Advanced Process Development in Gene and Cell Therapies

QC 2025-02-19

Abstract

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.

urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-360154