DROSHA Knockout Leads to Enhancement of Viral Titers for Vectors Encoding miRNA-Adapted shRNAs

Hee Ho Park; Robinson Triboulet; Martin Bentler; Swaroopa Guda; Peng Du; Haiming Xu; Richard I Gregory; Christian Brendel; David A Williams

doi:10.1016/j.omtn.2018.07.002

DROSHA Knockout Leads to Enhancement of Viral Titers for Vectors Encoding miRNA-Adapted shRNAs

Mol Ther Nucleic Acids. 2018 Sep 7:12:591-599. doi: 10.1016/j.omtn.2018.07.002. Epub 2018 Jul 9.

Authors

Hee Ho Park¹, Robinson Triboulet², Martin Bentler³, Swaroopa Guda⁴, Peng Du², Haiming Xu⁴, Richard I Gregory⁵, Christian Brendel⁶, David A Williams⁷

Affiliations

¹ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Program of Biotechnology and Bioengineering, Kangwon National University, Chuncheon, Gangwon-do 24341, Republic of Korea.
² Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Stem Cell Program, Boston Children's Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
³ Institute of Experimental Hematology, Hannover, Germany.
⁴ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
⁵ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Stem Cell Program, Boston Children's Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Harvard University, Boston, MA, USA; Harvard Initiative for RNA Medicine, Harvard Medical School, Boston, MA, USA.
⁶ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Harvard University, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
⁷ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Harvard University, Boston, MA, USA; Harvard Initiative for RNA Medicine, Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. Electronic address: dawilliams@childrens.harvard.edu.

Abstract

RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNA^miR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNA^miR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin-containing Venus transcripts in DROSHA KO producer cells consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin-containing constructs. We confirmed the absence of significant shRNA^miR processing by northern blot analysis and showed that this correlated with an increase in the amount of full-length vector genomic RNA. These findings may have important implications in future production of viral shRNA^miR-containing vectors for RNAi-based therapy.

Keywords: CRISPR/Cas9; DROSHA microprocessor; RNAi; producer cell line; recombinant viral vector titer.

Abstract

Grants and funding