Synthetic materials at the forefront of gene delivery

If chemistry is used to design innovative synthetic materials with rationally engineered properties—such as stimuli-responsive disassembly, membrane fusion, ionizable lipids, dynamic covalent linkers, self-assembly, and targeting—then non-viral vectors can overcome the biological barriers of gene delivery (protection, cellular uptake, endosomal escape, cargo release, and nuclear entry) and become viable alternatives to viral vectors for therapeutic gene delivery.

• Secondary Aims:
• To categorize synthetic carriers according to their chemical structure to highlight its importance for specific functional applications.
• To outline the major biological obstacles in gene delivery and the strategies used to overcome them (Table 2).
• To review non-viral delivery methods, including self-delivering oligonucleotides, physical methods, protein-, peptide-, lipid-, polymer-, and nanoparticle-based carriers.
• To compare the clinical potential, scalability, and limitations of different non-viral vectors (Table 4).
• To identify remaining challenges and future directions for synthetic gene delivery materials.

Category: Self-delivering oligonucleotides; Examples Discussed: Phosphorothioate (PS) ASOs, 2′-F-ANA, 2′-O-MOE, LNA, cEt, tcDNA, PS-tcDNA; charge-reduced phosphotriester siRNAs; aptamer–siRNA/miRNA chimeras; rolling-circle amplification/transcription DNA/RNA nanoparticles; PEG-brush pacDNA; DARTs (DNA-assembled recombinant transcription factors)

Category: Physical methods; Examples Discussed: Electroporation, osmotic shock, microinjection, microfluidic cell deformation, nanoneedles, ultrasound-triggered microbubble collapse, acoustic transfection

Category: Protein-based; Examples Discussed: Virus-like particles (VLPs), dsRNA-binding proteins (PKR, p19), albumin/antibody fusions, ARC protein, polyomavirus VLPs

Category: Peptide-based; Examples Discussed: CPPs, PIP peptides, arginine-rich peptides with non-proteinogenic amino acids, CLIP6, amphipathic RALA, peptide nanofibres/nanosheets, foldamers, guanidinocarbonylpyrrole peptides, PepM

Category: Lipid-based; Examples Discussed: Cationic lipids (DOTAP, DOTMA), DOPE, ionizable lipids (DLin-MC3-DMA, DOSPA), cuboplexes, SNARE-inspired fusogenic liposomes, exosomes, sterosomes, lipid nanoparticles

Category: Polymers, dendrimers, micelles; Examples Discussed: PDPA, PDMAEA, CARTs, PEI, cyclodextrin-based polymers, polyhydrazones, fluorinated PAMAM dendrimers, polymetformin, PBAEs

Category: Nanoparticles; Examples Discussed: Carbon nanotubes, fullerenes, nanodiamonds, gold nanoparticles, spherical nucleic acids (SNAs), mesoporous silica nanoparticles, metal–organic frameworks (MOFs)

Category: Payloads; Examples Discussed: Plasmid/minicircle DNA, mRNA, replicon RNA, ASOs/splice-correcting oligos, siRNA, miRNA, DNAzymes/RNAzymes/MNAzymes, CRISPR-Cas9 components

Category: Targeting ligands; Examples Discussed: Aptamers, GalNAc, RGD peptides, antibodies, transferrin, EGF, cRGD, TCP-1

• Categorizes synthetic carriers by chemical structure.
• Discusses in vitro, preclinical, and clinical examples.
• Includes tables summarizing nucleic acid cargoes (Table 1), obstacles and strategies (Table 2), peptide examples (Table 3), and non-viral vector categories (Table 4).
• Covers model systems such as immortalized cell lines, primary cells, spheroids, and mouse models (cancer, Duchenne muscular dystrophy, spinal muscular atrophy, diabetes, leukaemia, etc.).
• Notes clinical-stage examples, including patisiran (lipid nanoparticle siRNA for hereditary transthyretin-mediated amyloidosis) and approved ASOs.

• Chemical synthesis and modification: Click chemistry, disulfide exchange, hydrazone/acetal formation, phosphotriester synthesis, fluorination, ring-opening polymerization.
• Biophysical characterization: X-ray scattering (gyroid cubic phases), dynamic light scattering, zeta potential, electron microscopy.
• Biological assays: Transfection efficiency (luciferase, GFP), siRNA knockdown, mRNA expression, splice correction, CRISPR-Cas9 editing.
• Imaging and tracking: Live-cell confocal microscopy, single-particle tracking, fluorescence correlation spectroscopy.
• In vivo efficacy: Tumour growth inhibition, survival, protein expression, immune responses in mouse models.
• Clinical evaluation: Phase I–III trials (e.g., patisiran).

• No primary experimental data; conclusions are synthesized from existing literature.
• No systematic search strategy, inclusion/exclusion criteria, or meta-analysis.
• Space constraints limited coverage of all relevant research.
• Focus is primarily on synthetic materials; viral vectors and some emerging technologies are discussed but not exhaustively.

Limitations of the field highlighted by the authors:

• In vitro–in vivo gap: Most in vitro studies use serum-free conditions and immortalized cell lines with altered DNA/RNA sensing and survival pathways, which can overestimate delivery efficiency and underestimate toxicity.
• Endosomal escape remains a limiting factor for many carriers, including protein-mediated delivery systems.
• Non-biodegradable nanoparticles (carbon nanotubes, fullerenes, metal nanoparticles) may accumulate in the liver and kidneys, especially with repeated administration.
• Clinical translation challenges: Some cyclodextrin-based polymers showed toxicity in phase I trials; Glybera was withdrawn due to high production costs and lack of demand.
• Scalability and manufacturing: Complex formulations (e.g., some polymeric carriers, exosomes) can be difficult to scale up reproducibly.
• Stability vs. cargo release: Many systems struggle to balance protection of nucleic acids with timely release at the target site.
• Immune response and toxicity: Cationic carriers can be cytotoxic or immunogenic; modified oligonucleotides can cause complement activation, thrombocytopenia, or hepatotoxicity.
• Limited clinical validation: Most synthetic vectors remain preclinical; only a few non-viral approaches have reached clinical approval.