Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform.

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.

Delivery of antisense oligonucleotides using multi-layer coated gold nanoparticles to methicillin-resistant S. aureus for combinatorial treatment.

The spread of multidrug-resistant (MDR) bacterial infections has become a serious global threat. We introduce multi-layer coated gold nanoparticles (MLGNPs) delivering antisense oligonucleotides (ASOs) targeting the resistance gene of methicillin-resistant Staphylococcus aureus (MRSA), as a selective antimicrobial by restoring susceptibility. MLGNPs were prepared by multi-step surface immobilization of gold nanoparticles (GNPs) with polyethylenimine (PEI) and loaded with ASO targeting the mecA gene. The MLGNPs were shown to be efficiently internalized into various types of Gram-positive bacteria, including MRSA, Staphylococcus epidermidis, and Bacillus subtilis, which was superior to single-layer coated GNPs and free PEI polymer. The delivery of MLGNPs into MRSA resulted in up to 74% silencing of the mecA gene with high selectivity, in a dose-dependent manner. The treatment of MLGNPs to MRSA in the presence of oxacillin, a beta-lactam antibiotic, showed major suppression (~71%) of bacterial growth, due to the recovery of antibacterial sensitivity. Furthermore, the treatment of MLGNPs in a complex system showed preferential uptake into bacteria over mammalian cells, demonstrating the suitable characteristics of MLGNPs for selective delivery into bacteria. The current approach can be potentially applied for targeting various types of MDR bacterial infections by specific silencing of a resistance gene, as a combinatorial therapeutic used with conventional antibiotics.

Modulation of ACD6 dependent hyperimmunity by natural alleles of an Arabidopsis thaliana NLR resistance gene.

Plants defend themselves against pathogens by activating an array of immune responses. Unfortunately, immunity programs may also cause unintended collateral damage to the plant itself. The quantitative disease resistance gene ACCELERATED CELL DEATH 6 (ACD6) serves to balance growth and pathogen resistance in natural populations of Arabidopsis thaliana. An autoimmune allele, ACD6-Est, which strongly reduces growth under specific laboratory conditions, is found in over 10% of wild strains. There is, however, extensive variation in the strength of the autoimmune phenotype expressed by strains with an ACD6-Est allele, indicative of genetic modifiers. Quantitative genetic analysis suggests that ACD6 activity can be modulated in diverse ways, with different strains often carrying different large-effect modifiers. One modifier is SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1), located in a highly polymorphic cluster of nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes, which are prototypes for qualitative disease resistance genes. Allelic variation at SNC1 correlates with ACD6-Est activity in multiple accessions, and a common structural variant affecting the NL linker sequence can explain differences in SNC1 activity. Taken together, we find that an NLR gene can mask the activity of an ACD6 autoimmune allele in natural A. thaliana populations, thereby linking different arms of the plant immune system.