**FEDERICO BRUNELLO - HTGAA 2026**

title: ‘(“Pharmatech Academy” postmasters specialization - mRNA pharmaceuticals and gene therapy - Federico II University of Naples, Italy, 2025)’ weight: 10

title: ‘Week 1 HW: Principles and Practices’ weight: 10

(Fig 1 - Synthetic Biology artist impression: a blue fluorescently-labeled double-stranded DNA molecule is on display, electronic circuits background - Source: Helmholtz Munich Foundation, copyright: Adobe Premium Stock)

WEEK 1 QUESTIONS 1) Describe a biological engineering application or tool you want to develop and why According to the WHO International Agency for Research on Cancer (IARC), 2024 Global Cancer Burden News Release, in 2022, there were an estimated 20 million new cancer cases and approximately 9.7 million cancer deaths worldwide. (1,2) Due to the inherent ability of cancer cells to evade the traditional approaches to cancer therapy (surgery, chemotherapy, and radiation) due to the dynamically evolving, heterogeneous nature of the tumoral mass as well as via mechanisms as resistance mutation acquisition, genetic heterogenicity, microenvironment interactions, and immune evasion, several types of cancer (including pancreatic cancer, triple negative breast cancer, acute myeloid leukaemia) are able to successfully develop subsequent mutations and evade both the immune system of the host, causing eventually cancer relapse and the death of the patient. It is therefore necessary to devise a potential approach to treating emerging cancer mutations and the mutation of the corresponding epitopes in by being able to produce dynamically adjustable biotherapeutics that are able to target previously not known emerging epitopes that might not be recognized immediately by the host immune system. I am strongly interested in the applications of synthetic biology and artificial intelligence-mediated sequence optimization to improve the performance of innovative, targeted cancer-directed biotherapeutics (including monoclonal antibodies and CAR-T, or anti-cancer mRNA vaccines) to successfully address several kinds of cancer and provide personalized and targeted treatment against metastatic cancer in patients. Monoclonal antibodies are target-specific antibodies derived from the cloning of identical immune cells. The ability to use synthetic biology and genome editing tools to potentially customize the variable regions in monoclonal antibodies, coupled with the possibility of precisely editing genes, and the ability to perform in-silico prediction of protein folding and antigen binding properties, allows to potentially generate multiple strains of monoclonal antibodies that can be dynamically adjusted to the emerging mutations of the cancerous mass. CAR-T cell therapy is also prone to optimization with synthetic biology, allowing for the improvement of antigen recognition by the re-engineered T-cells so that they can be dynamically optimized to target emerging cancer mutations in the target cancer mass. mRNA vaccines can also be used in the fight against cancer, allowing for the insertion of an mRNA sequence encoding for a sequence able to either elicit an immune response or to directly kill the target cell; however, limitations include LNP being subject to first pass and degradation in the liver. As the process involves a continuous adjustment of therapy to the continuous evolution of the tumoral mass epitope configuration, it is necessary to insert predictive AI tools that can devise the most likely mutations that the tumoral mass may give rise to after each round of treatment and to potentially devise a follow-up optimized antibody / CAR-T, however ethical and cost considerations on costs of manufacturing, AI de-novo drug design capability, safety assessment and screening must be made. (1) WHO - Feb 2024 - Global cancer burden growing, amidst mounting need for services https://www.who.int/news/item/01-02-2024-global-cancer-burden-growing--amidst-mounting-need-for-services (2)Cancer Today Report - WHO IARC - https://gco.iarc.fr/en

2) Describe one or more governance/policy goals related to ensuring that this application or tool contributes to an “ethical” future, like ensuring non-malfeasance (preventing harm). Break big goals down into two or more specific sub-goals The main governance policy regards the prevention of toxicity of engineered antibodies to the patient and the preventive assessment of possible side effects that might be caused to the patient when treated with synthetic biology engineered mAbs. AI shall be used to screen the engineered mutations for possible side effects and out of target bindng of the mABs and ensure patient safety. Potentially, modelling of engineered monoclonal antibodies should be first tested by assessing their specificity to cancer cells from a biopsy or by employing virtual assessment to check against the presence of structurally similar off-target binding sites in other regions of the patient.

3) Describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”):

1. Purpose: The use of AI-aided design for synthetic biology enhanced biopharmaceuticals able to counter the evolution of cancer cells has great promise to produce customized and personalized medicine for patients suffering from the most untreatable conditions. –> Actions: a framework regulating the use of AI in drug discovery shall be agreed upon internationally. Scientists shall abide by an ethical code and follow a “do-not-harm” policy.
2. Design: Patients subject to a synthetic biology-enhanced monoclonal antibody therapy shall fully understand the potential consequences of treatment. Multiple quality control checks must be implemented in the process of design and optimization of monoclonal antibodies or CAR-T variable antigen binding site, as well as for the integrity of the mRNA sequence inserted in potential mRNA vaccines against cancer. Manufacturing errors and contaminations must be excluded by applying strict quality control checks. –> ACTIONS: Companies/Researchers: meet strict quality control checks, abide to GMP practices when manufacturing, perform GLP-grade R&D. Medical Practitioners: Provide information on potential risks to patients, administer advanced therapies only under medical supervision in adequate premises. International regulators: a framework on the use of AI for drug design shall be laid down to prevent accidental or intentional damages to patient.
3. Assumptions: What could you have wrong (incorrect assumptions, uncertainties)? Synthetic biology-enhanced mABs or CAR-T cells might bind to areas not relevant to the intended therapeutic site, hence causing off target inflammatory responses, damage to the patient’s non-tumoral tissue and potentially, his death. Malicious agents or hallucinations by AI may potentially introduce unwanted mutations in the engineered therapeutic agent, hence causing it not to work as intended or, worse, cause harm to the patient. –> ACTIONS: World regulatory authorities shall regulate the sequences that AI is able to synthesize when creating synthetic biology-enhanced biotherapeutics. A system of automated screening shall be put in place to rapidly score and assess the potential dangers and unwanted side effects (i.e. binding to a different location than the one they are devised for causing damage to patient) of such AI designed biotherapeutics.
4. Risks of Failure & “Success”: How might this fail, including any unintended consequences of the “success” of your proposed actions? Despite the first round of treatment being successful, the ablation of certain cancerous cells in the tumor mass via synthetic biology enhanced mABs or CAR-T gives free rein to the surviving cells to proliferate. Unless another round of targeted mABs is found quickly, the patient may still die due to the metastasis of surviving cells. Issues: therapeutic fairness, costs, and ethical considerations on the worthiness of treatment (how would the patient live if he/she has to be subject to continuous therapy of multiple rounds of synthetic biology evolved mAbs targeting specific sites with no certainty of a 100% absence of relapse?). -–> ACTIONS Ethical considerations and cost/benefit analysis shall be used based on internationally recognized guidelines.

The regulatory taken in consideration are as follows: 1) Regulate AI use in R&D 2) Use strict QC and R&D protocols 3) Regulate Hospital Administration Of AI-Generated Drugs

Next, score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals. The following is one framework but feel free to make your own:

Last, drawing upon this scoring, describe which governance option, or combination of options, you would prioritize, and why. Outline any trade-offs you considered as well as assumptions and uncertainties.

I would prioritize the international regulation of AI in performing research, taking into consideration that AI-generated research and pharmaceuticals shall always be checked for safety to the patient, as well as subject to concurrent human evaluation. It is also key to make sure biomanufacturing facilities in labs and hospitals are able to guarantee high GMP quality in compliance with manufacturing regulations.
. Reflecting on what you learned and did in class this week, outline any ethical concerns that arose, especially any that were new to you. Then propose any governance actions you think might be appropriate to address those issues. This should be included on your class page for this week.

Synthetic biology has a great potential to advance research, however, the emergence of AI agents that can potentially emulate the research abilities of a scientist poses questions about the ability to correctly manage and vet the results of AI-generated synthetic sequences, both for hallucinations/unintended results and for intentionally dangerous sequences that might be introduced for malicious purposes.

It is important that a international authority like the WHO and regional authorities are able to produce a framework to be able to channel the use of AI in research for positive outcomes.

Homework Question from George Church Google: searched for list of 10 essential amino acids in all animals, checked on Wikipedia. Used ChatGPT-4o Mini to generate a list of the 10 non-essential and 10 essential amino acids in some of the most representative animal species. Prompts: “Essential Amino Acids in Different Animals”, “List the essential and non-essential aminoacids” Amino acids are organic compounds constituted by structures containing of amino and carboxylic acid functional groups. (1) Of the more than 500 chemically synthesizable aminoacid structures (2), 22 amino acids are named “proteinogenic amino acids”, as they constitute the fundamental building blocks of proteins in living organisms. The 22 proteinogenic amino acids (alpha-aminoacids) include: 20 standard aminoacids (3) used to produce protein in the translation process, with the addition of pyrrolyisine and selenocysteine, found in certain methanogenic microorganisms as part of the methyltransferase enzyme aiding the conversion of carbon dioxide into methane (4)
In the translation process, the 20 standard amino acids are encoded and lined on the protein primary structure based on the encoding correspondence relation with one or more three mRNA base pair sequences (the theoretical amount of single amino-acids encodable by each different mRNA triplet is 64 = 4 base permutations ^ length of triplet, however, the genetic code is degenerate as multiple base pair sequences can be translated to the same aminoacid). During the process of mRNA translation into proteins, the ribosome machinery, based on the genetic code translates triplets of mRNA into a strand of concatenated corresponding aminoacids (linked by peptide bonds), giving rise to the primary structure of proteins, which is a polypeptide chain. The primary structure of proteins gives rise, via hydrogen bonds interactions among different amino acid residue groups, to the proteins secondary structure (alpha helix and beta-sheet). Interactions between the polypeptide chain and the environment (as hydrophobic interactions), hydrogen bonds, ionic bonds, and disulfide bridges give rise to the tertiary structure of the protein. Tertiary structure is fundamental to defining the specificity of enzymes and their functionality (e.g., defining the shape of the enzymatic active site). Of the 20 aminoacids generally used for protein synthesis, 10 are termed “essential” as they cannot be produced endogenously and must be acquired by diet (Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine, Arginine), absence of such amino-acids in the diet can negatively affect vital functions as enzyme activity, hormone synthesis and muscle development. The other 10 aminoacids (Alanine, Asparagine, Aspartic Acid, Glutamic Acid, Serine, Cysteine, Tyrosine, Glycine, Proline, Glutamine) are termed non-essential as they can be both acquired via diet and biosynthesized endogenously. Interestingly, most common animal species (including humans, cows, chickens, pigs, fish, dogs, cats, sheep, horses, cat, sheep, rabbits) share the same list of essential amino acids, which cannot be produced endogenously. Being Lysine an essential aminoacid that must be acquired through diet, “Lysine Contingency” refers to the issue that, in the practice of industrial animal livestock rearing, the absence or reduced quantity of lysine in animal feed may adversely impact the yield of livestock and poultry. (5) This scientific issue has inspired (despite being twisted) the story in the “Jurassic Park” book, where the captively held dinosaurs had been genetically engineered to inhibit the endogenous production of lysine (which in reality does not get produced endogenously) by inhibiting a putative lysine-producing enzyme, and depend on the constant supply of lysine in their diet, preventing them from fleeing in absence of food supplements provided to them in the park (6)

Interestingly, this approach could provide a hint to a the possible engineering of meat-producing animals via synthetic biology approaches as to be able to endogenously synthesize lysine, which is the limiting aminoacid in grain-fed cattle, such an approach could usher in higher livestock productivity.

References: (1) Lehninger, A. L. (1975). Biochemistry: The Molecular Basis of Cell Structure and Function (2nd ed.). New York: Worth Publishers. (2) Ye, C.-X., Dansby, D. R., Chen, S., & Meggers, E. (2023). Expedited synthesis of α-amino acids by single-step enantioselective α-amination of carboxylic acids. Nature Synthesis, 2, 645–652. https://doi.org/10.1038/s44160-023-00267-w (3) Stoye, E. (2019). Why does all life use the same 20 amino acids? Chemistry World. Retrieved from Chemistry World (4) Rother M, Krzycki JA (August 2010). “Selenocysteine, pyrrolysine, and the unique energy metabolism of methanogenic archaea”. Archaea. 2010 (5) Ronald O. Ball, Kristine L. Urschel, Paul B. Pencharz, “Nutritional Consequences of Interspecies Differences in Arginine and Lysine Metabolism123, The Journal of Nutrition, Volume 137, Issue 6, 2007”, Pages 1626S-1641S, ISSN 0022-3166

**FEDERICO BRUNELLO - HTGAA 2026**

title: ‘Week 2 HW: Reading and Writing DNA’ weight: 10

(Fig 1 - Synthetic Biology artist impression: a blue fluorescently-labeled double-stranded DNA molecule is on display, electronic circuits background - Source: Helmholtz Munich Foundation, copyright: Adobe Premium Stock)

WEEK 2 QUESTIONS Part 1 - Benchling & In-silico Gel Art a) Make a free account at benchling.com *b) * The main governance policy regards the prevention of toxicity of engineered antibodies to the patient and the preventive assessment of possible side effects that might be caused to the patient when treated with synthetic biology engineered mAbs. AI shall be used to screen the engineered mutations for possible side effects and out of target bindng of the mABs and ensure patient safety. Potentially, modelling of engineered monoclonal antibodies should be first tested by assessing their specificity to cancer cells from a biopsy or by employing virtual assessment to check against the presence of structurally similar off-target binding sites in other regions of the patient.

3) Describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”):

1. Purpose: The use of AI-aided design for synthetic biology enhanced biopharmaceuticals able to counter the evolution of cancer cells has great promise to produce customized and personalized medicine for patients suffering from the most untreatable conditions. –> Actions: a framework regulating the use of AI in drug discovery shall be agreed upon internationally. Scientists shall abide by an ethical code and follow a “do-not-harm” policy.
2. Design: Patients subject to a synthetic biology-enhanced monoclonal antibody therapy shall fully understand the potential consequences of treatment. Multiple quality control checks must be implemented in the process of design and optimization of monoclonal antibodies or CAR-T variable antigen binding site, as well as for the integrity of the mRNA sequence inserted in potential mRNA vaccines against cancer. Manufacturing errors and contaminations must be excluded by applying strict quality control checks. –> ACTIONS: Companies/Researchers: meet strict quality control checks, abide to GMP practices when manufacturing, perform GLP-grade R&D. Medical Practitioners: Provide information on potential risks to patients, administer advanced therapies only under medical supervision in adequate premises. International regulators: a framework on the use of AI for drug design shall be laid down to prevent accidental or intentional damages to patient.
3. Assumptions: What could you have wrong (incorrect assumptions, uncertainties)? Synthetic biology-enhanced mABs or CAR-T cells might bind to areas not relevant to the intended therapeutic site, hence causing off target inflammatory responses, damage to the patient’s non-tumoral tissue and potentially, his death. Malicious agents or hallucinations by AI may potentially introduce unwanted mutations in the engineered therapeutic agent, hence causing it not to work as intended or, worse, cause harm to the patient. –> ACTIONS: World regulatory authorities shall regulate the sequences that AI is able to synthesize when creating synthetic biology-enhanced biotherapeutics. A system of automated screening shall be put in place to rapidly score and assess the potential dangers and unwanted side effects (i.e. binding to a different location than the one they are devised for causing damage to patient) of such AI designed biotherapeutics.
4. Risks of Failure & “Success”: How might this fail, including any unintended consequences of the “success” of your proposed actions? Despite the first round of treatment being successful, the ablation of certain cancerous cells in the tumor mass via synthetic biology enhanced mABs or CAR-T gives free rein to the surviving cells to proliferate. Unless another round of targeted mABs is found quickly, the patient may still die due to the metastasis of surviving cells. Issues: therapeutic fairness, costs, and ethical considerations on the worthiness of treatment (how would the patient live if he/she has to be subject to continuous therapy of multiple rounds of synthetic biology evolved mAbs targeting specific sites with no certainty of a 100% absence of relapse?). -–> ACTIONS Ethical considerations and cost/benefit analysis shall be used based on internationally recognized guidelines.

The regulatory taken in consideration are as follows: 1) Regulate AI use in R&D 2) Use strict QC and R&D protocols 3) Regulate Hospital Administration Of AI-Generated Drugs

Next, score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals. The following is one framework but feel free to make your own:

Last, drawing upon this scoring, describe which governance option, or combination of options, you would prioritize, and why. Outline any trade-offs you considered as well as assumptions and uncertainties.

I would prioritize the international regulation of AI in performing research, taking into consideration that AI-generated research and pharmaceuticals shall always be checked for safety to the patient, as well as subject to concurrent human evaluation. It is also key to make sure biomanufacturing facilities in labs and hospitals are able to guarantee high GMP quality in compliance with manufacturing regulations.
. Reflecting on what you learned and did in class this week, outline any ethical concerns that arose, especially any that were new to you. Then propose any governance actions you think might be appropriate to address those issues. This should be included on your class page for this week.

Synthetic biology has a great potential to advance research, however, the emergence of AI agents that can potentially emulate the research abilities of a scientist poses questions about the ability to correctly manage and vet the results of AI-generated synthetic sequences, both for hallucinations/unintended results and for intentionally dangerous sequences that might be introduced for malicious purposes.

It is important that a international authority like the WHO and regional authorities are able to produce a framework to be able to channel the use of AI in research for positive outcomes.

Homework Question from George Church Google: searched for list of 10 essential amino acids in all animals, checked on Wikipedia. Used ChatGPT-4o Mini to generate a list of the 10 non-essential and 10 essential amino acids in some of the most representative animal species. Prompts: “Essential Amino Acids in Different Animals”, “List the essential and non-essential aminoacids” Amino acids are organic compounds constituted by structures containing of amino and carboxylic acid functional groups. (1) Of the more than 500 chemically synthesizable aminoacid structures (2), 22 amino acids are named “proteinogenic amino acids”, as they constitute the fundamental building blocks of proteins in living organisms. The 22 proteinogenic amino acids (alpha-aminoacids) include: 20 standard aminoacids (3) used to produce protein in the translation process, with the addition of pyrrolyisine and selenocysteine, found in certain methanogenic microorganisms as part of the methyltransferase enzyme aiding the conversion of carbon dioxide into methane (4)
In the translation process, the 20 standard amino acids are encoded and lined on the protein primary structure based on the encoding correspondence relation with one or more three mRNA base pair sequences (the theoretical amount of single amino-acids encodable by each different mRNA triplet is 64 = 4 base permutations ^ length of triplet, however, the genetic code is degenerate as multiple base pair sequences can be translated to the same aminoacid). During the process of mRNA translation into proteins, the ribosome machinery, based on the genetic code translates triplets of mRNA into a strand of concatenated corresponding aminoacids (linked by peptide bonds), giving rise to the primary structure of proteins, which is a polypeptide chain. The primary structure of proteins gives rise, via hydrogen bonds interactions among different amino acid residue groups, to the proteins secondary structure (alpha helix and beta-sheet). Interactions between the polypeptide chain and the environment (as hydrophobic interactions), hydrogen bonds, ionic bonds, and disulfide bridges give rise to the tertiary structure of the protein. Tertiary structure is fundamental to defining the specificity of enzymes and their functionality (e.g., defining the shape of the enzymatic active site). Of the 20 aminoacids generally used for protein synthesis, 10 are termed “essential” as they cannot be produced endogenously and must be acquired by diet (Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine, Arginine), absence of such amino-acids in the diet can negatively affect vital functions as enzyme activity, hormone synthesis and muscle development. The other 10 aminoacids (Alanine, Asparagine, Aspartic Acid, Glutamic Acid, Serine, Cysteine, Tyrosine, Glycine, Proline, Glutamine) are termed non-essential as they can be both acquired via diet and biosynthesized endogenously. Interestingly, most common animal species (including humans, cows, chickens, pigs, fish, dogs, cats, sheep, horses, cat, sheep, rabbits) share the same list of essential amino acids, which cannot be produced endogenously. Being Lysine an essential aminoacid that must be acquired through diet, “Lysine Contingency” refers to the issue that, in the practice of industrial animal livestock rearing, the absence or reduced quantity of lysine in animal feed may adversely impact the yield of livestock and poultry. (5) This scientific issue has inspired (despite being twisted) the story in the “Jurassic Park” book, where the captively held dinosaurs had been genetically engineered to inhibit the endogenous production of lysine (which in reality does not get produced endogenously) by inhibiting a putative lysine-producing enzyme, and depend on the constant supply of lysine in their diet, preventing them from fleeing in absence of food supplements provided to them in the park (6)

Interestingly, this approach could provide a hint to a the possible engineering of meat-producing animals via synthetic biology approaches as to be able to endogenously synthesize lysine, which is the limiting aminoacid in grain-fed cattle, such an approach could usher in higher livestock productivity.

References: (1) Lehninger, A. L. (1975). Biochemistry: The Molecular Basis of Cell Structure and Function (2nd ed.). New York: Worth Publishers. (2) Ye, C.-X., Dansby, D. R., Chen, S., & Meggers, E. (2023). Expedited synthesis of α-amino acids by single-step enantioselective α-amination of carboxylic acids. Nature Synthesis, 2, 645–652. https://doi.org/10.1038/s44160-023-00267-w (3) Stoye, E. (2019). Why does all life use the same 20 amino acids? Chemistry World. Retrieved from Chemistry World (4) Rother M, Krzycki JA (August 2010). “Selenocysteine, pyrrolysine, and the unique energy metabolism of methanogenic archaea”. Archaea. 2010 (5) Ronald O. Ball, Kristine L. Urschel, Paul B. Pencharz, “Nutritional Consequences of Interspecies Differences in Arginine and Lysine Metabolism123, The Journal of Nutrition, Volume 137, Issue 6, 2007”, Pages 1626S-1641S, ISSN 0022-3166

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project