New nanoparticles may correct genetic defects in lungs

MIT researchers hopeful their method will treat a range of genetic diseases

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by Steve Bryson, PhD |

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Researchers are developing new inhalable nanoparticles designed to deliver RNA-based gene-editing therapeutics directly to the lungs, a study reports.

Now shown to be effective in mice, the ultimate goal is to develop inhaled therapeutics to correct genetic defects in conditions affecting the lungs, such as cystic fibrosis (CF).

“This is the first demonstration of highly efficient delivery of RNA to the lungs in mice,” Daniel Anderson, PhD, co-lead of the study at the Massachusetts Institute of Technology (MIT), said in a university press release. “We are hopeful that it can be used to treat or repair a range of genetic diseases, including cystic fibrosis.”

Details of the new development were published in the journal Nature Biotechnology, in the study, “Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing.”

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In CF, inherited genetic defects result in deficient or absent CFTR protein, leading to a thick, sticky mucus coating various organs, including the airways.

While several approved therapies improve lung function in CF patients, such as CFTR modulators that work by correcting CFTR protein defects, they do not address the underlying genetic cause.

The gene-editing tool CRISPR-Cas9, delivered to the airways, represents a promising approach to correct genetic defects in CF, and similar lung conditions, according to the scientists.

In 2019, Anderson’s lab created inhalable nanoparticles that delivered messenger RNA (mRNA) to the lungs of mice. mRNA is the molecule that carries the information stored in genes to make proteins.

To further optimize the approach to deliver mRNA encoding the components of CRISPR-Cas9, allowing gene editing in the lungs, Anderson collaborated with Wen Xue, PhD, at the University of Massachusetts Medical School’s RNA Therapeutics Institute.

Lipid nanoparticles

The team first generated a library of 720 new potential lipid nanoparticles (LNPs) by combining 10 distinct lipids with 72 positively-charged head groups. The fat-like lipids help LNPs to pass through the cell membrane, while the positive charge on the head group binds to negatively charged mRNA. LNPs are tiny sacs composed of fat-like lipids that encapsulate the mRNA, a strategy shown effective by the success of mRNA vaccines for COVID-19.

Screening of the LNPs in cell-based tests identified those best at delivering mRNA into the cells, which was validated by intratracheal administration (into the airways) in mice.

Among the top nine new LNPs, six best carried the CRISPR-Cas9 mRNA into cells and altered a target gene. Notably, the LNP, dubbed RCB-4-8, which was best at delivering mRNA to the cells in mouse lungs, also had the highest efficiency (about 95%) in altering the target gene in cells.

In dose-response tests, RCB-4-8 was about 100 times more effective at delivering mRNA to mouse lungs than another lipid formulation (MC3) approved for RNA delivery. Moreover, less than 30% of RCB-4-8 remained in the lungs days after treatment compared with more than 90% retention of MC3, “suggesting lower toxicity,” the team noted.

Further experiments showed their LNP was amenable to repeat dosing. This may give them an advantage over using a modified version of harmless adenoviruses to deliver mRNA due to the potential immune response from repeated viral doses.

Efficient delivery

“This means that the cells we were able to edit are really the cells of interest for lung disease,” said Bowen Li, a former MIT postdoc who now is an assistant professor at the University of Toronto, Canada. “This lipid can enable us to deliver mRNA to the lung much more efficiently than any other delivery system that has been reported so far.”

Finally, RCB-4-8 was intratracheally administered in mice that were modified such that the CRISPR/Cas9 components cut a gene resulting in a fluorescent signal in lung cells. Three doses over four days generated a signal in almost 60% of lung cells. About 15% of the club and ciliated epithelial cells, two major cell types that line the airways, were effectively edited.

“Our proof-of-concept work convincingly demonstrates delivery approaches based on RCB-4-8 LNP for efficient CRISPR–Cas9 gene editing in the lung,” the team wrote.

Working on stability

The researchers noted they are working to improve the stability of LNP formulations for nebulization to “enable patients to receive gene therapy by simply inhaling mRNA LNPs via a nebulizer.” Also, because CF is characterized by thick mucus, future work will focus on LNP formulations in animal models with abnormal mucus.

“This achievement paves the way for promising therapeutic lung gene delivery applications for various lung diseases,” said Dan Peer, PhD, director of the Laboratory of Precision NanoMedicine at Tel Aviv University, in Israel. Peer also was among the first to demonstrate delivery of RNA molecules using nanocarriers.

“This platform holds several advantages compared to conventional vaccines and therapies, including that it’s cell-free, enables rapid manufacturing, and has high versatility and a favorable safety profile,” Peer added.

Funding for this study was provided by Translate Bio, the National Institutes of Health, the American Cancer Society, the Cystic Fibrosis Foundation, as well as the Leslie Dan Faculty of Pharmacy startup fund and a PRiME Postdoctoral Fellowship, both from the University of Toronto.

Translate Bio ran a Phase 1/2 trial (NCT03375047) called RESTORE-CF, testing MRT5005, an experimental inhaled mRNA therapy to deliver functional CFTR protein directly to CF lungs. Repeated dosing appeared to be generally safe and well tolerated in an early analysis.