Small Molecule Endosomal Disruptor for Biotherapeutic Delivery

The inventors have developed a new endosomolytic molecule, termed ED, for the intracellular delivery of macromolecule therapeutics. This is the first example of a small molecule that can perform endosomal disruption. The compound was designed with polyethylene glycol (PEG) moieties, which mask the hydrophobic membrane-disruptive portion of the molecule. Upon entering the endosomal compartment (pH~5) the acetal group hydrolyzes and triggers endosomal disruption, allowing cytosolic release of any co-delivered therapeutics. The membrane disruptive ability of ED was shown to be acid-dependent. Kinetic studies confirmed the pH-dependent hydrolysis with half-lives of >4h and 2.5 min at pH 7.4 and 5.0, respectively. This allows selective disruption of endosomal membrane compartments. Protein delivery was demonstrated using Saporin, a ribosome inactivation protein that has no mechanism of endosomal disruption3, and its toxic effect is therefore dependent on induced cytosolic delivery. Addition of 5 mg/mL endosomal disruptor to 10μg/mL Saporin in HEK293T cells caused complete cell death, compared to no cell death in cells treated with only Saporin or only ED.

Cas9 delivery

Bio-macromolecules such as proteins and nucleic acids have the potential to revolutionize therapeutics through their superior specificity and activity compared to classic small molecule drugs. However, despite extensive research in the use of bio-therapeutics there are currently no commercially available macromolecule drugs that target the intracellular compartment, due to delivery problems. The greatest barrier in this delivery of macromolecular drugs is the uptake of large molecules into endosomes and the ensuing disintegration in lysosomes. Disruption of the endosomal compartment has been shown to increase the therapeutic ability of macromolecular drugs. The nanoparticle, protein, and peptide based strategies currently available for disrupting endosomes are challenging to assemble with macromolecular drugs, due to their large size and chemical complexity.