation of the Lama1 gene, wherein the expression vector can transcriptionally activate the Lama1 gene for dysregulated CRISPRa system mediated by AAV9. H / dy H Mouse.

[0011] The second technical problem to be solved by the present invention is to provide the use of the above-mentioned expression vector for targeted transcriptional activation of the Lama1 gene for the preparation of a drug for treating LAMA2-related muscular dystrophy. Studies have found that early intervention upregulation of Lama1 can prolong dystrophy. H / dy H The study showed that the new gene therapy could extend the lifespan of mice, improve motor function, and halt pathological progression, and has great therapeutic potential for human LAMA2-MD.

[0012] In order to solve the above technical problems, the present invention discloses an expression vector for targeted transcriptional activation of the Lama1 gene, which is an AAV9-mediated transcriptional activation of the Lama1 gene expression vector constructed based on a dual AAV9 vector delivery CRISPRa system.

[0013] Specifically, the expression vector for targeted transcriptional activation of the Lama1 gene comprises an expression vector 1 for AAV9-mediated transcriptional activation of the Lama1 gene;

[0014] The expression vector 1 comprises the promoter miniCMV, the nuclease-inactive SpdCas9 derived from Streptococcus pyogenes, and the transcription activator VP64, and the expression vector is recorded as AAV9-miniCMV-SpdCas9-VP64.

[0015] Specifically, the expression vector for targeted transcriptional activation of the Lama1 gene comprises an expression vector 2 for AAV9-mediated transcriptional activation of the Lama1 gene;

[0016] The expression vector 2 contains promoter U6, gRNA2 and gRNA3 targeting the Lama1 gene promoter sequence, and the expression vector is recorded as AAV9-U6-gRNA2-U6-gRNA3-CAG-MPH-GFP.

[0017] Specifically, in the expression vector for targeting transcriptional activation of the Lama1 gene, the characteristic sequence of the gRNA2 targeting the promoter sequence of the Lama1 gene is:

[0018] 5′-GCGCCCAGGTCTGTCCTCCA-3′.

[0019] Specifically, in the expression vector for targeting transcriptional activation of the Lama1 gene, the characteristic sequence of the gRNA3 targeting the promoter sequence of the Lama1 gene is:

[0020] 5′-GTCCGAAGGGCCTGCGCACC-3′.

[0021] The present invention also discloses a method for constructing the expression vector for targeted transcriptional activation of Lama1 gene, comprising the steps of using plasmid to transfect 293T cells, and collecting the transfected virus solution, purifying, concentrating, ultrafiltration desalting, filtering and sterilizing to obtain the target vector.

[0022] The present invention also discloses the use of the expression vector for targeted transcriptional activation of Lama1 gene for preparing a drug for treating LAMA2-related muscular dystrophy symptoms.

[0023] Preferably, the LAMA2-related muscular dystrophy symptoms include LAMA2-related congenital muscular dystrophy (LAMA2-CMD) and / or autosomal recessive limb-girdle muscular dystrophy type 23 (LGMDR23).

[0024] Specifically, the LAMA2-related muscular dystrophy includes LAMA2-related congenital muscular dystrophy LAMA2-CMD type.

[0025] The present invention also discloses a pharmaceutical preparation for treating LAMA2-related muscular dystrophy, comprising the expression vector and gRNA for targeted transcriptional activation of the Lama1 gene.

[0026] The present invention also discloses the use of the expression vector for targeted transcriptional activation of Lama1 gene for preparing a transcriptional activation Lama1 gene drug.

[0027] The present invention also discloses a pharmaceutical preparation for transcriptionally activating the Lama1 gene, comprising the expression vector and gRNA for targeted transcriptionally activating the Lama1 gene.

[0028] The expression vector and pharmaceutical preparation of the targeted transcriptional activation Lama1 gene of the present invention are used to screen the gRNA with the best transcriptional activation effect of Lama1 gene at the cell level. C2C12 mouse myoblasts are transfected with a lentivirus targeted for transcriptional activation of Lama1, flow sorted and cultured, and the expression level of Lama1 after transfection is detected to screen the gRNA(s) with the best transcriptional activation effect.

[0029] The expression vector of the targeted transcriptional activation of the Lama1 gene described in the present invention constructs an expression vector of the AAV9-mediated transcriptional activation alternative gene Lama1, and compares the changes in survival, body weight, motor function, skeletal muscle MRI, muscle pathology, biochemical indicators, etc. of LAMA2-CMD mice intervened with AAV9-mediated transcriptional activation of the alternative gene Lama1, LAMA2-CMD mice without intervention, and wild-type (WT) mice, further confirming the improvement effect of AAV9-mediated transcriptional activation of Lama1 on the disease phenotype of LAMA2-CMD mice.

[0030] The expression vector of the targeted transcriptional activation of the Lama1 gene of the present invention was injected subcutaneously with appropriate amounts of expression vectors 1 and 2 (1:1) into P3 newborn mice, and transcriptomic analysis was performed on the muscle tissues of LAMA2-CMD mice, LAMA2-CMD mice without intervention, and WT mice with Lama1 transcriptional activation intervention on the 21st day after birth, to compare the changes in related genes in the key pathogenic pathways of LAMA2-CMD mice after Lama1 transcriptional activation. Through the above studies, it was confirmed that AAV9-mediated transcriptional activation of Lama1 can rescue the key pathogenic pathways abnormally regulated in LAMA2-CMD mice, revealing the molecular mechanism of Lama1 transcriptional activation therapy.

[0031] The present invention started from the forefront of international related research, starting from clinical problems, conducting basic research exploration, intending to solve potential clinical treatment problems, and taking the lead in conducting gene therapy related research for LAMA2-CMD in China. The present invention uses AAV9-mediated CRISPR / Cas9 transcriptional activation technology to study the improvement of LAMA2-CMD mice by early post-natal transcriptional activation of Lama1, especially the extension of survival, and provides a preclinical theoretical basis for the clinical application of alternative gene therapy in LAMA2-CMD.

[0032] The expression vector for targeted transcriptional activation of the Lama1 gene of the present invention utilizes the CRISPRa transcriptional activation system to independently design three gRNAs targeting the promoter region within 200 bp upstream of the transcription start site of the mouse Lama1 gene, and the effectiveness of the system has been demonstrated through cell experiments and animal experiments.

[0033] The present invention acts on the promoter region of Lama1 through a fusion vector of CRISPR / Cas9 and the transcriptional activation domain VP64, thereby promoting the transcriptional activation of Lama1 and increasing the expression of endogenous laminin-α1 protein. We use AAV9-mediated SpdCas9-VP64 / gRNAs to simultaneously introduce the homologous gene Lama1, which can efficiently transcriptionally activate the homologous gene, increase the expression of laminin-α1 protein in LAMA2-CMD mice, and replace the function of laminin-α2 protein to a certain extent, thereby improving the severe phenotype of LAMA2-CMD mice in terms of survival, weight gain, motor function, muscle pathology and pathogenic pathways, thereby treating LAMA2-MD. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0035] Figure 1 The results of in vitro and in vivo experiments on the transcriptional activation of Lama1 in Example 1;

[0036] Figure 2 In Example 2, upregulating Lama1 can prolong dy H / dy H lifespan, improved weight gain, and increased limb strength outcomes in mice;

[0037] Figure 3 In Example 3, upregulating Lama1 can improve dy H / dy H Muscle imaging, biochemical indices, and muscle pathology results in mice;

[0038] Figure 4 In Example 4, Lama1 is upregulated to make dy H / dy H Results of restoring expression levels of some genes in mice. DETAILED DESCRIPTION

[0039] Example 1 In vitro and in vivo results of transcriptional activation of Lama1

[0040] In this example, in order to study the therapeutic effect of transcriptionally activating the LAMA1 gene on LAMA2-CMD mice and correct the key pathogenic pathways of the model mice, an in vitro experiment was first performed.

[0041] In this example, a fusion vector of SpdCas9 and the transcriptional activation domain VP64 acts on the promoter region of the mouse Lama1 gene to promote Lama1 transcriptional activation and increase the expression of endogenous laminin-α1 protein.

[0042] like Figure 1 In the process shown in a, according to the CRISPR / Cas9 target design principle, three gRNAs were designed to target the promoter region within 200 bp upstream of the transcription start site of the mouse Lama1 gene, that is, three gRNAs (gRNA1-gRNA3) were designed to target the mouse Lama1 proximal promoter region, which is located within 200 bp upstream of the transcription start site (ATG represents the translation start site). The arrows indicate the direction of each gRNA. The sequence of each gRNA is adjacent to the PAM sequence (3'NGG), and lowercase letters and uppercase letters represent intron and exon sequences, respectively.

[0043] like Figure 1In the process shown in b, C2C12 mouse myoblasts were transfected with a lentiviral-packaged transcription activation system vector (pLV-U6-gRNA-CMV-SpdCas9-VP64-T2A-mScarlet) containing SpdCas9-VP64 and the corresponding gRNA. Three lentiviruses with gRNA (gRNA1-gRNA3) and a control lentivirus without mouse gRNA (no guide) were transfected into C2C12 mouse myoblasts, and the transfected C2C12 cells were further selected by flow cytometry for cell culture and total RNA extraction. RT-qPCR was used to detect the relative mRNA level of Lama1 / Gapdh (data are expressed as "mean ± standard deviation"; n = 3 independent samples).

[0044] In this embodiment, Figure 1 The RT-qPCR results shown in middle b showed that the lentivirus containing gRNA2 had the most significant increase in Lama1 mRNA expression in C2C12 cells, followed by the lentivirus containing gRNA3.

[0045] like Figure 1 The results shown in c and d are based on the high-frequency mutation region of LAMA2 in Chinese patients found in clinical practice. We used CRISPR / Cas9 technology to successfully construct a model mouse with homozygous exon 3 deletion and exon 4 5' end frameshift mutation that caused Lama2 out-of-frame mutation. We named it dy H / dy H (Subsequently, "KO" will be used to represent "dy H / dy H ”). Previous studies have shown that this is a new LAMA2-CMD mouse model that is suitable for studying mouse survival, body weight, movement, muscle pathology, imaging, and molecular mechanisms before and after treatment, such as the scheme described in Chinese patent CN116814687A.

[0046] According to the results of in vitro experiments, two gRNAs (gRNA2 and gRNA3) with strong transcriptional activation effects were selected in this example. Due to the limited packaging space of AAV9, the transcriptional activation systems SpdCas9-VP64 and gRNA2-gRNA3 were packaged in two AAV9s (AAV9-miniCMV-SpdCas9-VP64 and AAV9-U6-gRNA2-U6-gRNA3-CAG-MPH-GFP). Figure 1 In the process shown in middle e, mice were intervened by a single subcutaneous injection of AAV9 on the third day after birth (P3), and samples were collected on P21.

[0047] In this example, Western blot was used to analyze the expression of laminin-α1, GFP, and Gapdh in the biceps femoris of P21 WT mice, untreated KO mice, and two guides treated KO mice. Figure 1 The Western blot results shown in middle f showed that laminin-α1 protein was not expressed in the biceps femoris of WT mice and untreated KO mice, and was expressed only after the intervention of two guides. The expression of GFP represented the successful transfection of AAV9.

[0048] like Figure 1 The immunofluorescence results shown in middle g (scale bar, 20 μm) analyzed the expression of laminin-α2 protein and laminin-α1 protein in the biceps femoris and diaphragm of P21 WT mice, untreated KO mice, and two guides-treated KO mice. Immunofluorescence showed that laminin-α2 protein was absent in the cell membrane of the biceps femoris and diaphragm of KO mice, and laminin-α1 protein was expressed after two guides intervention.

[0049] Example 2 Upregulation of Lama1 can prolong dy H / dy H Longevity, improved weight gain, and increased limb strength in mice

[0050] In the experimental scheme of this embodiment, the experimental mice were divided into the following 5 groups:

[0051] (1) WT group;

[0052] (2) untreated KO group;

[0053] (3) no guide treats KO group (1.0×10 11 vg of AAV9-miniCMV-SpdCas9-VP64);

[0054] (4) Low-dose two guides treatment KO group (0.5×10 11 vg of AAV9-miniCMV-SpdCas9-VP64 and 0.5×10 11 vg of AAV9-U6-gRNA2-U6-gRNA3-CAG-MPH-GFP, the total dose was 1.0 × 10 11 vg);

[0055] (5) High-dose two guides treatment KO group (1.0×10 11 vg of AAV9-miniCMV-SpdCas9-VP64 and 1.0×10 11vg of AAV9-U6-gRNA2-U6-gRNA3-CAG-MPH-GFP, the total dose is 2.0×10 11 vg).

[0056] As attached Figure 2 The process shown in a, P3 newborn dy H / dy H Mice were subcutaneously injected with no guide AAV9 (1.0×10 11 vg), low-dose two guides AAV9 (divided into two vectors; total dose 1.0×10 11 vg), or high-dose twoguides AAV9 (split into two vectors; total dose 2.0 × 10 11 Specifically, (1) and (2) mice were not intervened, (3)-(5) mice were intervened by a single subcutaneous injection of AAV9 on postnatal day 3 (P3), and all KO mice were observed until natural death, with a total observation period of 1 year.

[0057] As attached Figure 2 The results of the Kaplan-Meier survival analysis described in b showed that the median survival time of untreated KO mice was 25.5 days (n=10, range 4-44 days), and there was no death in WT mice during the observation period (n=10). The median survival times of the KO groups treated with no guide, low-dose two guides, and high-dose two guides were 33 days (n=16, range 5-51 days), 50 days (n=10, range 10-86 days), and 53 days (n=16, range 6-114 days), respectively. It can be seen that the survival curves of the untreated KO group and the no guide treated KO group are very close, and there is no significant difference in survival time. In contrast, the median survival time of the KO group treated with low-dose two guides was increased to 50 days (range 10-86 days). When the total dose of AAV9 reached 2.0×10 11 vg (high-dose two guides treatment KO group), the survival period showed a two-level differentiation (range 6-114 days), that is, nearly half of the mice died within the first two weeks, and the survival period of the other half of the mice was greatly improved, and the survival time was extended to about 100 days.

[0058] As attached Figure 2The ANOVA test shown in c shows that the difference in survival time between the untreated KO group and the low-dose two guides-treated KO group (*p<0.05), and between the low-dose two guides-treated KO group and the no guide-treated KO group (*p<0.05) is statistically significant. There is no significant difference between the untreated KO group and the no guide-treated KO group (ns, p≥0.05).

[0059] In this example, while observing the survival period, the body weight and gripping strength were measured twice a week. The gripping strength test of each mouse was repeated 3 times and the average value was taken (using "SuperGSM" software to collect and analyze data).

[0060] like Figure 2 The results shown in middle d are line graphs showing the weight changes of mice in the WT group (n=10), untreated KO group (n=8), no guide treated KO group (n=9), low-dose two guides treated KO group (n=10) and high-dose two guides treated KO group (n=14) from 1 to 17 weeks. The data are expressed as "mean ± standard deviation", measured twice a week, and "n" is the number of mice in each group at the first measurement. It can be seen that without treatment, the weight of KO mice reached a peak at 2 weeks, with an average weight of up to 6.05g, and then gradually decreased. The no guide treated KO group was almost exactly the same. The high-dose two guides treated KO group reached a peak platform at 5-8 weeks, with a maximum average weight of 10.97g. The low-dose two guides treated KO group reached a peak platform at 5-6.5 weeks, with a maximum average weight of 8.18g.

[0061] like Figure 2 The results shown in middle e are line graphs showing the changes in the grasping strength of the limbs of mice in the WT group (n=10), untreated KO group (n=8), no guide treated KO group (n=9), low-dose two guides treated KO group (n=9), and high-dose two guides treated KO group (n=9) from 2 to 17 weeks. The data are expressed as "mean ± standard deviation", measured twice a week, and "n" is the number of mice in each group at the first measurement. It can be seen that in terms of grasping strength, the untreated KO group and the no guide treated KO group maintained similar low levels, with a maximum value of approximately 20 gf. In contrast, the grasping strength of KO mice in the high-dose and low-dose two guides treated groups increased to approximately 30 gf.

[0062] like Figure 2Results shown in middle d and e show that, in general, the weight and strength differences between the groups gradually widened from week 3. From week 3 until the end of observation, the weight and grip strength of the two guides-treated KO group were higher than those of the untreated KO group and the no guide-treated KO group.

[0063] like Figure 2 The results shown in middle f, the same littermates received different interventions at P3 and were photographed at P21. It can be seen that in appearance, among the same littermates at P21, the two guides-treated KO mice were significantly larger than the untreated KO mice and the no guide-treated KO mice, and there was no significant difference between the high-dose and low-dose two guides-treated KO mice.

[0064] Example 3 Upregulation of Lama1 can improve dy H / dy H Muscle imaging, biochemical parameters, and muscle pathology results in mice

[0065] In this example, considering the safety issues in the high-dose group and the fact that the improvement in muscle function was not better than that in the low-dose group, this example selected a single subcutaneous injection of AAV9 (low-dose two guides) at P3 as an intervention measure for further study.

[0066] KO mice were subcutaneously injected with a low dose of two guides AAV9 (divided into two vectors; total dose of 1.0×10 11 vg), considering that the phenotypic differences between the untreated group and the two guides treatment group began to become obvious at week 3, P21 was selected as the sampling time.

[0067] In this example, muscle MRI was used to study the improvement of skeletal muscle in KO mice after two guides treatment.

[0068] P3 KO mice were injected subcutaneously with AAV9, and hindlimb muscle MRI was performed on WT mice, untreated KO mice, and two guides-treated KO mice at P21. Image J software was used to analyze the muscle MRI images of mice, with n = 4 in each group, and ANOVA test was used for statistical analysis.

[0069] like Figure 3The results shown in a in the middle show that on T1-weighted (T1W) MRI, adipose tissue showed high signal. The relative muscle area (muscle area / fat area) of untreated KO mice was significantly lower than that of WT mice (**p<0.01). The relative muscle area of ​​KO mice treated with two guides was smaller than that of WT mice (*p<0.05), but larger than that of untreated KO mice (*p<0.05). On T2-weighted (T2W) MRI, high signal indicated muscle edema (WT vs untreated KO, **p<0.01; untreated KO vs two guides treated KO, *p<0.05; WT vs two guides treated KO, **p<0.01). Significantly increased high signal areas were found in untreated KO mice, indicating muscle edema. After two guides treatment, fat infiltration and edema in the hindlimb muscles of KO mice were improved.

[0070] Blood was collected from mice on P21, and serum was separated after centrifugation. Serum creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other biochemical indicators related to skeletal muscle injury were detected. The results are shown in the attached Figure 3 As shown in middle b. Among them, CK: WT vs untreated KO, *p<0.05; untreated KO vs two guides treated KO, *p<0.05; WT vs two guides treated KO, ns, p≥0.05. ALT: WT vs untreated KO, *p<0.05; untreated KO vs two guides treated KO, *p<0.05; WT vs two guides treated KO, *p<0.05. AST: WT vs untreated KO, *p<0.05; untreated KO vs two guides treated KO, *p<0.05; WT vs two guides treated KO, *p<0.05. n=6 per group, statistical analysis was performed using ANOVA test. It can be seen that compared with WT mice, serum CK in untreated KO mice was significantly increased. In two guides treated KO mice, serum CK was lower than that in untreated KO mice. ALT and AST were significantly increased in untreated KO mice and partially restored in two guides treated KO mice.

[0071] In this embodiment, Figure 3H&E staining shown in c shows (scale bar, 20 μm) H&E staining (left) and Sirius red staining (right) of the biceps femoris and diaphragm of WT mice, untreated KO mice, and two guides-treated KO mice at P21. H&E staining showed that the muscle fibers of untreated KO mice were unequal in size and infiltrated with inflammatory cells (arrows on the left). Sirius red staining showed that the red-stained collagen in the skeletal muscle of untreated KO mice increased (arrows on the right). These pathological changes were partially restored in twoguides-treated KO mice. The analysis results of all mouse samples (n=3 per group) were similar. It can be seen that H&E staining showed that the muscle fibers of the biceps femoris and diaphragm of untreated KO mice were unequal in size, with degeneration, necrosis, regeneration, connective tissue hyperplasia and inflammatory cell infiltration, which were consistent with typical muscular dystrophy-like pathological changes. Sirius red staining showed that the red-stained collagen fibers in the extracellular matrix of the skeletal muscle of untreated KO mice increased, indicating pathological fibrosis. The above pathological changes were partially restored in KO mice treated with two guides. The myocardium of KO mice was relatively unaffected.

[0072] Taken together, these results suggest that upregulation of Lama1 improves muscle imaging, biochemical parameters, and muscle pathology in Lama2-deficient mice.

[0073] Example 4 Upregulation of Lama1 can make dy H / dy H Restoration of some gene expression levels in mice

[0074] In this example, RNA sequencing (RNA-seq) was further performed on the biceps femoris of P21 mice, and the data were from n=3 mice in each group.

[0075] The results are attached Figure 4 As shown in Figures ac, according to |log2FoldChange|>=0.5 and padj<0.05, the most differentially expressed genes (DEGs) (blue marks) between the groups were "untreated KO vs WT" (1402 DEGs), followed by "two guides treated KO vs WT" (449 DEGs), and finally "two guides treated KO vs untreated KO" (72 DEGs). The upregulation of Lama1 was verified in two guides treated KO mice compared with untreated KO mice or WT mice.

[0076] As attached Figure 4Heat map of DEGs shown in d, according to |log2FoldChange|>=0.5 and p<0.05, WT mice or two guides treated KO mice compared with untreated KO mice, red represents high expression, blue represents low expression. The above results show that compared with untreated KO mice, 41 genes were consistently and significantly overexpressed or underexpressed in two guides treated KO and WT mice, of which 9 genes were upregulated and 32 genes were downregulated.

[0077] Further, as attached Figure 4 Gene Ontology (GO) enrichment analysis shown in Figure e shows that compared with untreated KO mice, the downregulated genes in two guides-treated KO mice were mainly enriched in the following pathways: (1) fatty acid metabolism; (2) butyrate metabolism; (3) carboxylic acid transmembrane transport; (4) adipocyte differentiation; (5) cAMP signaling pathway; (6) glucose homeostasis. The downregulation of these metabolism-related pathways to near WT levels may partly explain the decrease in dysregulated genes after two guides treatment. H / dy H Weight recovery of mice.

[0078] In summary, the present invention uses AAV9-mediated SpdCas9-VP64 / gRNAs to activate Lama1 gene transcription for dysgenesis H / dy H mice. At low doses, dy H / dy H The median survival of mice was extended to nearly twice that of the untreated group, body weight and grip strength were improved throughout the life cycle, and muscle pathology, muscle imaging and serum biochemical indicators were significantly improved. When the dose was doubled, the survival of mice was polarized. Nearly half of the mice died within the first two weeks of administration, and the survival of the other half of the mice was extended to four times that of the untreated group. Overall, low-dose treatment is safe and effective, and high-dose treatment has both benefits and risks. It can be seen that the research of the present invention shows that early intervention to upregulate Lama1 can prolong dy H / dy H The study showed that the new gene therapy could extend the lifespan of mice, improve motor function, and halt pathological progression, and has great therapeutic potential for human LAMA2-MD.

[0079] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An expression vector for targeted transcriptional activation of Lama1 gene, characterized in that: The expression vector is an AAV9-mediated transcriptional activation Lama1 gene expression vector constructed based on a dual AAV9 vector delivery CRISPRa system.

2. The expression vector for targeted transcriptional activation of Lama1 gene according to claim 1, characterized in that: The expression vector includes an expression vector 1 for AAV9-mediated transcriptional activation of the Lama1 gene; The expression vector 1 comprises the promoter miniCMV, the nuclease-inactive SpdCas9 derived from Streptococcus pyogenes, and the transcription activator VP64, and the expression vector is recorded as AAV9-miniCMV-SpdCas9-VP64.

3. The expression vector for targeted transcriptional activation of Lama1 gene according to claim 1, characterized in that: The expression vector includes an expression vector 2 for AAV9-mediated transcriptional activation of the Lama1 gene; The expression vector 2 contains promoter U6, gRNA2 and gRNA3 targeting the Lama1 gene promoter sequence, and the expression vector is recorded as AAV9-U6-gRNA2-U6-gRNA3-CAG-MPH-GFP.

4. The expression vector for targeted transcriptional activation of Lama1 gene according to claim 3, characterized in that: The characteristic sequence of the gRNA2 targeting the Lama1 gene promoter sequence is: 5′-GCGCCCAGGTCTGTCCTCCA-3′.

5. The expression vector for targeted transcriptional activation of Lama1 gene according to claim 3, characterized in that: The characteristic sequence of the gRNA3 targeting the Lama1 gene promoter sequence is: 5′-GTCCGAAGGGCCTGCGCACC-3′.

6. A method for constructing an expression vector for targeted transcriptional activation of the Lama1 gene according to any one of claims 1 to 5, characterized in that: The method comprises the steps of using plasmid to transfect cells, and collecting the virus solution after transfection, purifying, concentrating, ultrafiltration desalting, filtering and sterilizing to obtain the target vector.

7. Use of the expression vector for targeted transcriptional activation of the Lama1 gene according to any one of claims 1 to 5 for preparing a drug for treating LAMA2-related muscular dystrophy symptoms; Preferably, the LAMA2-related muscular dystrophy symptoms include LAMA2-related congenital muscular dystrophy (LAMA2-CMD) and / or autosomal recessive limb-girdle muscular dystrophy type 23 (LGMDR23).

8. A pharmaceutical preparation for treating LAMA2-related muscular dystrophy, characterized in that: It comprises the expression vector and gRNA for targeted transcriptional activation of the Lama1 gene as described in any one of claims 1 to 5.

9. Use of the expression vector for targeted transcriptional activation of Lama1 gene according to any one of claims 1 to 5 for preparing a drug for transcriptional activation of Lama1 gene.

10. A pharmaceutical preparation for transcriptionally activating the Lama1 gene, characterized in that: It comprises the expression vector and gRNA for targeted transcriptional activation of the Lama1 gene as described in any one of claims 1 to 5.

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