Non-viral strategies for delivering genome editing enzymes.

Genome-editing tools such as Cre recombinase (Cre), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biomedical research, agriculture, microbial engineering, and therapeutic development. Direct delivery of genome editing enzymes, as opposed to their corresponding DNA and mRNA precursors, is advantageous since they do not require transcription and/or translation. In addition, prolonged overexpression is a problem when delivering viral vector or plasmid DNA which is bypassed when delivering whole proteins. This lowers the risk of insertional mutagenesis and makes for relatively easier manufacturing. However, a major limitation of utilizing genome editing proteins in vivo is their low delivery efficiency, and currently the most successful strategy involves using potentially immunogenic viral vectors. This lack of safe and effective non-viral delivery systems is still a big hurdle for the clinical translation of such enzymes. This review discusses the challenges of non-viral delivery strategies of widely used genome editing enzymes, including Cre recombinase, ZFNs and TALENs, CRISPR/Cas9, and Cas12a (Cpf1) in their protein format and highlights recent innovations of non-viral delivery strategies which have the potential to overcome current delivery limitations and advance the clinical translation of genome editing.

Engineered Polymer Nanoparticles with Unprecedented Antimicrobial Efficacy and Therapeutic Indices against Multidrug-Resistant Bacteria and Biofilms.

The rapid emergence of antibiotic-resistant bacterial "superbugs" with concomitant treatment failure and high mortality rates presents a severe threat to global health. The superbug risk is further exacerbated by chronic infections generated from antibiotic-resistant biofilms that render them refractory to available treatments. We hypothesized that efficient antimicrobial agents could be generated through careful engineering of hydrophobic and cationic domains in a synthetic semirigid polymer scaffold, mirroring and amplifying attributes of antimicrobial peptides. We report the creation of polymeric nanoparticles with highly efficient antimicrobial properties. These nanoparticles eradicate biofilms with low toxicity to mammalian cells and feature unprecedented therapeutic indices against red blood cells. Most notably, bacterial resistance toward these nanoparticles was not observed after 20 serial passages, in stark contrast to clinically relevant antibiotics where significant resistance occurred after only a few passages.

Cross-Linked Polymer-Stabilized Nanocomposites for the Treatment of Bacterial Biofilms.

Infections caused by bacterial biofilms are an emerging threat to human health. Conventional antibiotic therapies are ineffective against biofilms due to poor penetration of the extracellular polymeric substance secreted by colonized bacteria coupled with the rapidly growing number of antibiotic-resistant strains. Essential oils are promising natural antimicrobial agents; however, poor solubility in biological conditions limits their applications against bacteria in both dispersed (planktonic) and biofilm settings. We report here an oil-in-water cross-linked polymeric nanocomposite (∼250 nm) incorporating carvacrol oil that penetrates and eradicates multidrug-resistant (MDR) biofilms. The therapeutic potential of these materials against challenging wound biofilm infections was demonstrated through specific killing of bacteria in a mammalian cell-biofilm coculture wound model.