A product and method for producing endogenous brain-derived trophic growth factors
By using viral vectors carrying genes for chemical or light-sensitive proteins within the brain region, combined with fiber optic or electrode stimulation, the precise release and concentration control of endogenous brain-derived growth factor (BDGF) have been achieved. This solves the problems of short half-life and difficulty in controlling expression levels in existing technologies, and achieves highly efficient spatiotemporally specific therapeutic effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
- Filing Date
- 2021-12-15
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies for brain-derived growth factor (BDGF) have drawbacks, including short half-life, difficulty in brain region diffusion, uncontrollable expression levels, poor spatiotemporal specificity, and exogenous administration and gene editing methods.
By using viral vectors carrying chemically or photosensitive protein genes, neurons in brain regions are activated through fiber optic or electrode stimulation, enabling precise release and concentration control of endogenous brain-derived growth factor. Adeno-associated virus vectors such as AAV-Camk2-ChR2-mCherry or AAV-Camk2-hM3dq-mCherry, combined with fiber optics or chemicals such as clozapine, achieve millisecond-level time control and high spatial precision release.
It achieves the immediate production and precise release of endogenous brain-derived growth factor with high temporal and spatial control precision, resulting in good efficacy and avoiding the metabolic problems of exogenous drug administration and the uncontrolled expression of gene editing.
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Figure CN116262140B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a product and method for producing endogenous brain-derived nutritional growth factor. Background Technology
[0002] Alzheimer's disease, also known as senile dementia, is a common neurodegenerative disease characterized by progressive cognitive decline. With the rapid increase in the global aging population, the number of Alzheimer's patients is rising dramatically year by year. Traditional treatment theory focuses on clearing β-amyloid protein, but this method has consistently failed clinically. Therefore, there is an urgent need to find new treatment methods. Brain-derived growth factor (BDNF) has shown significant efficacy in treating Alzheimer's disease; however, existing methods of producing BDNF in the brain through exogenous administration or gene editing have many drawbacks. Exogenous BDNF has a short half-life, is rapidly metabolized in the blood and cerebrospinal fluid, making it difficult to reach the target brain region. Furthermore, due to its high affinity, it is difficult to diffuse within the brain. Gene editing methods for producing BDNF in the brain have drawbacks such as uncontrolled protein expression, long and uncontrolled duration of action, and poor spatial specificity. Summary of the Invention
[0003] To address the problems of short half-life, difficulty in brain region diffusion, uncontrollable expression levels, and poor spatiotemporal specificity of existing brain-derived growth factor (BDGF), this invention provides a method for generating endogenous BDGF that can generate and exert its effects instantly, can be manipulated and released at any time with millisecond-level timing control precision, can simultaneously manipulate target brain regions, has high spatial precision and high spatiotemporal specificity, and produces endogenous BDGF with high affinity and better efficacy.
[0004] One aspect of the present invention provides the use of a viral vector carrying a gene for a chemically sensitive protein or a light-sensitive protein in the preparation of a reagent for controlling the release of endogenous brain-derived growth factor in the brain region or increasing the concentration of endogenous brain-derived growth factor in the brain region.
[0005] Furthermore, the light-sensitive protein gene was selected from ChR2; the chemical-sensitive protein gene was selected from hM3dq.
[0006] Furthermore, the viral vector was selected from adeno-associated virus.
[0007] In one specific embodiment, the viral vector carrying the light-sensitive 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).
[0008] Furthermore, the brain region is selected from the downstream brain region corresponding to the upstream brain region that is capable of releasing endogenous brain-derived growth factor.
[0009] Furthermore, 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.
[0010] Furthermore, there is a functional and anatomical mapping connection between the upstream and downstream brain regions.
[0011] Another aspect of the present invention provides a toolkit for preparing and controlling the release of endogenous brain-derived growth factor in brain regions, the toolkit comprising the aforementioned viral vector carrying a gene for a chemically sensitive protein or a light-sensitive protein and a stimulation activation tool.
[0012] The stimulation activation tool is selected from optical fibers for implantation in the brain, chemicals for activating chemosensitive proteins, or electrodes for stimulating neurons to generate activity.
[0013] Furthermore, the toolkit also includes an excitation light source capable of stimulating the photosensitive protein corresponding to the photosensitive gene.
[0014] Furthermore, when the chemosensitizing protein gene is selected from hM3dq, the chemical substance that activates the chemosensitizing protein is selected from clozapine.
[0015] Furthermore, the excitation source is capable of emitting a 473nm blue laser.
[0016] In another aspect, the present invention provides the use of the above-described toolkit in the preparation of medical devices for controlling the release of endogenous brain-derived growth factor in brain regions or increasing the concentration of endogenous brain-derived growth factor in brain regions.
[0017] In another aspect, the present invention provides a method for producing endogenous brain-derived growth factor, the method comprising the following steps:
[0018] S11) Fiber optic cables are deployed in the upstream brain region;
[0019] S12) Inject a formulation containing a viral vector carrying a light-sensitive protein gene into the upstream brain region;
[0020] S13) Uses an excitation source to emit a laser to initiate the release of endogenous brain-derived growth factor in downstream brain regions.
[0021] Furthermore, the upstream brain region is selected from the paraventricular nucleus of the thalamus.
[0022] Furthermore, the downstream brain region is selected from the lateral entorhinal cortex.
[0023] Furthermore, the excitation source emits a blue laser at 473nm.
[0024] Furthermore, the photosensitive protein gene in the viral vector carrying the photosensitive protein gene is selected from ChR2.
[0025] Furthermore, the viral vector carrying the light-sensitive protein gene is selected from adeno-associated virus.
[0026] In another aspect, the present invention provides a method for producing endogenous brain-derived growth factor, the method comprising the following steps:
[0027] S21) Injecting a formulation containing a viral vector carrying a gene for a chemically sensitive protein into the upstream brain region;
[0028] S22) Electrodes are placed in the upstream brain region or chemicals that activate chemosensitive proteins are released to initiate the release of endogenous brain-derived growth factor in the downstream brain region.
[0029] Furthermore, the upstream brain region is selected from the paraventricular nucleus of the thalamus.
[0030] Furthermore, the downstream brain region is selected from the lateral entorhinal cortex.
[0031] Furthermore, the chemically sensitive gene in the viral vector was selected from hM3dq.
[0032] The above methods are not for diagnostic or therapeutic purposes.
[0033] This invention provides a method for generating endogenous brain-derived growth factor (BDGF). It mainly releases BDGF by specifically manipulating gene-edited neurons in the brain through illumination of a certain wavelength of light. The overall method can take effect immediately and can be manipulated at any time with time control precision down to the millisecond level. It can also manipulate target brain regions in any space with high spatial precision and spatiotemporal specificity.
[0034] Beneficial effects
[0035] This method can manipulate the production of endogenous brain-derived growth factor at any time. The production of endogenous brain-derived growth factor is instantaneous, it is not rapidly metabolized, and the duration of action can be controlled. At the same time, the production of endogenous brain-derived growth factor can be precisely manipulated spatially, and the expression level can be controlled, resulting in good efficacy. Attached Figure Description
[0036] Figure 1 Schematic diagram of the experimental protocol for releasing endogenous brain-derived growth factor.
[0037] The diagrams show: A) Injection of the retrograde tracer dye Cholera Toxin Subunit B (CTB) into the lateral entorhinal cortex (LEC); B) Injection of the tracer virus (AAV-Camk2-mCherry) into the lateral entorhinal cortex (PVT); C) Injection of the optogenetic virus (AAV-Camk2-ChR2-mCherry) into the paraventricular nucleus of the thalamus and the placement of recording electrodes in the lateral entorhinal cortex (LEC); and D) Injection of the optogenetic virus (AAV-Camk2-ChR2-mCherry) into the paraventricular nucleus of the thalamus and the placement of optical fibers in the paraventricular nucleus of the thalamus.
[0038] Figure 2 Fluorescence expression of neurons in the lateral entorhinal cortex (LEC) and the upstream brain region that were retrogradely traced by the dye cholera toxin subunit B (CTB).
[0039] Figure 3 Fluorescent expression of tracer virus (AAV-Camk2-mCherry) injected into the lateral entorhinal cortex (LEC).
[0040] Figure 4 To study the expression of brain-derived growth factor in different brain regions using in situ hybridization technology.
[0041] Figure 5 A flowchart of the technology of photogenetics.
[0042] Among them, A. the light-sensitive protein ChR2 gene sequence is inserted into the adeno-associated virus vector; B. a schematic diagram of adeno-associated virus injection into the paraventricular nucleus of the thalamus; C. a schematic diagram of fiber optic implantation in the paraventricular nucleus of the mouse thalamus; D. Na+ during neuronal excitation. + Diagram showing the passageway opening.
[0043] Figure 6 The current delivery recorded by the patch-clamp technique.
[0044] Figure 7 Fluorescent expression of optogenetic virus (AAV-Camk2-ChR2-mCherry) injected into the paraventricular nucleus of the thalamus.
[0045] Figure 8 A map showing the expression of endogenous brain-derived growth factor in the lateral entorhinal cortex as detected by Western blot. Detailed Implementation
[0046] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.
[0047] Example 1: Determining the projection relationship between neurons in the lateral entorhinal cortex and the paraventricular nucleus of the thalamus.
[0048] The experimental protocol involved injecting the retrograde tracer dye cholera toxin subunit B (CTB) into the lateral entorhinal cortex of mice, as follows: Figure 1 As shown in A, the distribution of tracer dyes in the upstream brain region was then observed.
[0049] The experimental results are shown in Figure 2 The experimental results showed that tracer dyes were distributed in multiple upstream brain regions, such as the paraventricular nucleus of the thalamus, the lateral ventricular nucleus of the thalamus, the piriform cortex, and the amygdala. The distribution of tracer dyes in the paraventricular nucleus of the thalamus was the most dense.
[0050] Then, the anterograde tracer virus (AAV-Camk2-mCherry) was injected into the paraventricular nucleus of the mouse thalamus, as per the experimental protocol. Figure 1 As shown in Figure B, the distribution of fluorescent expressions in the downstream brain regions was then observed to determine whether excitatory neurons in the paraventricular nucleus of the thalamus projected to the lateral entorhinal cortex of the downstream brain regions. Experimental results are shown in [Figure B]. Figure 3 After injection of the tracer virus (AAV-Camk2-mCherry), a large number of fluorescent tags identical to those in the parathalamic nucleus were observed in the lateral entorhinal cortex.
[0051] The above experimental results demonstrate that there is a direct projection relationship between the paraventricular nucleus of the thalamus and the lateral entorhinal cortex.
[0052] Example 3: Screening of upstream brain regions capable of expressing brain-derived growth factor.
[0053] First, in situ hybridization was used to determine the expression of brain-derived growth factor.
[0054] The experimental results are shown in Figure 4 The results of in situ hybridization experiments in the paraventricular nucleus of the thalamus showed that excitatory neurons in the paraventricular nucleus of the thalamus expressed abundant brain-derived growth factor.
[0055] Example 3: Experiment to verify the functional connectivity of upstream and downstream brain regions
[0056] like Figure 1 As shown in C and D, optogenetics and electrophysiological techniques were then used to analyze whether there were functional connections between upstream and downstream brain regions. The optogenetics procedure is as follows: Figure 5As shown in the diagram, a virus carrying the light-sensitive gene was first prepared. In this embodiment, the adeno-associated virus (AAV-Camk2-ChR2-mCherry), which contains the light-sensitive gene Channelrhodopsin-2, was used. The AAV-Camk2-ChR2-mCherry virus carrying the light-sensitive gene was injected into the paraventricular nucleus of the thalamus in the upper brain region of the mouse. Then, light stimulation at a wavelength of 473 nm was applied to the lateral entorhinal cortex of the mouse, and the neuronal electrical signals in the lateral entorhinal cortex were recorded using patch-clamp.
[0057] The experimental results are shown in Figure 6 The experimental results showed changes in neuronal electrical signals at the axon terminals of neurons in the lateral entorhinal cortex. This was because a virus carrying a light-sensitive gene, injected into the paraventricular nucleus of the thalamus, could express light-sensitive proteins in the excitatory neurons of the paraventricular nucleus. Simultaneously, light-sensitive proteins were also expressed at the axon terminals of neurons in the lateral entorhinal cortex downstream of the paraventricular nucleus. When 473 nm light was applied to the axon terminals, the cation Na+ outside the ion channels on the axon terminals increased. + K + Ca 2+ H + Significant influx of neurons alters the membrane potential across the cells, generating action potentials and exciting neurons. This excites neurons in the downstream lateral entorhinal cortex. Experimental results demonstrate that excitatory neurons in the upstream paraventricular nucleus of the thalamus can functionally activate neurons in the downstream lateral entorhinal cortex, indicating a functional connection between the upstream and downstream brain regions.
[0058] Example 4: Experiment on the release of brain-derived growth factor from upstream brain regions to downstream brain regions by light stimulation.
[0059] The viral vector AAV-Camk2-ChR2-mCherry carrying the light-sensitive gene Channelrhodopsin-2 was injected into the paraventricular nucleus of the thalamus. Then, an optical fiber was implanted above the excitatory neurons in the paraventricular nucleus of the thalamus. The fluorescence expression of the genetic virus (AAV-Camk2-ChR2-mCherry) injected into the paraventricular nucleus of the thalamus is shown in the image. Figure 7 Then, a 473nm blue laser was transmitted through an optical fiber to excitatory neurons expressing light-sensitive proteins in the paraventricular nucleus of the thalamus. Western blot was used to detect the expression of endogenous brain-derived growth factor in the lateral entorhinal cortex, a downstream brain region. A schematic diagram of the experimental method is shown below. Figure 1 D.
[0060] A control group was set up, and the experimental method was simply to replace AAV-Camk2-ChR2-mCherry with AAV-ChR2-mCherry.
[0061] The experimental results are shown in Figure 8The experimental results showed that, compared to the control group which received AAV-ChR2-mCherry injection but no light, the release of endogenous brain-derived growth factor (BDGF) from the lateral entorhinal cortex in the experimental group was significantly increased, twice that of the control group. This indicates that when excitatory neurons in the paraventricular nucleus of the thalamus are excited by light stimulation, they release BDGF to the downstream lateral entorhinal cortex. This demonstrates that the method of this invention can release BDGF from brain regions with high BDGF expression to the downstream lateral entorhinal cortex, thereby nourishing and protecting neurons in the lateral entorhinal cortex.
Claims
1. The use of a viral vector carrying a light-sensitive protein gene in the preparation of a reagent for controlling the release of endogenous brain-derived growth factor in the brain region or increasing the concentration of endogenous brain-derived growth factor in the brain region. The light-sensitive protein gene is selected from ChR2; the viral vector carrying the light-sensitive protein gene is adeno-associated virus AAV-Camk2-ChR2-mCherry; The brain region is selected from the downstream brain region corresponding to the upstream brain region that can release endogenous brain-derived growth factor; the upstream brain region is selected from the paraventricular nucleus of the thalamus, and the corresponding downstream brain region is selected from the lateral entorhinal cortex. There is a functional and anatomical mapping connection between the upstream and downstream brain regions.
2. A toolkit for preparing and controlling the release of endogenous brain-derived growth factor in brain regions, the toolkit comprising a viral vector carrying a light-sensitive protein gene and a stimulation activation tool; The viral vector carrying the light-sensitive protein gene is adeno-associated virus (AAV-Camk2-ChR2-mCherry). The stimulation activation tool is selected from optical fibers used for implantation in the brain; The toolkit also includes an excitation light source capable of stimulating the photosensitive protein corresponding to the photosensitive gene; The excitation source is capable of emitting 473nm blue laser light.
3. Use of the toolkit of claim 2 in the preparation of a medical device for controlling the release of endogenous brain-derived growth factor in the brain region or increasing the concentration of endogenous brain-derived growth factor in the brain region.