RNA delivery biomaterials for the treatment of genetic and rare diseases

Purpose: Genetic and rare diseases (GARDs) affect more than 350 million patients worldwide, and many lack effective treatments. RNA-based therapeutics—siRNA, mRNA, miRNA, lncRNA, and RNA-containing genome editing systems—are promising, but naked RNA has poor cellular penetration, is degraded in biological fluids, and does not accumulate in target organs after systemic administration. Non-viral RNA delivery biomaterials are needed to enable systemic applications and improve therapeutic windows.

Hypothesis: No formal experimental hypothesis. Central thesis: non-viral delivery biomaterials, especially lipid nanoparticles (LNPs), ligand-RNA conjugates, polymeric nanoparticles, and cell-derived vehicles, can overcome RNA delivery barriers and enable RNA-based gene therapies for GARDs by targeting organs such as liver, lung, brain, muscle, skin, and eye.

Aims: Summarize non-viral biomaterials developed for RNA therapeutic delivery, including siRNA, mRNA, RNA-containing genome editing systems, miRNA, and lncRNA. - Categorize delivery systems by RNA payload and target organ. - Highlight clinical trials and therapeutic applications in GARDs. - Discuss advantages, limitations, and future perspectives for different delivery vehicles.

Delivery system: RNA payloads: siRNA, mRNA, CRISPR-Cas9 mRNA/sgRNA/donor DNA, miRNA mimics/antimiRs, lncRNA. - Lipid and lipid-derived nanoparticles (LNPs): ionizable lipids (DLin-DMA, DLin-KC2-DMA, DLin-MC3-DMA/MC3, C12-200, 304O13, cKK-E12, TT3, C12-(2-3-2), ATX lipid, LP01, 5A2-SC8 dendrimer-based DLNP, LUNAR); helper lipids (DSPC, cholesterol, PEG-lipid); cationic lipids (DOTMA, DOTAP, DOPE, lipofectamine). - Ligand conjugates: GalNAc-siRNA conjugates targeting ASGPR on hepatocytes; cholesterol-conjugated miRNA; RVG-SSPEI for brain; PECAM-1 antibody-modified LNPs for lung; aptamers and peptides. - Polymers: PEI, PBAEs, hyperbranched PBAEs, poly(glycoamidoamine) brushes, poly(aspartamide) PAsp(DET), PEG-PAsp(DET), chitosan/PLGA, PAMAM dendrimers, charge-altering releasable transporters (CARTs), polypeptide-polyamine nanoparticles. - Cell-derived vehicles: exosomes. - Inorganic materials: SiO2 capsules, gold nanoparticles. - Routes: intravenous, subcutaneous, intratracheal/intranasal, subretinal, intramyocardial, topical. - Target organs/diseases: liver (hemophilia B, hATTR, primary hyperoxaluria type 1, acute hepatic porphyria, β-thalassemia, OTCD, Fabry disease, AIP, HT-1, MMA, hypercholesterolemia, alpha-1 antitrypsin deficiency); lung (cystic fibrosis, SP-B deficiency); brain (Machado-Joseph disease, Huntington’s disease, fragile X syndrome); muscle (Duchenne muscular dystrophy); skin (Williams-Beuren syndrome); ear (TMC1-related deafness); eye (inherited retinal degeneration).

Approach: Review of preclinical and clinical literature; no primary experiments. In vitro systems include HeLa, HepG2, MCF-7, mesenchymal stem cells, and others. In vivo models include mice, rats, nonhuman primates, and pigs. Disease contexts include GARDs listed above. Clinical trials discussed include Patisiran (approved), Givosiran, Fitusiran, Inclisiran, Lumasiran, ALN-CC5, ALN-AT3, MRT5201 (OTCD), mRNA-3704 (MMA), MRT5005 (CF), and LUNAR-CF.

Key methods: Nanoparticle characterization: size, zeta potential, pKa, encapsulation efficiency. - Gene silencing/knockdown: qPCR, Western blot, serum protein levels. - Protein expression/activity: ELISA, enzymatic activity assays. - Genome editing: indel rates, T7E1 assays, sequencing. - Biodistribution and histology. - Safety: cytokine levels, liver damage/inflammation markers.

Key results: Patisiran became the first FDA-approved siRNA drug in 2018; LNP delivers siRNA to hepatocytes, targeting TTR for hATTR. - DLin-KC2-DMA SNALPs showed in vivo activity at 0.01 mg/kg in mice and TTR silencing ED50 ~0.3 mg/kg in nonhuman primates. - TT3 hFIX mRNA produced plasma FIX 1740 ng/mL and activity 791 mIU/mL at 6 h. - C12-200 hFIX mRNA at 0.5 mg/kg yielded 1800 ng/mL FIX; ATX hFIX mRNA at 2.0 mg/kg maintained therapeutic FIX up to 6 days. - hADAMTS13 mRNA for TTP: 140 ng/mL at 24 h, 90 ng/mL at 48 h, maintained up to 5 days. - hMUT mRNA with MC3 reduced plasma methylmalonic acid by up to 90% at 24 h; repeated dosing improved growth and survival. - hGLA mRNA C12-200 LNPs at 1.0 mg/kg increased hGLA 10-fold in heart/kidney versus rhGLA; Gb3 and lyso-Gb3 decreased 92% and 88% vs 67% and 61% for rhGLA. - MC3 hGLA mRNA restored α-GLA activity up to 6 weeks. - hPBGD mRNA MC3 normalized porphyrin precursors; liver expression lasted 10 days. - hCFTR mRNA MC3 intranasal restored chloride secretion to 55% of healthy at day 3, maintained >15 days. - PECAM-1-targeted LNPs showed ~200-fold inhibition in liver and 25-fold elevation of luciferase in lungs vs non-targeted. - LP01 LNP Cas9 mRNA/sgRNA reduced serum TTR >90%, persisting 12 months in mice/rats. - C12-200 + AAV donor for HT-1 achieved 6% hepatocyte editing and 24.1% indel rate. - RVG-SSPEI delivered miR-124a to neurons across the BBB.

Interpretation: Non-viral RNA delivery biomaterials—especially LNPs and GalNAc conjugates—have strong potential for GARD therapy. LNPs can deliver diverse RNAs from siRNA to mRNA and CRISPR components, but most accumulate in the liver, limiting non-liver applications. GalNAc-siRNA conjugates enable subcutaneous, liver-targeted delivery and are clinically advanced. New ligands, linker chemistries, and chemical modifications of large RNAs are needed. More delivery systems should be explored for non-liver GARDs. Overall, RNA therapeutics are promising, but expanded biomaterial chemistry and deeper disease understanding are required.

Limitations: Review article; no primary data. - Most RNA delivery biomaterials remain preclinical; only a small number in clinical trials. - LNPs predominantly accumulate in liver, limiting non-liver targeting. - GalNAc conjugates are mainly limited to liver and small RNAs; chemical modification of large RNA is difficult. - Delivery to target organs/cell populations beyond liver remains challenging. - Genome editing delivery is mostly limited to topical administration except liver. - Potential off-target effects and immune responses. - Many rare diseases still lack effective treatments and validated targets. - Need more categories of delivery systems and deeper understanding of GARD biology.