Application of Krit1 mRNA in treatment of cerebral cavernous malformation

Abstract

The invention relates to the technical field of biomedicine and gene therapy, in particular to application of Krit1 mRNA in treatment of cerebral cavernous malformation. The KRIT1 mRNA forms LNP (at) Krit1 mRNA through the lipid nanoparticles for delivery, and can be applied to preparation of a reagent for preventing and / or treating cerebral cavernous malformation, preparation of a barrier function reagent for recovering cerebral vascular endothelial cells and preparation of a reagent for reducing or inhibiting leakage and bleeding of brain blood vessels; the Krit1 function can be effectively recovered in both in-vitro and in-vivo CCM models, vasculopathy is remarkably improved, and the Krit1 gene has good development prospects and clinical transformation value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical and gene therapy, and particularly relates to application of Krit1 mRNA in treatment of cerebral cavernous malformations. BACKGROUND

[0002] Cerebral cavernous malformations (CCMs) is a common central nervous system vascular malformation, with a prevalence of about 0.5%. Its pathological features are the aggregation of thin-walled sinusoidal vessels in the brain, which lack normal vascular wall structure, and are prone to cause repeated microhemorrhage, epilepsy, focal neurological dysfunction and fatal stroke. CCMs can be divided into sporadic and familial two kinds, among which familial CCMs is an autosomal dominant genetic disease, which is caused by embryonic mutation of one of three key genes CCM1 (KRIT1), CCM2 (Malcavernin) and CCM3 (PDCD10).

[0003] KRIT1 (Krev interaction trapped protein 1) gene is located on human chromosome 7q21.2, containing 16 exons, and the protein encoded by it is called KRIT1 or CCM1 protein. KRIT1 protein is mainly composed of N-terminal FERM domain, multiple NPxY / F motifs and C-terminal PTB domain binding region. KRIT1 as a key scaffold protein, plays a core role in maintaining the homeostasis of brain endothelial cells.

[0004] In about 40%-50% of familial CCM cases, pathogenic mutations occur in the KRIT1 gene. Most of these mutations are nonsense mutations, frameshift mutations or splice site mutations, which lead to the production of truncated, non-functional KRIT1 protein or haploinsufficiency of the allele, ultimately triggering CCM lesions. At present, the treatment means for CCMs is very limited. For single lesion with symptoms or repeated hemorrhage, surgical resection is the preferred solution; for deep, multiple or inoperable lesions, there is a lack of effective drug treatment methods. SUMMARY

[0005] In view of the above problems of the prior art, the present application aims to provide application of Krit1 mRNA in treatment of cerebral cavernous malformations, which is delivered by forming LNP@Krit1 mRNA through lipid nanoparticles, to restore Krit1 function.

[0006] In order to solve the above problems, the present application adopts the following technical solutions: In a first aspect, the present application provides a composition for treating cerebral cavernous malformations, comprising KRIT1 mRNA.

[0007] Further, the KRIT1 mRNA is delivered by forming a LNP@Krit1 mRNA through a lipid nanoparticle.

[0008] Further, the nucleotide sequence of the KRIT1 mRNA is shown as SEQ ID NO. 1.

[0009] Further, the preparation of the LNP@Krit1 mRNA comprises: cholesterol, Dlin-MC3-DMA, DMG-PEG2000, DOPE are mixed and dissolved into anhydrous ethanol according to the molar ratio of 46.5:35:2.5:16; Krit1A mRNA is dissolved in a citrate buffer of 10 mM and pH=3.0; the water phase and the oil phase are mixed according to the volume ratio of 3:1 to prepare LNP@Krit1A mRNA, and the product is dialyzed in a PBS buffer prepared with DPEC water at 4℃ for 2 h.

[0010] In a second aspect, the present application provides an application of KRIT1 mRNA and / or KRIT1 protein, comprising: application in preparing an agent for preventing and / or treating cerebral cavernous malformations; application in preparing an agent for restoring the barrier function of cerebral vascular endothelial cells; application in preparing an agent for reducing or inhibiting the leakage and hemorrhage of cerebral blood vessels.

[0011] Further, the KRIT mRNA is delivered by forming a LNP@Krit1 mRNA through a lipid nanoparticle.

[0012] Further, the KRIT mRNA increases the Krit1 mRNA and KRIT1 protein levels in mouse models hCMEC / D3 and HUVEC cells.

[0013] In a third aspect, the present application provides an application of Krit1 mRNA in treating cerebral cavernous malformations.

[0014] The present application has the beneficial effect that the delivery by forming a LNP@Krit1 mRNA through a lipid nanoparticle can effectively restore Krit1 function in vitro and in vivo CCM models, significantly improve vascular lesions, and has good development prospects and clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A graph for verifying the expression level of Krit1 gene in Krit1 knockdown hCMEC / D3 cells after LNP@Krit1 mRNA transfection by qPCR.

[0016] Figure 2Figure for verification of the expression level of Krit1 gene after LNP@Krit1 mRNA transfection in Krit1 knockdown HUVEC cells by qPCR.

[0017] Figure 3 Figure for verification of the expression level of KRIT1 protein after LNP@Krit1 mRNA transfection in KRIT1 knockdown hCMEC / D3 cells by Western Blotting.

[0018] Figure 4 Figure for verification of the expression level of KRIT1 protein after LNP@Krit1 mRNA transfection in KRIT1 knockdown HUVEC cells by Western Blotting.

[0019] Figure 5 Figure for comparison of the number and area of brain hemorrhagic spots in LNP@Krit1 mRNA treatment group compared with control group for brain tissue imaging; left is empty LNP control group, right is LNP@Krit1 mRNA treatment group.

[0020] Figure 6 Figure for comparison of the number and area of brain hemorrhagic spots in LNP@Krit1 mRNA treatment group compared with control group for microCT three-dimensional reconstruction image of mouse brain tissue; left is empty LNP control group, right is LNP@Krit1 mRNA treatment group.

[0021] Figure 7 Figure for quantitative statistical analysis of the number and area of hemorrhagic spots.

[0022] Figure 8 Figure for quantitative results of Evans Blue dye (EBD) leakage experiment.

[0023] Figure 9 Figure for immunofluorescence staining results. DETAILED DESCRIPTION

[0024] The application will be further described below in conjunction with specific examples.

[0025] It should be noted that these examples are only used to illustrate the present application, and are not intended to limit the present application, and simple improvements of the present method under the concept of the present application all belong to the scope of the present application.

[0026] MATERIALS AND METHODS Synthesis of Krit1 mRNA The DNA fragment containing T7 promoter, CDS sequence of the target protein (mouse Krit1A) and tail was used as a template, and in vitro transcription was performed according to the instructions of T7 RNA synthesis kit (New England Biolabs, E2040S) (containing pseudouridine and m5C modification). The transcription process was co-transcriptional capping (Cap1 cap). The synthesis product was digested with DNase I (Promega, M6101) to remove the template DNA. Finally, the digested product was purified by RNA purification kit (New England Biolabs, T2050S). The concentration of the final product was quantified by nanodrop.

[0027] The Krit1 sequence is as follows (SEQ ID NO. 1):

[0028] Preparation of lipid nanoparticles (LNPs) Cholesterol (Avanti Polar Lipids, 700000P), Dlin-MC3-DMA (MedChemExpress, HY-112251), DMG-PEG 2000 (Avanti Polar Lipids, 880151P), and DOPE (Avanti Polar Lipids, 850725P) were dissolved in anhydrous ethanol and mixed in an anhydrous ethanol at a molar ratio of 46.5:35:2.5:16. Krit1A mRNA was dissolved in 10 mM citrate buffer (pH=3.0). The aqueous and oil phases were rapidly mixed at a volume ratio of 3:1 to prepare LNP@Krit1A mRNA (20 μg mRNA / 100 μL LNP). The product was dialyzed against PBS buffer prepared with DPEC water at 4°C for 2 h.

[0029] Cell experiments Cell transfection and expression validation Human brain microvascular endothelial cell line D3 clone hCMEC / D3 and human umbilical vein endothelial cells HUVEC were used. LNP@Krit1 mRNA was transfected into the cells at doses of 1 ng, 10 ng, and 100 ng. Subsequently, Krit1 gene expression levels were detected by qPCR, and KRIT1 protein expression was detected by Western blotting using an anti-KRIT1 antibody.

[0030] qRT-PCR experimental procedure: Follow the reagent instructions sequentially. First, extract cellular RNA using Trizol reagent. Then, detect the RNA concentration using a NanoDrop instrument and reverse transcribe the RNA into cDNA using a kit. The program was set as follows: 50℃, 15 min; 85℃, 5 s. Finally, amplify the gene using the SYBR qPCR Master Mix kit. Set the qPCR instrument to Target and Sample, and set the amplification program as follows: first, perform pre-denaturation at 95℃ for 30 s; then, proceed to the amplification phase with 40 cycles. Each cycle begins with denaturation at 95℃ for 10 s, followed by annealing and extension at 60℃ for 30 s. After the cycles, perform melting curve analysis using the following program: 95℃ for 15 s; 60℃ for 60 s; and finally, return to 95℃ for 15 s. GAPDH was used as an internal control gene. The mRNA expression level of the target gene was expressed as a normalized relative fold change with respect to GAPDH. See Appendix Table 1 for the relevant primer sequences.

[0031] Table 1 Primer sequence listing

[0032] Western blot experimental procedure After the cells were washed with PBS, the cells were lysed with a protein lysis solution on ice. After lysis, the lysis solution was collected in a centrifuge tube and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected and the protein was quantified according to the instructions of the protein quantification kit. According to the volume ratio of 4:1, 5x loading buffer was added and mixed well, and the protein was denatured in a dry constant temperature metal bath. Gel electrophoresis: according to the instructions of the precast gel, the gel was prepared, the corresponding denatured protein sample was added to the well, and the protein electrophoresis was performed under the set electrophoresis conditions. After electrophoresis, the band was transferred to the PVDF membrane, and the PVDF membrane was blocked with BSA for 2 h, then the primary antibody was added and incubated at 4°C overnight. The next day, the secondary antibody was added after washing away the primary antibody and incubated at room temperature for 2 h. The whole PVDF membrane was placed in the gel imaging system for protein development. The obtained protein band was quantified by ImageJ software, and the corresponding quantitative graph was drawn according to the obtained results.

[0033] Animal experiment Animal feeding The animal experiment was performed in accordance with the guidelines for laboratory animal care and use approved by the Nanjing University of Chinese Medicine Review Committee. CCM1ECKO mice were donated by the Australian Institute of Centenary. The mice were bred under specific pathogen-free conditions, with a light-dark cycle of 14 hours light / 10 hours dark. The environmental temperature was 21-23°C, and the humidity was 50%. The mice had free access to water and food.

[0034] Animal model Krit1 endothelial-specific inducible knockout mice (Krit1.c) were injected intragastrically with 4-hydroxytamoxifen (25-μg / mouse) on day 1 after birth to induce Krit1 gene knockout.

[0035] Dosing regimen The mice were randomly divided into two groups: (1) LNP@Krit1 mRNA treatment group; (2) empty LNP control group. On postnatal day 6 and day 13, the mice were injected subcutaneously in the neck with LNP-encapsulated Krit1 mRNA (0.1 μg / mouse), n=5.

[0036] Efficacy evaluation Micro-CT imaging and lesion analysis Fresh cerebellar tissue was first fixed with 4% paraformaldehyde for 24 h, followed by immersion in Lugol's iodine solution for 48 h. The samples were then removed with forceps, surface liquid was aspirated, and the samples were scanned using a Micro-CT imaging system. Finally, Analyze 12.0 software was used to perform three-dimensional reconstruction and quantitative analysis of the lesion areas in the obtained images.

[0037] Functional recovery experiment Vascular leakage assessment EBD vascular leakage experiment: 1% EBD (100 μl / mouse) solution was injected into the fundus of mice and entered the systemic circulation of the mice. After 20 minutes of systemic circulation, the mice were euthanized, and the brain tissue was quickly collected and photographed under a fluorescence stereomicroscope.

[0038] Vascular integrity assessment Fresh mouse brain tissue samples were collected for immunofluorescence staining, embedded in OCT gel, and rapidly frozen on dry ice. The samples were then stored at -80°C. For frozen sectioning of the brain tissue, the section thickness was set to 15 μm. The immunofluorescence staining procedure was as follows: First, the brain tissue sections were fixed with methanol, then permeabilized with 0.3% Triton solution, followed by blocking with BSA solution at room temperature for 2 h. The blocking solution was discarded, and staining was performed using antibodies against VE-cadherin, Claudin-5, NG2, and α-SMA. The expression of cell junction proteins was observed using a confocal microscope. The sections were incubated overnight at 4°C. The next day, the primary antibody was discarded, and the corresponding fluorescent secondary antibody was added. Incubation was continued at room temperature for 2 h. After nucleus staining with DAPI, the sections were mounted, and the entire section was photographed under a fluorescence microscope. Quantitative fluorescence analysis of the brain tissue sections was performed using ImageJ software.

[0039] LNP@Krit1 mRNA effectively restored Krit1 gene and protein expression in vitro. In hCMEC / D3 and HUVEC cells, qPCR showed that Krit1 mRNA levels were significantly upregulated compared to the empty control group after LNP@Krit1 mRNA transfection. Figure 1 , Figure 2 Western blotting confirmed that in a KRIT1 knockdown cell model, LNP@Krit1 mRNA treatment could restore KRIT1 protein expression to near-normal levels. Figure 3 , Figure 4 ).

[0040] LNP@Krit1 mRNA significantly reduced brain lesions in a CCM mouse model. In the Krit1 mouse model, brain tissue imaging and microCT 3D reconstruction results showed that, compared with the empty LNP control group, the number of hemorrhage points and the total hemorrhage area in the brain of mice treated with LNP@Krit1 mRNA were significantly reduced. Figures 5-7 ).

[0041] LNP@Krit1 mRNA improves vascular integrity and reduces leakage.

[0042] EBD leakage experiments showed that the amount of dye leakage into the brain tissue of the treatment group was significantly lower than that of the control group. Figure 8 Immunofluorescence results showed enhanced tight junctions and adhesion junctions of endothelial cells, increased pericyte coverage, and significantly improved vascular integrity. Figure 9 ).

[0043] This invention presents an effective and feasible treatment strategy for CCMs via LNP delivery of Krit1 mRNA. Its mechanism of action involves the transient expression of functional KRIT1 protein in cerebral vascular endothelial cells, thereby reversing the pathological progression of CCM.

[0044] Compared to traditional gene therapies (such as viral vectors), mRNA therapy offers advantages such as high safety (no risk of insertional mutations), rapid production, and controllable expression. The LNP formulation of this invention demonstrates good biocompatibility and therapeutic efficacy, providing novel drug candidates and treatment options for the clinical treatment of CCMs.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A composition for treating cavernous malformation of the brain, characterized in that, comprises KRIT1 mRNA.

2. The composition of claim 1, wherein, The KRIT1 mRNA is delivered by forming a LNP@Krit1 mRNA through a lipid nanoparticle.

3. The composition of claim 1, wherein, The nucleotide sequence of the KRIT1 mRNA is shown as SEQ ID NO.

1.

4. The composition of claim 2, wherein, The preparation of the LNP@Krit1 mRNA comprises: cholesterol, Dlin-MC3-DMA, DMG-PEG 2000, DOPE are mixed and dissolved into anhydrous ethanol according to the molar ratio of 46.5:35:2.5:16; Krit1A mRNA is dissolved in a citrate buffer of 10 mM, pH=3.0, and the water phase is mixed with the oil phase according to the volume ratio of 3:1 to prepare LNP@Krit1A mRNA, and the product is dialyzed in a PBS buffer prepared with DPEC water at 4℃ for 2h.

5. Use of KRIT1 mRNA and / or KRIT1 protein, characterized in that, comprises: application in preparing an agent for preventing and / or treating cerebral cavernous malformations; or, application in preparing an agent for restoring the barrier function of cerebral vascular endothelial cells; or, application in preparing an agent for reducing or inhibiting the leakage and hemorrhage of cerebral blood vessels.

6. Use according to claim 5, characterized in that, The KRIT1 mRNA is delivered by forming a LNP@Krit1 mRNA through a lipid nanoparticle.

7. Use according to claim 6, characterized in that, The KRIT1 mRNA increases the Krit1 mRNA and KRIT1 protein levels in mouse models hCMEC / D3 and HUVEC cells.

8. Application of Krit1 mRNA in treating cerebral cavernous malformations.

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