CN115120706B

CN115120706B - Application of Netrin-3 in preparation of products for preventing and treating neuropathic pain

Info

Publication number: CN115120706B
Application number: CN202210922462.3A
Authority: CN
Inventors: ***; 白戈; 潘伟萍
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-08-02
Filing date: 2022-08-02
Publication date: 2023-07-21
Anticipated expiration: 2042-08-02
Also published as: CN115120706A

Abstract

The invention provides application of Netrin-3 in preparation of products for preventing and treating neuropathic pain, relates to the technical field of genetic engineering, and discovers that Netrin-3 participates in sensory functions of a nervous system, and Netrin-3 overexpression can inhibit the sensory nerve hyperplasia of dorsal root ganglion and radically relieve pain from pathogenic causes. Meanwhile, experiments prove that in the diabetic peripheral neuropathic pain model, the Netrin-3 treatment of the mice basically disappears, and the Netrin-3 has a strong pain relieving effect and high curative effect. In addition, the intrathecal injection of Netrin-3 mainly infects dorsal root ganglion located in peripheral nervous system, has low efficiency on spinal cord infection, hardly causes any influence on brain, and therefore has no central side effect of common medicines.

Description

Application of Netrin-3 in preparation of products for preventing and treating neuropathic pain

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of Netrin-3 in preparation of a product for preventing and treating neuropathic pain.

Background

Neuropathic pain is an inadaptive response of the nervous system to injury, and is a typical clinical symptom and sign, including hyperalgesia (pain manifested by innocuous stimuli), hyperalgesia (increased pain response to noxious stimuli), spontaneous pain (no pain arising from visible stimuli), and occasionally sustained burning pain. Severely affects learning, work, diet and sleep, reducing quality of life. The etiology of neuropathic pain is numerous, including post-traumatic nerve injury, spinal or brain injury (including stroke), diabetes, aids, post-herpetic neuralgia, multiple sclerosis, cancer, and the toxic effects of chemotherapeutic drugs, among others. Epidemiological studies have shown that neuropathic pain is between 7% and 10% prevalence in the population.

There is no clinical treatment for the etiology of neuropathic pain, and currently, clinically common therapeutic drugs include anticonvulsants, tricyclic antidepressants (Tricyclic antidepressants, TCAs), selective 5-hydroxytryptamine and norepinephrine reuptake inhibitors (Selective serotonin and norepinephrine reuptake inhibitors, SSNRIs), and opioids (see table 1), among others, the main problems are: the curative effect is limited; the adverse reaction is more, especially the central adverse reaction such as dizziness is more.

TABLE 1 current clinical drugs for neuropathic pain treatment

In view of this, the present invention has been made.

Disclosure of Invention

It is an object of the present invention to provide an application of Netrin-3 in the preparation of a product for preventing and/or treating neuropathic pain, so as to solve at least one of the technical problems existing in the prior art.

It is a second object of the present invention to provide a recombinant adenovirus that overexpresses Netrin-3.

In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

according to a first aspect of the present invention there is provided the use of Netrin-3 in the manufacture of a product for the prevention and/or treatment of neuropathic pain.

Further, the Netrin-3 comprises Netrin-3 protein and/or Netrin-3 gene vector.

Further, the gene vector comprises a Netrin-3 over-expression vector;

preferably, the Netrin-3 overexpression vector is an adenovirus overexpression vector, preferably an AAV2/9-CAG vector.

Further, the product includes a medicament.

Further, the administration mode of the drug includes injection administration, preferably intrathecal injection and plantar skin injection;

preferably, the drug is administered at a dose of 1X 10 viral titer ¹³ vg/mL, intrathecal injection of 10-50 microliters; the plantar skin is injected 10-50 microliters.

Further, the neuropathic pain includes diabetic peripheral neuropathic pain, post-traumatic nerve injury, spinal or brain injury, diabetes, aids, post-herpetic neuralgia, multiple sclerosis, cancer, and neuropathic pain caused by toxicity of chemotherapeutic drugs.

According to a second aspect of the present invention there is provided an adenovirus vector overexpressing Netrin-3, the adenovirus vector comprising a Netrin-3 gene and an AAV2/9-CAG vector.

According to a third aspect of the present invention, there is also provided a method for constructing the above-described Netrin-3 overexpressing adenovirus vector, comprising inserting cDNA of Ntn-3 into a recombinant AAV2/9 vector downstream of a CAG promoter to obtain the Netrin-3 overexpressing adenovirus vector;

preferably, the recombinant AAV2/9-CAG vector has a viral titer of 1X 10 ¹³ vg/mL。

In addition, the invention also provides a recombinant adenovirus for over-expressing Netin-3, which comprises the adenovirus vector.

Compared with the prior art, the invention has the following beneficial effects:

a large number of experiments show that the excessive proliferation of the dorsal root ganglion sensory nerves in the disease process is the root cause of pain, and Netrin-3 is expressed in the dorsal root ganglion, so the inventor of the invention discovers that Netrin-3 participates in the sensory function of the nervous system for the first time, and the Netrin-3 over-expression can inhibit the excessive proliferation of the dorsal root ganglion sensory nerves and relieve the pain from the root of the pathogenic cause. Meanwhile, experiments prove that in the diabetic peripheral neuropathic pain model, the Netrin-3 treatment of the mice basically disappears, and the Netrin-3 has a strong pain relieving effect and high curative effect. In addition, the intrathecal injection of Netrin-3 mainly infects dorsal root ganglion located in peripheral nervous system, has low efficiency on spinal cord infection, hardly causes any influence on brain, and therefore has no central side effect of common medicines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of expression of Netrin-3 (Ntn-3) in adult DRG primary sensory neurons provided in example 1 of the present invention;

FIG. 2 is a statistical chart of mechanical pain degree of Netrin-3 (Ntn-3) gene knockout mice provided in example 2 of the present invention in a sciatic nerve injury model;

FIG. 3 is a statistical chart of pathological pain degree of Netrin-3 (Ntn-3) knockout mice provided in example 2 of the present invention in a diabetic peripheral neuropathic pain model;

FIG. 4 is a graph showing the results of AAV-Ntn-3-induced Netrin-3 (Ntn-3) overexpression in diabetic peripheral neuropathy model mouse DRG provided in example 3 of the present invention;

FIG. 5 is a graph showing the results of the DRG over-expression of Netrin-3 (Ntn-3) in the present invention in example 3 to significantly relieve diabetic neuropathic pain.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Netrin-3 is an axonal guidance factor Netrin family member. The inventor of the present invention found that Netrin-3 was expressed predominantly in dorsal root ganglions (Dorsal Root Ganglion, DRG) of adult mice and humans through a large number of experimental studies. DRG is the site of primary sensory neuron cell bodies, thus suggesting that Netrin-3 is involved in sensory function. There is no report on the function of Netrin-3 in the nervous system. The inventor of the present invention, based on the research on the function of the gene, found that the gene is involved in the occurrence of neuropathic pain under pathological conditions and is a new therapeutic target for intervention pain. Based on this, the invention provides the use of Netrin-3 for the preparation of a product for the prevention and/or treatment of neuropathic pain.

The inventor of the invention discovers that Netrin-3 participates in the sensory function of the nervous system for the first time, and the overexpression of Netrin-3 can inhibit the sensory nerve hyperplasia of the dorsal root ganglion, so that the pain is relieved fundamentally from the pathogenic cause. The Netrin-3 can be applied to the preparation of medicines for preventing and/or treating neuropathic pain, so that neuropathic pain can be effectively linked, a basis is provided for clinical experiments, and a new way is provided for the research and development of new medicines. In addition, the intrathecal injection of Netrin-3 in the invention mainly infects dorsal root ganglion positioned in peripheral nervous system, has low efficiency on spinal cord infection and hardly causes any influence on brain, thus having no central side effect of common medicines.

In the invention, the Netrin-3 comprises Netrin-3 protein and/or Netrin-3 gene vector, and in the practical application of preventing and/or treating neuropathic pain, the Netrin-3 protein can be directly used, or the Netrin-3 gene vector can be used, or the Netrin-3 gene vector and the Netrin-3 gene vector can be combined.

In some preferred embodiments, in order to increase the expression efficiency of the Netrin-3 gene, it is preferred that the Netrin-3 overexpression vector is used as a gene vector, and it is further preferred that the adenovirus overexpression vector is used. The adenovirus overexpression vector comprises an adenovirus packaging system, such as AAV2/9-CAG-Netrin-3 (AAV 2/9-CAG-Ntn-3).

In some preferred embodiments, the product comprises a medicament. The active ingredients of the medicine comprise Netrin-3, and the medicine can effectively play an obvious role in preventing and/or treating neuropathic pain due to the inhibition effect of the Netrin-3 on the neuropathic pain, and is safe, nontoxic and small in side effect.

Preferably, the administration mode of the medicine comprises injection administration, and when the medicine is administrated in an injection form, the medicine can be prepared into any injection acceptable preparation form, for example, but not limited to injection or powder injection.

Intrathecal injection and plantar skin injection are preferred for more efficient inhibition of neuropathic pain.

In some preferred embodiments, the drug is administered at a dose having a viral titer of 1X 10 ¹³ The dose of vg/mL is 10-50 microliters, which may be, for example, but not limited to, 10 microliters, 20 microliters, 30 microliters, 40 microliters, or 50 microliters, with an injection volume of preferably 20 microliters when intrathecal injection is used, and with an injection volume of preferably 10 microliters when plantar subcutaneous injection is used.

The amount of the agent to be administered varies depending on the half-life of the agent in the body of the subject, and may vary depending on whether the agent is treated prophylactically or therapeutically. In prophylactic applications, relatively low doses are administered at relatively low frequency intervals over a long period of time. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until the progression of the disease is delayed or stopped, and preferably until the individual exhibits a partial or complete improvement in the symptoms of the disease, after which a patient prophylactic regimen may be administered.

In some preferred embodiments, the neuropathic pain comprises diabetic peripheral neuropathic pain, post-traumatic nerve injury, spinal or brain injury, diabetes, aids, post-herpetic neuralgia, multiple sclerosis, cancer, and neuropathic pain resulting from toxicity of chemotherapeutic drugs.

The invention also provides an adenovirus vector for over-expressing Netrin-3, wherein the adenovirus vector comprises a human Netrin-3 gene and AAV2/9-CAG.

The adenovirus vector for over-expressing Netrin-3 is constructed by the following method:

comprises inserting cDNA of Netrin-3 into recombinant AAV2/9 vector at downstream of CAG promoter to obtain adenovirus vector of over-expression Netrin-3;

In addition, recombinant adenoviruses comprising the above-described adenoviral vectors over-expressing Netin-3 are also within the scope of the invention.

The invention is further illustrated by the following examples. The materials in the examples were prepared according to the existing methods or were directly commercially available unless otherwise specified.

Example 1 determination of Netrin-3 expression in adult DRG Primary sensory neurons

Human DRG is obtained from the brain tissue resource library of Chinese people through ethical application, and the slice thickness is 10 μm. Mice were post-fixed with 4% PFA post-cardiac perfusion medium and harvested from the lumbar region L4-L6 DRG, frozen tissue sections, thickness 10 μm. Antigen development was completed with fluorescent-labeled goat anti-rabbit IgG (h+l) (cat No. a11008,1:1000, invitrogen, usa) and fluorescent-labeled goat anti-mouse IgG (h+l) (cat No. a11029,1:1000, invitrogen, usa) at room temperature for 1 hour after incubation of the antibodies overnight at 4 degrees with Netrin-3 antibody (rabbit antibody, cat No. PA5-62249, invitrogen, usa, 1:1000) and NeuN antibody (murine antibody, cat No. MAB377, millipore, usa, 1:500).

The results are shown in FIG. 1, wherein A is adult DRG staining, B is adult mouse DRG staining, and NeuN is neuron-specific indicator protein. NeuN expression by both Nerin-3 (Ntn-3) positive cells can be seen, indicating that Nerin-3 is specifically expressed in DRG neurons.

Example 2Netrin-3 knockout resulted in mice with significantly increased pain levels in various neuropathic pain models

1. Modeling method for mouse sciatic nerve injury model

SNI (Spared nerve injury) mouse modeling: the mice were anesthetized and the sciatic nerve was exposed, the tibial nerve and the common fibular nerve in the three branches of the sciatic nerve were ligated, leaving the sural nerve intact. Lesions cause pronounced allergies in the lateral areas of the paw, which are innervated by the retained sural nerve.

CCI (chronic constriction injury) mouse modeling: the sciatic nerve was exposed after the mice were anesthetized and three rings were ligated with 6-gauge suture at 1mm intervals at the femoral terminal of the sciatic nerve. CCI models induce pain in mice by injury-induced local inflammation.

2. Modeling method for pathological pain model of peripheral nerve of mouse diabetes

(1) Mice were fasted overnight (14-16 h).

(2) Streptozotocin (STZ, sigma) was formulated at a concentration of 10mg/mL with a citric acid buffer solution at pH 4.5 and administered by intraperitoneal injection at 100mg/kg based on the body weight of the mice for two consecutive days. It should be noted that: the STZ water solution is unstable, light-proof and ready-to-use, the operation should be completed as soon as possible, and the STZ water solution is put back into the ice box for preservation when the operation interval is longer.

(3) Blood glucose was measured, blood was collected from the tail of the mice, and the mice with blood glucose concentration exceeding 15mmol/L for two consecutive weeks were screened out using a fish-strike glucometer test, which was a diabetic mouse model, and blood glucose body weight per week was weighed and recorded.

3. Knocking-out method

The knockout is performed by inserting a fragment of beta-galactosidase gene (lacZ) and neomycin gene (NEO) into exon 2 of Netrin-3 (Ntn-3), and inserting loxP and FRT sites (UC Davis) at both sides, respectively, so that Ntn-3 cannot be expressed normally, resulting in functional loss.

It was found through experiments that Netrin-3 knockout (Ntn-3 KO) mice showed an increased degree of mechanical pain in the sciatic nerve injury model as compared to Wild Type (WT) mice, and the statistical results are shown in fig. 2, in which fig. a is the mechanical pain result in the SNI model, and fig. B is the mechanical pain result in the CCI model. The statistical method is two-way ANOVA; * P <0.05, < P <0.01; ns, have no meaning; error bars are expressed as mean±sem. a-B, each group n=8-12.

Meanwhile, in the diabetic peripheral neuropathic pain model, the pain degree of the Ntn-3KO mice is increased, and the statistical result is shown in fig. 3, wherein A is that the pain threshold of the Ntn-3KO mice is found to be significantly lower than that of the WT mice by mechanociception; the results of the acetone plantar cold pain score for B showed that Ntn-3KO mice were more sensitive to acetone-induced cold stimulus responses. The statistical method is two-way ANOVA; error bars are expressed as mean±sem with P <0.05, P <0.01; ns, have no meaning; error bars are expressed as mean±sem. B-C, each group n=8-12; D-F, n=5-26 per group.

Example 3Netrin-3 overexpression significantly reduces pain in mice in a diabetic peripheral neuropathic pain model

Overexpression method

1. AAV2/9-CAG-Ntn-3 with a viral titer of 1X 10 ¹³ vg/mL. After anesthesia of the mice, the mice were injected with virus in a volume of 10. Mu.l in the L4-L5 spinal cavity using a microsyringe.

The map of the constructed over-expression plasmid (CAG-Ntn-3-V5-P2A-EGFP) is shown as A in FIG. 4.

The schedule and schematic diagram of this embodiment are shown as B in fig. 4.

After four weeks of virus expression, qPCR (quantitative polymerase chain reaction) detects the expression condition of Netrin-3 (Ntn-3) in the DRG after over-expression, and the result is shown as C in FIG. 4, wherein the statistical method is Student's t test; error bars are expressed as mean±sem, P <0.001. Each group n=6-8. The graph shows that the over-expression amount of the Netrin-3 is extremely obvious from the difference between the over-expression amount of the Netrin-3 and that of the control group, which indicates that the over-expression plasmid constructed by the embodiment can effectively realize the over-expression of the Netrin-3.

The immunofluorescent staining pictures of DRG sections for four weeks of virus expression are shown as D in fig. 4, where the V5 tag protein, scale bar: 50 μm. From the figure, it can be seen that Netrin-3 can be efficiently expressed in the DRG by using the over-expression plasmid provided by the embodiment.

2. After 4 weeks of virus injection, mice were injected with STZ to induce a diabetic pathologic pain model. Mice were tested behaviourally 4 weeks after STZ injection.

The results of the mechanical pain behavioural analysis of each group of mice are shown as A in FIG. 5, and from the graph, it can be seen that the AAV2/9-CAG-Ntn-3 (AAV-Ntn-3) treatment can significantly relieve mechanical hyperalgesia of DN animals.

The results of the acetone induced cold pain behavioural analysis of each group of mice are shown as B in FIG. 5, and it can be seen from the figure that AAV-Ntn-3 treatment can alleviate DN-induced cold pain hypersensitivity.

C and D in FIG. 5 are immunofluorescent staining and quantitative statistics of hind paw skin GAP-43 of each group of mice, from which it can be seen that abnormal regeneration of sprouting of intraepidermal nerve fibers (intra-epidermal nerve fiber, IENF) projected from DRG sensory neurons was inhibited in diabetic mice overexpressing Ntn-3 compared to diabetic mice treated with AAV control group. Scale bar: 50 μm.

FIG. 5 statistical method is two-way ANOVA test; error bars are expressed as mean±sem with P <0.05 and P <0.01.A and B: each group n=7-12; c and D: each group n=7-9.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. Use of a substance overexpressing Netrin-3 for the preparation of a product for the treatment of neuropathic pain;

the neuropathic pain is diabetic peripheral neuropathic pain or sciatic nerve injury neuropathic pain.

2. The use according to claim 1, wherein the Netrin-3 overexpressing substance comprises Netrin-3 protein and/or a Netrin-3 gene vector.

3. The use according to claim 2, wherein the Netrin-3 gene vector is an adenovirus overexpression vector.

4. The use according to claim 3, wherein the Netrin-3 gene vector is an AAV2/9-CAG vector.

5. The use according to claim 3, wherein the product comprises a medicament.

6. The use of claim 5, wherein the mode of administration of the medicament comprises injection administration.

7. The use according to claim 6, wherein the medicament is administered by intrathecal injection and plantar skin injection.

8. The use of claim 6 wherein the medicament is administered in a dose of 10 to 50 microliters.

9. The use according to claim 8, wherein the medicament is administered in a dose of 20 microliters.

10. The use according to claim 3, wherein the viral titer is 1 x 10 ¹³ vg/mL。