Multistage Sensitive NanoCRISPR Enable Efficient Intracellular Disruption of Immune Checkpoints for Robust Innate and Adaptive Immune Coactivation

Abstract

Synergistic immune checkpoints blockade (ICB) has the potential to surmount the heterogeneity of tumors that are resistant to conventional therapeutics and result in better treatment outcomes than monotherapy targetting of to a single immune checkpoint. However, current stategies to combining multi-target ICB are mainly focused on blockades on the cell surface which are incomplete, temporary, and inefficient. Here, a multistage sensitive nanocomplex (MUSE) loaded with programmed death-ligand 1/CD47 multiple targeting clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (MT-CRISPR/Cas9) system for complete intracellular immune checkpoint blockade are conducted. The core-shell structure not only endows the MUSE with prolonged circulation capacity and precise tumor recognition, but also facilitates cell uptake and endosomal escape when confronted with the tumor extra/intracellular environment, leading to efficient delivery of MT-CRISPR/Cas9 payloads with minimal toxity. It is demonstrated that the MUSE treatment in tumor models can activate robust CD8 + T-cells and M1 macrophages-mediated adaptive and innate anti-tumor immune responses and trigger a persistent immune memory effect, which results in significantly inhibition of tumor growth and improved survival rate with almost undetectable off-target delivery effect. Taken together, the MUSE-nanoCRISPR system opens a new avenue for combinative blockade of multiple immune checkpoints, in turn bringing clinical benefits for oncological treatment.

Improvement of the anticancer efficacy of PD-1/PD-L1 blockade via combination therapy and PD-L1 regulation

“…Targeting PD-L1 regulation can also produce good therapeutic effects. A multistage sensitive nanocomplex (MUSE) loaded with PD-L1/CD47 multiple targeting CRISPR/Cas9 system was developed for coactivation of both T cells and macrophages-mediated antitumor immune response [ 237 ]. The prepared MUSE has some beneficial characteristics, including prolonged blood circulation, rapid response to the MMP-9-rich TME, enhanced lysosomal escape, rapid nuclear localization and high transfection efficiency.…”

“…MUSE synergistically boosting CD8 + T cells and M1 macrophages-mediated adaptive and innate anticancer immunity 2. The MUSE-nano-CRISPR system showed efficient disruption efficiency of PD-L1 and CD47 in vitro and in vivo [ 237 ] Multispecific Platinum (IV) Complex DNP Downregulation Breast cancer (BC) Not applicable 1. COX-2 plays an important role in the progression of breast cancer, correlating with the levels of PD-L1 2.…”

A programmable hierarchical-responsive nanoCRISPR elicits robust activation of endogenous target to treat cancer

“…The TFA was removed by rotary evaporation at 40 °C for 10 min. The mixture was dialyzed against DMSO for 24 h, and then dialyzed against distill water for another 48 h. The grating ratios of Phe and Tyr on PEI 1.8K were confirmed by Ninhydrin assay according to our previous research 30 , and calculated through the formula: X = (16 - 1.8 × C) / (0.196 × C + 1), where X is the number of fluorinated groups on PEI 1.8K, and C is the concentration of primary amine groups, mmol/L.…”

Carrier strategies boost the application of CRISPR/Cas system in gene therapy

“…[ 183 ] For example, they developed a programmable delivery system of CRISPR/Cas9 for efficient tumor immunotherapy. [ 184 ] Cas9 plasmid simultaneously targeting PD‐L1 and CD47 was first condensed by the fluorinated poly(ethylamine) to form a core, which was subsequently coated with an MMP‐9 cleavable polymer shell to afford the multistage sensitive nanocomplex (MUSE). The shell polymer consisted of hyaluronan backbone, PEG branches, and shielded cell‐penetrating peptide branches.…”