| 학회 |
한국화학공학회 |
| 학술대회 |
2018년 가을 (10/24 ~ 10/26, 대구 EXCO) |
| 권호 |
24권 2호, p.1447 |
| 발표분야 |
생물화공(Biochemical Engineering) |
| 제목 |
Enhancement of Viral Titers for Vectors Encoding shRNAmiRs via DROSHA Knockout |
| 초록 |
RNAi-based gene therapy using shRNAmiR is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNAmiR vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. We designed gRNA CRISPR/Cas9 constructs targeting DROSHA and successfully generated knockout (KO) HEK293T cells. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus using DROSHA KO packaging cells. We observed an increase in the hairpin-containing Venus transcripts in DROSHA KO consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin vectors. We confirmed the absence of shRNAmiR processing by Northern blot and this correlated with an increase in the full-length vector genomic RNA. From rescue experiment, re-expression of WT DROSHA in DROSHA KO cells led to reduction in viral titer. These findings may have important implications in production of viral shRNAmiR vectors for RNAi-based therapy. |
| 저자 |
박희호1, Robinson Triboulet2, Martin Bentler3, Swaroopa Guda4, Peng Du5, Haiming Xu6, Richard I. Gregory3, Christian Brendel4, David A. Williams5
|
| 소속 |
1강원대, 2화학생물공학부 / Harvard Medical School, 3Boston, 4MA, 5USA, 6Harvard Medical School |
| 키워드 |
생물화공 |
| E-Mail |
|
| 원문파일 |
초록 보기 |
