Human Practices
Influenced by the Human Practices Cycle, we began by forming an interdisciplinary and capable team that could meet the demands and timelines of the competition. We started by researching everyday problems to which we could offer a solution through our research. We ultimately focused on a complex and challenging issue: the treatment of Alzheimer's Disease.
The Human Practices Team, in order to ensure that our project is responsible and beneficial to society, engaged with a wide network of people as part of the Integrated Human Practices. Each stage of our research has received positive feedback from expert scientists and researchers, as well as from the general public and all stakeholders, whose opinions have been key components of our project. We succeeded in making our research centered on the stakeholders and their needs, which we have aimed to address.
Additionally, as the Human Practices Team, we wanted to directly address the needs and challenges of patients and their families. To achieve this, we participated in and organized various activities. Our goal was not only to raise awareness and gain real-time insight into the problems these individuals face so that we could focus our research on the right areas, but also to offer our support and assistance.
For our iGEM team's Human Practices section, we focused on creating impactful activities designed to engage the public and spread awareness about Alzheimer's disease, while promoting inclusivity and education on synthetic biology. Our efforts were aimed not only at raising awareness, but also at integrating therapeutic interventions and fostering a supportive environment for patients and their families. Below is a summary of the key initiatives we implemented:
Art Exhibition: Bridging Generations through Creativity
We organized an art exhibition featuring paintings created by individuals with Alzheimer's alongside artwork from children. This initiative aimed to compare their artistic expressions while raising public awareness of the fact that people with Alzheimer's can still produce meaningful art. By displaying these works, patients experienced a sense of inclusion and belonging, as they saw their creations valued by society. This initiative highlighted the emotional depth of their artwork and underscored that their contributions still matter. The exhibition also included talks on Synthetic Biology, Alzheimer's research, active aging, and associated risk factors, spreading awareness about Alzheimer’s Disease and the use of Synthetic Biology.
Children's Storybook: Understanding Dementia through a Child's Eyes
A poignant part of our outreach was a storybook written by a child whose grandmother died from Alzheimer’s Disease. The book reflects the child's emotional experience and perspective on living with a loved one who suffers from Alzheimer's, teaching both youngsters and adults how they should be patient, loving, and understanding. Through this story, we aimed to showcase how young individuals perceive the disease and cope with its challenges, making it relatable for both children and adults and illustrating what they can do differently as far as their behavior is concerned.
Guides for Care and Communication
To support caregivers and family members, we created a Guide to Managing and Communicating with Alzheimer's Patients, helping them better understand and care for their loved ones. Additionally, we provided a Synthetic Biology Concepts Guide to explain the fundamentals of synthetic biology in simple terms, making it accessible to the general public and showcasing how this innovative field contributes to Alzheimer's research.
Interactive Games for Children: Learning through Play
We developed a series of games designed to teach children about Synthetic Biology and Alzheimer's disease in a fun and engaging way. These activities included card games, painting biological components and Alzheimer’s-related drawings, pantomime, and crosswords that not only entertained but also educated young participants about important concepts in science and health.
School Visits: Raising Awareness in All Educational Levels
Our team visited schools of all levels, from kindergartens to high schools, spreading awareness about Alzheimer's and synthetic biology. Through interactive workshops, games, and hands-on activities, we engaged students and teachers in conversations about the disease, its effects, and the potential of Synthetic Biology to offer solutions.
Inclusive Activities at a Special Needs School
In an effort to promote inclusivity, we visited a special education school and conducted activities for children with cognitive disabilities. Using interactive games, we fostered an environment of creative expression and learning, ensuring that these children felt included and engaged.
Simulating Life with Disabilities for Empathy and Inclusion
To deepen public understanding of the challenges faced by individuals with disabilities, we organized a unique simulation where children experienced what it was like to be blind. In this context, we also presented Synthetic Biology and its potential applications for these communities. This simulation helped foster empathy and inclusion, encouraging participants to better understand the daily struggles of people with disabilities.
Intergenerational Board Game Sessions
We initiated a program where children and elderly patients with Alzheimer's played board games together, promoting intergenerational bonding. This activity aimed to strengthen social connections across generations and enhance cognitive stimulation for patients.
Patient Engagement Application
To further support Alzheimer's patients, we developed a mobile application designed to assist with daily activities. The app features brain-stimulating games, medication reminders, physical exercise prompts, and personalized music playlists to soothe patients emotionally. It also includes profiles for both the patient and their family members, fostering intergenerational connection and care by keeping all relevant information and activities in one accessible place.
Environmental Clean-Up Event: Connecting Generations through Social Action
In collaboration with a local seniors' group (KAPI) in Patras and a local group that does environmental projects, Project Kapa, we organized a community park clean-up event where elderly citizens and students worked together to improve the environment. This activity not only encouraged physical exercise—known to benefit both physical and mental health—but also fostered social interaction, giving elderly participants a sense of purpose and inclusion. The beautified park now serves as a welcoming space where elderly individuals, including Alzheimer's patients, can enjoy nature, socialize, and take calming walks.
These activities formed a comprehensive approach to raising awareness about Alzheimer's Disease, promoting public understanding of Synthetic Biology, and fostering intergenerational relationships. We aimed to create a lasting impact by engaging with different demographics and ensuring that patients and their families felt included, supported, and empowered.
Creating an Accessible Lab Environment for Scientists with Disabilities
In the lab, we took bold and innovative steps to ensure that people with disabilities can actively participate in scientific research, fostering inclusivity and accessibility for all. For individuals who are visually impaired, we have developed a comprehensive navigation kit designed to make the laboratory experience seamless and engaging. This kit includes Braille labels for all essential lab equipment, tactile maps of the lab layout to facilitate movement, and detailed instructions in Braille, ensuring that visually impaired scientists can confidently carry out experiments.
Moreover, we have proposed crucial changes to the interior design of the lab, aiming to create a fully immersive and accessible research environment. These changes focus on improving the overall spatial arrangement and ensuring clear, navigable pathways for safe and efficient movement within the lab. By prioritizing universal design principles, we are ensuring that the scientific community becomes more inclusive, allowing individuals with disabilities to thrive and contribute meaningfully to groundbreaking research.
Integrated Human Practices
The following photographs were published, only after the respective individuals we got in touch with provided their consent.
Our journey began with one fundamental question: how could we use our knowledge to make a tangible impact on human health? As students living in a post-pandemic world, we had witnessed firsthand how the COVID-19 crisis revealed critical weaknesses in global healthcare systems. This experience made us realize the urgent need for innovative solutions in preserving human health and we considered Synthetic Biology as the key to driving this change.
The breakthrough came during an unexpected moment at a lecture on Human Physiology by Dr. Ioannis Sotiropoulos at the Department of Biology of the University of Patras, that sparked the inspiration we needed. More specifically, he described the tremendous challenges of Alzheimer's Disease, having a great experience in the research field of the diagnosis of the disease.
This resonated deeply with our team, because many of us had personal connections to the disease, with family members or close friends struggling with Alzheimer’s. Unfortunately, we witnessed firsthand the devastating impact it had on their lives and families, making this the pivotal moment that solidified our resolve to make Alzheimer’s disease the focus of our project.
Thus, we took matters into our own hands and, inspired by Dr. Sotiropoulos' work, we concluded that the therapeutic agent targeting the pathogenesis of Alzheimer’s must be delivered via a system capable of crossing the blood-brain barrier and reaching the target cells with precision, without inducing side effects in the patient. This led us to the idea of using exosomes as the delivery vehicle. After modification, these exosomes would be engineered to specifically target neural cells after successfully crossing the blood-brain barrier.
To validate our idea, we reached out to experts with the first one being Dr. Spyridon Mourtas, an experienced researcher in Alzheimer’s therapies due to his groundbreaking work with nanoparticles to target amyloid plaques. As an expert in this field, he approved of our core idea and instilled confidence that exosomes could indeed be the solution we're looking for. This interaction helped us solidify the core framework of our project, which led to Dr. Mourtas being our primary Principal Investigator (PI).
We then connected with
Dr. Zoe Piperigkou, Assistant Professor of Biochemistry at the Department
of Chemistry at the University of Patras, who was highly
enthusiastic about sharing her research expertise and
scientific knowledge with us. She also offered access to
her laboratory, as the initial analysis of our project
indicated that her lab had the necessary equipment and
reagents to help us bring this idea to life.
With the support of these two esteemed professors, we concluded that our optimal target should be hyperphosphorylated tau protein, due to the fact that recent findings show that this target is more closely associated with the pathogenesis of Alzheimer's Disease than amyloid plaques and, at the same time, remains an underexplored avenue in current research. This made it a promising yet scientifically challenging focus for our project, that we were determined to research as far as possible.
Armed with this knowledge, we were ready to delve deeper into the world of Synthetic Biology and explore how it could offer a solution to one of the most complex and significant challenges in human health, that of Alzheimer’s Disease…
What problems does Alzheimer's Disease cause?
In our effort to gain a comprehensive understanding of the issue, we consulted Mr. Kostis Prouskas, a psychologist and CEO of the care center "Aktios." Mr. Prouskas thoroughly explained the challenges and difficulties faced daily by patients and their families.
Specifically, he mentioned that, in addition to motor difficulties, patients often experience emotions such as melancholy, loneliness, obsessive behaviors, denial, fear, and a significant lack of spatial and temporal orientation. He pointed out that their daily lives change significantly, leading them to seek stability in their routines, as any change can cause them agitation and discomfort.
On the other hand, the role of caregivers, usually taken on by family members, is particularly exhausting. Initially, they experience intense doubt and denial, which can lead to delayed diagnosis and treatment, as well as conflicts between the caregivers and the patients. Moreover, caregivers often experience symptoms of depression, making their role even more challenging. They are required to adapt their daily lives to the needs of the patients, which can create problems for the entire family.
Additionally, Mr. Prouskas offered some general advice on how to care for patients and encouraged us to give them the necessary attention, engage them with games and stories, and promote intergenerational interaction, which can alleviate the feelings of loneliness they often experience.
In conclusion, we realize the difficult situation that patients and their families face daily. As a result, Alzheimer's Disease impacts society as a whole, especially considering the high average age in Greece, which further motivated us to intensify our focus on addressing the disease.
The need of Accomodation Structures to ease the Burden of Alzheimer’s
As part of our iGEM team's efforts to integrate community perspectives into our Alzheimer's therapeutic project, we visited "Frodizo", an accommodation structure for elderly individuals with dementia. During this visit, we had the opportunity to speak with a social worker who is the Director of the structure, Mr. Theodoropoulos and a psychologist Mrs Tselepi , sharing valuable insights into the therapeutic exercises and mental strengthening activities they use with patients. These exercises, designed based on the severity of dementia, focus on tasks related to language, such as sentence construction, vocabulary, antonyms, synonyms, proverbs, concept recall, reading comprehension, and critical thinking.
One key takeaway from our conversation was how these activities are tailored to the individual's cognitive level. For example, patients with mild cognitive impairment engage in more intensive exercises, while those in the early stages of dementia require simpler tasks with detailed explanations. For individuals in more advanced stages, the focus shifts to maintaining basic skills, such as copying text when reading and writing become too difficult. Proverbs are particularly effective at this stage to aid recall. They also stressed the importance of routines, as breaking them increases anxiety levels, especially for those in later stages.
A vital lesson we learned was the emphasis on preserving patients' dignity throughout their care. Even as abilities decline, modifying activities to suit their mental and emotional capacity is essential for maintaining their sense of self-worth. This focus on dignity underpins all interactions, and the exercises are structured to foster collaboration and positive feelings, even in cases where language abilities are lost.
We also discussed the emotional aspects of dementia, particularly the feelings of sadness, frustration, and depression that can arise when individuals realize their cognitive abilities are declining. Being part of a supportive structure like Frodizo helps patients feel connected and understood, reducing isolation and improving their emotional well-being. Furthermore, Mr Theodoropoulos emphasized the need of for greater awareness of such supportive structures to the general public about their services, mentioning that they are only known through word-of-mouth of patients’ families and social media.
To sum up, inspired by this conversation, our team has committed to supporting Frodizo's efforts. We participated in their activities, promote their work through our social media platforms, and offer seminars on AI tools that could enhance their therapeutic exercises. Additionally, to address the broader issue of underestimating dementia care facilities, we raised awareness by incorporating public talks in our art exhibition, helping more people understand the critical role these structures play in the lives of individuals with dementia.
Are modern pharmaceutical practices sufficient for addressing Alzheimer's Disease?
To understand the pathological background of the disease and contemporary therapeutic approaches, we consulted with the following experts: Dr. Panagiotis Ioannidis, Neurologist and Associate Professor at the Aristotle University of Thessaloniki, Dr. Konstantinos G. Lyketsos, Professor of Psychiatry and Behavioral Sciences at the Johns Hopkins University School of Medicine in the USA and Dr. Dimitrios Kapogiannis, Neuroscientist at the National Institute on Aging in the USA and Associate Professor of Neurology at Johns Hopkins University School of Medicine.
Initially, Dr. Ioannidis informed us that the existing drugs on the market show only a 23% effectiveness, with prices ranging from $23,000 to $52,000. These medications primarily aim to relieve symptoms but do not reverse the ongoing neurodegenerative progression. As he pointed out, "The difficulty lies in the fact that by the time a diagnosis is made, which can be through clinical observation, lumbar puncture, or imaging techniques, the disease is usually in an advanced stage, and the damage that has occurred years prior is irreversible." Dr. Ioannidis also expressed doubts about the accumulation of amyloid as a definitive causal factor of the disease. Moreover, he mentioned that, alongside pharmaceutical treatments, he advocates for a focus on the Mediterranean diet and enhanced social interaction, suggesting that non-pharmaceutical interventions can also improve disease management.
Thus, we conclude that, since diagnosis is particularly challenging in the early stages of the disease, the development of a therapeutic drug that could halt its progression, even in advanced stages, would be highly beneficial. Additionally, non-pharmaceutical interventions should also be emphasized for optimal outcomes.
Dr.Lyketsos added that, in the past year, besides symptomatic drugs, a monoclonal antibody vaccine targeting accumulated amyloid was introduced to the market. However, it shows significant side effects and limited progress and effectiveness. He further emphasized that combination therapies might prove more effective in the future, targeting both pathological causes of the disease: amyloid accumulation and tau protein hyperphosphorylation.
Therefore, we find that targeting amyloid alone is not a sufficient treatment. Future research should explore targeting tau protein hyperphosphorylation or even employing a combined approach to offer maximum effectiveness with the fewest possible side effects.
In Alzheimer’s there are things we don’t know and things we don’t know we don’t know them
After our discussion with the renowned neuroscientist Dr. Dimitrios Kapogiannis, Clinical Researcher at the Laboratory of Neurosciences of the National Institute on Aging in the U.S. and Assistant Professor of Neurology at Johns Hopkins University, we gained a deeper understanding of the complexity of Alzheimer's Disease, the philosophy behind the current therapeutic approaches, and the direction of our own proposal. Dr. Kapogiannis explained that existing drugs fall into two categories: (a) symptomatic treatments, which alleviate symptoms but do not address the disease itself, leading to minimal side effects but inability to halt disease progression, and (b) monoclonal antibodies, which target amyloid plaques but exhibit serious side effects and limited efficacy in disease progression. Based on this, he informed us that our therapeutic approach is cutting-edge due to its focus on tau phosphorylation targeting, which aims to increase effectiveness, and the use of engineered exosomes as a delivery method, minimizing side effects. He also emphasized that Alzheimer's is a highly multifactorial disease, even more so than cancer, with lifestyle factors such as diet and psychology playing a crucial role in its progression.
In conclusion, Dr. Kapogiannis confirmed that our therapeutic approach fills a significant gap in the market, characterizing it as cutting-edge. This solidified our core idea of targeting tau phosphorylation through engineered exosomes for greater efficacy with fewer side effects. At the same time, he encouraged us to take a holistic view of the therapy, leading us to develop actions that improve patients' lifestyles, such as our app for cognitive strengthening and engagement, thus reinforcing non-pharmaceutical interventions as well.
Which areas should we focus our research on?
In our effort to develop a viable solution that addresses the needs and gaps in effective Alzheimer's medications, we consulted with Verisfield Pharmaceuticals and its General Director, Mr. Christos Koutsodimos. Mr. Koutsodimos answered our questions in detail and helped us organize our research to arrive at the best possible solution.
When asked how to determine the appropriate method of drug administration for Alzheimer's patients, he responded:"The selection of the pharmaceutical form and the route of administration should take into account the following:
- Ease of use: The medication (and its device, if applicable) should be easy to administer, with simple and clear instructions that an Alzheimer's patient can follow without difficulty.
- Route of administration: The route should be convenient and comfortable for the patient while ensuring the drug’s stability and tolerability.
- Pharmaceutical form: The form should be chosen based on the properties of the drug, such as solubility and stability, its absorption characteristics, and the preferences of the target population.
- Safety: Since the drug is intended for use by Alzheimer's patients, any potential safety issues arising from misuse (e.g., self-injury, overdose, packaging errors resembling other pharmaceutical products) should be minimized."
Finally, Mr. Koutsodimos noted that the pharmacological target, pharmacokinetics and pharmacodynamics of the drug should be clarified. The drug must reach its target with appropriate sensitivity and specificity, remain stable and deliver sufficient quantities for the desired therapeutic outcome, while minimizing potential adverse side effects. Additionally, it should be specified in which populations the drug produces clinically significant results (e.g., at which stage of the disease) and its place within existing therapeutic protocols, alongside other treatments.
We concluded that the components we use for drug development must meet all these specifications. Furthermore, their characteristics must be thoroughly studied to ensure maximum efficacy, low toxicity, and that they fill a gap in the market for Alzheimer's treatments.
Following these valuable insights, our team began intensive research and reviewed relevant papers to define the appropriate therapeutic framework that satisfies all these criteria. We reached the following conclusions:
- Tau protein hyperphosphorylation is a critical factor in the progression of Alzheimer's Disease.
- Once hyperphosphorylation begins, severe symptoms appear, and the disease rapidly progresses.
- The link between amyloid accumulation and disease progression is widely debated.
- Most available drugs focus on inhibiting amyloid plaques rather than tau protein hyperphosphorylation.
- Exosomes are a valuable therapeutic tool, acting as carriers and offering low toxicity and numerous advantages.
- We identified the microRNA-195 molecule, known to be associated with various pathological processes in Alzheimer's. A reduction in microRNA-195 is linked to tau protein hyperphosphorylation via the deactivation of the PP2A phosphatase.
- The PP2A phosphatase is involved in the dephosphorylation of tau proteins. Thus, upregulating PP2A would inhibit tau protein phosphorylation and further disease deterioration.
Through our targeted research, we gained deeper knowledge about the characteristics and pathogenesis of the disease and focused on the key points recommended by the experts to design a viable solution to the problem. It became clear that we needed to target tau protein hyperphosphorylation and explore the further use of exosomes, as they seem to meet most of the expert-recommended criteria.
Although still in its early stages, our idea began to evolve and expand as we continued researching. The next step was to have relevant scientists and researchers validate our initial idea and help us refine it in the best possible way.
Integrated Synthetic Biology into Medicine
Throughout the development of our experiment, we engaged with former iGEM participants to ensure that our project adhered to the principles of Synthetic Biology, while gaining a deeper understanding of the competition’s guidelines. It was crucial for us to explore the scope of Synthetic Biology and fully comprehend the opportunities it offers.
In our discussions with Dr. Galateia Kallergi, Assistant Professor at the Department of Biology at the University of Patras and primary PI of the Patras Medicine iGEM 2022 team, with the project "Syn-PNOIA", she confirmed that our project aligns with the core principles of Synthetic Biology. Specifically, it involves the modification and creation of new biological molecules such as the engineering of exosomes, and the incorporation of microRNA into AAV vectors. This was further validated by Mr. Asterios Arabatzis, a former member of Greece's first iGEM team, who explained that Synthetic Biology encompasses a wide range of possibilities, and our project clearly fits within this spectrum.
With this positive feedback, we continued our efforts, now confident that Synthetic Biology is our most powerful tool in this challenging endeavor.
Wet lab experts
Are exosomes the right choice?
To answer this question, we not only conducted a literature review but also consulted with Dr. Galateia Kallergi, Assistant Professor of Biochemistry at the University of Patras’ Department of Biology. Dr. Kallergi highlighted the following advantages of exosomes: low toxicity, the ability to cross the blood-brain barrier, no immunological response, modifiability via synthetic biology methods, and the ability to transport cargo. Additionally, she mentioned that exosomes can be easily isolated in large quantities from patients' blood and can be produced and isolated from all normal cells in the body. A reliable and straightforward isolation technique is through the Exosome Isolation Kit.
Finally, Dr. Kallergi emphasized that exosomes are ideal for carrying cargos such as RNA, DNA, proteins, and lipids of various sizes, making them well-suited for transporting AAV containing the active microRNA-195 molecule.
In conclusion, we confirmed that exosomes are the optimal choice for specialized, side-effect-free delivery of microRNA-195, while their modification in our experiment aligns with the principles of Synthetic Biology. Furthermore, they are readily available in large quantities from all cell types, and their isolation is an easy and precise process using the Exosome Isolation Kit.
How can we ensure that exosomes specifically bind to target nerve cells?
To provide a clear and comprehensive answer to this question, we consulted Dr. Spyridon Mourtas, Assistant Professor at the University of Patras’ Department of Chemistry.
Dr. Mourtas approved incorporating RVG membrane glycoprotein into exosomes as the most suitable and effective method for targeting nerve cells, as this peptide has a high binding affinity for neuronal cells. He further explained that there is a highly effective method to incorporate RVG protein into the membrane of nerve cells, called click chemistry, which has been proven in the literature to yield positive results.
In conclusion, we clarified that exosomes must contain RVG protein on their surface to effectively target and bind to nerve cells. Additionally, we concluded that click chemistry should be used to correctly place the peptide on the surface of the exosomes.
Which pathological process of Alzheimer's should we target?
Comparing the pros and cons of targeting amyloid plaques and tau proteins, we felt it was necessary to seek Dr. Mourtas’ opinion. He told us that amyloid is not a good target for a therapeutic approach like ours, and that current drugs on the market targeting amyloid plaques do not provide maximum effectiveness against the disease, often causing severe side effects in patients.
He urged us to focus our experiment on tau proteins, which, either directly or indirectly, would offer a more feasible approach to treating the disease. We also agreed that targeting tau proteins could represent an innovative therapeutic approach, as no treatments based on tau proteins have been developed so far.
Does PP2A phosphatase play an important role in treating Alzheimer's Disease?
To answer this question, we consulted Dr. Zoe Piperigkou, Assistant Professor at the University of Patras’ Department of Chemistry.
Dr. Zoe Piperigkou emphasized that the pathway involving PP2A phosphatase is crucial to the progression of the disease and recommended that we focus on it intensively. However, since the processes in which the phosphatase is involved are not fully clarified, she suggested we focus on microRNA-195, which is known to regulate PP2A activity. In other words, microRNA-195 is a key regulatory molecule that can indirectly influence phosphatase levels.
In conclusion, Dr. Zoe Piperigkou strongly supported the importance of PP2A phosphatase in the pathogenesis of Alzheimer's Disease and emphasized microRNA-195 as a crucial biological unit for regulating phosphatase levels. She specifically noted that microRNA-195 could serve as a "promising therapeutic tool" for future efforts to eliminate the disease.
Why should we target PP2A phosphatase?
Following an extensive review of the relevant literature and discussions with our Principal Investigators (PIs), new questions emerged regarding our approach: Is the GSK3β kinase inhibitor the most innovative molecule to define our method? Or should we focus on a molecule that not only halts the progression of the disease but also reverses it through the dephosphorylation of tau proteins?
These questions, among others, were addressed during an in-person meeting with Dr. Konstantina Nika, Assistant Professor of Biochemistry at the University of Patras Medical School, following the recommendation of Dr. Mourtas, because of her extensive experience in kinase and phosphatase pathways and function.
Dr. Nika confirmed that the choice of a phosphatase over a kinase is more innovative and likely more effective for treating Alzheimer's disease. Specifically, she suggested targeting the PP2A phosphatase, which is known to be impaired in Alzheimer's patients.
Additionally, Dr. Nika emphasized the importance of carefully considering the method of introducing the phosphatase into exosomes, given its large size. She recommended consulting with Dr. Kallergi, a Biochemist with extensive experience of exosomes.
Finally, when asked about the most reliable method for analyzing our results, Dr. Nika explained that flow cytometry (FACS) is preferred due to its higher accuracy and reduced likelihood of false positives or ambiguous results compared to immunofluorescence.
Our meeting with Dr. Nika confirmed the strategic direction of our research, endorsing PP2A phosphatase as a more innovative and effective choice for Alzheimer's therapy. Her guidance on enhancing the expression of an introduced phosphatase, careful consideration of its delivery via exosomes, and the preference for FACS in result analysis, strengthened our approach and guided the next steps of our research project.
Is microRNA-195 the right choice for our treatment among the available microRNAs used as therapeutic tools?
To answer this question, we consulted Dr. Zoe Stamatopoulou, Assistant Professor in the Department of Medicine at the University of Patras.
Initially, Dr. Stamatopoulou advised that we should investigate the off-target genes that microRNA-195 might interact with, to ensure no unwanted reactions occur. Identifying off-target genes can be done through alignment in an online database. She also emphasized the need to check the literature to determine whether microRNA-195 is toxic to cells, and whether it requires any modifications—such as methylation—for its activity. This would help us ascertain whether we need to introduce genes for possible modifying factors.
Equally important, she mentioned, is examining whether microRNA-195 should be introduced in its pre-microRNA form rather than its mature form, allowing the cell to modify and mature it using its own mechanisms. Finally, she stressed the importance of identifying activation and deactivation mechanisms for microRNA-195 in clinical trials, so that it can act as a switch, enabling us to turn its expression on or off when needed.
In conclusion, Dr. Stamatopoulou helped us determine that microRNA-195 is the best choice among the other microRNAs used for therapeutic purposes. The reason for this conclusion is that microRNA-195 meets the criteria outlined by the professor, making it a suitable therapeutic tool.
Delivery vehicle of microRNA-195 into nerve cells: Plasmids or rAAVs?
Through a thorough literature review, we could not determine the appropriate plasmid vector to achieve repeated expression of the factor that would indirectly result to overexpression of PP2A. As a result, we decided to consult again with Dr. Nika to seek guidance on plasmid selection. During our meeting, it became clear that using a plasmid to express microRNA-195 was an incorrect approach, as Dr. Nika explained that its short lifespan (48 hours) would prevent sufficient overexpression of the phosphatase. She emphasized that this is why such plasmids are not used in fully developed drugs, but rather in the experimental phase of drug development. Instead, she recommended using a viral carrier, because they are more suitable for the integration and expression of desired sequences.
In conclusion, our consultation with Dr. Nika confirmed that using a plasmid to express microRNA-195 was not viable due to its short lifespan, hindering PP2A overexpression. She recommended switching to viral carrier as a more effective vector for gene expression, providing us with a clear direction for continuing our experimentation:that of using rAAV(recombinant Adeno-Associated Virus) as a carrier.
Approaching a more realistic experiment:
In order to evaluate the progress of our experiment and explore new ways for its improvement and implementation, we decided to consult the Professor of Physiology Μr. Stavros Taraviras from the University of Patras Medical School, due to his expertise in neurodegenerative diseases. After reviewing our idea, he advised against using neural cells to test tau dephosphorylation, as their cultivation requires specific conditions and more time than we have available. Instead, he suggested evaluating dephosphorylation in other human cells that could simulate the diseased state with hyperphosphorylated tau. Finally, he confirmed that qPCR is the most appropriate method for checking AAV entry with embedded microRNA-195.
In conclusion, recognizing the challenges of using neural cells for tau dephosphorylation due to time constraints, we shifted our approach to using alternative human cells that mimic the diseased state. Additionally, we followed the advice to use qPCR as the most suitable method for verifying AAV entry with microRNA-195.
How Lethe turned into a mass production therapy:
During our meeting with Dr. Kapogiannis, apart from gaining valuable insight into Alzheimer's disease, we also wanted to ask some key questions regarding the direction of our project. First and foremost, he highlighted the innovation behind our proposal, characterizing it as cutting-edge due to the focus on tau phosphorylation, a highly legitimate target, and the use of engineered exosomes as a delivery method, maximizing effectiveness and minimizing side effects, respectively. He then addressed the dilemma that had been troubling the team: personalized gene therapy or mass production? Dr. Kapogiannis recommended that, at this early stage, it is preferable to pursue a mass production therapy. This approach allows the treatment to be widely available and inclusive, especially for countries like Greece, where equipment for personalized therapies is limited, suggesting that personalized approaches could be explored as a future step.
Additionally, he explained that exosomes can be diluted using specialized bioreactors to avoid the presence of MHC, thus preventing immune responses and side effects in patients. When asked about the most suitable type of cells for exosome isolation, he recommended using blood lymphocytes or even skin biopsies. He also advised against using neural cells due to the challenges in their cultivation, emphasizing that we are already targeting effectively neurons via the RVG glycoprotein, which ensures uptake. This allows us to successfully perform the proof of concept using HEK cells, while leaving the exploration of neural cells for future steps as well.
Finally, when asked about the best method of administration that combines maximum effectiveness with minimal invasiveness for patients, Dr. Kapogiannis recommended intravenous administration. He assured us that this method is highly effective for drug absorption and would not cause significant discontent for patients, as monoclonal antibodies are already administered intravenously. However, he encouraged us to consider subcutaneous administration as a potential future step.
In conclusion, Dr. Kapogiannis provided invaluable information that led us to decide on a mass production therapy to reach a larger group of patients, as compatibility issues with exosomes will not arise. We also decided to use blood lymphocytes derived exosomes and avoid the use of neural cells to save time and to minimise the cost without compromising the validity of our experiment's results. Additionally, we opted for intravenous administration of our product for maximum effectiveness. Ultimately, we reaffirmed that the core of our idea remains cutting-edge, taking the opportunity to have already begun shaping future steps for further development of our product.
Critical Insights in AAV Gene Therapy: Optimizing Our Approach for Maximum Efficiency
In our initial meeting with Ms. Konstantina Tzimou, a PhD student at DTU specializing in AAV Gene Therapy, her insights played a crucial role in shaping the direction of our project, regarding AAV.
She provided us with the ITR parts for the plasmid, which would be included in our parts section. Konstantina advised us against bacterial amplification of the plasmid, warning that the ITRs could be lost during the process, significantly reducing the virus yield. Based on her recommendation, we decided to order the entire plasmid from Genescript upfront, ensuring the integrity of the ITRs.
Additionally, she offered valuable feedback on the design of our three cassettes and helped us finalize the second design. She emphasized that there are base limitations between the two ITRs, which guided our decision-making. She also suggested that we request an "ITR guarantee" from Genescript to ensure quality and stability.
In summary, Ms. Konstantina Tzimou's expertise in AAV Gene Therapy was pivotal in refining our project strategy. Her guidance helped us avoid potential pitfalls, such as the loss of ITRs during bacterial amplification, and ensured the stability and quality of our plasmid by recommending Genescript's services. Additionally, her feedback on cassette design and the base limitations between ITRs allowed us to make informed decisions, ultimately optimizing the efficiency and success of our AAV-based therapy.
Dry lab experts
"How can Agent-Based Modeling and Algorithmic Optimization improve simulations of complex biological systems like the Blood-Brain Barrier? "
We recently held an insightful ZOOM meeting with Mr. Grigoris Kapadoukas, MEng in Computer and Informatics Engineering and an incoming Ph.D. candidate at the Department of Computer Engineering and Informatics, University of Patras. Mr. Kapadoukas, an esteemed alumnus of this department, provided valuable guidance on enhancing the efficiency of our NetLogo-based model. Our discussion focused on optimizing algorithmic structures, reducing runtime execution, and improving the clarity and explainability of the model’s objectives.
Mr. Kapadoukas highlighted several strategies for optimizing the performance of our dry lab simulations. His primary recommendation was to integrate cutting-edge agent-based modeling (ABM) techniques into our workflow. He emphasized that ABM provides a more suitable framework for modeling the complexity of biological systems—such as the blood-brain barrier (BBB)—which are challenging to simulate using traditional machine learning languages like Python. This is especially relevant given the limited presence of such models in current research literature.
While machine learning, particularly neural networks and deep learning architectures like Transformers, have shown significant potential in modeling various biological processes, Mr. Kapadoukas reinforced our decision to use NetLogo. He agreed that NetLogo's strength lies in its ability to model complex, dynamic systems with a level of granularity that surpasses classical machine learning algorithms. This capability makes it an ideal choice for simulating the intricate biological interactions within the BBB, a system that remains underrepresented in the current body of research.
Drawing on his extensive experience with advanced machine learning architectures (i.e. Graph Neural Networks and BERT) and agent-based modeling during his undergraduate and Ph.D. studies, Mr. Kapadoukas provided invaluable feedback on refining our modeling approach. His contributions were critical in addressing both the computational challenges we faced and the biological complexities inherent in our project. He stressed the importance of thorough validation of machine learning outcomes through both wet and dry lab experiments to ensure biological accuracy, particularly in projects with time and resource constraints.
In summary, Mr. Kapadoukas’ expertise in agent-based modeling and neural networks significantly advanced the development of our computational model. His recommendations, particularly the adoption of agent-based techniques, positioned our approach closer to real-world biological systems and offered a promising alternative to traditional machine learning methods. We are confident that these insights will greatly enhance the efficacy and accuracy of our model in simulating the complex biological processes of the BBB.
"How can interdisciplinary expertise in EEG, Brain Biology and Bioinformatics possibly elevate our modeling approach?"
Our journey in developing a comprehensive model for our project would have been significantly less robust without the invaluable contributions of Mr. Sabbir Ahmed Sibli. Mr. Sabbir Ahmed Sibli previously served as a Lecturer in the Department of Computer Science and Engineering at Fareast International University, Dhaka, Bangladesh, from September 2020 to September 2023. His expertise and commitment to the advancement of the field of Computer Science and Engineering make him a pioneering figure in this discipline. His academic achievements are equally noteworthy—he graduated cum laude from the University of Asia Pacific, Dhaka, Bangladesh, with a CGPA of 3.82/4.00. In addition, he holds a Master of Science degree in Computer Science from BRAC University, Dhaka, Bangladesh, where he also graduated with cum laude honors, achieving a CGPA of 3.95/4.00. Currently, he is pursuing an M.Sc. in Biomedical Engineering through the prestigious Erasmus Mundus Joint Masters (EMJM) program.
Throughout his academic career and during his tenure as a lecturer, Sabbir has consistently contributed to the research community, actively publishing papers in various international conferences and journals. His research primarily focuses on EEG, Brain-Computer Interfaces, and bioinformatics, with an emphasis on machine learning applications in these fields. His extensive theoretical knowledge, combined with practical experience, positions him as an expert in machine learning and bioinformatics, particularly in the context of biological systems.
What makes Sabbir’s contributions to our project particularly invaluable is his interdisciplinary expertise. His deep understanding of both Computer Science and Biology allowed him to offer insights that bridged the gap between these fields, guiding us through complex technical and biological aspects of our work. Moreover, his deep knowledge of molecular physics and mathematical analysis served extensively in our mathematical model, enabling us to accurately simulate the various biological processes involved. His contribution in this area was critical for ensuring that our model captured the subtleties of brain behavior at both the molecular and systemic levels.
Sabbir’s profound understanding of brain function and neurobiological systems proved instrumental in shaping the direction of our discussions, particularly around modeling the intricate functionalities of the human brain and the Blood-Brain Barrier. Together, we explored the critical parameters that must be accounted for in our modeling approach, ensuring a more accurate and biologically relevant outcome.
In conclusion, Mr. Sabbir Ahmed Sibli’s expertise, dedication, and interdisciplinary approach were crucial to the development of our project. His ability to integrate computational and biological characteristics played a key role in enhancing the overall scope and depth of our research. We are deeply grateful for his significant contributions, which have undoubtedly enriched our work and propelled it toward successful completion.
Stochastic Modeling of Exosome Movement: Insights into Transport Mechanisms
One issue identified during simulations using the Agent-Based Model (ABM) for exosome passage is the random movement of exosomes, which may cause part of the drug payload to never reach the Blood-Brain Barrier (BBB) due to diffusion within the bloodstream. This challenge was highlighted in discussions with Mr. Georgios Moschidis, a prominent Greek scientist and Assistant Professor of Mathematical General Relativity at EPFL, renowned for proving the instability of anti-de Sitter (AdS) space. After reviewing our simulations and the outlined problem, Moschidis offered valuable insights, particularly emphasizing the importance of incorporating time as a crucial factor when calculating the probability of an exosome crossing a specific radius to interact with the BBB during its transit through the bloodstream.
Following this meeting, we developed a theoretical framework that accounts for both the geometry of the artery and the time elapsed. This framework models the exosome’s position across the artery’s cross-sectional dimensions—its height and width—rather than its longitudinal progression. As a result, we arrived at a more refined two-dimensional probability calculation that more accurately represents the exosome's interaction with the BBB over time.
About ethic values…
To apply ethical values and ensure bioethics in our experiment, we consulted Dr. Stavroula Tsinorema, Professor of Modern and Contemporary Philosophy and Bioethics. With Dr. Tsinorema, we discussed how to source the biological materials for our experiment from licensed and reliable sources. Additionally, she emphasized the importance of repeatability and advancing the experiment using models that better simulate human biology. This would help us integrate ethical values into our experimental model by demonstrating both the efficacy and safety necessary for future human applications. However, she clarified that the use of more specialized experimental models requires a detailed study of bioethical regulations and their strict application.
Dr. Tsinorema also pointed out that our research is ethically sound, not only because it aims to develop a new scientific treatment but also because it contributes to scientific knowledge regarding the causes of Alzheimer's Disease. Our findings on the role of microRNA-195 and tau protein phosphorylation as causes of the disease add new value to our research. Furthermore, she emphasized the importance of ensuring that our treatment adheres to Principles of Distributive Justice, guaranteeing equitable access and fair distribution despite the potential costs of the medication.
Finally, she emphasized that our research holds significant value and should be promoted, as it can provide a solution to a major problem while also exploring and clarifying the causes of a complex disease, provided it adheres to established bioethical rules and standards.
In conclusion, the guidance from Dr. Tsinorema underscores the critical intersection of ethics and scientific research. By prioritizing bioethics in our work, we not only contribute to the advancement of scientific knowledge but also uphold the values of equity and responsibility in healthcare. This holistic approach ensures that our findings can lead to meaningful solutions for those affected by Alzheimer's Disease while fostering trust and integrity in the scientific community.
Seeking to further ensure that our project meets the criteria outlined in bioethics and to broaden our outreach to nationwide organizations, we contacted the Hellenic Alzheimer’s Society.
During an online meeting, the bioethics committee of the society approved that our experiments and the dimensions they have reached in the context of the project's development comply with bioethical standards. The written certification provided to us confirms the correctness of our experimental course in accordance with bioethics regulations. This certification was crucial for our team, as it not only gave us the green light but also provided additional motivation for the proper continuation of our experiments within the bioethics framework.
In conclusion, the bioethics committee of the Hellenic Alzheimer’s Society approved our experimental approach based on bioethics regulations, and its members unanimously approved the Society's participation in conducting our study.
About Biosafety…
During our conversation with Mrs. Angeliki Roboti, an expert from the National Organization for Medicines (ΕΟΦ) in Greece, she provided invaluable insights into biosafety and cost reduction strategies for our Alzheimer’s therapeutic project. She noted that our therapy is not only highly innovative but also has the potential for minimal side effects, particularly due to the use of AAVs and exosomes, which have shown no significant side effects so far.
She emphasized the importance of adhering to Good Manufacturing Practices (GMP) to ensure safe production and highlighted the role of hospital laboratories in monitoring clinical use. Additionally, Mrs. Roboti mentioned that laboratory techniques can help identify and exclude high-risk patients early, although clinical trials remain essential to confirm safety and effectiveness. That’s why in this first preclinical stage of drug development, we can trust the literature about the minimum side effects as well as have a first say of the efficiency of the product from the first results of our experiment. She then stressed the need for ongoing safety evaluations, especially for biologics like our gene therapy, which require long-term monitoring for immune responses and side effects, emphasizing that most of the results will be concluded after the distribution of our product.
In terms of cost reduction, she recommended focusing on specific patient subgroups identified through clinical trials. This targeted approach would make the treatment more attractive for reimbursement by healthcare providers, further enhancing accessibility for patients, whose the administration of the drug will be really effective for the treatment of Alzheimer’s.
In conclusion, Mrs. Roboti's expert guidance provided us with a deeper understanding of the key considerations for biosafety and cost reduction in our Alzheimer’s therapeutic project. At first we understood that in this stage our product seems really promising as far as minimum side effects and high efficiency are concerned. Then, her insights into cost reduction, particularly through targeted patient subgroups and innovative reimbursement models, offer a strategic path to make our innovative therapy more accessible while maintaining a strong safety profile. Her input has been instrumental in shaping our approach as we advance the development of this promising treatment.
Interview with Patients
We first visited the most directly involved individuals, the patients themselves, at the "Frodizo" Care Facility. Together with the facility's supervisors, we had a constructive discussion. The group we spoke to was suffering from a mild form of Alzheimer's Dementia, and thus the interview proceeded smoothly without significant difficulties. Initially, we introduced ourselves, and the beneficiaries briefly presented themselves, their families, and their interests. This general conversation aimed to help us get to know each other better and create an environment of security and friendliness. From our general discussion, we noticed that they frequently mentioned a sense of loneliness, especially when talking about their children, who, due to their obligations, dedicate very little time to spending with them. Additionally, they emphasized how pleasantly they spend their time when visiting the facility and how satisfied and happy they feel after each session. During our conversation, they were very talkative and willing to assist us by answering all of our questions.
However, they also shared that they often experience social exclusion, as friends and relatives have changed their behavior towards them and distanced themselves. They feel marginalized by society, as social events rarely promote inclusivity, making it difficult for them to participate. They voiced their deep concerns about the future, expressing a sense that no one truly understands their problems or empathizes with their situation.
When we inquired about the treatment they were receiving, they indicated that they did not notice significant improvements. Many described the process as stressful, often accompanied by unwanted side effects. Conversely, they expressed a greater appreciation for non-pharmacological interventions, which they find more enjoyable and beneficial.
Next, we presented our intention to treat Alzheimer's Disease in simple terms. Their reaction was moving, as they encouraged us to continue our efforts and expressed how grateful they feel when young people strive for something so important, while also noting how hopeful our idea sounded to them .
In conclusion, our visit to the "Frodizo" Care Facility highlighted the profound challenges faced by individuals living with Alzheimer's disease, particularly their feelings of loneliness and social exclusion. The patients expressed significant concerns regarding the medications they are prescribed, noting a lack of noticeable improvement and the stress caused by unwanted side effects. These worries emphasize the need for more effective treatment options and a greater focus on non-pharmacological interventions, which they found more enjoyable and beneficial.
Interview with Relatives
After conducting several meetings with expert scientists regarding our experimental progress and the societal impacts of Alzheimer's disease, we deemed it essential to hold an open discussion with Mrs. Poulheria, Mrs. Aristea, Mrs. Pepi and Mrs. Gogo who are caregivers and family members. This was to gain deeper insight into the effects of the disease and tailor both our project and social initiatives to better address the needs of patients and their families.
The caregivers emphasized the complete lack of support from the state,financial, psychological, and even legal,due to the government's underestimation of the disease's severity. They noted that the facilities for engagement and care of individuals with memory disorders are insufficient, especially compared to the increasing number of patients in Greece, with rural areas being particularly underserved.
During our discussion, they explained that there is a significant lack of awareness and sensitivity toward Alzheimer's, both from society and doctors. This makes interactions with others and obtaining a diagnosis difficult. They highlighted the stigma and covert ridicule that patients face due to their behavioral changes, underscoring the need for societal guidance and awareness on how to properly treat people with the disease. Moreover, they stressed the importance of inclusion and acceptance for patients, who are often marginalized by their social circles.
In terms of family dynamics, caregivers reported that this marginalization also extends to other family members, leading to tension and placing the full burden of care on a few individuals, which takes a severe toll on their mental health.
Finally, when asked whether the high cost of a potential treatment would be a barrier, caregivers assured us that price would not be an obstacle, as long as the drug is effective, even if there are some side effects. They also clarified that intravenous administration of the drug would be an acceptable and manageable method for patients .
Support and Initiatives of the Region for Individuals
with Alzheimer's Disease: Collaboration and Prospects
Having interacted with patients and their relatives, we fully understood their needs and decided to speak with Ms. Anna Mastorakou, the Deputy Regional Governor for Public Health, to learn about the Region's contribution to supporting individuals with mental disorders and their families.
Initially, Ms. Mastorakou informed us that the Region has developed an action plan for Alzheimer's disease, which includes a caregiver training program that has been submitted to the World Health Organization for approval. Additionally, there is a plan in collaboration with the University of Patras that leverages artificial intelligence to detect behavioral differences in individuals with Dementia. Another pioneering initiative from the Region is the telephone helpline service that provides assistance to elderly individuals needing care. She emphasized that this service is free, but certain criteria must be met for elderly individuals to benefit from it.
She was also asked whether the Region could support our efforts, to which she responded positively, suggesting that they could provide us with space for a private Dementia Clinic that we would establish after approval from the 6th Health Region. She emphasized that we are young people with vision and a plan, and the Region will always stand by us, along with the entire state, which we occupy with a positive impact.
Another question raised in the discussion was the existence of Research Investment Programs for projects like ours. She informed us about the European research programs "Orizon", which allow partnerships between research entities and best practices with foreign centers.
Moreover, she made it clear that the Region's budget is not sufficient for individuals with mental disorders. According to a study from 2005, she stated that the cost of Dementia for an independent person amounts to €341, while for someone dependent on others, it rises to €957. When the patient is hospitalized in an institution, the cost increases to €1,267. In today's times, she argued that expenses for a family with individuals with mental disorders exceed €30,000. These amounts are exorbitant for the Region to cover, but they make every possible effort to provide financial support to patients and their families. However, they do provide benefits to patients who have a significant percentage of disability.
Additionally, Ms. Mastorakou informed us that the Region is active in seeking facilities that do not exist in Patras for Dementia patients, having submitted a request to the ministry and awaiting suitable announcements.
When asked whether our team could raise public awareness about our treatment, she replied that citizens often exhibit skepticism towards pioneering drugs like ours, similar to the reactions towards coronavirus vaccines. However, if there were a serious public awareness campaign regarding our treatment, backed by scientific data and clinical study results, then every citizen could be convinced of the therapeutic approach's contribution to Alzheimer’s Disease. On this matter, she stressed that the Region would support us in public awareness regarding the medication we will promote, as exosomes represent a "revolution" in medicine. Her words were a hopeful message for our support from the Region.
In conclusion, the Deputy Regional Governor for Public Health, Ms. Anna Mastorakou, emphasized that although the Region cannot meet all the financial requests of individuals with Alzheimer’s and their patients, it makes every possible effort to provide financial assistance to them. Furthermore, it strives to find suitable facilities for these individuals and for any other actions related to improving their health and quality of life. It was also significant that she would assist our team in establishing a private dementia clinic in the future, as well as in our efforts to raise awareness and inform the public about our treatment.
Engaging Society’s Perspective on Alzheimer’s Disease
In addition to understanding the needs of those directly affected by Alzheimer's, we sought to capture the broader societal pulse on how people experience and perceive the disease. Specifically, we created a questionnaire that was distributed to the general public via social media and local shops. The respondents were asked to share their views on Alzheimer’s awareness, how informed they felt about the disease, and how they behave when interacting with those affected.
The responses were highly insightful. We found that a majority of people interact with Alzheimer's patients on a daily basis. Most agreed that these individuals are often treated with indifference by society, and many expressed that patients face stigma, even mockery. However, an overwhelming majority indicated they would try to help an Alzheimer’s patient in need - yet there was a problem: they didn’t know how. They agreed that society is not well-trained in how to support or interact with Alzheimer’s patients.
Interestingly, 93.6% of respondents stated that a behavior guide for interacting with Alzheimer’s patients is necessary. In response, we decided to create such a guide, providing not only instructions on appropriate behavior but also advice on environmental factors, such as lighting and space adjustments, to improve patient interactions.
In coclusion, our questionnaire revealed that while many people are willing to help Alzheimer’s patients, they often lack the knowledge on how to do so effectively. With 93.6% of respondents supporting the creation of a behavior guide, we developed a comprehensive resource to provide clear guidance on proper interactions and environmental adjustments, promoting greater understanding and support for Alzheimer’s patients.
Find the results here...
What is the process and cost of producing our treatment?
For the market research we conducted, we contacted Mr. Christos Koutsodimos, Director of the pharmaceutical company Verisfield. With Mr. Koutsodimos, we discussed the production process and the cost of developing such a drug. The following questions were raised: "How much time should pass between the recommended doses of the drug?" The response was: “The dosing regimen must be determined through the pharmacokinetic and pharmacodynamic investigation of the drug". Preclinical dose-determination studies in animals should be conducted, which will lead to proposed doses for investigation in humans. Subsequently, the final dose will be determined through Phase I and II clinical trials, while its safety and efficacy will be confirmed in Phase III studies.”
Regarding the cost of developing and producing a drug for Alzheimer's Disease, Mr. Koutsodimos pointed out that: “The cost of developing biologic drugs varies and depends on the requirements of each development stage. It can range from a few million to billions of euros. The production cost per unit, based on the price list of already approved and circulating similar products, ranges from 100 to 1,000 euros.”
When asked if prescription is required for such drugs, the answer was as follows: “Over-the-counter (OTC) drugs require the ability of self-diagnosis by the patient and a safety level such that, even in cases of overdose or misuse, the patient remains safe. Given the proposed indication and the available research material, treatments for neurological diseases such as AD, if they fall under the broader category of biologic drugs, will require a prescription.”
As for how long it will take for a molecular therapeutic to be available on the market, Mr. Koutsodimos responded: “The preclinical study stage lasts approximately 1-2 years (with a 7% success rate). This is followed by Phase I clinical trials, also lasting 1-2 years, Phase II trials lasting 2-4 years, and Phase III trials lasting 3-5 years (with an overall clinical success rate of 10-15%). Additionally, time must be accounted for the production of the drug, stability studies (2-3 years), the preparation of the dossier, and the approval process by regulatory authorities. However, some stages can occur in parallel, so the overall time amounts to 10 years.”
Summarizing, Mr. Koutsodimos provided us with valuable advice regarding the production process, the required time and production cost, as well as the characteristics our drug must possess to be approved for widespread use. His insights were particularly helpful for the next steps in our research.
Product Evaluation and Communication with the Director of GENESIS Pharma
During our effort to estimate the cost of our product and assess its market suitability, we reached out to Mr. Georgios Karachalios, the Director of GENESIS Pharma. Mr. Karachalios, who leads a company specializing in the distribution of therapeutic drugs for serious diseases like Alzheimer's, in collaboration with leading biotech pharmaceutical companies, explained to us that our product is characterized by innovation. This makes it attractive for future investment from pharmaceutical companies.
He also pointed out that our product addresses a significant gap in the Alzheimer's treatment market, where most available therapies focus on symptom relief. Mr. Karachalios emphasized that current therapies primarily target amyloid plaques with monoclonal antibodies, which present significant side effects, regarding their reduced efficiency. This observation aligns with the scientific community's skepticism as far the role of amyloid plaques is concerned in the disease.
When asked if the high cost of the drug might pose a barrier to future investment or consumer access, he assured us that price would not deter caregivers as long as the drug proves effective. This assertion is also supported by the literature on which our idea is based.
Finally, he advised us to protect the intellectual property of our technology in a more sophisticated manner, similar to the approach used in Gene Therapy and CAR T-cell Technology. He also suggested that, as an initial step, we should consider establishing a start-up based at the University of Patras.
Our communication with Mr. Karachalios highlighted the innovation and market potential of our product in Alzheimer's treatment. His positive remarks regarding the market gap, the product's efficacy, and his encouragement to adopt a strategic approach in protecting our intellectual property reinforce our confidence in the success of this project. The establishment of a start-up at the University of Patras represents an important next step in realizing the potential of our technology.
First steps of financial implementation
Ms. Marina Makri played a pivotal role in guiding our team during its early stages. As the Project Coordinator for the Greek Association of Alzheimer’s Disease and Related Disorders and with her vast experience as a Fundraising Consultant at Alzheimer Hellas, she brought invaluable insights to our journey. With a background in biology, just like most of our team, she immediately understood the essence of our project. Her extensive experience in the Alzheimer’s Disease field allowed her to provide us with crucial information about the landscape of Alzheimer’s in Greece, including specific organizations and sponsors that could support our cause.
Ms. Marina was the first to set us on the right path for fundraising, facilitating connections with key contacts and organizations, and helping us communicate with the right people when we needed it most. She was a key factor in starting our fundraising journey, enabling us to quickly understand the environment of communicating with experts in this field. This allowed us to present our project in a more effective way.
In addition, she emphasized the importance of keeping our product affordable, given the economic situation in Greece. Her advise was to find ways to reduce costs so that our project would have a competitive advantage over the existing ones in the market. In this way, we would combine the innovation of our project with cost-effectiveness, enhancing our chances of success.
Her guidance has been instrumental in shaping the course of our efforts.
Optimizing our Pricing Strategy
During our call with the marketing consultant at NN Hellas, Mr. George Agapiou, his expertise in mathematics played a crucial role in refining our pricing strategy. He helped us craft a balanced pricing model that not only covers our production costs but also reflects the healthcare market’s varying ability to pay for innovative Alzheimer’s treatments. Using a highly analytical, data-driven approach, he introduced us to price elasticity of demand , enabling us to predict how different pricing tiers would influence market penetration and revenue generation. His guidance in implementing a tiered pricing strategy , differentiating between high-income and low-income regions, ensured that our therapy remains accessible to a global audience. This approach allows us to maximize profitability in wealthier markets while maintaining affordability in lower-income countries, ultimately expanding the reach of our solution.
His insights have been instrumental in aligning our pricing model with our broader business objectives, ensuring scalability in both developed and emerging markets.
Business Plan Creation
During the creation of our Business Plan, we faced numerous challenges, particularly in understanding how to approach our project from a business perspective and how to position it within the highly competitive pharmaceutical market. We needed to start step by step, reflecting on the problem at hand, the source of our treatment, and how we could ultimately arrive at the solution: treating patients effectively.
How could our drug reach elderly patients and lead to their healing? To ensure our product is accessible to those in need, we had to navigate the complexities of bringing it to market, which begins with a well-structured plan - the Business Plan.
This is where Mr. George Makris stepped in as a pivotal ally. His expertise as a Business Analyst at PwC was crucial in guiding us through this intricate process. Mr. George’s insights allowed us to tackle the fundamental questions surrounding our project with clarity. Specifically, he helped us refine our approach to the SWOT and PESTEL analyses, ensuring our evaluations were realistic and actionable.
As we progressed as a team, we realized that beyond the scientific refinement of our treatment, it was of immense importance how our product would be integrated into the market. The commercial viability of our therapy would play a significant role in its success. Mr. George’s feedback on our strategies not only helped us identify potential challenges but also allowed us to explore innovative solutions that would facilitate our entry into the market.
With his meticulous attention to detail, our initial draft transformed into a comprehensive roadmap, complete with a solid foundation that resonated with our vision. His recommendations provided the necessary adjustments that added credibility to our business model.
Moreover, Mr. George's guidance on the various funding options illuminated the path to financial support, enabling us to navigate the complex landscape of pharmaceutical financing. By assisting us in constructing our financial cost sheet, he empowered us to accurately project the expenses involved in launching our startup, especially as we strive to introduce innovative treatment delivery methods, such as our dedicated treatment van and specialized treatment space.
In summary, Mr. George’s contributions were instrumental in not only completing our Business Plan but also in ensuring it was infused with a sense of realism and strategic insight. His support has been vital in shaping our project, leading us toward a robust proposal ready to make a significant impact in the healthcare sector.
We also received invaluable support from Ms. Anna Kalogridi, particularly in terms of the business approach to our idea and the realistic calculation and design of our cost sheets. As the General Manager at the Medical Center Cretan Medicare, she possesses extensive knowledge in financial management, which proved to be instrumental for our team.
Ms. Anna provided us with insights into how startups operate, guiding us through the process of calculating the cost of our product on our initial steps while helping us understand how our company can gradually become profitable over the years. Our discussions were not only enlightening but also incredibly helpful in assessing our resources and developing an effective budget allocation strategy.
Her practical advice and expertise allowed us to visualize the financial landscape more clearly, ensuring that we could make informed decisions about our project. The collaboration with Ms. Anna complemented the efforts of George Makris, as both their contributions have significantly enhanced our understanding of the financial aspects necessary for our startup's success. Together, they helped us build a solid foundation for our Business Plan, ensuring that we are well-prepared to navigate the complexities of the pharmaceutical market.
The road to the implentation of Application 'LETHE'
Driven by our commitment to make a meaningful impact on the lives of Alzheimer's patients and to benefit society in the most positive way, we developed a mobile application aimed at assisting patients with their daily activities, providing them with additional support.
However, this idea and its implementation did not happen overnight. We engaged with multiple experts in the fields of healthcare and patient care to ensure that our application was developed with the highest level of scientific accuracy and professionalism, while carefully considering the specific needs of the patients."
Initially, through discussions with the accommodation structures for people with dementia, 'Frodizo', they expressed their frustration about the limited number of such organizations available in the country's rural areas. This scarcity prevents patients from engaging in cognitive empowerment exercises and socialization activities, which are essential for their smooth daily functioning. This concern was further confirmed in our conversations with caregivers and family members, who shared that they need a tool to engage patients in a way that simultaneously enhances their cognitive abilities and keeps them productively occupied. Many of these patients live in areas outside of Patras or in villages where there are no organizations to support them, and some patients are unwilling to leave their homes.
Moreover, during our meeting with Mrs. Aivazoglou, a graduate psychology student specializing in the implementation of diagnostic tools for Alzheimer's disease, she presented her thesis— a 3D diagnostic tool that can also be used for cognitive improvement. This inspired us to visualize and design our application accordingly."
Once we had finalized the concept of the application, we needed to determine its features and goals. Initially, we decided to include cognitive empowerment exercises in the form of games, such as pantomime, card pair games, crosswords, jigsaw puzzles, and chess, to enhance memory, sharpen critical thinking, and provide opportunities for engagement.
To better implement these features, we visited 'Frodizo', where psychologist Mrs. Limos showed us the types of exercises they include in their program, helping us understand what would be most appropriate for cognitive empowerment and ensure that the exercises would not overwhelm patients if they were too difficult.
At the same time, in our discussion with psychologist Mr. Prouskas, he emphasized the importance of intergenerational relationships, highlighting how crucial it is to strengthen family bonds, especially between elderly patients and their grandchildren, due to the generational gap. This led us to include a feature allowing family members, such as a grandchild, to switch profiles and manage the patient's progress, as well as a section where family and close individuals' details are stored for constant reminders.
Additionally, following our conversation with occupational therapist Ms Moraiti, who has experience working with elderly care institutions, we gained a deeper understanding of how vital physical exercises are for both mental health and overall well-being, as well as for keeping the mind alert. This prompted us to add another feature specifically designed for exercises suitable for elderly patients with dementia.
Another significant contribution to the design of the app came from neurologist Mr. Ioannidis, who informed us that cognitive exercises are often used to monitor the progression of the disease over six-month periods, based on the scores generated from each session. As a result, we decided to integrate a feature that tracks scores after each completed task, allowing patients to experience a sense of achievement and encouraging them to continue, while caregivers can monitor the patient's progress and intervene as needed.
Lastly, our discussions with music therapist Mrs. Batsila, who specializes in dementia, helped us understand the importance of music for these patients. She explained that patients can perform daily tasks more effectively when listening to songs with which they have a strong emotional connection. This inspired us to add a feature allowing family members to create a playlist of the patient’s favorite songs.
To sum up, through our discussions with experts and families, we understood the need for an application focused on cognitive empowerment and patient engagement. With their valuable insights into the disease, we were able to successfully implement the most suitable features, ensuring that the app meets the specific needs of Alzheimer's patients.
Lethe ApplicationEngaging with Children
Recognizing the importance of inclusivity for all social groups, we visited the 1st Special Primary School of Patras. We believed it was essential to consult with Ms. Sfyri E., a teacher and Master student in Psychology and Ms. Papanikolaou A., a psychologist with experience working with students with intellectual disabilities. Their insights were crucial in helping us understand how to effectively convey the concepts of Synthetic Biology and Alzheimer’s disease in a way that would be accessible and easily comprehensible for the children.
Thanks to their advice, we adapted our communication approach, ensuring that our educational material was engaging and appropriately simplified to meet the cognitive needs of the students. This allowed us to present complex scientific ideas in a more meaningful and impactful way for this audience.
Specifically, Ms. Sfyri highlighted the importance of making the children feel comfortable, suggesting we start with an interactive icebreaker game. She also emphasized the need for activities that allow the children to express their creativity more freely, such as drawing, which can help them better understand biological concepts. Additionally, she pointed out that it would be easier to convey our information through a more interactive and creative medium, rather than a simple PowerPoint presentation. This led us to incorporate storytelling, using the storybook our team had prepared.
She also stressed the importance of being encouraging, praising the children for their efforts even when they didn’t find the correct answers, helping them feel good about themselves. At the same time, she advised us to avoid memory-based games, as they might make some children feel uncomfortable.
When we shared our educational materials and previous school activities with Ms. Papanikolaou, she provided valuable feedback that helped us refine our approach to make it more accessible and engaging for children. She noted that our diverse range of activities was inclusive of all types of children, thanks to its variety. However, she suggested we avoid using pantomime, which we had done in other schools, to create a more comfortable environment for the kids. She also emphasized the importance of adjusting our tone of voice, ensuring we were neither too exaggerated nor too formal.
Respecting personal space was another key aspect she highlighted, advising us to avoid activities that required excessive physical interaction. Additionally, she helped us organize the activities in a more cohesive manner. For example, she suggested following up the card-based game with coloring pages related to Alzheimer’s and biology, which would help reinforce the concepts visually by connecting the images from the game to the coloring activity.
Both experts provided crucial insights that helped us refine and optimize our activities, allowing us to include children more effectively in the world of science, Synthetic Biology, and Alzheimer’s disease. Their advice ensured that our message was not only accessible but also engaging and respectful of the children's individual needs.
A Comprehensive Guide for Caregivers & Society:
One of the main goals of our project is to provide accurate information regarding the needs of patients and their families while also addressing the daily challenges they face. To understand the obstacles these individuals encounter, we engaged with both the patients and their families, as well as with numerous scientists who informed us about the situation.
Specifically, Mr. Prouskas mentioned that patients are often disregarded by society and their surroundings; their needs are not recognized, and the way they are treated can evoke negative feelings and confusion in them. The remarks made by Mr. Prouskas were confirmed by the relatives themselves. They pointed out that society does not provide the help they need, while they are often looked down upon and treated disrespectfully. Furthermore, they highlighted that their social circles, such as friends and other relatives, do not offer the necessary support to cope with the challenges of caregiving, and they fail to understand the patients' conditions and provide the compassion they require.
Sensitive to these findings, we created a Questionnaire to confirm the prevailing ignorance in society regarding issues related to Alzheimer’s disease.
Therefore, immediate awareness of societal issues concerning the treatment of patients and the support for their relatives, who have taken on the role of caregivers, is essential. For this reason, we have developed a comprehensive guide ,with the assistance of psychologist Ms. Vasiliki Georgousi and architect Ms. Maria Zisi, outlining appropriate behavior and interactions with patients suffering from Alzheimer’s disease. This guide serves as an essential resource for caregivers, family members, and healthcare professionals, promoting general education about the unique needs and challenges faced by individuals with this condition. By providing clear instructions and insights, the guide helps to foster understanding and empathy, ensuring that everyone involved in the care of Alzheimer’s patients is better equipped to offer support.
In conclusion, our communications with specialists as well as with the patients themselves were particularly useful in understanding the problem holistically and in identifying the serious shortcomings of society in addressing this critical issue. Their insights helped us create a guide that we believe will change the way patients and their families are treated.
Behavior Guide
After completing our initial experiments, we consulted again with Konstantina Tzimou and Dr. Kapogiannis to refine our approach based on their expert insights.
Ms. Tzimou emphasized that the issue might have stemmed from either the dosage of the treatment or the duration we applied it. She highlighted the possibility that many viral capsids could be empty, and therefore recommended that we determine a full-to-empty ratio using techniques such as ELISA or flow cytometry to quantify the capsid numbers, and qPCR to measure the DNA content inside the capsids. She also advised us to explore ways to optimize the virus production process, such as by reviewing the cell line and transfection reagents used. Another critical point was the proper mixing of the transfection mix to ensure even distribution of the reagent. For the production of a pharmaceutical product, she noted that exosomes must be purified using chromatography.
Dr. Kapogiannis provided additional feedback, suggesting that we could use iPSCs (induced pluripotent stem cells) as they are more cost-effective. He reassured us that even though the transfection was performed with a single replicate and we could not conduct a statistical analysis, it was acceptable since this was a proof-of-concept experiment. Due to limitations in media availability, further replicates weren’t possible at this stage, but this can be revisited in future steps.
One issue Dr. Kapogiannis pointed out was the lack of exosome characterization, which was due to technical difficulties. Moving forward, he advised conducting PCR on the exosomes to confirm the presence of the virus. He also stressed that the iPSCs should be in a highly pluripotent state to isolate the exosomes effectively. Standardizing the dose is critical, and this can be achieved by determining the concentration of AAV. Previously, we estimated the dose based on literature and analogical data, but now, we need to refine this through direct measurements.
The next steps he outlined include detailed AAV characterization, determining the concentration of recovered AAVs, assessing the purity (free from contaminants), performing a dose-response curve, quantifying PP2A, using neuron cells as recipients, and ultimately evaluating the effects on tau proteins. This last step would bring us closer to understanding the broader impact of our treatment on the disease mechanism.
To explore the future possibilities of our project and the necessary steps that must be experimentally validated for its advancement, we contacted Ph.D. student Mr. Asterios Arampatzis. We discussed our future plans in detail and received positive feedback, which further inspired us to aim in new directions. Specifically:
- First, we will ensure the efficacy of our treatment through the repeatability of our experiments.
- In addition, we will use other models (such as animal models) to verify the effectiveness of our treatment in humans.
- Furthermore, we will research our personalized treatment approach to provide the best possible results at the lowest cost compared to other treatments.
- However, personalized treatment is expected to be particularly costly. Thus, as an intermediate solution, we can leverage the benefits of personalized therapy while significantly reducing costs. This solution may involve obtaining exosomes not from each patient individually but from a stem cell bank.
In addition to Mr. Arampatzis, other experts have inspired us to plan our future steps. Specifically, Mr. Lyketsos suggested:
- To investigate in the future the combined targeting of both β-amyloid plaques and hyperphosphorylation of tau proteins for greater therapeutic efficacy.
- In addition, we will investigate the combined action of our treatment with non-pharmacological practices such as diet and social activities.
The advice of Mr. Kapogiannis led us to consider:
- To explore alternative ways of the administration of the treatment such as intranasal or subcutaneous administration.
- To explore personalized gene therapy for individualized treatment options.
- To try using neuron cells, instead of HEK for more targeted research.
- To see if we can use cells from skin biopsy instead of lymphocytes.
- To check what happens after the dephosphorylation, regarding the stage.
- To try detailed characterisation of AAV, such as determination of the concentration of the recovered ones and purity.
- To try making a dose-response curve, for determination of the dose.
- To check the quantitation of PP2A.
- To check the effect of our product on tau proteins, as it is one step further than the mechanism.
- To buy the iPSCs cells, from which we will isolate exosomes, instead of making them in the lab
Also, through market research, we decided:
- To create a startup company that will secure funding for the completion and perfection of our patent.
- To establish a mobile treatment unit so that everyone can access our treatment, including people with mobility difficulties, as our treatment does not require special storage conditions (storage at -20°C is recommended) and administration.
Finally, our goal is to further reduce the cost of treatment so that more people can access it.
