Cell-type-specific CRISPRization of mitochondrial DNA using bifunctional biodegradable silica nanoparticles.

We report cell-type-specific and CRISPR/Cas9-mediated mtDNA editing platform by using bifunctional biodegradable silica nanoparticles, which were capable of selective intracellular delivery to CD44-overexpressed cells and subsequent mitochondrial localization, followed by glutathione-responsive biodegradation and release of Cas9/sgRNA to realize precise mtDNA editing.

Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.

Mitochondrial DNA (mtDNA) plays an essential role in maintaining normal cellular activities. Its heteroplasmic mutations are known to cause various genetic diseases. Current genetic engineering strategies, such as those based on RNA interference (RNAi) and antisense technology, are difficult to genetically alter mtDNA, however, due to the inability of highly negatively charged oligonucleotides to translocate across the double-membrane mitochondria. We report herein a universal mitochondria-targeted gene-delivery approach by using cell-penetrating poly(disulfide)s (CPDs). Novel CPD-based mitochondrial transporters, named Mito-CPDs, were synthesized by using triphenylphosphonium (TPP)-fused propagating monomers containing either disulfide or diselenide backbones. Upon spontaneous complex formation with an oligonucleotide (single- or double-stranded), the resulting nanoscale Mito-CPD@Oligo exhibited excellent properties in common biological media. While the intracellular gene-delivery efficiency of these Mito-CPDs was comparable to that of commercial transfection agents, their unique mitochondria-localized properties enabled effective release of the loaded cargo inside these organelles. Subsequent mitochondrial delivery of siRNA and antisense oligonucleotides against suitable mtDNA-encoded proteins showed successful down-regulation of target protein expression, leading to profound effects on mitochondrial functions. Mito-CPDs thus provide a useful tool for future investigations of mitochondrial biology and treatment of mitochondria-related diseases.

Recent advances in the development of EGFR degraders: PROTACs and LYTACs.

Epidermal Growth Factor Receptor (EGFR), a transmembrane tyrosine kinase receptor, belongs to the ErbB receptor family, also known as HER1 or ErbB1. Its abnormal expression and activation contribute to tumor development, especially in non-small cell lung cancer (NCSCL). The first-to fourth-generation inhibitors of EGFR were developed to solve mutations at different sites, but the problem of resistance has not been fundamentally addressed. Targeted protein degradation (TPD) technologies, including PROteolysis Targeting Chimeras (PROTACs) and LYsosome Targeting Chimeras (LYTACs), take advantages of protein destruction mechanism in cells, which make up for shortcomings of traditional small molecular occupancy-driven inhibitors. PROTACs based heterobifunctional EGFR degraders were recently developed by making use of wild-type (WT) and mutated EGFR inhibitors. These degraders compared with EGFR inhibitors showed better efficiency in their cellular potency, inhibition and toxicity profiles. In this review, we first introduce the structural properties of EGFR, the inhibitors that have been developed against WT/mutated EGFR, and then mainly focuses on the recent advances of EGFR-targeting degraders along with its limitations and unlimited prospects.

"Clickable" ZIF-8 for Cell-Type-Specific Delivery of Functional Proteins.

Protein therapy provides a powerful alternative to small-molecule-based therapy, especially on cellular targets that are normally considered to be less druggable. Intracellular protein delivery, in particular, in a cell-type-specific manner, is still highly challenging. At present, few general strategies are available for the robust and selective intracellular delivery of proteins. In this Letter, by using zeolitic imidazolate framework-8 (ZIF-8) as protein-encapsulated nanoparticles and simultaneous doping with norbornene-modified imidazole (MIM-Nor), followed by surface attachment of the resulting nanoparticles with cetuximab (Cet) through click chemistry, we successfully synthesized Cet@protein@ZIF-8N, which was subsequently used for the selective intracellular delivery of functional proteins to epidermal-growth-factor-receptor (EGFR)-overexpressed cells. Both in-cell and in vivo experiments proved that Cet@RNase A@ZIF-8N can effectively deliver RNase A with the retention of selective inhibition. Furthermore, the same strategy was successfully applied to cell-type-specific gene editing through the delivery of a Cas9/sgRNA complex to knockdown the endogenous expression of glutathione peroxidase (GPX4), a key protein in ferroptosis. Our new system thus has potential implications in future cancer treatment and the development of precision medicine.