Protein therapeutics, engineered bacteria for diagnosis &therapeutics

[research555]

Here are two papers, one on protein therapeutics and one review. I am currently reading two additional papers; one to do with cell lysis through quorum sensing and one which is talking about automation. I also posted a CRISPR system controlled by redox on the gene editing folder of our drive. it would be cool if we looked through that as it explains how to create a promoter which is electrically inducible.

I guess a question to really ask ourselves is how do we want this biosensor to work? Do we want protein dependent or DNA dependent sensing?

Minimizing side effects and maximizing returns

Paper:

https://www.researchgate.net/publication/336437172_Minimizing_side_effects_maximizing_returns_what_makes_a_smart_therapeutic_design

Current protein therapeutics

Advantages of protein therapies

• Protein - protein interactions are highly specific and potent
• Can be engineered to have better properties for targeting and specificity
• We can design protein therapeutics with desired properties due to knowledge of evolution and structure function relationships
• Easily predictable
• Manufacturing is simpler than small molecules

  Classes

  Cytokines

• Eg Tumor necrosis factor alpha
• These are signalling molecules that modulate immune response
• Cytokines act locally
• Can provide a replacement for endogenous protein that patient lacks or is missing due to genetics

  Hormones

• Signalling molecule that modulates cell to cell communication, and proliferation
• All of the above applies

  Monoclonal antibodies

• Proteins produced by wbc to attack specific targets
• Can be used to block undesired native signalling or trigger immune response
• Long half life in body
• Highly specific so avoids off targets

  Issues

• Drug administration is usually systemic, not targeted which can lead to cytotoxicity or bad side effects
• Synthetic biologists can manipulate their designs to provide safer more biologically inspired alternatives

  Fusion proteins for cell targeted delivery

• By creating fusion proteins composed of two or more different proteins put together synthetically, you can increase efficiency, safety and specificity of certain drugs
• An example of this is successful targeting via cytokine - antibody fusion

  Chimeric Activators

• Type of antibody fusion protein that incorporates a mechanism to prevent activating the cytokine/antibody domain to non target cell receptors
• Dont understand fully

Engineering bacteria for diagnostic and therapeutic applications

Paper

https://www.nature.com/articles/nrmicro.2017.172

One sentence overview

• Review paper taking a holistic approach to the question and exploring different key events in the field

  Advantages

• Bacteria can sense, and respond to changing environment
• Can deliver DNA or RNA to host cells
• Can provide a way to deliver therapy locally rather than systemically which can put strain on the body and result in unintended negative side effects
• Chemo for example damages all the cells in the body
• Can direct sensing or secretion of therapeutic to specific tissues or cells
• Can detect subclinical levels of biomarkers and thus be more effective than our current methods of detecting disease states
• Can detect biomarkers that are usually broken down quickly by for example the gut
• Can secrete therapeutics with low biostability

  Things to consider in a strain

• Only few strains have been used for this research
• Must be easy to genetically engineer
• Must grow rapidly in the lab
• Must have an environmental niche
• Ability to colonize the environmental niche in vivo
• Durability in niche or perhaps non durability when that might be advantageous
• Non toxicity upon colonization and control of proliferation

  Salmonella Typhimurium

• Targets mouse gut mucosa as well as hypoxic tissue at the core of tumors
• Engineered to release anti cancer drugs
• It can grow successfully in tumors and has been shown to stop metastasis
• Engineered to have reduced virulence so no off target infection

  Synthetic Genetic Circuits

• Able to intricately control gene expression responses

  Can be used to create memory devices and counters to record cellular events or biomarker presence/absence

• Via feedback loops

  Via permanent genetic changes via recombinases

• Perhaps something like CRISPR can be used to encode each peak of biomarker concentration
• That way you can determine amount of peaks and troughs given a time period

  Genetic logic gates

• Similar to electronic boolean logic gates
• Turn on expression of a specific element only after presence of a determined combination of inputs

  AND

• Only when two or more inputs are given

  OR

• One when one differential input is given

  State machines

• State machines are gates that read a certain input and switches their states accordingly
• It depends not only on signal but also their order
• Could be used to record experience plus order of events

  Genetic oscillators

  Quorum Sensing

• Sensing of quorum sensing molecules can be used to detect the presence of pathogens
• Quorum sensing itself can be used to control gene expression and restrict it to certain body sites
• This can reduce off target effects as well as spatial-temporal control of action

  Challenges and Technologies

  Key factors in success

  Functional stability (Circuit burden)

• The burden that the circuit places upon the bacteria by cost of expression can reduce proliferation and viability
• This could select for loss of function mutations
• Especially in stressful or competitive environments
• Components that encode cell death mechanisms like kill switches are particularly negatively selected for
• Burden can however be small enough for the selective pressure to be low
• Computational and experimental methods can aid in finding out the burden
• A CRISPR-CAS9 system was used to halt the expression of a synthetic circuit once burden increased.
• This made viability and proliferation better

  Clinical potency

• In vitro these organisms are usually given ideal medium to grow in
• In the body there is the possibility of suboptimal oxygen and nutrient environments as well as competition
• Realistic in vitro testing is necessary

  Some systems

• Human Intestinal Microbial Ecosystem fermenter system (SHIME)
• organ-on-chip microfluidics
• Ex vivo organ growth models
• These systems are complex and hard to set up though

  Safety

• Creating these microorganisms mean they will end up in the environment

  Things to consider

• Preventing transfer between individuals
• Control of growth and expression
• Prevention of gene transfer outside the engineered bacteria
• Kill switches that respond to temperature changes have worked somewhat successfully but they have negative selective pressure

  Synthetic auxotrophy

• One codon was recoded to match a non native tRNA so when theres no access to that tRNA
• This was done to several essential proteins and therefore would not survive outside the environment that we put it in
• Administering this environment is hard in humans though cause you need to continually add it

  Conclusion

• They believe memory circuits, logic gates and state machines are vital going forward
• Synthetic DNA synthesis advances can help us create synthetic pathways

[JulietteB-cri]

I really like the idea of quorum sensing - thanks for the article sharing -it was really clear and complete! Do you have some idea in mind on how we could exploit quorum sensing?

[research555]

@JulietteB-cri I have a good paper called bacterially speaking that I posted on discord about quorum sensing. The cool thing about it is that we can control when the circuit should be triggered via cell number determination. There is another paper that can show which generation the cell is in in relation to the first GMO, it's called cell counting or something like that. I'll post a summary tomorrow

[JulietteB-cri]

I found a really interesting paper on the development of temperature-dependant kill switches - they conducted a really nice experiment (and proposed a really nice diagnosis-related application at the end: detection host's fever!) https://www.nature.com/articles/nchembio.2233

I'll do a summary during the day in my page :)

[research555]

Sounds good. I've actually read this paper and I presented this when I presented kill switches. Its really cool. I might as well add some reviews I've written before as well. One is on kill switches

[JulietteB-cri]

I also read about Deadman and Passcode kill switches, on a really nice paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718764/#!po=13.8889) after last time-kill switch discussion (both circuits are modular and could be interested and + or - easily applied to our project). i'll write a summary as well, espect if you already read it too (LacI/tetR w/ aTc and IPTG as inhibitors)