Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.
| Original language | English |
|---|---|
| Article number | e2205318 |
| Journal | Small |
| Volume | 19 |
| Issue number | 2 |
| Early online date | 18 Nov 2022 |
| DOIs | |
| Publication status | Published - 11 Jan 2023 |
Funding
The authors acknowledge support by the UPGRADE (Unlocking Precision Gene Therapy) project that has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 825825. This work was also supported by the German Research Foundation (DFG) SFB1032 (project-ID 201269156) sub-project B4. Y.L., X.L., and H.M. appreciate the fellowship of the China Scholarship Council that supports their Ph.D. studies. U.L. appreciates support from the Galenus Foundation (Vienna, Austria). The authors thank \u00D6zg\u00FCr \u00D6zt\u00FCrk for performing TEM measurements, and Teoman Benli-Hoppe for performing MALDI-TOF mass spectrometry measurements. TOC figure and some graphical elements in Figure\u00A06c, Figure\u00A07a, and Figure\u00A08a were created with Biorender.com. The authors acknowledge support by the UPGRADE (Unlocking Precision Gene Therapy) project that has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 825825. This work was also supported by the German Research Foundation (DFG) SFB1032 (project\u2010ID 201269156) sub\u2010project B4. Y.L., X.L., and H.M. appreciate the fellowship of the China Scholarship Council that supports their Ph.D. studies. U.L. appreciates support from the Galenus Foundation (Vienna, Austria). The authors thank \u00D6zg\u00FCr \u00D6zt\u00FCrk for performing TEM measurements, and Teoman Benli\u2010Hoppe for performing MALDI\u2010TOF mass spectrometry measurements. TOC figure and some graphical elements in Figure 6c , Figure 7a , and Figure 8a were created with Biorender.com.
Austrian Fields of Science 2012
- 301213 Drug targeting
Keywords
- cellular delivery
- clustered regularly interspaced short palindromic repeats/ clustered regularly interspaced short palindromic repeats-associated protein 9
- folate receptors
- gene editing
- nanocarriers
