Electronic Resource
Development of transposon-mediated gene delivery systems with improved safety for the use in gene and cell therapy
| العنوان: | Development of transposon-mediated gene delivery systems with improved safety for the use in gene and cell therapy |
|---|---|
| المؤلفون: | Amberger, Maximilian |
| بيانات النشر: | 2024-01-08 |
| نوع الوثيقة: | Electronic Resource |
| مستخلص: | Chapter I of this work addressed the piggyBac (PB) transposon system, a non-viral genome engineering tool that is capable of efficiently performing stable integration of DNA sequences into a target cells genome and has already been used in clinical trials. However, the PB transposase has the problematic property of preferentially integrating transposons near transcriptional start sites (TSSs). This increases the likelihood of causing genotoxic effects, limiting its potential use as a tool in clinical applications. It has been shown in the past that the PB transposase shows physical interactions with BET proteins (e.g. BRD4) through Co-IP experiments. Representatives of these proteins are part of the transcriptional activation complex and are abundant at TSSs. Accordingly, it was previously proposed that this interaction is the underlying cause for the biased integration preference. For the first chapter of this thesis, the goal was to disrupt this interaction potentially modifying said integration preference. A secondary structure hypothesized to be mainly responsible for said interaction was extensively mutated resulting in several PB variants that were analyzed for their interaction capacity through a series of Co-IP experiments with BRD4. In total, seven substitutions were identified (E380F, V390K, T392Y, M394R, K407C, K407Q, and K407V) which exhibited reduced interaction capacity with BRD4. Each of the aforementioned mutants were used to generate integration libraries and, through NGS, it was determined if the integration preferences of the respective mutants had changed. In the immediate range 200 base pairs up- and downstream from known TSSs all mutants used exhibited a reduced integration bias. At a wider observation window 3 kbp up- and downstream from TSSs, further mutants with the substitutions M394R, T392Y and V390K showed a reduction in integration frequency of 17.3%, 1.5% and 5.4%, respectively, compared to the wildtype. Of particular note was the M394R |
| مصطلحات الفهرس: | ddc:570, doctoralthesis, doc-type:doctoralThesis |
| URL: | |
| الاتاحة: | Open access content. Open access content http://publikationen.ub.uni-frankfurt.de/home/index/help#policies info:eu-repo/semantics/openAccess |
| ملاحظة: | application/pdf English |
| Other Numbers: | DEJWG oai:publikationen.ub.uni-frankfurt.de:81592 urn:nbn:de:hebis:30:3-815924 1417380158 |
| المصدر المساهم: | JOHANN WOLFGANG GOETHE UNIV From OAIster®, provided by the OCLC Cooperative. |
| رقم الانضمام: | edsoai.on1417380158 |
| قاعدة البيانات: | OAIster |
| الوصف غير متاح. |