The problem the researchers set out to solve is rooted in the physics of how lipid nanoparticles form. These particles assemble spontaneously when ionizable lipids, helper lipids, cholesterol, polyethylene glycol lipids and RNA are mixed in carefully controlled conditions. The ionizable lipid is the star of the show: it is positively charged at acidic pH, which allows it to bind and encapsulate negatively charged RNA, but neutral in the bloodstream, which limits toxicity. Decades of optimization have produced lipids such as ALC-0315, used in the Pfizer-BioNTech vaccine, and LP-01, a benchmark for in vivo editing studies. But those lipids were largely selected using short reporter RNAs, and their efficiency drops measurably when the transcript grows to the size of a full-length Cas9 mRNA, which can exceed four kilobases.

That size penalty matters enormously for genome editing. Unlike small interfering RNAs or compact vaccine transcripts, the mRNAs that encode CRISPR nucleases and base editors are among the largest RNAs anyone would want to deliver. Adenine base editor mRNA, for example, stretches to roughly 5.7 kilobases, and it must be co-delivered with a guide RNA and then translated into a functional protein inside the target cell. Viral vectors such as adeno-associated virus struggle to fit such cargo into a single genome, and preexisting immunity to Cas9 proteins and to viral capsids raises additional safety concerns. Nonviral delivery by lipid nanoparticles avoids those constraints, but only if the particle can actually get its bulky payload into the cytoplasm.

To find lipids suited to large transcripts, the Toronto group, led by Bowen Li with co-first authors Songtao Dong, Fanglin Gong and Tyler Thomson, built a combinatorial library of 384 ionizable lipids and screened them against a 5.7-kilobase reporter mRNA encoding an adenine base editor fused to NanoLuc, a luminescent protein. Crucially, the screen was conducted with the large cargo in hand, so the selection pressure reflected the real-world payload rather than a convenient stand-in. The top performer from that screen, LC-1, outshone both LP-01 and ALC-0315 when the same formulations were challenged with large mRNAs, and the advantage held across multiple cell types in culture.

The in vivo results are the heart of the study. When LC-1 nanoparticles carrying Cas9 mRNA and guide RNAs were injected intravenously into Ai9 reporter mice, which light up fluorescently when Cre-mediated recombination occurs, the team measured knockout of the reporter gene in up to 79 percent of liver cells. More remarkable still, LC-1 achieved 48 percent editing in the brain and 27 percent in the lung, depending on the route of administration, which included intrathecal injection into the spinal fluid and intratracheal delivery to the airways. Those figures represent up to fourfold improvements over the benchmark lipids LP-01 and ALC-0315 under the same conditions. For a field in which extrahepatic delivery has long been the bottleneck, editing nearly half of the relevant cells in brain tissue is a striking result.

The researchers then pushed the platform toward therapeutic targets. LC-1 supported base editing of PCSK9 in the liver, a gene whose disruption durably lowers LDL cholesterol and has already been validated in nonhuman primates as a potential one-time treatment for cardiovascular disease. In the lung, the team edited CFTR carrying the R55X mutation, a nonsense variant relevant to cystic fibrosis, where even partial restoration of functional protein is expected to ameliorate disease severity. And in the brain, delivered via intrathecal injection, LC-1 enabled editing at the Ube3a-ATS locus, the long noncoding RNA that silences the paternal copy of Ube3a in neurons. Reactivating that paternal allele is a leading strategy for treating Angelman syndrome, a severe neurodevelopmental disorder. Across all three routes of administration, LC-1 also produced higher correction rates in the LumA reporter mouse model of adenine base editing than the comparison lipids.

Why does LC-1 succeed where established lipids falter? The mechanistic studies in the paper offer an answer grounded in lipid physical chemistry. Using structural analyses, the team found that LC-1 forms stronger interactions with RNA and, critically, retains an ordered, fusogenic inverted-hexagonal phase as mRNA size increases. The inverted hexagonal phase is a nonbilayer lipid arrangement long associated with membrane fusion: lipids in this geometry promote contact between the nanoparticle and the endosomal membrane, allowing the RNA to escape into the cytoplasm rather than being routed to the lysosome for degradation. LC-1 also preserves pH-responsive membrane disruption as the cargo grows. The benchmark lipids, by contrast, become structurally disordered when loaded with large transcripts, losing the very geometry that enables endosomal escape. In other words, big RNA destabilizes ordinary particles but leaves LC-1’s fusogenic architecture intact.

The authors traced these properties to specific subchemical features of the lipid structure, showing how variations in the lipid’s building blocks influence both endosomal escape and the internal organization of the particle. That structure-function insight is arguably the study’s most consequential contribution, because it converts cargo size from an afterthought into a rational design variable. Screening libraries against a representative large transcript, the work suggests, should become standard practice for anyone developing ionizable lipids intended for genome editing, self-amplifying RNA, or other oversized payloads. It also complements a growing toolkit of approaches for tissue targeting, from selective organ targeting formulations to ligand-decorated particles, by ensuring that once a particle reaches its destination cell, it can actually deliver the goods.

Caveats remain before such particles reach the clinic. The results are in mice, and serum factors are known to create species-specific barriers to lipid nanoparticle delivery, meaning performance in larger animals and humans must be demonstrated. Dosing, biodistribution, immune responses to repeated administration, and long-term safety all require further study. The inventors have filed an invention disclosure through the University of Toronto, and the corresponding author serves as an advisor to biotechnology companies, so commercial development is likely to follow. Still, the demonstration that a single optimized lipid can mediate efficient editing in liver, brain and lung through intravenous, intrathecal and intratracheal routes marks a meaningful advance. If the large-cargo-informed design principle generalizes, it could widen the path toward nonviral, potentially redosable gene-editing medicines for diseases of the heart, the airway and the nervous system.

Subject of Research: Design of large-cargo-optimized ionizable lipids for lipid nanoparticle delivery of genome-editing RNA in vivo

Article Title: Lipid nanoparticles optimized for large RNA cargo and tissue targeting enhance in vivo genome editing

Article References: Dong, S., Gong, F., Thomson, T., Cai, Y., Healy, L., Lu, R. X. Z., Zhou, Z., Xu, Y., Chen, J., Savguira, M., Fu, X., Luozhong, S., Zhou, M., Kirtley, P., Wilder, B. K., & Li, B. (2026). Lipid nanoparticles optimized for large RNA cargo and tissue targeting enhance in vivo genome editing. Nature Biotechnology. https://doi.org/10.1038/s41587-026-03298-8

Image Credits: AI Generated

DOI: 10.1038/s41587-026-03298-8

Keywords: lipid nanoparticles, ionizable lipids, genome editing, CRISPR, Cas9, base editing, mRNA delivery, endosomal escape, PCSK9, CFTR, Angelman syndrome, drug delivery

BEST4⁺ cells are defined by a distinctive combination of genes, including BEST4, OTOP2, CA7 and GUCY2C. In the small intestine, they also show particularly high expression of CFTR, the chloride and bicarbonate channel best known for its role in cystic fibrosis. Together, these markers suggest a specialized role in moving negatively charged ions across the epithelium. Chloride and bicarbonate transport draw water into or out of the intestinal lumen and help regulate the chemical environment surrounding epithelial cells and mucus. OTOP2 adds a sensing capability: this proton-selective channel can respond to changes in extracellular acidity. CA7 may support intracellular bicarbonate production, while GUCY2C encodes guanylyl cyclase C, a receptor that converts extracellular signals into cyclic GMP. The resulting molecular profile is unusually coherent for a relatively small cell population.

The cells were first associated with the intestinal epithelium through studies of bestrophin expression, but modern single-cell sequencing made their identity much clearer. These analyses separate individual epithelial cells according to their RNA profiles, allowing researchers to identify populations that conventional staining can overlook. BEST4⁺ cells appear early in human intestinal development, reportedly as early as gestational week 11, and generally represent less than 5 percent of the fetal epithelial compartment. In adults, their abundance varies by region, with reported enrichment in parts of the jejunum, ileum and colon. Small-intestinal cells are concentrated toward the upper and middle villus, whereas colonic cells tend to occupy apical crypt regions. The regional differences suggest that a shared core program may be adapted to local pH, microbial exposure, mucus and transport demands.

The developmental identity of BEST4⁺ cells is still being debated. Several lines of evidence place them near the end of an absorptive differentiation pathway. They occupy post-mitotic compartments, lack conventional proliferation markers and express genes associated with mature enterocytes and colonocytes, including VIL1, AQP8 and SLC26A3. Human organoid experiments indicate that NOTCH signaling is required for their emergence, and the transcription factor SPIB appears indispensable: removing SPIB with CRISPR-based gene editing prevented BEST4⁺ cell generation even when NOTCH signaling remained active. Yet other findings point toward connections with the secretory lineage. Trajectory analyses in human tissue have identified a low-probability link to ATOH1-positive secretory progenitors, while zebrafish lineage-tracing experiments suggest that related cells can arise from secretory precursors. These differences may reflect species, anatomical region or inflammatory state rather than a single universal developmental route.

Under normal conditions, BEST4⁺ cells may act as coordinated pH and ion-transport units. BEST4 belongs to the bestrophin family of calcium-activated anion channels, which can conduct chloride and bicarbonate when intracellular calcium rises. CFTR provides another major route for apical anion secretion. The cells also express guanylin and uroguanylin, the endogenous ligands for GUCY2C, alongside the receptor itself. This arrangement could create an autocrine circuit in which locally produced ligands stimulate cyclic GMP, activate downstream protein kinases and increase CFTR activity. Bicarbonate secretion is important beyond fluid balance: it helps neutralize acidity near the epithelial surface and allows newly released MUC2 mucin to hydrate and expand into an effective protective layer. The review therefore proposes that BEST4⁺ cells may support mucus-barrier assembly in cooperation with goblet cells, although direct proof that these cells provide the critical bicarbonate flux is still lacking.

The same machinery can be exploited during secretory diarrhea. Bacterial heat-stable enterotoxin, or STa, binds and activates GUCY2C, raising intracellular cyclic GMP and stimulating CFTR through protein kinase G. Cholera toxin and the heat-labile toxin of enterotoxigenic Escherichia coli activate adenylate cyclase through persistent cyclic AMP signaling, leading to protein kinase A-mediated CFTR activation. In both cases, excessive chloride and bicarbonate secretion promotes water movement into the lumen. The review highlights evidence that BEST4⁺ cells are unusually equipped for this response because they co-express GUCY2C and CFTR at functionally relevant levels. Human intestinal organoids exposed to these pathways swell as fluid accumulates. Investigational inhibitors of GUCY2C or CFTR can reduce secretion in experimental systems, but broad suppression carries risks because basal GUCY2C signaling also contributes to barrier integrity, mucus hydration and epithelial maintenance.

Viral diarrhea may involve BEST4⁺ cells more indirectly. Porcine epidemic diarrhea virus preferentially damages villus absorptive enterocytes and can impair NHE3, a sodium-hydrogen exchanger needed for sodium-coupled water absorption. Studies in infected piglets have reported reduced expression of several water and nutrient transporters, together with increased ileal CFTR transcripts. Electrical measurements of infected jejunal tissue also indicate enhanced secretory responses. These observations are consistent with a shift away from absorption and toward secretion, and the presence of CFTR-rich BEST4⁺ cells makes them plausible contributors. But the available evidence does not show that the virus infects BEST4⁺ cells or that the cells are responsible for the altered current. Rotavirus offers another possible route: its NSP4 protein disturbs calcium signaling and triggers ADP-dependent calcium waves in neighboring uninfected cells. BEST4⁺ cells could respond as bystander effectors through calcium-sensitive anion channels, but this remains a testable hypothesis.

Norovirus likewise causes changes that could intersect with the BEST4⁺ program, including reduced epithelial resistance and increased electrogenic chloride secretion. The virus can replicate in differentiated enterocytes and some enteroendocrine cells, but there is no direct evidence that mature BEST4⁺ cells support norovirus replication. Inflammation may nevertheless alter their numbers or activity. In human organoids, interferon-gamma increases BEST4⁺ cell differentiation through a SPIB-dependent mechanism, and the resulting cells show stronger CFTR-dependent secretion after toxin exposure. If antiviral inflammation produces a similar response in living intestine, it could amplify fluid loss during acute disease. Conversely, bicarbonate secretion and mucus hydration might aid barrier repair during recovery. The review also connects BEST4⁺ cells to inflammatory bowel disease, where their abundance and expression of transport and metal-buffering genes appear altered, and to cystic fibrosis, in which defective CFTR trafficking may leave these high-CFTR cells unable to regulate anion transport, luminal acidity and mucus hydration.

These possibilities make BEST4⁺ cells attractive but challenging therapeutic targets. A drug that blocks pathological GUCY2C or CFTR activation could reduce fluid loss, yet permanent or systemic inhibition might undermine normal mucosal defense. Experimental CFTR inhibitors have reduced toxin-induced secretion in rodents, although some show limited solubility, rapid washout, narrow dosing windows or off-target effects on mitochondria. A more selective strategy may involve ADRA2A, an adrenergic receptor enriched in human BEST4⁺ cells; activating it suppresses cyclic AMP secretion and reverses cholera-toxin-induced swelling in enriched organoids. Such findings remain preclinical. The next steps will require lineage-specific genetic tools, direct electrophysiological measurements and disease experiments in animals that actually possess a conserved BEST4⁺ population. Conventional laboratory mice lack a canonical intestinal Best4 lineage, making rats, pigs, zebrafish and human organoids complementary rather than interchangeable models. Until researchers can manipulate these cells in vivo, BEST4⁺ cells should be viewed not as a confirmed master switch for viral diarrhea, but as a promising framework for understanding how infection, inflammation and epithelial ion transport converge.

At the molecular level, the proposed hub function depends on the way several transport systems are colocated rather than on BEST4 alone. Bestrophin channels are described as pentameric calcium-sensitive anion channels with a calcium-binding region, a hydrophobic gate and a cytoplasmic regulatory segment. This architecture provides a potential link between intracellular calcium signals and rapid changes in chloride or bicarbonate permeability. In a BEST4⁺ cell, such calcium-dependent conductance could complement CFTR, whose activity is controlled primarily through cyclic-nucleotide signaling. The two routes therefore offer distinct but potentially convergent means of regulating apical anion movement, while OTOP2 and CA7 could help couple that transport activity to the chemical conditions at the epithelial surface.

That arrangement also helps explain why anatomical location matters. BEST4⁺ cells are reported in the proximal small intestine and at the colonic surface, but their associated transport programs are not identical in every region or species. Villus-associated small-intestinal cells encounter different nutrient, acid and fluid gradients from cells near colonic crypt openings. Cross-species conservation supports a shared cellular program, yet conservation of marker genes does not establish conservation of net ion flux. Differences in epithelial architecture, microbiota, mucus organization and channel abundance could alter the physiological contribution of the same transcriptional cell type. Functional comparisons will therefore need to measure transport in defined regions rather than treating all BEST4⁺ cells as equivalent.

A central experimental challenge is separating correlation from cell-specific causation. High BEST4, GUCY2C or CFTR expression identifies a candidate effector population, but whole-organoid swelling, tissue short-circuit current and bulk transporter measurements integrate responses from many epithelial cells. Stronger tests would combine selective deletion or activation of BEST4⁺ cells with live measurements of intracellular pH, calcium, cyclic GMP, bicarbonate flux and mucus expansion. These experiments could determine whether BEST4 itself is the principal anion pathway, whether it mainly amplifies CFTR-mediated secretion, or whether its greatest contribution is sensing and coordinating responses among neighboring cells. They could also clarify whether toxin-induced secretion requires the endogenous guanylin–uroguanylin circuit or is driven predominantly by pharmacological stimulation of GUCY2C.

Therapeutic development will depend on preserving the distinction between pathological hypersecretion and protective basal transport. GUCY2C signaling and bicarbonate movement may contribute to epithelial maintenance and mucus function even while excessive cyclic GMP or cyclic AMP drives diarrhea. This argues for approaches that limit abnormal signal amplitude, duration or cellular targeting instead of eliminating the pathway entirely. Cell-type-resolved physiology, supported by species with a conserved BEST4⁺ population, should help identify that therapeutic window and establish whether the proposed hub is a druggable controller or primarily a useful map of interacting intestinal transport mechanisms.

Subject of Research: BEST4⁺ intestinal epithelial cells and their role in ion transport and diarrheal mechanisms

Article Title: BEST4⁺ cells: a potential hub of intestinal ion transport and diarrhea manipulation

Article References: Qu, H.-Z., & Wang, X.-Q. (2026). BEST4⁺ cells: a potential hub of intestinal ion transport and diarrhea manipulation. Advanced Biotechnology, 4(3), Article 33. https://doi.org/10.1007/s44307-026-00126-7

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00126-7

Keywords: BEST4⁺ cells, intestinal epithelium, ion transport, CFTR, GC-C signaling, secretory diarrhea, viral gastroenteritis, mucus barrier, BEST4, cells, potential, intestinal