Investigators at the University of Texas MD Anderson Cancer Center have used a novel cellular nanoporation method to produce microscopic extracellular vesicles (EVs) from human skin fibroblasts as a natural delivery vehicle for therapeutic mRNA. The researchers have demonstrated the use of these RNA loaded EVs by replacing lost collagen protein in photoaged skin in an animal model, to reduce the formation of wrinkles.

These findings were published in the journal Nature Biomedical Engineering, on January 12, 2023 “Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy.” This is the first study to demonstrate the successful use of EVs as an mRNA delivery vehicle for a pharmaceutical therapy.

“This is an entirely new modality for delivering mRNA,” said Betty Kim, MD, PhD, an associate professor of neurosurgery at the University of Texas MD Anderson Cancer Center and a co-corresponding author of the study. “In this example we used it to initiate collagen production in cells, but it has the potential to be a delivery system for a number of mRNA therapies that currently have no good method for being delivered.”

A better delivery vehicle

mRNA is an inherently brittle biomolecule. Therefore, to successfully harness its therapeutic potential and translate it into protein workhorses requires thoughtful packaging to ensure safety, stability, and target specificity.

“The widespread deployment of mRNA vaccines to fight COVID-19 has greatly catalyzed the advancement and translation of mRNA technologies in medicine. Delivery of mRNA, such as the mRNA vaccine, often requires a carrier to safeguard the fragile mRNA molecules into the target cells. This is typically done by synthetic lipid nanoparticles,” said Joseph Wu, MD, PhD, Director of the Stanford Cardiovascular Institute. (Wu was not involved in this study).

However, lipid nanoparticles (LNPs) frequently cause irritation and inflammation. They are not always biocompatible and are difficult to direct to a specific tissue. These drawbacks do not create a significant problem for the use of LNPs for vaccines, but they can be limiting in the delivery of other therapies.

Viral vectors, especially adeno-associated viruses (AAV), have been the major delivery vehicle for gene therapies, including mRNA, and have been approved by the US FDA (Food and Drug Administration) for two drugs. Unlike LNPs, the issues with AAV delivery involve immunogenicity, toxicity, and limited cargo size. Most human genes are too large to fit into an AAV.

EV delivery of mRNA can potentially overcome many obstacles that promising mRNA therapies currently face. Unlike LNPS and AAVs, EVs, including exosomes and micro-vesicles, are produced by the body’s own cells and biologically function to transport a variety of biomolecules, including nucleic acids such as RNA. Therefore, they are intrinsically biocompatible and endowed with the ability to cross biological barriers without triggering any serious immune response. This allows EVs to be administered repeatedly without resulting in inflammation. Moreover, EVs can be produced from cells in a simple and cost-effective manner, in substantial quantities, and are big enough to hold even the largest human genes and proteins.

“In this study, the authors reported a new mRNA delivery technology that overcomes many practical limitations associated with LNPs. The authors produced EVs loaded with mRNA by transfecting donor cells via nanoporation. These EVs have an improved safety profile, increased stability at room temperature, and high transfection efficiency,” said Wu.

Application in protein replacement

Kim’s team used EV-loaded mRNA to program skin cells to produce collagen in an in-vivo animal model and reduced wrinkle formation. The encapsulated mRNA encoded for COL1A1 (extracellular-matrix α1 type-I collagen) which replaced collagen in the deep dermal layers of the skin in mice that was experimentally aged using light.

The therapeutic EVs were delivered into the deep layers of the skin using a microneedle array patch that was applied to the skin for 15 minutes and delivered the biologic uniformly.  The single injection improved collagen production in the targeted area for nearly two months.

“When coupled with a microneedle injection platform, these mRNA loaded EVs achieved impressive long-term efficacy in collagen replacement and wrinkle reduction,” said Wu. “More importantly, their performance was shown to be superior to LNPs, the standard tool for mRNA delivery.”

Future directions

In earlier studies the team had developed a cellular nanoporation (CNP) method, which creates transient nanometer scale pores on the surface of source cells to enable the large-scale loading of full-transcript mRNAs into secreted EVs. In the current study, they explore the application of their CNP technology to produce a targeted therapeutic. Pending further refinements and clinical validation, this delivery option could prove to be a boon for mRNA-based drugs.

“Admittedly, there is still much room for improvement. These EVs cannot target specific tissue/cell populations. Consistently manufacturing these EVs on a large scale might be challenging in comparison with making synthetic LNPs. The molecular content of these EVs may require more in-depth and comprehensive characterizations before moving into human trials,” said Wu.

“mRNA therapies have the potential to address a number of health issues from protein loss as we age to hereditary disorders where beneficial genes or proteins are missing or under-expressed,” said Kim. “There is even the potential for delivering tumor-suppressing mRNA as a cancer therapy. So, finding a new avenue to deliver mRNA is exciting. There is a still work to be done to bring this to the clinic, but these early results are promising.”

Wu added, “I think this technology has the promise to serve as a universal mRNA delivery platform to treat various diseases beyond collagen replacement. I look forward to their follow-up studies.”

The research was funded by the MD Anderson Cancer Center.