Nucleic acid and protein therapeutics hold significant promise for the treatment of a wide range of diseases. These macromolecular drugs can engage intracellular targets, drive endogenous protein expression, and enable genome editing. However, realizing their full potential requires delivery systems that can efficiently transport these cargos into the cytosol of target cells. Existing viral and non-viral platforms face persistent challenges, including limited cell specificity, immunogenicity, and manufacturing scalability.

This project began with the idea that we could build an entirely protein-based, nonviral delivery system that is biocompatible, customizable, and scalable using recombinant protein production methods. We were inspired by years of work in our lab focused on elastin-like polypeptides (ELPs), which are protein polymers derived from human tropoelastin. We have designed ELPs that self-assemble into micellar nanoparticles under physiological conditions, and we saw an opportunity to evolve them into a platform for delivering gene editors and other intracellular therapeutics.

The project was launched with support from the NIH Common Fund’s Somatic Cell Genome Editing Program. Our goal was to build a modular protein nanoparticle system that could deliver macromolecular drugs like mRNA, siRNA, and gene editing ribonucleoproteins into cells. We knew this would require addressing two major challenges that have plagued previous protein material-based nanoparticle design efforts: particle instability and inefficient endosomal escape.

Over four generations of design, we introduced features to our ELP nanoparticles to overcome these challenges. To promote cargo release inside cells, we incorporated histidine residues to help the particles disassemble in maturing endosomes as they acidify. These histidines act as a "proton sponge" that buffers protons as they are shuttled into the endosome, ultimately promoting endosomal swelling and rupture, releasing the cargo into the cytosol. To further improve endosomal escape efficiency, we added cationic, membrane-disruptive endosomal escape peptides (EEPs) identified through computational screening of helical peptide libraries. Fusing these EEPs to ELPs enabled electrostatic complexation of nucleic acid cargo and dramatically improved intracellular delivery while reducing toxicity, by shielding these membrane-disruptive EEPs until the particles disassemble within the endosome.

While our fourth-generation ELP-EEP nanoparticles were originally designed to facilitate the encapsulation and delivery of nucleic acids,  they also performed remarkably well in delivering protein cargo intracellularly. We tested delivery of Cre recombinase in mRNA, plasmid DNA, and protein forms, as well as CRISPR-based gene editors and siRNAs, and observed strong activity across a range of cell types, including primary cells. In vivo, we demonstrated that intranasal administration of ELP-EEP nanoparticles loaded with a Cre recombinase protein cargo enabled robust gene editing in the lung epithelium of reporter mice.

To our knowledge, ENTER (Elastin-based Nanoparticles for Therapeutic delivERy) represents the first fully recombinant, non-viral protein nanoparticle system capable of intracellular delivery of both nucleic acid and protein therapeutics. This modular delivery platform offers the ability to recombinantly integrate functional domains, such as targeting ligands, endosomal escape peptides, or cargo-binding motifs, directly to the nanoparticle scaffold. In addition to pulmonary gene editing applications, we are now exploring the use of ENTER for therapeutic delivery of gene editors to hematopoietic stem cells, with the goal of enabling in vivo genome editing for the treatment of inherited diseases such as cystic fibrosis and sickle cell disease.

Looking ahead, our efforts are focused on improving systemic delivery by optimizing nanoparticle formulations for intravenous administration and engineering recombinant ligands for receptor-mediated targeting. While substantial work remains, we believe ENTER represents a promising and versatile platform for intracellular drug delivery. We are particularly grateful for the support of the NIH Common Fund’s Somatic Cell Genome Editing Program, which enabled us to take this concept from an early-stage idea to a functioning delivery system through multiple rounds of design, iteration, and in vivo validation. This project has been shaped by both unexpected discoveries and focused engineering, and we are excited to continue advancing it toward therapeutic applications.