| RRC ID |
59752
|
| 著者 |
Li C, Mishra AS, Gil S, Wang M, Georgakopoulou A, Papayannopoulou T, Hawkins RD, Lieber A.
|
| タイトル |
Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors.
|
| ジャーナル |
Mol Ther
|
| Abstract |
Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.
|
| 巻・号 |
27(12)
|
| ページ |
2195-2212
|
| 公開日 |
2019-12-4
|
| DOI |
10.1016/j.ymthe.2019.08.006
|
| PII |
S1525-0016(19)30366-1
|
| PMID |
31494053
|
| PMC |
PMC6904827
|
| MeSH |
Adenoviridae / genetics*
Animals
CRISPR-Cas Systems
Dependovirus / genetics*
Female
Genes, Reporter
Genetic Therapy
Genetic Vectors / genetics*
Hematopoietic Stem Cell Mobilization
Hematopoietic Stem Cells / cytology
Hematopoietic Stem Cells / metabolism*
Humans
Mice
Mice, Inbred C57BL
Mice, Transgenic
Transduction, Genetic*
Transgenes / physiology*
Virus Integration*
gamma-Globins / antagonists & inhibitors
gamma-Globins / genetics
|
| IF |
8.402
|
| 引用数 |
5
|
| リソース情報 |
| ヒト・動物細胞 |
HUDEP-2(RCB4557) |
