aimofcontact:[<p>When we realized that the creation of a nickase from the endonucleases in use was a desired outcome, it became necessary to talk to an expert in the field of enzyme engineering. Our first contact was Kai Schülke, a former iGEMer and PhD student under the guidance of Prof. Dr. Hammer[Link Hammer], who is the leader of the working group organic chemistry and bioanalytics at Bielefeld University.</p>],
aimofcontact:[<p>When we realized that the creation of a nickase from the endonucleases in use was a desired outcome, it became necessary to talk to an expert in the field of enzyme engineering. Our first contact was Kai Schülke, a former iGEMer and PhD student under the guidance of <HPLinktoOtherHPTabtab="hammer"text="Prof. Dr. Hammer"/>, who is the leader of the working group organic chemistry and bioanalytics at Bielefeld University.</p>],
insights:[<p>In the process of our interaction with Kai, we learned about the various methods employed in enzyme engineering. He demonstrated the complexity of this field of research and emphasized the importance of choosing the right approach. As a former iGEMer, Kai, inspired by his past experiences, is highly motivated and determined to develop an outstanding project. He pointed out that we cannot rely on classical methods such as directed evolution, but instead should use a rational approach to select mutation candidates. His insights and enthusiasm have encouraged us to think critically and pursue innovative solutions in our work. </p>],
implementation:[<p>We incorporated Kai's insights into our project by shifting our approach to enzyme engineering. By focusing on a more targeted approach, we were able to refine our enzyme optimization process, ensuring that the modifications we made were based on informed, calculated decisions. This not only streamlined our research but also improved the chances of success by reducing the trial-and-error inherent in traditional methods. </p>],
summary:"The team reached out to Kai Schülke, a former iGEM participant and enzyme engineering expert, for guidance on developing a nickase from the endonucleases in use. Kai emphasized the need for a rational, targeted approach rather than traditional methods like directed evolution. His insights helped the team refine their enzyme optimization process, making it more strategic and efficient. This shift reduced trial-and-error efforts and improved the chances of success, driving innovation in their project.",
aimofcontact:[<p>During our interview with Makoto Saito[Linkinterview] about fanzor[link fanzor], it became evident that the expression of our fanzor nickases in yeast is very promising. We then refined our expression strategy for the nickases and approached Nils Berelsmann, who is currently working on his PhD thesis with the yeast strain Pichia pastoris (SMD1163). This particular strain could be ideal for expressing the SpuFz1 nickase variants. Our main aim in contacting Nils was to gain insight and advice on yeast expression and he generously shared his expertise with us. Not only did he give us valuable advice, but he also provided us with the yeast strain itself, along with a corresponding expression vector for possible experiments. He also provided us with detailed protocols and the plasmid map of the vector and gave us practical tips on how to optimize the expression process. His support was invaluable in moving our work forward. </p>],
aimofcontact:[<p>During our interview with <HPLinktoOtherHPTabtab="saito"text="Makoto Saito"/> about fanzor[link fanzor], it became evident that the expression of our fanzor nickases in yeast is very promising. We then refined our expression strategy for the nickases and approached Nils Berelsmann, who is currently working on his PhD thesis with the yeast strain Pichia pastoris (SMD1163). This particular strain could be ideal for expressing the SpuFz1 nickase variants. Our main aim in contacting Nils was to gain insight and advice on yeast expression and he generously shared his expertise with us. Not only did he give us valuable advice, but he also provided us with the yeast strain itself, along with a corresponding expression vector for possible experiments. He also provided us with detailed protocols and the plasmid map of the vector and gave us practical tips on how to optimize the expression process. His support was invaluable in moving our work forward. </p>],
insights:[<p>Pichia pastoris (SMD1163) is a promising option for expressing SpuFz1 nickase variants. Refining expression strategies based on expert insights is crucil for success. Nils provided practical tips on yeast expression, including optimizing growth conditions and fine-tuning induction protocols.</p>],
implementation:[<p>We adapted our expression strategy for Fanzor nickases in yeast by incorporating the Pichia pastoris strain (SMD1163) and the provided expression vector into our experiments. Following Nils' detailed protocols and plasmid map, we optimized key steps, enhancing expression efficiency and protein yield.</p>],
summary:"The team sought expert advice from Nils to optimize yeast expression for Fanzor nickases. Nils provided invaluable guidance on addressing potential challenges and troubleshooting the process. He supplied the Pichia pastoris (SMD1163) strain along with a suitable expression vector, crucial for expressing SpuFz1 nickase variants. Additionally, he shared detailed protocols for yeast transformation and growth optimization, enabling the team to replicate his methods effectively for their experiments.",
quote:"Minimum requirement: Acute toxicity study on animals - important for calculating the starting dose for humans. In addition, pharmacological studies, genotoxic studies and immunotoxic studies. In your case also chronic toxicity studies.",
aimofcontact:[<p>The aim of our contact with GxP expert Dr. Marcus Berger [Link https://gxpexpert.de/] was to gather insights and ask questions about how to proceed with the Precyse project, clarifying the next steps required for its continued development, particularly concerning regulatory strategy, quality management, and clinical development. GxP, which stands for ‘Good Practice’ guidelines, encompasses various regulations and standards intended to ensure quality and compliance in the development and manufacturing of pharmaceutical and biopharmaceutical products. </p>],
aimofcontact:[<p>The aim of our contact with GxP expert <ahref="https://gxpexpert.de/">Dr. Marcus Berger</a> was to gather insights and ask questions about how to proceed with the Precyse project, clarifying the next steps required for its continued development, particularly concerning regulatory strategy, quality management, and clinical development. GxP, which stands for ‘Good Practice’ guidelines, encompasses various regulations and standards intended to ensure quality and compliance in the development and manufacturing of pharmaceutical and biopharmaceutical products. </p>],
insights:[<p>From the discussion with Dr. Berger expert, we gained several key insights into how to proceed with the Precyse project. First, it became clear that obtaining a Scientific Advice from the Paul-Ehrlich-Institut (PEI) [Link https://www.pei.de/DE/home/home-node.html] is essential to get qualified feedback on our development project and align our regulatory strategy. The expert emphasized the need to collect and document preclinical data, including acute and chronic toxicity studies, genotoxicity, immunotoxicity, and carcinogenicity studies, as well as conducting safety pharmacology and biodistribution studies to ensure a thorough understanding of the substance's mechanism of action.
The expert also provided guidance on how to establish a GxP-compliant quality management system (QMS), stressing the importance of setting up a robust SOP system for manufacturing, preclinical, and clinical development. This system must adhere to ICH Q10 guidelines, ensuring that the quality control processes are in line with regulatory expectations. Furthermore, we learned about the critical initial steps for validating and verifying the manufacturing process, including defining the physical and chemical properties of the active substance and excipients, developing the pharmaceutical formulation, and identifying critical quality attributes (CQAs) and critical material attributes (CMAs).
The ICH-GCP guidelines, which apply to all clinical trials, are essential for ensuring the integrity and safety of clinical development. These guidelines include E2A-E2F, which cover topics like clinical safety data management and adverse event reporting, E3 for clinical study reports, E5 for handling ethnic factors, and E9 for statistical principles. Additionally, E14 is crucial for managing cardiovascular risks, an especially important consideration for gene therapies, where monitoring for heart-related side effects is critical. Guidelines like E15 and E16 address biomarkers and pharmacogenomics, helping tailor clinical development to the specifics of the treatment being tested. Implementing these GCP requirements ensures compliance, patient safety, and data integrity throughout the trial process.
