Gene therapy for STAT3-Associated Autosomal-Dominant Hyper-Ige-Syndrome (AD-HIES) using RNA-guided nucleases
Abstract: Autosomal dominant hyper-IgE syndrome (AD-HIES) due to mutations in the STAT3 locus is characterized by high serum IgE, recurring staphylococcal skin abscesses, and recurrent pneumonia. While allogeneic hematopoietic stem cell transplantation is generally not recommended due to potential severe side effects, adoptive transfer of autologous T cells, in which the mutated STAT3 allele is inactivated or corrected, may represent a promising alternative. We have designed allele-specific CRISPR/Cas nucleases to target the DNA sequence corresponding to some of the most common STAT3 mutations (H58Y, C328_P330dup, R382W, V463del and V637M). After identification of the best performing CRISPR/Cas candidates in an episomal reporter assay, the selected designer nucleases were tested on genomic level in a human reporter cell line harboring integrated copies of the mutated STAT3 locus. Allele-specific nucleases disrupted 30%-70% of the mutant sequences without altering the endogenous wild-type STAT3 gene. When applied to patient-derived peripheral blood mononuclear cells (PBMCs), the mutation-specific CRISPR/Cas nucleases were able to selectively disrupt up to 62% of the mutated STAT3 alleles, as assessed by next generation sequencing. A thorough analysis of STAT3 target gene expression by ddPCR suggests that monoallelic STAT3 expression is not sufficient to restore functionality. These results are in line with a recent report of a patient harboring a STAT3 mutation resulting in haploinsufficiency, suggesting that the inactivation of the dominant-negative allele in AD-HIES cells might not restore functionality. To overcome this limitation, we have established an alternative strategy based on the correction of the selected dominant negative STAT3 mutations by combining the mutation-specific CRISPR/Cas we have developed and single stranded oligodeoxynucleotides (ssODNs) as templates for correction. While we could edit up to 12% of the alleles in healthy cells, this approach led to unexpectedly high toxicity in patient-derived cells. Further experiments will be crucial to establish the proper conditions for safe manipulation and editing of patient-derived cells in order to reveal the potential of this approach to restore the functionality of affected cells and to represent a novel therapeutic option for AD-HIES patients in the future
- Standort
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Deutsche Nationalbibliothek Frankfurt am Main
- Umfang
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Online-Ressource
- Sprache
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Englisch
- Anmerkungen
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Universität Freiburg, Dissertation, 2019
- Schlagwort
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Nucleases
Gene therapy
- Ereignis
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Veröffentlichung
- (wo)
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Freiburg
- (wer)
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Universität
- (wann)
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2019
- Urheber
- Beteiligte Personen und Organisationen
- DOI
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10.6094/UNIFR/151206
- URN
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urn:nbn:de:bsz:25-freidok-1512062
- Rechteinformation
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Der Zugriff auf das Objekt ist unbeschränkt möglich.
- Letzte Aktualisierung
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25.03.2025, 13:54 MEZ
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Beteiligte
Entstanden
- 2019
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