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TY  - THES
AU  - Hüllen, Fabian
TI  - Development and implementation of a genome-wide CRISPR/Cas9 screen for identification of cellular factors affecting recombinant adeno-associated virus (rAAV) production
PB  - RWTH Aachen University
VL  - Dissertation
CY  - Aachen
M1  - RWTH-2024-10018
SP  - 1 Online-Ressource : Illustrationen
PY  - 2024
N1  - Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
N1  - Dissertation, RWTH Aachen University, 2024
AB  - Advances in the development of gene therapy pharmaceuticals offer a great opportunity to treat or potentially cure previously untreatable diseases. Recombinant adeno-associated viral vectors (rAAV) are emerging as the most promising gene delivery tools for in vivo gene therapies, as demonstrated by numerous recent approvals of rAAV-based therapeutics. Despite advantages such as low immunogenicity and the ability for long-term episomal expression, the limited scalability of existing rAAV production systems is a persistent challenge. To address this problem, we have developed a screening method to identify cellular genes that either supports or repress rAAV production in a fully stable, plasmid- and helper virus-free rAAV suspension packaging cell line. We created an innovative genome-wide Lenti-AAV-CRISPR plasmid screening library (pLAC library) containing 68,563 unique sgRNAs. After packaging this library into lentiviral particles, we transduced a human stable AAV packaging cell line, adapted for the constitutive expression of Cas9. The rAAVs produced by the transduced packaging cells were then analysed by next generation sequencing. This revealed a large number of over- or underrepresented sgRNAs, indicating that inactivation of the corresponding cellular genes had a positive or negative impact on rAAV production. To validate the screening method initially, genes with a putative positive impact on rAAV production were selected and inactivated in individual rAAV packaging cell lines. Subsequently, an AAV transfer vector was integrated into the cells by lentiviral transduction to generate stable rAAV production cell lines, which allowed the investigation of the potential target genes in a fully stable and inducible production system. In a second project, we have screened the AAV producer cells to identify novel integration sites for targeted and reusable integration of transgene cassettes enabling strong and stable overexpression. Such integration sites could be used, for example, to overexpress cellular genes which could have a potentially beneficial effect on rAAV production. Stable integration of transgenes into host cells is a widely used strategy for cell line development. Typically, this is accomplished through random transgene integration, requiring an elaborate and time-consuming screening process to identify clones with long-term stability and high expression levels. In contrast, utilizing a well-characterized chromosomal locus allows a much faster and reliable generation of such clones. Using CRISPR/Cas9-mediated homologous recombination of a reporter cassette, we identified a genomic region immediately downstream of the highly conserved ACTB locus encoding for actin beta. To assess the relative expression strength compared to other loci, additional clones with single copy integrations were generated in a non-targeted screening approach employing a random expression cassette integration. Analysis of different clones revealed relatively high expression of single copy transgene insertions into the ACTB locus, demonstrating stability over multiple passages. Subsequent single copy insertion of a C1 esterase inhibitor expression cassette into this site resulted in strong expression. In conclusion, the results pave the way for the development of an optimized, fully scalable rAAV production cell line. The targets investigated so far show great potential for the targeted optimization of rAAV productivity. In addition, the over- or under- represented sgRNAs identified in the screening provide further promising targets that still need to be investigated. With the identification of the region downstream of the ACTB locus, we have also created the basis for the targeted integration of transgenes such as genes with a positive influence on rAAV production, to optimize cellular production systems. Moreover, both the screening method and the described locus offer potential applications for other cell lines and viruses, which makes the development interesting beyond the AAV field.
LB  - PUB:(DE-HGF)11
DO  - DOI:10.18154/RWTH-2024-10018
UR  - https://publications.rwth-aachen.de/record/995603
ER  -