New Delivery Methods Boost mRNA Therapy 20-Fold and Enable CRISPR Gene Drives in Bacteria

Scientists have identified a simple metabolic strategy that dramatically improves the delivery of mRNA therapies and CRISPR gene editing tools, while separate research teams have developed advanced delivery systems for gene editing in both human cells and bacterial populations. According to a study published in Science Translational Medicine, researchers led by Daniel Zongjie Wang and Shana O. Kelley at Biohub discovered that giving three common amino acids together with lipid nanoparticles (LNPs) can increase mRNA delivery by as much as 20 times and raise CRISPR gene editing efficiency from about 25 percent to nearly 90 percent after a single treatment.

Lipid nanoparticles are best known as the delivery system used in the COVID-19 mRNA vaccines given to billions of people worldwide. Scientists are now exploring their potential to carry therapeutic mRNA into cells to treat cancer and inflammatory diseases, and to deliver CRISPR gene editing tools that can repair harmful genetic mutations. A major challenge has slowed progress: for LNP therapies to work, the particles must release their cargo by fusing with cell membranes, a step that happens efficiently in laboratory experiments but far less reliably inside the human body.

The amino acids methionine, arginine, and serine produced dramatic improvements when added alongside LNP treatments. Across many types of cells, the method increased target protein production by five to 20 times in both cell experiments and living animals. The effect was consistent across several delivery methods, including intramuscular, intratracheal, and intravenous administration. The improvement also did not depend on the specific lipid formulation or the type of mRNA cargo being delivered.

The finding grew out of examining whether the cells themselves might be limiting the process rather than focusing on the nanoparticles. When researchers grew cells in a special human plasma-like medium that closely mimics the body's metabolic environment, LNP uptake fell sharply, dropping by 50 to 80 percent. Cells grown in the plasma-like environment showed reduced activity in several metabolic pathways related to amino acids. The researchers concluded that cells inside the body operate under leaner metabolic conditions, which reduces their ability to internalize nanoparticles.

In one experiment, researchers used a mouse model of acetaminophen-induced acute liver failure. Further experiments showed that the amino acid mixture activates a specific cellular uptake pathway, making it easier for nanoparticles to enter cells.

Separately, engineered virus-like particles (eVLPs) have emerged as a distinct delivery platform that packages preassembled CRISPR-Cas protein complexes together with guide RNA and releases them directly into the cytoplasm through virus-inspired membrane fusion. This approach bypasses endosomal trafficking and avoids sustained nuclease expression in recipient cells, resulting in a more transient editing window.

Structurally, eVLPs are nanoscale particles measuring 100–200 nm in diameter. They assemble around the retroviral Gag polyprotein, which forms an inner capsid core surrounded by a lipid envelope derived from the producer cell membrane. Production typically occurs in HEK293T cells via transient co-transfection with plasmids encoding Gag, a Gag–editor fusion, the guide RNA, and an envelope glycoprotein. Vesicular stomatitis virus glycoprotein (VSV-G) is commonly used to confer broad tropism, whereas alternatives such as the baboon retroviral envelope (BaEV) can redirect targeting toward haematopoietic cells.

The essential eVLP platform for CRISPR-Cas delivery, called Nanoblades, was first described by Mangeot et al. in 2019 using Moloney murine leukaemia virus (MMLV) Gag fused to Streptococcus pyogenes Cas9 (SpCas9). It demonstrated editing in primary cells and in mouse embryos in vivo. Subsequent development focused on systematically removing bottlenecks through successive base editor eVLP (BE-eVLP) versions.

By v4, a single intravenous injection achieved 63% editing of the Pcsk9 gene in mouse liver, resulting in a sustained 78% reduction in circulating PCSK9 protein levels. In the central nervous system, local administration produced up to 60% editing in transduced cells, while subretinal injection corrected the pathogenic Rpe65 mutation in the rd12 retinal degeneration model with efficiencies of up to 30 percent, sufficient to restore partial visual function.

The transition to v5 BE-eVLPs represented a shift from rational engineering to library-based directed evolution. Because eVLPs lack a viral genome, they do not naturally link genotype and phenotype as AAV capsid screens do. Each Gag capsid mutant was tagged with a unique barcode embedded within the co-packaged sgRNA. Screening identified two cooperative substitutions, Q226P and C507V, that remodelled the capsid interior to better accommodate large RNP cargoes. Together, they delivered a two- to fourfold increase in editing potency over v4.

Prime editing introduced additional complexity. The prime editor–reverse transcriptase fusion protein is substantially larger than base editors, and the prime editing guide RNA (pegRNA) contains a long 3′ extension that is particularly vulnerable to degradation. In the v3b PE-eVLP, direct Gag–PE fusion was replaced with a smaller scaffold protein recruited via a coiled-coil interaction to improve cargo loading. The MS2/MCP aptamer system was substituted with the higher-affinity COM/Com pair to stabilise pegRNA capture. In vivo, a single subretinal injection corrected approximately 15% of the pathogenic Mfrp mutation in the rd6 model.

In a separate application of CRISPR technology, researchers at the University of California San Diego have developed a gene drive system to combat antibiotic resistance in bacteria. Antibiotic resistance has escalated rapidly in recent years, growing into a serious global health emergency, with projections suggesting that by 2050 drug resistant superbugs could be responsible for more than 10 million deaths worldwide each year.

Professors Ethan Bier and Justin Meyer of the UC San Diego School of Biological Sciences created a second generation Pro-Active Genetics (Pro-AG) system called pPro-MobV. This updated technology is designed to spread through bacterial communities and disable the genes that make them resistant to antibiotics. According to findings published in the Nature journal npj Antimicrobials and Resistance, the researchers demonstrated that the system can travel through a natural mating channel formed between bacteria, distributing the resistance disabling elements across populations.

The foundation for this work began in 2019, when Bier's lab partnered with Professor Victor Nizet's team at UC San Diego School of Medicine to design the original Pro-AG system. That earlier version introduced a genetic cassette into bacteria, allowing it to copy itself between bacterial genomes and shut down antibiotic resistance genes. This cassette specifically targets resistance genes carried on plasmids, which are small circular DNA molecules that replicate inside bacterial cells. By inserting itself into these plasmids, the cassette disrupts the resistance genes and makes the bacteria vulnerable to antibiotics again.

The newer pPro-MobV system expands on that concept by using conjugal transfer, a process similar to bacterial mating, to move CRISPR components from one cell to another. The team showed that this method works inside biofilms, dense communities of microbes that cling to surfaces and are notoriously difficult to eliminate with standard cleaning methods. They are involved in most serious infections and help bacteria survive antibiotic treatment by forming a protective barrier that limits how easily drugs can penetrate.

The researchers also discovered that elements of their active genetic system can be transported by bacteriophage, or phage, viruses that naturally infect bacteria. Phage are already being engineered to fight antibiotic resistance by slipping past bacterial defenses and delivering disruptive genetic material into cells. These dangerous bacteria often thrive in hospitals, wastewater treatment facilities, livestock operations, and fish farms.