Gene therapy, as conceptualised in the early 1970s, is based on the use of genetically engineered DNA to treat disease. Whilst most current approaches to gene therapy rely upon replacing a mutated gene with a correctly functioning gene in the nucleus of
Gene therapy, as conceptualised in the early 1970s, is based on the use of genetically engineered DNA to treat disease. Whilst most current approaches to gene therapy rely upon replacing a mutated gene with a correctly functioning gene in the nucleus of a cell, there is another approach that can turn the cells of the body into miniature drug factories. Using an appropriately designed synthetic DNA it is possible but challenging to penetrate the outer membrane of a cell and transfect the cell nucleus. Once this has occurred, the biological processes in the cell will begin to replicate the DNA thereby expressing therapeutic protein that can be targeted to fight the disease that the patient is suffering from. This is the intent behind DNA vaccines.
The DNA molecule is delivered to the cell through the use of a vector, a biological unit that contains the key fragment of DNA required for protein expression and a larger component that provides the structure of the molecule. Additional features of the vector can include components to encourage the expression of the gene and ensure that it attaches to the correct point on the host DNA. Delivering genetic material from one organism to another (a transgene) has been successfully, and commercially, developed for a number of applications including disease resistant crops, and the use of laboratory rodents to explore different human pathologies.
The key problem to producing cellular transfection is that cells don’t really want alien material to pass through the cell boundary into the nucleus and have developed protective structures to prevent this. In response, viruses have developed approaches to pass through the cell wall and infect the cell nucleus and it is by attaching the desired section of synthetic DNA to a virus that a lot of the early success in genetic engineering has been achieved. However, even when the virus has been selected and modified to reduce any undesired issues, tying the gene for replication to a virus can produce unwanted side effects with active immune responses, cancerous mutation and toxicity. Even though the virus is deactivated when used in this way, there is still concern that the virus could mutate in an uncontrolled way and deliver a new disease to the patient.
Using ‘naked’ DNA in the form of plasmids is the other main way of delivering a synthetic DNA. A typical plasmid is a circular section of DNA that naturally occurs in bacteria. As with the virus vectors, a section of the bacterial plasmid DNA is removed and replaced with the gene, and with enough supporting material to enable replication, to complete the synthetic plasmid. Plasmids present several advantages over viral vectors as they can be produced in large scale and present fewer issues with immunogenicity. The biggest problem with plasmid vectors is persuading the target cell to transfect and commence production of the therapeutic proteins which will attack the disease the patient is suffering from.
The delivery mechanisms being explored rely upon introducing energy to the cells to disrupt the function of the lipid bilayer and allow the dna into the Cell nucleus.
If DNA plasmids are injected into tissue with a regular hypodermic needle the therapeutic response is so slight it can barely be measured and this resistance to transfection is largely down to the lipid bilayer. Found on the outside of a cell, the lipid bilayer is an extremely thin membrane of fatty molecules that controls the passage of material into and out of the cell.
Getting the modified plasmids past the lipid bilayer and into the cell nucleus is now the focus of much attention and the techniques being considered now require the skills of (or input from) the medical device designer.
The delivery mechanisms being explored rely upon introducing energy to the cells to disrupt the function of the lipid bilayer and allow the DNA into the cell nucleus. There have been several approaches adopted including:
– The Gene Gun (or bioballistic) method; this uses small metal particles (usually gold or tungsten) which are coated with the desired DNA and fired into the target tissue.
– Needle-free jet injectors; these have been used to deliver vaccines to intramuscular and sub-cutaneous sites for years but the significant pressures that result can cause shearing of the DNA, tearing the plasmid into ineffective DNA fragments.
– Sonoperation; this relies on delivering ultrasonic energy to disrupt the cell membrane structure and improve take up of the DNA molecules.
– Magnetofection; magnetic nanoparticles coated with the DNA vectors are guided under directed magnetic fields to specific structures in the body.
– Electroporation; the most widely used way of improving DNA take up, this is the application of localised high voltage pulses produced by electrodes placed into muscle tissue.
Electroporation is used to improve the take up of cancer drugs. As these drugs can be highly toxic, the ability to target affected tumours with reduced dose quantities offers far better outcomes for patients.
In addition to this work on enhanced mechanical systems, one of our clients – Professor Matti Sallberg of ChronTech Pharma – has developed a novel approach to injecting a synthetic DNA vaccine into muscle tissue. The 