Introduction
Priority of Ethical Principles
Ethical principles hold paramount importance in every project, and their prioritization may vary among iGEM teams on their distinct objectives and aspirations. These moral tenets present value to each scientific endeavor and serve as an indicator of its trustworthiness and responsibility to the global community. Hence, our foremost emphasis revolves around safeguarding the well-being and security of all individuals participating in the production processes of our therapy, as well as ensuring the safe administration of our product to patients. Below, we describe the methodologies employed to guarantee the safety of our experimental procedures within the laboratory and elaborate upon the necessary criteria meticulously incorporated into our proposed therapy to augment the overall safety profile of our product.
Ensuring safety during product manufacturing in the laboratory
Our safety initiatives
Our team upholds the principles and rigorous standards of iGEM, with a strong commitment to biosafety and security. Our project strictly adheres to the safety regulations and guidelines presented by the iGEM competition, our university, and all applicable governing bodies.
The experiments of our team were conducted at the Institute of Cell Therapies, located within the Research Center of the University of Patras. We have successfully completed specialized training in laboratory procedures within a Biosafety Level 2 (BSL-2) environment, encompassing the practice of safety protocols while handling cell cultures, both in the Institute's laboratory and the Developmental Biology Laboratory of the Department of Biology at the University of Patras. We performed our experiments in accordance with established protocols that have been previously validated, and we ensured that our supervisors were informed and consulted us before proceeding. During the project, our team utilized the following biological material: K562 cell line, HEK 293T cell line, NK-92 cell line, AsPC-1 cell line, iPSCs-derived NK cells, and NK cells from peripheral blood, which we exclusively managed within specialized workspaces equipped with the necessary safety gear, such as biosafety cabinets and chemical ventilation hoods.
Our laboratory’s biosafety level
Determining a laboratory's biosafety level represents a fundamental principle of Biosafety. Stringent and level-specific protocols must be diligently observed when dealing with high-concentration infectious substances within laboratory settings.
Our research endeavors were conducted at the Institute of Cell Therapies, situated within the esteemed Research Center of the University of Patras. Notably, this Institute accommodates a facility compliant with Good Manufacturing Practice (GMP) standards. This facility is dedicated to processing grafts and producing cellular therapies, ensuring adherence to international standards and guidelines. These efforts are integral to the more extensive transplantation program managed by the Bone Marrow Transplantation Unit (BMTU). The laboratory unit comprises specialized Cell Processing Rooms (B and C), alongside a dedicated Cryopreservation Room (F). These spaces are seamlessly interconnected through the utilization of pass boxes, facilitating the safe and controlled movement of materials and samples."
Click here to learn more about the Institute of Cell Therapies, in which we conducted our experiments.
Click here to learn more about the three-dimensional representation of the interior of the laboratory.
Our laboratory had the Biosafety Level 2 (BSL-2): Biosafety Level 2 is appropriate for clinical, diagnostic, teaching, and other laboratories that handle moderate-risk agents that cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Examples are the Hepatitis B virus, HIV, Salmonella, and Toxoplasma. These microorganisms may be used on the open bench as long as there is low production of aerosols. Biosafety Level 2 laboratory includes Standard Microbiological Practices: GMT plus protective clothing (PPE), Biohazards sign, Special Practices: Separate bin for general waste, biohazards and sharps, Safety Equipment: Open bench and Biosafety Cabinet for potential aerosols and Laboratories Facilities: Primary health services, diagnostic services and research.[1]
ATMPs (Advanced Therapy Medicinal Products)
At this point, it is pertinent to underscore the legislative framework about Biopharmaceutical Safety, which governs the realm of Advanced Therapy Medicinal Products (ATMPs), the category that our proposed cellular immunotherapy falls within. We are undertaking this analysis within our project, recognizing that a comprehensive description of the regulatory guidelines is essential to the product's implementation, including products’ manufacturing and administration to the patient. In addition, we seek to expound upon the considerations of safety and efficacy, that should be addressed in the risk management plan, which will be established as a facet of the marketing authorization process.
The product we propose, which involves CAR-NK cells derived from iPSCs originating from somatic cells of a healthy donor, falls within the classification defined by the European Medicines Agency (EMA) as Advanced Therapy Medicinal Products (ATMPs). This category encompasses a spectrum of therapeutic innovations, including gene therapies, somatic cell therapies, and tissue engineering products. These introduce new possibilities for storage, modification, and enhancement of physiological functions. However, their innovative nature, inherent complexity, and technical specificity can introduce new considerations about patient safety.[2]
Safety concerns for Αdvanced Τherapy Μedicinal Products (ATMPs)
Outlined below are the risks associated with the entire process, from the manufacturing of an ATMP to its administration, according to the Guideline on safety and efficacy follow-up and risk management of Advanced Therapy Medicinal Products'established by the European Medicines Agency (EMA).
The patient-related risks associated with product’s quality, storage, and distribution, are presented here.
- Risk of transmission of diseases which depends on:
1. Origin of cells or tissues (Autologous vs. Allogeneic)
2. Characteristics of the cell type used (The characteristics of the cell type used can influence the disease transmission risk).
3. Ability of cells to proliferate and differentiate (some cells like embryonic stem cells and induced pluripotent stem cells have a high proliferative and differentiation capacity which can lead to a high risk of mutations and genetic alterations.
- Risk of Tumorigenicity
1. Cells that have a high proliferative potential increase the risk of tumorigenicity. Extended culture for proliferating cells, like mesenchymal stem cells, can impact their differentiation capacity and potentially lead to tumorigenicity. This risk depends on gene editing techniques that are used, as they can introduce "off target" mutations or unintended "on target" mutations.
- Risk related to transport, storage and distribution of ATMPs
1. Preservation and Freezing/Thawing.
2. Cold Chain management
3. Product Stability
Risks related to patient associated conditions/disease or underlying disease, or concomitant treatment /interactions with other medicinal products, are displayed here.
- Risks of unwanted Immunogenicity and its consequences(anaphylaxis, graft-versus-host disease (GVHD), graft rejection, cytokine release syndrome, inflammation).
- Risks related to a patient's condition/preparation (In certain cases, patients need to be prepared for ATMP treatment in order for the effectiveness of the therapy to be increased).
- Consequences of homing, grafting, differentiation, migration, and proliferation (the administered cells can lead to inflammation or tissue damage to the patient).
- Risks related to infection with vector in gene therapy medicinal products (potential for latency and reactivation of the vector, potential integration of genetic material into the host’s genome, altered expression of the host’s genes)
There are also risks to patients which are related to reconstitution procedures, showed below.
Reconstitution procedures in healthcare typically involve mixing a medication or substance with a liquid (usually sterile water or saline) to prepare it for administration to a patient. Possible risks related to reconstitution procedures include contamination, dosage errors, chemical incompatibility and allergic reactions.
Risks to patients which are related to administration procedures and re-administration, are described here.
Refer to the potential adverse outcomes or complications that can occur during the process of giving a treatment or medication to a patient. These risks include medication errors (wrong dose, wrong route), allergic reactions, pain, resistance and psychological effects.
Risks related to persistence of the product in the patient
The persistence of a product, such as a medication in a patient’s body can lead to various risks and potential complications. These risks can be influenced from many factors like the product’s half-life, the route of administration, the patient’s characteristics and the specific nature of the product. Some risks related to the persistence of a product in a patient are the toxicity, the prolonged side-effects, the tolerance and the reduced efficacy, the carcinogenic effects and the mental health conditions.
Risks to healthcare professionals, care givers, offspring and other close contacts and its risks to the environment, are outlined below.
Risks for Healthcare professionals and caregivers (infections, physical injuries, mental health), risks for close contacts (psychological impact), risks to the environment (medical waste, chemicals exposures) [2] conditions.
A more detailed description of all safety aspects of our therapeutic project can be found in the Safety form by selecting our team’s name, which is Patras-Med.
All safety aspects of the possible implementation of our project are outlined on the implementation page
Other Initiatives
In the following you may find other initiatives of ours, concerning Biosafety and Biosecurity.
Safety Guide
With a paramount emphasis on safety regulations, we proceeded on the development of a Safety Guide, which forms an integral part of our collaborative efforts with the iGEM Toulouse team. This guide provides a description of the risks associated with the utilization of cancer cell lines within laboratory settings. Furthermore, it comprehensively presents the biosafety protocols aimed at safeguarding the well-being of all individuals involved. Developing our project, it is imperative to underscore the meticulous handling of pancreatic cancer cell lines. The significance of this cautiousness arises from the potential adverse repercussions associated with any mishandling of these cell lines. Take a look here for the Safety Guide.
Post World Day for Safety and Health at Work on Social Media - April 28
On the 28th of April, our team disseminated a post via social media platforms to raise awareness about laboratory safety regulations. The emphasis of this initiative was to underscore the critical significance of biosafety protocols in the context of experimental procedures. Paramount among our concerns is the safeguarding and well-being of all individuals participating in laboratory experiments. In particular, we provided a detailed presentation of the biosafety measures encompassing pre-entry, in-laboratory, and post-experiment procedures.
References
[1]. 9010018_Guide to Biosafety and Biological Safety Cabinets_A4_vD_041321.pdf (escolifesciences.com)