Long term space flight is quickly becoming technologically possible, however the limiting factor will soon be the human body’s ability to survive the flight.l. Space exploration takes a toll on the human body and comes with many risks. A major health issue that arises with space exploration is Osteoporosis.
What is Osteoporosis?
Osteoporosis, more commonly known as brittle bone disease, is a condition that causes the bone to become more porous due to a decrease in bone density. This results in the bone becoming more fragile, which can increase the risk of bone fracturing [1]. Studies have shown that the human body not being under the influence of gravity causes the bones to lose density at a rate of over 1% per month. The elderly on Earth lose bone density at a rate of 1-1.5% per year, so a new and more efficient treatment is sorely needed in both circumstances [2].How does loss of bone density occur?
Calcitonin is a hormone that strongly inhibits Osteoclast function. Osteoclasts are involved in the breakdown of bone into calcium, whereas Osteoblasts increase bone density [3]. The parathyroid hormone inhibits Osteoblast function and stimulates the activation of Osteoclasts. Calcitonin opposes the effects of this by lowering calcium levels via suppression of Osteoclast function and stimulating Osteoblasts [3]. After the age of 35 years, the ageing process occurs which results in difference in levels of Osteoclasts and Osteoblasts. This results in loss of bone density. This change in the bones results in the bones becoming more fragile in old age and the risk of fractures becoming more common, additional effects occur, such as the increased risk of kidney stones, due to the elevated levels of calcium in the bloodstream.How does this occur in astronauts?
The answer is simple: lowered gravity. A typical space mission forces astronauts to endure three fields of gravity, all with different strengths: the travel to the destination is where astronauts are weightless. On the surface of their destination, the gravity acting upon the body is far less than as if you were on Earth, and finally when you return to Earth, the body has to readapt to the gravity here [4]. The body is constantly adjusting to suit its environment, this means that when in an environment where there is a reduced gravitational field, it adjusts, in this case the stress that bones normally go through every day supporting our bodies is reduced/ removed. This means the body does not need the bones to be as strong, and so reduces the bone density. This becomes a problem when transitioning to an environment that has a stronger gravitational field than your body has adapted to, say when astronauts return to Earth after a long time in space. The changes that the body has to go through in order to adjust itself to each situation causes bones to lose density at over 1% per month[4]. Even though when astronauts return to Earth they go through rehabilitation, there is a risk that bone loss may not be fully corrected, therefore astronauts are at greater risk of Osteoporosis-related fractures in the future.Current Treatments
The most common treatment for Osteoporosis is calcitonin, however it is not very efficient for space travel due to weight.
Calcitonin is usually administered in 2 ways: orally or via a nasal spray. The calcitonin used in both methods are from salmon. Although it can be effective, there is a 50% difference in the protein sequence between human and salmon calcitonin, this could mean that the salmon analogue is not as effective, also, salmon calcitonin is known to cause nausea and vomiting [5]. Furthermore, calcitonin nasal sprays are ineffective and slow acting while oral administration does not work effectively as the peptide needs to be absorbed into the bloodstream in order to work properly [5].
Our Project
We planned to use the CRISPR associated protein 9 (Cas9) protein, however it is a mutant, and is known as dCas9 targeted to the human Calcitonin promoter region in order to increase and decrease the expression of downstream elements. The dCas9 has a mutation in amino acid 840 causing the substitution of an alanine, this mutation cause the Cas9 to be unable to cleave DNA, instead the dCas9 will bind to the DNA and remain there until it is removed. To target the dCas9 to the promoter region we are using synthetic gRNAs, these are short scaffolds of RNA that have the complementary sequence to your target site. We designed 2 gRNAs targeted to a similar region of the calcitonin promoter,with complementary sequences 20 base pairs long, as this was the recommended length.
It would be technically very complex to assay the production of calcitonin in the E.coli, so therefore we decided to insert the calcitonin promoter into a GFP reporter setup, this comprised of the same elements in each track, but in different orders
Upregulation
In order to achieve a site targeted increase of expression we planned to fuse a transcriptional regulator to Cas9. We went through multiple different potential molecules, and decided to utilise stringent starvation protein A (sspA). sspA functions as a dimer and occurs naturally in E.coli. It is expressed when the cells undergo amino acid starvation, and binds to the RNA polymerase holoenzyme to induce expression. However sspA’s dimeric function posed a challenge to us as we were not confident in being able to express dCas9 and two sspA molecules all together. Instead we implemented a third plasmid that also expresses sspA, and that the sspA in the cytoplasm would dimerise with the fused sspA-dCas9 protein.
From figure 2A you can see that the upregulation track features 3 plasmids:
The upregulation construct.
The sspA plasmid
The upregulation test
Only the sspA plasmid and the upregulation test will be present. This is to assess the level of expression of GFP when the system is ‘inactive’ (the sspA is added in case it has any abnormal actions upon the E.coli or our plasmids). This allowed us to have a fully comparative assay for the effect of our system. We transformed the upregulation construct into the host cell, and the system will then get to work.
First, the gRNAs will bind to the target site on the calcitonin promoter region (fig 2A.1) Then the dCas9-sspA fusion will bind to the gRNA, and upon detecting an adjacent protospacer adjacent motif (PAM) it will also then bind to the DNA (fig 2a.2). The penultimate step in the system is that the cytoplasmic sspA will collide with and dimerise with the fused sspA, and recruit the holoenzyme, from there the transcripts will be expressed (albeit with larger RNA strands), this happens at the same time as the sigma 70 promoter, so will increase the levels of expression
Downregulation
The downregulation track (fig 2B) was started when we were questioned about implementation inside the cells of the parathyroid and whether there would be a way to switch off the system once the astronauts reached earth. This led to us creating a second track of experimentation, as not only would it answer that question, but it is also a useful tool to develop for future use. The concept was to use dCas9 to reversibly block the path of the RNAP and slow down expression. This would be achieved using two plasmids:
From figure 2A you can see that the upregulation track features 3 plasmids:
The downregulation plasmid.
The downregulation test
This system was tested in a similar way to the upregulation track, i.e, we measured absorbance and emission levels of liquid cultures of E.coli containing just the test plasmid. This gave us a baseline of GFP expression. Then the construct was implemented, and the assays was repeated.
The mechanism of action is a few steps simpler, first, with fig 2B.1
Future Programmable medicine in space
The future for this technology rests in being able to communicate a DNA sequence from Earth into space, and then implement that sequence in bacteria, or even yeast, to make any medicine that the astronauts would need. Using this system it could be done, as all you would need in space is a culture of microbes and a DNA synthesis machine. In space, they would already have the upregulation construct and the sspA construct implemented, then the sequence for the ‘test’ plasmids (that contain the code for the medicine/hormone) would be sent with other communications. This sequence would be created using the sequencer and then much like our project they use gibson assembly to form the full plasmid. There is the option to add a constitutive promoter to the gRNA synthesis, this would mean that the medicine is only created when needed. These would then be transformed, turned on and then the microbe could produce the medicine. There are obvious improvements that need to be made before this point, primarily the efficient transport into space, as currently a lot of room and weight is used in shielding from ionising radiation.
References
Sources [1] Reynolds, T. (2016) Osteoporosis handout on health. Available at: http://www.niams.nih.gov/health_info/Osteoporosis/default.asp (Accessed: 19 September 2016)[2] Ohshima, H (2015) Preventing bone loss in space flight. Available at: http://www.nasa.gov/mission_pages/station/research/benefits/bone_loss.html (Accessed: 19 September 2016).
[3] Caetano-Lopes, J., Canhão, H. and Fonseca, J. (2007) ‘Osteoblasts and bone formation’, Acta reumatológica portuguesa., 32(2), pp. 103–10
[4] Gushanas, T. (2016) The human body in space. Available at: http://www.nasa.gov/hrp/bodyinspace (Accessed: 19 September 2016)
[5] Calcitonin in the prevention and treatment of osteoporosis Available at: http://www.uptodate.com/contents/calcitonin-in-the-prevention-and-treatment-of-osteoporosis (Accessed: 19 September 2016)
