Natural killer (NK) cell therapies hold great promise for cancer treatment; however, donor-to-donor heterogeneity in the ex vivo expansion process remains a critical bottleneck in their supply. This study aimed to identify factors influencing donor variability in a 2-week-long ex vivo NK cell expansion from peripheral blood mononuclear cells, analyzed across three donors. Single-cell transcriptomics was applied to investigate the distribution of cell types and phenotypes, as well as trajectory inference and differential gene expression. Our results identified that several factors were associated with the variability in the final NK cell fraction and expansion, and that their influence was prevalent between culture days 3 and 8. Compared to a high final NK cell expansion, a culture with a low final NK cell expansion exhibited an upregulation of some stress and inflammatory genes and an increase in one specific subcluster of NK cells already on culture day 3. It showed a low score of CD56^Bright CD16^– phenotype and a high score of CD56^Dim CD16⁺ phenotype. It also had a decreased presence of cytotoxic CD8⁺ Tm cells. Among the observed subclusters of CD8⁺ Tm cells, it exhibited a higher presence of a subcluster associated with a less differentiated and less cytotoxic phenotype, as well as a lower prevalence of a subcluster associated with chemokine and cytotoxic genes. Finally, it had a major expansion of one of the CD8⁺ Tm cells subclusters annotated as NK-like T cell and characterized by a high CCR5 mRNA expression, while the levels of CCL3, CCL4 and CCL5 mRNA were downregulated. The present findings point toward a potential link between CCL signaling and improved NK cell expansion performance, including possible markers for further investigations, and suggest future strategies to increase the final NK cell fraction and expansion based on donor-specific markers.

A paradigm shift in the treatment of many genetic and acquired diseases is

underway. At the heart of this change are gene and cell therapies. They offer the

potential to cure many conditions that previously carried a poor prognosis. These

life changing therapeutics make use of complex biological modalities to target

underlying disease mechanisms, offering precise and effective treatments. The

complexity of these products, however, presents a barrier to their widespread

accessibility, in part due to the high cost of manufacturing.

Gene therapies use heavily modified viruses, called viral vectors, to insert

genetic material into a patient’s cells to restore normal function. The most used

viral vector in gene therapy is based on Adeno-associated virus (AAV). Currently

a major bottleneck for AAV-based therapeutics is their production. Not only does

the high manufacturing cost impact the accessibility of these treatments, the

limited production capacity reduces their availability.

Cell therapy is a broad category of innovative treatments that use cells as the

therapeutic substance. A category within cell therapy is immune cell therapy,

which uses cells from the body’s immune system to combat a wide range of

conditions, such as cancer, infectious disease, and autoimmune disorders. A

promising candidate in immune cell therapy are natural killer (NK) cells. These

cells are highly effective in the recognition and elimination of tumor cells, making

them a valuable tool in cancer immunotherapy. Though, like AAV-based

therapeutics, inefficiencies in their production limits their accessibility and

availability.

The aim of this thesis is to investigate these production bottlenecks and

provide potential solutions to overcome them. The first section investigates

methods to improve the scalability and efficiency of recombinant AAV (rAAV)

production, using techniques such as continuous manufacturing and

intensification. Continuous production is particularly well suited to the production

of rAAVs due to its ability to address critical challenges encountered during the

manufacturing process. Intensification offers an interesting complementary

approach to increasing the efficiency of rAAV manufacturing, by producing more

in the same amount of space. In papers I and II proof-of-concept systems were

developed that enabled the several fold increase in rAAV production.

The second part of this thesis focuses on the use of single-cell RNA

sequencing (scRNA-seq) to study processes in rAAV and NK cell production.

scRNA-seq is an advanced tool that gives an immense wealth of data that can be

used to gain deep insights into production processes. Previously, this tool has seen

limited use in process development, but the outcomes of papers III and IV show 

that it can be highly effective in this setting. Paper III highlighted a phenomenon

in the production of rAAV that severely limits production efficiency. In fact,

strategies were proposed that could potentially improve the production capacity

40-fold. Paper IV studied the donor-to-donor heterogeneity of a manufacturing

process for NK cells and identified key parameters that have the potential to

predict manufacturing performance. Additionally, these parameters could

potentially be used to not only monitor but to control the process, improving

yields.

This thesis investigates a wide array of topics in the field of gene and cell

therapies, from adherent cell culture to single-cell transcriptomics. It covers

aspects in process development of both gene and cell therapies, provides strategies

for the several-fold improvement of current rAAV manufacturing systems,

highlights a phenomenon holding back further advances in rAAV production and

suggests key process parameters that can be used to track and potentially improve

the performance of NK cell manufacturing.

Det pågår just nu ett paradigmskifte i behandlingen av många genetiska och

förvärvade sjukdomar. I centrum för denna förändring står gen- och cellterapier.

Dessa terapier har potential att bota många tillstånd som tidigare hade en dålig

prognos. Dessa livsförändrande terapier använder komplexa biologiska

modaliteter för att rikta sig mot underliggande sjukdomsmekanismer, vilket

möjliggör mer precisa och effektiva behandlingar. Komplexiteten hos dessa

produkter utgör dock en barriär för deras breda tillgänglighet, delvis på grund av

de höga tillverkningskostnaderna.

Genterapier använder kraftigt modifierade virus, så kallade virala vektorer,

för att införa genetiskt material i en patients celler och därmed återställa normal

funktion. Den mest använda virala vektorn i genterapi är baserad på

adenoassocierat virus (AAV). För närvarande är produktionen en stor flaskhals för

AAV-baserade terapier. De höga tillverkningskostnaderna påverkar inte bara

dessa behandlingars tillgänglighet, utan den begränsade produktionskapaciteten

minskar också tillgången.

Cellterapi är en bred kategori av innovativa behandlingar där celler används

som den terapeutiska substansen. En underkategori inom cellterapi är

immuncellterapi, där celler från kroppens immunsystem används för att bekämpa

en rad olika tillstånd, såsom cancer. En lovande kandidat inom immuncellterapi är

så kallade natural killer-celler (NK-celler). Dessa celler kan mycket effektivt

känna igen och eliminera tumörceller, vilket gör dem till ett värdefullt verktyg

inom cancerimmunterapi. Precis som för AAV-baserade terapier begränsar dock

produktionen tillgängligheten av och tillgången på dessa livsförändrande

behandlingar.

Syftet med denna avhandling är att undersöka dessa

produktionsbegränsningar och föreslå potentiella lösningar för att övervinna dem.

Den första delen undersöker metoder för att förbättra skalbarheten och

effektiviteten hos rekombinanta AAV (rAAV)-vektorer genom teknikerna för

kontinuerlig produktion och intensifiering. Kontinuerlig produktion kan vara

särskilt väl lämpad för produktionen av rAAV på grund av utmaningar vid

uppskalning. Intensifiering erbjuder en intressant parallell metod för att öka

effektiviteten i rAAV-tillverkning genom att producera mer i samma volym. I

artikel I och II utvecklas proof-of-concept-system som möjliggör en flera gånger

större rAAV-produktion.

Den andra delen av denna avhandling fokuserarfokuserade på användningen

av så kallad singelcellstranskriptomik för att studera processer inom rAAV- och

NK-cellsproduktion. Singelcells-RNA-sekvensering (scRNA-seq) är ett avancerat verktyg

som ger en enorm mängd data och kan användas för att få djup insikt i

produktionsprocesser. Tidigare har detta verktyg använts i begränsad omfattning

inom processutveckling, men resultaten från artikelartiklarna III och IV visar att

det kan vara mycket effektivt i detta sammanhang. Artikel III belyser ett fenomen

inom rAAV-produktion som begränsar produktionseffektiviteten. Här föreslås

strategier som potentiellt kan göra produktionskapaciteten 40 gånger större.

Artikel IV studerarstuderade variabiliteten i en produktionssprocess för NK-celler.

Nyckelparametrar identifieras som har potential att förutsäga

tillverkningsprocessens prestanda flera veckor i förväg. Dessa nyckelparametrar

skulle dessutom kunna justeras för att förbättra produktionsprocessen.

Denna avhandling berör en rad ämnen inom gen- och cellterapifältet, från

adherent cellodling till singelcellstranskriptomik. Den täcker aspekter av

processutveckling för både gen- och cellterapier, presenterarpresenterat strategier

för mångdubbel förbättring av nuvarande rAAV-tillverkningssystem, belyser ett

fenomen som begränsar ytterligare framsteg inom rAAV-produktion och föreslår

nyckelparametrar som kan användas för att övervaka och potentiellt förbättra

prestandan för produktionen av NK-celler.

The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successfully demonstrated in a process where single cells were first expanded, then cell aggregates were formed by dilution into a different medium 1 day before triple plasmid transfection was conducted. An alternative approach for the STR inoculation using a seed taken from a high cell density perfusion (HCDP) culture was also investigated. It was, however, found that the spent medium of the HCDP inhibited the transfection of HEK293T cell aggregates, which was confirmed when testing with single-cell suspension culture. The formation of aggregates in shaken multi-well plates was also investigated to develop a screening system using the average power input as a scale-down criterion, which revealed that cell aggregates could be generated in 12-well plates, however with a larger size than in a STR. Taking into account the reported higher rAAV production of adherent cells in comparison with single cells for triple-plasmid transfection, HEK293T cell aggregates can possibly surpass single-cell suspension in space–time rAAV yield. The formation of HEK293T cell aggregates in a STR system offers a promising approach for scaling up and intensifying rAAV production by triple-plasmid transfection, in comparison with traditional 2D scale-up methods.

The popularity of rAAV vectors in gene therapy are placing a burden on current production systems. To improve the accessibility of these life changing treatments, increases in production yields and a reduction in the cost-of-goods are needed. Transient transfection is the most common way to introduce rAAV-encoding plasmids to producer cells but it suffers from significant drawbacks such as low and inconsistent yields as well as high cost due to its need for plasmid DNA. This study aims to address the low yield of transient transfection-based rAAV production through advanced methods in process characterization. Adherent and suspension cultures of a HEK293T cell line were triple-transfected for rAAV9 production using polyethylenimine (PEI). Samples were taken at various times post-transfection for analysis with bulk and single-cell transcriptomics. It was revealed that 46% of the cells lacked transcripts of genes from at least one plasmid, indicating that a significant proportion of the cells did not have the genes necessary for rAAV9 production. Among the remaining 54% of the cells expressing genes from all three plasmids, only 8% showed high plasmid gene expression. Flow cytometric analysis of intracellular rAAV9 confirmed these results by showing that only similar to 3% of cells contained assembled rAAV9 capsids. Titre analysis by qPCR of the supernatant and lysate of the producer cells indicated an average culture performance of 10(13) vg/L. Analysis of the single-cell transcriptomic data showed that a significant proportion of cells that had high plasmid gene expression were in the S-phase. Trajectory inference highlighted that genes involved in the G2-M phase transition, immune response, and protein unfolding were differentially expressed at the branch point between high and low plasmid expression. This study reveals a significant bottleneck in the transient transfection-based production of rAAV. With less than 5% of cells producing rAAV, significant improvements in titres can be achieved if this fraction can be increased. Moreover, regulation of the cell-cycle, inhibition of the immune response, and alleviating protein misfolding all potentially offer the key to enabling these life changing treatments to reach a wider audience.

Adeno-associated virus vectors (AAV) are reported to have a great potential for gene therapy, however, a major bottleneck for this kind of therapy is the limitation of production capacity. Higher specific AAV vector yield is often reported for adherent cell systems compared to cells in suspension, and a microcarrier-based culture is well established for the culture of anchored cells on a larger scale. The purpose of the present study was to explore how microcarrier cultures could provide a solution for the production of AAV vectors based on the triple plasmid transfection of HEK293T cells in a stirred tank bioreactor. In the present study, cells were grown and expanded in suspension, offering the ease of this type of operation, and were then anchored on microcarriers in order to proceed with transfection of the plasmids for transient AAV vector production. This process was developed in view of a bioreactor application in a 200 mL stirred-tank vessel where shear stress aspects were studied. Furthermore, amenability to a continuous process was studied. The present investigation provided a proof-of-concept of a continuous process based on microcarriers in a stirred-tank bioreactor.

The popularity of rAAV vectors in gene therapy are placing a burden on current production systems. To improve the accessibility of these life changing treatments, increases in production yields and a reduction in the cost-of-goods are needed. Transient transfection, while the most popular method of rAAV production, suffers from significant drawbacks such as low and inconsistent yields as well as high cost due to its need for plasmid DNA. This study aims to address the low yield of transient transfection-based rAAV production through advanced methods in process characterization. Adherent and suspension cultures of a HEK293T cell line were triple-transfected for rAAV9 production using polyethylenimine (PEI) as transfection reagent. Samples were taken at various times post transfection for analysis with bulk and single cell transcriptomics. It was revealed that 46% of cells lacked transcripts of genes from at least one plasmid, indicating that a significant proportion of the cells did not have the genes necessary for rAAV9 production. Among the remaining 54% of the cells expressing genes from all three plasmids, only 8% showed high plasmid gene expression. Flow cytometric analysis of intracellular rAAV9 confirmed these results by showing that only ~3% of cells had assembled rAAV9 capsids. Titre analysis by qPCR in supernatant and lysate indicated average culture performance of 1013 vg/L. Analysis of the single cell transcriptomic data showed that a significant proportion of cells that had high plasmid gene expression were in the S-phase. Trajectory inference highlighted that genes involved in the G2-M phase transition, immune response, and protein unfolding were differentially expressed at the branch point between high and low plasmid expression. This study reveals a significant bottleneck in the transient transfection-based production of rAAV. With less than 5% of cells producing rAAV, significant improvements in titres can be achieved if this fraction can be increased. Regulation of the cell-cycle, inhibition of the immune response, or alleviating protein misfolding all potentially offer the key to enabling these life changing treatments to reach a wider audience.

Natural killer (NK) cell therapies hold great promise for cancer treatment; however, donor-to-donor heterogeneity in the ex vivo expansion process remains a critical bottleneck in their supply. This study aimed to identify factors influencing donor variability in a two-week long ex vivo NK cell expansion from PBMCs, analyzed across three donors. Single-cell transcriptomics was applied to investigate the distribution of cell types and phenotypes, as well as trajectory inference and differential gene expression. Our results identified that several factors were associated with the variability in the final NK cell fraction and expansion, and that their influence was prevalent between culture day 3 and 8. Compared to high final NK cell fraction, a culture with low final NK cell fraction exhibited an upregulation of some stress and inflammatory genes and an increase in one specific subcluster of the NK cells already on culture day 3. It showed a low score of CD56 Bright CD16- phenotype and high score of CD56Dim CD16+ phenotype. It had also an increased presence of cytotoxic CD8+ Tm cells. Among the observed subclusters of CD8+ Tm cells, it exhibited a higher presence of a subcluster associated with a less differentiated and less cytotoxic phenotype as well as a lower prevalence of a subcluster associated with chemokine and cytotoxic genes. Finally, it had  a major expansion of one of the CD8+ Tm cells subclusters annotated as NK-like T cell and characterized by a high CCR5 mRNA expression while the levels of CCL3, CCL4, and CCL5 mRNA were downregulated. The present findings point towards a potential link between CCL signaling and improved NK cell expansion performance, including possible markers for further investigations, and suggest future strategies to increase the final NK cell fraction and expansion based on donor-specific markers.