CN118028371A

CN118028371A - Expression vector for targeted transcriptional activation of Lama1 gene and application thereof

Info

Publication number: CN118028371A
Application number: CN202410177113.2A
Authority: CN
Inventors: 熊晖; 刘艺丹; 谈丹丹; 张弘
Original assignee: Peking University First Hospital
Current assignee: Peking University First Hospital
Priority date: 2024-02-08
Filing date: 2024-02-08
Publication date: 2024-05-14

Abstract

The invention belongs to the technical field of biological gene engineering, and particularly relates to an expression vector of a targeted transcription activation Lama1 gene and application thereof in preparing medicines for treating Lama 2-related muscular dystrophy. The expression vector of the targeted transcription activation Lama1 gene is prepared by constructing AAV 9-CMV-SpdCas-VP 64 and AAV9-U6-gRNA2-U6-gRNA3-CMV-GFP of AAV 9-mediated transcription activation substitution gene Lama1, and the improvement effect of AAV 9-mediated transcription activation Lama1 on Lama2-CMD mouse disease phenotype is proved.

Description

Expression vector for targeted transcriptional activation of Lama1 gene and application thereof

Technical Field

The invention belongs to the technical field of biological gene engineering, and particularly relates to an expression vector of a targeted transcription activation Lama1 gene and application thereof in preparing medicines for treating Lama 2-related muscular dystrophy.

Background

LAMA2-related muscular dystrophy (LAMA 2-related muscular dystrophy, LAMA 2-MD) is an autosomal recessive inherited myopathy due to pathogenic variations in the LAMA2 gene. Depending on the age of onset and the severity of the clinical manifestations, it is generally classified into two subtypes, namely LAMA2-related congenital muscular dystrophy (LAMA 2-related congenital muscular dystrophy, LAMA 2-CMD) and autosomal recessive genetic limb banding muscular dystrophy type 23 (autosomal recessive limb-girdle muscular dystrophy-23, lgmdr 23). Among them, LAMA2-CMD is the most predominant subtype of LAMA2-MD, and is also the most common congenital muscular dystrophy (congenital muscular dystrophy, CMD) subtype, accounting for about one third of worldwide CMD patients. LAMA2-MD is a serious disabling and fatal rare disease, and clinically presents as extensive symmetric muscle weakness, reduced muscle tension, abnormal brain white matter signals, joint and spinal deformity and the like, so that diagnosis and treatment of LAMA2-MD still have a plurality of challenges, and an effective treatment method is not yet clinically available.

The study showed that the human LAMA2 gene was located at 6q22.23, containing 65 exons, and the cDNA was approximately 9.4kb in length. The gene codes laminin-alpha 2 (laminin-alpha 2) and consists of 3122 amino acids, and is an important extracellular matrix protein. Laminin-alpha 2 is expressed primarily in muscle, skin, trophoblasts, peripheral nerves and cerebrovascular endothelial cells. The Laminin-alpha 2, beta 1 and gamma 1 proteins are assembled together to form a heterotrimeric protein Laminin-211, and participate in the formation of skeletal muscle basement membrane, playing an important role in muscle cell contraction, regeneration, differentiation and migration. LAMA2 pathogenicity changes cause complete or partial deletion of laminin-alpha 2, which can lead to insufficient assembly of laminin-211, unstable extracellular matrix structure, connection damage of cell membranes and extracellular matrix, separation of basement membrane from sarcoplasmic membrane, and damage of contraction load of muscle fibers and abnormal cell signal transduction pathways. Patient muscle biopsies can see pathological changes in muscular dystrophy, myofiber dysplasia, degeneration, necrosis and regeneration, and interstitial connective tissue hyperplasia, resulting in LAMA2-MD.

According to the possible pathogenesis and intervention links of the muscle lesions of LAMA2-MD, the therapeutic studies of LAMA2-MD are mainly classified into 3 categories: the LAMA2 gene is interfered by various technical means, for example, the LAMA2 transgene overexpression is carried out on a knock-out mouse dy ^W/dy^W, or the nonsense reading is carried out on the LAMA2 nonsense mutation at the cellular level, and the LAMA2 splicing mutation can be corrected by the methods of antisense oligonucleotide mediated LAMA2 exon 4 jumping, CRISPR/Cas9 and the like; secondly, the method aims at the transgenic overexpression of the connexin or the homologous protein substitution, such as the connexin transgene of mini-agrin, integrin-alpha 7, the transgenic overexpression of the homologous protein laminin-alpha 1, alpha LNNd and the like aiming at laminin-alpha 4, or exogenous supplement laminin-111 and the like; thirdly, downstream events such as Bcl-2 overexpression, omigapil inhibition of apoptosis, N-acetylcysteine, vitamin E and other antioxidation, prednisone anti-inflammatory, losartan improvement of muscle tissue fibrosis, insulin-like growth factor-1 overexpression promotion of dy ^W/dy^W mouse muscle regeneration and the like are improved.

At present, although the therapeutic research on LAMA2-MD is increasingly paid attention to, many problems remain unsolved. On the one hand, although correcting LAMA2 gene mutation appears to be the most fundamental treatment regimen, since LAMA2 cDNA is about 9.4kb in its entire length, no suitable vector can achieve exogenous introduction of full-length LAMA2 cDNA, nor functional truncated cDNA sequences are found, making LAMA2 gene replacement therapy difficult to develop in basic research and clinical trials; on the other hand, research shows that nonsense read-through fails to improve laminin-alpha 2 expression at the cellular level, exon 4 skipping does not improve LAMA2-MD mice, CRISPR/Cas9 has a certain effect on correcting splicing mutation, but LAMA2 mutation types are numerous, no hot spot mutation exists, and gene modification aiming at a certain LAMA2 mutation has great limitation in application. In addition, therapeutic studies aimed at increasing the expression of connexins and homologous proteins, improving secondary cellular events, and the like have only partially obtained a degree of improvement of motor function and muscle pathology in animal models, whereas studies on methods for evaluating improvement of motor function and prolongation of survival are relatively few. Thus, it is unknown how to select a suitable carrier, route of administration, and when to administer therapeutic interventions, etc., and the treatment of LAMA2-MD remains a challenge and difficulty.

Currently, only laminin- α1 and laminin- α2 are most similar in structure and function among the 5 homologous laminin chains known. Wherein, laminin-alpha 1 is encoded by LAMA1 gene and is also an important extracellular matrix component, which plays an important role in regulating and controlling cell adhesion, cell migration, tissue organ development, nerve axon growth and the like in the embryo development period. Laimin-alpha 1, beta 1 and gamma 1 form a Laminin-111 trimer. Laimin-gamma 1 is connected with nidogen-1 protein, so that the Laminin-111 is indirectly connected with IV type collagen and perlecan protein, a loose Laminin network becomes stable, and a stable basement membrane is formed. LAMA1 is also known as a homologous gene of LAMA2, and its encoding product laminin- α1 has a growing role in the treatment of LAMA 2-MD. Studies have shown that the laminin-111 protein or recombinant human laminin-111 protein extracted from Engelbreth-Holm-Sarm (EHS) mouse tumors can improve the muscular dystrophy phenotype of dy ^W/dy^W mice. However, this method requires multiple doses in succession, rather than a single dose for life. Since laminin- α1 is expressed mainly during embryonic development, postnatal expression is rapidly reduced and hardly expressed in the adult neuromuscular system. However, the two homologous genes LAMA1 and LAMA2 are similar in size, and the full-length substitution cannot be realized. Therefore, how to increase endogenous full-length laminin- α1 expression becomes an important therapeutic target.

With the development and application of AAV vectors and CRISPR/Cas9 technology, gene-related therapies for monogenic genetic diseases have come to be new, especially with breakthrough progress in gene-related therapies for hereditary neuromuscular diseases. The expression of the homologous gene is increased to achieve the aim of treatment, for example, in the gene modification treatment of spinal muscular atrophy (spinal muscular atrophy, SMA) caused by the pathogenic variation of the SMN1 gene, the transcription of the homologous gene SMN2 exon 7 is promoted through shearing regulation, so that the expression of the full-length SMN2 is increased, the full-length SMN protein is obtained, and two medicines are marketed in batches and are applied in the global scope. Thus, it is possible to achieve alternative therapeutic effects by activating LAMA1 expression by transcription early in life. Previous studies have demonstrated the efficacy of CRISPR/Cas9 transcriptional activation system on dy ^2J/dy^2J mice, however, the effect of this approach on Lama2 deficient mice survival cannot be studied because dy ^2J/dy^2J mice have normal life.

Thus, an appropriate disease model is needed for the assessment of transcriptional activation LAMA1 therapy. The current mouse models for the foreign study of LAMA2-MD are dy/dy, dy ^2J/dy^2J、dy^3K/dy^3K and dy ^W/dy^W constructed in the last century. Wherein the Lama2 mutation of dy/dy is unknown; dy ^2J/dy^2J is due to deletion of the 2 nd exon of Lama2 by a cleavage mutation, affects the phenotype slightly and hardly affects the survival; dy ^3K/dy^3K and dy ^W/dy^W, although both had severe muscular dystrophy phenotypes, were generated by mutations at the 3 'end of exon 4 and at the 5' end of exon 1 of Lama2, respectively, according to the old animal model construction method. However, there is no related animal model in China.

The Lama2 gene knockout mouse model disclosed in Chinese patent CN116814687A is a model mouse which is based on a clinically found region of Lama2 suffering from high frequency mutation in China, and is successfully constructed by adopting CRISPR/Cas9 technology to cause homozygous deletion of 3 rd exon and frame shift mutation of 5' end of 4 th exon of Lama2 frame mutation, and is named dy ^H/dy^H. The early research shows that the LAMA2-CMD mouse model is suitable for the research on the survival time, weight, exercise, muscle pathology, imaging, molecular mechanism and other aspects of mice before and after treatment. Based on the model, how to simultaneously introduce a transcriptional activation domain and accurately reach a mouse Lama1 gene needs to be solved, and gRNA (guide RNA) aiming at a Lama1 promoter needs to be designed, which is also a problem to be solved in the field.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide an expression vector for targeted transcriptional activation of a Lama1 gene, wherein the expression vector can transcriptionally activate the Lama1 gene to treat dy ^H/dy^H mice based on an AAV 9-mediated CRISPRa system.

The second technical problem to be solved by the invention is to provide the application of the expression vector of the targeted transcription activation Lama1 gene in preparing medicines for treating Lama 2-related muscular dystrophy, and research shows that early intervention and upregulation of Lama1 can prolong the life of dy ^H/dy^H mice, improve motor functions, prevent pathological progression and have great therapeutic potential for human Lama 2-MD.

In order to solve the technical problems, the expression vector of the targeted transcription activation Lama1 gene is constructed based on a double AAV9 vector delivery CRISPRa system, and is an AAV 9-mediated expression vector of the transcription activation Lama1 gene.

Specifically, the expression vector of the targeted transcription activation Lama1 gene comprises an AAV9 mediated transcription activation Lama1 gene expression vector 1;

The expression vector 1 comprises promoters CMV, streptococcus pyogenes source SpdCas without nuclease activity and a transcriptional activator VP64, and is named AAV9-CMV-SpdCas9-VP64.

Specifically, the expression vector of the targeted transcription activation Lama1 gene comprises an AAV9 mediated transcription activation Lama1 gene expression vector 2;

the expression vector 2 comprises a promoter U6, gRNA2 and gRNA3 for targeting the promoter sequence of the Lama1 gene, and is named AAV9-U6-gRNA2-U6-gRNA3-CMV-GFP.

Specifically, the expression vector of the targeted transcription activation Lama1 gene is characterized in that the gRNA2 of the targeted Lama1 gene promoter sequence has the following sequence:

5’-GCGCCCAGGTCTGTCCTCCA-3’。

Specifically, the expression vector of the targeted transcription activation Lama1 gene is characterized in that the gRNA3 of the targeted Lama1 gene promoter sequence has the following sequence:

5’-GTCCGAAGGGCCTGCGCACC-3’。

The invention also discloses a method for constructing the expression vector of the targeted transcription activation Lama1 gene, which comprises the steps of transfecting 293T cells by using plasmids, collecting transfected virus liquid, and purifying to obtain a target vector. Specifically, the transfected virus liquid is purified, concentrated, ultrafiltered, desalted, filtered and sterilized to obtain the target carrier.

The invention also discloses application of the expression vector of the targeted transcription activation Lama1 gene in preparing medicines for treating Lama 2-related muscular dystrophy symptoms.

Preferably, the LAMA 2-related muscular dystrophy symptoms include LAMA 2-related congenital muscular dystrophy (LAMA 2-CMD) and/or autosomal recessive genetic limb banding muscular dystrophy type 23 (LGMDR).

In particular, the LAMA 2-related muscular dystrophy includes LAMA2-CMD type LAMA 2-related congenital muscular dystrophy.

The invention also discloses a pharmaceutical preparation for treating LAMA 2-related muscular dystrophy, which comprises the expression vector and gRNA of the targeted transcription activation LAMA1 gene.

The invention also discloses application of the expression vector of the targeted transcription activation Lama1 gene in preparing a transcription activation Lama1 gene medicine.

The invention also discloses a pharmaceutical preparation of the transcription activation Lama1 gene, which comprises the expression vector and gRNA of the targeted transcription activation Lama1 gene.

The expression vector and the pharmaceutical preparation of the targeted transcription activation Lama1 gene screen gRNA with the best transcription activation Lama1 gene effect from the cellular level. C2C12 mouse myoblasts are transfected by lentivirus for targeted transcriptional activation of Lama1, flow sorting and culture are carried out, the expression level of the transfected Lama1 is detected, and gRNA(s) with the best transcriptional activation effect is screened.

The expression vector of the targeted transcription activation Lama1 gene is constructed, and the improvement effect of AAV 9-mediated transcription activation Lama1 on Lama2-CMD mice disease phenotype is further verified by comparing the change conditions of survival time, weight, exercise function, skeletal muscle MRI, muscle pathology, biochemical indexes and the like of AAV 9-mediated transcription activation substitution gene Lama1 transcription activation intervention Lama2-CMD mice, non-intervention Lama2-CMD mice and Wild Type (WT) mice.

The expression vector of the targeted transcription activation Lama1 gene is prepared by injecting proper amount of expression vectors 1 and 2 (1:1) into P3 neonatal mice by subcutaneous injection, performing transcriptome analysis on muscle tissues of Lama1 transcription activation intervening Lama2-CMD mice, non-intervening Lama2-CMD mice and WT mice on 21 st day after the birth, and comparing the change condition of related genes in key pathogenic pathways of Lama2-CMD mice after the Lama1 transcription activation. Through the above study, it is demonstrated that AAV 9-mediated transcriptional activation of Lama1 can rescue key pathogenic pathways abnormally regulated by LAMA2-CMD mice, revealing the molecular mechanism of action of Lama1 transcriptional activation therapy.

The invention starts from the international relevant research front, starts from clinical problems, performs basic research and aims to solve the clinical potential treatment problem, and firstly develops the related research of the LAMA2-CMD gene treatment in China. The invention adopts AAV9 mediated CRISPR/Cas9 transcriptional activation technology, researches improvement conditions of Lana 1 on LAMA2-CMD mice in early postnatal transcriptional activation, particularly prolongation conditions of survival time, and provides a preclinical theoretical basis for clinical application of substitution gene therapy in LAMA 2-CMD.

According to the expression vector for targeted transcription activation of the Lama1 gene, a CRISPRa transcription activation system is utilized, 3 gRNAs are independently designed to target a promoter region within 200bp upstream of a transcription initiation site of the mouse Lama1 gene, and the effectiveness of the system is verified through a cell experiment and animal experiments.

The fusion vector of CRISPR/Cas9 and the transcriptional activation structural domain VP64 acts on the promoter region of the Lama1 to promote the transcriptional activation of the Lama1 and increase the expression of endogenous laminin-alpha 1 protein. AAV9 mediated SpdCas-VP 64/gRNAs are simultaneously introduced to efficiently transcribe and activate homologous genes Lama1, so that the expression of Lama2-CMD mouse laminin-alpha 1 protein is increased, the functions of laminin-alpha 2 protein are replaced to a certain extent, and the serious phenotype of the Lama2-CMD mouse is improved from the aspects of life cycle, weight growth, exercise function, muscle pathology, pathogenic pathways and the like, thereby treating Lama2-MD.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,

FIG. 1 shows the results of in vitro and in vivo experiments for transcription activated Lama1 of example 1;

FIG. 2 shows the results of example 2 in which upregulation of Lama1 increases the longevity, weight gain, and strength of the extremities of dy ^H/dy^H mice;

FIG. 3 shows that upregulation of Lama1 in example 3 improves muscle imaging, biochemical index and muscle pathology results in dy ^H/dy^H mice;

FIG. 4 shows the results of the recovery of expression levels of a portion of the genes from dy ^H/dy^H mice by upregulation of Lama1 in example 4.

Detailed Description

Example 1 results of in vitro and in vivo experiments for transcriptional activation of Lama1

In this example, in order to study the therapeutic effect of the transcription activated LAMA1 gene on LAMA2-CMD mice and correct key pathogenic pathways in model mice, in vitro experiments were first performed.

In this example, the fusion vector SpdCas and the transcriptional activation domain VP64 act on the promoter region of the mouse Lama1 gene to promote Lama1 transcriptional activation so as to increase endogenous laminin-alpha 1 protein expression.

As shown in the process a in fig. 1, according to the CRISPR/Cas9 target design principle, 3 gRNAs were designed to target the promoter region within 200bp upstream of the transcription start site of the mouse Lama1 gene, i.e., three gRNAs (gRNA 1-gRNA 3) were designed to target the proximal promoter region of the mouse Lama1, which is located within 200bp upstream of the transcription start site (ATG represents translation start site). Arrows indicate the direction of each gRNA. The sequence of each gRNA is adjacent to the PAM sequence (3' ngg), with lower case and upper case letters representing the intron and exon sequences, respectively.

C2C12 mouse myoblasts were transfected with lentiviral packaging transcription activation system vector (pLV-hU 6-gRNA-CMV-dCAS9-VP 64-T2A-mScarlet) containing SpdCas-VP 64 and corresponding gRNA, as shown in FIG. 1 b. And 3 lentiviruses with gRNA (gRNA 1-gRNA 3) and a control lentivirus without gRNA (no guide) are respectively transfected into C2C12 mouse myoblasts, and the C2C12 cells positive to transfection are further screened by using flow sorting for cell culture and total RNA is extracted. The RT-qPCR assay was used to detect the relative mRNA levels of Lama1/Gapdh (data expressed as "mean.+ -. Standard deviation; n=3 independent samples).

In this example, the results of RT-qPCR as shown in FIG. 1b show that the increase in expression of Lana 1 mRNA was most pronounced in C2C12 cells by lentiviruses containing gRNA2, followed by lentiviruses containing gRNA 3.

As shown in fig. 1c and d, based on the clinically found LAMA2 high frequency mutation region of chinese patient, we successfully constructed a model mouse with homozygous 3 rd exon deletion and 4 rd exon 5' frameshift mutation, which caused LAMA2 out-of-frame mutation, using CRISPR/Cas9 technology, we named dy ^H/dy^H (subsequently "dy ^H/dy^H" as "KO"). The previous research shows that the LAMA2-CMD mouse model is a new LAMA2-CMD mouse model, and is suitable for various researches on the survival time, weight, exercise, muscle pathology, imaging, molecular mechanism and the like of the mice before and after treatment, such as a scheme recorded in Chinese patent CN 116814687A.

According to the results of in vitro experiments, two gRNAs with stronger transcriptional activation (gRNA 2 and gRNA 3) were selected in this example. Because of the limited packaging space of AAV9, the transcriptional activation systems SpdCas-VP 64 and gRNA2-gRNA3 are packaged within 2 AAV9 (AAV 9-CMV-SpdCas9-VP64 and AAV9-U6-gRNA2-U6-gRNA 3-CMV-GFP), respectively. As shown in the procedure of fig. 1 e, mice were intervened on postnatal day 3 (P3) by a single subcutaneous injection of AAV9, drawing material at P21.

In this example, P21 WT mice, untreated KO mice and two guides treated KO mice were analyzed by Western blot for expression of biceps femoris laminin-alpha 1, GFP and Gapdh. As shown in FIG. 1 f, western blot results showed that laminin- α1 protein was not expressed in both the WT mice and the biceps femoris of untreated KO mice, and only after two guides days, GFP expression represented AAV9 transfection was successful.

The immunofluorescence results (scale bar, 20 μm) shown in FIG. 1 g were analyzed for the expression of laminin- α2 protein and laminin- α1 protein in P21 WT mice, untreated KO mice, and two guides-treated KO mice in biceps femoris and diaphragm. Immunofluorescence showed that the KO mice were deficient in the laminin- α2 protein of the biceps femoris and diaphragm cell membranes and that laminin- α1 protein was expressed following two guides intervention.

EXAMPLE 2 upregulation of Lama1 increases the longevity, weight gain and limb strength in dy ^H/dy^H mice

In the experimental scheme of this example, experimental mice were divided into the following 5 groups:

(1) WT group;

(2) Untreated KO group;

(3) no guide treatment of KO group (AAV 9-CMV-SpdCas9-VP64 of 1.0X10 ¹¹ vg);

(4) The KO group (0.5X10 ¹¹ vg of AAV 9-CMV-SpdCas-VP 64 and 0.5X10 ¹¹ vg of AAV9-U6-gRNA2-U6-gRNA3-CMV-GFP, total dose 1.0X10 ¹¹ vg) was treated with low dose two guides;

(5) High dose two guides treatment of KO group (1.0X10 ¹¹ vg AAV 9-CMV-SpdCas-VP 64 and 1.0X10 ¹¹ vg AAV9-U6-gRNA2-U6-gRNA3-CMV-GFP, total dose 2.0X10 ¹¹ vg).

As shown in the procedure of fig. 2a, P3 neonatal dy ^H/dy^H mice were subcutaneously injected with no guide AAV9 (1.0×10 ¹¹ vg), low dose two guides AAV (split into two vectors; total dose 1.0×10 ¹¹ vg), or high dose two guides AAV9 (split into two vectors; total dose 2.0×10 ¹¹ vg) and death was observed. Specifically, (1) and (2) mice were not interfered with, and (3) - (5) mice were interfered with by a single subcutaneous injection of AAV9 on day 3 (P3) after birth, all KO mice observed natural death for a total observation period of 1 year.

The Kaplan-Meier survival analysis results as depicted in fig. 2b showed that the median survival time of untreated KO mice was 25.5 days (n=10, range 4-44 days) and WT mice did not die during the observation period (n=10). Median survival times for no guide, low dose two guides and high dose two guides treatment KO groups 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 untreated KO group and no guide treated KO group were very similar, with no significant difference in survival time. In contrast, the median survival time of the low dose two guides treatment KO group was increased to 50 days (range 10-86 days). When the total AAV9 dose reaches 2.0X10 ¹¹ vg (KO group is treated by high dose two guides), the survival period shows two-stage differentiation (range 6-114 days), namely, nearly half of mice die in the first two weeks, the survival period of the other half of mice is greatly improved, and the survival time is prolonged to about 100 days.

As shown in fig. 2c, the unpaired t-test shows that the difference in survival time was statistically significant in untreated KO group compared to low dose two guides KO group (p=0.0039, ×p < 0.01), untreated KO group compared to high dose two guides KO group (p=0.028, ×p < 0.05), low dose two guides KO group compared to no guide KO group (p=0.0056, ×p < 0.01). The untreated KO group was not significantly different from the no guide treated KO group (p=0.7, ns, p. Gtoreq.0.05).

In this example, body weight and grip were measured twice a week while observing lifetime. The grip test was repeated 3 times for each mouse and averaged (data collected and analyzed using "SuperGSM" software).

As shown in fig. 2d, the line graph shows weight changes for 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) mice for 1-17 weeks. Data are expressed as "mean ± standard deviation" measured twice a week, "n" being the number of mice per group at the time of the first measurement. It can be seen that in the untreated case, the body weight of the KO mice reached a peak at 2 weeks, and the average body weight was 6.05g at the maximum, and gradually decreased thereafter. no guide treatment KO group was almost completely identical thereto. The high dose two guides treatment KO group reached peak plateau at 5-8 weeks with a maximum average body weight of 10.97g. The low dose two guides treatment KO group reached peak plateau at 5-6.5 weeks with a maximum average body weight of 8.18g.

As shown in fig. 2 e, the line graph shows the limb grip changes in 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), high dose two guides treated KO group (n=9) mice over 2-17 weeks. Data are expressed as "mean ± standard deviation" measured twice a week, "n" being the number of mice per group at the time of the first measurement. It can be seen that the untreated KO group and the no guide treated KO group remained at a similarly low level, with a maximum of about 20gf in terms of grip. In contrast, the grip of the high and low dose two guides treated group KO mice was increased to about 30gf.

As a result of d and e in fig. 2, overall, the weight and strength differences between the groups gradually increased from week 3. Starting at week 3 and ending with observations, two guides treated KO groups had a higher body weight and grip than untreated KO groups and no guide treated KO groups.

As a result of f in fig. 2, littermates received different interventions at P3 and were photographed at P21. It can be seen that, in appearance, two guides treated KO mice were significantly larger than untreated KO mice and no guide treated KO mice, with no significant difference between high and low dose two guides treated KO mice.

EXAMPLE 3 upregulation of Lama1 improves the muscle imaging, biochemical index and muscle pathology results in dy ^H/dy^H mice

In this example, the P3 subcutaneous single injection AAV9 (low dose two guides) was chosen for further study as an intervention, considering the safety issues with the high dose group and that the improvement in muscle function was not superior to the low dose group.

KO mice were subcutaneously injected with low dose two guides AAV (split into two vehicles; total dose 1.0X10 ¹¹ vg) at P3, and P21 was chosen as the time of draw, given that the differences in phenotype of untreated versus two guides treated groups were apparent at week 3.

In this example, skeletal muscle improvement in KO mice after two guides treatment was studied by muscle MRI.

P3 KO mice were subcutaneously injected with AAV9, WT mice, untreated KO mice and two guides treated KO mice were subjected to hind limb muscle MRI examination at P21. The mice muscle MRI images were analyzed using Image J software, n=4 for each group, and statistically analyzed using t-test. As a result shown in fig. 3 a, adipose tissue exhibits a high signal on T1-weighted (T1W) MRI. The relative muscle area (muscle area/fat area) of untreated KO mice was significantly lower than WT mice (p= 0.000026, < p < 0.0001). The relative muscle area of two guides treated KO mice was smaller than WT mice (p=0.0082, × p < 0.01), but larger than untreated KO mice (p=0.0053, × p < 0.01). On T2-weighted (T2W) MRI, a high signal suggests muscle edema (WT vs untreated KO, p=0.0038, < p <0.01; untreated KO vs two guides treated KO, p=0.0073, < p <0.01;WT vs two guides treated KO, p=0.035, < p < 0.05). Untreated KO mice found significantly increased areas of high signal, indicating muscle edema. After two guides treatment, the fat infiltration and edema of the hind limb muscles of the KO mice were improved.

Blood of the mice was collected at P21, and serum was separated after centrifugation, and biochemical indexes related to skeletal muscle injury such as serum Creatine Kinase (CK), glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) were detected, and the results are shown in FIG. 3 b. Wherein, CK: WT vs untreated KO, p=0.0052, <0.01; untreated KO vs two guides treated KO, p=0.011, p <0.05; WT vs two guides treatment KO, p=0.024, p <0.05.ALT: WT vs untreated KO, p=0.0046, <0.01; untreated KO vs two guides treated KO, p=0.0095, <0.01; WT vs two guides treat KO, p=0.011, p <0.05.AST: WT vs untreated KO, p=0.0058, <0.01; untreated KO vs two guides treated KO, p=0.014, < p 0.05; WT vs two guides treatment KO, p=0.017, p <0.05. N=6 for each group, and the statistical analysis used t-test. It can be seen that untreated KO mice had significantly elevated serum CK compared to WT mice. In two guides treated KO mice, serum CK was reduced compared to untreated KO mice. ALT and AST were significantly elevated in untreated KO mice and partially recovered in two guides treated KO mice.

In this example, H & E staining as shown in FIG. 3c (scale bar, 20 μm) shows that at P21, WT mice, untreated KO mice and two guides treated KO mice were H & E stained for biceps femoris and diaphragm (left) and sirius red stained (right). H & E staining showed that untreated KO mice were not equal in myofiber size and inflammatory cell infiltration (left arrow). Sirius red staining showed increased collagen red staining in skeletal muscle of untreated KO mice (right arrow). These pathological changes were partially restored in two guides treated KO mice. The results of the analysis were similar for all mouse samples (n=3 per group). As can be seen, H & E staining showed that the untreated KO mice had biceps femoris and diaphragmatic muscle fibers of unequal size, myofibrosis, necrosis, regeneration, connective tissue hyperplasia and inflammatory cell infiltration, consistent with typical muscular dystrophy-like pathological changes. While sirius red staining showed an increase in red-stained collagen fibers in the extracellular matrix of skeletal muscle of untreated KO mice, indicating pathological fibrosis. The above pathological changes were partially restored in two guides treated KO mice. The myocardium of KO mice is relatively unaffected.

Taken together, these results indicate that upregulation of Lama1 can improve muscle imaging, biochemical index and muscle pathology in Lama2 deficient mice.

EXAMPLE 4 upregulation of Lama1 restored the expression level of the dy ^H/dy^H mouse partial Gene

In this example, further RNA sequencing (RNA-seq) was performed on the biceps femoris of P21 mice, data from n=3 mice per group.

As a result, as shown in fig. 4 a-c, the most Differentially Expressed Genes (DEGs) (blue label) between groups were "untreated KO vsWT" (1402 DEGs), followed by "two guides treated KO vs WT" (449 DEGs), and finally "two guides treated KO vs untreated KO" (72 DEGs) according to |log FoldChange | > =0.5 and padj < 0.05. The upregulation of Lama1 was confirmed in two guides treated KO mice compared to untreated KO mice or WT mice.

As shown in figure 4d by DEGs heat map, red represents high expression and blue represents low expression in comparison to untreated KO mice, wt mice or two guides treated KO mice according to |log FoldChange | > =0.5 and p < 0.05. The above results show that 41 genes are consistently and significantly over-expressed or under-expressed in two guides treated KO and WT mice, with 9 genes up-regulated and 32 genes down-regulated, compared to untreated KO mice.

Further, gene Ontology (GO) enrichment analysis as shown in fig. 4 e shows that the two guides down-regulated genes of treated KO mice are mainly enriched in the following pathways compared to untreated KO mice: (1) fatty acid metabolism; (2) a butyrate metabolic process; (3) carboxylic acid transport across a membrane; (4) adipocyte differentiation; (5) cAMP signaling pathway; (6) glucose homeostasis. Down-regulation of these metabolic-related pathways to near WT levels might explain, in part, the recovery of body weight in dy ^H/dy^H mice following two guides treatments.

Taken together, AAV 9-mediated SpdCas-VP 64/gRNAs transcriptional activation was used in the present protocol to treat dy ^H/dy^H mice using the Lama1 gene. At low doses, it was observed that median survival in dy ^H/dy^H mice was prolonged to nearly twice that of untreated groups, and that body weight and grip strength were improved over the full life cycle, with significant advances in muscle pathology, muscle imaging and serum biochemical index. When the dose was doubled, the mice survived in two stages of differentiation. Nearly half of the mice die within the first two weeks of dosing, and the other half of the mice survive four times longer than the untreated group. Overall, low dose therapies are safe and effective, and high dose therapies benefit and risk. Therefore, the research of the invention shows that early intervention and upregulation of Lama1 can prolong the service life of dy ^H/dy^H mice, improve the exercise function, prevent pathological progress and have great therapeutic potential for human Lama 2-MD.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. An expression vector of a targeted transcription activation Lama1 gene, which is characterized in that the expression vector is an AAV 9-mediated transcription activation Lama1 gene constructed based on a double AAV9 vector delivery CRISPRa system.

2. The expression vector of claim 1, wherein the expression vector comprises expression vector1 of AAV 9-mediated transcription activation Lama1 gene;

3. The expression vector of claim 1, wherein the expression vector comprises expression vector2 of AAV 9-mediated transcription activation Lama1 gene;

4. The expression vector of claim 3, wherein the sequence of the gRNA2 targeting the promoter sequence of the Lama1 gene is characterized by:

5’-GCGCCCAGGTCTGTCCTCCA-3’。

5. the expression vector of claim 3, wherein the sequence of the gRNA3 targeting the promoter sequence of the Lama1 gene is characterized by:

5’-GTCCGAAGGGCCTGCGCACC-3’。

6. A method of constructing an expression vector for targeted transcriptional activation of Lama1 gene according to any one of claims 1 to 5, comprising the steps of transfecting cells with a plasmid, and collecting the transfected virus solution, and purifying the virus solution to obtain the target vector.

7. Use of an expression vector of a targeted transcriptional activation Lama1 gene according to any one of claims 1-5 for the preparation of a medicament for the treatment of Lama 2-related muscular dystrophy symptoms;

8. A pharmaceutical formulation for the treatment of LAMA 2-related muscular dystrophy comprising an expression vector and a gRNA of any one of claims 1-5 that targets the transcription activated LAMA1 gene.

9. Use of an expression vector of a targeted transcription activated Lama1 gene according to any one of claims 1-5 for the preparation of a medicament for transcription activated Lama1 gene.

10. A pharmaceutical formulation of a transcription activated Lama1 gene, comprising an expression vector and a gRNA of the targeted transcription activated Lama1 gene according to any one of claims 1-5.