Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

Yvonne Baron; Johanna Sens; Lucas Lange; Larissa Nassauer; Denise Klatt; Dirk Hoffmann; Marc-Jens Kleppa; Philippe Vollmer Barbosa; Maximilian Keisker; Viviane Steinberg; Julia D Suerth; Florian W R Vondran; Johann Meyer; Michael Morgan; Axel Schambach; Melanie Galla

doi:10.1016/j.omtn.2021.12.033

Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

Mol Ther Nucleic Acids. 2022 Jan 1:27:810-823. doi: 10.1016/j.omtn.2021.12.033. eCollection 2022 Mar 8.

Authors

Yvonne Baron¹, Johanna Sens¹, Lucas Lange¹, Larissa Nassauer¹, Denise Klatt¹, Dirk Hoffmann¹, Marc-Jens Kleppa¹, Philippe Vollmer Barbosa^{1

2}, Maximilian Keisker¹, Viviane Steinberg¹, Julia D Suerth¹, Florian W R Vondran^{3

4}, Johann Meyer¹, Michael Morgan¹, Axel Schambach^{1

5}, Melanie Galla¹

Affiliations

¹ Institute of Experimental Hematology, Hannover Medical School, Hannover 30625, Germany.
² Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover 30625, Germany.
³ ReMediES, Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover 30625, Germany.
⁴ German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover Medical School, Hannover 30625, Germany.
⁵ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Abstract

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34⁺ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

Keywords: CRISPR-Cas9; Gag.MS2 chimera; alpharetroviral vector; gene editing; homology directed repair; multiplexing; transient expression; virus-like particles.