WO2023108489A1 - Product and method for producing endogenous brain-derived neurotrophic factor - Google Patents

Abstract

The present invention provides a product and method for producing endogenous brain-derived neurotrophic factor. Specifically disclosed in the present invention is a use of a viral vector carrying a light-sensitive protein gene or a chemical-sensitive protein gene in the preparation of a reagent for controlling the release of endogenous brain-derived neurotrophic factor in a brain area or increasing the concentration of endogenous brain-derived neurotrophic factor in the brain area. The light-sensitive protein gene is selected from ChR2, and the chemical-sensitive protein gene is selected from hM3dq. Also disclosed is a method for producing endogenous brain-derived neurotrophic factor, comprising: arranging an optical fiber in an upstream brain area and injecting a preparation containing a virus vector carrying a light-sensitive protein gene; and using an excitation light source to emit laser light to activate the release of endogenous brain-derived neurotrophic factor in a downstream brain area.

Description

A product and method for producing endogenous brain-derived trophic growth factors technical field

The invention belongs to the field of biotechnology, and in particular relates to a product and a method for producing endogenous brain-derived nutritional growth factors.

Background technique

Alzheimer's disease, also known as senile dementia, is a common neurodegenerative disease, mainly manifested as progressive cognitive decline. With the rapid increase of the aging population around the world, the number of Alzheimer's patients is increasing year by year. The traditional treatment theory is to clear β-amyloid protein, but the current method of clearing β-amyloid protein has been clinically failed. Therefore, it is urgent to find a new treatment method. Brain-derived trophic growth factor (BDNF) has a significant effect in the treatment of Alzheimer's disease, but the currently used BDNF is produced in the brain by exogenous administration or gene editing. Sexual vegetative growth factors have many disadvantages. Exogenous administration of brain-derived trophic growth factor has a short half-life and is rapidly metabolized in blood and cerebrospinal fluid, making it difficult to reach the target brain area. At the same time, due to its high affinity, it is difficult to spread in the brain area. The method of producing BDGF in the brain by gene editing has the disadvantages of uncontrolled protein expression, long and uncontrolled action time, and poor spatial specificity.

Contents of the invention

Aiming at the problems of short half-life of brain-derived trophic growth factor in the prior art, difficult diffusion in brain regions, difficult control of expression, and poor spatio-temporal specificity and the like. The present invention provides a method for producing endogenous brain-derived trophic growth factor, which can produce and exert effects immediately, and can be controlled and released at any time. The time control accuracy can reach millisecond level, and at the same time, the target brain area can be controlled. The spatial precision is high, the spatiotemporal specificity is high, and the endogenous brain-derived trophic growth factor produced has a high affinity and a better effect.

One aspect of the present invention provides a virus vector carrying a chemical sensitive protein or light sensitive protein gene to control the release of endogenous brain-derived vegetative growth factor in the brain region or increase the release of endogenous brain-derived vegetative growth factor in the brain. Use of the reagent in the area of concentration.

Further, the photosensitive protein gene is selected from ChR2; the chemosensitive protein gene is selected from hM3dq.

Further, the viral vector is selected from adeno-associated virus.

In a specific embodiment, the viral vector carrying the photosensitive protein gene is adeno-associated virus AAV-Camk2-ChR2-mCherry, and the viral vector carrying the chemical sensitive protein gene is adeno-associated virus AAV-Camk2-hM3dq-mCherry .

Further, the brain region is selected from the downstream brain region corresponding to the upstream brain region capable of releasing endogenous brain-derived trophic growth factors.

Further, the upstream brain region is selected from the paraventricular nucleus of the thalamus, and its corresponding downstream brain region is selected from the lateral entorhinal cortex.

Further, there is a functional and anatomical mapping connection relationship between the upstream brain region and the downstream brain region.

Another aspect of the present invention provides a tool set for preparing and controlling the release of endogenous brain-derived vegetative growth factors in the brain region, the tool set includes the above-mentioned virus vector carrying the chemical sensitive protein or light sensitive protein gene and the stimulus activation tool .

The stimulus-activating means is selected from optical fibers used to implant in the brain, chemicals used to activate chemosensitive proteins, or electrodes used to stimulate neurons to generate activity.

Further, the tool set also includes an excitation light source capable of stimulating the light-sensitive protein corresponding to the light-sensitive gene.

Further, when the chemosensitive protein gene is selected from hM3dq, the chemical substance that activates the chemosensitive protein is selected from clozapine oxide.

Further, the excitation light source can emit 473nm blue laser light.

Another aspect of the present invention provides the use of the tool set above in the preparation of medical devices for controlling the release of endogenous brain-derived trophic growth factors in brain regions or increasing the concentration of endogenous brain-derived trophic growth factors in brain regions.

Another aspect of the present invention provides a method for producing endogenous brain-derived vegetative growth factors, the method comprising the following steps:

S11) arranging optical fibers in the upstream brain region;

S12) Injecting a preparation comprising a viral vector carrying a photosensitive protein gene into the upstream brain region;

S13) Using an excitation light source to emit laser light to initiate the release of endogenous brain-derived trophic growth factor in downstream brain regions.

Further, the upstream brain region is selected from the paraventricular nucleus of the thalamus.

Further, the downstream brain region is selected from the lateral entorhinal cortex.

Further, the excitation light source emits a blue laser with a wavelength of 473nm.

Further, the light-sensitive protein gene in the viral vector carrying the light-sensitive protein gene is selected from ChR2.

Further, the viral vector in the viral vector carrying the photosensitive protein gene is selected from adeno-associated virus.

Another aspect of the present invention provides a method for producing endogenous brain-derived vegetative growth factors, the method comprising the following steps:

S21) Injecting a preparation comprising a viral vector carrying a chemosensitive protein gene into the upstream brain region;

S22) arranging electrodes in the upstream brain area or releasing chemical substances that activate chemosensitive proteins, so as to initiate the release of endogenous brain-derived trophic growth factors in the downstream brain area;

Further, the upstream brain region is selected from the paraventricular nucleus of the thalamus.

Further, the downstream brain region is selected from the lateral entorhinal cortex.

Further, the chemically sensitive gene in the viral vector is selected from hM3dq.

The methods described above are not intended for diagnostic or therapeutic purposes.

The present invention provides a scheme for producing endogenous brain-derived vegetative growth factors, which mainly releases endogenous brain-derived vegetative growth factors by specifically manipulating gene-edited neuron cells with light of a certain wavelength to the brain of the organism. , the overall solution can exert immediate effects, and can be manipulated at any time, with a temporal control accuracy of millisecond level, and can manipulate target brain regions in any space, with high spatial precision and spatiotemporal specificity.

Beneficial effect

This method can be manipulated at any time to produce endogenous brain-derived vegetative growth factors. The production of endogenous brain-derived trophic growth factors is instant and will not be rapidly metabolized. The action time can be controlled, and at the same time, it can be precisely spaced. Manipulate the production of endogenous brain-derived trophic growth factors, and control the expression level, and the effect is good.

Description of drawings

Figure 1 Schematic diagram of the experimental scheme for the release of endogenous brain-derived vegetative growth factors.

Among them, A reverse tracer dye cholera toxin subunit B (Cholera Toxin Subunit B, CTB) is injected into the lateral entorhinal cortex (LEC) schematic diagram; B tracer virus (AAV-Camk2-mCherry) is injected into the lateral entorhinal cortex (PVT ) schematic diagram; C schematic diagram of optogenetic virus (AAV-Camk2-ChR2-mCherry) injected into paraventricular nucleus of thalamus and recording electrode embedded in lateral entorhinal cortex (LEC); D optogenetic virus (AAV-Camk2-ChR2- mCherry) injected into the paraventricular nucleus of the thalamus and the optical fiber embedded in the paraventricular nucleus of the thalamus.

Figure 2 is a graph showing the fluorescent expression of reverse traced dye cholera toxin subunit B (CTB) injected into the lateral entorhinal cortex (LEC) and neurons in the upstream brain region that was reverse traced.

Figure 3 is a graph showing the fluorescent expression of the tracer virus (AAV-Camk2-mCherry) injected into the lateral entorhinal cortex (LEC).

Figure 4 shows the in situ hybridization technique to study the expression of BDGF in different brain regions.

Figure 5 is a flow chart of optogenetics technology.

Among them, A, the light-sensitive protein ChR2 gene sequence is inserted into the adeno-associated virus vector; B, the schematic diagram of the injection of the adeno-associated virus into the paraventricular nucleus of the thalamus; C, the schematic diagram of the optical fiber embedded in the paraventricular nucleus of the mouse thalamus; D, the Na ⁺ when neurons are excited Schematic diagram of channel opening.

Figure 6 shows the current discharge recorded by the patch clamp technique.

Fig. 7 is a photogenetic virus (AAV-Camk2-ChR2-mCherry) injected into the paraventricular nucleus fluorescence expression map of the thalamus.

Figure 8 shows the expression of endogenous brain-derived trophic growth factor in the lateral entorhinal cortex detected by Western blot.

Detailed ways

In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention will be described in detail below, but they should not be construed as limiting the scope of implementation of the present invention.

Example 1 Determining the projection relationship between neurons in the lateral entorhinal cortex and the paraventricular nucleus of the thalamus

By using the retrograde tracer dye Cholera Toxin Subunit B (CTB) to inject into the lateral entorhinal cortex of mice, the experimental scheme is shown in Figure 1A, and then observe the tracer dye distribution in the upstream brain area.

The experimental results are shown in Figure 2. The experimental results showed that the tracer dye distribution was observed in multiple upstream brain regions, such as the paraventricular nucleus of the thalamus, the medial intraventricular nucleus of the thalamus, the piriform cortex, and the amygdala. Among them, the tracer dye was most densely distributed in the paraventricular nucleus of the thalamus.

Then the forward tracer virus (AAV-Camk2-mCherry) was injected into the paraventricular nucleus of the mouse thalamus, the experimental scheme is shown in Figure 1B, and then the distribution of fluorescent expression in the downstream brain regions was observed to determine the excitability of the paraventricular nucleus of the thalamus Whether neurons project to the downstream brain region of the lateral entorhinal cortex. The experimental results are shown in Figure 3. After injection of the tracer virus (AAV-Camk2-mCherry), a large number of fluorescent labels identical to those of the parathalamic nucleus could be observed in the lateral entorhinal cortex.

The above experimental results prove that there is a direct projection relationship between the paraventricular nucleus of the thalamus and the lateral entorhinal cortex.

Example 3 Screening the upstream brain region capable of expressing brain-derived trophic growth factor

First, the method of in situ hybridization was used to determine the expression of brain-derived vegetative growth factor.

The experimental results are shown in Fig. 4. The in situ hybridization experiment results of the paraventricular nucleus of the thalamus indicated that the excitatory neurons of the paraventricular nucleus of the thalamus had abundant expression of BDGF.

Example 3 Verification of functional connectivity experiments of upstream and downstream brain regions

As shown in Figure 1C and D, optogenetics and electrophysiological techniques were then used to analyze whether there is a functional connection between the upstream and downstream brain regions. The technical process of optogenetics is shown in Figure 5. First, a virus carrying a light-sensitive gene was prepared. In this example, an adeno-associated virus AAV-Camk2-ChR2-mCherry containing the light-sensitive gene Channelrhodopsin-2 was used. The virus AAV-Camk2-ChR2-mCherry carrying the light-sensitive gene was injected into the paraventricular nucleus of the thalamus in the upper brain region of the mouse, and then the light stimulation of 473nm wavelength was given to the lateral entorhinal cortex of the mouse, and the lateral internal medial was recorded by patch clamp. Neuronal electrical signaling in the olfactory cortex brain region.

The experimental results are shown in Figure 6. The experimental results show that there are changes in the electrical signals of neurons at the axon terminals of neurons in the lateral entorhinal cortex. This is because the virus with a light-sensitive gene injected at the paraventricular nucleus of the thalamus can Excitatory neurons express light-sensitive proteins. At the same time, the axon terminals of the neurons in the lateral entorhinal cortex downstream of the paraventricular nucleus of the thalamus also express light-sensitive proteins. When light stimulation with a wavelength of 473nm is given to the axon terminals, the positive ion Na ⁺ on the outside of the ion channel on the axon terminals, A large amount of K ⁺ , Ca ²⁺ , and H ⁺ flow inward, which changes the membrane potential on both sides of the cell, generates an action potential, excites neurons, and activates neurons in the downstream outer entorhinal cortex at the same time. The experimental results prove that the upstream excitatory neurons of the paraventricular nucleus of the thalamus can functionally activate the neurons of the downstream lateral entorhinal cortex, and there is a functional connection between the upstream and downstream brain regions.

Example 4 The experiment of light stimulating the upstream brain area to release brain-derived trophic growth factor to the downstream brain area

The viral vector AAV-Camk2-ChR2-mCherry carrying the light-sensitive gene Channelrhodopsin-2 was injected into the paraventricular nucleus of the thalamus, and then the optical fiber was implanted above the excitatory neurons of the paraventricular nucleus of the thalamus. Genetics virus (AAV-Camk2-ChR2- mCherry) is injected into the paraventricular nucleus of the thalamus for the fluorescence expression diagram shown in Figure 7, and then a 473nm blue laser is transmitted to the excitatory neurons expressing light-sensitive protein in the paraventricular nucleus of the thalamus through an optical fiber channel, and the downstream brain is detected by Western blot. Endogenous brain-derived trophic growth factor expression in the lateral entorhinal cortex. A schematic diagram of the experimental method is shown in Figure 1D.

At the same time, a control group was set, and the experimental method was only to replace AAV-Camk2-ChR2-mCherry with AAV-ChR2-mCherry.

The experimental results are shown in Figure 8. The experimental results show that compared with the control group injected with AAV-ChR2-mCherry but not given light, the release of endogenous brain-derived trophic growth factor in the lateral entorhinal cortex of the experimental group is greatly increased, which is the control group. Group 2 times, there is a significant difference. This shows that when the excitatory neurons in the paraventricular nucleus of the thalamus are excited by light stimulation, they release brain-derived trophic growth factor to the lateral entorhinal cortex in the downstream brain region. This shows that by the method of the present invention, the brain-derived vegetative growth factor in the high-expression brain region of the brain-derived vegetative growth factor can be released to the lateral entorhinal cortex of the downstream brain region for nutrition and protection of the lateral entorhinal cortex nerves. Yuan.

Claims (10)

A virus vector carrying a photosensitive protein or chemosensitive protein gene is used in the preparation of a reagent for controlling the release of endogenous brain-derived vegetative growth factor in the brain area or increasing the concentration of the endogenous brain-derived vegetative growth factor in the brain area the use of; Preferably, the photosensitive protein gene is selected from ChR2; the chemosensitive protein gene is selected from hM3dq; Preferably, the viral vector is selected from an adeno-associated virus; More preferably, the viral vector carrying the photosensitive protein gene is adeno-associated virus AAV-Camk2-ChR2-mCherry, and the viral vector carrying the chemosensitive protein gene is adeno-associated virus AAV-Camk2-hM3dq-mCherry. The use according to claim 1, wherein the brain region is selected from the downstream brain region corresponding to the upstream brain region capable of releasing endogenous brain-derived trophic growth factors; Preferably, the upstream brain region is selected from the paraventricular nucleus of the thalamus, and its corresponding downstream brain region is selected from the lateral entorhinal cortex; Preferably, there is a functional and anatomical mapping connection relationship between the upstream brain region and the downstream brain region. A tool set for preparing and controlling the release of endogenous brain-derived vegetative growth factors in the brain region, the tool set includes virus vectors carrying light-sensitive protein and chemosensitive protein genes and stimulation and activation tools; Preferably, the photosensitive protein gene is selected from ChR2; the chemosensitive protein gene is selected from hM3dq; Preferably, the viral vector is selected from an adeno-associated virus; More preferably, the viral vector carrying the photosensitive protein gene is adeno-associated virus AAV-Camk2-ChR2-mCherry, and the viral vector carrying the chemosensitive protein gene is adeno-associated virus AAV-Camk2-hM3dq-mCherry. The tool set according to claim 3, wherein the stimulation activation tool is selected from optical fibers for implanting in the brain, chemicals for activating chemosensitive proteins, or electrodes. The tool set according to claim 3 or 4, wherein the tool set also includes an excitation light source capable of stimulating light-sensitive genes corresponding to light-sensitive proteins; Preferably, the excitation light source can emit 473nm blue laser light. The tool set according to claim 3 or 4, wherein when the chemosensitive gene is selected from hM3dq, the chemical substance activating the chemosensitive protein is selected from clozapine oxide. Use of the tool set according to claim 3 or 4 in the preparation of medical devices for controlling the release of endogenous brain-derived vegetative growth factors in brain regions or increasing the concentration of endogenous brain-derived trophic growth factors in brain regions. A method for producing endogenous brain-derived vegetative growth factors, characterized in that the method comprises the following steps: S11) arranging optical fibers in the upstream brain region; S12) Injecting a preparation comprising a viral vector carrying a photosensitive protein gene into the upstream brain region; S13) using an excitation light source to emit laser light to initiate the release of endogenous brain-derived trophic growth factors in downstream brain regions; Preferably, the light-sensitive protein gene in the viral vector carrying the light-sensitive protein gene is selected from ChR2, Preferably, the viral vector in the viral vector carrying the photosensitive protein gene is selected from adeno-associated virus; More preferably, the viral vector carrying the light-sensitive protein gene is an adeno-associated virus AAV-Camk2-ChR2-mCherry. The method of claim 8, wherein the upstream brain region is selected from the paraventricular nucleus of the thalamus; Preferably, the downstream brain region is selected from the lateral entorhinal cortex; Preferably, the excitation light source emits blue laser light of 473nm. A method of producing endogenous brain-derived vegetative growth factor, said method comprising the steps of: S21) Injecting a preparation comprising a viral vector carrying a chemosensitive protein gene into the upstream brain region; S22) Arranging electrodes in the upstream brain area or intraperitoneally injecting chemical substances that activate chemosensitive proteins to initiate the release of endogenous brain-derived trophic growth factors in the downstream brain area; Preferably, the upstream brain region is selected from the paraventricular nucleus of the thalamus; Preferably, the downstream brain region is selected from the lateral entorhinal cortex; Preferably, the chemosensitive protein gene in the viral vector is selected from hM3dq.

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