Nonviral Methods:
At meetings of such medical associations as the American Society for Gene Therapy, debate often centers on which vectors will prevail. The newer field of nonviral vectors suggests several novel approaches to gene therapy:[17]
Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses.
There are three main types of non-viral methods:
a) Gene Gun: Particle bombardment through a gene gun is an ideal method for gene transfer to skin, mucosa, or surgically exposed tissues within a confined area. DNA is deposited on the surface of gold particles, which are then accelerated by pressurized gas and expelled onto cells or a tissue. The momentum allows the gold particles to penetrate a few millimeters deep into a tissue and release DNA into cells on the path. Such a simple and effective method of gene delivery is expected to have important applications as an effective tool for DNA-based immunization. Further improvements could include chemical modification of the surface of the gold particles to allow higher capacity and better consistency for DNA coating, and fine-tuning of the expelling force for precise control of DNA deposition into cells in various tissues.[40] |
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b) Microinjection: This technique is useful for embryonic cells, primary cells and cells established in culture. 10-30 ng of DNA can be transferred into the nucleus of target cell. The method is tedious. Only one cell at a time can be handled, so laborious too. Integration is random and aberrations in adjacent DNA are common. Frequency of stable integration is higher than any other method by several orders of magnitude. |
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c) Electroporation: This technique is becoming popular very fast, and the main advantage is large number of cells can be transformed in a shorter time with more ease. 80-90% cells are killed but out of survivors, 30% are stably transformed. The method is suitable for primary cells such as hepatocytes, epidermal cells, pancreatic B cells, pre-B cells and haematopoietic stem cells.[46] |  |
a) Calcium Phosphate Mediated Transfection: In this technique, copies of healthy genes are mixed with charged substances. Calcium phosphate, dextran, certain lipids may be used to make mixture with DNA, which is then dumped onto recipient cells. A fraction of cells take up the DNA by endocytosis. The frequency is very low- only one cell in 106-107 cells gets transfected. However, the mechanism of incorporation of transfected DNA is not clear. Low frequency and aberrations caused in DNA makes the method less applicable in gene therapy protocols. It is mainly used for gene transfer into cultures cells.[47] |  |
a) Liposomes (Facilitated DNA): Direct administration of DNA or DNA complexes such as liposomes in vivo is in its infancy. Here an artificial lipid sphere with an aqueous core is created that can carry therapeutic DNA. The vector could carry genes of unlimited size, but its low efficiency compared with viruses and the absence of a mechanism to maintain the therapeutic effect are drawbacks. Recently, newer molecules—lipoplexes and polyplexes—have been created that can protect DNA from degrading during the insertion process. The most common use of lipoplexes has been in gene transfer into cancer cells.[46] Intravenous injection of liposomes to deliver rat insulin gene to rat liver and spleen, as model for diabetic treatment, has also been tried.[47] |
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a) Naked DNA: The simplest of all methods involves use of naked DNA, which is easy to prepare in large quantities and has high safety levels in tests thus far. This vector is injected into the muscle. The amount of naked DNA that can be injected is unlimited because it can deliver larger chunks of DNA and cause relatively less severe immune reactions. However, the usefulness of naked DNA appears to be limited by a low rate of gene transfer and lower gene efficiency compared to viruses. Because naked DNA does not integrate into the cells, its projected use may be for mechanical and topical applications and for accessible areas such as skin, vascular, pulmonary, and endothelial cells. Use by in vivo vaccination appears to be promising. A newer method of delivery has been the gene gun, which shoots DNA-coated gold particles into cells using high-pressure gas.[46][41] |
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b) Human artificial chromosomes: Researchers also are experimenting with introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations.[8] The idea of building a chromosome from the ground up—using a set of telomeres, a centrosome, and therapeutic material—could mimic one advantage of the herpes-based vector without the toxicity. This chromosome would be capable of carrying substantial amounts of genetic code and scientists anticipate that, because of its construction and autonomy the immune system would not attack it. It could carry a large insertion of multiple genes, which of course would be the downfall of this method—its delivery of such a large molecule to the nucleus of a target cell.[46] |  |
c) Infectious mammalian chromosomes: This technique represents a synthesis of viral and nonviral approaches. Researchers produced a component of the Epstein-Barr virus (EBV) in the form of a large circular molecule that shows stable expression for longer than a year. EBV is a very large virus belonging to the herpes family. In early transgenic studies, the efficiency of this method was 25 percent higher than strictly nonviral vectors. |  |
d) Starburst dendrites and new polymers: These polymers are shaped like a star. The fundamental building blocks to this new polymer class are referred to as “dendrimers”, which grow in a geometrically progressive fashion. Chemically bridging these dendrimers leads to the new class of topological macromolecules—”starburst polymers”. Partially fractured versions have the ability to release DNA from the endosomes, a quality lacking in some nonviral approaches. |
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e) Endothelial cells: Modifying endothelial cells into vectors could provide a specific delivery system. Harvested from subcutaneous fat or even the bloodstream, endothelial cells are readily available. Human umbilical vein endothelial cells (HUVECs) were evaluated for utility as a vector to achieve a bystander effect and killing of ovarian carcinoma cell lines. |  |
