DESIGNING HOW WE AGE
By using a synthetic biology approach to our gene therapy solutions we set the foundation for enabling modulation of our treatment over time as required. Through our discussions with the general public, Parliament, world leading academics and industrial experts, we have completed a comprehensive analysis of the social, economic and ethical implications of our application. We have incorporated our findings from human practices to design our BioBrick devices to be safer and controlled for human use.
LUNGS: Gene Therapy
Whilst ageing is a universal process, there is complexity below the surface. Even though animal models suggest that intervening with ageing has had a positive impact on the healthy lifespan of the animals, we are still not able to guarantee the same effects on humans. Biosynthage wanted to make sure that even though we are proposing novel synthetic biology products to solve a real world problem, that we are still addressing the negative issues that our products also raise. Therefore, it was even more important that we have incorporated safety into the design of each idea.
After designing our SOD3 system, we wanted to find out what the experts in the ageing field think of our ideas We talked to William Bains, who is an ageing researcher and knows a lot about emerging therapies within the field. He told us that he thought that the gene therapy was a good idea, rapid and interesting. We also discussed that research indicates that oxidative stress is actually good for the body, and that we neede to conside this in our design. William bains suggested that it would be a good idea to titrate our system in a way in which SOD3 is only released when it senses high levels of oxidative stress. Therefore, in the future we would change the design such that our system will be activated by nfkb induced promotor. Nfkb is a transcription factor that is only released when it senses reactive oxidative species.
It is possible that we could add more nfkb binding sites to the existing promotor in the registry made by a previous 14_ATOMS-Turkiye team such that high levels of nfkb are required to release SOD3. This would make our gene therapy safer for the body, and more controlled.
Why did we chose to do gene therapy
There has been attempts of making an oral supplement with pure SOD enzyme to act as an ageing therapy, but it's been found that the SOD protein molecule is easily deactivated by harsh acids and enzymes contained in the digestive tract. No-one has yet made a SOD gene therapy. Also the lungs is highly susceptible to oxidative stress hence we combined the two ideas for a novel approach.
The generation of free radicals and their involvement in ageing have been a concern in the past years. These agents are atoms or molecules with an unpaired electron in the outer shell, which makes most of them highly reactive towards several relevant biomolecules. The proposed free radical theory of ageing establishes that the progression of ageing is because the accumulation of damage caused by these radicals, over a period of time. One example is the free radical called superoxide (O2-), which is a by-product of the respiratory cycle in every aerobic organism. The unregulated presence of this oxidative agent could lead to substantial cell damage. Fortunately, there are ways to prevent this damage. The antioxidants are reducing agents capable of limiting this damage through the passivation of important biomolecules. One example of these agents is the enzyme Superoxide dismutase (SOD). The mechanism of SOD is based on the partitioning (dismutation) of the superoxide into an oxygen (O2) or hydrogen peroxide (H2O2) molecule. Considering this information and as a way to prevent the harmful effects of ageing, we propose a novel gene therapy based on the regulated expression of SOD on respiratory cells.
We have design a 3rd Generation Lentiviral Systems for our gene therapy. We would lentivirus, a type of retroviruses which store their genomes as RNA to reverse transcribe it later to DNA. The DNA is incorporate into host cells’ genome. The use of lentivirus as the vector of the gene therapy give the opportunity to integrate large amounts of cDNA (~ 10 Kb) into the host genome, the virus can be used in animal models since it avoids the immune response and its rate of transfection is very high which is relevant since many important cell types are highly resistant to transfection.
This strategy known as Transient tripartite transfection requires three different plasmids:
Transfer plasmid: contains the genetic material of the genetic therapy, in this case Superoxide dismutase (SOD) + GFP, which it would be integrated into the host genome through the long terminal repeats (LTRs)
Packaging plasmid: Contains the HIV-1 core proteins. HIV derives its virulance from 9 genes, however just the main three are necessary for conserve its high transferability (gag, pol and int)
Envelope plasmid: determines the specificity of the virus, it contains the envelope of another virus (non HIV-1), normally VSV. Determines the specificity of the virus: what types of cells can be infected by the virus, etc.
The only plasmid to be inserted in the host genome it would be the one containing LTRs: the transfer plasmid that contains the Superoxide dismutase (SOD) + GFP. The sequences of the vector require extra security modifications to avoid the dangerous formation of viral particles packing viral genomes which could be produce by recombination of the different plasmids or with genes already present in the genome host.
Self-inactivating (SIN) Lentivirus is the name of the approach use for the Transfer plasmid which gives more security and a better transfection. The 5’ LTR is partially deleted and fused to a heterologous promoter such as CMV or RSV. SIN plasmids also harbour a large deletion in the U3 region of the 3’LTR which results in the virus being unable to reconstitute its promoter. The deletion is duplicated into the 5’LTR during the reverse transcription process, leaving the 5’LTR incapable of promoting transcription process, leaving the 5’LTR incapable of promoting transcription of full-length viral genomic RNA-making the virus replicant incompetent.
This therapy will use an inhaler to introduce SOD enzyme in the organism since SOD enzyme cannot be administrated efficiently as an oral supplement or intravenously due to reported problems with immune response, proteases, etc. Besides, an advantage of this approach is the application of the therapy directly in the lung cells which are very exposed to oxidizing agents.