Dissertation
Development of a clinically relevant insulated foamy viral vector for hematopoietic stem cell gene therapy
Doctor of Philosophy (PhD), Washington State University
01/2016
Handle:
https://hdl.handle.net/2376/112408
Abstract
Retroviral vector mediated stem cell gene therapy is a promising approach for the treatment of hematopoietic disorders. Therapeutic gene containing retroviral vectors integrate into the host cell genome, thus allowing for permanent delivery of a corrective gene to hematopoietic stem cell (HSC). This approach has already been successful in several clinical trials, though enhancers within integrated retroviral vectors can interact with the surrounding host genome and cause dysregulation of proto-oncogenes leading to leukemia. This is a major hurdle for the use of retroviral vectors in the clinic. The choice of retroviral vector is important for safety.
FV vectors have an integration profile more conducive to safety than other retroviral vectors and have more efficient polyadenylation. Promoters necessary to express therapeutic genes may still interact with the host genome and are of concern. Adding genomic elements which block the interaction of enhancers and promoters (enhancer blocking insulators) can significantly increase the safety of retroviral vectors. Here we have developed insulated FV vectors to reduce the effects of internal promoters on the surrounding host genome and improve the safety of retroviral vector mediated gene therapy.
Six insulators were evaluated for activity, effects on FV vector titer, and retention of the insulator following integration. Three of these insulators, a 650 bp version of the well-studied chicken hypersensitivity site 4 (650cHS4) insulator and two insulators (A1 and A2) from the human genome were most promising for FV vectors and assessed for safety in human CD34+ cord blood cells. Retroviral vector integration (RIS) sites were evaluated for the presence of dominant clones, tight groupings of integration sites (hotspots) and proximity to the transcription start sites (TSS) of proto-oncogenes. In vitro, insulators reduced the capture of integrations within hotspots. In vivo, the A1 insulated vector significantly reduced clonal dominance and reduced the proportion of RIS near proto-oncogene TSS compared to control FV vectors. The 650cHS4 insulator had no effect on clonal dominance. The A1 insulated FV vector is promising for further evaluation in disease treatment models. The work presented here is critical pre-clinical data for future clinical use of insulated FV vectors.
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Details
- Title
- Development of a clinically relevant insulated foamy viral vector for hematopoietic stem cell gene therapy
- Creators
- Diana Lynn Browning
- Contributors
- Grant D Trobridge (Advisor)Maragaret E Black (Committee Member)Weihang Chai (Committee Member)Alan Goodman (Committee Member)Anthony Nicola (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 138
- Identifiers
- 99900581835201842
- Language
- English
- Resource Type
- Dissertation