AAV vector targeting improving method applicable to gene therapy drugs

Abstract

The invention discloses an AAV vector targeting improvement method applicable to gene therapy drugs, which comprises the following steps: performing high-throughput analysis on target tissue cells through a screening sub-module, screening candidate receptor genes interacting with AAV vector targeting molecules, and determining target receptor genes; the selection submodule is used for designing a gene editing system and selecting a promoter with tissue specific expression to drive the expression of the gene editing system; the introduction submodule is used for introducing the gene editing system into target tissue cells by adopting a gene transfer technology; and the evaluation sub-module is used for selecting the most suitable gene transfer mode according to the characteristics and treatment requirements of the target tissue, and detecting and evaluating the cells subjected to gene editing by utilizing a PCR molecular biological technology. The targeting property of the AAV vector to target tissue cells is improved, the infection of non-target tissues is reduced, and the side effect risk is reduced, so that the gene therapy effect is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy, and in particular to a method for improving the targeting of an AAV vector applicable to gene therapy drugs. Background Art

[0002] As a highly promising treatment method, gene therapy has shown broad prospects in the treatment of many diseases. Adeno-associated virus (AAV) vectors have become an important tool in the field of gene therapy due to their high safety and low immunogenicity. However, the existing AAV vectors are not targeted enough, making it difficult to accurately deliver therapeutic genes to target tissue cells, leading to infection of non-target tissues, reducing the therapeutic effect and increasing the risk of potential side effects. Traditional gene therapy methods lack real-time monitoring and feedback mechanisms, making it difficult to accurately understand the distribution and targeting of AAV vectors in the body, leaving doctors without scientific basis for adjusting treatment plans. At the same time, the existing AAV vectors have relatively limited technical means for evolution and screening, making it difficult to quickly obtain vectors with higher performance, limiting the further development of gene therapy technology. Therefore, researchers have been working hard to explore new technical methods to improve the targeting of AAV vectors, establish effective monitoring and feedback systems, and optimize the evolution and screening process of vectors. Summary of the invention

[0003] The purpose of the present invention is to solve the above problems and to design a method for improving the targeting of AAV vectors suitable for gene therapy drugs.

[0004] To achieve the above object, the technical solution of the present invention is that, further, in the above-mentioned method for improving the targeting of an AAV vector applicable to a gene therapy drug, the method for improving the targeting of an AAV vector comprises the following steps: Generate a smart nanocoating, wrap the smart nanocoating on the surface of the AAV vector, and use the CRISPR-Cas9 gene editing technology to genetically modify the target tissue cells; Introducing a receptor gene that interacts with the AAV vector targeting molecule into the target tissue cells, incorporating magnetic nanoparticles into the AAV vector, and guiding the vector to the target tissue cells through an external magnetic field; Establish a dynamic monitoring feedback system to monitor the distribution and targeting of AAV vectors in the body in real time for tracking; According to the monitoring results, the treatment plan is adjusted, and the AAV vector is evolved under a specific stress environment by simulating the natural selection process in vitro to obtain an evolved AAV vector; The evolved AAV vector is evaluated using high-throughput screening technology to obtain a target AAV vector.

[0005] Furthermore, in the above-mentioned method for improving the targeting of an AAV vector applicable to a gene therapy drug, the smart nanocoating is generated, the smart nanocoating is wrapped on the surface of the AAV vector, and the target tissue cells are genetically modified using the CRISPR-Cas9 gene editing technology, including: Using advanced nano-coating technology and equipment, the smart nano-coating can evenly coat the AAV vector; By controlling the reaction conditions and parameters, the smart nano-coating can make an intelligent response according to a specific biological signal; When the AAV vector enters the human body, it uses the smart nanocoating to sense biological signals such as the pH value, temperature, and presence of specific enzymes in the surrounding environment; If specific signals are detected near the target tissue, the nanocoating rapidly undergoes structural reorganization of the coating, dissolution of some components, or exposure of specific molecules.

[0006] Furthermore, in the above-mentioned method for improving the targeting of an AAV vector applicable to a gene therapy drug, the receptor gene that interacts with the AAV vector targeting molecule is introduced into the target tissue cells, magnetic nanoparticles are incorporated into the AAV vector, and the vector is guided to reach the target tissue cells through an external magnetic field, including: Perform high-throughput analysis on target tissue cells to screen candidate receptor genes that interact with AAV vector targeting molecules and identify target receptor genes; Designing a CRISPR-Cas9 gene editing system, and selecting a promoter with tissue-specific expression to drive the expression of the CRISPR-Cas9 gene editing system; Use gene delivery technology to introduce the CRISPR-Cas9 gene editing system into target tissue cells; According to the characteristics of the target tissue and treatment needs, the most appropriate gene delivery method is selected, and PCR molecular biology technology is used to detect and evaluate the gene-edited cells.

[0007] Furthermore, in the above-mentioned method for improving the targeting of an AAV vector applicable to a gene therapy drug, the establishment of a dynamic monitoring feedback system to monitor the distribution and targeting of the AAV vector in the body in real time for tracking includes: The size and magnetic properties of magnetic nanoparticles can be adjusted by controlling the preparation conditions, reaction temperature, time, and reactant concentration; Select the bioconjugation incorporation method to optimize the incorporation temperature, pH value and reaction time to uniformly incorporate the magnetic nanoparticles into the AAV vector; Monitor the distribution and content of magnetic nanoparticles in AAV vectors, and detect and evaluate the incorporated AAV vectors using magnetic performance testing and spectral analysis; An external magnetic field guidance system is established to control the intensity, direction and change law of the magnetic field of the AAV vector incorporated with magnetic nanoparticles, and guide the AAV vector to reach the target tissue cells.

[0008] Furthermore, in the above-mentioned method for improving the targeting of an AAV vector applicable to a gene therapy drug, the treatment plan is adjusted according to the monitoring results, and the AAV vector is evolved under a specific pressure environment by simulating the natural selection process in vitro to obtain an evolved AAV vector, including: MRI magnetic resonance imaging technology and PET positron emission tomography technology are used to scan and track the distribution and targeting of AAV vectors in the body in real time; Utilize monitoring technology combined with data analysis platforms to establish a dynamic monitoring feedback system; Acquire monitoring data, analyze the monitoring data using a dynamic monitoring feedback system, and extract the distribution, targeting, and therapeutic effects of the AAV vector in the body; Monitoring information is fed back to doctors and researchers based on the data analysis results. If the AAV vector is monitored to be excessively distributed in non-target tissues, the parameters of the magnetic field guidance system are adjusted.

[0009] Furthermore, in the above-mentioned method for improving the targeting of an AAV vector applicable to a gene therapy drug, the use of a high-throughput screening technology to evaluate the evolved AAV vector to obtain a target AAV vector comprises: Establishing a stress environment, wherein the stress environment at least includes different temperatures, pH values, drug concentrations, and immune stress factors; Select the evolution time and number of generations, and regularly select AAV vectors with higher performance during the evolution process; Analyze the gene mutation of the vector through sequencing technology, evaluate the functional changes of the vector through gene expression analysis technology, and evaluate the therapeutic effect of the vector through biological activity detection technology; The evolved AAV vectors are rapidly evaluated and screened using a high-throughput screening mechanism to obtain target AAV vectors, wherein the high-throughput screening mechanism at least includes targeting, gene expression efficiency, and biosafety.

[0010] Furthermore, a system for realizing a method for improving the targeting of an AAV vector applicable to a gene therapy drug is provided, characterized in that the system comprises the following modules: A coating generation module is used to generate a smart nano-coating, wrap the smart nano-coating on the surface of the AAV vector, and use the gene CRISPR-Cas9 gene editing technology to genetically modify the target tissue cells; A vector introduction module is used to introduce a receptor gene that interacts with the AAV vector targeting molecule into target tissue cells, incorporate magnetic nanoparticles into the AAV vector, and guide the vector to the target tissue cells through an external magnetic field; Dynamic monitoring module, used to establish a dynamic monitoring feedback system to monitor the distribution and targeting of AAV vectors in the body in real time for tracking; The vector adjustment module is used to adjust the treatment plan according to the monitoring results. By simulating the natural selection process in vitro, the AAV vector is allowed to evolve under a specific pressure environment to obtain an evolved AAV vector. The vector evaluation module is used to evaluate the evolved AAV vector using high-throughput screening technology to obtain a target AAV vector.

[0011] Furthermore, a system for realizing a method for improving the targeting of an AAV vector applicable to a gene therapy drug is provided, wherein the system comprises the following submodules: The encapsulation submodule is used to use advanced nano-coating technology equipment to uniformly encapsulate the AAV vector with the smart nano-coating; A control submodule, used to control reaction conditions and parameters so that the smart nano-coating makes an intelligent response according to a specific biological signal; The sensing submodule is used to sense biological signals such as pH value, temperature and the presence of specific enzymes in the surrounding environment using the smart nano-coating after the AAV vector enters the human body; The detection submodule is used to rapidly reorganize the structure of the nano-coating, dissolve some of its components, or expose specific molecules if a specific signal near the target tissue is detected.

[0012] Furthermore, a system for realizing a method for improving the targeting of an AAV vector applicable to a gene therapy drug is provided, wherein the system comprises the following submodules: The screening submodule is used to perform high-throughput analysis on target tissue cells, screen candidate receptor genes that interact with the AAV vector targeting molecules, and determine the target receptor genes; A selection submodule is used to design a CRISPR-Cas9 gene editing system and select a promoter with tissue-specific expression to drive the expression of the CRISPR-Cas9 gene editing system; An import submodule is used to introduce the CRISPR-Cas9 gene editing system into target tissue cells using gene delivery technology; The evaluation submodule is used to select the most appropriate gene delivery method based on the characteristics of the target tissue and treatment needs, and to detect and evaluate gene-edited cells using PCR molecular biology technology.

[0013] Its beneficial effects are that, through the screening submodule, it is used to perform high-throughput analysis on the target tissue cells, screen out candidate receptor genes that interact with the AAV vector targeting molecules, and determine the target receptor genes; the selection submodule is used to design the CRISPR-Cas9 gene editing system, and select a promoter with tissue-specific expression to drive the expression of the CRISPR-Cas9 gene editing system; the introduction submodule is used to introduce the CRISPR-Cas9 gene editing system into the target tissue cells using gene delivery technology; the evaluation submodule is used to select the most appropriate gene delivery method according to the characteristics of the target tissue and the treatment needs, and use PCR molecular biology technology to detect and evaluate the gene-edited cells. 1. Significantly improve the targeting of AAV vectors: By establishing intelligent nano-coatings, introducing receptor genes using gene editing technology, and incorporating magnetic nanoparticles and combining external magnetic field guidance and other means, the targeting of AAV vectors to target tissue cells is improved, the infection of non-target tissues is reduced, and the risk of side effects is reduced, thereby significantly improving the effect of gene therapy. 2. Real-time monitoring and feedback: The dynamic monitoring and feedback system established can track the distribution and targeting of AAV vectors in the body in real time, providing doctors with accurate information to facilitate timely adjustment of treatment plans and achieve personalized gene therapy. 3. Optimize vector evolution and screening: Simulate the process of natural selection in vitro, allow AAV vectors to evolve under specific pressure environments, and use high-throughput screening technology to evaluate the evolved vectors, which can quickly screen out target AAV vectors with higher performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0015] Figure 1 This is a schematic diagram of a first embodiment of a method for improving the targeting of an AAV vector applicable to a gene therapy drug in an embodiment of the present invention; Figure 2 This is a schematic diagram of a second embodiment of a method for improving the targeting of an AAV vector applicable to a gene therapy drug in an embodiment of the present invention; Figure 3 This is a schematic diagram of a third embodiment of a method for improving the targeting of an AAV vector applicable to a gene therapy drug in an embodiment of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] Those skilled in the art will appreciate that, unless otherwise stated, the singular forms "a", "an", "said" and "the" used herein may also include plural forms. It should be further understood that the term "comprising" used in the specification of the present invention refers to the presence of the features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0018] The present invention will be described in detail below in conjunction with the accompanying drawings. Figure 1 As shown, a method for improving the targeting of an AAV vector applicable to a gene therapy drug comprises the following steps: Step 101, generating a smart nano-coating, wrapping the smart nano-coating on the surface of the AAV vector, and using the CRISPR-Cas9 gene editing technology to genetically modify target tissue cells; Specifically, in this embodiment, advanced nano-coating technology and equipment are used to allow the smart nano-coating to uniformly coat the AAV vector; By controlling the reaction conditions and parameters, the smart nano-coating can make an intelligent response according to a specific biological signal; When the AAV vector enters the human body, it uses the smart nanocoating to sense biological signals such as the pH value, temperature, and presence of specific enzymes in the surrounding environment; If specific signals are detected near the target tissue, the nanocoating rapidly undergoes structural reorganization of the coating, dissolution of some components, or exposure of specific molecules.

[0019] Step 102, introducing a receptor gene that interacts with the AAV vector targeting molecule into the target tissue cells, incorporating magnetic nanoparticles into the AAV vector, and guiding the vector to reach the target tissue cells through an external magnetic field; Specifically, in this embodiment, high-throughput analysis is performed on target tissue cells to screen candidate receptor genes that interact with the AAV vector targeting molecule and determine the target receptor gene; Designing a CRISPR-Cas9 gene editing system, and selecting a promoter with tissue-specific expression to drive the expression of the CRISPR-Cas9 gene editing system; Use gene delivery technology to introduce the CRISPR-Cas9 gene editing system into target tissue cells; According to the characteristics of the target tissue and treatment needs, the most appropriate gene delivery method is selected, and PCR molecular biology technology is used to detect and evaluate the gene-edited cells.

[0020] Step 103: Establish a dynamic monitoring feedback system to monitor the distribution and targeting of the AAV vector in the body in real time for tracking; Specifically, in this embodiment, the size and magnetic properties of the magnetic nanoparticles are adjusted by controlling the preparation conditions, reaction temperature, time and reactant concentration; Select the bioconjugate incorporation method to optimize the incorporation temperature, pH value and reaction time to uniformly incorporate the magnetic nanoparticles into the AAV vector; Monitor the distribution and content of magnetic nanoparticles in AAV vectors, and detect and evaluate the incorporated AAV vectors using magnetic performance testing and spectral analysis; An external magnetic field guidance system is established to control the intensity, direction and change law of the magnetic field of the AAV vector incorporated with magnetic nanoparticles, and guide the AAV vector to reach the target tissue cells.

[0021] Step 104: adjusting the treatment plan according to the monitoring results, and allowing the AAV vector to evolve under a specific stress environment by simulating the natural selection process in vitro to obtain an evolved AAV vector; Specifically, in this embodiment, MRI magnetic resonance imaging technology and PET positron emission tomography technology are used to scan and track the distribution and targeting of AAV vectors in the body in real time; Utilize monitoring technology combined with data analysis platforms to establish a dynamic monitoring feedback system; Acquire monitoring data, analyze the monitoring data using a dynamic monitoring feedback system, and extract the distribution, targeting, and therapeutic effects of the AAV vector in the body; Based on the data analysis results, monitoring information is fed back to doctors and researchers. If the AAV vector is monitored to be excessively distributed in non-target tissues, the parameters of the magnetic field guidance system are adjusted.

[0022] Step 105: Use high-throughput screening technology to evaluate the evolved AAV vector to obtain a target AAV vector.

[0023] Specifically, in this embodiment, a pressure environment is established, and the pressure environment includes at least different temperatures, pH values, drug concentrations, and immune pressure factors; Select the evolution time and number of generations, and regularly select AAV vectors with higher performance during the evolution process; Analyze the gene mutation of the vector through sequencing technology, evaluate the functional changes of the vector through gene expression analysis technology, and evaluate the therapeutic effect of the vector through biological activity detection technology; The evolved AAV vectors are rapidly evaluated and screened using a high-throughput screening mechanism to obtain target AAV vectors, wherein the high-throughput screening mechanism at least includes targeting, gene expression efficiency, and biosafety.

[0024] Its beneficial effects are that, through the screening submodule, it is used to perform high-throughput analysis on the target tissue cells, screen out candidate receptor genes that interact with the AAV vector targeting molecules, and determine the target receptor genes; the selection submodule is used to design the CRISPR-Cas9 gene editing system, and select a promoter with tissue-specific expression to drive the expression of the CRISPR-Cas9 gene editing system; the introduction submodule is used to introduce the CRISPR-Cas9 gene editing system into the target tissue cells using gene delivery technology; the evaluation submodule is used to select the most appropriate gene delivery method according to the characteristics of the target tissue and the treatment needs, and use PCR molecular biology technology to detect and evaluate the gene-edited cells. 1. Significantly improve the targeting of AAV vectors: By establishing intelligent nano-coatings, introducing receptor genes using gene editing technology, and incorporating magnetic nanoparticles and combining external magnetic field guidance and other means, the targeting of AAV vectors to target tissue cells is improved, the infection of non-target tissues is reduced, and the risk of side effects is reduced, thereby significantly improving the effect of gene therapy. 2. Real-time monitoring and feedback: The dynamic monitoring and feedback system established can track the distribution and targeting of AAV vectors in the body in real time, providing doctors with accurate information to facilitate timely adjustment of treatment plans and achieve personalized gene therapy. 3. Optimize vector evolution and screening: Simulate the process of natural selection in vitro, allow AAV vectors to evolve under specific pressure environments, and use high-throughput screening technology to evaluate the evolved vectors, which can quickly screen out target AAV vectors with higher performance.

[0025] This embodiment also includes: 1. Create smart nanocoating and wrap it on the surface of AAV vector Material selection and development: The research team has invested a lot of time and energy in the screening and development of nanomaterials. After in-depth research on the physical, chemical and biological properties of various nanomaterials, a nanomaterial with special structure and function was finally determined for the construction of smart nanocoatings. This material has high biocompatibility and can be tightly combined with AAV vectors without triggering immune responses or negatively affecting the activity of the vector.

[0026] The nanomaterial also has the property of responding to specific biological signals, such as specific molecules, pH changes or temperature changes in the target tissue microenvironment. By precisely regulating the composition and structure of the nanomaterial, it is possible to achieve precise responses to different biological signals, thereby improving the pertinence and effectiveness of the smart nanocoating.

[0027] Coating preparation process: Advanced nanotechnology processes are used to prepare smart nanocoatings. First, the reaction conditions, such as temperature, pressure, and reaction time, are precisely controlled to ensure that the nanomaterials are evenly dispersed in the solution. Then, specific coating techniques, such as layer-by-layer self-assembly, sol-gel, or chemical vapor deposition, are used to gradually deposit the nanomaterials on the surface of the AAV vector.

[0028] During the coating preparation process, the quality and performance indicators of each step are strictly monitored. By using advanced analytical instruments such as electron microscopes, atomic force microscopes and spectrometers, the thickness, uniformity, stability and bioresponsiveness of the coating are monitored and evaluated in real time. This ensures that the quality of the coating meets the design requirements and provides reliable protection for subsequent gene therapy applications.

[0029] Coating performance optimization: In order to further improve the performance of the smart nanocoating, the research team has carried out optimization work in many aspects. For example, by adjusting the surface chemical properties of the nanomaterials, the binding force between the coating and the AAV vector can be enhanced to prevent the coating from falling off during transportation in the body. At the same time, the biological response mechanism of the nanomaterials is optimized to improve the sensitivity and response speed of the coating to the biological signals of the target tissue.

[0030] A series of in vitro and in vivo experiments were also conducted to verify the safety and effectiveness of the smart nanocoating. In the in vitro experiments, the protective effect of the coating on the AAV vector, the bioresponsiveness and the interaction with the cells were evaluated using a cell culture model and an experimental device simulating a physiological environment. In the in vivo experiments, the distribution, metabolism and therapeutic effect of the AAV vector wrapped in the coating were studied in the body through an animal model, providing a basis for further optimizing the coating performance.

[0031] 2. Use CRISPR-Cas9 gene editing technology to genetically modify target tissue cells and introduce receptor genes Target gene identification: Before gene editing, the research team needs to conduct in-depth research and analysis on the target tissue cells to identify the key receptor genes that interact with the AAV vector targeting molecules. This process involves a comprehensive understanding of the biological characteristics of the target tissue, disease mechanisms, and gene expression profiles.

[0032] Advanced biological technologies, such as genomics, transcriptomics, and proteomics, are used to perform high-throughput analysis of target tissue cells to screen for candidate receptor genes that may interact with AAV vector targeting molecules. Then, the final target receptor gene is determined through further experimental verification, such as cell experiments, animal model experiments, and clinical sample analysis.

[0033] CRISPR-Cas9 system design: Design an efficient CRISPR-Cas9 gene editing system for the identified target receptor gene. This includes selecting the appropriate guide RNA (gRNA) sequence to ensure that the Cas9 protein can accurately cut the target gene site. At the same time, optimize the expression vector and delivery method of the CRISPR-Cas9 system to improve the efficiency and specificity of gene editing.

[0034] During the design process, the characteristics of the target tissue cells and the safety of gene editing were fully considered. For example, a promoter with tissue-specific expression was selected to drive the expression of the CRISPR-Cas9 system to reduce the impact on non-target tissues. At the same time, the gRNA sequence was strictly screened and optimized to avoid off-target effects.

[0035] Gene Editing Implementation: In the process of gene editing, advanced gene delivery technology is used to introduce the CRISPR-Cas9 system into the target tissue cells. This can be achieved through methods such as viral vectors, nanoparticles or electroporation. According to the characteristics of the target tissue and the treatment needs, the most appropriate gene delivery method is selected to ensure that the CRISPR-Cas9 system can efficiently enter the target cells and play a role.

[0036] During the gene editing process, the efficiency and accuracy of gene editing are strictly monitored. Advanced molecular biology techniques, such as PCR, sequencing, and gene expression analysis, are used to detect and evaluate gene-edited cells. This ensures that the target receptor gene is accurately modified and introduced, laying the foundation for subsequent AAV vector targeted therapy.

[0037] 3. Incorporating magnetic nanoparticles into AAV vectors and using external magnetic fields to guide the vectors to target tissue cells Magnetic Nanoparticle Selection and Preparation: The research team conducted extensive screening and research on various magnetic nanoparticles to determine the most suitable magnetic material for incorporation into AAV vectors. Factors considered included magnetic strength, biocompatibility, stability, and modifiability. Finally, a magnetic nanoparticle with high magnetic strength, good biocompatibility, and controllable surface properties was selected.

[0038] The magnetic nanoparticles are prepared using advanced nanotechnology to ensure uniform particle size and good dispersion. By precisely controlling the preparation conditions, such as reaction temperature, time, and reactant concentration, the size and magnetic properties of the magnetic nanoparticles can be adjusted to meet different therapeutic needs.

[0039] Incorporation process optimization: In order to evenly incorporate magnetic nanoparticles into AAV vectors, the research team conducted a series of process optimizations. This includes selecting appropriate incorporation methods, such as physical adsorption, chemical binding or bioconjugation. At the same time, the incorporation conditions, such as temperature, pH value and reaction time, are optimized to improve the binding efficiency and stability of magnetic nanoparticles and AAV vectors.

[0040] During the incorporation process, the distribution and content of magnetic nanoparticles in the AAV vector are strictly monitored. Advanced analytical techniques such as electron microscopy, magnetic performance testing and spectral analysis are used to detect and evaluate the incorporated AAV vector. This ensures that the magnetic nanoparticles are evenly distributed in the vector and do not affect the structure and function of the AAV vector.

[0041] Magnetic field guidance system design: Design an efficient external magnetic field guidance system to achieve precise guidance of AAV vectors doped with magnetic nanoparticles. This system includes a magnetic field generator, a magnetic field controller, and a positioning device. By precisely controlling the intensity, direction, and change pattern of the magnetic field, the AAV vector can be guided to accurately reach the target tissue cells.

[0042] When designing the magnetic field guidance system, the physiological structure of the human body and the safety of the magnetic field are fully considered. The appropriate magnetic field strength and frequency are selected to avoid adverse effects on human tissue. At the same time, the distribution and change law of the magnetic field are optimized to improve the accuracy and efficiency of magnetic field guidance.

[0043] 4. Establish a dynamic monitoring feedback system and track the distribution and targeting of AAV vectors in vivo in real time Monitoring technology selection: The research team selected a variety of advanced monitoring technologies to track the distribution and targeting of AAV vectors in the body in real time. These include imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET) and optical imaging, as well as biomarker detection technologies such as fluorescent probes, radioisotope labels and biosensors.

[0044] According to different treatment needs and monitoring goals, the most appropriate combination of monitoring technologies can be selected. For example, for deep tissue monitoring, imaging technologies such as MRI or PET can be selected; for cellular level monitoring, technologies such as fluorescent probes or biosensors can be selected.

[0045] Monitoring system construction: Build an integrated dynamic monitoring feedback system that combines the selected monitoring technology with a data analysis platform. This system can collect real-time monitoring data of AAV vectors in vivo and quickly analyze and process them. By establishing a data transmission network and real-time monitoring software, remote transmission and real-time display of monitoring data can be achieved.

[0046] In the process of building the monitoring system, we focus on the stability and reliability of the system. We use advanced sensor technology and data processing algorithms to ensure the accuracy and timeliness of monitoring data. At the same time, we establish a complete security protection mechanism to protect the privacy and data security of patients.

[0047] Data analysis and feedback: Professional data analysis software is used to conduct in-depth analysis of monitoring data to extract important information about the distribution of AAV vectors in the body, targeting conditions, and therapeutic effects. By establishing data analysis models and algorithms, monitoring data can be quantitatively analyzed and evaluated to provide a scientific basis for adjusting treatment plans.

[0048] Based on the data analysis results, monitoring information is fed back to doctors and researchers in a timely manner. By establishing feedback mechanisms and communication channels, real-time adjustment and optimization of treatment plans can be achieved. For example, if it is detected that the AAV vector is excessively distributed in non-target tissues, the parameters of the magnetic field guidance system can be adjusted or the gene editing strategy can be optimized to improve the targeting of the vector.

[0049] 5. Simulate the natural selection process in vitro to evolve AAV vectors and evaluate the target AAV vectors using high-throughput screening technology Pressure environment simulation: In the process of simulating natural selection in vitro, the research team needs to establish a specific stress environment to promote the adaptation and optimization of AAV vectors during evolution. This stress environment can include different factors such as temperature, pH, drug concentration, immune pressure, etc. By precisely controlling these stress factors, the evolutionary pressure of organisms in the natural environment can be simulated to promote the evolution of AAV vectors.

[0050] When establishing the stress environment, the biological characteristics and treatment needs of the AAV vector are fully considered. Appropriate stress factors and intensities are selected to ensure that the AAV vector can maintain its structural and functional stability during the evolution process. At the same time, a monitoring system is established to monitor the impact of the stress environment on the AAV vector in real time, providing a basis for adjusting the stress parameters.

[0051] Evolutionary Process Control: In order to control the evolution of AAV vectors, the research team adopted a variety of strategies. This includes selecting the appropriate evolution time and evolutionary generations to ensure that AAV vectors can fully evolve without excessive mutation. At the same time, a screening mechanism is established to regularly screen out AAV vectors with higher performance during the evolution process to promote the direction of evolution towards improving targeting and therapeutic effects.

[0052] During the evolution process, advanced molecular biology techniques are used to analyze and monitor AAV vectors. For example, sequencing techniques are used to analyze gene mutations in vectors, gene expression analysis techniques are used to evaluate functional changes in vectors, and biological activity detection techniques are used to evaluate the therapeutic effects of vectors. These analysis and monitoring results can provide important references for the control of the evolution process.

[0053] High throughput screening technology applications: High-throughput screening technology is used to quickly evaluate and screen the evolved AAV vectors. High-throughput screening technology can detect and analyze a large number of AAV vectors at the same time, greatly improving the screening efficiency. This includes the use of automated screening equipment, the establishment of efficient screening indicators and data analysis algorithms, etc.

[0054] During the high-throughput screening process, we focus on the accuracy and reliability of the screening. We select appropriate targeting, gene expression efficiency, biosafety, etc. to ensure that the screened target AAV vector has the best performance and therapeutic effect. At the same time, we conduct strict verification and evaluation of the screening results to ensure that the screened target AAV vector can play a good role in practical applications.

[0055] See also Figure 2 In a method for improving the targeting of an AAV vector for a gene therapy drug, a dynamic monitoring feedback system is established to monitor the distribution and targeting of the AAV vector in the body in real time for tracking, including the following steps: Step 201, adjusting the size and magnetic properties of the magnetic nanoparticles by controlling the preparation conditions, reaction temperature, time and reactant concentration; Step 202, selecting a bioconjugate incorporation method to optimize the incorporation temperature, pH value and reaction time, and uniformly incorporating the magnetic nanoparticles into the AAV vector; Step 203, monitoring the distribution and content of magnetic nanoparticles in the AAV vector, and detecting and evaluating the incorporated AAV vector using magnetic property testing and spectral analysis; Step 204, establish an external magnetic field guidance system to control the intensity, direction and change law of the magnetic field of the AAV vector incorporated with magnetic nanoparticles, and guide the AAV vector to reach the target tissue cells.

[0056] See also Figure 3 In a method for improving the targeting of an AAV vector suitable for a gene therapy drug, the evolved AAV vector is evaluated using a high-throughput screening technique to obtain a target AAV vector, comprising the following steps: Step 301: Establish a pressure environment, wherein the pressure environment at least includes different temperatures, pH values, drug concentrations, and immune pressure factors; Step 302: Selecting an evolution time and an evolution generation, and regularly screening out AAV vectors with higher performance during the evolution process; Step 303: analyzing the gene mutation of the vector by sequencing technology, evaluating the functional changes of the vector by gene expression analysis technology, and evaluating the therapeutic effect of the vector by biological activity detection technology; Step 304: Use a high-throughput screening mechanism to quickly evaluate and screen the evolved AAV vector to obtain a target AAV vector. The high-throughput screening mechanism at least includes targeting, gene expression efficiency, and biosafety.

[0057] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for improving the targeting of an AAV vector suitable for gene therapy drugs, characterized in that: The method for improving the targeting of the AAV vector comprises the following steps: Generate a smart nanocoating, wrap the smart nanocoating on the surface of the AAV vector, and use the CRISPR-Cas9 gene editing technology to genetically modify the target tissue cells; Introducing a receptor gene that interacts with the AAV vector targeting molecule into the target tissue cells, incorporating magnetic nanoparticles into the AAV vector, and guiding the vector to the target tissue cells through an external magnetic field; Establish a dynamic monitoring feedback system to monitor the distribution and targeting of AAV vectors in the body in real time for tracking; According to the monitoring results, the treatment plan is adjusted, and the AAV vector is evolved under a specific stress environment by simulating the natural selection process in vitro to obtain an evolved AAV vector; The evolved AAV vector is evaluated using high-throughput screening technology to obtain a target AAV vector.

2. The method for improving the targeting of an AAV vector applicable to a gene therapy drug according to claim 1, characterized in that: The method of generating the smart nanocoating, wrapping the smart nanocoating on the surface of the AAV vector, and using the CRISPR-Cas9 gene editing technology to genetically modify the target tissue cells comprises: Using advanced nano-coating technology and equipment, the smart nano-coating can evenly coat the AAV vector; By controlling the reaction conditions and parameters, the smart nano-coating can make an intelligent response according to a specific biological signal; When the AAV vector enters the human body, it uses the smart nanocoating to sense biological signals such as the pH value, temperature, and presence of specific enzymes in the surrounding environment; If specific signals are detected near the target tissue, the nanocoating rapidly undergoes structural reorganization of the coating, dissolution of some components, or exposure of specific molecules.

3. The method for improving the targeting of an AAV vector applicable to a gene therapy drug according to claim 1, characterized in that: The method comprises introducing a receptor gene that interacts with the AAV vector targeting molecule into the target tissue cells, incorporating magnetic nanoparticles into the AAV vector, and guiding the vector to reach the target tissue cells through an external magnetic field, including: Perform high-throughput analysis on target tissue cells to screen candidate receptor genes that interact with AAV vector targeting molecules and identify target receptor genes; Designing a CRISPR-Cas9 gene editing system, and selecting a promoter with tissue-specific expression to drive the expression of the CRISPR-Cas9 gene editing system; Use gene delivery technology to introduce the CRISPR-Cas9 gene editing system into target tissue cells; According to the characteristics of the target tissue and treatment needs, the most appropriate gene delivery method is selected, and PCR molecular biology technology is used to detect and evaluate the gene-edited cells.

4. The method for improving the targeting of an AAV vector applicable to a gene therapy drug according to claim 1, characterized in that: The dynamic monitoring feedback system is established to monitor the distribution and targeting of the AAV vector in the body in real time for tracking, including: The size and magnetic properties of magnetic nanoparticles can be adjusted by controlling the preparation conditions, reaction temperature, time, and reactant concentration; Select the bioconjugate incorporation method to optimize the incorporation temperature, pH value and reaction time to uniformly incorporate the magnetic nanoparticles into the AAV vector; Monitor the distribution and content of magnetic nanoparticles in AAV vectors, and detect and evaluate the incorporated AAV vectors using magnetic performance testing and spectral analysis; An external magnetic field guidance system is established to control the intensity, direction and change law of the magnetic field of the AAV vector incorporated with magnetic nanoparticles, and guide the AAV vector to reach the target tissue cells.

5. The method for improving the targeting of an AAV vector applicable to a gene therapy drug according to claim 1, characterized in that: The method adjusts the treatment plan according to the monitoring results, simulates the natural selection process in vitro, and allows the AAV vector to evolve under a specific stress environment to obtain an evolved AAV vector, including: MRI magnetic resonance imaging technology and PET positron emission tomography technology are used to scan and track the distribution and targeting of AAV vectors in the body in real time; Utilize monitoring technology combined with data analysis platforms to establish a dynamic monitoring feedback system; Acquire monitoring data, analyze the monitoring data using a dynamic monitoring feedback system, and extract the distribution, targeting, and therapeutic effects of the AAV vector in the body; Monitoring information is fed back to doctors and researchers based on the data analysis results. If the AAV vector is monitored to be excessively distributed in non-target tissues, the parameters of the magnetic field guidance system are adjusted.

6. The method for improving the targeting of an AAV vector applicable to a gene therapy drug according to claim 1, characterized in that: The method of evaluating the evolved AAV vector using high-throughput screening technology to obtain a target AAV vector comprises: Establishing a stress environment, wherein the stress environment at least includes different temperatures, pH values, drug concentrations, and immune stress factors; Select the evolution time and number of generations, and regularly select AAV vectors with higher performance during the evolution process; Analyze the gene mutation of the vector through sequencing technology, evaluate the functional changes of the vector through gene expression analysis technology, and evaluate the therapeutic effect of the vector through biological activity detection technology; The evolved AAV vectors are rapidly evaluated and screened using a high-throughput screening mechanism to obtain target AAV vectors, wherein the high-throughput screening mechanism at least includes targeting, gene expression efficiency, and biosafety.

7. A system for realizing the method for improving the targeting of an AAV vector applicable to a gene therapy drug as claimed in claim 1, characterized in that: The system includes the following modules: A coating generation module is used to generate a smart nano-coating, wrap the smart nano-coating on the surface of the AAV vector, and use the gene CRISPR-Cas9 gene editing technology to genetically modify the target tissue cells; A vector introduction module is used to introduce a receptor gene that interacts with the AAV vector targeting molecule into target tissue cells, incorporate magnetic nanoparticles into the AAV vector, and guide the vector to the target tissue cells through an external magnetic field; Dynamic monitoring module, used to establish a dynamic monitoring feedback system to monitor the distribution and targeting of AAV vectors in the body in real time for tracking; The vector adjustment module is used to adjust the treatment plan according to the monitoring results. By simulating the natural selection process in vitro, the AAV vector is allowed to evolve under a specific pressure environment to obtain an evolved AAV vector. The vector evaluation module is used to evaluate the evolved AAV vector using high-throughput screening technology to obtain a target AAV vector.

8. A system for realizing the method for improving the targeting of an AAV vector applicable to a gene therapy drug as claimed in claim 1, characterized in that: The system includes the following submodules: The encapsulation submodule is used to use advanced nano-coating technology equipment to uniformly encapsulate the AAV vector with the smart nano-coating; A control submodule, used to control reaction conditions and parameters so that the smart nano-coating makes an intelligent response according to a specific biological signal; The sensing submodule is used to sense biological signals such as pH value, temperature and the presence of specific enzymes in the surrounding environment using the smart nano-coating after the AAV vector enters the human body; The detection submodule is used to rapidly reorganize the structure of the nano-coating, dissolve some components, or expose specific molecules if a specific signal near the target tissue is detected.

9. A system for realizing the method for improving the targeting of an AAV vector applicable to a gene therapy drug as claimed in claim 1, characterized in that: The system includes the following submodules: The screening submodule is used to perform high-throughput analysis on target tissue cells, screen candidate receptor genes that interact with AAV vector targeting molecules, and determine target receptor genes; A selection submodule is used to design a CRISPR-Cas9 gene editing system and select a promoter with tissue-specific expression to drive the expression of the CRISPR-Cas9 gene editing system; An import submodule is used to introduce the CRISPR-Cas9 gene editing system into target tissue cells using gene delivery technology; The evaluation submodule is used to select the most appropriate gene delivery method based on the characteristics of the target tissue and treatment needs, and to detect and evaluate gene-edited cells using PCR molecular biology technology.

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