Overview of Microinjection-based Gene Delivery

Gene therapy is a method of treating inherited or acquired diseases by delivering specific genetic material to target cells. Gene therapy can achieve therapeutic effects by interfering with gene function, restoring missing functions, or introducing new functions. A key step in gene therapy is to efficiently deliver gene vectors to target tissues/cells, which can be achieved by viral or non-viral vectors. Over the past 40 years, the field of gene therapy has undergone many transformations, many advances in treating human diseases, and many setbacks. Over the past 5 years, the field of gene therapy has seen regulatory approvals for a number of viral vector-based drugs spanning a variety of designs and purposes, including treating cancer, single-gene inherited diseases, and more.

Fig.1 Summary of Viral Gene Therapy Modalities

Microinjection is a gene transfer method that injects naked DNA directly into the nucleus. Microinjection can bypass cytoplasmic degradation, resulting in higher expression levels than cytoplasmic injection. Microinjection has the advantages of high efficiency, precision, and controllability. It can be used to deliver different types of gene vectors, such as plasmids and viral vectors, and can also be used to target different types of cells and tissues, such as embryos, stem cells, and nerve cells. Microinjection has high efficiency and stability in gene transfer and expression, so it has an important role and potential in gene therapy.

The Method, Principle and Mechanism of Microinjection in Gene Therapy

The principle of microinjection is to use micromanipulation technology to insert a glass microtube with a diameter of 1-10 microns into the nucleus or cytoplasm of the target cell, and then push the liquid containing the gene carrier into the cell through pressure or voltage. The operation steps of microinjection mainly include the following aspects: Firstly, put the liquid containing the gene carrier into the microtube, and seal the microtube opening with oil or air; Secondly, fix the target cells under the microscope and clamp them with a pipette or electrodes cells; then, connect the microtube with a pressure or voltage controller, and use the operating rod to insert the microtube into the nucleus or cytoplasm; Finally, inject the liquid into the cell through the pressure or voltage controller, and slowly withdraw the microtube.

Fig.2 Microinjection of DNA Into the Nucleus of a Single Cell

Microinjection can be used to deliver different types of gene vectors, including naked DNA, RNA, proteins, antibodies, etc. Among them, naked DNA is the most commonly used gene carrier because of its simple preparation, high stability, and low immune response. Naked DNA can directly enter the nucleus and undergo transcription and translation, thereby expressing the target gene. Viral vectors are another commonly used gene carrier, which utilizes the ability of viruses to infect host cells and integrate into the host genome to achieve gene transfer. Viral vectors usually need to remove the pathogenicity and replication ability of the virus itself, retain only the infection and integration functions, and carry the target gene fragment. Commonly used viral vectors include retroviruses, adeno-associated viruses, and adenoviruses.

Microinjection can be used to target different types of cells and tissues, including animal and plant germ cells, stem cells, and differentiated cells. Of these, germ cells are the most commonly targeted cell type because they can produce genetic changes and pass the gene of interest to offspring. For example, in animals, transgenic animals can be produced by microinjection of eggs or fertilized eggs. Likewise, in plants, transgenic plants can be produced by microinjection of pollen or seeds. Stem cells are another cell type that is often targeted because of their ability to self-renew and multilineage, and can be preserved and expanded for long periods of time. By performing microinjection on stem cells, their fate or function can be changed and applied in fields such as regenerative medicine or tissue engineering. Differentiated cells are the most difficult cell type to target because they are generally more stable and less proliferative, and may suffer from issues such as immune rejection or senescence. However, in some cases, microinjection of differentiated cells can also achieve therapeutic effects. For example, in the nervous system, through microinjection of neurons or neural stem cells, nerve damage can be repaired or neurodegenerative diseases can be treated.

Clinical Research Progress of Microinjection in Disease Treatment

As a method of gene therapy, microinjection has been verified and applied in clinical research in the treatment of various diseases. These disorders include genetic disorders, cancer, and other types of diseases.

Hereditary diseases are a group of diseases caused by gene mutations or defects, which are usually hereditary, irreversible and difficult to cure. Gene therapy can restore missing or damaged functions by delivering normal or repaired genes to a patient's cells, thereby achieving a cure or remission. As an efficient, precise, and controllable gene transfer method, microinjection can be used to deliver the desired gene carrier to patient cells or tissues. For example, in the treatment of hemophilia, injecting AAV vectors containing coagulation factor genes into the patient's liver cells or muscle cells can restore the patient's coagulation ability and reduce the risk of bleeding. In the treatment of muscular dystrophy, by injecting AAV vector containing micromolecule RNA or CRISPR/Cas9 system into patient muscle cells, the expression of mutated dystrophin gene can be reduced or the mutation site can be repaired, thereby improving muscle function and delaying progression.

Cancer is a kind of malignant tumor caused by abnormal cell proliferation and metastasis, and it is one of the most serious health threats facing human beings. Gene therapy can achieve various therapeutic strategies such as killing cancer cells, immune regulation or gene editing by delivering specific genes to cancer cells or immune cells. As a direct, efficient and flexible method of gene transfer, microinjection can be used to inject desired gene vectors into cancer cells or immune cells. For example, in immunotherapy, by injecting retrovirus or adeno-associated virus vectors containing specific receptor genes or inhibitory molecular genes into patient T cells, T cells can recognize and attack cancer cells and resist immunosuppressive signals. In gene editing, by injecting plasmids or AAV vectors containing specific nucleases such as TALENs or CRISPR/Cas9 systems into cancer cells, the targeted modification or excision of cancer cell genomes can be achieved, thereby affecting the growth, invasion or apoptosis.

Other types of diseases refer to a broad class of human common or rare diseases other than genetic diseases and cancers, such as neurodegenerative diseases, infectious diseases, metabolic diseases, etc. Gene therapy can achieve various therapeutic effects such as functional supplementation, regulation or change by delivering specific genes to patient cells or tissues. Microinjection can be used to inject the desired gene vector into patient cells or tissues. For example, in neurodegenerative diseases, by injecting AAV vectors containing genes such as neurotrophic factors, inhibitory molecules, or editing molecules into patient neurons or neural stem cells, it is possible to protect neurons from damage, promote nerve regeneration, or repair mutation sites. In infectious diseases, injecting plasmids or AAV vectors containing genes such as resistance molecules, clearance molecules or editing molecules into the patient's blood or target organs can enhance the body's defense against infectious agents, remove infectious agents or repair the damage caused by infectious agents.

Advantages, Disadvantages and Prospects of Microinjection in Gene Therapy

Microinjection has some obvious advantages and disadvantages, and also faces some challenges and opportunities.

The advantages of microinjection over other gene delivery methods are mainly in several aspects. First of all, microinjection is highly efficient and can achieve high levels of gene transfer and expression, thereby achieving a strong therapeutic effect. Second, microinjection is precise and can target specific cells or tissues, thereby reducing the impact on normal cells or tissues. Third, microinjection is controllable, which can achieve precise control of the dose, time and location of the gene carrier, thereby optimizing the treatment parameters. Fourth, microinjection is versatile and can be used to deliver different types of gene carriers, such as naked DNA, RNA, proteins, antibodies, and can also be used to target different types of cells and tissues, such as germ cells, stem cells, differentiated cells.

Microinjection has several major disadvantages and faces some challenges. First, microinjection is operationally complex and requires specialized equipment and technicians, which increases cost and time. Second, microinjection has safety issues that may cause adverse reactions such as cell damage, infection, or an immune response. Third, microinjection has limitations; it is difficult to apply to large-scale or systemic treatment, and it is also difficult to achieve long-term or sustained gene expression. Fourth, microinjection is uncertain and may be affected by factors such as the stability, integration, and toxicity of the gene carrier.

Based on the above, the future development direction and possibility of microinjection in gene therapy have several aspects. First, combining other technologies, such as nanotechnology, optical technology, and microfluidic technology, can improve the efficiency, precision, safety, and convenience of microinjection. Second, optimizing operating parameters, such as pressure, voltage, time, and temperature, can improve the success rate, stability, and reproducibility of microinjection. Third, the development of new gene carriers, such as nucleases, nucleic acid drugs, and nucleic acid modifications, can improve the versatility, specificity, and flexibility of microinjection. Fourth, expanding the scope of application, such as animal models and human clinical trials, can improve the effectiveness, feasibility, and popularization of microinjection.

Table 1. A summary of microinjection as a gene therapy method

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