Activation of the cGAS-STING pathway combined with CRISPR-Cas9 gene editing triggering long-term immunotherapy
Introduction
The discovery of immune checkpoint blockade (ICB) has had quite a huge effect on cancer treatment, with numerous antibodies targeting the immune checkpoints programmed cell death-1(PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) received FDA approval [[1], [2], [3]]. However, these immune checkpoint antibodies have some drawbacks, including 1) restricted availability due to few patients responding, 2) high price, 3) vulnerability to drug resistance, 4) side effects from off-target injection, 5) tedious and continuous administration, and 6) low patient compliance [[4], [5], [6], [7], [8], [9], [10]]. As a result, in clinical practice, ICB is widely used as a supplement or in conjunction with other medications to activate effective immunotherapy [11,12]. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology may suppress immunological checkpoints, which is exceedingly selective and irreversible, and compensates for the inadequacies listed above [[13], [14], [15], [16]]. Furthermore, except for other advantages, the technology has the advantages of great efficiency, simplicity, low cost, and ease of use [17]. Although both strategies of small interfering RNA and CRISPR-Cas9 could down regulate gene editing, knockdown strategy based on CRISPR-Cas9 has the advantages of permanently silencing the target gene, high effectiveness for accurate gene editing, and low off-target likelihood [18,19]. CRISPR-Cas9 gene editing method, in particular, uses sgRNA (Guide RNA) to target genome sequence and leads the Cas9 protease to effectively cut the target gene [20,21].
Recent research discovered that combining the stimulator of interferon genes (STING) pathway with ICB could stimulate type I interferons to improve immunological function [22]. STING is a cytoplasmic adaptor protein that works as a homodimer in the endoplasmic reticulum. It is also the receptor for 2, 3-cycloguanosine monophosphate (cGAMP) and is found in high concentration in T cells and antigen-presenting cells (APCs) [[23], [24]]. As a result, researchers are committed to developing and screening effective STING agonists, and some have even conducted clinical trials. According to the method of administration, it could be broadly classified into three categories: 1) cyclic dinucleotide natural ligand derivatives are used for intratumoral delivery due to poor stability, clinical trials have been carried out like NCT02675439, NCT03172936, and NCT03010176, 2) small molecules of intravenous administration, clinical trials have been conducted including NCT03843359, NCT04420884 and NCT04096638, and 3) and oral administration of MSA-2 and SR-717 [25,26], which are highly likely to be the next generation of clinical molecules, although toxicity and efficacy have not yet been studied in clinical trials [27].
Because of its low toxicity, excellent responsiveness to tumor microenvironment (TME), and high loading capacities, hollow manganese dioxide (HMn) is a suitable nanocarrier for drug delivery [28,29]. Herein, we created a nanoplatform based on HMn loaded with the novel STING agonist MSA-2 and CRISPR-Cas9 plasmid for PD-L1 silencing, which was further coated with hyaluronic acid (HA). The nanoplatform was called HMnO2-MSA-2-PD-L1@HA (termed as HMnMPH, which was displayed in Scheme 1a). HMnPMH nanoplatform could get into cancer cells via the CD44 receptor and the EPR effect and degrade to release Mn2+, MSA-2, and Cas9/sg-PD-L1 plasmid in the TME in tandem with GSH/pH (Scheme 1b). Mn2+, on the one hand, could be used for magnetic resonance imaging (MRI) to guide therapy, and on the other hand, could act together with MSA-2 to trigger downstream signaling events through recruitment and activation of tank-bound kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). The expression and secretion of type I interferon (IFN) and proinflammatory cytokines such as IFN-β and interleukin-6 (IL-6) were induced to activate the STING pathway, and type I interferon subsequently promoted dendritic cell maturation and antigen presentation. In addition, CRISPR-Cas9 knocked down the PD-L1 gene and awaken immunosuppressed T cells to differentiate into cytotoxic T lymphocytes, triggering a series of powerful cellular immune responses to suppress cancer cells.
Section snippets
Results and discussion
Here, HMnMPH was fabricated beginning with the synthesis of HMn nanoparticles, followed by loading with STING agonists MSA-2 (termed as HMnM). After being treated with a cationic polymer polyallylamine hydrochloride (PAH), it is electrostatically attached with a negatively charged Cas9/sg-PD-L1 plasmid (termed as HMnMP). Finally, hyaluronic acid (HA), with high affinity for CD44 receptor, was coated onto the surface of HMnMP (termed as HMnMPH). A slight modification of the hard template method
Conclusion
In summary, we successfully constructed a TME responsive nanoplatform named HMnMPH for dual-activated cGAS-STING pathway in combination with CRISPR-Cas9 gene editing for cancer immunotherapy. HMnMPH was generated by HMnO2 loaded with STING agonist MSA-2 and Cas9/sg-PD-L1 plasmid with outer modification of HA. The nanoplatform has been proven to inhibit both primary and distal cancers, as well as to generate memory T cell proliferation in order to induce long-term immunity to eliminate tumors.
Credit author statement
Y·S., and X.H., designed the research and supervised the project.; Q.L., synthesized and characterized of nanoparticles. S.D., and C.C. performed the Cas9/sg-PD-L1 plasmid constructions and validation; Q.L., R.C., S.D., and Y·P., performed celluar and animal experiment. X.L., J.Y., and F.Z. assisted in the experiments. Q.L., analyzed data and wrote the manuscript with feedback. B, H and Y.S. assisted in revising the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This research was funded by the National Natural Science Foundation of China (21874066), the National Key R&D Program of China (2019YFA0709200), the Jiangsu Province Key Research and Development Program (BE2021373), the Jiangsu Province Natural Science Foundation (BK20200336), the Fundamental Research Funds for Central Universities, and the Jiangsu Program for Innovative Talents and Entrepreneurs.
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