Since December 2019, I have been working towards my PhD in single-cell multi-omics as an Early Stage Researcher (ESR1) in the STACCATO training network. As I head towards the end of my time as a PhD researcher, I wanted to take this opportunity to think and reflect on this learning experience.
Almost three years ago, I exchanged my life in England for one in Ireland. I was fortunate to be accepted to study at University College Dublin with a full-time industrial placement at multinational medical technology company Becton Dickinson (BD). This was all thanks to the encouragement and support of my team at Horizon Discovery, where I had worked for the past two years as a junior scientist in the bioprocessing industry. It was here that my love for science flourished.
Arrival in Ireland
I made the exceptionally tiring journey across England, the Irish Sea, and Ireland to my new home in Limerick, where I was met with rain. It was not long, though, before I set out to explore and discovered the beauty that Ireland had to offer. Fast forward to my first week at the lab, I was thrown into work to meet the team, BD’s technology, sample processing, and best of all, a mini technology fair where I discussed my PhD project with some of BD’s leadership team, as the only PhD researcher at BD’s Limerick site.
My PhD Project
As part of the STACCATO consortium, my project addresses human disease, cell therapies and single-cell RNA sequencing in the context of solid and haematological cancers. My research utilises many of BD’s reagents, technology and expertise, to address some of the current gaps in scientific knowledge in this field. The most exciting technology that I get to use is a powerful, high throughput microwell-based single-cell partitioning system known as the BD Rhapsody, which was developed by the many brilliant scientists in BD that I get to work with. So why would someone choose an industrial-based PhD?
Learnings from an industrial PhD
There are many advantages to completing your PhD in industry, from gaining professional experience to forming industry-based contacts. The main advantage for me is the exposure to leading researchers, scientists and engineers that form the BD workforce, both as part of the BD Research Centre Ireland and further afield in America and beyond. Nevertheless, a PhD is no easy task. Whilst I still have quite a way to go, it is important to celebrate each goal and victory, however small they may be. Every project, meeting, task, or experiment each contributes to learning and developing as an early-stage researcher. It is these learnings that I hope to continue to develop in future, wherever that may be, to progress not only my career, but the greater scientific field in as many ways as possible.
Attending scientific conferences is a key way for young researchers to receive feedback from other scientists, explore possible collaborations and establish a professional network for future opportunities. Thus, I was very happy that I and other ESRs (Marco, Kelly, James, Brian, Ryan and Alan) attended this year’s “European Society of Animal Cell Technology” (ESACT) Meeting, which took place in my beautiful host city Lisbon (Portugal) from the 26th to the 29th of June. The event organised under the banner of “Advanced Cell Technologies: Making Protein, Cell, and Gene Therapies a Reality” brought together worldwide experts from the animal cell technology field. You can get more information on the conference or the society following these links.
The scientific committee approved my research being presented in poster format during several sessions throughout the conference. My work “Single-cell and Bulk RNA sequencing of insect Sf9 cells during rAAV production” was also selected for a 3-minute “flash presentation” in the main auditorium in the “Poster Student Highlights” session. While it was nerve-wracking to speak in front of an auditorium of more than 400 people, I was also very happy for the great opportunity to make people aware of the research we conduct at iBET within the STACCATO project.
In the poster session, several people showed interest in my research approaching me with questions. I enjoyed the scientific discussions, insightful questions, valuable feedback and suggestions to collaborate.
Additionally, STACCATO organized a workshop on “Single cell omics” under the lead of our PI’s Colin Clarke and António Roldão. In this workshop important experimental and data analysis aspects of single-cell RNA-seq were presented. This was followed by ESR-led discussions in working groups on how to apply the method in the fields of recombinant protein production, gene therapy or cell therapy. Overall, the workshop was a great success with more than 70 attendees interested in how to include single cell RNA-seq in their projects.
STACCATO’s single cell omics workshop at ESACT 2022
Next to all the inspiring and insightful scientific talks, ESACT was also an unique opportunity to network and to catch up with my fellow ESR’s, some of which I haven’t seen in person for more than 2 years due to the COVID-19 pandemic.
Overall, I think the conference was a great success and an amazing opportunity to highlight the research we do within STACCATO. I can only recommend every early-stage researcher attending scientific conferences in fields of their interest.
“Success is not final; failure is not fatal; it is the courage to continue that counts”.
Winston S. Churchill
My name is Marco Silvano. It’s been nearly 3-years since I first stepped into the Cell-based Vaccines Development Lab, where I am a PhD student at Instituto de Biologia Experimental e Tecnológica (iBET) in Portugal. Just a few months away from the finish line, I take time to reflect on my experiences and the journey of the past 3 years.
If you ask me
I’ve always felt admiration for those who spend years pursuing doctoral research. It’s a journey of extremes. Through the struggles and the acknowledgments – when you develop a deep bond to your project through commitment and self-motivation. Before my adventure began, a former colleague of mine told me: “It’s the best and the worst time of your life.” While I immediately understood his point, the duality deeply intrigued me.
Something that is necessary but unspoken is that you know what you leave behind, but you do not know what you take. A reason why I was perplexed by the idea of leaving a safe job and my comfort zone to start over somewhere else. However, my concerns were quickly replaced by excitement and the urge to prove myself. I was enrolled in a top-quality consortium – the promises and ambitions were immediately high.
My expectations have been partially met. Just when my work required a significant effort and networking between research institutes, the Covid-19 outbreak began to gain momentum. Unfortunately, for this reason, I have the feeling that I have not fully benefited from my position. It’s left a slightly bitter taste in my mouth as I am a few months away from the completion of my research. Nevertheless, I am blessed and glad to have been part of this journey. It doesn’t matter if things did not turn out the way completely as planned – things do not always go as expected, though it is a hard and beautiful truth about life.
I have been blessed with the good fortune to be surrounded by amazing people. They have been my strength and have definitely added value to my PhD experience. I have a core group of friends who, although interested in my work, are far from my research area and can take me away from work-related thoughts. In the end, it’s all about the balance between carrying on the research project successfully and knowing how to enjoy life. Though it’s not always spontaneous and often comes with experience. I cannot say I am there but I know I am getting closer. Something which has guided me from my first day: the awareness of wanting to live these years to the fullest, while striving to improve myself both as a person and scientist. As long as doctoral studies may seem while participating in the program, these experiences are short in the grand scheme of life and will pass in the blink of an eye. In no time you think about what you did and what you could have done – yet one cannot be everything and be everywhere. Nevertheless, it’s important to live so that there will be fewer regrets when all is said and done.
But the “end” word has not yet been pronounced. On the contrary, more than ever, it’s time for the final push to complete my work in the short remaining time. Recently, the days seem to run and chase each other. I often asked myself “how will I make it out of here alive.” But all jokes aside, these years have taught me to never back down and that, with the right attitude, things will fall into place. It will be challenging, but the satisfaction will be undoubtedly great.
We’ve heard a lot about viruses lately, and I bet you’ve never thought that we should have more of them, right?
Well, this is exactly my goal, I want to make as many viruses as possible.
My name is Brian Ladd and while it may sound like I am some kind of a supervillain I am actually a PhD student working to cure a wide range of diseases from Alzheimer’s to Muscular Dystrophy. To do this I am going to need viruses, a lot of viruses. A virus is just a set of instructions that your body follows wrapped up in a special package called the capsid. In a wild virus, this set of instructions makes your body produce more of that virus, and this is why a wild virus can be dangerous because your body will keep making more and more of them in a chain reaction.
A wild virus
It is possible to hack into these instructions and swap them out with something that you want your body to do, to fight back against Alzheimer’s for example.
When you swap out the set of instructions inside the capsid you transform the wild virus to a viral vector.
A wild virus to a viral vector
There are many new viral vector therapies under development. The vector I am working with, Adeno-associated-virus (AAV), has a number of very exciting candidates. Including possible treatments for Alzheimer’s and Hemophilia.
The problem though is that currently there is not a good way to make a lot of these viral vectors, and to treat these diseases we are going to need A LOT of them. This is the goal of my PhD project.
In order to swap out the “wild” viral instruction for the ones that we want I use special chemicals that I treat lab grown human cells with to make them produce the viral vectors.
These chemicals though, are a 1000x more expensive than gold so it’s really important to use them in the best way possible.
To do this I use a variety of sophisticated tools to study how these chemicals interact with each other and with the cells. With this knowledge, I can create mathematical models of how they interact with each other and by using engineering principles I aim to increase the number of viral vectors that we can produce.
The strategies and tools that I use are high cell density cultivations, continuous manufacturing, and single cell transcriptomics. With these I hope to increase the efficiency of viral vector production, enabling the testing of novel treatments that have the potential to improve the lives of millions.
Viral vectors offer the potential for a cure
I hope by now I have convinced you that I am not some kind a super villain but a researcher working together with many other scientists in the fight against a wide range of diseases and hopefully, one day, improve the quality of life for millions. But if you still aren’t convinced, please check out the following links.
On the 22nd of March 2022, I had the privilege of providing a scientific workshop to a group of excited 5-7 year olds, at an international kindergarten in Helsinki, Finland. The day started with a short presentation about my work (germs, viruses and bacteria), including answers to a list of six interesting questions, the children had prepared beforehand. I was impressed by their challenging questions, which included ‘What colour can viruses be?’ and ‘How do they form and what is the process to find them?’. I was also happy to see how impressed the students were (“wowww”), when they learned that dogs see fewer colours than humans, whilst birds see more.
Having got the kids thinking, we moved straight into the ‘laboratory demonstration’, where the students were asked to perform a serial dilution using food colourings.
Kids performing “laboratory research” with food colourings as part of the demonstration.
This was followed by a scientific illustration class where they used crayons to draw their favourite viruses from a list of well-known viruses.
At this point, they were focused and subsequently created a series of signed pictures, including a very pretty Adenovirus and some hazardous looking Ebola! Some of these are now decorating our lab and have been inspiring our team ever since. Lastly, we had a microscope demonstration, where they had the opportunity to look close up at pollen and different parts of plants.
At the end one of the students came up to me, whilst wearing an oversized lab coat and said to me “I’m a scientist, now you ask me questions!”. At this point, I was happy to know that maybe one of these students would end up contributing to science, perhaps even being a Nobel Prize winner.
Alan Foley is a PhD student within the EU funded Marie Sklodowska-Curie STACCATO project. His research focusses on single cell mitochondrial analysis in the biopharmaceutical industry.
As an Early Stage Researcher within the STACCATO project, I engage in scientific research every day. However, because of the vastness of science, I am uncomfortable calling myself a scientist in any field other than the area I work in. Nevertheless, we’ve endlessly seen during the last pandemic the word “scientist” used by governments to give credence to their public health measures.
“Scientist’s say…” is a faceless term that neglects the broad range of credence in the scientific community. The truth is science can be “good” and “bad” for many reasons. Here I’ll focus on a key tenant: is the research reproducible?
An example of “good science” is the development of the CRISPR-Cas9 genome editing system. In the last 2 decades, it has emerged as the bona fide mode of choice for genome editing. The ability to change DNA sequences with ease and precision has huge implications across science. Interestingly, the story starts with a yoghurt researcher wanting to protect yoghurt bacteria from viruses. They uncovered an innate bacterial immune system that cuts viral DNA based on memorised sequences (ASCB, 2014). “Scientists” created a CRISPR-Cas9 system that could be programmed to cut specific sequences of DNA by changing the memorised sequence. Later, this was adapted to introduce precise changes to the DNA sequence at a specific location (Adli, 2018). The best way to confirm science is “good” is by observing its reproducibility. In other words, does it work? Over the last decade, CRISPR-Cas9 has introduced targeted DNA editing to cure cancer, treat genetic diseases, improve food crop production, develop sustainable fuels with many many more applications (Nidhi et al., 2021). We see it as “good” science because it has been proven.
Since science can be called “good” when it’s reproducible, it can also be called “bad” when it’s not. One such example is the paper “Efficient Mitochondrial Genome Editing by CRISPR/Cas9” by Areum Jo et al., 2015 (Jo et al., 2015). They claimed CRISPR-Cas9 DNA editing of mitochondrial DNA; something that had never before been performed. One difficulty is RNA sequences vital for CRISPR/Cas9 do not spontaneously localise to mitochondria; however, Jo et al. appeared to observe this localisation, in direct contradiction to other papers in the field (Gammage et al., 2018) (Rai et al., 2018). A journalist writing about the paper might easily conclude “Scientists have adapted CRISPR-Cas9 to edit mitochondrial DNA”. And to be honest, I wouldn’t blame them. It’s published in a respectable journal, it’s cited many times and displays its data for all to see. A lot of science is based on this “trust”. We believe the authors’ methods are exactly what they said, and their data is produced to scientific standards. In this case, the doubt over the paper’s veracity is best described by the lack of follow up studies. Since this 2015 paper, there are no publications demonstrating use of CRISPR/Cas9 efficiently in the mitochondria. Only failed attempts have been published (Mullaly, 2019) (Verechshagina et al., 2018). To me (and others in the field), this suggests the Jo et al. paper is not reproducible and is therefore “bad” science.
In this brief look at 2 examples, I hope to highlight one of science’s great gifts to the world: doubt. If we believe everything we read, even if a “scientist” said it, we lose our ability to critically analyse for ourselves. When we doubt, we can investigate, then innovate and ultimately create some “good” reproducible science. Perhaps it is here we can justifiably refer to ourselves as a “scientist”.
I am Kelly Blust (ESR 11) working at KTH (Royal Institute of Technology) in Stockholm. I was born in Dresden, Germany and I have completed my education in Germany at the University of Rostock and Halle. Now enjoying Sweden very much! I like walking with my dog in the pure Swedish landscape, it’s a perfect way to relax after work; as well as swimming in the sea during summer, which is warmer than you think. 😉 During the cold and dark winter, I am enjoying cross-country skiing in the north of Sweden where, if lucky enough, you can see some reindeers, I was!
My project within STACCATO
My PhD topic is about stem cell differentiation of human embryonic stem cells to pancreatic tissue in a 3D cell culture system based on recombinant spider silk. With my project, I aim to develop methods for 3D culturing of human embryonic stem cells and differentiation to pancreatic tissue using recombinant spider silk. I perform validation on differentiated pancreatic tissue using single cell analysis to establish a protocol for the maturation of the different pancreatic cells (alpha, beta, etc). STACCATO gives me this great opportunity to use a novel technique of single cell transcriptome analysis BD RhapsodyTM to analyse gene expression of every single cell in differentiated pancreatic. I´m looking forward to going to NIRBT in Dublin for my secondment to learn more bioinformatic. I am very glad to be a part of this European Industrial PhD program due to good networking, many interesting courses e.g. about Entrepreneurship and finding collaborations and friends for life!
Falling WALLS lab Marie Skłodowska-Curie Actions (MSCA)
“Which are the next walls to fall in science and society? Discover future breakthroughs!“ #Falling Walls
The Falling Walls competition is a world-class pitching event, networking forum and global platform for outstanding innovators from multidisciplinary research fields. It’s part of the international Falling Walls Labs occurring all over the world and including the Falling Walls Marie Skłodowska-Curie Actions (MSCA). At Falling Walls MSCA lab 15 candidates, MSCA fellows from across a wide array of scientific disciplines, have competed for a spot at the global Falling Walls Lab Finale in Berlin (7-9 November 2021). With this getting the chance to become a Breakthrough winner in Emerging Talents. All of the 15 candidates address with their research for greatest global challenges our time in health care, climate, engineering technologies and environmental issues.
I would like to tell you more about this exceptional experience of participating in the Falling Walls MSCA in 2021. From many applicants, 15 Marie Curie fellows were selected to present their innovative idea. We all had the chance to participate in a group coaching session with Alexandra Smith from Debatrix about Storytelling based on TED talks which was very helpful to prepare the pitch. Additionally, all of us had an individual coaching session to improve the speech for the pitch. The coach helped me to present myself better and get more confidence in myself and my research as well as learn how to talk about research in a way that people can relate to it! So quite a lot 😉
# Breaking the Wall of Curing Diabetes
In my pitch, I presented a Novel cell therapy to cure Diabetes using Spider silk!
In the process, pancreatic islets are generated from induced pluripotent stem cells and incorporated into a 3D network of spider silk for transplantation to diabetic patients. Providing this 3D network, pancreatic islets will be incorporated in spider silk foams to get mature pancreatic cells to enhance insulin and glucagon production and protect pancreatic islets. With this personalized treatment, a patient would be able to produce insulin on its own.
It was a thrilling competition and the first place went to Giulia Rocco, who works on a new way to analyse the cerebellum. The second place went to Anwesh Bhattacharya, who develops an infrared camera using complex scattering media to look through walls. Both will continue to compete for the Breakthrough winner in Emerging Talents in Berlin.
I encourage everyone to apply for a Falling Walls lab competition next year and take this great opportunity to share your research with the public!!
Any resemblance to other scientists is purely coincidental.
The author of this blog, aka blogger, introduces himself as Filip (Φίλιππος, φίλος: lover, friend; ίππος: horse). He belongs to the Hominidae family (phylum: Chordata, class: Mammalian), particularly genus Homo, species sapiens. The subject’s genotype classifies the gender to be male, red/green colorblind and blood type O+. His phenotype seems to be a typical Caucasian male with some mixes from eastern European parts, light blue/grey eye colored, brown hair, 169cm short, weighing around 60-70kg (fluctuates by his diet and general mobility/activity). He was born in Kavala (Greece) in 1992, raised in Eleftheroupoli, and lived for a while in Veroia (Greece).
The natural habitat of the biologist comprises many elegant and some not that much elegant devices, i.e. flow cytometer and thermoblock, respectively. He is familiar with the sounds of his surroundings, and he can communicate with the machines and identify if something is wrong. The surfaces tend to be sterile most of the time, while alcohol disinfectant or UV light help him to clean his mess. In principle, there are two types of items in the lab: the “don’t touch it, it’s expensive” and the “don’t drink it, it’s deadly”. Miraculously, he successfully recognizes both.
The dried coffee remnant in multiple cups found on his desk is a rule of thumb. There are rumors about an actual map that shows exactly the coordinates of his experimental records. If a notebook is found, please provide it directly to a specialized professional for translating the hieroglyphics. Don’t be afraid, the office is the place where the biologist can spend his time hibernating. You can safely approach him by that time to admire this wild-type species. Please, do not ask him if he’s procrastinating…you should have already known that this is quite obvious. Other strictly forbidden questions include: “How far have you reached with your experiments?”, “Do you have any interesting results?”, “When are you going to publish?”, “When are you finishing your PhD?”, “What are your next steps after your PhD?”, “How do you see yourself after 10 years?”, “When will I become a grandma?”.
Running with a slippery agarose gel late in the afternoon is not fun. Stepping on the slippery agarose gel is not fun either. His favorite reaction in the lab is staring surprisingly at his samples, while holding the pipette and shouting “Noooooo!”.
The conversation with his labmates comprises usually of two sentences: “Good morning” and “See you tomorrow!”. However, there are days when overwhelming discussion among them can be observed. Ready for goosebumps? They call it “Lab Meetings”.
The manager is a substantial figure in his habitat. When the employer is around, our biologist quickly grasps a pipette and pretends that he’s busy. Don’t try this at your workplace. It requires advanced acting skills. Also, don’t try that while having lunch, it’s not realistic. Trust me, he tried.
All together are having great fun during their weekly break-out. Apparently, the place they’re going for partying is quite famous among other scientific groups as well. It’s a pity though that alcoholic drinks are not served in the “Journal Club”. At least, the music is quite chill. Major aim is to not fall asleep while listening to the lethargic ballads one of the same species colleague mumbles among them.
Getting through my bachelor studies at the Aristotle University of Thessaloniki (Greece), I timidly carved my professional path to where I am now. My Erasmus Internship at the Lund University (Sweden) was the beginning of a big journey of studies and exploring abroad. Since then, I had the opportunity to finish my masters at the University of Copenhagen (Denmark) and join the Marie Curie European Industrial Doctorate programme at Paul Ehrlich Institut (Germany).
Chimeric antigen receptor (CAR) T cell therapy has emerged due to the beneficial outcome particularly in B cell malignancies. However, the complicated and multifactorial manufacturing process involving gene delivery often by retro- or lentiviral vectors (LVs) could potentially affect the results in clinics. Hence, more contemporary monitoring methods are required to precisely investigate the molecular insights of CAR T cell generation in order to optimize therapeutic effects and minimize adverse events.
By additionally implementing detection tools for the expressed transgene, we managed to distinguish transduced from non-transduced cells in a targeted gene approach in scRNA seq. In our experimental set up, we incubated human PBMCs with either non-cell-specific LV or with receptor-targeted LVs in order to explore their unique fingerprints on the transduced and untransduced T cells.
Our mission is to provide fundamental elements for the manufacturing optimization of CAR T cell products for clinical use. Together with our partner institutes, we have set a goal of completing our tasks by utilizing state-of-the-art technologies to explore in-depth genes and proteins that are responsible for the enhanced production of the desirable biologic, for the optimized differentiation of stem cells to transplant cells, for the improved production of vaccines and tumor-targeted viruses, and hijacking cellular pathways for amplified gene delivery. Overall, we aspire to ameliorate manufacturing procedures of certain biopharmaceuticals in order to minimize side effects and costs of production.
After finishing my master’s degree in Iran, I was ready to make a change in my life. I have to say that this was not a spontaneous decision; I was always thinking about living and studying in a different country, enthusiastic about being in a new society and experiencing a new culture. All things considered, Germany is the first choice for many students, and I was no exception.
I was happy to be accepted in the STACCATO (European Industrial Doctorates programme) project available at the PEI (Paul-Ehrlich-Institut). This is an international collaborative project in the field of immunotherapy (my special interest), it offers the opportunity of a secondment in another country, and lots of bioinformatics! Overall, the position in the STACCATO programme covered all my interests.
So I moved to the city of skylines and was ready to start a new chapter in my life. Moving to Germany was both challenging and pleasing. For the first few days I had to do lots of official paperwork which now I know it is typical in Germany, but apart from that I found myself enjoying life in Frankfurt.
During the first days at the Lab, I met my nice colleagues and another ESR, Theofilos Filippos Charitidis. We are both working in the same research group, and since Filip had started his Ph.D. prior to me, he kindly helped me to get oriented in the lab. And we still have close collaboration in our projects, which is indeed encouraging. What specifically attracted my attention on a walking tour around Frankfurt is the mix of both historical buildings and the modern, futuristic skylines. Nevertheless, there are also many attractive natural areas and Frankfurt is a very green city. Soon after starting my PhD, it was Christmas time! Hanging around the warm and shiny Christmas Market was all that a newcomer needs to do in the grey German winter!
But unfortunately, the new year started with a global pandemic and, as for everyone around the world, our work was affected. Soon after the crisis became worse, the work in the lab stopped and we were working from home. But during that period I actually found time to learn more about bioinformatics analysis and got to know some of the software tools in this field, which is very helpful. Luckily, under stringent safety measures, we have now started the lab work and I hope everybody will stay safe.
Now, after sharing a bit of my story, I would like to tell you what this journey is all for….
Amazing CAR T cells!
Chimeric antigen receptor T cells, so-called CAR T cells are considered as an evolutionary type of cancer immunotherapy. So far the FDA has approved CAR T-cell therapy for adult patients with certain types of lymphoma, as well as children and young adults with acute lymphoblastic leukemia. Beyond that, there are many clinical trials ongoing assessing CAR T-cell therapy for other types of blood cancer, as well as research projects regarding their efficacy in the management of solid tumors.
But what are CAR T cells? One can imagine them as intelligent T cells that go throughout the body, recognize tumors, and kill them. CAR T cells are actually genetically manipulated T cells, with the ability to express a chimeric antigen receptor (CAR), specific for a surface protein on cancer cells. So the cells gain the ability to recognize cancer cells via their receptor.
In the process of making T cells intelligent enough to recognize tumor cells we need to send the genetic information of the CARs to the cells, then cells will translate this information for tumor cell recognition. This genetic information is transferred to the cells by specific lentiviruses. The virus binds to the cell and injects its genetic information (which in this case is the CAR gene) into the T cells.
This might sound straightforward but it requires a complicated manufacturing process. T cells are isolated from blood samples and activated in cell culture and subsequently transduced with viral particles. After the expansion of these genetically modified T cells, they can be used for clinical purposes. The lentiviral vectors used for transduction of T cells are mostly pseudotyped with the glycoprotein G of vesicular stomatitis virus (VSV) which mediates a broad tropism and thus CAR gene delivery to various cell types. In the Molecular Biotechnology and Gene Therapy group of Paul-Ehrlich-Institut, which I am working in, receptor-targeted lentiviral vectors have been developed. This type of vectors is designed in a way that they can specifically bind to CD4 or CD8 T cell subtypes and transduce them. These vectors can potentially facilitate CAR T cell production by eliminating the need for previous isolation and manipulation of the cells and also direct in vivo generation of the CAR T cells.
What is the purpose of my project?
Despite the successful results obtained with CAR T cell therapy, still some limitations exist. The complicated generation process of CAR T cells which includes delivery of the CAR gene to the T cells often via lentiviral vectors, can affect the functionality of the cells and decrease the clinical efficacy of CAR T cell therapy. The aim of my project is to have a close look at the CAR T cells after production and perform molecular analysis to get an insight about the alterations of the cells during lentiviral vector mediated gene transfer on their efficacy and functionality.
I will try to dissect the CAR T cell characteristics with single-cell profiling. In particular, single-cell transcriptome analysis will be performed along with barcoded antibodies, enabling a comprehensive immunophenotypic and transcriptomic analysis of the cells. This is important to follow the fate of vector particles sticking to cells, and, because often the RNA levels versus protein levels are not correlative.
During this project, conventional or receptor-targeted lentiviral vectors will be used to transduce T cells. Different transduction and manufacturing conditions of CAR T cells will be applied and the overall effect on the functionality and exhaustion of the cells will be measured. Based on the data obtained in this project, I hope we would be able to find solutions for the optimization of the CAR T cell manufacturing process and ultimately improve the functionality of CAR T cells. After almost 8 months working in the lab and living in my cosy apartment in a nice and green neighborhood of Frankfurt, I can say that so far, Germany has been a place for me to learn and experience many new things, and STACCATO not only supports me in pursuing my Ph.D. career, but also provides me an opportunity to meet great people in the field and be part of an international collective that will broaden my horizons. Along with other research groups in STACCATO and in direct collaboration with other brilliant ESRs, I hope that we will answer some fundamental questions in the field.
I always wanted to experience some time abroad. I lived my whole life in Austria, and after studying for six years in Vienna, I was ready for a change. So, when I heard about the eleven job openings of STACCATO (an European Industrial Doctorate program under the scope of the European Horizon 2020 programme) from a colleague of mine, I thought that this was the perfect opportunity to step out of my comfort zone and try something new. I applied for the ESR (Early Stage Researcher) position 8 hosted at iBET (Instituto de Biologia Experimental e Tecnológica) in Portugal. The topic “Combining single cell RNA sequencing with systems biology to fine-tune the production of recombinant adeno-associated virus vectors (rAAVs)” excited me the most. Moreover, I must admit that the idea of living close to the sea and the nice climate of Portugal were additional benefits to the position itself.
I was very excited when I heard that I was accepted. Even though it was hard for me to say good-bye to all my friends and family, I packed my bags and was eager to start this new chapter in my life.
I used the next couple of days after my arrival to explore Lisbon, some nearby beaches and the neighbourhood around iBET. Even though it wasn’t easy to communicate at first (my Portuguese was limited to two words: bom dia (“Good morning”) and obrigado (“Thank you”), the beauty of Portugal was staggering. I am very glad to be able to call this country home for the next couple of years.
On my first day at iBET, I immediately felt welcomed by all the people of the “Animal Cell Technology Unit”. During this time, I also met my new “partner in crime” Marco Silvano (ESR 7). Marco S. is also conducting his PhD at iBET under the scope of STACCATO and is trying to improve the production of influenza virus-like particles (VLPs) using single cell RNA sequencing. Even though we both have our separate projects, we have several tasks in our PhD where we cooperate or complement each other. I have to say that having someone to share the whole STACCATO experience, the work, travels and ups and downs of a PhD life is very nice.
But before I continue talking too much about my life here in Portugal let’s talk some science…
What are rAAVs, and why do we need them?
Recombinant adeno-associated virus (rAAVs) vectors are promising delivery tools in gene therapy, a procedure in which nucleic acids are delivered directly to patient’s cells to treat or prevent diseases.
Recombinant adeno-associated virus vectors are believed to be specifically suitable for gene therapy applications due to their high transduction efficiency and their good safety profile. Despite the high potential of AAV-based products, gene therapy is still far from being a mainstream clinical practice, mainly due to the high production cost and poor vector quality. The approval of uniQure’s Glybera® in the EU in 2012, the first-ever rAAV based gene therapy product, encouraged other manufacturers in pursuing the development of their own rAAV-based products, leading to increased demand. Nonetheless, current production platforms are not able to fulfil this increased demand in a timely and cost-effective manner, leading to a lack of vectors and resulting in financially painful delays in clinical trials.
What does this have to do with my STACCATO project?
I mentioned that current production platforms are not able to fulfil the current demand of rAAV vectors in a cost-efficient manner. But what are the production platforms currently used?
There are several different platforms available to produce rAAV vectors. The first-ever production was done in adherent HEK293 cells. However, as this system was hard to scale-up, alternative platforms were created such as stable producer cell lines, the herpes simplex virus system and the insect cell baculovirus expression vector system (IC-BEVS). All these platforms have their pros and cons but describing them would exceed the scope of this post. We will focus more on the IC-BEVS system as my work is based on it. However, if you want to read about it in more detail, I recommend a review by Otto-Wilhelm Merten, who wrote a good comparison of the different systems and a nice summary of remaining challenges (Mertens, et al. 2016).
But back to the IC-BEVS…
As adeno-associated virus (AAV) is a small single-stranded DNA virus which depends on the presence of a helper virus for replication, the introduction of helper genes is essential to produce functional virus vectors. The insect cell baculovirus expression vector system is suitable for introducing those helper functions and expressing capsid and replicase genes for successful assembly. Major advantages of the systems are mostly simple scalability and fast production, leading to their popularity as a production platform. Nevertheless, drawbacks of this system still occur, namely low vector titers, quality and potency. Furthermore, the use of the system in a continuous production system is limited due to the lytic effect of the baculovirus.
To tackle those drawbacks and improve the production of rAAVs with IC-BEVS, many scientists try to engineer baculovirus or improve the production process itself. Having said this, we, however, believe that the overall knowledge of the underlying biological mechanisms inside the insect cells should be increased in order to improve production. This leads me to the scope of my project:
Increasing the knowledge of the underlying biological mechanisms of the IC-BEVS
As mentioned before, during my PhD I am going to explore the molecular signatures of insect cells during baculovirus infection using a multi-omics approach. First, I will use single cell RNA sequencing in order to explore the single cell transcriptome and molecular heterogeneity of this cell factory during the infection process. Next, I will supplement this information with bulk metabolomics and fluxomics data, acquired from metabolic flux analysis. Here I will explore changes in flux distributions and nutrient availability throughout the production process. As the last step, I will try to combine the data in a multi-omics approach in order to investigate limiting steps further and deliver metabolic engineering strategies to increase productivity.
Overall, I hope that my PhD will contribute to a better understanding of the insect cell-baculovirus expression vector system and my proposed strategies will allow for an increase in production titers and vector quality in order to make recombinant adeno-associated virus vectors more available.
Concluding this blog post, I would like to summarise my STACCATO experience so far, which was amazing. Next to the outstanding resources STACCATO provides me for my research, I also had the opportunity to spend one week in Dublin to meet our STACCATO coordinator Colin Clarke and to get my first experience in bioinformatics. During this time, I met my fellow ESR’s Alan, Marco R. and Ryan, which showed me around in Dublin. Later we met again during the 1st Annual Industry-Academia Network Training Week in Dublin, where I was also introduced to all the other ESRs for the first time, an amazing group of young scientists.
I would like to encourage everyone to think about pursuing his/her PhD under the scope of a European funded project, and I am looking forward to great years doing my PhD with STACCATO.