From 018aa39bb7b3c0fe1ed97d0c8de80d098180050d Mon Sep 17 00:00:00 2001
From: Kaya <kaya.lange@uni-bielefeld.de>
Date: Wed, 25 Sep 2024 20:32:52 +0200
Subject: [PATCH] changes timeline 3
---
src/data/hptimelinedata.tsx | 10 +++++-----
1 file changed, 5 insertions(+), 5 deletions(-)
diff --git a/src/data/hptimelinedata.tsx b/src/data/hptimelinedata.tsx
index b30da5e9..35520f7e 100644
--- a/src/data/hptimelinedata.tsx
+++ b/src/data/hptimelinedata.tsx
@@ -425,15 +425,15 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
interviewtabid: "mattijsinv",
cardtext: "",
language: "en",
- quote: "I believe the challenge lies in the process when your cell has to repair the new DNA strand",
- aimofcontact: "Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research1. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K). ",
- insights: "The insights gained from this interview proved invaluable in shaping our subsequent mechanistic approaches. Besides his comprehensive perspective of the significant advancements in the field of prime editing, Mattijs engaged us in a constructive dialogue, offering a critical assessment of our own ideas and approaches. ",
- implementation: "Mattijs explained why he did not try to target F508del. Of all the possible applications of Prime Editing in gene therapy, he told us, the CFTR F508del mutation is the most obvious target, but no substantial editing efficiency could be achieved at this target yet. He further elaborated that this might be due to DNA packaging, low cutting efficiencies at that locus, the AT rich region surrounding F508del and also possible intramolecular binding of the pegRNA, but we do not really understand the mechanistic reason yet. Additionally, he mentioned a talk given by David Liu,[Link] the principal investigator behind prime editing that helped us to consider further novel advancements in F508del-specific CFTR-treatment and include them into our project. Beside the clinical context, we discussed different methods of testing edited cells for CFTR reconstitution, such as measurements using the Ussing chamber, organoid swelling assays and halide sensitive eYFP fluorescence assays. Additionally, we discussed the use of stem loops for protection of the pegRNA, which we then included into our own pegRNA design. Lastly, we officially cooperated with the research group and the university: Mattijs was willing to share two different HEK293T cell lines [Link] stably overexpressing the CFTR wild-type and F508del proteins. We also visited Mattijs in his laboratory in Leuven to discuss this further. During our visit we discussed different downstream applications on proteomic, genomic and electrophysiological levels. Besides antibody staining of the CFTR-3HA protein, we asked for his insights on patch clamp measurements and DNA sequencing as well as the advantages and disadvantages of different delivery systems for prime editors. Additionally, Mattijs provided more details into the CFTR overexpressing cell lines, and we received guidance on handling of these kinds of cells. Until the end of the project, we maintained communication with Mattijs Bulcaen via email for further questions and gave updates concerning our project to implement the feedback loop according to our integrated Human Practice Framework.",
+ quote: "[…] Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure.",
+ aimofcontact: "Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research[1]. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K).  ",
+ insights: "The interview with Mattijs was valuable for us in a lot of ways. At that point in the project we were starting to design the components of our prime editor, but we were lacking a broader overview over the state of the field. Mattijs gave us this insight, mentioning techniques like PE3b systems, dsgRNAs and a talk given by David Liu,[Link] the principal investigator behind prime editing that helped us to consider further novel advancements in in Prime Editing and include them into our project. He discussed with us the difficulties that might await us when targeting the CFTR F508del deletion and mentioned that insertions of all the edits possible with prime editing are the hardest to make, the recognition of edits in the region might attract mismatch repair systems and the chromatin organization might negatively impact prime editing efficiency. Also, we learned a lot about how to design our pegRNAs, with important inputs being the 3’ stem loop motif trevopreQ1 used by Mattijs in his publication and the suggestion to use prediction tools to evaluate sgRNA spacer cutting efficiency. We reviewed our approach of testing pegRNAs using the PEAR reporter system and Mattjis recommended to use HEK cell lines for screening because of their easy handling and naturally impaired mismatch repair system. ",
+ implementation: "The inputs given by Mattijs directly impacted our design choices for multiple parts of the project. For the pegRNA design, we decided to use the same 3’ motif as Mattijs had used and also, like he suggested, checked our spacer candidates for predicted cleavage efficiency. Also we used HEK cells for screening our pegRNAs. We looked further into PE systems that influence cellular mismatch repair (such as PE4) and tried to include these into our design. ",
/* references:
<ol>
/*<!-- Citation num 1--> */
},
- {
+ {
vorname: "Nicole",
nachnname: "Friedlein",
job: "Research group on fundamental rights",
--
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