Methods for delivering genes to odontoblast cells in vivo in mice using recombinant adeno-associated viral vectors and expression

Abstract

The application provides a method for delivering genes to mouse dentinoblasts in vivo by using a recombinant adeno-associated virus vector and expressing the genes, screening of an adeno-associated virus vector which can safely and efficiently deliver genes to mouse dentinoblasts, and a method for successfully expressing the genes in the dentinoblasts by local injection through a submandibular approach, which can be used for prevention and treatment of dentin development defects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for delivering and expressing genes into odontoblasts in mice using a recombinant adeno-associated virus vector. Background Technology

[0002] Dentin is the main hard tissue that makes up teeth and plays a vital role in their normal function. Odontoblasts are responsible for dentin formation, and mutations or deletions of various genes can affect odontoblast differentiation, leading to irreversible dentin developmental defects in animals and humans. Clinically, these patients often present with symptoms such as tooth discoloration, severe wear, root fracture, and even infection and pain. However, dentin developmental defects are irreversible, and conventional treatments can only repair and protect the remaining tooth structure, with unsatisfactory results. They cannot fundamentally prevent dentin developmental abnormalities or restore normal dentin development.

[0003] Therefore, it is necessary to develop a formulation to prevent dentin developmental abnormalities. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for delivering and expressing genes into odontoblasts in mice using a recombinant adeno-associated virus vector. The adeno-associated virus is applied to the treatment of hereditary dentinogenesis imperfecta, and the serotype with the highest infection efficiency, AAV6, is selected. The pathogenic gene causing dentin developmental defects is loaded into the aforementioned adeno-associated virus vector, and the drug is administered via local injection into the mesenchyme surrounding the molars through a submandibular approach, thereby treating and alleviating hereditary dentinogenesis imperfecta.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect of the invention, a method is provided for delivering and expressing a gene into mouse odontoblasts using a recombinant adeno-associated virus vector, the method comprising:

[0007] The gene was cloned into an adeno-associated virus vector to obtain a recombinant adeno-associated virus vector;

[0008] The recombinant adeno-associated virus vector is delivered to odontoblasts in mice via local injection through a submandibular approach, where the gene is expressed.

[0009] The gene can be Mdm2 or other genes.

[0010] In a second aspect of the invention, the use of recombinant adeno-associated virus vector AAV6-Mdm2 in the preparation of formulations for the prevention or treatment of hereditary dentin developmental defects is provided, the nucleotide sequence of said recombinant adeno-associated virus vector AAV6-Mdm2 being shown in SEQ ID NO:2.

[0011] The recombinant adeno-associated virus vector AAV6-Mdm2 is a recombinant vector formed by inserting the Mdm2 nucleotide sequence into the adeno-associated virus vector AAV6. Specifically: the nucleic acid molecule shown in SEQ ID NO:1 is amplified by PCR using primers shown in SEQ ID NO:2-SEQ ID NO:3 to obtain the target gene fragment; the AAV6 expression vector GPAAV WPRE (i.e., pAAV-ITR) is digested with Sal I and Nde I and then ligated with the target gene fragment to obtain the recombinant adeno-associated virus vector AAV6-Mdm2. The nucleotide sequence of Mdm2 is shown in SEQ ID NO:1.

[0012] The recombinant adeno-associated virus was obtained by infecting host cells with the recombinant adeno-associated virus vector AAV6-Mdm2, the helper plasmid pAdDeltaF6, and the serotype capsid plasmid pAAV R / C.

[0013] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0014] This invention provides a method for delivering and expressing genes into odontoblasts in mice using a recombinant adeno-associated virus vector. The adeno-associated virus is applied to the treatment of hereditary dentinogenesis imperfecta, and the serotype with the highest infection efficiency, AAV6, is selected. The pathogenic gene causing dentin developmental defects is loaded into the aforementioned adeno-associated virus vector, and the drug is administered via local injection into the mesenchyme surrounding the molars through a submandibular approach, thereby treating and alleviating hereditary dentinogenesis imperfecta. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The expression level of the reporter gene GFP carried by AAV after mouse OLCs were infected with different serotypes of AAV.

[0017] Figure 2After AAV6 serotype virus infection of OLCs, the TUNEL assay was used to detect the apoptosis level of OLCs in the AAV6-infected group and the control group (a), where red represents apoptotic cells; qRT-PCR was used to detect the expression levels of apoptosis-related marker genes Bax and Bcl-2 in AAV6-infected cells and control cells (b); qRT-PCR was used to detect the expression levels of odontoblast differentiation markers Dspp and Dmp1 (c).

[0018] Figure 3 The image shows the local injection sites of the developing ink and its distribution after injection. Red dots indicate the injection sites (a), and red arrows indicate the distribution of ink in the odontoblast layer (c). (c) is an enlarged view of the boxed area in (b).

[0019] Figure 4 Three days after AAV6 injection, the reporter gene GFP carried by AAV6 was observed to be positively expressed in odontoblasts of mouse molars.

[0020] Figure 5 Two and three weeks after injection of AAV6-Mdm2, Mdm2 expression in the odontoblast layer of mouse molars was restored.

[0021] Figure 6 Micro-CT and H&E staining show the recovery of mineralized dentin thickness in mouse molars at 2 and 3 weeks after AAV6-Mdm2 injection. Red dashed lines indicate the boundary between dentin and enamel. White dashed lines indicate the boundary between mineralized dentin and pre-dentin.

[0022] Figure 7 This study investigated the expression of the reporter gene GFP carried by human OLCs after infection with different serotypes of AAV.

[0023] Figure 8 This refers to the expression of AAVR and EGFR receptors in the odontoblast layer of human teeth. Detailed Implementation

[0024] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0027] The present invention adopts the following technical solution:

[0028] AAV vectors expressing the GFP reporter gene were packaged with six widely used serotype capsids (AAV1, 5, 6, 8, 9, and DJ), and then used to infect mouse odontoblast-like cells (OLCs) in vitro. Infection efficiency was compared by calculating the percentage of GFP-positive cells and the mean fluorescence intensity, demonstrating that the multiplicity of infection (MOI) was 1.25 × 10⁻⁶. 6 Or 2.5×10 5 The AAV6 serotype adeno-associated virus had the highest infection efficiency at a rate of vg / cell.

[0029] After AAV6 serotype virus (AVV) infection of OLCs, apoptotic cells were detected by terminal deoxyuridine triphosphate (TUNEL) labeling. The expression of the pro-survival marker Bcl-2 and the pro-apoptotic marker Bax was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), both demonstrating that AAV6 AVV infection did not affect the survival ability of OLCs. The expression of the odontoblast differentiation markers Dspp and Dmp1 was detected by qRT-PCR, confirming that AAV6 AVV infection did not affect the differentiation ability of OLCs.

[0030] Two μl of AAV6 virus or PBS (control) was locally injected into the molar papilla tissue of mice via the submandibular approach. The effectiveness of local injection of recombinant adenovirus vector to infect odontoblasts was confirmed by detecting GFP expression in odontoblasts after drug administration. Two μl of recombinant adeno-associated virus vector AAV6-Mdm2 was also injected into Mdm2 gene knockout mice (Dmp1-Cre; Mdm2). flox / flox In the molar papilla tissue, the mouse exhibited defects in odontoblast differentiation and dentin formation due to the absence of Mdm2. After a period of time, the expression of Mdm2 in the odontoblast layer and dentin formation were observed to be restored, demonstrating that this method can successfully deliver the gene to odontoblasts in mice and enable them to function.

[0031] The effects of this application will be described in detail below with reference to embodiments and experimental data. Unless otherwise specified, the molecular cloning methods, protein expression and purification methods, cell culture methods, and various detection methods mentioned in the following schemes are all traditional experimental methods, which can be obtained by consulting the literature; the relevant reagents used can be purchased from the corresponding reagent suppliers.

[0032] Example 1: Highly efficient infection of cultured mouse OLCs with AAV6 serotype

[0033] OLCs were seeded into 96-well plates at a density of 10,000 cells per well, and different serotypes were infected with OLCs at different multiples of infection (MOIs). Infection efficiency was compared by calculating the percentage of GFP-positive cells and the mean fluorescence intensity.

[0034] Experimental results are as follows Figure 1 As shown, the expression level of GFP in OLCs infected with the AAV6 serotype was higher than that in other groups, indicating that the infection efficiency of AAV6 is higher than that of other AAV serotypes.

[0035] Example 2: After AAV6 serotype virus infection of mouse OLCs, TUNEL assay was used to detect cell apoptosis, and qRT-PCR was used to detect the expression of pro-survival marker Bcl-2 and pro-apoptotic marker Bax, as well as the expression of odontoblast differentiation markers Dspp and Dmp1.

[0036] To test whether AAV6 infection has adverse effects on mouse OLCs, we used the TUNEL assay to detect cell apoptosis. Total RNA was extracted from mouse OLCs using an RNA extraction kit, and the mRNA transcription levels of Bax, Bcl-2, Dspp, and Dmp1 were detected using qRT-PCR. Gapdh was used as an internal control, and the relative expression levels of each gene's mRNA were standardized.

[0037] Experimental results are as follows Figure 2 As shown, compared with the positive control cells treated with DNase I, very few apoptotic cells were observed in the AAV6-infected group; there was no significant difference in the mRNA transcription levels of Bcl-2, Bax, Dspp, and Dmp1 between AAV6-infected cells and normal cells. These data indicate that infection with the AAV6 serotype virus does not affect the survival and differentiation ability of mouse OLCs.

[0038] Example 3: Local injection administration via submandibular approach in mice

[0039] To test the feasibility of local injection via the submandibular approach, we first injected the molars of mice born on the day of birth using a developing ink. The specific injection procedure was as follows: Before injection, the mice were placed in a supine position to expose the mandible. The injection point was located on the long axis of the mandible, 3 mm from the corner of the mouth. When injecting the left molar, using the mouse's sagittal plane as a reference, the needle was inserted at an angle of approximately 30° to the operator's right, to a depth of approximately 1 mm; the right molar was injected in the same manner, but the needle was inserted at an angle to the operator's left. A diagram of the injection procedure and the post-injection effects are shown below. Figure 3 As shown.

[0040] We further injected 2 μl of AAV6 virus solution or PBS (control) locally into the molar papilla tissue of mice via the submandibular approach, and detected GFP expression in odontoblasts of molars on day 3 post-administration. The experimental results are as follows: Figure 4 As shown, significant GFP expression was observed in molar odontoblasts injected with AAV6, while no GFP expression was observed in molar odontoblasts injected with PBS.

[0041] Example 4: Construction of AAV6-Mdm2 delivery vector and local injection administration in mice

[0042] To demonstrate that AAV6 can successfully deliver and express genes into odontoblasts in vivo, we plan to use the above-mentioned verification method to deliver the Mdm2 gene into mouse odontoblasts via AAV6.

[0043] The specific construction method of AAV6-Mdm2 is as follows:

[0044] The mouse Mdm2 gene sequence was synthesized and cloned into the AAV6 expression vector GPAAV WPRE. The AAV vector expressing the GFP reporter gene under the regulation of the EF1α promoter was purchased from BrainV TA Biotechnology Co., Ltd. Sal I and NdeI were used as cloning insertion sites. The upstream primer sequence was 5'-ACCGG ATCCTCTAGAGTCGACATGTGCAATACCAACATGTCTGTG-3' (SEQ ID NO.3), and the downstream primer sequence was 5'-AGTAGCTCCGCTTCCCATATGGTTGAAGTAAGTTAGCACAATCATTTGG-3 (SEQ ID NO.4).

[0045] The AAV viral vector carrying Mdm2 was packaged with an AAV6 serotype capsid. Specifically, the AAV-Mdm2 viral vector and the helper plasmid pAdDeltaF6, along with the AAV6 serotype capsid plasmid, were co-transfected into 293T cells using Lipofectamine 2000 reagent (Invitrogen). The culture medium was then collected to obtain the viral supernatant, which was subsequently purified. The obtained AAV viral titer was further quantified by qRT-PCR.

[0046] The Kunming mice used were purchased from the Hubei Provincial Center for Disease Control and Prevention. Mdm2 conditional knockout (Mdm2 cKO; Dmp1-Cre; Mdm2) was employed. flox / flox The mice consisted of Dmp1-Cre mice and Mdm2 mice. flox / flox The virus was obtained through mouse hybridization. Mice were genotyped and hypothermia-anesthetized before injection. All injections were performed using a 10μl microsyringe and a 32-gauge needle. 2μl of AAV6-Mdm2 virus was injected locally into the perimolar papilla tissue via a submandibular approach. Mice were sacrificed 2 and 3 weeks after administration to observe the recovery of Mdm2 expression in the odontoblast layer. Micro-CT and H&E staining were used to observe the recovery of the width of mineralized dentin.

[0047] Experimental results are as follows Figure 5 and Figure 6 As shown, local injection of AAV6-Mdm2 successfully restored Mdm2 expression in the odontoblast layer, and restored Mdm2 expression and mineralized dentin width in the odontoblast layer, indicating that local injection of the AAV6-Mdm2 viral vector can improve the dentin developmental defects in mice caused by Mdm2 gene defects.

[0048] Example 5: Acquisition of human OLCs and observation of their expression receptors

[0049] Primary human dental pulp cells (hDPCs) were isolated from healthy human dental pulp tissue, and cells from passages 3-5 were used for research. hDPCs were induced to differentiate into oral lactate cells (OLCs) in differentiation medium (DM) containing 10% fetal bovine serum, 10 mM β-glycerophosphate, 10 nM dexamethasone, and 50 μg / mL ascorbic acid. The expression of corresponding receptors was observed by immunofluorescence.

[0050] To further evaluate the infection efficiency of different AAV serotypes in human OLCs, human OLCs were infected with six AAV serotypes respectively. After 72 hours, the infection efficiency of each serotype in human OLCs was evaluated by observing the average fluorescence intensity of GFP.

[0051] The results are as follows Figure 7 As shown, the average expression level of GFP was the highest in human OLCs infected with AAV6, indicating that AAV6 has a higher infection efficiency in human OLCs than other AAV serotypes.

[0052] The surface receptors AAVR and EGFR, which recognize AAV6 during host cell infection, are strongly expressed in the odontoblast layer of developing human molars, as shown in the results. Figure 8 As shown, this indicates that the AAV6 viral vector also has the potential for application in human odontoblasts.

[0053] In summary, the method of local injection of AAV6 recombinant adenovirus vector into the perimolar papilla tissue of mice via the submandibular approach can safely and efficiently deliver genes to mouse odontoblasts and enable gene expression and function.

[0054] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0055] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0056] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of recombinant adeno-associated virus vector AAV6-Mdm2 in the preparation of formulations for treating hereditary dentin developmental defects, characterized in that, The nucleotide sequence of the recombinant adeno-associated virus vector AAV6-Mdm2 is shown in SEQ ID NO:2.

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