Note from symposium: mRNA as genetic medicines: the next-generation biologics toward ATMPs and vaccines

I learn together with Gemini during the symposium,  need to remind some concepts in structural mRNA and immunology - I used to work with adoptive T-cell transfer to fight against Blastomyces infection. During that time we usually used peptide (tetramer), and live-attenuated as the antigens to initiate the cellular immune response. With the successfulness of mRNA vaccine during the COVID, there is a huge development on using mRNA instead of protein/peptide/whole cells to initiate immune response.

Hand-written note

Gemini 2.5 Flash

Prompt: What is cap 2 structure in mRNA

Ans: The "Cap2" structure in mRNA refers to a specific modification found at the 5' end of messenger RNA molecules in eukaryotes. It's a more elaborate version of the basic mRNA cap.

Here's a breakdown:

• The Basic Cap (Cap0): All eukaryotic mRNAs start with a 5' cap, which is an inverted 7-methylguanosine (m7G) linked to the first transcribed nucleotide (N1) via a 5'-5' triphosphate bridge (m7GpppN1). This is sometimes called Cap0.

• Cap1: In most eukaryotes, the first transcribed nucleotide (N1) immediately adjacent to the m7G cap undergoes a 2'-O-methylation. This means a methyl group is added to the 2' hydroxyl group of the ribose sugar of N1. This modified structure is called Cap1 (m7GpppN1mp).

• Cap2 (m7GpppN1mpN2mp): The Cap2 structure takes this a step further. In addition to the 2'-O-methylation on the first transcribed nucleotide (N1), the second transcribed nucleotide (N2) also undergoes 2'-O-methylation. So, Cap2 has 2'-O-methylation on both the first and second nucleotides (N1 and N2) following the 7-methylguanosine cap.

Functions of the mRNA Cap (including Cap2):

The mRNA cap structure, including its various modifications like Cap2, plays several crucial roles in mRNA metabolism and gene expression:

1. Protection from Degradation: The 5' cap protects the mRNA from degradation by 5'-3' exonucleases, which would otherwise rapidly chew away at the mRNA from the 5' end.
2. Efficient Translation: The cap is essential for efficient initiation of protein synthesis (translation). It serves as a recognition site for cap-binding proteins (e.g., eIF4E), which recruit the ribosomes to the mRNA.
3. Nuclear Export: The cap is involved in the export of mRNA from the nucleus to the cytoplasm, where translation occurs.
4. Pre-mRNA Processing: It plays a role in various pre-mRNA processing events, such as splicing and polyadenylation.
5. Immune Evasion (Self vs. Non-Self Recognition): The presence of 2'-O-methylation (as seen in Cap1 and Cap2) is crucial for distinguishing "self" (host) mRNA from "non-self" (e.g., viral) RNA. Unmethylated or less methylated caps can trigger an innate immune response (e.g., through RIG-I activation). Cap2, in particular, has been shown to reduce activation of the innate immune response and enhance translation efficiency.
6. mRNA Ageing: Recent research suggests that Cap2 modification accumulates on mRNAs as they age in the cytosol, converting from Cap1 to Cap2. This means Cap2 is often enriched on long-lived mRNAs.

In summary, Cap2 is a specific modification at the 5' end of eukaryotic mRNA where both the first and second transcribed nucleotides are 2'-O-methylated, building upon the basic 7-methylguanosine cap. This modification is important for mRNA stability, translation efficiency, and distinguishing cellular mRNA from foreign RNAs, thereby contributing to immune evasion.

Prompt: What could be the major obstacles for producing therapeutic mRNA

Ans: While mRNA therapeutics, especially vaccines, have shown tremendous promise, several major obstacles need to be overcome for their widespread and effective production and application:

1. mRNA Instability and Degradation:

• Highly Fragile Molecule: mRNA is an inherently unstable molecule, susceptible to degradation by ubiquitous RNases (enzymes that break down RNA) in the body and environment. This fragility makes it challenging to handle, store, and deliver without losing its integrity and function.
• Short Half-Life: Even within cells, mRNA has a relatively short half-life, meaning it's rapidly broken down. While this is beneficial for transient protein expression (e.g., for vaccines), it can be a challenge for sustained therapeutic effects needed for some chronic diseases.
• Storage and Cold Chain: The instability necessitates ultra-cold storage temperatures (e.g., -70°C for some vaccines), posing significant logistical and cost challenges for global distribution, especially in regions with limited cold chain infrastructure.

2. Delivery Challenges:

• Size and Charge: mRNA is a large, negatively charged molecule, making it difficult for it to passively cross the negatively charged cell membrane.
• Targeting Specific Cells/Tissues: Delivering mRNA to the desired target cells or tissues in the body (e.g., specific cancer cells, brain cells) while avoiding off-target effects remains a significant hurdle. Current delivery systems often accumulate in organs like the liver or spleen.
• Endosomal Escape: Even if the mRNA delivery vehicle gets into a cell, it often gets trapped within endosomes (small vesicles). It needs to "escape" these endosomes to reach the cytoplasm where ribosomes can translate it into protein. Inefficient endosomal escape is a major bottleneck.
• Vehicle Limitations:
  • Lipid Nanoparticles (LNPs): While highly successful (used in COVID-19 vaccines), LNPs can have their own challenges, including potential for systemic toxicity, immunogenicity, and limitations in precise targeting.
  • Viral Vectors: While efficient, viral vectors have limitations such as pre-existing immunity, immunogenicity (triggering an immune response against the vector itself), payload capacity limits, and challenges with re-dosing.
  • Non-LNP/Viral Systems: Other delivery systems like polymeric nanoparticles are being explored but also face their own set of challenges in terms of efficiency, safety, and scalability.

3. Immunogenicity and Safety:

• Innate Immune Response: The body's immune system can recognize foreign mRNA as a pathogen and trigger an innate immune response (e.g., interferon production), which can lead to inflammation and reduce the therapeutic effect. While nucleoside modifications (like pseudouridine) help mitigate this, it's still a concern, especially for high-dose or repeated administrations.
• Off-Target Effects: Unintended expression of the therapeutic protein in non-target cells or tissues can lead to undesirable side effects.
• LNP Toxicity: While generally safe, the lipid components of LNPs can sometimes cause inflammatory responses or other toxicities at higher doses.
• Autoimmune Reactions: Although largely mitigated by current mRNA technologies, there's always a theoretical concern of triggering autoimmune reactions, especially with long-term or repeated administration.

4. Manufacturing and Scalability:

• Complexity of Production: The production of mRNA (in vitro transcription, capping, purification) is a complex, multi-step process requiring precise optimization to ensure quality, efficacy, and scalability.
• Yield Limitations: Achieving high yields of high-purity mRNA can be challenging due to enzyme inefficiencies and the generation of unwanted byproducts during in vitro transcription.
• Purification Bottlenecks: Removing impurities (e.g., DNA template, enzymes, incomplete transcripts, double-stranded RNA contaminants) to meet stringent purity requirements for clinical use is a significant challenge and can impact yield and cost.
• Quality Control and Consistency: Ensuring consistent batch-to-batch quality, purity, and potency for large-scale production is crucial and requires advanced analytical techniques.
• Raw Material Sourcing: The availability and quality of GMP-grade (Good Manufacturing Practice) raw materials for mRNA production can be a limiting factor.
• Cost of Production: Currently, the production of therapeutic mRNA can be expensive, limiting accessibility, especially for global health initiatives.

5. Regulatory and Clinical Development:

• Efficacy vs. Safety: Balancing the desired therapeutic efficacy with an acceptable safety profile is paramount, particularly for novel mRNA applications beyond vaccines.
• Long-Term Data: For many therapeutic applications, long-term safety and efficacy data are still being gathered, which is critical for regulatory approval and widespread adoption.
• Specific Challenges for Different Diseases: The optimal mRNA design, delivery system, and dosing regimen will vary significantly depending on the target disease, adding complexity to clinical development.

Addressing these obstacles through continued research and development in mRNA design, delivery technologies, manufacturing processes, and regulatory frameworks will be key to unlocking the full potential of therapeutic mRNA.

Prompt: Why are there trends to replacing peptide drugs using mRNA?

Ans: There's a significant trend towards replacing peptide drugs with mRNA therapeutics for several compelling reasons, largely stemming from the inherent advantages of mRNA technology and the limitations of traditional peptide-based approaches.

Limitations of Peptide Drugs:

Peptides are short chains of amino acids that can act as signaling molecules, hormones, or enzyme inhibitors. While they offer high specificity and potency, they have several inherent drawbacks:

1. Poor Stability and Short Half-Life: Peptides are highly susceptible to enzymatic degradation by proteases in the body. This leads to a very short half-life in circulation, often requiring frequent injections and limiting their therapeutic window.
2. Poor Bioavailability: Due to their size and susceptibility to degradation in the gastrointestinal tract, most peptide drugs cannot be administered orally and require parenteral (injectable) administration, which can be inconvenient for patients.
3. Limited Cell Permeability: Many therapeutic targets are located inside cells. Peptides often struggle to cross cell membranes efficiently, making it difficult to target intracellular pathways.
4. Manufacturing Complexity and Cost: Synthesizing peptides, especially longer or modified ones, can be a complex and expensive process. Each amino acid addition requires a separate chemical step, increasing the likelihood of errors and purification challenges.
5. Immunogenicity: While generally less immunogenic than larger proteins, some peptides can still elicit an immune response, leading to reduced efficacy or adverse reactions.
6. Challenges with Large-Scale Production: Scaling up peptide synthesis to meet commercial demands can be difficult and costly.

Advantages of mRNA Therapeutics Over Peptides:

mRNA offers a paradigm shift by leveraging the body's own cellular machinery to produce therapeutic proteins in situ. This addresses many of the limitations of peptides:

1. "On-Demand" Protein Production: Instead of delivering the protein itself, mRNA delivers the instructions for making the protein. This allows the body's cells to produce the therapeutic protein continuously for a period, potentially leading to more sustained effects and less frequent dosing compared to direct peptide administration.
2. Intracellular Target Access: Since mRNA is translated into protein inside the cell, it can effectively target intracellular proteins and pathways that are inaccessible to most peptide drugs. This opens up possibilities for treating diseases caused by deficiencies in intracellular enzymes or proteins.
3. Versatility and Rapid Design: The genetic code is universal. Once a protein sequence is known, the corresponding mRNA sequence can be quickly designed and synthesized. This makes mRNA a highly versatile platform for producing virtually any protein, including large, complex proteins that are difficult or impossible to synthesize chemically as peptides.
4. Simplified Manufacturing (Relative to Protein/Peptide Drugs): mRNA production relies on in vitro transcription, a cell-free enzymatic process. While it has its own complexities (as discussed in the previous answer), it can be more scalable and potentially less expensive in the long run than complex chemical peptide synthesis or large-scale recombinant protein production.
5. Reduced Immunogenicity (with Modifications): While unmodified mRNA can be immunogenic, advanced mRNA technologies incorporate nucleoside modifications (like pseudouridine) that "trick" the immune system into recognizing the mRNA as "self" RNA. This reduces the innate immune response and enhances translation efficiency, making the therapeutic more tolerable.
6. No Genomic Integration Risk: mRNA does not enter the cell nucleus, so there's no risk of it integrating into the host genome, a concern with some DNA-based gene therapies. This contributes to a favorable safety profile.
7. Rapid Development and Adaptability: The modular nature of mRNA allows for incredibly rapid development. The COVID-19 mRNA vaccines are a prime example, demonstrating how quickly an mRNA therapeutic can go from concept to clinical trials and approval. This adaptability is crucial for responding to emerging threats or for personalized medicine approaches.

Specific Applications Where mRNA Outperforms Peptides:

• Protein Replacement Therapy: For genetic disorders where a missing or dysfunctional protein is needed (e.g., enzyme deficiencies, cystic fibrosis), mRNA can instruct cells to produce the correct protein internally, which is often more effective than delivering the external protein.
• Vaccines: mRNA vaccines instruct cells to produce antigens, triggering a robust immune response. Peptides, while used in some vaccine approaches, often require strong adjuvants and can be less effective at eliciting a broad and sustained immune response compared to full-length protein production.
• Cancer Immunotherapy: mRNA can encode tumor-specific antigens, training the patient's immune system to recognize and attack cancer cells.
• Intracellular Targets: For diseases affecting intracellular proteins or pathways, mRNA is a powerful tool to express therapeutic proteins directly within the affected cells.

In essence, mRNA therapeutics represent a shift from delivering the drug product (peptide) to delivering the drug blueprint (mRNA), allowing the body to become its own drug factory. This fundamentally changes the pharmacokinetics, targetability, and manufacturing profile, making mRNA a highly attractive and rapidly developing alternative for a wide range of therapeutic applications where peptides face significant limitations.

Prompt: Compare between mRNA and circular RNA for therapeutic use

Ans: Both linear mRNA (messenger RNA) and circular RNA (circRNA) hold immense promise for therapeutic applications, leveraging the cell's machinery to produce desired proteins or exert other regulatory functions. However, they have distinct structural and functional characteristics that lead to different advantages and limitations for therapeutic use.

Here's a comparison:

Linear mRNA for Therapeutic Use

Structure:

• A single-stranded RNA molecule with a defined 5' end (capped with 7-methylguanosine, m7G) and a 3' end (terminated by a poly-A tail).
• The 5' cap and 3' poly-A tail are crucial for translation initiation, mRNA stability, and protection from exonucleases.

Key Advantages:

• Established Technology: mRNA therapeutics, particularly vaccines, are clinically validated and have shown tremendous success (e.g., COVID-19 mRNA vaccines). The manufacturing processes, delivery systems (e.g., LNPs), and regulatory pathways are relatively more established.
• Transient Expression: The inherent instability of linear mRNA leads to transient protein expression, which is desirable for applications like vaccines (where a short burst of antigen production is needed) or acute treatments where sustained expression might be undesirable.
• Rapid Development: The modular nature of mRNA allows for rapid design and synthesis of new constructs, making it highly adaptable for emerging threats (like pandemics) or personalized medicine.
• No Genomic Integration Risk: mRNA remains in the cytoplasm and does not enter the nucleus, eliminating the risk of integration into the host genome.

Key Limitations:

• Instability and Degradation: Linear mRNA is highly susceptible to degradation by RNases (exonucleases) from both the 5' and 3' ends. This necessitates specific modifications (e.g., nucleoside modifications, optimized UTRs) and often ultra-cold storage conditions, impacting logistics and cost.
• Immunogenicity: Unmodified linear mRNA can trigger a strong innate immune response, which can lead to inflammation and reduced protein expression. While nucleoside modifications (like pseudouridine) significantly reduce this, it's still a factor, especially for high-dose or repeated administrations.
• Sustained Expression Challenges: Achieving prolonged therapeutic protein expression can be challenging due to its rapid degradation, often requiring repeated dosing for chronic conditions.
• Delivery Efficiency: Despite advances in LNP delivery, getting mRNA efficiently into target cells and avoiding off-target accumulation remains an active area of research.

Primary Therapeutic Applications:

• Vaccines: Infectious diseases (COVID-19, flu, RSV), cancer vaccines.
• Protein Replacement Therapy: For genetic disorders causing protein deficiencies (e.g., enzyme replacement).
• Gene Editing Tools: Delivery of mRNA encoding nucleases (CRISPR-Cas9) or base editors for transient gene editing.
• Cancer Immunotherapy: Encoding tumor antigens or immunostimulatory cytokines.

Circular RNA (circRNA) for Therapeutic Use

Structure:

• A single-stranded RNA molecule that forms a covalently closed loop, lacking free 5' cap and 3' poly-A tail.
• Translation is often initiated via an Internal Ribosome Entry Site (IRES) or other cap-independent mechanisms within the circRNA.

Key Advantages:

• Enhanced Stability: The closed-loop structure makes circRNA highly resistant to degradation by exonucleases (which act on free ends). This significantly increases its half-life in cells and in circulation, potentially leading to more sustained protein expression.
• Reduced Immunogenicity: Due to the absence of free ends, circRNAs are less likely to be recognized as foreign RNA by cellular innate immune sensors, potentially leading to a milder immune response compared to unmodified linear mRNA. This could allow for higher doses or more frequent administration.
• Sustained Protein Expression: The superior stability directly translates to prolonged protein production, making circRNA highly attractive for chronic diseases requiring continuous therapeutic protein levels.
• Potentially Lower Dosing/Cost: Longer half-life and sustained expression might mean lower required doses or less frequent administration, potentially reducing overall treatment costs and patient burden.
• Novel Functional Roles: Beyond protein coding, endogenous circRNAs have diverse regulatory functions (e.g., miRNA sponges, protein scaffolds), opening up possibilities for non-coding therapeutic applications.

Key Limitations:

• Nascence of Technology: CircRNA therapeutics is a much newer field compared to linear mRNA. Manufacturing processes for producing high-purity, therapeutic-grade circRNA are still being optimized and scaled.
• Circularization Efficiency: Efficient and precise circularization of linear RNA precursors into desired circRNA is a significant technical challenge in manufacturing, often leading to linear RNA impurities.
• Translation Efficiency: While circRNAs can be translated, the efficiency of cap-independent translation (e.g., via IRES) can sometimes be lower or less predictable than cap-dependent translation in linear mRNA. Optimizing IRES elements or other translation enhancers is crucial.
• Delivery Challenges: Similar to linear mRNA, effective and targeted delivery of circRNA to specific cells/tissues remains a hurdle, although the enhanced stability might offer more flexibility in delivery vehicle design and storage.
• Regulatory Pathway: The regulatory pathway for novel circRNA therapeutics is less defined than for linear mRNA, potentially leading to longer approval times.

Primary Therapeutic Applications (Emerging):

• Next-Generation Vaccines: Leveraging sustained antigen expression for potentially stronger and longer-lasting immune responses.
• Chronic Protein Replacement Therapies: For conditions requiring sustained therapeutic protein levels (e.g., enzyme deficiencies, hormone replacement).
• Gene Editing: Delivering nucleases or base editors for more sustained editing activity.
• Cancer Therapy: Encoding therapeutic proteins or manipulating cellular pathways through non-coding circRNA functions.
• Modulating miRNA Activity: As miRNA sponges to regulate gene expression in various diseases.

Summary Comparison

Feature	Linear mRNA	Circular RNA (circRNA)
Structure	Linear, with 5' cap and 3' poly-A tail	Covalently closed loop, no free ends
Stability (in vivo)	Lower; susceptible to exonuclease degradation	Much higher; resistant to exonuclease degradation
Protein Expression	Transient; cap-dependent translation	Sustained; cap-independent translation (e.g., via IRES)
Immunogenicity	Can be immunogenic (mitigated by modifications)	Generally lower, due to lack of free ends
Manufacturing	More established (IVT, capping, purification)	Still developing (efficient circularization is a challenge)
Development Stage	Clinically validated (e.g., vaccines), widespread	Emerging; strong preclinical and early clinical interest
Storage	Often requires ultra-cold storage (-70°C)	Potentially less stringent due to higher stability
Cost	High (due to cold chain, raw materials, purification)	Potentially lower in future (due to stability, less frequent dosing)

In conclusion, while linear mRNA has paved the way for RNA therapeutics and is currently dominant, circRNA is a compelling "next-generation" RNA platform. Its superior stability and potentially lower immunogenicity offer significant advantages for applications requiring sustained protein expression or long-term therapeutic effects. However, challenges in manufacturing efficiency and clinical translation still need to be addressed for circRNA to fully realize its potential.

Prompt: Explain on the translational switch for the RNA based therapeutic approach

Ans: A "translational switch" in the context of RNA-based therapeutics refers to engineered mechanisms that allow for the controlled and inducible expression of a therapeutic protein from an RNA molecule (typically mRNA or circRNA). Instead of the RNA being constantly translated into protein once delivered, a translational switch enables the therapeutic protein to be produced only when a specific trigger is present.

This concept is crucial for enhancing the safety, efficacy, and precision of RNA therapeutics, especially for diseases that require:

• Spatial control: Protein expression only in specific cells or tissues.
• Temporal control: Protein expression only at specific times or for a defined duration.
• Dose control: Adjusting the amount of protein produced based on therapeutic need.
• Reduced off-target effects: Preventing unintended protein production in healthy cells.

Why is a Translational Switch Needed?

Currently, most therapeutic mRNA (like in COVID-19 vaccines) is designed for constitutive expression, meaning it's translated as long as the mRNA is stable within the cell. While this is effective for vaccines, it has limitations for other applications:

• Toxicity concerns: Continuous, high-level expression of a potent therapeutic protein can be toxic if not carefully controlled.
• Off-target effects: If the RNA is delivered systemically, it might be translated in cells where it's not needed, leading to side effects.
• Precise dosage: For many diseases, a precise and adjustable level of protein is required, which is hard to achieve with constitutive expression.
• Dynamic disease states: Some diseases require protein production only during certain phases or in response to specific biomarkers.

Mechanisms of Translational Switches in RNA Therapeutics

Translational switches primarily operate by controlling the initiation of protein synthesis from the therapeutic RNA. Here are some key strategies:

1. Riboswitch-based Systems:

   • Mechanism: These involve RNA elements (riboswitches) that directly bind to a small molecule (inducer or repressor). This binding causes a conformational change in the RNA, either exposing or occluding the ribosome binding site (RBS) or other translational control elements (like the start codon or an IRES).
   • How it works:
     • "ON" switch: In the presence of a specific ligand, the riboswitch changes conformation to allow ribosome access and translation initiation.
     • "OFF" switch: In the presence of a ligand, the riboswitch changes conformation to block ribosome access, thereby inhibiting translation.
   • Therapeutic relevance: Could allow protein production to be turned ON/OFF by administering a specific small molecule drug.
   • Example: Imagine an mRNA encoding an anti-cancer protein that is only translated when a non-toxic small molecule is administered, allowing for precise control of therapy.

2. miRNA-Responsive Switches:

   • Mechanism: These systems leverage the endogenous microRNA (miRNA) machinery of the cell. Therapeutic mRNA is engineered with target sequences in its 3' untranslated region (3' UTR) that are complementary to specific cellular miRNAs.
   • How it works:
     • If the target cell expresses a high level of a specific miRNA that matches the sequence in the therapeutic mRNA's 3' UTR, the miRNA will bind, leading to degradation of the mRNA or translational repression. This acts as an "OFF" switch in cells where that miRNA is abundant.
     • Conversely, if the miRNA is absent or at low levels, the mRNA is freely translated.
   • Therapeutic relevance: Can enable cell-type specific expression (e.g., only in cancer cells that have altered miRNA profiles, or avoiding expression in healthy cells that express specific miRNAs). It can also create "ON" switches where a miRNA sponge (an RNA sequence that "soaks up" a repressive miRNA) is delivered, thereby de-repressing translation of the therapeutic mRNA.
   • Example: Designing an mRNA that is silenced in healthy lung cells (due to a specific miRNA) but active in lung cancer cells (where that miRNA is downregulated).

3. Protein-Responsive Switches (e.g., RNA-binding proteins, RBPs):

   • Mechanism: The therapeutic RNA is designed with binding sites for specific RNA-binding proteins. The presence or absence of these RBPs, or their modified state (e.g., phosphorylation), can regulate translation.
   • How it works: An RBP might bind to a specific sequence in the UTR of the therapeutic mRNA, either blocking or enhancing translation initiation.
   • Therapeutic relevance: Allows protein expression to be controlled by the presence or activity of specific cellular proteins, which can be indicators of disease states or cellular differentiation.

4. Toehold Switches (eToeholds):

   • Mechanism: These are sophisticated synthetic RNA constructs that can be designed to respond to specific RNA "trigger" sequences. The toehold contains a sequestered ribosome binding site (RBS) or start codon. When a complementary trigger RNA binds, it unfolds the toehold structure, exposing the RBS and allowing translation to begin.
   • How it works: An "ON" switch is activated by the presence of a specific trigger RNA (e.g., a viral RNA, a cancer-specific RNA).
   • Therapeutic relevance: Offers highly programmable and specific control. Could enable therapeutic protein production only in cells infected by a virus, or in cancer cells expressing a unique RNA signature.

5. ADAR-mediated editing switches:

   • Mechanism: Leverages the endogenous adenosine deaminase acting on RNA (ADAR) enzyme. Therapeutic RNA is engineered with a premature stop codon that can be "edited out" by ADAR, converting an adenosine to an inosine (which is read as guanosine by the ribosome), thereby allowing full-length protein translation.
   • How it works: The presence or activity of ADAR in a specific cell type, or under certain conditions, can trigger the "ON" switch by removing the stop codon.
   • Therapeutic relevance: Enables context-dependent protein expression based on ADAR activity.

Benefits of Translational Switches

• Enhanced Safety: Reduces off-target effects and potential toxicity by limiting protein expression to desired locations and times.
• Improved Efficacy: Ensures protein is produced when and where it's most needed, potentially increasing therapeutic benefit.
• Personalized Medicine: Allows for highly specific responses to individual patient biomarkers or disease states.
• Reduced Dosing Frequency: If control is achieved through sustained sensing of a signal, it might allow for less frequent administration compared to constant expression.
• Complex Logic Operations: Advanced designs can combine multiple RNA inputs to create sophisticated "AND," "OR," or "NOT" logic gates for highly precise control.

Challenges

While promising, developing robust translational switches for therapeutic RNA still faces challenges:

• Efficiency and Dynamic Range: Achieving a high "ON" signal and a low "OFF" signal (high dynamic range) is critical to avoid leaky expression or insufficient therapeutic effect.
• Specificity: Ensuring the switch responds only to the intended trigger and not to endogenous molecules that could lead to off-target activation.
• Delivery to Target Cells: The underlying challenge of delivering the therapeutic RNA to the correct cells remains.
• Immunogenicity: The engineered RNA elements themselves should not trigger an unwanted immune response.
• Scalability and Manufacturing: Producing these more complex RNA constructs efficiently and at scale can be more challenging.

Overall, translational switches represent a significant advancement in RNA therapeutic design, moving beyond simple constitutive expression to enable smarter, safer, and more precisely controlled treatments.

Prompt: Please explain this slide

Ans: This slide outlines an experimental strategy for screening mRNA or lncRNA candidates that can act as "translational switches" for circular RNA (circRNA), specifically aiming to control the expression of a reporter protein (RFP). The core idea is to find RNA molecules that can activate or inhibit translation from a circRNA in a targeted manner.

Let's break down the slide into its key components:

1. The Goal: "Screening mRNA or lncRNA for the translation switch of circular RNA."

• The objective of the experiment is to identify specific linear RNA molecules (either messenger RNA or long non-coding RNA) that can influence the translation of a separate circular RNA.
• The "translation switch" implies that the identified mRNA/lncRNA will either turn on or turn off protein production from the circRNA.

2. The System: Cre-loxP and RFP Expression

• Cre circular mRNA: This is the therapeutic circular RNA construct being tested. It's engineered to encode the Cre recombinase enzyme.
• Cre expression $\to$ RFP expression: The slide shows a crucial link. When the Cre circular mRNA is translated and produces Cre protein, this Cre protein will then act on a Cre-loxP mouse model. In this model, Cre recombinase catalyzes recombination between loxP sites, which often leads to the activation of a reporter gene. In this specific setup, the activation leads to RFP (Red Fluorescent Protein) expression.
• Why RFP? RFP is a common reporter gene because its fluorescence is easy to detect, allowing researchers to quantify the amount of protein produced by the circRNA (indirectly, via Cre activity).

3. The Screening Library: "Design sequences targeting tens of thousands of lncRNAs (~60,000 library) - Harada Lab"

• The "Harada Lab" is designing a large library of potential translational switch candidates.
• These candidates are either mRNA or lncRNA.
• They are specifically designing sequences that are expected to target (i.e., interact with or influence) the Cre circular mRNA, aiming to modulate its translation. The sheer size of the library (~60,000 types) indicates a high-throughput, discovery-oriented screening approach.

4. The Experimental Workflow (In Vitro and In Vivo):

• Top Left: Oligo array (targeting mRNA and lncRNA) & DNA Library:

  • An "Oligo array" (likely referring to oligonucleotide synthesis array) is used to synthesize a vast number of DNA sequences (60,000 types). These DNA sequences represent the designed mRNA or lncRNA candidates.
  • This DNA is then used to create a "DNA library." This DNA library will serve as the template for producing the mRNA or lncRNA candidates.

• Middle: "Injection to Cre-loxP mouse by using carriers such as LNP"

  • The core experiment involves injecting these RNA molecules (the Cre circular mRNA and the screening candidates) into a mouse model.
  • Cre-loxP mouse: This mouse strain is genetically engineered such that RFP expression is activated by Cre recombinase.
  • Carriers such as LNP (Lipid Nanoparticles): LNPs are a common and effective delivery system for RNA therapeutics, facilitating their entry into cells. Both the Cre circular mRNA and the screening mRNA/lncRNA candidates would likely be delivered via LNPs.
  • The idea is that a specific mRNA/lncRNA candidate from the library, when co-delivered with the Cre circular mRNA, will act as a translational switch, affecting how much Cre protein is made, and thus how much RFP is expressed.

• Right Side: Organ Collection and Analysis

  • Collect organs: Heart, Brain, Lungs, Liver, Spleen: After injection and a suitable incubation period, various organs are collected from the mice. This suggests they are looking for tissue-specific effects or general systemic activity.
  • RFP fluorescence: Initial assessment involves checking for RFP fluorescence in the collected organs. This is a direct readout of Cre activity and, by extension, the translational activity of the Cre circular mRNA in the presence of the screening candidate.
  • Liver cells, Spleen cells $\to$ RFP+ cells: Specific cell types from the liver and spleen are isolated and sorted for RFP positivity (RFP+ cells). This allows for quantification of cells where the translational switch was active.
  • DNA extraction: DNA is extracted from the RFP+ cells.
  • PCR amplification + Index addition: The DNA corresponding to the specific mRNA/lncRNA candidates that caused RFP expression is amplified. Indexing allows for distinguishing between different candidates if multiple were delivered simultaneously or in pools.
  • NGS (Next-Generation Sequencing): NGS is used to identify and quantify the specific mRNA/lncRNA candidates that led to RFP expression. By sequencing the amplified DNA from RFP+ cells, researchers can determine which of the 60,000 candidates successfully functioned as a translational switch.

In summary, the slide describes a high-throughput screening platform designed to discover novel mRNA or lncRNA molecules that can regulate the translation of circular RNA. By using a Cre-loxP/RFP reporter system in mice, the researchers can identify which of their 60,000 candidate RNAs effectively "switch on" (or off) the production of a desired protein from a circRNA, paving the way for more controllable and targeted RNA therapeutics.

Prompt: Please compare between mRNA vaccine vs therapeutic mRNA

Ans: While both mRNA vaccines and therapeutic mRNA leverage the same fundamental principle – using messenger RNA to instruct cells to produce a specific protein – their goals, mechanisms, and design considerations differ significantly.

Here's a comparison:

mRNA Vaccines

Primary Goal: To prevent infectious diseases or treat cancer by inducing an immune response against specific antigens.

Mechanism:

1. Antigen Production: The mRNA encodes a foreign antigen (e.g., a viral spike protein, a tumor-specific protein).
2. Immune System Activation: Once the mRNA is translated into protein in the host cells (often antigen-presenting cells like dendritic cells), this protein is recognized as foreign.
3. Immune Response: The immune system then mounts a response, generating antibodies and T-cells that can recognize and neutralize the actual pathogen or cancer cells if encountered in the future.
4. Transient Expression: The mRNA is designed to be relatively transiently expressed, providing a short burst of antigen production to trigger immunity, after which the mRNA is degraded.

Key Design Considerations:

• Immunogenicity: The mRNA needs to be sufficiently immunogenic to provoke a strong and lasting immune response. Sometimes, a mild innate immune response is even desirable to act as an adjuvant.
• Antigen Selection: The encoded antigen must be highly specific and accessible to the immune system.
• Delivery: Typically delivered via intramuscular injection, often encapsulated in Lipid Nanoparticles (LNPs), to muscle cells or draining lymph nodes.
• Dose: Relatively low doses are usually sufficient as the goal is a robust immune response, not continuous high-level protein production.
• Safety: Focus is on eliciting a protective immune response without causing significant side effects or autoimmune reactions.

Examples: COVID-19 mRNA vaccines (Pfizer-BioNTech, Moderna), ongoing development for influenza, RSV, HIV, and various cancer vaccines.

Therapeutic mRNA (Non-Vaccine)

Primary Goal: To treat a disease by producing a functional protein in situ to correct a deficiency, modify a cellular process, or deliver a therapeutic agent. This is broadly protein replacement or functional protein delivery.

Mechanism:

1. Functional Protein Production: The mRNA encodes a therapeutic protein (e.g., an enzyme, an antibody, a growth factor, a gene-editing enzyme).
2. Disease Modulation: This produced protein then performs its intended biological function to directly treat the underlying cause of the disease or its symptoms.
3. Sustained Expression (often desired): For many therapeutic applications, prolonged and sometimes precisely controlled protein expression is critical for efficacy.

Key Design Considerations:

• Stability: Often requires higher mRNA stability to ensure sustained protein production, potentially involving more extensive modifications (e.g., circular RNA, self-amplifying mRNA) to prolong its half-life.
• Reduced Immunogenicity: For many therapeutic applications, an innate immune response against the mRNA itself is undesirable as it can reduce protein expression and cause inflammation. Extensive nucleoside modifications (e.g., pseudouridine) are often used to minimize immunogenicity.
• Targeting: Precise delivery to specific cells, tissues, or organs is often paramount to maximize efficacy and minimize off-target effects and toxicity. This is a major area of research (e.g., modified LNPs, cell-specific ligands).
• Dose: Can vary widely depending on the desired protein level and duration, often requiring higher doses than vaccines.
• Safety: Focus is on delivering a functional protein safely, with minimal systemic toxicity or immune reactions against the mRNA or the produced protein.
• Translational Control: For some applications, "translational switches" (as discussed previously) are being explored to allow for inducible or cell-specific protein production.

Examples:

• Protein Replacement Therapy: For genetic disorders where a specific protein is missing or dysfunctional (e.g., cystic fibrosis, enzyme deficiencies, hemophilia).
• Cancer Therapy (non-vaccine): Encoding antibodies, cytokines, or other anti-cancer agents.
• Cardiovascular Diseases: Encoding growth factors to promote blood vessel formation or repair.
• Gene Editing: Delivering mRNA for CRISPR-Cas9 components to achieve transient gene editing.
• Regenerative Medicine: Encoding factors to promote tissue repair or regeneration.

Key Differences Summarized

Feature	mRNA Vaccine	Therapeutic mRNA (Non-Vaccine)
Primary Goal	Prevent disease; Induce immune response	Treat disease; Produce functional protein
Encoded Protein	Foreign antigen (e.g., viral spike)	Functional protein (e.g., enzyme, antibody, growth factor)
Immune Response	Desired and central to mechanism	Undesired (for the mRNA itself), can hinder efficacy and cause inflammation
Expression	Transient, burst	Often aims for sustained, controlled expression
Dose	Generally lower	Can be higher, varied based on need
Targeting	Immune cells (e.g., APCs), muscle cells	Specific diseased cells/tissues (e.g., liver, lung, tumor)
Key Challenge	Eliciting broad, durable immunity	Efficient, targeted delivery & sustained, controlled protein expression with minimal immunogenicity

In essence, while both utilize mRNA as a blueprint, mRNA vaccines aim to teach the immune system, whereas therapeutic mRNA aims to provide a missing or beneficial biological function directly. This fundamental difference drives their distinct design, delivery, and application strategies.

Prompt: Make the comparison table for both vaccine purpose vs therapeutic app

Ans: Okay, here's a comparison table distinguishing mRNA vaccines from therapeutic mRNA applications.

Comparison: mRNA Vaccines vs. Therapeutic mRNA Applications

Feature	mRNA Vaccines	Therapeutic mRNA Applications (Non-Vaccine)
Primary Goal	Prevent disease (infectious, cancer); Induce a protective immune response.	Treat disease; Produce a functional protein to correct a deficiency, modify a pathway, or provide a therapeutic agent.
Encoded Protein	Foreign antigen (e.g., viral spike protein, tumor-specific antigen).	Functional, therapeutic protein (e.g., enzyme, antibody, cytokine, gene-editing component, growth factor).
Desired Immune Response	Desired and central to the mechanism. The immune system is the "effector."	Undesired against the mRNA itself (to avoid inflammation and reduce expression); protein-specific immune response may be desired (e.g., for antibodies) or undesirable (e.g., for enzyme replacement).
Protein Expression Duration	Transient burst (hours to days); enough to prime the immune system.	Often aims for sustained or controlled expression (days to weeks/months) for continuous therapeutic effect.
mRNA Stability	Moderate to high; sufficient for transient expression.	Often requires higher stability (e.g., using circRNA, self-amplifying mRNA) to achieve prolonged expression.
mRNA Immunogenicity	Can be moderate; sometimes a degree of innate immune activation is beneficial as an adjuvant.	Minimized (e.g., through extensive nucleoside modifications like pseudouridine) to avoid inflammation and maximize protein output.
Dose	Generally lower (e.g., tens to hundreds of micrograms).	Can vary widely; potentially higher for sustained effects or to achieve high protein levels in target tissue.
Targeting/Delivery	Often intramuscular injection to muscle cells and draining lymph nodes; systemic delivery generally not needed.	Often requires precise targeting to specific cells, tissues, or organs to maximize efficacy and minimize off-target effects. This is a major area of research.
Mechanism of Action	Indirect: instructs cells to make an antigen, which then trains the immune system to fight disease.	Direct: instructs cells to make a therapeutic protein that directly performs a function to treat the disease.
Re-dosing Frequency	Typically infrequent (e.g., 1-3 doses, boosters).	Can be frequent for acute conditions or less frequent for chronic conditions if sustained expression is achieved.
Current Clinical Examples	COVID-19 vaccines (Pfizer-BioNTech, Moderna), ongoing trials for flu, RSV, cancer vaccines.	Hemophilia, cystic fibrosis, metabolic disorders, cardiovascular repair, cancer immunotherapies (non-vaccine), gene editing tools.
Key Challenge	Eliciting robust, broad, and durable protective immunity.	Efficient, highly targeted delivery; achieving sustained, controlled protein expression; minimizing unwanted immune responses to the mRNA itself or the therapeutic protein.

Prompt: Tell me more in the current trend of therapeutic mRNA through inhalation

Ans: The delivery of therapeutic mRNA via inhalation is a rapidly emerging and highly promising trend, particularly for the treatment of pulmonary (lung) diseases. This approach offers several compelling advantages over traditional systemic (e.g., intravenous) administration.

Why Inhalation for Therapeutic mRNA?

1. Direct and Targeted Delivery:

   • The most significant advantage is the ability to deliver the mRNA directly to the target organ – the lungs. This maximizes the concentration of the therapeutic protein in the affected tissue, potentially increasing efficacy.
   • It minimizes systemic exposure and potential off-target side effects that can occur with intravenous administration, where the mRNA might accumulate in other organs like the liver or spleen.

2. Large Surface Area:

   • The lungs have an extensive surface area (alveoli) for absorption, facilitating efficient uptake of the mRNA.
   • The thin alveolar epithelium provides a relatively short diffusion distance for the mRNA to reach target cells.

3. Reduced Systemic Toxicity:

   • By localizing the therapy to the lungs, the risk of systemic side effects from the therapeutic protein itself (if it's potent) or the delivery vehicle (e.g., lipid nanoparticles) is significantly reduced.

4. Bypassing First-Pass Metabolism:

   • Inhaled therapies bypass the gastrointestinal tract and first-pass metabolism in the liver, which can degrade or alter drugs administered orally or intravenously.

5. Convenience and Patient Compliance:

   • Inhalation can be a more patient-friendly and less invasive administration route compared to injections, potentially improving compliance, especially for chronic conditions. Nebulizers or dry powder inhalers could make self-administration at home feasible.

Key Applications Driving the Trend

The primary focus areas for inhaled therapeutic mRNA are lung-related diseases:

1. Cystic Fibrosis (CF):

   • CF is caused by mutations in the CFTR gene, leading to a dysfunctional CFTR protein.
   • Inhaled mRNA encoding a functional CFTR protein aims to deliver the genetic instructions directly to lung epithelial cells, allowing them to produce the correct protein and restore ion channel function, thereby improving mucus clearance and lung health.
   • This is a leading application, with several companies (e.g., ReCode Therapeutics, Arcturus Therapeutics, Vertex/Moderna collaboration) in preclinical and early clinical stages.

2. Alpha-1 Antitrypsin Deficiency (AATD):

   • A genetic disorder leading to a deficiency of alpha-1 antitrypsin, which primarily protects the lungs from inflammation.
   • Inhaled mRNA can instruct lung cells to produce functional AAT, protecting the lungs from progressive damage.

3. Lung Cancer:

   • Inhaled mRNA can deliver therapeutic proteins directly to lung tumors.
   • Immunotherapy: mRNA encoding cytokines (e.g., IL-12) or tumor-associated antigens can be inhaled to stimulate a local anti-tumor immune response within the lung, potentially reducing systemic side effects of these potent molecules.
   • Tumor Suppressors: mRNA encoding tumor suppressor genes (e.g., p53) can be delivered to restore anti-cancer mechanisms in lung cancer cells.

4. Idiopathic Pulmonary Fibrosis (IPF) and other Fibrotic Lung Diseases:

   • mRNA could encode proteins that inhibit fibrosis or promote lung tissue repair.

5. Acute Lung Injury/ARDS:

   • mRNA could deliver anti-inflammatory or regenerative factors to mitigate damage and promote recovery.

Challenges and Current Research Focus

Despite the promise, several challenges are being actively addressed in research:

1. Delivery Vehicle Optimization:

   • Traditional LNPs, successful for intramuscular vaccines, may not be ideal for inhalation due to instability during nebulization, interactions with mucus, or inefficient cellular uptake in lung specific cell types.
   • New lipid formulations, polymeric nanoparticles, or even extracellular vesicles (exosomes) are being developed that can withstand aerosolization, penetrate the mucus layer, and efficiently deliver mRNA to target lung cells (e.g., epithelial cells, macrophages, cancer cells).
   • Ensuring uniform distribution of the inhaled particles throughout all lung lobes is also critical.

2. Overcoming Lung Barriers:

   • Mucus Layer: The thick mucus layer in diseased lungs (e.g., CF) can act as a significant barrier. Formulations need to be designed to penetrate or bypass this layer.
   • Mucociliary Clearance: The natural clearance mechanisms (coughing, cilia) can rapidly remove inhaled particles.
   • Alveolar Macrophages: These immune cells in the lungs can engulf and degrade foreign particles, including mRNA delivery vehicles.

3. mRNA Stability in the Lung Environment:

   • Despite the lack of gastric degradation, the lung environment still contains nucleases that can break down mRNA. The design of the mRNA sequence (e.g., nucleoside modifications, optimized UTRs, use of circRNA) is crucial for stability.

4. Manufacturing and Scale-Up:

   • Producing inhalable mRNA formulations (especially dry powder formulations for inhalers) at scale, with consistent quality and particle size distribution, is a significant manufacturing challenge.
   • Developing formulations that are stable at room temperature would greatly simplify storage and distribution.

5. Regulatory Pathway:

   • As a relatively new therapeutic modality and delivery route, the regulatory pathways are still evolving.

The trend towards therapeutic mRNA through inhalation is strong, driven by the advantages of direct lung targeting and the potential for transformative treatments for a wide range of debilitating respiratory diseases. While challenges remain, significant progress in delivery technologies and RNA engineering suggests a promising future for this approach.

Promtpt: Can you explain this slide, why there is a trend to avoid peg as the mRAN carrier?

Ans: This slide focuses on the use of hydrophilic polymers to create "stealth" surfaces for various biomedical applications. The central theme is to prevent unwanted interactions of surfaces with biological components, thereby improving performance and biocompatibility.

Let's break down the slide:

Title: "Hydrophilic polymer providing "stealth" surfaces for various application"

• Hydrophilic polymer: A polymer that has a strong affinity for water.
• "Stealth" surfaces: Surfaces designed to evade detection or unwanted interactions with biological molecules (like proteins, cells, or immune components) when introduced into a biological environment. They essentially make the material "invisible" to the body.
• Various applications: The slide then details several areas where such stealth surfaces are beneficial.

Left Side: "Chemistry of stealth surfaces of hydrophilic polymer" This section illustrates the underlying principle of how these polymers create stealth surfaces:

• Material Surface: This is the base material that needs to be made "stealthy."
• Hydrophilic Polymer Coating: The hydrophilic polymer is coated onto this material surface.
• Hydration Layer: The key mechanism. Because the polymer is hydrophilic, it attracts and holds a layer of water molecules around its surface. This "hydration layer" effectively acts as a physical and chemical barrier.
• Blocking Biomolecules: This hydration layer physically repels or prevents biomolecules (like proteins, enzymes, cells) from directly adsorbing onto or interacting with the material surface.
• Neutral Charge vs. Zwitterionic:
  • Neutral charge: Some hydrophilic polymers achieve this stealth effect simply by being neutral and highly hydrated.
  • Zwitterionic: A particularly effective type of hydrophilic polymer. Zwitterionic molecules contain both positive and negative charges in their structure, but with a net zero charge. This creates strong electrostatic interactions with water molecules, forming an even more robust and stable hydration layer, making them exceptionally resistant to protein adsorption.
• H-bond: Hydrogen bonds are shown to be crucial for establishing and maintaining the hydration layer around the hydrophilic polymer.

Right Side: Applications of Stealth Surfaces

The slide presents three main applications:

1. "Coated on SPR chips/paper-based sensors for... Detection of specific target molecules... Reduced non-specific protein adsorption"

   • SPR (Surface Plasmon Resonance) chips/paper-based sensors: These are analytical tools used to detect and quantify molecular interactions (e.g., drug-target binding, biomarker detection).
   • Problem: In typical sensors, non-specific binding of unwanted proteins from a complex biological sample (like blood serum) can lead to false positives or reduced sensitivity.
   • Solution: Coating the sensor surface with a hydrophilic "stealth" polymer prevents these non-specific interactions. This ensures that only the specific target molecule being sought binds, leading to more accurate and sensitive detection.
   • Benefit: "Reduced non-specific protein adsorption."
   • Citations: Relevant scientific publications are listed, indicating this is an active research area.

2. "Coated on substrates as universal biofilm-resistance coating"

   • Substrates: Refers to various materials, both inorganic (e.g., Si for silicon, used in medical devices, implants) and organic (e.g., PE for polyethylene, used in catheters, tubing).
   • Problem: Microorganisms (bacteria, fungi) can adhere to surfaces, forming biofilms. Biofilms are notoriously difficult to remove and can cause persistent infections (e.g., on medical implants) or fouling in industrial settings.
   • Solution: Coating these substrates with a hydrophilic polymer creates a surface that bacteria struggle to adhere to, thus preventing biofilm formation.
   • Images: The images clearly illustrate this: "Without coating" shows significant bacterial growth (green fluorescence indicating bacteria), while "With coating" shows very few bacteria, demonstrating effective biofilm resistance.
   • Benefit: Acts as a "universal biofilm-resistance coating" against both inorganic and organic substrates.
   • Citations: Again, supporting scientific literature is provided.

3. "Hydrogel platform for enzyme immobilization in electrochemical/colorimetric sensors"

   • Enzyme immobilization: Attaching enzymes to a solid support to retain their activity and allow for reuse in biosensors or industrial processes.
   • Problem: Enzymes can lose activity when immobilized or be susceptible to denaturation or non-specific interactions within a complex sample.
   • Hydrogel platform: A hydrophilic polymer-based hydrogel (a 3D network that holds water) can be used to encapsulate or embed enzymes.
   • Benefit: The hydrophilic nature of the hydrogel creates a protective, biocompatible environment for the enzymes, maintaining their activity and preventing interference from other molecules in the sample. This enhances the performance of electrochemical or colorimetric sensors that rely on enzyme activity.
   • Diagram: Shows a "PBM hydrogel" encapsulating enzymes, which then react with a substrate, producing a product detectable by the sensor.
   • Citations: Further evidence from publications.

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Why the Trend to Avoid PEG as an mRNA Carrier?

The latter part of your question, while not directly addressed by the slide, is highly relevant to the concept of "stealth" surfaces in drug delivery, particularly for mRNA carriers like Lipid Nanoparticles (LNPs).

PEG (Polyethylene Glycol) is a hydrophilic polymer that has long been the gold standard for creating "stealth" surfaces on nanoparticles, including early LNP formulations for mRNA. It works precisely by forming a hydration layer, preventing opsonization (coating by immune proteins) and subsequent rapid clearance by the reticuloendothelial system (RES), thereby increasing the circulation time of nanoparticles.

However, there's a growing trend to reduce or replace PEG in mRNA carriers for several reasons:

1. Anti-PEG Antibodies and Accelerated Blood Clearance (ABC) Phenomenon:

   • Pre-existing Antibodies: A significant portion of the human population (estimates vary, but can be as high as 25-70%) has pre-existing antibodies against PEG. This is due to widespread exposure to PEG in various consumer products, cosmetics, and other medications.
   • Immune Response: When PEGylated nanoparticles are introduced, these pre-existing antibodies can bind to the PEG chains.
   • ABC Phenomenon: This binding triggers an accelerated blood clearance (ABC) phenomenon. The PEGylated nanoparticles are rapidly recognized and removed from circulation by the immune system, leading to:
     • Reduced efficacy: The therapeutic payload (mRNA) doesn't reach its target efficiently.
     • Increased toxicity: The nanoparticles accumulate in organs like the liver and spleen where they are cleared, potentially causing adverse effects.
     • Hypersensitivity Reactions: In some cases, severe allergic or anaphylactic reactions (like those observed in a small percentage of recipients of early PEGylated LNP COVID-19 vaccines) can occur due to immune complex formation.

2. Reduced Dosing Flexibility/Repeat Dosing Issues:

   • The ABC phenomenon also means that with repeated dosing of PEGylated formulations, subsequent doses are cleared even faster than the first, making it difficult to achieve consistent therapeutic effects. This is a major concern for chronic conditions requiring multiple administrations.

3. Limitations for Intravenous Delivery:

   • For therapeutic mRNA requiring systemic (intravenous) delivery to reach various organs, the ABC phenomenon is a significant hurdle.

4. Desire for Improved Biocompatibility and Biodegradability:

   • While PEG itself is generally considered safe, there's a push for next-generation polymers that offer even better biocompatibility, biodegradability, and a lower propensity for immune reactions, especially for long-term or repeated therapeutic applications.

What are the alternatives to PEG?

Researchers are actively exploring and developing alternative hydrophilic polymers and surface modifications to achieve stealth properties without the drawbacks of PEG. These often include:

• Zwitterionic polymers: As highlighted in the slide, zwitterionic polymers are excellent candidates because their strong hydration layer and net neutral charge make them highly resistant to protein adsorption and immunogenicity.
• Polysaccharides: Certain natural or modified sugars can create stealth surfaces.
• Other synthetic hydrophilic polymers: Various other synthetic polymers with unique architectures are being investigated.

In essence, while PEG revolutionized stealth delivery, its widespread use has revealed limitations related to pre-existing immunity. The trend is to find equally effective but less immunogenic "stealth" coatings for advanced therapeutic mRNA carriers, ensuring better safety and efficacy, especially for chronic treatments and repeated dosing. The research presented in this slide on zwitterionic and other hydrophilic polymers directly contributes to this effort to find superior alternatives to PEG.

Prompt: Why there is a trend to develop the TB vaccine for adult, especially using mRNA based vaccine, why dont they use BCG?

Ans: This slide focuses on "PART 1: Target Antigen Selection" for a new TB vaccine, highlighting a multi-antigen, multi-stage approach. It provides a strong context for understanding why there's a trend to develop new TB vaccines for adults, particularly using mRNA-based platforms, instead of solely relying on BCG.

Let's break down the slide and then address your question directly:

Slide Analysis: PART 1: Target Antigen Selection

Background & Rationale (Left Side):

• "TB antigens are expressed at different stages of infection (early, active, and latent)." This is a critical point. Mycobacterium tuberculosis (M.tb), the bacterium causing TB, changes its gene expression and thus the antigens it presents depending on whether it's in the initial infection, active disease, or latent (dormant) state.
• "Multi-stage targeting is essential for broad and durable protection." Because M.tb presents different faces at different stages, a vaccine needs to target antigens from all these stages to provide comprehensive and long-lasting immunity. A vaccine only targeting active disease antigens might not protect against initial infection or latent TB, and vice-versa.
• "Combination approach for better protection." This directly follows from the multi-stage concept. Combining multiple antigens is likely to provide broader and more robust protection than a single antigen.
• "Prior vaccine studies (e.g., H56; IC31) used Ag85B, ESAT-6, and Rv2660c - Advanced to Phase 2b trials, showing safety and immunogenicity." This indicates that these specific antigens have a track record of being safe and stimulating an immune response in human trials. This provides a scientific basis for their selection in the current study.

Antigens Selected for This Study (Right Side): This section details the specific antigens chosen and their characteristics:

• Ag85B (Rv1886c):
  • Expressed in early infection.
  • Involved in cell wall biosynthesis.
  • Stimulates humoral and cellular immunity (both antibody and T-cell responses).
• ESAT-6 (Rv3875):
  • Expressed consistently across all stages of infection.
  • Stimulates T-cell activation via TLR-2 (Toll-like Receptor 2, an innate immune sensor, suggesting it can trigger a strong immune response).
  • High immunogenicity and diagnostic value (also used in TB diagnostic tests).
• Rv2660c:
  • Predominantly expressed during latent infection.
  • Triggers pro-inflammatory responses via TLR-2.
  • Potential for post-exposure vaccine development (meaning it could be effective even if someone has already been exposed to TB, possibly by boosting their immune response against latent infection).
• Fusion Construct: Ag85B-ESAT6-Rv2660c:
  • This is the innovative part: combining all three antigens into a single construct.
  • Designed to combine benefits of all three antigens. This aims to achieve the "multi-stage targeting" and "combination approach" mentioned in the rationale.
  • Aims to stimulate broad immune response. By presenting multiple antigens, the immune system is more likely to generate a comprehensive and protective response.

---

Why the Trend to Develop TB Vaccines for Adults (Especially mRNA-based), and Why Not Just Use BCG?

The slide's content directly explains the limitations of BCG and the rationale for new, advanced TB vaccines like those potentially based on mRNA.

1. Limitations of BCG (Bacille Calmette-Guérin) Vaccine:

• Variable Efficacy Against Pulmonary TB in Adults: BCG is the only licensed TB vaccine, introduced over 100 years ago. Its primary limitation is its highly variable efficacy, particularly against pulmonary (lung) TB in adolescents and adults, which is the most common and transmissible form of the disease. While it's relatively effective at preventing severe forms of TB (like disseminated TB and TB meningitis) in infants and young children, its protection wanes over time and is inconsistent against the adult pulmonary form.
• Impact of Environmental Mycobacteria: Prior exposure to non-tuberculous mycobacteria (NTM), common in many environments, can interfere with BCG's effectiveness.
• Safety in Immunocompromised Individuals: BCG is a live-attenuated vaccine, meaning it contains a weakened form of the bacteria. This makes it unsafe for individuals with compromised immune systems (e.g., HIV-infected individuals), who are ironically at high risk for TB.
• Interference with TB Diagnostics: BCG vaccination can cause a positive tuberculin skin test (TST), making it difficult to differentiate between vaccinated individuals and those truly infected with M.tb.

2. Why Develop New TB Vaccines for Adults?

• Global Health Crisis: TB remains a leading cause of death from an infectious disease globally (over 1.6 million deaths annually). The majority of these deaths occur in adults.
• Need for Adult Protection: Since BCG offers limited protection against adult pulmonary TB, there's a critical unmet need for a vaccine that can effectively prevent or reduce the burden of active TB in adults.
• Addressing Latent TB: A significant portion of the global population (estimated 1/4) has latent TB infection (LTBI). These individuals are at risk of developing active TB later in life. New vaccines targeting antigens like Rv2660c (as seen on the slide) could help prevent progression from latent to active disease.
• Combating Drug Resistance: The rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB strains makes effective new vaccines even more urgent as treatment options become limited.
• Breaking the Chain of Transmission: Adults with active pulmonary TB are the primary drivers of transmission. An effective adult vaccine would significantly reduce community transmission and help achieve global TB elimination goals.

3. Why the Trend Towards mRNA-based TB Vaccines?

mRNA technology offers several compelling advantages for new TB vaccine development:

• Multi-Antigen Delivery (as shown on the slide): mRNA can easily encode multiple antigens within a single molecule (like the "Fusion Construct" of Ag85B-ESAT6-Rv2660c). This allows for the "multi-stage targeting" and "combination approach" deemed essential for TB, which is harder to achieve with traditional protein-based or live-attenuated vaccines.
• Potent Immune Response: mRNA vaccines are known to elicit strong cellular (T-cell) immune responses, which are crucial for controlling intracellular pathogens like M.tb. They also induce robust humoral (antibody) responses.
• Speed and Flexibility: The rapid design, manufacturing, and scalability of mRNA vaccines (demonstrated by COVID-19 vaccines) are highly attractive. This allows for faster development cycles and potential rapid adaptation to new M.tb strains or evolving understanding of TB immunology.
• Safety Profile (Non-Live): Unlike BCG, mRNA vaccines are non-live. They do not carry the risk of causing disease in immunocompromised individuals, making them a safer option for vulnerable populations.
• Adjuvant Effect: The mRNA itself (especially with certain modifications) can act as an innate immune stimulant, essentially self-adjuvating, reducing the need for external adjuvants that might have their own side effects.
• Ability to Elicit Specific T-cell Responses: For TB, a strong and appropriate T-cell response (e.g., Th1 response) is vital. mRNA vaccines are well-suited to generate these types of responses.

In conclusion, the slide perfectly illustrates the scientific rationale for moving beyond BCG: the need for a multi-stage, multi-antigen vaccine to provide broader and more durable protection against TB, especially against the pulmonary form in adults. mRNA technology offers a highly versatile, potent, and safe platform to achieve these complex vaccine design goals, addressing the critical unmet need in adult TB prevention.

Prompt: Why the company engineer both 5’ and 3’ UTR on mRNA

Ans: This slide provides a high-level comparison of the mRNA constructs used in the Pfizer-BioNTech (BNT162b2) and Moderna (mRNA-1273) COVID-19 vaccines. It clearly shows that both companies engineered not only the coding sequence (for the Spike protein) but also the 5' and 3' Untranslated Regions (UTRs).

The reason companies engineer both the 5' and 3' UTRs on mRNA is to optimize the overall performance of the mRNA molecule for therapeutic (or vaccine) purposes. These regions, despite not coding for protein themselves, play crucial roles in regulating the mRNA's life cycle within the cell:

1. mRNA Stability:

   • Protection from Degradation: Cellular enzymes called exonucleases can degrade mRNA from its 5' and 3' ends. The UTRs, along with the 5' cap and 3' poly-A tail, act as protective elements. Engineering them can make the mRNA more resistant to degradation, thus prolonging its lifespan in the cytoplasm.
   • Example from Slide: For Moderna's mRNA-1273, the 3' UTR uses the "Human α-globin gene" which is known to confer good mRNA stability and translation efficiency. Pfizer's BNT162b2 uses a "Human amino-terminal enhancer of split (AES)UTLE5 + mitochondrial 12S rRNA (mtRNR1)" sequence for its 3' UTR, also chosen for stability and translation.

2. Translational Efficiency (How much protein is made):

   • Ribosome Recruitment: The 5' UTR is critical for ribosome binding and initiation of translation. Sequences within the 5' UTR can influence how efficiently the ribosome loads onto the mRNA.
   • Example from Slide: Both companies use the "Human α-globin gene" for their 5' UTRs. The α-globin 5' UTR is a well-known sequence that promotes high levels of cap-dependent translation.
   • Open Reading Frame (ORF) Scanning: The 5' UTR needs to allow the ribosome to efficiently scan and find the correct start codon (AUG). Secondary structures within the 5' UTR can sometimes impede this process.
   • Poly-A Tail Interaction (3' UTR): The 3' UTR interacts with the poly-A tail and certain proteins (e.g., poly(A)-binding protein, PABP) which can circularize the mRNA molecule. This circularization (a pseudo-circularization, not a true circRNA) is thought to enhance ribosome recycling and re-initiation, thereby boosting translation efficiency.

3. Immunogenicity:

   • Avoiding Innate Immune Activation: Untranslated regions contain sequences that can be recognized by cellular innate immune sensors (e.g., Toll-like receptors, RIG-I). Such recognition can trigger an inflammatory response and lead to mRNA degradation, reducing protein expression.
   • Codon Optimization & UTR Selection: By carefully selecting or modifying UTR sequences, companies aim to reduce these "danger signals" that could trigger an unwanted immune response against the mRNA itself. This allows the mRNA to "hide" from the immune system for longer, leading to more efficient protein production.
   • Combined with Nucleoside Modifications: This optimization of UTRs works in conjunction with the "Modified nucleosides" shown in the diagram (e.g., pseudouridine instead of uridine in the coding sequence and UTRs) to further dampen immunogenicity.

4. Codon Optimization (Relevant to coding sequence, but influences UTRs):

   • While not strictly UTRs, codon optimization of the coding sequence (e.g., using more common codons for efficient translation by host ribosomes) also influences the overall mRNA design and often necessitates careful UTR selection to complement these changes.

Specific Examples from the Slide:

• Pfizer (BNT162b2):
  • 5'-UTR: Human α-globin gene. Chosen for its strong translational efficiency.
  • 3'-UTR: Human amino-terminal enhancer of split (AES)UTLE5 + mitochondrial 12S rRNA (mtRNR1). These are selected for their roles in enhancing stability and potentially contributing to translation.
• Moderna (mRNA-1273):
  • 5'-UTR: Human α-globin gene. Similar to Pfizer, indicating its known efficacy in promoting translation.
  • 3'-UTR: Human α-globin gene. Re-using the α-globin gene for the 3' UTR suggests its strong properties for stability and translation.

In summary, engineering both the 5' and 3' UTRs in mRNA therapeutics is crucial for:

• Maximizing mRNA stability and preventing premature degradation.
• Optimizing translational efficiency to produce high levels of the desired protein.
• Minimizing unwanted innate immune responses against the mRNA itself.

These UTRs act as critical regulatory elements, fundamentally impacting the overall performance and safety of the mRNA therapeutic in vivo.

Prompt: Can you tell me why mRNA vaccine seems to initiate immune response better than peptide or protein?

Ans: You've hit on a critical advantage of mRNA vaccines that has been a major driver of their success, particularly evident with the COVID-19 vaccines. While peptide and protein vaccines have their place, mRNA vaccines often initiate a more robust and comprehensive immune response for several key reasons:

1. Endogenous Antigen Presentation (Mimicking Natural Infection):

• mRNA: When an mRNA vaccine is delivered, it enters the host cell (e.g., a muscle cell or an antigen-presenting cell like a dendritic cell). The cell's own machinery then translates the mRNA into the desired antigen (e.g., the viral spike protein). This antigen is produced inside the cell.
  • MHC Class I Presentation: Critically, proteins produced endogenously (inside the cell) are processed and presented on the cell surface via MHC Class I molecules. This presentation pathway is essential for activating CD8+ T-cells (cytotoxic T lymphocytes or "killer T-cells"). CD8+ T-cells are crucial for recognizing and killing infected cells, providing cellular immunity.
  • MHC Class II Presentation: The exogenously delivered mRNA (or the produced protein that is then taken up by APCs) is also processed and presented via MHC Class II molecules, activating CD4+ T-cells (helper T-cells). These helper T-cells are vital for orchestrating the overall immune response, including B-cell activation (for antibody production) and supporting CD8+ T-cell function.
• Peptide/Protein: Peptide or protein vaccines deliver the pre-formed antigen directly.
  • MHC Class II Presentation: Primarily, these exogenous antigens are taken up by antigen-presenting cells (APCs) through endocytosis or phagocytosis. They are then processed and presented almost exclusively on MHC Class II molecules, leading predominantly to the activation of CD4+ helper T-cells and subsequent antibody production by B-cells.
  • Limited MHC Class I Presentation: While there are some mechanisms for cross-presentation (where exogenous antigens can be presented on MHC Class I), it's generally less efficient and robust than the endogenous pathway initiated by mRNA. This means peptide/protein vaccines are often less effective at generating strong CD8+ T-cell responses.

2. Adjuvant Effect / Innate Immune Activation:

• mRNA: The mRNA molecule itself, particularly with certain modifications (or lack thereof in some self-amplifying mRNA designs), can act as an "intrinsic adjuvant."
  • Pattern Recognition Receptors (PRRs): mRNA molecules can be recognized by intracellular Pattern Recognition Receptors (PRRs) like Toll-like Receptors (TLRs, especially TLR7 and TLR8) and RIG-I-like receptors (RLRs) in endosomes and the cytosol.
  • Inflammatory Cytokines: This recognition triggers an innate immune response, leading to the production of inflammatory cytokines (e.g., interferons). These cytokines create an inflammatory environment that signals "danger" to the immune system, enhancing the activation and maturation of APCs (like dendritic cells).
  • Robust Immune Priming: This innate immune activation is critical for efficiently priming adaptive immune responses (both T-cell and B-cell), leading to more potent and durable immunity.
• Peptide/Protein: Peptide and protein vaccines are often poorly immunogenic on their own and require the addition of external adjuvants (e.g., alum, AS04) to stimulate the innate immune system and generate a strong response. Without adjuvants, the immune system might not perceive the protein as a threat.

3. "Self-Amplifying" Potential (for some mRNA platforms):

• Self-amplifying mRNA (saRNA): Some advanced mRNA vaccine designs include genes for viral replicase enzymes. Once inside the cell, this saRNA not only encodes the antigen but also makes multiple copies of itself.
• Prolonged Antigen Expression: This in situ amplification leads to significantly higher and more prolonged antigen expression from a single, lower dose of mRNA. More antigen over a longer period can lead to a stronger and more sustained immune response.
• Peptide/Protein: These vaccines deliver a fixed amount of antigen that is then gradually degraded. There's no inherent amplification.

4. Broad Epitope Presentation:

• mRNA: By encoding the full-length antigen, the cell produces the entire protein. This protein is then processed into numerous different peptides (epitopes) that can be presented to the immune system. This leads to a broader repertoire of T-cell and B-cell responses, making the immune response less susceptible to viral mutations.
• Peptide: Peptide vaccines usually present only a few specific, short peptide sequences (epitopes). If the pathogen mutates these specific epitopes, the vaccine might lose efficacy.
• Protein: While protein vaccines present the full protein, their processing pathway often biases towards certain epitopes and MHC presentation pathways.

In summary, mRNA vaccines initiate a better immune response than many peptide or protein vaccines primarily because they:

• Leverage endogenous protein production, leading to robust CD8+ T-cell (killer T-cell) activation via MHC Class I, in addition to CD4+ T-cell and antibody responses via MHC Class II.
• Act as their own adjuvants, stimulating innate immunity and better priming the adaptive immune system without the need for external additives.
• (For saRNA) Can amplify antigen expression within the cell, leading to prolonged and higher levels of antigen presentation.
• Present a broader range of epitopes as the full protein is made and processed by the host cell.

These factors combine to generate a more comprehensive, potent, and often more durable immune response, which has been a game-changer in vaccinology.

Prompt: Why there are some concerns on mRNA vaccine? especially on the metastasis in cancer.

Ans: It's important to clarify that the primary mRNA vaccines currently in widespread use (like those for COVID-19) are designed to prevent infectious diseases and generally do not raise concerns about promoting cancer metastasis. In fact, mRNA technology is being actively explored for cancer therapy, often aiming to reduce metastasis.

However, when discussing "concerns" with mRNA technology, especially in the context of cancer, there can be a few angles, some of which are misinterpretations or relate to the broader field of genetic medicines rather than specifically to the infectious disease vaccines:

1. Misinformation and Lack of Understanding:

• Confusion with Gene Therapy: A common misconception is that mRNA vaccines alter a person's DNA or genetic code. This is incorrect. mRNA stays in the cytoplasm and is quickly degraded; it does not enter the nucleus or integrate into the genome. However, this misunderstanding can fuel unsubstantiated fears about cancer development or progression.
• Rapid Development and Novelty: The rapid development and deployment of mRNA vaccines during the pandemic, while a scientific triumph, also meant that some individuals were wary of a "new" technology without extensive long-term data. This novelty can be exploited by misinformation.

2. Theoretical Concerns (largely disproven for current vaccines):

• Inflammation: Some theories initially suggested that the inflammatory response triggered by mRNA (even modified mRNA) could theoretically contribute to an environment that might promote cancer in susceptible individuals. However, the immune response is transient and localized, and there is no evidence from extensive clinical trials or real-world data (billions of doses) that COVID-19 mRNA vaccines cause or promote cancer. In contrast, chronic inflammation is a known risk factor for cancer, but the acute, controlled inflammation from a vaccine is fundamentally different.
• Lipid Nanoparticle (LNP) Accumulation: Early concerns sometimes focused on the delivery vehicle (LNPs), suggesting they could accumulate in certain organs or induce inflammation there. While LNPs do distribute to various organs (like liver and spleen), they are designed to be biodegradable and are cleared from the body. Again, no evidence supports a link between LNP accumulation from current vaccines and cancer metastasis.
• Off-target protein expression: A hypothetical concern might be if the mRNA was somehow mistranslated into an unintended, harmful protein. However, mRNA sequences are meticulously designed and verified, and quality control during manufacturing is stringent.

3. Context of Therapeutic mRNA for Cancer Treatment:

• Targeting Tumors and Metastasis: Ironically, the goal of therapeutic mRNA in cancer is often to fight tumors and prevent or treat metastasis. For example:
  • Cancer Vaccines: mRNA cancer vaccines aim to train the immune system to recognize and attack cancer cells, including metastatic ones.
  • mRNA-encoded therapeutic proteins: mRNA can deliver instructions for proteins that directly kill cancer cells, inhibit their growth, or block pathways involved in metastasis.
  • mRNA-encoded gene editing tools: mRNA can deliver CRISPR-Cas9 components to correct oncogenes or activate tumor suppressor genes.
• Potential for Immune Activation to Counter Metastasis: Metastasis is a complex process involving immune evasion by cancer cells. By stimulating a strong anti-tumor immune response, mRNA-based cancer therapies could actually help clear metastatic cells.

4. Misinterpretation of Research (e.g., self-amplifying mRNA or specific targeting):

• Some advanced mRNA platforms, like self-amplifying mRNA (saRNA), or specific targeted delivery systems for cancer might be misunderstood. For example, if an saRNA is designed to produce a highly potent molecule, concerns might arise about its long-term activity if not strictly controlled. However, this is about the design of specific therapeutics, not a general property of all mRNA vaccines.

Conclusion on Concerns about Metastasis and mRNA Vaccines:

For the widely used COVID-19 mRNA vaccines, there is no scientific evidence or clinical data to suggest they initiate or promote cancer metastasis. These vaccines are designed to elicit a transient immune response to a viral protein and are rapidly cleared from the body. Major regulatory bodies (FDA, EMA, CDC) continuously monitor safety data, and no such link has been established.

Concerns about "metastasis" often stem from:

• Misinformation or a fundamental misunderstanding of how mRNA works.
• Extrapolating theoretical risks without considering the specific design, modifications, and transient nature of the current vaccines.
• Confusing the preventive nature of current mRNA vaccines with the much broader and more complex field of therapeutic mRNA for cancer, where the technology is being used to fight cancer and metastasis.

The trend in mRNA technology, especially in oncology, is actually toward harnessing mRNA to prevent and treat cancer, including its spread, rather than causing it.