Chemical Methods
Advances in gene transfer technology can make a breakthrough in the field of gene therapy. When gene transfer is used in the treatment of diseases, the purpose is to prevent genetic diseases by introducing genes encoding functional proteins into cells to normalize cells and even organs. Gene transfer is not only used to treat genetic diseases, but it can also produce large amounts of secreted proteins for direct use in therapy or vaccine production. It is well known that due to the size of nucleic acids and/or their physicochemical properties, such as hydrophilicity, it is difficult for nucleic acids to diffuse directly through the plasma membrane.
Therefore, various strategies for transferring nucleic acids, especially genes, have been developed. The ideal gene delivery and transfection system should have high transfection efficiency, low cytotoxicity and single-cell specificity, and be able to handle heterogeneous systems with many different cells simultaneously. The current gene transfer methods can be divided into three categories: viral methods, chemical methods and physical methods.
Chemical Methods for Gene Delivery
Although viral systems and physical methods have attracted great attention from researchers, they still have many disadvantages. Because of this, since the 1960s, many chemical transfection systems have been developed for gene transfer. The more commonly used are calcium phosphate, lipids and cationic polymers, including polyamideamide dendrimers and polyethylene Imine (PEI) and so on. Chemical gene delivery methods were initially seen as alternatives to viral delivery methods because of their potential to avoid certain problems related to viral systems. We can adjust the molecular structure or other characteristics to improve the binding ability of these chemical agents to biomolecules and the transfection efficiency, and ensure that their toxicity will not cause damage to the body. At present, a variety of synthetic transfection reagents have been commercialized and used for in vitro gene transfer, and have shown excellent efficacy in many experimental projects.
Liposomes and Cationic Lipids (Lipofection)
Among non-viral vectors, cationic lipid-based liposomes are by far the most commonly used gene delivery systems. Liposomes are synthetic lipid spheres composed of fatty acids on a polymer that has one or more bilayer membrane structures surrounding a water core and can be used to encapsulate small molecules. The direct action of cationic lipids with DNA leads to the formation of complexes and self-assembly of liposomes. The research of liposome gene delivery technology generally focuses on its formation efficiency. The relevant parameters include the mixing ratio, DNA concentration, the size of the cationic liposome used, and the ionic strength of the buffer.
Figure 1. Schematic representation of the structure of liposomes. (Jin, 2014)
Cationic Polymers
Cationic polymer is an ideal gene delivery medium that can appear as a substitute for viral gene therapy. It relies on endocytosis of synthetic polymer-based carriers bio-conjugated to the targeted gene or other biological molecules. Compared with liposomes, cationic polymer-based gene carriers (multi-chains) have good biodegradability, low toxicity, structural diversity and relatively high transfection efficiency. The gene transfer process is shown in Fig.2. Cationic fragments, organelle escape units and degradable fragments are essential for polymer-based gene delivery vectors. Gene carriers for ion polymerization such as chitosan, PEI, polylysine, and polyurethane are mostly derived from polyamines. These cationic polyamines not only protect DNA from degradation, but also promote endocytosis of the carrier by the endosomal membrane. Once the complex enters the cytoplasm, degradable fragments are necessary for the vector to release DNA. The organelle escape unit is one of the key factors affecting the transfection efficiency.
Figure 2. The schematic presentation of gene delivery process of polymers. (Jin, 2014)
Proteins
The advantage of protein and peptide vectors over other non-viral gene delivery strategies is that they can be prepared as monodisperse materials with precise molecular weight by solid-phase synthesis or genetic engineering methods. That is, large-scale production of viral vectors is still a limiting step, but the synthesis of therapeutic amounts of recombinant peptides for gene therapy can be more easily accomplished. In addition, the use of peptides as gene vectors also has the unique advantage that cellular obstacles in individual constructs can be overcome through precise control of molecular-level design. Considering the advantages of peptide-based vectors and understanding intracellular trafficking, we can prepare genetically engineered fusion peptides with different domains to overcome cellular barriers during gene delivery.
Figure 3. The schematic image of the fusion peptide with related sequences. (Sadeghian, 2012)
In current gene delivery methods, there are two difficulties that need to be overcome. One is the need to carry nucleic acids into target cells without potential risks; the other is that the nucleic acids need to penetrate into the cells through the plasma membrane. In order to obtain an effective vector system and achieve high rate of cell transfection, we need to continue to study the transfection process including the endocytosis of different molecular vectors and the release of DNA from the complex. Only by deeply understanding its mechanism can we purposefully discover and screen materials with excellent biodegradability and biocompatibility, which is an important process to obtain effective gene delivery vectors in the near future.
References
- Jin, L.; et al. (2014). Current progress in gene delivery technology based on chemical methods and nano-carriers. Theranostics. 4(3): 240.
- Sadeghian, F.; et al. (2012). Design, engineering and preparation of a multi-domain fusion vector for gene delivery. International journal of pharmaceutics. 427(2): 393-399.
