LAMP2B overexpression vector and application thereof

Abstract

The invention discloses an overexpression vector of LAMP2B and application thereof, which can effectively delay the disease progress of DMD, and comprises: (a) an AAV vector which is an adeno-associated virus vector and has high muscle tissue targeting and low liver tropism; (b) a CMV (cytomegalovirus) promoter, wherein the CMV promoter is a cytomegalovirus promoter; (c) a coding sequence of an hLAMP2B gene, namely a coding sequence of a human LAMP2B gene, and the coding sequence of the hLAMP2B gene is operably linked to the CMV promoter; wherein the vector is configured to overexpress LAMP2B protein in muscular tissue, and the vector is self-complementary AAV, namely scAAV, the concept is ingenious, the novel AAV vector with high muscle transformation efficiency is utilized to deliver a complete LAMP2B gene (LAMP2B is a gene related to autophagy regulation) into muscle cells of all organs of a mouse (AAV-hLAMP2B for short), and the autophagy regulation function of the mouse is improved. The muscle tissue function of the mouse DMD disease model is comprehensively assisted to recover, and the disease progress of the DMD is delayed.

Description

Technical Field

[0001] This invention belongs to the field of viral vector technology, and relates to an overexpression vector of LAMP2B and its application. Background Technology

[0002] Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive genetic disorder caused primarily by mutations in the DMD gene that result in a deficiency of dystrophin, leading to the loss of the intact skeletal structure of muscle cell membranes and progressive destruction of muscle cells. Due to muscle weakness, patients with DMD may be unable to breathe or cough normally, eventually leading to progressive respiratory failure. Furthermore, their heart muscle may also be affected, causing decreased cardiac function, heart failure, and other cardiac problems. Therefore, most DMD patients ultimately die from complications such as cardiopulmonary failure.

[0003] Currently, there is no cure for Duchenne muscular dystrophy (DMD). Existing treatments focus on improving quality of life and slowing the progression of disease-related symptoms. Emerging treatments widely utilize adeno-associated virus (AAV) as a vector and exon skipping agents for gene therapy, which focuses on compensating for the impact of mutations on protein synthesis and function at the gene level, thereby restoring the expression and partial function of dystrophin proteins. Current DMD gene therapies depend on the type of mutation carried by the patient, requiring different treatment regimens targeting different DMD gene mutation sites, thus making it difficult to establish a universal, universal, and standardized clinical treatment approach.

[0004] Previous studies have shown that the LAMP2B gene is the main subtype in muscle tissue, and human LAMP2B can also be normally expressed and function in mice. Therefore, an overexpression vector for LAMP2B is needed to help intervene in the course of Duchenne muscular dystrophy. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes an overexpression vector for LAMP2B and its application, which effectively solves the problems in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An overexpression vector for LAMP2B, the vector comprising: (a) An AAV vector, wherein the AAV vector is an adeno-associated virus vector with high muscle tissue targeting and low liver tropism; (b) The CMV promoter, wherein the CMV promoter is a cytomegalovirus promoter; (c) The hLAMP2B gene coding sequence, i.e. the human LAMP2B gene coding sequence, wherein the hLAMP2B gene coding sequence is operatively linked to the CMV promoter; The vector is configured to overexpress LAMP2B protein in muscle tissue, and the vector is a self-complementary AAV, i.e., scAAV.

[0007] Preferably, the vector further comprises an HA tag sequence, a WPRE enhancer sequence, and an SV40 polyA signal sequence.

[0008] Preferably, the CMV promoter sequence is as shown in SEQ NO.1.

[0009] Preferably, the encoding sequence of the hLAMP2B gene is shown in SEQ NO.2.

[0010] Preferably, the AAV vector is the AAV-P7 serotype, which is obtained through AAV9 directed evolution.

[0011] A pharmaceutical composition comprising the above-described LAMP2B overexpression vector and a pharmaceutically acceptable vector.

[0012] One use, as described above, is the use of a LAMP2B overexpression vector in the preparation of a medicament for the treatment of Duchenne muscular dystrophy.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a novel AAV vector with high conversion efficiency in muscle to deliver the complete LAMP2B gene (LAMP2B is a gene related to the regulation of autophagy) into muscle cells of various organs in mice (referred to as AAV-hLAMP2B), comprehensively assisting in the recovery of muscle tissue function in a mouse DMD disease model and delaying the progression of DMD. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the carrier structure of the present invention.

[0015] Figure 2 This is a schematic diagram showing the expression levels of hLAMP2B in major organs of mice in Example 1 of the present invention.

[0016] Figure 3 This is a schematic diagram of the mouse forelimb strength test results in Example 1 of the present invention.

[0017] Figure 4 This is a schematic diagram of the results of the mouse limb strength experiment in Example 1 of the present invention.

[0018] Figure 5 This is a schematic diagram of the mouse movement time verification results in Example 1 of the present invention.

[0019] Figure 6This is a schematic diagram of the mouse movement distance verification results in Example 1 of the present invention.

[0020] Figure 7 This is a schematic diagram of mouse plasma CK levels (UL) in Example 1 of the present invention.

[0021] Figure 8 This is a schematic diagram of HE staining of the heart in Embodiment 2 of the present invention.

[0022] Figure 9 This is a schematic diagram of HE staining of skeletal muscle in Embodiment 2 of the present invention.

[0023] Figure 10 This is a schematic diagram of liver HE staining in Example 2 of the present invention.

[0024] Figure 11 This is a schematic diagram of Masson staining of the heart in Embodiment 2 of the present invention.

[0025] Figure 12 This is a schematic diagram of Masson staining of skeletal muscle in Embodiment 2 of the present invention.

[0026] Figure 13 This is a schematic diagram of Masson staining of the liver in Embodiment 2 of the present invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The following is in conjunction with the appendix Figures 1 to 13 The specific embodiments of the present invention will be described in further detail below.

[0029] Example 1: Construction of LAMP2B overexpression vector: 1.1 Carrier Design: The AAV-P7 serotype was obtained through directed evolution of AAV9, and its capsid protein exhibits high muscle affinity and low liver tropism. It should be noted that the AAV-P7 capsid can be referenced in the Chinese invention patent with authorization announcement number CN116041443B, entitled "Adeno-associated Virus Mutants and Their Applications," which describes the development process and functional characteristics of the AAV-P7 capsid, including its specific sequence, high muscle targeting, and low liver targeting, etc., which will not be elaborated upon here.

[0030] The AAV-P7 vector skeleton includes: CMV promoter (SEQ NO.1): drives hLAMP2B gene expression; hLAMP2B coding sequence (SEQ NO.2): derived from the human LAMP2B gene; HA tag sequence (SEQ NO.3): used for protein detection; WPRE enhancer (SEQ NO.4) and SV40 polyA signal (SEQ NO.5): enhance expression stability.

[0031] Carrier structure such as Figure 1 As shown, this is a self-complementary AAV (scAAV), which can rapidly initiate gene expression.

[0032] 1.2 Carrier Assembly: Recombinant plasmids were constructed using molecular cloning technology: The CMV promoter, hLAMP2B sequence, HA tag, WPRE and SV40 polyA were sequentially inserted into the AAV-P7 vector backbone; Verification was performed by digestion and ligation with restriction endonucleases (such as SmaI and BshTI). SmaI or BshTI can be used as restriction endonucleases. Recombinant plasmids were transfected into HEK-293T cells and packaged to generate scAAV-P-hLAMP2B virus (titer ≥1×10⁻⁶). 12 GC / mL).

[0033] Virus purification was performed using iodixanol gradient centrifugation, and the titer was quantified by qPCR.

[0034] It should be noted that the specific construction process of this viral vector conforms to industry common sense and convention. In this embodiment, it was specifically produced by Guangzhou Paizhen Biotechnology Co., Ltd. using the company's π-Alpha™293 cell AAV platform system.

[0035] 1.3 Feasibility verification of vector infection of myocardium and other muscles: AAV-hLAMP2B virus was injected into 4-week-old male mice via tail vein injection, and the hLAMP2B gene expression level was detected 4 weeks later. hLAMP2B expression levels in major organs of mice are as follows: Figure 2 As shown: Preliminary experimental results showed that, compared with the control group, the AAV-hLAMP2B gene was significantly overexpressed in mouse myocardial tissue, and was also significantly expressed in respiratory muscles, tibialis anterior muscles, gastrocnemius muscles, and other muscles throughout the body, as well as the liver, demonstrating the high efficiency and feasibility of AAV-hLAMP2B viral vector infecting myocardium and other muscles.

[0036] 1.4 Feasibility verification of LAMP2 gene overexpression therapy for DMD: AAV-hLAMP2B virus was injected into 4-week-old male DMD mice via tail vein injection, and the phenotype of the DMD mice was examined 4 weeks later.

[0037] Since cardiomyopathy in DMD mice develops later than limb muscle lesions, the motor function of DMD mice was first assessed in this preliminary experiment: The results of the gripping force test are as follows Figure 3 and Figure 4 As shown, Figure 3 These are the results of a mouse forelimb strength test. Figure 4 Results of the mouse limb strength test: The results showed that, compared with the AAV-GFP control group DMD mice, the forelimb and limb strength of the AAV-hLAMP2B-treated DMD mice were significantly improved in the grip strength test (P<0.05).

[0038] The results of the treadmill experiment are as follows Figures 5-6 As shown, Figure 5 The results were used to validate the mouse movement time. Figure 6 Results of mouse movement distance verification: In the treadmill experiment, the exercise time and distance of DMD mice treated with AAV-hLAMP2B were significantly increased (P<0.05).

[0039] Mouse plasma CK levels (UL) Figure 7 As shown: Plasma CK levels, which reflect muscle injury, were also significantly reduced (P<0.01). Although the phenotypic improvement in DMD mice after AAV-hLAMP2B treatment had not yet reached the level of WT mice, it was already a significant improvement.

[0040] Example 2: Evaluation of the efficacy of the vector in DMD model mice: 2.1 Animal Models and Drug Administration: Four-week-old DMD model mice (mdx mice) were randomly divided into three groups: Treatment group: AAV-hLAMP2B injected via tail vein (dose 1×10⁻⁶) 12 GC / only); Model control group: AAV-GFP injected via tail vein (dose 1×10⁻⁶) 12 GC / only); Wild-type control group (WT): injected with PBS.

[0041] Four weeks after injection, tissue samples were collected from the heart, skeletal muscle, and liver for histological analysis.

[0042] 2.2 Histological analysis: HE staining results of tissues and organs Figures 8-10 As shown: Among them, cardiac HE staining, as shown Figure 8 As shown, compared with WT mice, the DMD model group mice showed obvious tissue structural disorder, inflammatory cell aggregation and fibrosis in their hearts, while the DMD treatment group significantly improved tissue structural disorder and inflammation. skeletal muscle HE staining as follows Figure 9 As shown, compared with the WT mouse skeletal muscle, the DMD model group mice showed obvious tissue structure disorder, inflammatory cell aggregation and fibrosis, while the DMD treatment group significantly improved tissue structure disorder and inflammation. Liver HE staining Figure 10 As shown, compared with the liver of WT mice, the liver of mice in the DMD model group showed obvious tissue structure disorder and inflammatory cell aggregation, while the DMD treatment group significantly improved tissue structure disorder and inflammation.

[0043] To further confirm the improvement in fibrosis, Masson staining results of tissues and organs were obtained as follows: Figures 11-13 As shown: Among them, cardiac Masson staining, such as Figure 11 As shown, compared with the heart of WT mice, the heart of mice in the DMD model group showed obvious fibrosis, while the fibrosis in the DMD treatment group was significantly reduced. Masson staining of skeletal muscle as follows Figure 12 As shown, compared with the skeletal muscle of WT mice, the skeletal muscle of mice in the DMD model group showed obvious fibrosis, while the fibrosis in the DMD treatment group was significantly reduced. Masson staining of the liver Figure 13 As shown, compared with the livers of WT mice, the livers of mice in the DMD model group showed obvious fibrosis, while the fibrosis in the DMD treatment group was significantly reduced.

[0044] This invention is ingeniously conceived and simple to operate. Through this design, a novel AAV vector with high conversion efficiency in muscle is used to deliver the complete LAMP2B gene (LAMP2B is a gene related to the regulation of autophagy) into muscle cells of various organs of mice (referred to as AAV-hLAMP2B), which comprehensively assists in the recovery of muscle tissue function in a mouse DMD disease model and delays the progression of DMD.

[0045] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0046] The amino acid or nucleotide sequences mentioned or used in this article are as follows.

[0047]

[0048]

Claims

1. An overexpression vector of LAMP2B, characterized by, The vector comprises: (a) an AAV vector, which is an adeno-associated virus vector, has high muscle tissue targeting and low liver tropism; (b) a CMV promoter, which is a cytomegalovirus promoter; (c) a hLAMP2B gene coding sequence, which is a human LAMP2B gene coding sequence, operably linked to the CMV promoter; wherein the vector is configured to overexpress LAMP2B protein in muscle tissue, and the vector is a self-complementary AAV, namely scAAV.

2. The LAMP2B overexpression vector according to claim 1, wherein: The vector further comprises a HA tag sequence, a WPRE enhancer sequence and a SV40 polyA signal sequence.

3. The LAMP2B overexpression vector of claim 1, wherein: The CMV promoter sequence is shown in SEQ NO.

1.

4. The LAMP2B overexpression vector of claim 1, wherein: The hLAMP2B gene coding sequence is shown in SEQ NO.

2.

5. The LAMP2B overexpression vector of claim 1, wherein: The AAV vector is an AAV-P7 serotype, which is obtained by AAV9 directed evolution.

6. A pharmaceutical composition, characterized by, A LAMP2B overexpression vector according to any one of claims 1-5, and a pharmaceutically acceptable carrier.

7. Use of a LAMP2B overexpression vector according to any one of claims 1-5 in the preparation of a medicament for treating Duchenne muscular dystrophy.

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