With the rapid advancement of molecular biology and genetic engineering, technologies including mRNA vaccines, siRNA therapeutics, ASO drugs, gene editing, cell therapy, and in vivo gene replacement therapy have matured progressively. Nucleic acid biopharmaceuticals have fully transitioned from fundamental research to clinical translation. Compared with traditional small-molecule drugs and antibody therapeutics, nucleic acid medicines intervene diseases at the genetic root, featuring abundant druggable targets, shortened R&D cycles, and precise therapeutic efficacy. They demonstrate tremendous application potential in rare diseases, metabolic disorders, oncology, and infectious disease prevention and control.
Nevertheless, native nucleic acid molecules carry inherent drawbacks. Their negatively charged physicochemical properties prevent them from crossing the negatively charged cell membrane barrier. Once in systemic circulation, nucleic acids are rapidly degraded by nucleases, readily trigger innate immune responses, and lack tissue tropism, which frequently leads to off-target effects and systemic toxicity. To address these challenges, selecting appropriate delivery vehicles and developing efficient delivery technologies are critical for nucleic acid therapeutics. An ideal drug delivery system should possess the following properties:
1.Favorable biocompatibility without eliciting host immune reactions;
2.Prolonged blood circulation capability to evade nuclease degradation;
3.Strong tissue targeting and specificity to enable accumulation at lesion sites;
4.High cellular permeability to facilitate cellular uptake;
5.Efficient endosomal escape to allow nucleic acids to exert intracellular functions.
After decades of technological iteration, nucleic acid delivery systems have evolved into three mature technical platforms: viral vectors, non-viral vectors, and physical delivery. Viral vectors dominate long-term gene therapy by virtue of superior transduction efficiency. Non-viral vectors serve as the mainstream platform for nucleic acid vaccines and short-acting nucleic acid drugs owing to high safety, scalable manufacturing, and broad versatility. Physical delivery is primarily applied in basic laboratory research and ex vivo cell engineering. This paper systematically compares the technical characteristics, core advantages and disadvantages, industrialization bottlenecks, and clinical application scenarios of various delivery technologies, providing comprehensive references for technology selection in academia and the biotech industry.
Viral Vector Delivery Technologies
1. Adeno-Associated Virus (AAV)
Adeno-associated virus is a small non-enveloped single-stranded DNA virus with low immunogenicity. In practical applications, its capacity to integrate into the host genome is eliminated to enhance vector safety. AAV boasts abundant serotypes with innate tissue tropism: AAV8 targets the liver, while AAV9 homes to cardiac tissue and crosses the blood-brain barrier. To date, AAV has been widely adopted for in vivo gene therapy, with representative approved products including Luxturna for retinal gene therapy and Zolgensma for spinal muscular atrophy.
Technical Advantages: AAV mediates predominantly non-integrated transgene expression, posing an extremely low risk of genomic insertional mutagenesis with outstanding safety profiles. It delivers high transduction efficiency and sustains stable long-term transgene expression in target tissues for several years, perfectly matching the therapeutic demands of long-term monogenic rare disease treatment.
Limitations: AAV exhibits restricted packaging capacity, with a maximum insert size of merely 4.7 kb, which cannot accommodate large transgenes. In addition, pre-existing neutralizing antibodies against AAV are prevalent in the human population, blocking vector transduction and prohibiting repeated administration. Its large-scale industrial production entails complex workflows and exorbitant costs, hindering widespread commercial adoption.
Derived from engineered HIV-1, lentivirus is an enveloped RNA virus capable of efficiently transducing both dividing and non-dividing cells, rendering it the core viral vector for ex vivo cell engineering. The hallmark feature of lentivirus is genomic integration, which stably inserts exogenous genes into the host cell genome to achieve permanent transgene expression, suitable for stable cell line construction and long-term cellular modification. Its packaging capacity reaches 8 kb, substantially exceeding that of AAV to accommodate longer foreign gene fragments, alongside low immunogenicity and controllable cytotoxicity.
Major Limitations: Its integrating nature carries potential risks of insertional mutagenesis, and its in vivo delivery efficiency is far inferior to AAV, making it unsuitable for systemic administration. Lentiviral packaging workflows require sophisticated processes and stringent quality control standards, accompanied by prohibitive manufacturing costs. Currently, lentivirus is predominantly utilized for ex vivo genetic modification, acting as the core delivery vector for CAR-T, CAR-NK and other cell therapy products, as well as for stem cell engineering and long-term neuronal transfection in basic and clinical research.
Adenovirus is a double-stranded DNA virus incapable of genomic integration, categorized as a transient expression vector. Its core strength lies in an oversized packaging capacity that accommodates large transgenes and multi-gene co-expression cassettes. Adenovirus yields high viral titers with short production cycles and potent transient transgene expression to drive rapid robust target gene expression. Accordingly, adenoviral delivery has been extensively deployed in vaccine development. Multiple adenovirus-vectored COVID-19 vaccines have achieved clinical translation, including candidates developed by CanSino Biologics, AstraZeneca, and Johnson & Johnson, validating its industrialization value.
Primary Drawbacks: Adenovirus induces robust innate and adaptive immune responses upon in vivo administration, triggering inflammatory reactions and organ toxicity. Vectors are rapidly cleared by the host, precluding repeated dosing, and transgene expression only persists for 1–2 weeks. Given these characteristics, adenovirus is rarely deployed for long-term gene therapy, and is mainly applied in vaccine development, transient oncogene therapy, and intratumoral local delivery.
Non-Viral Vector Delivery Technologies
1. Lipid Nanoparticles (LNPs)
Lipid nanoparticles represent the most clinically mature non-viral delivery vectors. Self-assembled from ionizable lipids, helper phospholipids, cholesterol, and PEGylated lipids, LNPs efficiently encapsulate diverse nucleic acids including mRNA, siRNA, and plasmid DNA. The core functional mechanism relies on protonation of ionizable lipids within acidic endosomes to enable robust endosomal escape, releasing nucleic acids into the cytoplasm to execute biological functions.
Core Advantages: LNPs demonstrate broad versatility with minimal constraints on nucleic acid cargo, compatible with all categories of nucleic acid therapeutics. They exhibit inherent hepatic tropism, accumulating efficiently in the liver following intravenous injection. LNPs feature controllable immunogenicity, zero genomic integration risk, and excellent safety profiles, paired with highly standardized manufacturing workflows amenable to large-scale industrial production, as fully validated by the global rollout of mRNA COVID-19 vaccines.
Key Limitations: Conventional LNPs suffer from poor formulation stability, requiring low-temperature cold chain storage and transportation. PEGylation may trigger the accelerated blood clearance (ABC) effect, compromising the efficacy of repeated dosing. Systemically administered LNPs predominantly accumulate in the liver, and achieving efficient extrahepatic tissue targeting remains a major research priority and technical bottleneck. Presently, LNPs are widely utilized in mRNA vaccines, siRNA lipid-lowering therapeutics, oncolytic gene therapy, and transient gene expression research.
Polymeric nanoparticle delivery systems utilize natural or synthetic polymers to bind nucleic acid therapeutics via adsorption, covalent conjugation, crosslinking, or encapsulation for in vivo transport. The principal merits of polymeric nanoparticles stem from their tunable chemical and physical properties; parameters including chemical composition, molecular weight, and polydispersity index can be rationally tailored to match different nucleic acid cargoes.
Centered on cationic polymers such as PEI, PLGA, and chitosan, polymeric vectors condense and adsorb negatively charged nucleic acids via electrostatic interactions to form stable nanocomplexes for cargo encapsulation and delivery. These materials feature robust chemical stability and flexible molecular engineering, enabling surface functionalization for targeted delivery. The proton sponge effect facilitates endosomal escape of nucleic acid therapeutics, alongside simple manufacturing procedures and low production costs.
Compared with LNPs, polymeric vectors exhibit elevated cytotoxicity—high-molecular-weight PEI in particular induces cellular damage. Biodegradable polymer materials mitigate persistent in vivo polymer aggregation and associated latent toxicity to improve biocompatibility. Polymeric vectors deliver inferior in vivo transfection efficiency relative to LNPs and viral vectors, and nanocomplexes are readily cleared by the reticuloendothelial system with suboptimal circulatory stability. At present, polymeric vectors are unsuitable for systemic clinical administration, and are primarily deployed for ex vivo transient cell transfection, cutaneous and mucosal local delivery, and DNA vaccine development in cost-sensitive scenarios with moderate efficiency requirements.
3. GalNAc-Conjugated Vectors
N-acetylgalactosamine (GalNAc) represents a targeted conjugate delivery technology. Covalent linkage of GalNAc small molecules to siRNA leverages the highly specific ASGPR receptors abundantly expressed on hepatocytes to achieve precise hepatic nucleic acid delivery. This carrier-free purely chemical conjugation platform eliminates vector-associated toxicity with exceptional biosafety.
Core Advantages: GalNAc conjugation delivers ultra-high hepatic targeting specificity, potent delivery efficiency, minimal immunogenicity, and compatibility with repeated dosing, establishing it as the gold-standard technology for liver-targeted siRNA therapeutics.
Application Limitations: This platform is only compatible with short-strand siRNA (≤21 nt) and cannot accommodate long nucleic acid cargo such as mRNA or plasmids. To date, GalNAc technology has been fully commercialized for siRNA therapeutics targeting hepatic metabolic diseases, hyperlipidemia, and liver-derived genetic disorders, with marketed representative products including Inclisiran and Givosiran.
Physical Delivery Technologies
Physical delivery employs external mechanical forces to disrupt cell membrane barriers and directly import nucleic acids into cells without auxiliary vectors, encompassing electroporation, microinjection, gene gun delivery, and other modalities. Electroporation applies high-voltage pulses to transiently perforate cell membranes for rapid nucleic acid loading with relatively high delivery efficiency, yet it inflicts severe cellular damage and high mortality rates, requiring specialized dedicated equipment. Microinjection and gene gun technology offer superior precision but feature cumbersome operation and extremely low throughput, incompatible with large-scale applications.
Overall, physical delivery imposes no biological toxicity or vector-mediated immune risks and imposes no restrictions on nucleic acid cargo types. However, its invasive nature, low throughput, and challenges for in vivo administration confine its application to niche scenarios including basic laboratory research, ex vivo cell engineering, and embryonic gene editing, lacking large-scale clinical industrialization potential.
Technology Selection Principles and Application Matching Strategies
Standardized technology selection frameworks can be formulated based on the technical profiles of various delivery platforms and combined clinical and industrial demands to achieve precise application matching:
1.Long-term in vivo gene therapy: AAV vectors are the primary choice for sustained gene replacement in ocular, neural, muscular, hepatic, and other tissues. Adenovirus vectors are selected for oversized transgene delivery where prolonged expression is unnecessary.
2.Ex vivo cellular genetic modification: Lentivirus is preferred for stable cell engineering such as CAR-T and stem cell modification; polymeric vectors or LNPs are adopted for low-cost transient transfection.
3.Nucleic acid therapeutics and vaccine development: LNPs serve as the universal platform for mRNA vaccines and multi-type nucleic acid drugs. GalNAc conjugation technology is prioritized for pure liver-targeted siRNA drugs to realize high-specificity and high-safety delivery.
4.Cost-sensitive and basic research scenarios: Low-cost polymeric vectors are favored for routine ex vivo transfection, mucosal local administration, and fundamental laboratory research. Physical delivery is adopted for small-scale precision cellular modification.
Existing Technical Bottlenecks and Future Development Trends
1. Current Core Technical Limitations
All nucleic acid delivery vectors retain prominent technical drawbacks. Viral vectors generally suffer from high manufacturing costs, limited packaging capacity, interference from pre-existing antibodies, and barriers to repeated dosing, failing to balance safety and large-scale production. Lipid carriers such as LNPs are plagued by insufficient formulation stability, weak extrahepatic targeting capacity, and mandatory cold chain logistics. GalNAc technology is restricted to narrow application scopes and cannot support diversified nucleic acid therapeutics. Polymeric vectors have yet to fully resolve the trade-off between delivery efficiency and cytotoxicity, while physical delivery cannot be translated for in vivo clinical use. Collectively, an integrated delivery platform combining high efficiency, superior safety, broad compatibility, robust stability, low cost, and repeatable administration remains unachieved.
2. Future Development Directions
Nucleic acid delivery vectors will evolve toward four major trajectories: precise targeting, multi-functional vector design, streamlined manufacturing workflows, and affordable production costs.
1.Vector engineering optimization: Capsid modification of AAV, innovation of LNP lipid materials, and surface targeting functionalization will break tissue tropism limitations to enable precise delivery to extrahepatic organs.
2.Development of hybrid composite vectors: Novel delivery systems integrating the strengths of multiple vector classes will be engineered to combine high transfection efficiency, low toxicity, and enhanced stability.
3.Formulation technology upgrading: Ambient-temperature stable nucleic acid formulations will be developed to eliminate cold chain reliance and cut storage and transportation expenses.
4.Industrial process optimization: Simplified production workflows for viral and nanocarrier vectors will lower manufacturing costs and advance the universal accessibility of nucleic acid therapeutics and gene therapy.
5.Expansion of repeatable administration technologies: Vector surface modification will circumvent host immune rejection and clearance effects to enable multi-cycle therapeutic dosing.
Nucleic acid delivery vectors constitute the core technical barrier of the nucleic acid drug and gene therapy industry. Viral and non-viral vectors form complementary technical systems catering to differentiated demands of long-term gene therapy versus short-acting nucleic acid therapeutics and vaccines. Sustained innovations in material science, vector engineering, and formulation technology will continuously elevate the safety, targeting performance, and stability of existing delivery platforms. Novel multi-functional delivery vectors will repeatedly overcome technical bottlenecks, extending nucleic acid therapeutic applications from rare diseases and infectious diseases to broader clinical fields including oncology and chronic disorders, delivering core support for precision medicine and the advancement of the biopharmaceutical industry.
