Week 1 HW: Principles and Practices

Week 1 Biological Engineering Application Governance Exercise

• I’m interested in developing phage chassis capable of targeting bacteria that commonly cause infections during spaceflight. I’m interested in developing these phage chassis because:

  • They would give astronauts and future space travelers more autonomy in countering ad hoc novel infections that may occur during long-duration missions
  • They would help contribute to greater personalization of medical care for broader ranges/more diverse spacefaring populations, who will likely travel, work, and live in space for extended periods of time/longer than traditional space missions to date
  • This development may help counter novel terrestrial infections for medically underserved populations

• Governance Goal 1 (G1): Preventing/Mitigating Malicious Dual-Use (i.e., ensuring)

  • Appropriate biosecurity (including cyberbiosecurity) controls
  • Safe deployment conditions

• Governance Goal 2 (G2): Empowering Autonmous and Equitable Use

  • ‘Plug-and-play’ functionality (i.e., any mission or crew should be able to intuitively use solution – advanced technical acumen not required)
  • Solution costs and distribution should develop with access across wide range(s) of demographics and socio-economic strata in mind

• Governance Action 1: Phage Safety Refusal (PSR)

  Purpose:

  • Current State: There are no proactive mechansisms preventing malicious dual-use phage development (i.e., developing a phage speicifcally designed to degrade host mRNA in healthy cells).
  • Proposed Changes: Easily implementable phage production host ‘kill switches’ that can be deployed if n^th concentrations of healthy cells accidentally or deliberately targeted by phage therapy

  Design:

  • Existing global space health biotechnology base (consisting of private firms, academia, and government-affiliated research centers) need to opt-in and actively invest in space-based phage therapies at adequate enough levels to ensure this notion of a phage defensive ‘kill switch’ can be validated and deployed, or invalidated and no longer pursued
  • Diverse space health regulators (consisting of a variety of agencies across nation-state governments) would need to approve said ‘kill switches’ (ex. Food and Drug Administration in the United States)
  • Patients participating in clinical trials of the ‘kill switch’ would need to feel comfortable enough with the solution to participate
  • Investors and members of private industry must see enough potential in the ‘kill switch that they see it as worthy of their time and investment
  • Astronauts and future spacefarers must be comfortable enough with the ‘kill switches’ to consent to their use

  Assumptions

  • The underlying notion of malicious dual-use phage applications, particularly in a space biotechnology context. Assumptions underneath this assumption include:
    • Viable malicious dual-use phage applications within a space health context and
    • Malicious actors would see and act on the benefits of phage interventions for targeted action against healthy cells in adversarial/antagonistic persons
  • That defensive anti-phage ‘kill switches’ even make sense to pursue technically

  Risks of Failure & “Success”

  • No viable malicious dual-use phage application concern (terrestrially or in a space context)
  • No interest in developing anti-phage ‘kill switches’
  • Developing anti-phage ‘kill switches’ is too technically intensive/not scalable to address the diverse needs of potential future users
  • Not enough funding to develop necessary research body of knowledge to make anti-phage ‘kill switches’ viable and safe to use in a space health context
  • Anti-phage ‘kill switches’ successfully developed and deployed and become another chapter in the never-ending story of phages v. bacteria (i.e., phages adapt to the ‘kill switches’ over time, rendering them ineffective)

• Governance Action 2: Space Medicine Access Consortia (SMAC)

  Purpose

  • Current State: The field of space medicine is still in its relative nascency. While space health consortia exist, there are no consortia explicitly aimed at making space medicine advancements as broadly accessible as possible to both spacefaring and terrestrial populations.
  • Proposed Changes: Charter and develop a consortia explicitly aimed at making space medicine advancements as broadly accessible as possible to both spacefaring and terrestrial populations. Ideally this consortia should comprise members from private industry, academia, government-affiliated research centers, and allied partners

  Design:

  • Potential and future committed consortia partners from across private industry, academia, government-affiliated research centers, and allied partners need to see the value of the consortia’s mission (i.e., they need to see and align with how the consortia’s explicit charter to make space medicine advancements as broadly accessible as possible to both spacefaring and terrestrial populations would benefit their organization)
  • Consortia partners need to agree on:
    • Rules of the road (this will likely need to be contractual)
    • Research scope(s) for their organizations
  • Concrete milestones indicating consortia success, stagnation, or failure in achieving its goals
  • Adequate funding to see consortia from inception through the point where most of its charter has been fulfilled (a somewhat analogous example might be the pool of institutions the Information Processing Technology Office (IPTO) at the Defense Advanced Research Projects Agency (DARPA) pooled together when creating the ARPANET, which later evolved to become the modern Internet, and the shifts in IPTO’s mission over time as a result of these developments)

  Assumptions

  • Critical mass of stakeholders:
    • cares about making space medicine advancements accessible to spacefaring and terrestrial populations
    • can see the value of a dedicated consortia to make space medicine advancements more accessible for spacefaring and terrestrial populations
    • can agree to the rules of the road necessary to make consortia viable
    • see the value of making phage chassis capable of targeting bacteria that commonly cause infections during spaceflight ‘plug-and-play’ and cost-effective for end-users (i.e., they wouldn’t prioritize some other space medicine intervention)
  • Consortia can achieve its charter, with greater phage chassis accessiblity as a step on its path to success

  Risks of Failure & “Success”

  • No or not enough interest in:
    • making space medicine advancements more accessible for spacefaring and terrestrial populations
    • developing consortia to make space medicine advancements more accessible for spacefaring and terrestrial populations
  • SMAC not viable due to:
    • Too much burden/not enough return on investment for potential consortia members (i.e., they don’t see the value)
    • Disagreeements over rules of engagement
    • Disagreement over research scope for participating members (i.e. who does what)
    • Inadequate implementation pathways (i.e., most of SMAC’s work languishes in the ‘valley of death’)
  • SMAC prioritizes other space medicine interventions over making phage chassis capable of targeting bacteria that commonly cause infections during spaceflight ‘plug-and-play’ and cost-effective for end-users
  • SMAC might ‘succeed’ too much, and as a result, the loudest or wealthiest SMAC members might eventually see value in exercising disproportionate control over the entity, diluting its charter

• Governance Action 3: Space Applied Biomedicine Repository (SABR)

  Purpose

  • Current State: Numerous space medicine guides have been developed by NASA, including the NASA Astronaut Medical Operations Handbook, Advanced Diagnostic Ultrasound in Microgravity (ADUM) Protocols, and OCHMO-STD-100.1A, otherwise known as the NASA Medical Standard. Commercial space missions largely lean on these guides for their missions
  • Proposed Changes: While NASA’s precedent is useful and organizations like the Translational Research Institue for Space Health (TRISH) have proposed tailored guidance for commercial space travelers, there is no existing repository explicitly focused on lessons learned and best practices for applied biomedicine in a space context. As opposed to a static guide, this work be more like a git-based repo, that could be updated with lessons learned, remaining customaizable for large numbers of future spacefarers and their medicial needs

  Design

  • User base:
    • willing to contribute to and derive value from SABR
    • with enough technical know-how to contribute to and derive value from SABR
  • Stakeholder(s) willing to pay subscription costs to run (likely git-based) SABR technical back-end
  • Easy to understand guidance and lessons learned that can be easily ingested in text, video, or audio form on (likely) simple, non-bandwidth intensive network connected devices

  Assumptions:

  • A (likely git-based) repository is an optimal vehicle for distributing recursively improving or community-based applied biomedicine lessons-learned for the space medicine community and future missions
  • This repository would contain accumulated, useful insights regarding how phage chassis capable of targeting bacteria that commonly cause infections during spaceflight can be deployed or administered:
    • safely (i.e. without malicious dual-use) across a wide variety of space missions
    • in a ‘plug-and-play’ or affordable manner
  • Enough of a user base:
    • willing to contribute to and derive value from SABR
    • with enough technical know-how to contribute to and derive value from SABR
  • Stakeholder(s) willing to pay subscription costs to run (likely git-based) SABR technical back-end
  • Easy to understand guidance and lessons learned that can be easily ingested in text, video, or audio form on (likely) simple, non-bandwidth intensive network connected devices (i.e., SABR can adequately work even in very bandwidth constrained conditions)

  Risks of Failure & “Success”

  • Not enough interest in SABR (potential users or contributors don’t see its value)
  • SABR is:
    • too:
      • early/the timing’s not right
      • abstract and remote in its guidance (i.e., the actual users or contributors who might derive value from its content see its content as too over their heads, technical, jargon-filled, or not applicable for their specific use cases)
    • a useful application, just not for the safe, easy-to-use, or cost-efective deployment of phage chassis capable of targeting bacteria that commonly cause infections during spaceflight
    • bandwidth-constrained to such a degree that it’s untenable for all intensive purposes
  • Can’t find stakeholder(s) willing to pay subscription costs to run (likely git-based) SABR technical back-end
  • SABR works so well and grows to such an extent that discerning actual mission-relevant content of value from the repository becomes a challenge for uninitiated users (i.e., filtering is a challenge – hard to separate filler from useful content)

• Biological Engineering Application Governance Scoring Rubric

• Biological Engineering Application: Phage chassis capable of targeting bacteria that commonly cause infections during spaceflight

• The rubric below works as follows: Policy goals and sub-goals are listed vertically, while each of the governance actions are listed next to the respective column header titled ‘Option’. Governance actions are scored from 1-3 based on how well they fulfil each policy goal and sub-goal. A score of 1 indicates a governance action does a poor job at fulfilling a policy goal, a 2 indicated a governance action does an OK job at fulfilling a policy goal, and a 3 indicates a governance action is the best at fulfilling a policy goal.

• Based on the scoring above, I’d probably prioritize SABR. Given the difficulties around space-based governance or governance in remote conditions, I think an organization like a TRISH or the Organization for Space Medicine, Engineering, and Design (OSMED) might be a good starting point, promoter, or convener to begin making something like SABR a reality. The more I think about it, the varied jurisdictions and varied, unclear regualtory regimes at the nation-state level make the idea of a more open-source/tribal knowledge-based self-governing solution appealing (or at bare minimum it shows a gap that could potentially be filled). I also think that if the timing was right (i.e., enough useful information could be added by a community of contributors) this could actually help fulfill some of the policy goals associated with the phage chassis project, not by a lot of sanctioned formal policy-making perhaps, but by community contribution, input, and/or agreed-upon best practices. That said, I can see and understand the trade-offs between formal policy-making at the nation-state level and more grassroots normative development of best practices as a result of doing this exercise. The glaring uncertainties that remain are whether or not the repository’s timing is right and/or if a dedicated user and contributor base could coalesce around it

• Ethical Considerations: Given the mechanics of phages, specifically their ability to hijack host cell tRNAs, ribosomes, and amino acids, I was somewhat surprised that there wasn’t more mention of potential deliberate malicious dual-use of phages. Maybe it’s my relative nascent understanding of the life sciences, the limitations of my research on this topic, or maybe the topic itself is either not researched or considered extensively. If this is true and the notion of potential deliberate malicious dual-use of phages might be a little bit left field, not well understood, or not well defined, perhaps convening working groups might be a sensible governance action, as these groups can often help map areas of concern for emerging dual-use technologies. Maybe distributing outputs from these working groups (i.e., white papers) to relevant academic journals, technical standards bodies, or policymakers might also be a worthwhile governance action.

All supporting prompts for the governance exercise above listed below:

Week 1 Homework Questions

Professor Jacobson Questions

• Two widely used polymerases are thermus aquaticus (Taq) and pyrococcus furiosus (Pfu) ¹. Taq has error rates ranging between 1 x 10^-5 to 2 x 10^-4 errors per base pair per doubling while Pfu has error rates of 1.3 x 10^{6 2 3}. Compared to the length of the human genome, 3 x 10⁹ base pairs, this comes out to apprxoimately 3.3 x 10^-13%, 6.6 x 10^-12%, and 3.3 x 10^-2% ⁴. Biology deals with this discrepancy during DNA replication through proofreading when it detects inaccurate nucleotides. When the polymerase detects that an inccorect base has been added, the polmyerase enzyme makes a cut in the chemical bond, releasing the incorrect nucleotide ⁵. If errors are made after replication, a mismatch repair is initiated. This is where enzymes recognize incorrectly added nucleotides and dispose of them. Nucelotide excision repair is another way nature corrects these errors. This occurs when ezymes remove and replace incorrect bases via cuts at the 3 and 5 prime ends of the incorrect base ⁶.
• An average human protein is approximately 375 amino acids long ⁷. As each codon consists of 3 letters, rough math indicates there are approximately 3³⁷⁵ number of potential coding sequences, an extremely large number of combinations ⁸. Some of the reasons all these different codons don’t code for the protein of interest are:
  • Codon Bias: Some codons are represented during transcription at a far greater level than others, traditionally due to more abundant transcription RNA (tRNA), ensuring higher levels of expression ^{9 10}.
  • mRNA Structure: Certain mRNA structures can be impacted by certain codon expression (i.e., become less stable), and therefore become more susceptible to degradation ¹¹.
  • Translation Accuracy Issues: Non-optimal codons decrease protein translation efficiency, due to a form of crowding in the ribosome, the area in the cell where protein production takes place ¹².

Dr. LeProust Questions

• Solid-phase phosphoramidite synthesis is the most common currently used oligo synthesis method ¹³.
• It’s difficult to make oligos longer than 200nt via direct synthesis because coupling errors/inefficiencies compound to the point where one ends up with lots of short, incomplete fragments ¹⁴.
• A 2000bp gene has 4000 nucleotides ¹⁵. Based on the answer to the previous question, creating a 2000bp is not currently feasible due to the accumulation of coupling errors/inefficiencies, even when stitching together smaller oligos or using novel enzymatic methods are taken into account ^{16 17}.

George Church Question

• Question 3 Response: ARPA-H Biostablization Sytem (BoSS) Grant Response

  All supporting prompts listed below answer:

Concept Summary

The team behind Pyrococcus furiosus-Inspired Molecular Staples (PFIMS) seeks to develop small organic molecules capable of binding to the ‘grooves’ of DNA and proteins across a variety of temperatures. If successfully developed, PFIMS would allow heat-proofing a biologic across a variety of temperatures without the need for dehydration. By locking protein folds through ionic pull, we can stablize biologics for longer periods of time (TA1). Our work will scale this system to scalable cell processing across an array of temperatures and use cases (TA2).

Innovation and Impact

PFIMS is inspired by extremophile biology. Pyrococcus furiosus can survive at temperatures of 100 degrees Celsius in ocean vents. By mimicking pyrococcus furiosus’ molecular heat shield, we can keep cells alive and functioning for exteneded periods of time at refrigerator or room temperature. Unlike modern cryopreservation methods that employ various forms of freezing that can harm cells during thawing, our stabilization solution stabilizes proteins using byproducts proteins naturally produce. Not only are cold temperatures avoided entirely, but once scaled this solution will slash costs for biologics shipping. Most importantly, PFIMS can work for any cell type, as it builds off fundamental biological features, such as protein folding and membrane strength.

Proposed Work

We plan to develop a novel, functioning bench-top bioprocessing system inspired by pyrococcus furiosus. We will create a polyamine-based stabilization medium that will power this bioprocessing system for a standard biologic (ex. cell therapy or antibodies). PFMIS’ approach is grounded in existing literature on stablization for high temperature DNA/protein stabilization via polyamines, small organic molecules with muliple amino groups (Bae, 2018) (Despotović, 2020) (Oshima, 2007). These polymaines act as a form of ‘molecular staples’ in preliminary modeling efforts (Vieille, C.,2001).

Key Milestones and Deliverables

Phase 1: Synthesize the branched polyamine formulation. Deliverable: Optimized medium prototype based on a standard biologic.

Phase 2: Integrate the stabilization medium into bioprocessing hardware, likely a singlee-use bioreactor to for initial prototype followed by a larger testing and deplouyment within a media/buffer prep mixer. Deliverable: Protoyped biostabilization device.

Phase 3: Validate stability metrics for a model biologic using PFIMS at room temperature over time. Deliverable: Validation report and delivery of biostabilization system capable of scaled biostablization across nth biologics

Technical Risks and Mitigations

Risk: Polyamines may exhibit toxicity at necessary concentratioons. Mitigation: Screening polyamines for reduced toxicity levels; introducing wash and resuspension steps into bioprocessing.

Risk: Stabilization mechanism may exhibit difficulty transferring from archaic single-celled microbes like pyrococcus furiosus to eukaryotic cells (cells containing nuclei and organelles). Mitigation: Tests across multiple cell types

Use Case

PFIMS enables rapid delivery of life-saving biologics and therapeutics in low-resourced or contested conditions. This allows for dramatically cheaper shipment of biologics to locations such as rural communities without robust public health infrastructure, remote or relatively isolated geographies, or active conflict zones.

Sources

• Bae DH, Lane DJR, Jansson PJ, Richardson DR. The old and new biochemistry of polyamines. Biochim Biophys Acta Gen Subj. 2018 Sep;1862(9):2053-2068. doi: 10.1016/j.bbagen.2018.06.004. Epub 2018 Jun 8. PMID: 29890242.
• Despotović Dragana, Longo Liam, et. al. Polyamines mediate folding of primordial hyperacidic helical peptides into stable amyloid-like fibrils. Biochemistry, 60(4), 257–267.
• Oshima T. Unique polyamines produced by an extreme thermophile, Thermus thermophilus. Amino Acids. 2007 Aug;33(2):367-72. doi: 10.1007/s00726-007-0526-z. Epub 2007 Apr 12. PMID: 17429571.
• Vieille C, Zeikus GJ. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev. 2001 Mar;65(1):1-43. doi: 10.1128/MMBR.65.1.1-43.2001. PMID: 11238984; PMCID: PMC99017.

All supporting prompts listed below: