addition, our research steered us in a different direction, particularly with regard to the alternative mechanism involving the XBP1 intron to regulate the prime editing system. This alternative seemed more promising and was based on an established regulatory mechanism that is triggered by cellular stress and specifically responds to misfolding processes.
</p>
<H4text="XBP1 Intron"></H4>
<p>After extensive research, we discovered a regulatory system in eukaryotic cells, the XBP1 mechanism. The activation of XBP1 is an important mechanism that occurs as part of the Unfolded Protein Response (UPR), a cellular stress response triggered by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER is a key cellular component responsible for protein folding and transport. When many misfolded proteins accumulate in the ER, a specific regulatory mechanism is activated to reduce the stress on the ER. XBP1 activation is controlled by a protein called IRE1α, which is embedded in the ER membrane. IRE1α acts as a sensor for protein misfolding stress in the ER. Once IRE1α detects misfolded proteins, it dimerizes and becomes activated through autophosphorylation. This activation switches on the endoribonuclease activity of IRE1α, which is a crucial step in the activation of XBP1. The mRNA for XBP1 is continuously transcribed in the nucleus and transported to the cytoplasm, where it contains an intron that is not normally spliced out. This intron contains a stop codon, preventing the translation of a functional XBP1 protein. However, when ER stress activates IRE1α, the endoribonuclease domain of IRE1α splices this intron out of the XBP1 mRNA. This is an unconventional splicing event, as it occurs in the cytoplasm rather than in the nucleus. Once the intron is removed, the spliced XBP1 mRNA can be translated into a functional XBP1 protein. This activated XBP1 acts as a transcription factor, turning on genes that increase the protein-folding capacity of the ER and promote the degradation of misfolded proteins. In this way, XBP1 helps the cell cope with ER stress and restore balance in the protein-folding process. Thus, this mechanism originally functions within the cell in the context of ER stress to maintain ER function when protein folding is disrupted. [5] [6] Our idea was therefore to integrate this intron into the mRNA encoding our prime-editing complex and thus use this mechanism to ensure that a functional prime editor is only synthesized when there is a high accumulation of misfolded proteins in the cell (similar to F508del). This would therefore represent an optimal safety aspect, as our fusion protein, which is essential for prime editing, cannot be fully synthesised as long as the genetic defect is not present in the cell. Accordingly, this provides the security that no healthy cells, as well as correctly edited cells, cannot be edited, which is an enormous contribution to biosafety. However, there was too much uncertainty about the extent to which other factors, such as misfolded proteins that are not associated with the CFTR protein, play a role in this mechanism. And since we could not and did not want to take the risk of such factors initiating the system, we decided against using it. To clarify this unknown correlation, we have considered a future experiment in which we want to switch this intron in front of a fluorescent marker and express it in cells with defective CFTR in order to confirm/investigate the dependence of intron splicing and the presence of CFTR F508del.</p>
<p>
After extensive research, we discovered a regulatory system in eukaryotic cells, the XBP1 mechanism. The activation of XBP1 is an important mechanism that occurs as part of the Unfolded Protein Response (UPR), a cellular stress response triggered by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER is a key cellular component responsible for protein folding and transport. When many misfolded proteins accumulate in the ER, a specific regulatory mechanism is activated to reduce the stress on the ER. XBP1 activation is controlled by a protein called IRE1α, which is embedded in the ER membrane. IRE1α acts as a sensor for protein misfolding stress in the ER. Once IRE1α detects misfolded proteins, it dimerizes and becomes activated through autophosphorylation. This activation switches on the endoribonuclease activity of IRE1α, which is a crucial step in the activation of XBP1. The mRNA for XBP1 is continuously transcribed in the nucleus and transported to the cytoplasm, where it contains an intron that is not normally spliced out. This intron contains a stop codon, preventing the translation of a functional XBP1 protein. However, when ER stress activates IRE1α, the endoribonuclease domain of IRE1α splices this intron out of the XBP1 mRNA. This is an unconventional splicing event, as it occurs in the cytoplasm rather than in the nucleus. Once the intron is removed, the spliced XBP1 mRNA can be translated into a functional XBP1 protein. This activated XBP1 acts as a transcription factor, turning on genes that increase the protein-folding capacity of the ER and promote the degradation of misfolded proteins. In this way, XBP1 helps the cell cope with ER stress and restore balance in the protein-folding process. Thus, this mechanism originally functions within the cell in the context of ER stress to maintain ER function when protein folding is disrupted. [5] [6] Our idea was therefore to integrate this intron into the mRNA encoding our prime-editing complex and thus use this mechanism to ensure that a functional prime editor is only synthesized when there is a high accumulation of misfolded proteins in the cell (similar to F508del). This would therefore represent an optimal safety aspect, as our fusion protein, which is essential for prime editing, cannot be fully synthesised as long as the genetic defect is not present in the cell. Accordingly, this provides the security that no healthy cells, as well as correctly edited cells, cannot be edited, which is an enormous contribution to biosafety. However, there was too much uncertainty about the extent to which other factors, such as misfolded proteins that are not associated with the CFTR protein, play a role in this mechanism. And since we could not and did not want to take the risk of such factors initiating the system, we decided against using it. To clarify this unknown correlation, we have considered a future experiment in which we want to switch this intron in front of a fluorescent marker and express it in cells with defective CFTR in order to confirm/investigate the dependence of intron splicing and the presence of CFTR F508del.
</p>
</Subesction>
<Subesctiontitle="Safety aspects of our Airbuddy"id="Biosafety2">
Our project focuses on the genetic disease cystic fibrosis, specifically targeting the Delta-508 mutation. The aim is to correct this mutation using Prime Editing, a precise genome-editing technique. We have explored different strategies to optimize the Prime Editing complex for this specific application.
</p>
<p>
The Prime Editing complex consists of a nickase, a reverse transcriptase, a pegRNA.. The pegRNA guides the editing process by directing the complex to the target DNA sequence, allowing for precise genetic modifications. For targeted delivery, we selected lipid nanoparticles (LNPs) to introduce the mRNA encoding the Prime Editing components specifically into lung epithelial cells, where the CFTR protein is highly expressed. Additionally, we investigated alternatives to the conventional Cas9 nickase, such as the smaller CasX and Fanzor, aiming to reduce the overall size of the Prime Editing complex. In our optimization efforts, we also explored smaller reverse transcriptases to enhance the efficiency of the system in human cells.
</p>
<p>
Furthermore, we have developed a modular plasmid that contains the backbone of our Prime Editing complex. The individual components can be cloned individually into the backbone. This plasmid allows us to either deliver the construct directly into target cells or transcribe the plasmid into RNA, enabling the delivery of the Prime Editing complex in the form of mRNA. The modularity of the plasmid is a key feature; specific restriction sites are included to facilitate the easy exchange of the complex's components. This design makes it straightforward to adapt the Prime Editing complex for various use cases and therapeutic requirements.
</p>
<p>
We have investigated safety mechanisms to control the prime-editing complex, including a riboswitch that responds to sodium ion concentrations, but have discarded it due to suspected insufficient sensitivity. We are currently investigating the use of the ER stress response to activate the prime editing complex only in cells with high ER stress, as is typical for cystic fibrosis. Further details can be found in the Biosafety section.
Given the sensitive nature of genome editing, our project presents specific biosecurity concerns that need to be assessed and mitigated.
</p>
<p>
<strong>Dual-Use Potential:</strong> One of the main biosecurity risks is the potential for dual-use of the Prime Editing technology. The system we are developing, while intended for therapeutic use, could be misused to target other genes or genomes for malicious purposes.1 This includes the possibility of weaponizing the technology to induce harmful genetic changes in crops, animals, or even humans. The modular design of our plasmid system, although intended to facilitate optimization, could be exploited to exchange components for harmful applications, thereby increasing the risk of misuse.
</p>
<p>
<strong>Unintendend Dissemination:</strong> Since our approach uses mRNA delivered via LNPs, there is a risk of unintended dissemination into the environment. If the LNPs are not adequately contained or disposed of, there is a possibility that they could be absorbed by non-target organisms, potentially leading to off-target genetic modifications.2 In addition, the mRNA itself could theoretically be transferred between cells, especially if taken up by unintended hosts, raising concerns about unintentional spread in the environment.
</p>
<p>
<strong>Unauthorized Access:</strong> The genetic constructs and the detailed methodology of our Prime Editing system must be securely stored and protected.3 If unauthorized individuals were to gain access to the plasmids, LNP formulations, or editing protocols, there is a risk of the technology being replicated or adapted for unintended, potentially harmful uses. This highlights the importance of proper biosecurity protocols in both physical and digital storage of our project materials.
</p>
<p>
<strong>Synthetic Biology and information Sharing:</strong> The ease of synthesizing genetic material means that our project information could potentially be used to order similar constructs from commercial synthesis providers.4 While these providers follow biosecurity guidelines, the increasing accessibility of synthetic biology raises the concern of our Prime Editing system being reproduced or modified without our knowledge. This includes potential attempts to bypass safety mechanisms or create variants that evade current regulatory frameworks.
</p>
<p>
<strong>Public Perception and Miscommunication:</strong> There is a biosecurity risk in how our project's technology is communicated to the public.5 Miscommunication or misunderstanding of the project’s intent and capabilities could lead to misinformation, fear, or even attempts to replicate the technology outside of controlled and regulated environments. This could undermine public trust in legitimate therapeutic uses of genome-editing technologies and potentially facilitate misuse.
To mitigate the risk of dual-use, where our Prime Editing technology could be exploited for unintended and potentially harmful purposes, we have implemented several strategies.
</p>
<p>
Firstly, we strictly control access to all our project data, including genetic sequences, plasmid designs, and protocols. Access is limited to authorized team members and collaborators who follow strict biosecurity guidelines. Further elaboration on these access controls will be provided in the "Unauthorized Access" section.
</p>
<p>
Secondly, we intend to incorporate a safety mechanism into our Prime Editing complex that significantly limits its potential misuse. By utilizing the ER stress response pathway, we would design our therapeutic mRNA to undergo unconventional splicing only in cells experiencing high levels of protein stress. Only this unconventional splicing would convert the mRNA into a form that can be translated into the final PE complex. This mechanism ensures that the Prime Editing complex becomes active primarily in cells under such stress conditions. While this does not exclusively limit the complex to cystic fibrosis-affected cells, it considerably narrows the range of cells where activation can occur, thus preventing arbitrary application of the editing system and reducing the risk of targeting unintended cells.
</p>
<p>
Together, these measures provide a layer of protection against dual-use risks, making it more difficult for the technology to be employed outside of its intended therapeutic context.
</p>
<p>
<strong>Unitended Dissemination</strong>
</p>
<p>
To minimize the risk of our construct being inadvertently released into the environment, we adhere to strict standard operating procedures (SOPs) in the laboratory. These SOPs are designed to ensure that all safety standards are maintained, particularly when handling LNPs) and genetic materials. This includes rigorous protocols for storage, disposal, and decontamination, reducing the chance of accidental dissemination of the materials we work with.
</p>
<p>
In addition to our laboratory practices, we have formulated our LNPs with specific chemical components that enable them to selectively fuse with lung epithelial cells, such as ionocytes and club cells, which express CFTR. This selective fusion ensures that even if the LNPs were to accidentally enter the environment, they would only interact with the intended cells. This approach significantly enhances the specificity of the LNPs and provides an additional layer of containment, thereby reducing the risk of unintended dissemination or environmental contamination.
</p>
<p>
Looking ahead, we plan to further enhance the specificity of our LNPs by incorporating antibodies on their surface. These antibodies would be engineered to bind exclusively to surface proteins present on CFTR-expressing lung epithelial cells, adding an extra layer of control. While this antibody-based targeting has not yet been implemented, it represents a crucial next step in our development process, reinforcing our commitment to biosecurity by limiting the potential for unintended dissemination.
</p>
<p>
Together, these practices and design strategies help to ensure that the risk of our constructs inadvertently affecting non-target organisms or environments is minimized.
</p>
<p>
<strong>Unauthorized Access</strong>
</p>
<p>
To prevent unauthorized access to our genetic sequences, plasmid constructs, and other sensitive information, we have implemented strict internal biosecurity policies. These policies ensure that we carefully manage which materials exist in physical form and their exact storage locations.
</p>
<p>
In terms of digital security, we strictly regulate access to our data storage systems through a combination of measures. We use a secure, password-protected cloud system, ensuring only authorized team members can access sensitive project information. Access to the lab is limited to team members with internal keys, and our server security includes stringent access rights management, allowing only approved individuals to view, modify, or share the data. These safeguards ensure that project information remains protected against unauthorized access.
</p>
<p>
Furthermore, we ensure that any sensitive data generated during our project is only published in the supplementary materials section, which is accessible exclusively to those with an official iGEM account. By taking this approach, we can limit the availability of sensitive information and reduce the risk of it being misused.
</p>
<p>
<strong>Synthetic Biology and Information Sharing</strong>
</p>
<p>
Managing the risks associated with synthetic biology and information sharing is a complex challenge. Many of the strategies we use to address other risks, such as "Unauthorized Access" and "Dual-Use Potential," are equally relevant in this context. For instance, our strict control over access to sensitive information and our safety mechanisms both help mitigate the risks posed by the accessibility of synthetic biology tools.
</p>
<p>
However, it is important to acknowledge that this risk is inherent to the field of synthetic biology. The ease of obtaining information and, with the right expertise, combining various components to create powerful and potentially hazardous tools is a fundamental concern.
</p>
<p>
To address this, we adhere strictly to all policies and regulations set forth by iGEM, our university, the German government, and the European Union. By aligning our practices with existing regulations on synthetic biology, we aim to conduct our work responsibly and reduce the risks associated with the field.
</p>
<p>
<strong>Public Perception and Miscommunication</strong>
</p>
<p>
One of the challenges in scientific research, especially in fields like synthetic biology, is that advancements can often outpace public understanding and discourse. This can lead to confusion, fear, or mistrust if the research is not communicated effectively. To address this issue, we believe that scientific progress should occur in constant dialogue with the public.
</p>
<p>
We have adopted a strong Human Practices approach to ensure transparency and foster public engagement. Our efforts include initiatives like "MUKOmoove" and "Teuto ruft," where we have worked directly with students, educational institutions, and public organizations. Through these initiatives, we aim to explain our project, discuss its implications, and answer any questions, thus maintaining an open line of communication. By collaborating with a variety of public entities, including patient associations, educational programs, and community groups, we ensure that our research remains accessible and understandable to a broader audience.
</p>
<p>
This proactive approach helps us address potential concerns, demystify our research, and contribute to a more informed public perception of synthetic biology.
