Biochemistry, Genetics and Molecular Biology · Research topic

Open research questions in RNA Interference and Gene Delivery

39 unresolved questions extracted from the limitations and future-work sections of 212 RNA Interference and Gene Delivery papers in our library. Each links back to the study that raised it.

What the literature leaves open

  • In the burgeoning field of therapeutic nucleic acid delivery, dendrimers offer a versatile and multifaceted platform endowed with a wealth of unique properties and possibilities for precision medicine. Dendrimers allow precise control over size, shape, flexibility, amphiphilicity and surface chemistry to enable the encapsulation, protection, release and delivery of nucleic acids to fulfil specific therapeutic needs while achieving favourable biocompatibility, biodegradability and pharmacokinectics, thus ensuring their safety in concert with efficacy. Compared to LNPs, dendrimers offer several intrinsic advantages for nucleic acids delivery. First, dendrimers can be engineered to reduce inflammatory response through surface modification15,57 and to overcome the ‘PEG dilemma’ via structural variation93.

    Precision chemical engineering of dendrimers for nucleic acid delivery · 2026 · DOI
  • siRNA miRNA saRNA DNA ASO mRNA A full list of affiliations appears at the end of the paper.

    Precision chemical engineering of dendrimers for nucleic acid delivery · 2026 · DOI
  • The application of RNAi technology in agricultural pest and pathogen control has vast potential, yet its large-scale commercialization and sustainable development still hinge on overcoming a series of critical technological bottlenecks. The main objective is the continuous optimization of dsRNA design and the enhancement of target prediction accuracy. Contrary to earlier assumptions, emerging evidence suggests that dsRNA length is not the definitive determinant of resistance. Instead, uptake efficiency and environmental context often outweigh intrinsic design parameters. These data must be integrated into efficient, automated design platforms to develop more potent dsRNA molecules. The transition from laboratory-scale yields to industrial-scale production remains a significant hurdle. Many laboratory-proven yields have yet to translate reliably to commercial manufacturing. Although engineered microbial and cell-free systems have reduced costs, scaling is hampered by plasmid instability and inducer toxicity during fermentation. Achieving consistent, low-energy, and standardized processes capable of meeting the agricultural demand for dsRNA requires further optimization. Most critically, the development of efficient and safe nanodelivery systems remains a key limitation. The performance of nanocarriers under field conditions often is inferior to results observed in controlled laboratory trials. Formulations that appear stable in vitro tend to degrade rapidly when exposed to environmental stressors such as UV radiation, precipitation, and temperature fluctuations.

    Rational dsRNA design, scalable production and nanodelivery to enhance spray-induced gene silencing · 2026 · DOI
  • It remains to be investigated if the signal loss was a consequence of the decrease in concentration caused by the buffer exchange to PBS and the following purification step for SAXS measurement.

    Fucoidan-Coated Layer-by-layer Lipid Nanoparticles for the Generation of CAR-Macrophages · 2026 · DOI
  • ABSTRACT Lipid nanoparticles (LNPs) are the cornerstone of RNA‐based vaccine delivery, yet their internal structure, particularly in the presence of large, single‐stranded messenger RNA (mRNA), remains poorly understood.

    Mapping mRNA Localization and Internal Structure in Lipid Nanoparticles through Solid‐State Dynamic Nuclear Polarization NMR and Proton Spin‐Diffusion Modeling · 2026 · DOI
  • ) Dormancy as a therapeutic goal: Shifts the clinical objective from "eradication" to "containment" -- an approach that has succeeded in HIV and CML but is under-explored in solid oncology Combination with liquid biopsy: ctDNA monitoring could identify patients with molecular relapse before radiographic recurrence, triggering mRNA dormancy therapy pre-emptively 9.

    mRNA-LNP Delivery of Endostatin and Angiostatin for Metastatic Dormancy Maintenance: A Proposed Therapeutic Strategy · 2026 · DOI
  • , acyclovir-resistant TK-deficient strains) remains to be evaluated. The MSN delivery system was not designed to selectively target macrophages, and therefore its antiviral activity in other relevant cell types remains to be characterized.

    Mesoporous silica nanoparticle-mediated delivery of a synergistic 5-miRNA combination inhibits HSV-2 infection · 2026 · DOI
  • mRNA vaccines have great potential in the field of immunotherapy for colorectal cancer. However, further research and clinical trials are essential to optimize their efficacy, safety, and long-term benefits. Potential future directions and advancements include: 5.1 Optimizing mRNA vaccine design mRNA can induce cytokine production by activating innate immune responses. However, excessive or sustained cytokine release may lead to severe side effects, including autoimmune reactions, and potentially interfere with specific immune responses against vaccine antigens (160). Excessive innate sensing may impair translation and shorten expression duration, whereas excessive suppression of intrinsic immunostimulatory signals may weaken dendritic cell activation and T-cell priming (161). Therefore, next-generation mRNA design should focus on fine-tuning, rather than eliminating, innate immune stimulation. Optimization at the RNA level should extend beyond nucleoside substitution to include coordinated engineering of the 5′ cap, untranslated regions, poly(A) tail, codon usage, and RNA secondary structure, with the goal of controlling not only expression intensity but also expression kinetics and immunological outcome. Additionally, the IVT preparation process often generates doublestranded RNA (dsRNA) by-products (162). These dsRNA impurities can activate intracellular immune sensing pathways, such as upregulating protein kinase R and oligoadenylate synthase, thereby triggering IFN-I-mediated immune responses that lead to rapid degradation of mRNA and compromise vaccine efficacy (163). While the removal of dsRNA via High-Performance Liquid Chromatography (HPLC) or RNase III enzymatic digestion is technically feasible in industrial settings, these downstream purification methods face significant challenges in terms of scalability, high operational costs (163). Therefore, future research should prioritize upstream process optimization—such as refining IVT parameters and engineering more precise RNA polymerases—to mitigate dsRNA formation at the source, thereby enhancing both the purity and potency of mRNA vaccines. Finally, delivery systems should be optimized not only to protect mRNA from degradation, but also to improve tissue distribution, antigen-presenting cell targeting, repeated-dose tolerability, and safety (164). Thus, future advances will likely depend on integrated strategies that combine RNA engineering, immune modulation, and precision delivery to generate more potent and clinically translatable mRNA cancer vaccines. 5.2 Innovations in mRNA vaccine storage methods The development of new technologies is crucial for stabilizing vaccines while addressing some limitations associated with traditional freeze-dried storage (78).

    The evolution of vaccine strategies for colorectal cancer: from conventional approaches to the mRNA era · 2026 · DOI
  • Synthesis of Findings The advent of lipid nanoparticle (LNP) technology has catalyzed a profound paradigm shift in modern medicine, acting as the critical translational bridge between fundamental molecular VOLUME 25 : ISSUE 04 (April) - 2026 Page No:364. YMER || ISSN : 0044-0477 https://ymerdigital.eu/ biology and clinical oncology. Historically, the clinical utility of nucleic acid therapeutics was severely bottlenecked by their inherent physicochemical vulnerabilities, including rapid enzymatic degradation by ubiquitous nucleases, unfavorable electrostatic properties preventing cellular uptake, and endosomal entrapment. By engineering a sophisticated, multi- component core-shell architecture integrating pH-responsive ionizable lipids, structural phospholipids, cholesterol, and stealth polymers LNPs have successfully overcome these formidable biological barriers. In the context of cancer immunotherapy, this platform has evolved from a simple protective carrier into an active, programmable immunomodulator. Today, LNPs uniquely facilitate the rapid translation of genomic data into precision therapeutics, enabling the in vivo generation of chimeric antigen receptor (CAR) T-cells, the targeted delivery of patient-specific neoantigen vaccines, and the spatiotemporal remodeling of the highly immunosuppressive tumor microenvironment (TME). The Road Ahead: Advancing RNA Architectures While conventional linear mRNA-LNP platforms have achieved landmark clinical validations, their inherently transient expression kinetics mandate repeated dosing regimens, which can exacerbate cumulative toxicities, induce immune exhaustion, and increase patient burden. To achieve durable anti-tumor immunity and expand the therapeutic window, the field must aggressively pivot toward advanced, next generation RNA formats, most notably circular RNA (circRNA) and self-amplifying RNA (saRNA). Circular RNA (circRNA): By utilizing a covalently closed-loop structure that lacks free 5' and 3' termini, circRNA is rendered highly resistant to exonuclease mediated degradation. When formulated within LNPs, this structural stability translates to exponentially prolonged intracellular half-lives, yielding superior and sustained antigen or CAR expression for weeks rather than days, all without the risks of genomic integration. Self-Amplifying RNA (saRNA): By incorporating viral replication machinery (e.g., alphavirus RNA-dependent RNA polymerase), saRNA constructs actively amplify the therapeutic transcript within the host cell cytosol. This self-replication mechanism significantly amplifies translational yield, exerting a profound dose sparing effect that requires 10- to 100-fold lower payload concentrations compared to conventional linear mRNA. Consequently, saRNA-LNPs mitigate the systemic reactogenicity and manufacturing costs associated with high lipid-to-RNA ratios while driving potent, long lasting T-cell responses. A Call for Multidisciplinary Collaboration The future of LNP-mediated cancer immunotherapy is intrinsically tied to our ability to orchestrate complex, multi-scale biological interventions. Overcoming the remaining clinical bottlenecks such as the physical barriers of desmoplastic solid tumors, the accelerated blood clearance (ABC) phenomenon, and the necessity for ultra-cold chain logistics demands unprecedented, multidisciplinary collaboration. The integration of artificial intelligence and machine learning is required to accelerate neoantigen discovery and automate the inverse design of optimized, organ-tropic lipid chemistries. Concurrently, materials scientists, immunologists, and clinical oncologists must unite to engineer stimuli-responsive and VOLUME 25 : ISSUE 04 (April) - 2026 Page No:365. YMER || ISSN : 0044-0477 https://ymerdigital.eu/ lyophilizable nanocarriers that bypass cold-chain dependencies. Through this synergistic convergence of scientific disciplines, the ultimate vision of precision oncology can be realized: the development of potent, "off-the-shelf", and universally accessible cancer immunotherapies that provide curative outcomes for patients worldwide.

    Translational Advances in Lipid Nanoparticle-Mediated Nucleic Acid Therapeutics for Cancer Immunotherapy: Overcoming Resistance and Engineering Immunity · 2026 · DOI
  • Bhumika Chakraborty1 · Taru Singh1 Received: 24 January 2026 / Accepted: 20 April 2026 © The Author(s), under exclusive licence to Springer Nature B.V.

    mRNA vaccines for viral diseases: mechanism, advances, and future perspective · 2026 · DOI
  • Limitations of this study: (1) The use of the miniUcp1 promoter enables specific over-expression in iWAT whereas it limits the use of systemic administration (e.g. intraperitoneal injection) which could lead to transgene expression in brown adipose tissue (2), also represent- ing a limitation during thermoneutrality experiments (3). The platform was not tested in obesity, where adipocyte hyperplasia could dilute AAV titers and require dose optimisation (4). Our system allows expression of the proteforms above its already existing endogenous levels, requiring careful consideration when evaluating physio- logical effects due to possible supraphysiological concen- trations. One can thus speculate about the utility of this system during rescue experiments (such as GoF experi- ments in Aoc3 knock-out background), although this was not tested in this study (5). Many shed proteins remain poorly characterized, with unknown sheddases or cleav- age sites [8, 9]. While this limits our ability to accurately mimic endogenous proteoforms, the system can be adapted to test alternative proteoforms lengths.

    EctoShed: a novel gene delivery platform for functional analysis of adipocyte-shed proteoforms · 2026 · DOI
  • Non-viral cationic systems are designed to enable efficient and safe nucleic acid delivery. Although viral vectors often achieve high transduction efficiency, they are limited by safety concerns, high immunogenicity, and a complex production process. In contrast, the non-viral vectors are generally considered safer, less immunogenic, and easier to produce.

    Emerging Trends and Recent Development in the Synthesis of Non-Viral Cationic Systems for Targeted Gene Delivery Applications · 2026 · DOI
  • Cationic triblock Mater., 2008, 2022, 2021, 2019, 225, 10, 62, 47, 8, 3, 2, https://doi.org/10.63654/icms.2026.03029 52 J. 73, 20, Mol. 359. Rev., 2001, 2023, 2001, Control. Release., radical 2921. the application J. Xia. Atom Maji et al. Innov. Chem. Mater. Sustain. 2026, 3(1), 29-54 gene delivery systems. J. Control. Release, 2016, 236, 1. therapy. https://doi.org/10.1016/j.jconrel.2016.06.023. https://doi.org/10.1016/S0168-3659(01)00295-4. 115. R. Bofinger, M. Zaw-Thin, N. J. Mitchell, P. S. Patrick, C. 97. G. Moad, B. E. Rizzardo, S. H. T. A, Living Radical Stowe, A. Gomez-Ramirez, H. C. Hailes, T. L. Kalber, A. B. Tabor. Polymerization by the RAFT Process – A Second Update. Aust. J. Chem., 2009, 62, 1402. https://doi.org/10.1071/CH09311. Development of lipopolyplexes for gene delivery: A comparison of the effects of differing modes of targeting peptide display on the 98. K. Parkatzidis, H. S. Wang, N. P. Truong, A. Anastasaki. structure and transfection activities of lipopolyplexes. J. Pept. Sci., Recent developments and future challenges in controlled radical 2018, 24, e3131. https://doi.org/10.1002/psc.3131. polymerization: A 2020 update. Chem., 2020, 6, 1575. 116. F. Liu, L. Huang. Electric gene transfer to the liver following https://doi.org/10.1016/j.chempr.2020.06.014. systemic administration of plasmid DNA. Gene Ther., 2002, 9, 99. G. Moad, E. Rizzardo, S. H. Thang. Living radical 1116. https://doi.org/10.1038/sj.gt.3301733. polymerization by the RAFT process a third update. Aust. J. Chem., 2012, 65, 985. https://doi.org/10.1071/CH12295. 117. S. Persano, M. L. Guevara, Z. Li, J. Mai, M. Ferrari, P. P. Pompa, H. Shen. Lipopolyplex potentiates anti-tumor immunity of transfer 100. K. Matyjaszewski, mRNA-based vaccination. Biomaterials, 2017, 125, 81. polymerization. 101, Chem. https://doi.org/10.1016/j.biomaterials.2017.02.019. https://doi.org/10.1021/cr940534g. 118. H. Yun, K. Wang, J. Zhang, G. Peng, H. Zhao. Construction 101. C. J. Hawker, A. W. Bosman, E. Harth. New polymer of peptide-lipoic acid cationic polymers with redox responsiveness synthesis by nitroxide mediated living radical polymerizations. and low toxicity for gene delivery. ACS Omega, 2023, 9, 3499. Chem. Rev., 2001, 101, 3661. https://doi.org/10.1021/cr990119u. https://doi.org/10.1021/acsomega.3c07194. 102. X. Jun, S. Jie, Y. Ting, and H. Teng. Co-delivery of drug and 119. M. -W. Hei, Y. -R. Zhan, P. Chen, R. -M. Zhao, X. -L. Tian, X. DNA from from cationic dual-responsive micelles derived poly(DMAEMA-co-PPGMA). Mater. Sci. Eng., C, 2013, 33, 4545. -Q. Yu, J. Zhang. Lipoic acid-based poly(disulfide)s as versatile in tumor biomolecule delivery vectors and https://doi.org/10.1016/j.msec.2013.07.011. 3210. Pharm., immunotherapy. 103. K. Matyjaszewski, Atom Transfer Radical Polymerization (ATRP): Current status and future perspectives. Macromol., 2012, https://doi.org/10.1021/acs.molpharmaceut.3c00231. 120. D. P. Walsh, A. Heise, F. J. O’Brien, S. A. Cryan, An efficient, 45, 4015. https://doi.org/10.1021/ma3001719. non-viral dendritic vector for gene delivery in tissue engineering. 104. F. Di Lena, K. Matyjaszewski. Transition metal catalysts for Gene Ther., 2017, 24, 681. https://doi.org/10.1038/gt.2017.58. controlled radical polymerization. Prog. Polym. Sci., 2010, 35, 959. 121. N. Sikhosana, L. C. du Toit, P. N. Fru, P. Walvekar, Y. E. https://doi.org/10.1016/j.progpolymsci.2010.05.001. Choonara. In vitro evaluation of a cationic polymer-lipid conjugate 105. S. Maji, F. Mitschang, L. Chen, Q. Jin, Y. Wang, S. Agarwal. as a potential nanosystem for gene delivery. Int. J. Pharm., 2026, Functional poly(dimethyl aminoethyl methacrylate) by combination 692, 126641. https://doi.org/10.1016/j.ijpharm.2026.126641. of radical ring-opening polymerization and click chemistry for biomedical applications. Macromol. Chem. Phys., 2012, 213, 122. C. Pegoraro, E. M. Sanchis, S. Đorđević, I. Dolz-Pérez, C. Huck-Iriart, L. Herrera, S. Esteban-Pérez, I. Conejos-Sanchez, M. 1643. https://doi.org/10.1002/macp.201200220. J. Vicent. Multifunctional polypeptide-based nanoconjugates for 106. Y. Zhang, M. Zheng, T. Kissel, S. Agarwal. Design and targeted mitochondrial delivery and nonviral gene therapy. Chem.

    Emerging Trends and Recent Development in the Synthesis of Non-Viral Cationic Systems for Targeted Gene Delivery Applications · 2026 · DOI
  • Scalable manufacturing and stable formulation protocols for clinical-grade LNP-ncRNA therapeutics require process optimization; specific manufacturing parameters for maintaining ncRNA payload integrity, controlling particle size distribution, and achieving reproducible loading efficiency across different ncRNA classes during scale-up from pilot to GMP production have not been detailed.

    Non-coding RNA delivery via lipid nanoparticles: a novel strategy for cancer immunotherapy · 2026 · DOI
  • Organ- and cell-selective delivery of LNP-ncRNA therapeutics remains a fundamental distribution challenge identified as mid-term priority; specific studies addressing active targeting mechanisms for preferential accumulation in cancer-associated fibroblasts, immune cells, and tumor endothelium versus off-target accumulation in liver and spleen have not been comprehensively reported.

    Non-coding RNA delivery via lipid nanoparticles: a novel strategy for cancer immunotherapy · 2026 · DOI
  • Synergistic co-delivery of diverse ncRNAs (siRNA, miRNA, circRNA, lncRNA combinations) via single LNP particles for tumor microenvironment remodeling has been proposed conceptually but lacks systematic optimization studies; rational design principles for multi-ncRNA payload selection and dosing ratios to achieve synergistic immunomodulation in specific cancer contexts remain undefined.

    Non-coding RNA delivery via lipid nanoparticles: a novel strategy for cancer immunotherapy · 2026 · DOI
  • The POST modular platform for organ-specific LNP surface modification has been proposed theoretically but lacks comprehensive experimental validation across multiple cancer types; specific studies comparing targeting efficacy of different surface modifications (ligands, antibodies, peptides) for selective accumulation in pancreatic cancer, colorectal cancer, and cholangiocarcinoma tissues are needed.

    Non-coding RNA delivery via lipid nanoparticles: a novel strategy for cancer immunotherapy · 2026 · DOI
  • lncRNA delivery systems lack established safety profiles for long-term clinical use, with unknown carcinogenic risks and poor stability; preclinical studies systematically evaluating lncRNA-LNP genotoxicity, off-target genome integration, and in vivo degradation kinetics across tumor microenvironment conditions are absent.

    Non-coding RNA delivery via lipid nanoparticles: a novel strategy for cancer immunotherapy · 2026 · DOI
  • circRNA production faces high-cost barriers and lacks standardized functional validation systems for large-scale manufacturing; specific protocols for circularization efficiency, copy number optimization, and batch-to-batch consistency assessment in GMP-compliant LNP-circRNA formulations have not been established.

    Non-coding RNA delivery via lipid nanoparticles: a novel strategy for cancer immunotherapy · 2026 · DOI
  • Off-target effects and immunogenicity of siRNA-LNP formulations remain inadequately characterized across different cancer types; systematic comparison of off-target binding profiles and immune activation signatures in hepatocellular carcinoma, pancreatic cancer, and non-small cell lung cancer is needed to optimize siRNA sequence design.

    Non-coding RNA delivery via lipid nanoparticles: a novel strategy for cancer immunotherapy · 2026 · DOI
  • Sequence-engineered mRNA without chemical nucleoside modifications has shown efficacy in large animal models, but direct comparative analysis between chemically modified and unmodified mRNA sequences delivered via LNPs, accounting for different ionizable lipid chemistries and surface modifications, is lacking for clinical translation.

    Lipid nanoparticles for mR delivery: from rational design and manufacturing to clinical translation · 2026 · DOI
  • Cap analog chemistry (CleanCap, modified Cap 1 structures) has been optimized for in vitro transcription, but comparative studies evaluating how different cap analogs perform when incorporated into diverse LNP formulations—particularly regarding protection against exonucleases and translation efficiency in vivo—are limited.

    Lipid nanoparticles for mR delivery: from rational design and manufacturing to clinical translation · 2026 · DOI
  • The manufacturing process variables affecting mRNA chemistry (as demonstrated by Nelson et al. 2020 on innate immune activation) require systematic integration with LNP manufacturing parameters; no comprehensive framework currently exists linking mRNA synthesis methods, purification strategies (HPLC), and subsequent LNP assembly on immunogenicity outcomes.

    Lipid nanoparticles for mR delivery: from rational design and manufacturing to clinical translation · 2026 · DOI
  • Recent advances in deep learning and neural network-based codon optimization (CodonTransformer, CodonBERT) show promise for enhancing mRNA translational capacity and stability, but their predictive accuracy and generalization across diverse tissue-specific delivery scenarios with different LNP formulations have not been validated in vivo.

    Lipid nanoparticles for mR delivery: from rational design and manufacturing to clinical translation · 2026 · DOI
  • The impact of mRNA poly(A) tail length on translation efficiency has been demonstrated, but the optimal poly(A) tail length for mRNA-LNP formulations in clinical applications and how tail length interacts with LNP-mediated cellular uptake and mRNA stability in different tissue types requires systematic investigation.

    Lipid nanoparticles for mR delivery: from rational design and manufacturing to clinical translation · 2026 · DOI

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39 open questions have been extracted from the limitations and future-work passages of 212 RNA Interference and Gene Delivery papers in our library. Each one below links back to the study that raised it, so you can read the original claim in context.

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