Nano-assembly of a Chemically Tailored Cas9 Ribonucleoprotein for In Vivo Gene Editing and Cancer Immunotherapy

Abstract

Cancer gene therapy based on the clustered regularly interspaced short palindromic repeat (CRISPR) system has been challenging due to the poor delivery and efficacy in vivo. Herein, we report the development of nanoassembled ribonucleoprotein complexes (NanoRNP), which can efficiently block the PD-L1 immune checkpoint and induce an anti-tumor effect in vivo. We utilize CRISPR-associated protein 9 (Cas9) that is chemically derivatized with a low-molecular weight polymer, which condenses with single-guide RNA and modified DNA oligonucleotides to form stabilized NanoRNPs. Delivery of the NanoRNP into B16 melanoma cells in vitro leads to efficient internalization and gene disruption with low cytotoxicity, leading to sustained downregulation of PD-L1. In vivo delivery in a mouse melanoma model demonstrates that the NanoRNP can induce indels in the target cells of the tumor at high frequencies, resulting in major suppression of tumor growth, without involving combinatorial treatment. Blockade of the PD-L1 checkpoint by the NanoRNP in tumor tissues promotes T cell infiltration and effector cytokine release, which are characteristics of the activation of anti-tumor immunity, and inhibition of immunosuppressive myeloid cells. The current system suggests a promising strategy as an in vivo gene editing platform for cancer immunotherapy.

Nitrogen-Doped Carbon Dots Facilitate CRISPR/Cas for Reducing Antibiotic Resistance Genes in the Environment

“…Direct internalization of Cas9/sgRNA into cells can take place with low efficiency. 12 The editing of bacterial tet, cat, and aph(3′)-Ia implies a possible internalization of Cas9/sgRNA without the assistance of NCDs, albeit with a significantly lower cleavage efficiency (11.9−25.7%). The cleavage of ARGs was further evaluated by qRT-PCR.…”

“…The continued widespread use of antibiotics in animal agriculture, as well as intense use of antibiotics and large-scale application of disinfectants during COVID-19 pandemic have accelerated the global spread of antibiotic resistance genes (ARGs) in the environment. − To reduce the persistence and related health risks of ARGs, − effective strategies are urgently needed to maintain both public and ecosystem health . Current attempts include degradation of extracellular ARGs, reduction of antibiotic-resistant bacteria (ARB), and inhibition of ARG transfer. , Given that levels of antibiotic resistance can be increased through gene amplification, elimination at gene level ( e.g ., genome editing) is necessary to truly minimize ARG propagation. − The clustered regularly interspaced short palindromic repeats (CRISPR) system and CRISPR-associated protein 9 (Cas9) have enabled a new era for gene editing. − Having the advantages of precise target-positioning, fast gene deletion, ease of customization, and no disturbance on nontarget populations, − the CRISPR/Cas system is regarded as a promising strategy for reducing ARGs in a sequence-specific manner …”

Emerging Trends in Nano-Driven Immunotherapy for Treatment of Cancer

“…The knockout also resulted in significant infiltration of CD4 + and CD8 + T lymphocytes in the tumour microenvironment indicating significant immune activation. In a similar strategy, PD-L1 was blocked by CRISPR-Cas9 derivatized with a low molecular weight branched poly(ethylene imine) [ 102 ]. The gene editing efficiency was confirmed by observing the down-regulation of PD-L1 and consequent suppression of tumour growth in melanoma bearing mice models.…”

The Future of Healthcare Materials

“…Programmable hydrogels have also been reported to improve the cargo loading and overall performance of the polymeric platforms, which is crucial for injectable biomaterials. , Brambilla et al also showed that polymer-based microneedles can be successfully used for individualized diagnostic and monitoring . Further, promising reports toward individualized therapy, however, pertain to using specialized nucleic acids and antibodies as cargo, such as the work of Li et al, Chung et al, and Caruso et al…”

“…14,15 Brambilla et al also showed that polymer-based microneedles can be successfully used for individualized diagnostic and monitoring. 16 Further, promising reports toward individualized therapy, however, pertain to using specialized nucleic acids and antibodies as cargo, such as the work of Li et al, 17 Chung et al, 18 and Caruso et al 19 It is clear that the road to the "perfect" delivery platform, if that ever exists, is not easy or in this sense straightforward, but we are learning valuable information along the way. This has already made the rapid production of vaccines for SARS-Cov-2 possible, and the next frontier is personalized targeted therapy.…”