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All the way from Iran to Germany

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.

An Austrian goes south: My life as a PhD student in Portugal

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.

Marco (ESR 7) and me (ESR 8) during a hiking tour in Sintra.

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.

Posted by Nikolaus.

Life of a Londoner studying and working in Finland.

I never imagined that I would find myself living in Helsinki, Finland but after almost eight months living here, I am now a full supporter and advocate for Finland. On the 5th of June, I packed my bags and boarded my first ever, one-way flight, to the land of Santa Claus and Saunas. I was lucky enough to be greeted by a family friend at the airport and was taken directly to an official shop to have my Finnish identification photo taken. It was summer and I remember being surprised that it was significantly warmer and sunnier than London. For some reason I thought Finland was permanently snowed in and this is a stereotype I have to now fight off myself. We then went to the family home and I was greeted by my housemate to be, a charming elderly Swedish man who proceeded to welcome me with a glass of Aquavit and baked salmon – when in Rome? This set the stage for what I imagined it was going to be like for the next 4 years!

I spent the first few days exploring Helsinki by running around the islands in the bay, this also helped me deal with the stress of not knowing anyone. Luckily, I joined a very friendly lab and was invited to join a midsummer social on Pihlajasaarito to meet other University of Helsinki students from Finland and abroad. Apparently, moving in during the summer was good idea as everyone is in a considerably better mood, the nights are long and the Finns are more social than usual. At the same time, during this time of year, a mass migration of Finns to the forest (summer houses in the countryside) for berry picking and traditional sauna occurs and Helsinki becomes populated only by tourists and people like me!

Having now met many people here, everyone seems to ask the question “Why Helsinki!?” as if I’m a lunatic. I always wanted to do my PhD outside the UK, learn a new language and get outside my comfort zone. I was thinking about applying to Germany but thought it was too classical of a scientific career pathway. The opportunity presented by STACCATO (European Industrial Doctorates programme), specifically TILT Biotherapeutics Ltd was also perfect, as it combined academia, industry and the chance to carry out a secondment in another country. STACCATO also aims to connect and support early-stage researchers throughout Europe and so I am glad to share this journey with other students who share similar aspirations and interests for life sciences entrepreneurship. 

With this, I now feel settled and even have Helsinki-withdrawal when I go back to London for too long. However, there are a few things that have taken some time to get used to, such as being fully nude in the sauna with colleagues or the ‘awkward silence’ that is typical of lunch hour, something the Finns call ‘ruokarauha’ (food peace). Nevertheless, I look forward to learning more about Finnish culture and I see a lot of potential from a relatively unknown country leading the world in terms of social welfare, education and equality.

Left: taken in December from the coastal line on Lauttasaari, one of Helsinkis’ largest islands, Middle: taken in autumn on a pathway in Munkkiniemi. Right: historical buildings of Finland as part of the museum on Seurasaari.

What’s all the hype around oncolytic viruses?

But back to the point. Why I moved to this Nordic country– to contribute to the advancement of immunotherapy, specifically oncolytic viruses. There is a lot of hype around this technology due to its potential as a cancer therapy, particularly in patients unresponsive to immune checkpoint inhibitors (antibodies that prevent useful immune cells from becoming exhausted). This is because they offer a versatile tool for killing cancer, in that they selectively replicate in tumour cells and can express transgenes that augment cytotoxicity and alter the tumour microenvironment to enhance immune-mediated tumour killing both at primary and secondary sites of disease.

 The lytic activity of oncolytic viruses comes with the natural life cycle of the virus, after several rounds of viral replication; the cell bursts resulting in the release of new virions and the infection of the surrounding cells. The cell lysis releases tumour-specific antigens that trigger both the innate and adaptive immune system, this process is described as altering the tumour microenvironment from immunologically cold to hot. This status change is associated with an improved therapeutic response and more durable remission in patients. There are almost endless amounts of possible modifications to the various families of viruses. Consequently, modified oncolytic viruses are attractive for use in combination with other systemically delivered therapies and agents as a treatment regime is tailorable to the malignant phenotype. This offers the benefit of providing resolution to the large, underserved population of patients who fail to respond to existing therapies.

As an example, I recently enjoyed reading about the use of a modified Vaccinia virus engineered to produce Leptin, a metabolic modifier usually associated with appetite. Metabolic insufficiency is a significant barrier for sustained antitumor immunity and is a typical profile of immune cells found in the tumour microenvironment. This is largely a result of the highly glycolytic profile of the tumour– also known as the Warburg effect. The paper showed that by using this leptin expressing virus, they were able to improve the metabolic capacity of T- cells and therefore put them back in the driver’s seat. This paper is one of a few that have taken a different approach to the traditional virus design of using cytokines or costimulatory molecules. I hope that this will trigger a wave of new generation of viruses that target the metabolic hallmark to improve antitumor efficacy.


TILT-123 for head and neck squamous carcinoma

But what exactly am I doing? I am funded by a Marie-Curie early-stage researcher EU training grant, designed with the goal of using state-of-the-art single cell sequencing technologies, to deepen our understanding of the therapeutic mechanisms behind oncolytic viruses and investigate ways to improve production. TILT Biotherapeutics Ltd is a spin out from the University of Helsinki, with a highly promising patented oncolytic adenovirus engineered to enhance T cell therapies – TILT-123.

The most significant modifications to TILT-123, includes the ability of the virus to produce two powerful cytokines, IL-2 and TNFa, which enhance T- cell proliferation and exert direct cytotoxicity to the tumour and associated vasculature respectively. This combined with the lytic life cycle of the virus, makes for a power weapon of mass destruction. The efficacy of TILT-123 has already been validated in other types of cancer using animal models, with the virus reaching up to a 100 % cure rate in combination with tumour infiltrating lymphocytes (local T- cells familiar with the tumour from the patient itself). Subsequently TILT Biotherapeutics Ltd will start a first-stage clinical trial this year for melanoma in combination with adoptive transfer of tumour infiltrating lymphocyte, so watch this space!

My first task will be to extend TILT123’s portfolio of different cancer types, starting with squamous cell carcinomas of the head and neck in combination with checkpoint inhibitors. I will validate its therapeutic efficacy using a syngeneic murine and Syrian Hamster model. The former provides us with a detailed insight into immunological mechanisms, while Syrian hamsters, as a semipermissive model, provides additional insight on how the lytic properties of TILT-123 contributes to the overall efficacy and immunological landscape. Additionally, I will look into optimising the manufacturing of this immunotherapy, by identifying genetic profiles of producer cells associated with an increased yield and vector quality. Some initial challenges have included developing the Syrian Hamster model. Since this is not a commonly used model, the availability of many tools required for experimental procedures and characterisation is limited.

Altogether, Finland has been a fantastic experience, both, as a culture shock but also the skills I have already learned as a scientific researcher. One of the most important traits for Finns is something called “sisu”, a concept described as stoic determination with no equivalent in English. Over the next four years, I hope to find my sisu and use this to work together with a bunch of eager scientists to contribute to the field and improve the world.

Posted by James.

What is STACCATO?

As the first post on the STACCATO Early Stage Researcher’s (ESRs) Blog, I think it is a good idea to introduce the premise of what this Marie Skłodowska-Curie Actions (MSCA) funded project is all about and what my role is as one of its 11 ESRs/PhD Students.

Before continuing, I want to introduce what essentially underpins STACCATO, which is single cell analysis. The definition of a staccato is musical composition performed with each note sharply detached or separated from the others. To bring this into context with our project: We are separating and taking measurements from thousands of individual cells to study their intricate differences just like the notes in a staccato.

STACCATO is an initiative funded by the EU’s prestigious Marie Skłodowska-Curie Actions (MSCA) Innovative Training Network (ITN) programme. Prof Colin Clarke designed it to bolster Europe’s innovation capacity and leadership in the biopharmaceutical manufacturing sector.

Growing Europe’s pool of skilled scientists

If Europe’s biopharmaceutical sector is to continue to flourish and remain competitive in a booming international market, it is urgent that we invest in expanding our pool of highly skilled scientists and pioneer new analytical approaches to enable rapid design of efficient bioprocesses for biopharmaceutical production.

As an ITN, STACCATO has enabled the partnership of industry and academic experts from all over Europe to provide 11 ESRs (PhD Student researchers), of which I am one, with world-class intersectoral training and upskilling. This is to prepare us to tackle some of the most significant challenges facing the biopharma manufacturing industry.

The STACCATO ESRs and a few of our brilliant supervisors at the 1st Annual Industry-Academia Network Training Week together in Dublin, Ireland.

One of the unique advantages of MSCA funded training networks is that ESRs have great opportunity to travel to partnered institutes for scientific collaboration throughout Europe, taking take part in training courses, presenting research at conferences and gaining invaluable industry experience during Secondments – extended international placements. I’m currently situated at Ireland’s National Institute for Bioprocessing Research and Training (NIBRT), but my first Secondment planned in 2020 will allow me to conduct some of my PhD research at the Paul-Ehrlich-Institut (PEI), another world-class institute for biomedicines research, in Langen, Germany.

What is single cell analysis and why do we care?

The underlying vision uniting the STACCATO ESRs, Principal Investigators and industry partners is to utilise high-resolution data captured from thousands of single cells to enhance and develop new manufacturing methods for biological medicines.

The majority of studies in cell biology are performed with large bulk numbers of cells, often over a million per sample. Although this is a powerful tool, by averaging over enormous cell populations we ignore the fact that no two cells are exactly alike even if they’re of the same type. Conventional bulk sample measurements overlook many important details that only become clear at single cell resolution. This could be likened to tasting a fruit smoothie and trying to identify everything in it; versus tasting each individual piece of fruit before everything was blended together.

Left Photo: by Tara Evans on Unsplash. Right Photo: by Rebecca Hansen on Unsplash

This ability to comprehensively profile the cellular heterogeneity of single cells is crucial to advancing our understanding of cell behaviours, disease mechanisms and identify previously elusive biomarkers and drug targets.

My project as a STACCATO ESR

Each ESR will focus on a different research aspect within STACCATO, such as developing single cell analysis methods or improving cell line performance, but all projects share the common goal to enhance bioprocessing development.
Here at NIBRT, I will be developing and applying methods for Single Cell Proteomics analysis to help in understanding the heterogeneity of industrially important cell lines utilized for biopharmaceutical manufacturing. The field of Proteomics encompasses all aspects of protein study, including protein identification, structural analysis, abundance and the qualitative and quantitative characterization of post translational modifications (PTMs). One of the most commonly used methods of doing this is called shotgun or bottom-up proteomics, whereby the proteins are first enzymatically digested into peptide fragments prior to separation and analysis by liquid chromatography-mass spectrometry (LC-MS), following this, specialised bioinformatics pipelines and vast databases are used to identify the proteins from their peptide sequences.

Mass Spectrometry warrants its own blog post, so I will go into detail on how this works and in relation to proteomics at a later time!

A science in its infancy

Single Cell Proteomics (SC-Proteomics) hasn’t quite been possible until recently, and it’s still in its infancy when compared to the powerful single cell RNA-sequencing methods that are used by thousands of labs around the world. SC-Proteomics is difficult because there is very little protein in a single cell (around 200 picograms!) and no equivalent of DNA amplification for proteins – a major bottleneck is losing what little protein you had throughout the sample preparation process.

Nikolai Slavov’s Lab at NEU Boston have developed SCoPE-MS (Single Cell ProtEomics by Mass Spectrometry), a method which tackles this issue by chemically labelling the individual cells and a ‘carrier channel’ of around 100 cells to absorb some of the protein losses during processing, which would otherwise be incurred by the single cell protein samples. However, there is still room for improvement here!

The different chemical labels of individual cells and carrier channel allows the relative quantification of protein for each cell to be seen in the MS data. I will apply similar methods to study CHO (Chinese Hamster ovary) cell heterogeneity, and attempt to make improvements in recovery of the cellular proteome and sample throughput (currently it takes around 10 days of instrument time to analyse around 1000 cells – that’s a long time!).

More to come

I hope that if you made it to the end of this; that you understand the gist of STACCATO, Single Cell Analysis and a little bit about my project at NIBRT. I will try to update here with posts that you may find interesting or educational on Mass Spectrometry, Single Cell Proteomics and gossip about the other ESRs. I am greatly looking forward to blog posts from the 10 talented and interesting people that I’ll be collaborating with over the next few years on the STACCATO Project.

Posted by Marco. R