Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs
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Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs. / Wichmann, Max; Maire, Cecile L; Nuppenau, Niklas; Habiballa, Moataz; Uhde, Almut; Kolbe, Katharina; Schröder, Tanja; Lamszus, Katrin; Fehse, Boris; Głów, Dawid.
In: INT J MOL SCI, Vol. 24, No. 14, 13.07.2023, p. 11399.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs
AU - Wichmann, Max
AU - Maire, Cecile L
AU - Nuppenau, Niklas
AU - Habiballa, Moataz
AU - Uhde, Almut
AU - Kolbe, Katharina
AU - Schröder, Tanja
AU - Lamszus, Katrin
AU - Fehse, Boris
AU - Głów, Dawid
PY - 2023/7/13
Y1 - 2023/7/13
N2 - The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.
AB - The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.
KW - Mice
KW - Animals
KW - Humans
KW - CRISPR-Cas Systems/genetics
KW - Retroviridae/genetics
KW - Gene Editing/methods
KW - Lentivirus/genetics
KW - Nanoparticles
U2 - 10.3390/ijms241411399
DO - 10.3390/ijms241411399
M3 - SCORING: Journal article
C2 - 37511168
VL - 24
SP - 11399
JO - INT J MOL SCI
JF - INT J MOL SCI
SN - 1661-6596
IS - 14
ER -