Overview of Naked DNA Delivery Systems

Naked DNA delivery systems are a non-viral gene therapy method that uses naked DNA molecules (not combined with any carrier or protective agent) to directly transfect target cells or tissues so as to achieve the purpose of treating or preventing diseases. Naked DNA delivery systems have some advantages, such as high safety, low preparation cost, good stability, a weak immune response, and high transfection efficiency. Naked DNA delivery systems can be used to treat various inherited or acquired diseases, such as heart disease, cancer, HIV, malaria, and others. Naked DNA delivery systems can also be used to prepare DNA vaccines to resist foreign pathogens or tumor cells by inducing a specific immune response in the body.

However, naked DNA delivery systems also have some limitations, such as that DNA molecules are easily degraded by nucleases in body fluids, DNA molecules are difficult to pass through the cell membrane and nuclear membrane, the distribution and location of DNA molecules in the body are not easy to control, and DNA molecules in the genome are unstable. To overcome these limitations, scientists have developed various methods and techniques, such as changing the structure and size of DNA molecules, using different pathways and devices for the delivery of DNA molecules, and using various physical or chemical stimuli to enhance the transfection efficiency of DNA molecules. These methods and technologies not only improve the therapeutic effect of naked DNA delivery systems but also expand the scope of their application.

Methods and Principles of Naked DNA Delivery Systems

The methods of naked DNA delivery systems are mainly divided into two categories: physical methods and chemical methods. Physical methods use various physical devices or stimuli, such as injection, electroporation, ultrasound, and gene guns, to directly deliver naked DNA into target cells or tissues. The advantages of physical methods are high transfection efficiency, simple operation, and being not limited by the size and structure of DNA. The disadvantages are that it may cause damage to cells or tissues, it is difficult to control the distribution and positioning of DNA, and it is difficult to achieve large-scale transfection. The chemical method is to use various chemical substances or carriers, such as liposomes, polymers, and peptides, to form complexes with naked DNA, thereby increasing the stability of DNA and its ability to penetrate cell membranes. The advantages of chemical methods are high transfection efficiency, simple operation, and less damage to cells or tissues. The disadvantages are that they may cause immune or toxic reactions, are limited by DNA size and structure, and are difficult to integrate into the genome.

The principle of naked DNA delivery systems mainly includes the following steps: 1) naked DNA forms a complex with a carrier or a stimulus or is activated; 2) naked DNA enters the target cell or tissue; 3) naked DNA passes through the cell membrane and nucleus; 4) naked DNA is integrated in the genome or expressed in the cytoplasm; 5) naked DNA induces therapeutic or preventive effects in the body. Different methods and techniques may affect the efficiency and outcome of these steps, thereby determining the therapeutic efficacy of naked DNA delivery systems.

Mechanisms of Naked DNA Delivery Systems

The mechanism of naked DNA delivery systems mainly involves the transfection, expression, and immune response of naked DNA. The transfection of naked DNA refers to the process by which naked DNA enters the target cell or tissue and reaches the nucleus, which is affected by many factors, such as the size, structure, sequence, dosage, purity of naked DNA, the transfection method, carrier or stimulus, cell or tissue type, state, and receptor. The transfection mechanism of naked DNA is not fully understood and may involve various pathways, such as phagocytosis, endocytosis, fusion, and electroporation. Generally speaking, the transfection efficiency of naked DNA is higher in vitro but lower in vivo, which may be related to the complexity and diversity of the in vivo environment. The expression of naked DNA is the process by which naked DNA is transcribed and translated into a target protein or RNA in the cell. It is affected by, for example, the sequence of naked DNA, promoter, enhancer, terminator, type, state, and metabolism of cells or tissues, and other factors. The expression mechanism of naked DNA is not yet fully understood and may involve various pathways, such as genome integration, cytoplasmic expression, and nucleoplasmic shuttling. Naked DNA usually has higher expression efficiency in vitro but lower expression efficiency in vivo, which may be related to the stability and selectivity of the in vivo environment. The immune response of naked DNA is the process of immune response and regulation caused by naked DNA in the body. Factors including the source, sequence, dosage, and purity of naked DNA, as well as the type, state, and genetic background of the organism, affect this process. The immune response mechanism of naked DNA is not yet fully understood and may involve various pathways, such as Toll-like receptor recognition, CpG island activation, and cytokine release. Possibly based on the tolerance and inhibition of the in vivo environment, the immune response to naked DNA is weaker in vivo but stronger in vitro.

Applications of Naked DNA Delivery Systems in Disease Treatment

There are two main categories of applications of naked DNA delivery systems in disease treatment: gene therapy and DNA vaccines. Gene therapy is the use of naked DNA to carry therapeutic or preventive genes, transfected into target cells or tissues, so as to repair or replace defective or missing genes or increase or decrease the expression of certain genes to achieve the purpose of treating or preventing diseases. DNA vaccines use naked DNA to carry antigenic or immunoregulatory genes and transfect them into immune cells or other cells in the body, thereby inducing the body to produce a specific immune response and thereby achieving the purpose of preventing or treating diseases.

Application examples of naked DNA delivery systems in gene therapy:

  • Heart repair: use naked DNA to carry genes related to cardiomyocyte differentiation, such as GATA4, MEF2C, and TBX5, transfect them into human umbilical cord mesenchymal stem cells (hUC-MSCs), and then implant these transfected stem cells into damaged cardiac tissue, thereby promoting the differentiation of cardiomyocytes and the recovery of cardiac function.
  • Cancer: use naked DNA to carry tumor suppressor genes or suicide genes, such as p53, p21, Bax, and HSV-TK, and transfect them into tumor cells or surrounding normal cells, thereby inducing apoptosis or suicide of tumor cells or inhibiting the growth of tumor cell proliferation and metastasis.
  • HIV: use naked DNA to carry HIV-related genes, such as env, gag, and pol, and transfect them into immune cells or other cells in the body to induce the body to produce specific antibodies and cytotoxic T lymphocytes (CTL) to achieve the inhibition of HIV infection and replication.
  • Malaria: use naked DNA to carry Plasmodium-related genes, such as CSP and MSP, and transfect them into immune cells or other cells in the body, thereby inducing the body to produce specific antibodies and CTLs, thereby inhibiting the infection and development of Plasmodium.

Application examples of naked DNA delivery systems in DNA vaccines:

  • Influenza: Use naked DNA to carry influenza virus-related genes, such as HA and NA, and transfect it into immune cells or other cells in the body to induce the body to produce specific antibodies and CTLs to achieve the purpose of preventing or treating influenza.
  • Rabies: Use naked DNA to carry rabies virus-related genes, such as G, and transfect it into immune cells or other cells in the body to induce the body to produce specific antibodies and CTLs, thereby preventing or treating rabies.
  • Liver cancer: Use naked DNA to carry liver cancer-related genes, such as AFP and MAGE, and transfect them into immune cells or other cells in the body to induce the body to produce specific antibodies and CTLs, thereby preventing or treating liver cancer.
  • HPV: Use naked DNA to carry human papilloma-related genes, such as E6 and E7, and transfect them into immune cells or other cells in the body to induce the body to produce specific antibodies and CTLs, thereby preventing or treating HPV-related cervical cancer.

Progress of Naked DNA Delivery Systems in Clinical Trials

In terms of cardiovascular diseases, naked DNA delivery systems are mainly used to treat or prevent ischemic myocardium or limbs. The formation of new blood vessels and the repair of ischemic tissue are promoted by transfecting vascular endothelial growth factor (VEGF) or other angiogenesis-related genes. In addition, naked DNA delivery systems are mainly used to treat or prevent various types of tumors. Transfection of anti-tumor-related genes, such as p53 and IL-12, can induce apoptosis or suicide of tumor cells or activate the body's immune system to fight against tumors. Moreover, naked DNA delivery systems are mainly used to treat or prevent diseases caused by gene defects or deletions in genetic diseases, such as cystic fibrosis and Duchenne muscular dystrophy. Transfection of defective or missing genes can restore or replace normal gene function. As far as vaccines are concerned, naked DNA delivery systems are mainly used to prevent or treat various infectious or non-infectious diseases, such as HIV, HPV, HBV, influenza, and rabies. The body produces specific antibodies, and CTLs are induced by transfecting disease-related genes to prevent or treat corresponding diseases. In conclusion, the application of naked DNA delivery systems in clinical trials covers many fields, such as cardiovascular diseases, tumors, genetic diseases, and vaccines. Some trials have shown that this technology has certain safety and effectiveness characteristics. However, there are still problems and challenges in transfection efficiency, expression level, expression time, and immune response that need further optimization and verification.

References

  1. Huang NC, et al. Effective naked plasmid DNA delivery into stem cells by microextrusion-based transient-transfection system for in situ cardiac repair. Cytotherapy. 2020 Feb;22(2):70-81.
  2. Mendes BB, et al. Nanodelivery of nucleic acids. Nat Rev Methods Primers. 2022;2:24.
  3. Herweijer H, et al. Progress and prospects: naked DNA gene transfer and therapy. Gene Ther. 2003 Mar;10(6):453-8.
  4. Wolff JA, et al. The mechanism of naked DNA uptake and expression. Adv Genet. 2005;54:3-20.
  5. Nayerossadat N, et al. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res. 2012;1:27.
  6. Li S, et al. Nonviral gene therapy: promises and challenges. Gene Ther. 2000;7(1):31-34.
  7. Somvanshi P, et al. Peptide-based DNA delivery system. Med Novel Technol Devices. 2021;11:100091.
  8. Li ZM, et al. Biosafety assessment of delivery systems for clinical nucleic acid therapeutics. Biosaf Health. 2022;4(2):105-117.
  9. Zhang YJ, et al. Recent advances in non-viral gene delivery systems for cancer therapy and immunotherapy. Cancer Lett. 2021;518:1-15.
For research use only. Not intended for any clinical use.