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Advanced Healthcare Materials2022ReviewNon-viral Gene Delivery

Toward Gene Transfer Nanoparticles as Therapeutics

Erin W. Kavanagh And Jordan J. GreenDOI 10.1002/adhm.202102145

Summary

Viral vectors dominate gene therapy but suffer from immunogenicity, insertional mutagenesis risk, cargo-size limits, and manufacturing challenges. Non-viral nanoparticles are safer, more economical, and easier to scale, but their clinical translation has been limited by inefficient delivery, especially for DNA and gene-editing cargoes that must reach the nucleus. There is a need to review non-viral nanomaterial design, delivery barriers, and. Viral vector dominance: Approximately 70% of gene therapy clinical trials to date have used viruses. - LNP gene editing in liver: A single administration of CRISPR-Cas9 LNPs achieved >97% reduction in target protein.

Keywords

NanoparticlesLipid nanoparticlePolymericmRNADNACRISPRGene editing
Purpose: Viral vectors dominate gene therapy but suffer from immunogenicity, insertional mutagenesis risk, cargo-size limits, and manufacturing challenges. Non-viral nanoparticles are safer, more economical, and easier to scale, but their clinical translation has been limited by inefficient delivery, especially for DNA and gene-editing cargoes that must reach the nucleus. There is a need to review non-viral nanomaterial design, delivery barriers, and emerging strategies for therapeutic gene transfer and gene editing.
Hypothesis: As a review article, this work does not test a single hypothesis. Its central thesis is:

If non-viral nanoparticles are engineered with biodegradable, biocompatible materials that can overcome sequential extracellular and intracellular delivery barriers—and are designed to deliver DNA, mRNA, or ribonucleoproteins for gene editing—then they can enable durable therapeutic gene transfer and gene editing, moving beyond transient mRNA expression toward cures for genetic diseases.

Aims: Primary Aim: To review the transition from viral to non-viral gene delivery, focusing on nanoparticle materials and their therapeutic potential.
  • Secondary Aims:
  • To describe natural, lipid, polymer, inorganic, and metal–organic framework nanomaterials for gene delivery.
  • To outline extracellular and intracellular barriers that nanoparticles must overcome.
  • To discuss gene-editing strategies (CRISPR-Cas9, base editors, RNPs, mRNA, DNA) and their delivery requirements.
  • To highlight clinical progress in blood diseases and cystic fibrosis, and to identify remaining engineering challenges.
Delivery system:

Component: Natural materials; Examples Discussed: Chitosan, cyclodextrins, peptides (lysine/arginine for binding; histidine for endosomal disruption; cell-penetrating peptides)

Component: Lipids; Examples Discussed: Cationic lipids, ionizable lipids, helper lipids, cholesterol, PEG-lipids; lipid nanoparticles (LNPs); liposomes; hybrid lipid–inorganic systems

Component: Polymers; Examples Discussed: Poly(ethyleneimine) (PEI), poly(beta-amino esters) (PBAEs), charge-altering releasable transporters (CARTs), polymersomes, dendrimers (PAMAM, PGA, PLL), PEGylated polymers

Component: Inorganic; Examples Discussed: Silica nanoparticles, gold nanoparticles, spherical nucleic acids (SNAs), mesoporous silica

Component: Metal–organic frameworks (MOFs); Examples Discussed: Metal ions/clusters (Pt, Zr, Fe, Zn, Cu, Ni) with organic bridging ligands

Component: Payloads; Examples Discussed: Plasmid DNA, mRNA, siRNA, ribonucleoproteins (RNPs), Cas9 protein, guide RNA, base editors, donor ssDNA

Component: Targeting ligands; Examples Discussed: Folate, RGD peptides, HER2 antibodies, insulin receptor antibodies, SORT lipid properties

Component: Routes; Examples Discussed: Intravenous, inhalation, local (eye, muscle, brain), ex vivo electroporation

Approach: This is a narrative review synthesizing preclinical and clinical literature. No primary experimental data are presented. The review covers:
  • In vitro studies: Primary human hematopoietic stem/progenitor cells, breast cancer cells, patient-derived organoids, cell lines.
  • In vivo models: Mice, non-human primates.
  • Disease contexts: Cystic fibrosis, hemophilia A/B, hereditary transthyretin amyloidosis, sickle cell disease, beta thalassemia, alpha-1 antitrypsin deficiency, cancer, COVID-19.
  • Clinical trials: NTLA-2001 (CRISPR LNP for ATTR amyloidosis), MRT5005 (mRNA LNP for CF), COVID-19 mRNA-LNP vaccines, ex vivo CRISPR-edited CD34+ cells for hemoglobinopathies.
Key methods: As a review, the “methods” are literature synthesis and comparative analysis. Headline data cited from primary studies were generated using:
  • Reporter gene assays: Luciferase, GFP.
  • Gene editing quantification: Indel rates, HDR efficiency, base editing efficiency, serum protein reduction (e.g., TTR, PCSK9, factor IX/VIII).
  • Clinical endpoints: Adverse events, serum protein levels, FEV1 (lung function), vaccine efficacy.
  • Biodistribution and targeting: SORT lipid delivery profiles, organ-specific expression.
  • Physicochemical characterization: Particle size, zeta potential, cryo-TEM.
  • Toxicity: Cell viability, apoptosis/necrosis, immunogenicity.
Key results: Viral vector dominance: Approximately 70% of gene therapy clinical trials to date have used viruses. - LNP gene editing in liver: A single administration of CRISPR-Cas9 LNPs achieved >97% reduction in target protein levels in a mouse model of alpha-1 antitrypsin deficiency. - Clinical CRISPR LNP (NTLA-2001): In six patients with hereditary transthyretin amyloidosis, a LNP containing Cas9 mRNA and gRNA targeting TTR (0.1–0.3 mg dose) produced only mild adverse events and led to an average 52–87% reduction in serum transthyretin at day 28, depending on dose. - COVID-19 mRNA-LNP vaccines: More than 1 billion mRNA nanoparticle doses administered worldwide; Pfizer/BioNTech and Moderna vaccines showed 94–95% efficacy. - Dendrimer-based LNPs: Achieved ~20% homology-directed repair (HDR) in vivo following direct injection of dLNPs carrying Cas9 mRNA, gRNA, and donor ssDNA. - Targeted polyplexes: HER-2 antibody-conjugated DNA/PEI polyplexes showed up to 20-fold higher gene delivery in breast cancer cells in vitro compared with non-functionalized PEI polyplexes. - Base editor efficiency: Cytosine base editors (CBEs) corrected point mutations with 35–75% target editing efficiency and ~5% indel rate, compared with CRISPR-Cas9’s 0.1–0.3% efficiency and 26–40% indel rate in the cited comparison. - Cystic fibrosis organoids: Base editing restored CFTR function with editing efficiency up to 9.3% without off-target effects. - Cystic fibrosis clinical trial (MRT5005): Nebulized mRNA encoding CFTR in LNPs entered Phase I/II; initial interim analysis showed increased lung function, but a second interim analysis showed no significant improvement in lung function; repeated doses were well tolerated. - Cationic liposome CF trial: Stabilization of FEV1 after 12 monthly doses of CFTR cDNA compared with a decline in controls.
Interpretation: The authors conclude that non-viral nanoparticles are becoming an exciting alternative to viral vectors due to safety, flexible cargo capacity, targeting, and scalable manufacturing. LNPs have demonstrated clinical success for RNA delivery and are promising for DNA and gene editing. To achieve long-term therapeutic genetic medicine, gene editing technologies hold the key. However, non-viral delivery remains far less efficient than viral vectors on a per-particle basis, limiting efficacy and therapeutic window. Overcoming extracellular barriers, endosomal escape, nuclear uptake, off-target accumulation, durability, and manufacturing/sterilization challenges will be necessary for broad clinical application.
Limitations: Limitations inherent to the review:
  • No primary experimental data; conclusions are synthesized from existing literature.
  • No systematic search strategy or meta-analysis.
  • Focus is primarily on non-viral nanoparticles; viral vectors are discussed mainly for comparison.
  • Most cited studies are preclinical or early clinical; limited late-stage clinical data.

Limitations of the field highlighted by the authors:

  • Efficiency gap: Non-viral nanoparticle delivery is far less efficient than the best viral vectors on a per-particle or per-nucleic-acid basis.
  • Endosomal escape bottleneck: A crucial challenge; many NPs are internalized but fail to escape the endosome.
  • Nuclear delivery for DNA: DNA and gene-editing cargoes must reach the nucleus, which is more difficult than cytosolic mRNA/siRNA delivery.
  • Off-target accumulation: Nanoparticles can accumulate in liver and macrophages, reducing potency.
  • Durability: Delivered mRNA and episomal DNA are short-lived; chronic diseases require repeated administration or gene editing for durable effects.
  • Immunogenicity and toxicity: PEG immunogenicity remains under investigation; cationic materials can be cytotoxic; repeat administration safety needs study.
  • Manufacturing challenges: Sensitive biomolecules do not tolerate heat; terminal sterilization and scale-up are challenging.
  • Cystic fibrosis barriers: Mucus penetration, inflammatory lung environment, and extracellular barriers limit delivery; clinical efficacy not yet demonstrated.
  • Clinical translation: Gene editing with non-viral nanoparticles is still early; most successes are preclinical or ex vivo.

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