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Hematopoietic stem cell genetherapy with CRISPR/Cas9 inmetachromatic leukodystrophy(MLD)

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  1. Tez No: 723868
  2. Yazar: MERVE KAFTANCIOĞLU
  3. Danışmanlar: Belirtilmemiş.
  4. Tez Türü: Yüksek Lisans
  5. Konular: Tıbbi Biyoloji, Medical Biology
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2018
  8. Dil: İngilizce
  9. Üniversite: Universiti Malaya UM
  10. Enstitü: Yurtdışı Enstitü
  11. Ana Bilim Dalı: Belirtilmemiş.
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 124

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Özet (Çeviri)

Background: Metachromatic leukodystrophy (MLD) is a familial progressive neurologic dysfunction due to Arylsulfatase A (ASA) enzyme deficiency. The unfunctional enzyme cannot carry out its normal lysosomal process for sulfatides, therefore it leads neurodegeneration with a temporal decline. The one of the propitious therapy option is through lentiviral-mediated autologous transplantation of Hematopoietic Stem Cells (HSC) that targets the primary nervous system manifestations; however insertional mutagenesis due to random is a risk that cannot be disregarded. Thus, we have chosen to study an approach that combines autologous HSC Transplantation (HSCT) with CRISPR/Cas9 system and non-integrating repair templates to ensure target integration and thereby create a safe gene therapy. Methods: We assessed the ASA expression and enzyme activity in CD34+ HSCs. We utilized plasmid-encoded CRISPR/Cas9 system to target three specific mutations in ARSA gene locus in K562 cells. In addition, we used CRISPR/Cas9 system (encoded as mRNA/protein) and single-stranded DNA (ssDNA) repair template to integrate reporter protein/ARSA cDNA into the endogenous locus in CD34+ HSCs. The efficiency of integration was measured by reporter protein expression. Results: Our results demonstrated that CD34+ HSCs expresses enzymatically active Arylsulfatase A. Our designed sgRNAs targeting the three mutation sites of ARSA revealed that targeted indels induction in K562 is lower (up to 30%) due to plasmid encoded CRISPR/Cas9 system. The mRNA/protein encoded CRISPR/Cas9 system exhibited the improved efficacy in both K562 (up to 86%) and CD34+ HSCs (up to 56%). We found that out of the five tested sgRNAs, three of them resulted in moderate knockout efficacy (> 40%). The gene-repair quantification by flow cytometry showed that up to 3% site-specific integration of iv reporter gene was achieved at ARSA locus using Cas9 protein and single stranded DNA (ssDNA) in CD34+ HSCs. Conclusion: Our approach provides guidance on non-viral gene-correction in CD34+ HSCs using Cas9 protein and ssDNA. However, further optimization is indispensable to increase the homology directed repair (HDR) to attain a clinical benefit for MLD.

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