Interdisciplinary Initiatives Program Round 11 - 2022

Project Investigators:

Steven Banik, Chemistry
Polly Fordyce, Bioengineering and Genetics


Numerous drivers of tumorigenesis and neurodegenerative disease are proteins which present substantial challenges for current therapeutic strategies. These drivers, such as Myc, KRAS, and TDP-43, have been deemed difficult-to-drug due to their lack of well-defined and unique binding pockets or interaction surfaces. In principle, the ability to deploy protein-based therapeutics could overcome the limitations of small molecules as they can be readily engineered to exhibit high affinity or selectivity for challenging disease targets. However, realizing the translational promise of these protein therapeutics requires solving the central challenge of how to deliver large protein molecules into the cell. Most current approaches to deliver proteins across endosomal membranes rely on protein transduction domains. However, these domains must present at very high concentrations to disrupt membrane bilayers, which can lead to non-specific targeting, and they are limited in the types and sizes of proteins they can deliver. The most effective of these domains on average results in 5% successful delivery in cell culture with lower efficacy in vivo. Thus, alternative strategies are urgently needed.

Every domain of life has developed classes of enzymes, called phospholipases, which can selectively cleave phospholipids, the primary component of endosomal membranes. As phospholipid-editing reagents, phospholipase (PLA) enzymes are an unexplored but potentially transformative means to drive the escape of protein therapeutics from endosomes by opening gaps in bilayer membranes. Reflecting this promise, clinically approved gene therapy agents derived from adeno-associated viruses use domains exhibiting this enzymatic activity to efficiently escape from endosomes and transduce host cells. Technologies which allow the exploration and optimization of the vast sequence space of PLA enzymes are necessary to apply these largely uncharacterized enzymes as new therapeutic agents. The ability to modulate PLA enzymes activity and stability is crucial to limiting activity to endosomes and mitigating off-target membrane cleaving. The goal of the proposed work is to develop PLAs into protein-transduction reagents that can be tuned and targeted to engage intractable targets, and to develop a map of targetable features that can enhance endosomal escape efficiency by PLAs. We will leverage techniques and strategies from chemical biology, protein engineering, cell biology, microfluidics and biochemistry. The realization of these goals could provide a novel mechanism to solve a longstanding challenge in therapeutic science.