Protein-based nanoparticles can effectively deliver DNA, RNA, proteins, and gene editors directly into multiple cell types while avoiding toxicity
By Lindsay Brownell
(BOSTON) — Getting medicines into the cells they’re designed to treat is a perennial problem for the medicine and pharmaceutical industries, and patients often suffer from side effects and ineffective treatments as a result. Current drug delivery vehicles carry a host of risks and limits: lipid nanoparticles can cause immune reactions and accumulate in the liver, viral envelopes can’t accommodate cargoes above a certain size, and membrane-disrupting peptides can cause collateral damage to healthy cells. And to make matters worse, many substances that are successfully delivered into a cell find themselves trapped inside endosomes – bubble-like structures that the cell membrane forms around molecules as they enter to “quarantine” them while the cell decides whether their contents should be kept or destroyed.
A multidisciplinary team of researchers has created a new type of drug delivery vehicle that overcomes all of these issues simultaneously and opens a new frontier for drug delivery. The team engineered their system, called ENTER, to break apart in the endosome’s acidic environment and puncture the endosome’s membrane, releasing its cargo directly into the cell. Studies in mice showed that ENTER was able to successfully deliver a gene editing tool that modified the genomes of cells in the animals’ airways, demonstrating the potential to treat a number of human conditions including cystic fibrosis.
“Previous peptide-based methods to deliver drug cargoes to cells didn’t fully address the challenge of getting things out of the endosome, which is a really big problem. RNA and proteins are degraded by enzymes inside the endosome, and while DNA is relatively stable within an endosome, it can’t do its job if it stays there. This is the first effort we know of that enables delivery of a full range of macromolecule therapies ranging from mRNA to siRNA, DNA, and proteins via a single system that solves the problem of endosomal escape,” said co-first author Feyisayo (Sayo) Eweje, an M.D./Ph.D. student in the Harvard-MIT Program in Health Sciences and Technology and member of the Wyss Institute.
ENTER is described in a new paper published in Nature Biotechnology.
Deflating the endosome’s balloon
ENTER, which stands for “elastin-based nanoparticles for therapeutic delivery,” was developed in the lab of corresponding author Elliot Chaikof, M.D., Ph.D., an Associate Faculty member at the Wyss Institute. The project began in 2019, when Chaikof and collaborator David Liu, Ph.D. were awarded a grant from the NIH’s Somatic Cell Genome Editing (SCGE) program to investigate better ways to deliver gene editors like CRISPR and other therapeutic molecules into cells.
The lab focused on a type of molecule called elastin-like polypeptides (ELPs), which are synthetic protein-based molecules inspired by the naturally occurring protein elastin. Like elastin, ELPs are flexible, can change their shape in response to temperature, and can self-assemble into nanoscale spheres that can be loaded with cargo molecules. However, previous efforts to use them to deliver biomolecules like mRNA and proteins to target cells have not been successful, likely because of ELPs’ failure to break out of endosomes after being engulfed by target cells.
To overcome this problem, Eweje and his co-authors set about designing an ELP-based system that delivered a one-two punch to the endosome. First, they engineered the ELP molecule itself to incorporate an amino acid called histidine. As hydrogen ions (protons) are pumped into the endosome to acidify it, the histidine acts like a sponge, soaking up free protons. This causes even more protons to enter the endosome, making it swell up like a balloon filled with air. Once the endosome reaches a certain acidity level (pH), the histidine also triggers the ELP structure to start to break apart.
But the team still needed a biological “needle” to puncture the swollen endosome’s membrane and release the drug cargo into the surrounding cytoplasm. They found their answer in another type of molecule called endosomal escape peptides (EEPs). EEPs are effective at disrupting the membrane of the endosome, but their use has been limited because they also disrupt the membranes of off-target cells and structures, causing toxicity.
By combining self-assembling ELPs with membrane-disrupting EEPs into ENTER, the researchers created a way to smuggle the proverbial needle in with the haystack. The EEP components cluster together at the core of the ENTER structure along with the drug cargo, while the ELPs form the outer “shell” to safely shepherd both components into an endosome. Once there, the influx of protons breaks open the ELP shell, exposing the EEPs, which puncture the endosome’s membrane. The drug cargo is then finally free to move into the cell.
From design to delivery
The team’s theory became reality when they tested their ENTER construct in a special line of human cells that had been genetically modified to include the gene for a red fluorescent protein as well as a genetic “stop switch” that prevents the red protein from being expressed under normal conditions. They constructed ELPs that contained a previously reported EEP known as S10 as well as an enzyme cargo called Cre recombinase. If ENTER behaved as intended, it would enter the cell, break out of the endosome, and enable the Cre recombinase to enter the cell’s nucleus and snip out the “stop switch” from the cell’s genome, allowing the red fluorescent protein to be produced.
And that is exactly what they observed: applying ENTER to the human cells turned them red.
What’s more, they observed the same effect when they modified ENTER to contain mRNA encoding Cre recombinase rather than the enzyme itself. These results demonstrated that ENTER is a flexible tool that enables various payloads to reach the right part of the cell and trigger a precise genetic response – overcoming critical challenges faced by modern gene editing platforms.
But the team knew that they could do even better. They created a machine learning model to comb through a database of more than 11,000 EEPs and identify those that could surpass S10’s ability to puncture the endosomal membrane and enable drug delivery. This analysis revealed seven top candidate EEPs, which were then tested in the lab, and the results were fed back into the model in an iterative process. One EEP, called EEP13, rose to the top as the most effective at delivering protein cargoes into the human cell line – it increased production of the red fluorescent protein by ~50%.
As a first use case for their enhanced ELP-EEP13 version of ENTER, the team tested its ability to deliver the protein form of the CRISPR-Cas9 gene editing system into the cells. Delivery of CRISPR-Cas9 in DNA or mRNA form, which instructs cells to produce the editing machinery themselves, has been widely used in research and is currently being tested in clinical trials. But this approach carries risks, namely that there is no “off” switch to stop the production of the CRISPR-Cas9 proteins, which can lead to unintended genetic consequences. Delivering the Cas9 protein itself offers more control over the gene editing process, but it is large, structurally complex, and prone to degradation, making it difficult to successfully deliver into cells.
The team found that ENTER effectively delivered CRISPR-Cas9 proteins into the human cell line, resulting in a gene editing efficiency of 65% — comparable to other experimental protein delivery methods. ENTER performed even better when delivering a different gene editing system called an adenine base editor, achieving 83% efficiency.
The improved version of ENTER was also able to deliver Cre recombinase mRNA and protein into multiple types of mouse cells, including lung fibroblasts, macrophages (white blood cells), T cells, and hematopoietic stem cells with high levels of genetic recombination and no observed cell toxicity.
Further in vitro studies demonstrated that ENTER also effectively delivered small interfering RNAs (siRNAs), messenger RNA (mRNA), and plasmid DNA (pDNA) into human and mouse cells with minimal toxicity, showcasing its versatility.
To test how ENTER performed in a living organism, the team formulated it to deliver Cre recombinase protein into thelungs of mice that carried the same red fluorescent protein gene controlled by the “stop switch” and red fluorescent protein genes as previously described. Five days after treatment, the percentage of lung cells that glowed red (indicating successful gene editing) in both the large and small airways had more than doubled in mice that received Cre recombinase via ENTER compared to Cre recombinase alone. They also found that this genetic change was induced in multiple types of cells, including ciliated epithelial cells, goblet cells, club cells, and stem cells. Importantly, the mice displayed minimal cell damage, showing that ENTER achieves its goals without the toxicity observed in other delivery methods.
“This project is incredibly exciting, because we have found a common pathway that allows ENTER to safely and effectively deliver both proteins and nucleic acids into many kinds of cells. This opens up the potential to treat a wide range of human diseases, from those that require the durable expression of a functional gene to those that need an mRNA molecule to transiently produce a protein, then disappear. As a physician and a scientist, I look forward to making patient’s lives better with this research,” said Chaikof, who is also the Chair of the Roberta and Stephen R. Weiner Department of Surgery and Surgeon-in-Chief at Beth Israel Deaconess Medical Center (BIDMC).
Given ENTER’s demonstrated ability to edit the genes of lung cells, Chaikof’s lab is exploring ENTER’s commercial potential as a treatment for cystic fibrosis through a 2024-2025 Wyss Validation Project in collaboration with the lab of Wyss Founding Director Don Ingber. Beyond cystic fibrosis, the Validation Project team is also investigating potential to deliver antigen molecules directly to antigen-presenting cells (APCs) to function as a cancer vaccine, and to deliver gene editors to hematopoietic stem cells to treat inherited blood disorders like sickle cell anemia and thalassemia. Chaikof’s team at BIDMC has also been named winners of the NIH’s TARGETED Phase 1 Challenge, securing $75,000 to further their research.
“This ability to deliver mRNA, protein, and CRISPR drugs into the cytoplasm of cells in a controlled and efficient manner is a major breakthrough that will open up entirely new therapeutic approaches to a broad range of diseases. ENTER is the first technology from BIDMC that has been named a Wyss Validation Project in recognition of its potential to improve the lives of patients, and I’m excited to see it progress from the lab to the market,” said Don Ingber, M.D., Ph.D., who is the Founding Director of the Wyss Institute as well as the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital and the Hansjörg Wyss Professor of Biologically Inspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.
Additional authors of the paper include co-first author Jiaxuan Chen, Vanessa Ibrahim, Aram Shajii, Michelle Walsh, Kiran Ahmad, Assma Alrefai, Dominie Miyasato, Hyunok Ham, Kaicheng Li, Carolyn Haller, and Michael Roehrl from BIDMC; and Jessie Davis and David Liu from Harvard University.
This research was supported by the NIH, Howard Hughes Medical Institute, the Harvard/MIT MD-PhD program, and the Ruth L. Kirchstein NRSA F31 Fellowship.