Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes
Abstract The loss‐of‐function of the proprotein convertase subtilisin–kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low‐density lipoprotein cholesterol (LDL‐C) levels. Both CRISPR/Cas9 and CRISPR‐based editor‐mediated Pcsk9 inactivation have successfully l...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2024-01-01
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| Series: | Journal of Extracellular Vesicles |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jev2.12389 |
| _version_ | 1797346238072356864 |
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| author | Nazma F. Ilahibaks Thomas A. Kluiver Olivier G. deJong Saskia C. A. deJager Raymond M. Schiffelers Pieter Vader Weng Chuan Peng Zhiyong Lei Joost P. G. Sluijter |
| author_facet | Nazma F. Ilahibaks Thomas A. Kluiver Olivier G. deJong Saskia C. A. deJager Raymond M. Schiffelers Pieter Vader Weng Chuan Peng Zhiyong Lei Joost P. G. Sluijter |
| author_sort | Nazma F. Ilahibaks |
| collection | DOAJ |
| description | Abstract The loss‐of‐function of the proprotein convertase subtilisin–kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low‐density lipoprotein cholesterol (LDL‐C) levels. Both CRISPR/Cas9 and CRISPR‐based editor‐mediated Pcsk9 inactivation have successfully lowered plasma LDL‐C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle‐mediated CRISPR delivery inactivating Pcsk9 affected low‐density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV‐mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin‐interacting heterodimer FK506‐binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12‐rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNP delivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low‐density lipoprotein receptor and LDL‐C. These readouts can be used in future experiments to assess the efficacy of vehicle‐mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV‐mediated CRISPR/Cas9 RNP delivery targeting Pcsk9. |
| first_indexed | 2024-03-08T11:30:09Z |
| format | Article |
| id | doaj.art-83cebf0ddaa745919861e6dd47a10bfc |
| institution | Directory Open Access Journal |
| issn | 2001-3078 |
| language | English |
| last_indexed | 2024-03-08T11:30:09Z |
| publishDate | 2024-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj.art-83cebf0ddaa745919861e6dd47a10bfc2024-01-26T03:51:18ZengWileyJournal of Extracellular Vesicles2001-30782024-01-01131n/an/a10.1002/jev2.12389Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytesNazma F. Ilahibaks0Thomas A. Kluiver1Olivier G. deJong2Saskia C. A. deJager3Raymond M. Schiffelers4Pieter Vader5Weng Chuan Peng6Zhiyong Lei7Joost P. G. Sluijter8Laboratory of Experimental Cardiology, Department Heart & Lungs University Medical Center Utrecht Utrecht The NetherlandsPrincess Máxima Center for Pediatric Oncology Utrecht The NetherlandsDepartment of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences Utrecht University Utrecht The NetherlandLaboratory of Experimental Cardiology, Department Heart & Lungs University Medical Center Utrecht Utrecht The NetherlandsCDL Research, University Medical Center Utrecht Utrecht The NetherlandsLaboratory of Experimental Cardiology, Department Heart & Lungs University Medical Center Utrecht Utrecht The NetherlandsPrincess Máxima Center for Pediatric Oncology Utrecht The NetherlandsLaboratory of Experimental Cardiology, Department Heart & Lungs University Medical Center Utrecht Utrecht The NetherlandsLaboratory of Experimental Cardiology, Department Heart & Lungs University Medical Center Utrecht Utrecht The NetherlandsAbstract The loss‐of‐function of the proprotein convertase subtilisin–kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low‐density lipoprotein cholesterol (LDL‐C) levels. Both CRISPR/Cas9 and CRISPR‐based editor‐mediated Pcsk9 inactivation have successfully lowered plasma LDL‐C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle‐mediated CRISPR delivery inactivating Pcsk9 affected low‐density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV‐mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin‐interacting heterodimer FK506‐binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12‐rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNP delivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low‐density lipoprotein receptor and LDL‐C. These readouts can be used in future experiments to assess the efficacy of vehicle‐mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV‐mediated CRISPR/Cas9 RNP delivery targeting Pcsk9.https://doi.org/10.1002/jev2.12389cholesterol‐lowering therapyCRISPR/Cas9 deliveryexosomesextracellular vesiclesgene therapyincreased LDL‐C uptake |
| spellingShingle | Nazma F. Ilahibaks Thomas A. Kluiver Olivier G. deJong Saskia C. A. deJager Raymond M. Schiffelers Pieter Vader Weng Chuan Peng Zhiyong Lei Joost P. G. Sluijter Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes Journal of Extracellular Vesicles cholesterol‐lowering therapy CRISPR/Cas9 delivery exosomes extracellular vesicles gene therapy increased LDL‐C uptake |
| title | Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes |
| title_full | Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes |
| title_fullStr | Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes |
| title_full_unstemmed | Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes |
| title_short | Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes |
| title_sort | extracellular vesicle mediated delivery of crispr cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin kexin type 9 pcsk9 in primary mouse hepatocytes |
| topic | cholesterol‐lowering therapy CRISPR/Cas9 delivery exosomes extracellular vesicles gene therapy increased LDL‐C uptake |
| url | https://doi.org/10.1002/jev2.12389 |
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