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@PHDTHESIS{Hllen:995603,
author = {Hüllen, Fabian},
othercontributors = {Huber, Michael and Scheer, Nico and Pradel, Gabriele},
title = {{D}evelopment and implementation of a genome-wide
{CRISPR}/{C}as9 screen for identification of cellular
factors affecting recombinant adeno-associated virus
(r{AAV}) production},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2024-10018},
pages = {1 Online-Ressource : Illustrationen},
year = {2024},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2024},
abstract = {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.},
cin = {513000-3 ; 924720 / 160000},
ddc = {570},
cid = {$I:(DE-82)513000-3_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2024-10018},
url = {https://publications.rwth-aachen.de/record/995603},
}
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