Week 1 HW: Principles and Practices

1. I would like to develop a transcription factor cocktail for the direct conversion of fibroblasts into megakarycytes - specifically platelet-producing megakaryocytes. Fibroblasts are an accessible cell type so this would be a scalable method to produce a more rare cell type for various therapeutic applications. It could also be a stepping stone for studying engineering platelets, creating a platform for stem-cell independent targeted drug delivery, clotting modulation, or tumor microenvironment sensing.

2. First, prevent clinical and biological harm by requiring long‑term follow‑up and pharmacovigilance frameworks for patients receiving induced platelets (e.g. registries, mandated adverse event reporting, standardized functional and thrombotic risk assays). Promote equity, prevent misuse, and ensure fair access by establishing specialized oversight bodies (e.g., under existing stem‑cell/gene‑therapy committees) that periodically review safety data, emerging uses, and social impacts of fibroblast‑to‑MK reprogramming. Also we can encourage open publication of both successes and failures, standardized reporting of protocols and outcomes, and regular public engagement to discuss benefits, risks, and value alignment for this technology.

3. a) GMP and indication‑specific regulatory pathway for induced platelet products:

   • Now: platelet products are regulated as blood components, and cell/gene therapies follow general advanced therapy or biologics pathways that are not tailored to reprogrammed platelet products.
   • Proposed change: to create a specific GMP and regulatory track for “engineered or reprogrammed platelet products,” with clear expectations for CMC, potency, and safety testing of fibroblast‑derived MKs and platelets.
   • Design: National regulators (e.g. FDA/CBER, EMA, Korean MFDS), WHO for guidance convergence, hospital transfusion services, industry manufacturers would be involved in for example, defining product category (reprogrammed platelets) with risk‑based classification.
   • Assumptions: Assumes regulators can carve out a special pathway rather than forcing products into existing, possibly ill‑fitting categories and that robust potency/safety assays for induced platelets are technically feasible and can be standardized across centers.
   • Failure risk: Over‑stringent requirements could make development prohibitively expensive, limiting innovation and access; under‑stringent rules could allow unsafe products or “stem‑cell clinic” style abuses.
   • Success risk: If this pathway is very efficient, it might incentivize rapid commercialization of borderline‑indicated uses (e.g. “anti‑aging” platelet infusions), stretching the system’s ethical and enforcement capacity.

   b) Mandatory institutional reprogramming oversight (IRO) for TF‑based conversion work

   • Now: Stem cell and gene‑therapy work is typically reviewed by IRBs, biosafety committees, and in some jurisdictions stem‑cell oversight committees, but direct reprogramming may fall into gray zones, especially for early‑stage TF cocktails developed in academic labs.
   • Proposed change: Require that any lab working on fibroblast‑to‑MK reprogramming (even preclinical) be overseen by a specialized “Institutional Reprogramming Oversight” body, modeled on stem‑cell research oversight committees.
   • Design: Academic institutions, research hospitals, funding agencies (NIH, national research foundations), professional societies. Protocol review for: source and consent of fibroblasts, genetic constructs used, downstream uses (clinical vs. research), sharing of TF cocktails and vectors, and plans for data governance. Reporting and auditing: annual reports to funders; flagging of concerning trends (e.g., attempts at unsupervised human self‑administration).
   • Assumptions: Assumes institutions have the capacity and expertise to run these committees without causing paralyzing bureaucracy and that that most significant TF cocktail development will occur in institutions that fall under these rules (i.e. not in fully private or informal labs).
   • Failure risk: IROs become a box‑ticking exercise, offering little real scrutiny, or alternatively become so conservative that they stifle exploratory work (e.g. basic TF screening).
   • Success risk: Strong oversight in formal institutions may push some high‑risk work into poorly regulated commercial or DIY settings, where there is less ethical review but strong financial or ideological incentives.

   c) Global access and anti‑exploitation compact for reprogrammed platelet technologies

   • Now: Advanced cell therapies often launch as ultra‑high‑cost products in high‑income settings, with slow trickle‑down to low‑ and middle‑income countries; unregulated “stem cell clinics” exploit patients’ desperation.
   • Proposed change: Create a multi‑stakeholder compact (funders, companies, regulators, NGOs) that ties support for fibroblast‑to‑MK technologies to commitments on equitable pricing, technology transfer, and restrictions on exploitative marketing or non‑therapeutic uses.
   • Design: International organizations (WHO, professional hematology societies), major public and philanthropic funders, large manufacturers, national health systems all could be involved in generating codes of conduct for marketing and indication selection (e.g. ban on advertising reprogrammed platelet infusions for unproven “wellness” indications; mandatory trial registration and data disclosure). Shared technical standards and open reference protocols to reduce duplication and support safe local manufacture where appropriate, analogous to WHO norms for cell and gene therapies.
   • Assumptions: Assumes that technology transfer of complex MK/platelet manufacturing processes is feasible and cost‑effective across diverse health systems and that early funders and key companies are willing to accept access conditions and codes of conduct as part of doing business.
   • Failure risk: Compact remains soft‑law, with major commercial players opting out, leading to fragmented and inequitable access; weak enforcement of marketing norms allows predatory clinics to persist.
   • Success risk: If access conditions are perceived as too demanding, investment may shift toward less regulated but potentially more ethically problematic interventions, or toward markets with weaker patient protections.

5. I would prioritize a combination of Action 1 (dedicated GMP/regulatory pathway) and Action 3 (access/anti‑exploitation compact), with Action 2 (institutional oversight) implemented in a lighter, targeted form, rather than as a heavy universal mandate. I would frame this recommendation to National regulators and health agencies (e.g., MFDS in Korea, FDA/CBER, EMA). For a national regulator or health ministry, my prioritized package would be to define product category, preclinical safety and potency benchmarks, post‑market surveillance, and harmonized standards with other regenerative products. This is because Action 1 did best on feasibility and promoting constructive applications, while being moderate on biosecurity and safety, and worst on cost/burden. That profile matches what regenerative medicine reviews identify as the central challenge: we need predictable pathways that both protect patients and make serious products investable.

Week 2 Lecture Prep

Professor Jacobson

1. Polymerase makes about 1 error per 10^{6 bases after proofreading, while the human genome is ~3.2×10}9 bases long, so raw copying would introduce thousands of errors per genome. Biology fixes this using polymerase proofreading and post‑replicative repair pathways that together push the effective mutation rate orders of magnitude lower.
2. An average human protein (~345 amino acids from ~1036 bp) could in principle be encoded by roughly 61^345 different DNA sequences because of codon degeneracy. In reality, most of these “codes” fail or perform poorly due to codon usage bias, problematic mRNA structures and processing (e.g., cryptic splice sites, unstable transcripts), and unintended regulatory motifs that affect expression or genome stability.

Dr. LeProust:

1. The most commonly used method for oligo synthesis today is solid‑phase phosphoramidite chemistry on a support such as CPG or silicon (the Caruthers phosphoramidite cycle: deprotect → couple → cap → oxidize, repeated N times).
2. Direct chemical synthesis is a stepwise process with <100% efficiency per coupling, so the fraction of full‑length product decays exponentially with length; beyond ~150–200 nt, most molecules are truncated or contain multiple errors, making long oligos low‑yield and low‑quality without extensive purification.
3. A 2000 bp gene would require 2000 coupling steps; with realistic coupling yields, the full‑length fraction becomes essentially zero, and the product is a complex mixture of truncated and error‑laden fragments. Instead, genes of this size are built by assembling multiple shorter, synthesized oligos or gene fragments using PCR assembly, Gibson assembly, or similar enzymatic methods.

Professor Church: The 10 nutritionally essential amino acids for animals are usually summarized as PVT TIM HALL: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine, leucine, and lysine. This already includes lysine as only one of many essential amino acids; animals cannot synthesize any of these carbon skeletons in sufficient amounts and must obtain them all from diet. The “Lysine Contingency” in Jurassic Park imagined controlling dinosaurs by making them lysine‑auxotrophs that die without a supplemented lysine source. Knowing that all animals already depend on a full set of essential amino acids, including lysine, makes this seem naive: in real ecosystems escaped animals would easily obtain lysine from natural protein sources (meat, plants, other animals), so targeting a single essential amino acid is neither robust nor effective as a containment strategy. AI prompt: What is the “Lysine Contingency”

Week 1 Lab: Pipetting

Individual Final Project

Abstract
Cold storage at 4 °C enhances platelet hemostatic function but induces a well-characterized storage lesion, including glycoprotein Ib (GPIb) clustering, desialylation, premature activation, and apoptosis. These alterations result in rapid clearance of transfused platelets, limiting their clinical utility despite improved function. Tardigrade-derived intrinsically disordered proteins (IDPs), such as CAHS, MAHS, and RAHS, form protective amorphous glass-like matrices under stress conditions, stabilizing proteins and membranes. This project aims to harness these properties to mitigate cold-induced platelet damage by engineering megakaryocytes and platelets to express IDPs. By incorporating optimized IDP gene constructs (synthesized via TWIST Bioscience), we seek to create platelets with enhanced structural resilience during cold storage, preserving both function and circulation time.

Specific Aim 1: Expression of Tardigrade IDPs in Mammalian Cells
Establish robust expression of CAHS, MAHS, and RAHS proteins in mammalian systems. Codon-optimized gene blocks will be synthesized and cloned into mammalian expression vectors under constitutive and inducible promoters. Expression will be validated using Western blotting, immunofluorescence, and subcellular localization analysis. Protein solubility and potential aggregation behavior will also be assessed to ensure compatibility with mammalian cellular environments.

Specific Aim 2: Optimization of IDP Constructs for Megakaryocyte and Platelet Expression
Refine IDP gene constructs for efficient expression in megakaryocytes and subsequent incorporation into platelets. This includes promoter selection (e.g., PF4 or CD41-specific promoters), transcript stability optimization, and evaluation of translational efficiency. Constructs will be tested in megakaryocytic cell lines (e.g., MEG-01) and primary differentiation systems. Functional assays will assess whether IDP expression alters baseline platelet activation, morphology, or viability.

Specific Aim 3: Generation of Engineered Megakaryocytes Producing IDP-Containing Platelets
Develop engineered megakaryocytes that stably express IDPs and produce modified platelets. Lentiviral or CRISPR-based integration strategies will be used to introduce IDP genes into hematopoietic progenitors or megakaryocyte precursors. Platelets derived from these cells will be analyzed for IDP retention, structural integrity, and resistance to cold-induced storage lesions. Functional assays will include aggregation, adhesion, and clearance studies in vitro and, if feasible, in vivo models.

Background and Significance
Platelet transfusion is critical in managing bleeding disorders, yet storage remains a major limitation. While cold storage enhances immediate hemostatic function, it accelerates clearance due to biochemical and structural changes collectively termed the storage lesion. These include receptor clustering (notably GPIb), loss of sialic acid residues, and activation of apoptotic pathways. Current strategies to mitigate these effects have had limited success.

Tardigrades, extremophiles capable of surviving desiccation and temperature extremes, utilize intrinsically disordered proteins (IDPs) to form reversible, glass-like matrices that stabilize cellular components. CAHS (cytosolic abundant heat-soluble), MAHS (mitochondrial), and RAHS (secretory) proteins are key mediators of this protection. Their ability to buffer macromolecular structures without requiring rigid folding makes them attractive candidates for preserving platelet integrity under cold stress. Introducing these proteins into platelets represents a novel bioengineering approach to extend platelet shelf life while maintaining function.

Experimental Design
The project will begin with synthesis of codon-optimized IDP gene blocks via TWIST Bioscience, followed by cloning into mammalian expression vectors. In Aim 1, transient transfection into HEK293 and other model cell lines will establish baseline expression, localization, and biophysical behavior of IDPs.

In Aim 2, constructs will be tailored for megakaryocyte-specific expression using lineage-restricted promoters and regulatory elements. These will be tested in megakaryocytic cell lines and stem cell-derived megakaryocytes, with evaluation of expression efficiency and cellular compatibility.

In Aim 3, stable integration strategies (lentiviral transduction or CRISPR/Cas9 knock-in) will be employed to generate engineered megakaryocytes. Platelets derived from these cells will be subjected to cold storage assays at 4 °C. Key readouts will include GPIb clustering (via microscopy), desialylation (lectin binding assays), activation markers (e.g., CD62P), apoptosis indicators, and functional assays such as aggregation and adhesion. Clearance potential may be assessed using macrophage uptake assays or animal models if available.

Together, these experiments will test whether IDP expression can act as a physical and molecular buffer against cold-induced platelet damage, providing a foundation for next-generation platelet storage technologies.

Group Final Project