Recombinant adeno-associated virus for regulating and controlling excitability of peripheral pain related neurons and application of recombinant adeno-associated virus

Abstract

The invention relates to a recombinant adeno-associated virus for regulating excitability of peripheral pain related neurons and application thereof. The recombinant adeno-associated virus comprises an rAAV (recombinant adeno-associated virus) vector, the peripheral pain related neurons and an excitability inhibiting unit. The recombinant adeno-associated virus disclosed by the invention performs specific intervention on peripheral pain neurons, reduces side effects of analgesia, achieves the effect of relieving chronic pain by using a simple and convenient administration means, and has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pain treatment, and specifically relates to a recombinant adeno-associated virus that regulates the excitability of peripheral pain-related neurons and its applications. Background Technology

[0002] The vast majority of pain, including acute and chronic pain, originates from the activation of peripheral pain-related neurons caused by peripheral injury, inflammation, etc. Subsequently, peripheral pain neurons become sensitized with continuously enhanced excitability, and acute pain transforms into persistent and difficult-to-treat chronic pain, tormenting patients. Early intervention targeting peripheral pain neurons to prevent over-excitation can theoretically effectively reduce the incidence of chronic pain. However, current methods mainly rely on medication or electrical stimulation for analgesia, lacking specific analgesic approaches that target key neurons encoding pain information. Summary of the Invention

[0003] This invention provides a recombinant adeno-associated virus that modulates the excitability of peripheral pain-related neurons and its application. The technical problems to be solved include: 1) improving specificity, specifically intervening only in pain neurons; 2) reducing side effects; 3) simple operation and minimal trauma; and 4) having a significant therapeutic effect on chronic pain.

[0004] This invention provides a recombinant adeno-associated virus (rAAV) that modulates the excitability of peripheral pain-related neurons, comprising an rAAV viral vector, peripheral pain-related neurons, and an inhibitory excitability unit, such as... Figure 1 As shown; the peripheral pain-related neurons include one or more of the following: calcitonin gene-related peptide (CGRP), neuropeptide (Substance P, SP), transient receptor potential vanilloid 1 (TRPV1), and transient receptor potential A1 (TRPA1) positive neurons; the inhibitory excitatory unit includes one or more of the following: modified human muscarinic acetylcholine receptor M4 (hM4D(Gi)) and shRNA that interferes with the Fos gene.

[0005] Preferably, the rAAV virus vector includes one or more of rAAV1, rAAV2, rAAV4, rAAV9, rAAV2Retro, and rAAV9Retro.

[0006] Furthermore, the recombinant adeno-associated virus may also include an expression unit.

[0007] More preferably, the recombinant adeno-associated virus is rAAV / 9-FDIO-shRNA (mFos), rAAV / 2Retro-hCGRP-hM4D (Gi), or rAAV / 9Retro-hCGRP-hM4D (Gi).

[0008] It should be noted that the three viruses are merely examples, and those skilled in the art can replace other rAAV viral vectors, peripheral pain-related neurons, and inhibitory excitatory units with conventional knowledge.

[0009] This invention also provides the application of a recombinant adeno-associated virus that regulates the excitability of peripheral pain-related neurons in the preparation of pain medications.

[0010] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipients.

[0011] Preferably, the pharmaceutically acceptable carrier and / or excipient includes at least one of diluent, binder, surfactant, humectant, adsorbent, lubricant, filler, and disintegrant.

[0012] Preferably, the drug is administered via DRG in situ injection or peripheral injection.

[0013] Beneficial effects

[0014] This invention utilizes recombinant adeno-associated virus to specifically intervene in peripheral pain neurons, reducing analgesic side effects and achieving the effect of relieving chronic pain through a simple and convenient administration method, which has good market application prospects. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the present invention.

[0016] Figure 2 In situ injection of targeted TRPV1 into the dorsal root ganglia of the spinal cord + / A1 + Statistical analysis results of the effects of neuronal AAV-shRNA (mFos) virus on mechanical pain threshold, thermal pain threshold, and open field behavior in CFA model mice. * indicates comparison between the WT-shRNA (mFos) group and the Flpo-shRNA (mFos) group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0017] Figure 3 Intra-articular injection of different doses of targeted CGRP in the knee joint +Statistical analysis results of the effect of neuronal AAV-hM4D(Gi) virus on the mechanical pain threshold of osteoarthritis model mice. * indicates comparison between the rAAV / 2R+CNO 3μl group and the rAAV / 2R group, *p<0.05, ***p<0.001, ****p<0.0001; # indicates comparison between the rAAV / 2R+CNO 6μl group and the rAAV / 2R group, #p<0.05, ##p<0.01, ####p<0.0001.

[0018] Figure 4 Intra-articular injection of targeted CGRP in the knee joint + Statistical analysis results of the effects of neuronal AAV-hM4D(Gi) virus on the mechanical and thermal pain thresholds in osteoarthritis model mice. * indicates comparison between the rAAV / 2R+CNO group and the rAAV / 2R group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; # indicates comparison between the rAAV / 9R+CNO group and the rAAV / 9R group, #p<0.05, ###p<0.001, ####p<0.0001.

[0019] Figure 5 Intra-articular injection of targeted CGRP in the knee joint + AAV-hM4D(Gi) virus in neurons affects CGRP in DRG. + Immunostaining results and statistical graphs showing the effect of neuronal activity. **p<0.01, ***p<0.001.

[0020] Figure 6 Intra-articular injection of targeted CGRP in the knee joint + Immunofluorescence staining results and statistical graphs showing the effects of AAV-hM4D(Gi) virus on the liver and kidneys. ****p<0.0001.

[0021] Figure 7 This is a schematic diagram of the viral structure of rAAV / 9-EF1α-FDIO-EGFP-5'miR-30a-shRNA(mFos)-3'miR-30a-WPREs.

[0022] Figure 8 This is a schematic diagram of the structure of the rAAV / 2Retro-hCGRP-hM4D(Gi)-P2A-EGFP-WPRE-hGH polyA virus.

[0023] Figure 9 This is a schematic diagram of the structure of the rAAV / 9Retro-hCGRP-hM4D(Gi)-P2A-mCherry-WPRE-hGH polyA virus. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] Example 1

[0026] 1) In situ injection of targeted TRPV1 into the dorsal root ganglion of the spinal cord + / A1 + AAV-shRNA (mFos) viruses in neurons can alleviate chronic inflammatory pain.

[0027] First, TRPV1 / A1-Flpo-tdTomato transgenic mice and wild-type mice were anesthetized with 1% isoflurane, and their body temperature was maintained using a 37°C heating blanket. Aureomycin was applied to the mice's eyes for moisturizing, and the surgical site was shaved and disinfected with povidone-iodine. The skin at L3-L6 of the spine was incised, and the muscles on both sides of the spinal processes were removed. The spine was fixed using a spinal cord adapter. Under a microscope, the left transverse processes of L4 and L5 were removed to expose the L4 and L5 DRGs. A glass electrode connected to a microsyringe was used to insert into the DRGs, and rAAV / 9-EF1α-FDIO-EGFP-5'miR-30a-shRNA(mFos)-3'miR-30a-WPREs virus (e.g., ) was injected. Figure 7(As shown) 800 nL (100 nL / min, needle retained for 10 min). Three weeks later, mice were anesthetized with 1% isoflurane, and 20 µL LCFA was injected into the left foot of the mouse using an insulin needle, with pressure applied to the injection site for 10 s to induce inflammatory pain. The 50% mechanical pain threshold, mechanical pain withdrawal rate, thermal pain withdrawal time, thermal pain withdrawal rate, and open field behavior of mice were measured before virus injection, 7 days before modeling, on the day before modeling, and 1, 3, 7, 10, and 14 days after modeling. For mechanical pain, mice were first placed in a transparent box on a raised metal mesh to acclimatize for 30 min, and then the surface of the mouse foot was vertically stimulated with von Frey fibers of different intensities for 2-3 s each time, with a 3-min interval. The 50% mechanical pain threshold was calculated using the up-and-down method. For the mechanical pain withdrawal rate, the mouse foot was stimulated with 0.6 g von Frey fibers for 2 s, repeated 10 times with the same pressure, and the proportion of mice exhibiting foot lifting or licking behavior was calculated. For heat-induced pain, mice were first placed in a transparent box containing an infrared heat therapy device for 30 minutes to acclimatize. The intensity of the device was set to 18%, and the maximum duration was 20 seconds to avoid burning the mice. Infrared light was directed at the soles of the mice's feet, and the time it took for the mice to lift or lick their feet was recorded. This was repeated three times, and the average value was taken. Each experiment was spaced 10 minutes apart. The heat withdrawal rate was measured to determine the proportion of mice that lifted or licked their feet in response to 12 seconds of infrared light at 15% intensity. This was repeated five times. For open field behavior, mice were first placed in a behavior room to acclimatize for 30 minutes. Then, each mouse was placed in the center of a white box with sides of 40 cm, and the mouse's behavior was recorded for 5 minutes. After each experiment, the box was cleaned with 75% alcohol and left to stand for 10 minutes.

[0028] Experimental results are as follows Figure 2 As shown in the results, the experimental group of transgenic mice experienced significant pain relief compared to the control group of wild-type mice.

[0029] 2) Intra-articular injection of targeted CGRP in the knee joint + AAV-hM4D(Gi) virus in neurons can alleviate chronic pain induced by knee osteoarthritis.

[0030] Wild-type mice were then anesthetized with 1% isoflurane, and their body temperature was maintained using a 37°C heating blanket. Chlortetracycline was applied to both eyes for moisturizing, and the surgical site was shaved and disinfected with povidone-iodine. The skin on the medial side of the mouse's knee was incised, and a glass electrode connected to a microsyringe was inserted into the knee joint to inject different doses (1 μL, 3 μL, 6 μL, and 8 μL, respectively) of rAAV / 2Retro-hCGRP-hM4D(Gi)-P2A-EGFP-WPRE-hGH polyA virus (2500 μL / min, needle retained for 10 min). Figure 8As shown in the figure), a mouse osteoarthritis model (KOA) was induced by intra-articular injection of 100 μL (5 mg / mL) of sodium monoiodoacetate (MIA) using an insulin needle. Simultaneously, CGRP was chronically inhibited by feeding the mouse with clozapine solution (CNO, 250 μg / L). + Neurons (control group fed with standard drinking water). The 50% mechanical pain threshold and heat withdrawal latency were measured every two days (measurement method as above). Experimental results are as follows: Figure 3 As shown. The results showed that, at week 5 after viral injection, compared with the control group, the pain threshold of mice in the 3 μL and 6 μL injection groups gradually recovered. This preliminary experiment suggests that the optimal viral dose is 3-6 μL, with an onset time of 5 weeks. Subsequently, 5 μL was selected as the viral injection dose, and rAAV / 2Retro-hCGRP-hM4D(Gi)-P2A-EGFP-WPRE-hGH polyA or rAAV / 9Retro-hCGRP-hM4D(Gi)-P2A-mCherry-WPRE-hGH polyA (e.g., 5 μL) was injected into the knee joint cavity of mice. Figure 9 (As shown) Virus (injection method as above), KOA modeling was performed 3 weeks later (modeling method as above). Two weeks after modeling, clozapine (CNO) solution or regular drinking water was continuously fed. The 50% mechanical pain threshold and thermal pain withdrawal time were measured on the day before modeling and on days 1, 3, 7, 10, 14, 15, 17, 19 and 21 after modeling (measurement method as above).

[0031] Experimental results are as follows Figure 4 As shown in the figure, the results indicate that the pain threshold of the experimental group mice was significantly restored.

[0032] 3) Intra-articular injection of targeted CGRP in the knee joint + AAV-hM4D(Gi) virus in neurons can inhibit the activity of this group of neurons in DRG.

[0033] Three weeks after intra-articular injection of rAAV / 2Retro-hCGRP-hM4D(Gi)-P2A-EGFP-WPRE-hGHpolyA or rAAV / 9Retro-hCGRP-hM4D(Gi)-P2A-mCherry-WPRE-hGHpolyA virus into the knee joint cavity of mice, a KOA model was established (using the same method as above). Two weeks after modeling, mice were continuously fed CNO solution or regular drinking water. One week later, mice were anesthetized with 1% isoflurane, exposing the thoracic cavity. 20 mL of 0.1 M phosphate-buffered saline (PBS) was injected into the left ventricle of the mice using a 20 mL syringe, followed by 20 mL of 4% paraformaldehyde (PFA). The lumbar spine was removed, cut along the dorsal and ventral sides, and the DRGs from both sides of the spinal cord were extracted and fixed in 4% PFA at room temperature for 3 h. The DRGs were then transferred to 30% sucrose solution and precipitated at 4°C for at least 24 h. After the DRGs settled, they were embedded in tissue embedding medium and sectioned to a thickness of 10 µm. After mounting the slides with adhesive slides, dry them in a 37°C oven for 1 hour. Then, place the slides in a washing box and wash three times with PBS for 10 minutes each time. Next, break the membrane with 1% Triton-100× PBST solution for 20 minutes, followed by washing with 0.3% PBST solution for 10 minutes. Remove the slides, wipe off the fluid around the tissue, circle the tissue with an immunohistochemistry pen, add immunofluorescence staining blocking solution (Beyotime, P0260), and incubate in the dark. After 1 hour, remove the blocking solution from the slides, add diluted c-Fos and CGRP primary antibodies, and incubate overnight at 4°C. The next day, warm the slides to room temperature for 20 minutes, then place them in a washing box and wash three times with 0.3% PBST solution for 10 minutes each time. Remove the slides, add diluted secondary antibody, and incubate in the dark for 90 minutes. Finally, wash four times with 0.3% PBST solution for 10 minutes each time, air dry at room temperature, and mount with mounting medium.

[0034] Experimental results are as follows Figure 5 As shown, the results indicated that, compared to the control group, CGRP levels were significantly lower in both the rAAV / 2Retro and rAAV / 9Retro experimental groups. + The proportion of c-Fos expression in neurons decreased significantly.

[0035] 4) Intra-articular injection of targeted CGRP in the knee joint + The rAAV / 2Retro-hM4D(Gi) virus in neurons is not expressed in the liver and kidneys.

[0036] In mice, rAAV / 2Retro-hCGRP-hM4D(Gi)-P2A-EGFP-WPRE-hGHpolyA or rAAV / 9Retro-hCGRP-hM4D(Gi)-P2A-mCherry-WPRE-hGHpolyA virus was injected intra-articularly into the knee joint cavity (injection method as above). Six weeks later, the mice were perfused (perfusion method as above). Liver and kidney tissues were collected and fixed in 4% PFA at 4°C for 12 h, then transferred sequentially to 20% and 30% sucrose solutions at 4°C for 24 h. After the tissues settled, they were embedded in tissue embedding medium and sectioned to a thickness of 20 µm. The sections were mounted on adhesive slides, dried, washed, and stained with GFP and mCherry (staining method as above).

[0037] Experimental results are as follows Figure 6 As shown, the results indicated that, compared with the blank control group, no AAV virus fluorescence expression was observed in the kidneys of mice, but significant mCherry fluorescence carried by rAAV / 9Retro was observed in the liver.

Claims

1. A recombinant adeno-associated virus that regulates the excitability of peripheral pain-related neurons, characterized in that, It includes an rAAV viral vector, peripheral pain-related neurons, and an inhibitory excitatory unit; the peripheral pain-related neurons include one or more of calcitonin gene-related peptide, neuropeptides, transient receptor potential vanillic acid subtype 1, and transient receptor potential A1 positive neurons; the inhibitory excitatory unit includes one or more of modified human muscarinic acetylcholine receptor M4 and shRNA that interferes with the Fos gene.

2. The recombinant adeno-associated virus according to claim 1, characterized in that, The rAAV virus vector includes one or more of rAAV1, rAAV2, rAAV4, rAAV9, rAAV2Retro, and rAAV9Retro.

3. The recombinant adeno-associated virus according to claim 1, characterized in that, It also includes expression units.

4. The use of a recombinant adeno-associated virus as described in claim 1, which modulates the excitability of peripheral pain-related neurons, in the preparation of pain medications.

5. The application according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable carriers and / or excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable carriers and / or excipients include at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

7. The application according to claim 4, characterized in that, The drug is administered via DRG in situ injection or peripheral injection.

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