In order to overcome problems associated with low scale production and high host immunogenicity that are encounted in viral methods, researchers have developed another means of transferring therapeutic genes into cells, however low levels of transfection and expression of the gene held non-viral methods at a disadvantage. Recent advances in vector technology have yielded molecule and techniques with transfection efficiencies similar to those of viruses (Murakami and Sunada, 2011).
Non-viral methods of gene transfer can also be called transfection and include:-
- Injection of Naked DNA
- Physical methods to enhance delivery which include; Electroporation
- Gene gun
- Chemical methods to enhance delivery which indlude;
- Lipoplexes and polyplexes
- Inorganic nanoparticles
Infection of Naked DNA:- This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular infection of a naked DNA plasmid have occurred with some success, however this has been very low when compared to other methods of transfection. This naked DNA can be inform of plasmid or transposon which when injected into cells can be transcribed into mRNA that will code for the functional protein, however cellular uptake of naked DNA is generally inefficient.
Electroporation:- Electroporation/Electropermeabilization is a method that uses short pulses of high voltage to carry DNA across the cell membrane. This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecular to pass through.
However, a high rate of cell death following electroporation has limited its use, including clinical applications. More recently, a newer method of electroporation termed electron-avalanche transfection has been used in gene therapy experimens, compared to electroporation, the technique resulted in greatly increased efficiency and less cellular damage.
Gene Gun:- The use of particle bombardment or the gene gun is another physical method of DNA transfection. In this techniques, the DNA which is the therapeutic gene is coated onto gold particles and loaded into a device which generates a force to achieve penetration of the DNA into the cells, leaving the gold behind on a “stopping disk”.
Sonoporation:- This method uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to move into cells.
Magnetofection:- In this method, the therapeutic gene/DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with cell monolayer (en.wikipedia.com).
Chemical Methods of Gene Transfer Oligonucleotides:- Oligonudeotides are short, single-stranded RNA molecules that have a wide range of applications in genetic testing research and forensics. In nature oligonucleotides are usually found as small RNA molecules that function in regulation of gene expression (eg. microRNA).
The use of synthetic oligonucleotides in gene therapy is to deactivate the genes involved in the disease process. There are several methods of achieving this:-
- Antisense gene therapy:- Specific to the target gene to disrupt the transcription of faculty gene.
- Use of small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faculty gene, disrupting translation of the faculty mRNA and therefore expression of the gene.
- Use of Oligodeoxynucleotides:- A further strategy uses double stranded Oligodeoxynucleotide as delay for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faculty gene, which reduces the transcription of the target gene, lowering expression.
- Additionally, single stranded DNA oligonucleotides have been used to direct a single base change within a mutant gene, this technique is referred to as Oligonucleotide mediated gene repair, target gene repair or targeted nucleotide alteration.
Other chemical methods of gene transfer includes; lipoplexes and polyplexes, dendrimers and inorganic nanoproteins.
CONVERTING A VIRUS INTO A VECTOR On Gene Therapy
- The initial step in converting a virus into a vector for use in gene therapy is to remove enough endogenous genetic material from the virus to render it harmless. This removal of genes from the virus is called deletion. A specific deletion within viral vectors ensures that it cannot cause undesired effect or trigger disease.
- The second step in converting a virus into a gene therapy vector is to replace the deleted viral genes with new genetic information useful to therapy. The viral vector containing the therapeutic genes must be designed so that it binds to and enters the cell. In most cases, it must make its way into the nucleus of the target cell. Once there, the vector genome must end up by expressing the therapeutic gene in the foreign environment of the target cell nucleus.
- The third and final step in generating a viral vector for effective gene therapy is to produce the viral vector in quantities and concentration sufficient for therapeutic use. This requires being able to gear up from small scale crafting of the viral vector to large-scale production of the vector.
PROCEDURE OF EX VIVO GENE THERAPY
Ex vivo/invitro gene therapy involves the following procedures;
- Somatic cells are collected from the affected individuals.
- The genetic defect is corrected by transferring the genes into the isolated cells.
- The genetically corrected cells are selected and grown.
- The genetically modified cells are transferred into the patients.
- During the process, the use of patients’ own cells (autologous cells) have no adverse immunological response after transplantation.
- Vectors derived from mouse retroviruses are mostly used; intact particles deliver the complete vector RNA to a host cell at a high frequency.
After transferring to the patient, the transferred target cells are tested to ensure that;
- The desired gene product is produced in the gene transferred humans.