Ageing of lungs: the problem

Vital for respiration, the lungs are responsible for keeping us alive. As we age, our lungs become less effective at carrying out their function and these age-related changes contribute somewhat to an older person's reduced ability to do vigorous exercise, especially intense aerobic exercise. More specifically, the effects of aging on the respiratory system are similar to those that occur in other systems: there is a decrease in peak airflow and gas exchange and a decline in the effectiveness of lung defence mechanisms.

It is also known that ageing is the main risk factor for major non-communicable chronic lung diseases, including chronic obstructive pulmonary disease (COPD), most forms of lung cancer and idiopathic pulmonary fibrosis. COPD leads to chronic inflammation and progressive destruction of lung tissues, and its prevalence increases with age. This is currently a major public health problem worldwide and is the fourth leading cause of death in the world.

With a large and growing aging population, it is critical to understand how the body changes with age and how this impacts the entire respiratory system. Understanding the aging process in the lung is necessary in order to provide optimal care to our aging population. Hence Biosynthage wanted to create a therapy, targeting the ageing lungs, to maximise quality of life of the ageing population and increase the healthy human lifespan by preventing the onset of these deadly age-related diseases.

Ageing Lungs: What is the cause?

In order to create a healthy ageing therapy for the lungs, we needed to understand what actually defines the ageing of a cell. We particularly liked the definition of ageing provided by Aubrey de Grey that states that:

“Aging is the set of side-effects of metabolism that alter the composition of the body over time to make it progressively less capable of self-maintenance and thereby, eventually, progressively less functional.”

The side effects of metabolism produce damage to our cells which eventually leads to the functional decline (pathology) of our cells. This understanding allowed us to then investigate what actually causes the deterioration of our lung cells as we age and how we could build our synthetic biology strategy for postponing aging and thereby extending healthy lifespan.

As our cells' mitochondria produce energy in the form of ATP via oxidative phosphorylation, chemical species are released as a side product. One class of chemical species is known as reactive oxygen species (ROS), which are free radicals. In small quantities, ROS act as signalling molecules and are involved in various physiological roles. However, as mentioned before, ROS are toxic to the cells, and so they are naturally removed rapidly from the body.

Many papers suggest that as we age, we accumulate higher levels of ROS which causes DNA damage, cell dysfunction and cell death which lead to the cell being stressed (oxidative stress) and ageing the cell. Overproduction of ROS is also associated with pathogenesis of some age-related diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases.

Because of their anatomy, location and function, the lungs are highly susceptible to oxidative damage. Increasing evidence suggests that oxidative damage of lung cells due to ageing leads to the onset of these age-related diseases like COPD.

Natural defences in the body

Naturally, antioxidants break down harmful ROS into less harmful substances like h202. It’s been found that in imbalance in the amount of ROS and antioxidants leads to the damage of a cell, therefore leading to molecular ageing of a cell. An imbalance between generation of ROS and antioxidant defences leads to oxidative stress in which cell antioxidants are at an insufficient level to keep ROS below a toxic threshold.

Superoxidise dismutase (SOD) is an important antioxidant enzyme which is present in nearly all living cells exposed to oxygen and is located inside and outside the cell membrane.

SOD enzymes catalyse the reaction O-2 + O-2 + 2H+ → H2O2 + O2.

There are three types of SOD enzymes:

• SOD1 (Soluble - CuZnSOD) – Involved in removing oxidative stress causing ischemia-reperfusion injury and ischemic heart disease.
• SOD2 (Mitochondrial - MnSOD) - SOD2 clears mitochondrial reactive oxygen species (ROS).
• SOD3 (Extracellular - ECSOD) - protect the brain, lungs, and other tissues from oxidative stress.

SOD3 is found in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS. It is also the primary extracellular antioxidant enzyme found in the lungs and protects the extracellular matrix during lung injury. It has been found that mutations in the SOD3 protein are associated with reduced lung function in adults and lung function decline in chronic obstructive pulmonary disease (COPD). Because of their anatomy, location and function, the lungs are highly susceptible to oxidative damage. One study also showed that SOD3 levels decreased as the mice lungs aged. ROS accumulate as we age, therefore the overexpression of SOD is one potential way to decrease the levels of free radicals that accumulate.

Biosynthage: Our approach

So what can we do to prevent the deterioration of lung cells that then prevent the onset of age-related diseases like COPD?

Biosynthage have built a gene therapy device that reduces the amount of free radicals exposed to the lung cells, to reduce the ageing of the cells that lead to COPD and other age-related diseases in the lungs. Our idea is to overexpress antioxidant enzyme SOD3 in the lungs through a gene therapy approach targeted towards people who are 30-40 years old. Many papers suggest that overexpression of SOD3 in the lungs is a good way to increase healthy lifespan and to prevent the onset of pulmonary disease in the lungs. Our system is composed of a lentivirus vector with the SOD3 gene. The idea would be that the patient inhales the lentivirus, which then incorporates the SOD3 gene into the DNA of the lung epithelial cells. SOD3 will always be expressed in the epithelial cells, and will always be available to break down harmful free radicals into less harmful chemicals. Our therapy can be given to anyone as a preventative method (mop up ROS as you age) of ageing.

Results

Our BioBrick was constructed to have SOD3 with GFP downstream, so that when mammalian cells are transfected, we can see which cells have successfully taken up our lentivirus system. Virus titration was done using pRRLSIN-cPPT-hPGK-eGFP-WPRE (pRPEW) packaged with VSV-G envelope. Please refer to our results page to find out more about our data.

Future improvements

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 the fact that research indicates that oxidative stress is actually good for the body, and that we needed to consider 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 an oxidative-stress-inducible promotor. This would make our gene therapy safer for the body, and more controlled. Have a look at our design and integrated human practices pages to find out more.

Integrated human practices

We wanted to understand more about our gene therapy, so we discussed the safety and pharmacokinetics (dosage, efficacy) with cell and gene therapy catapult, a company founded by the UK government to enable safe scale up of gene therapy technologies. Catapult highlighted that safety is a really important component of our gene therapy that we need to incorporate this into the design of our therapy. So we decided that to ensure that our SOD3 enzyme was only expressed in lung epithelial cells, that we have a tissue-specific (lung) promoter. This will ensure that even if the lentivirus transfects into other tissue types, expression of our SOD3 gene will only occur in lung epithelial cells. We have also thought about the mode of delivery of our gene therapy into the human lungs. And we have decided that the gene therapy will by inhaled using an inhaler device for direct transfesction of the virus making it a faster, more rapid mode of delivery. In addition, inhaler devices are currently designed to reduce aerosol formation, therefore reducing the chance of GMO being released into the environment.

CELL AND GENE THERAPY CATAPULT and Prof Chris Mason Chief Science Officer, AvroBio Inc, will be providing ONGOING oversight of the SOD gene therapy research post-Jamboree.