Application Note
siRNA Therapeutics and siRNA-LNP Applications
siRNA-LNP therapeutics represent the convergence of precise RNA interference technology with sophisticated lipid nanoparticle delivery systems, achieving eight FDA approvals through 2025 and demonstrating exceptional clinical potential. While current applications predominantly target hepatic diseases, breakthrough technologies including SORT LNPs, next-generation ionizable lipids, and microfluidic manufacturing are expanding delivery capabilities to extrahepatic tissues and improving therapeutic efficacy.
1. Working Mechanism of siRNA-LNPs
Small interfering RNA (siRNA) therapeutics delivered via lipid nanoparticles (LNPs) represent one of the most clinically validated approaches for nucleic acid medicine. siRNA molecules are synthetic double-stranded RNA constructs, typically 20-25 base pairs in length, that harness the endogenous RNA interference (RNAi) pathway to achieve sequence-specific gene silencing[1]. The mechanism proceeds through four sequential steps: Dicer-mediated processing of double-stranded RNA into siRNA duplexes; binding to the Argonaute-2 protein within the RNA-induced silencing complex (RISC); strand separation and passenger strand degradation; and guide strand-directed cleavage of complementary target mRNA[2].
Fig 1. Schematic of the mechanism of synthetic small interfering RNA (siRNA)-mediated knockdown. Image adapted from BioDrugs. 2022 Sep;36(5):549-71.
LNPs provide the critical delivery vehicle that protects siRNA from nuclease degradation and enables cellular uptake and endosomal escape[3]. The standard LNP formulation has four essential components: ionizable cationic lipids (typically 50 mol%), helper phospholipids (10 mol%), cholesterol (38.5 mol%), and PEGylated lipids (1.5 mol%)[4]. Of these, the ionizable lipids serve as the functional core with their pH triggered charge shift. Briefly, they are neutral at physiological pH (7.4) and positively charged in acidic environments (pH 5-6). This reduces LNP toxicity during circulation while facilitating membrane destabilization and RNA release in the endosomal environment[5][6].
Fig 2. Mechanism of LNP-Mediated RNA Interface. Image adapted from EBioMedicine. 2020 Feb 1;52.
2. FDA-Approved siRNA Therapeutics
The clinical validation of siRNA-LNP technology is exemplified by Patisiran (Onpattro®), the first FDA-approved siRNA therapeutic in August 2018[7]. This landmark approval demonstrated the safety and efficacy of LNP-mediated siRNA delivery in humans for treating hereditary transthyretin (TTR) amyloidosis. The APOLLO clinical trial showed an 81% reduction in serum TTR levels with significant improvements in polyneuropathy and quality of life measures[8].
Fig 3. Key milestones in the development of Patisiran for the treatment of hereditary transthyretin-mediated amyloidosis. Image adapted from Drugs. 2018 Oct;78:1625-31.
Among the eight FDA-approved siRNA drugs as of July 2025, Patisiran remains the only LNP-formulated therapeutic, while the others utilize GalNAc conjugation for hepatocyte-specific delivery. The predominance of GalNAc conjugates reflects their advantages for liver targeting: simplified synthesis, defined chemical composition, subcutaneous administration, and reduced immunogenicity compared to complex LNP formulations[9][10]. However, LNPs maintain advantages for certain applications, including protection from harsh endolysosomal environments and potential for extrahepatic delivery[8].
Fig 4. Mechanism of action of GalNAc-conjugated siRNAs. Image adapted from The Journal of Clinical Pharmacology. 2024 Jan;64(1):45-57.
As of July 2025, eight siRNA drugs have received FDA approval, all leveraging either lipid nanoparticles (LNPs) or GalNAc conjugation for hepatocyte targeting:
Therapeutic Name (Brand) | Indication | Delivery Platform | Approval Date |
|---|
Seven siRNA drugs are in pivotal Phase III trials, representing diverse therapeutic areas beyond hepatology:
Drug Name | Target mRNA | Indication | Delivery | Administration | Phase III Status |
|---|---|---|---|---|---|
Olpasiran | Lp(a) | Cardiovascular risk reduction | GalNAc | SC | Phase III (olpasiran HORIZON) |
Inclisiran | PCSK9 | Hypercholesterolemia | GalNAc | SC | Commercially approved; outcomes trials ongoing |
Tivanisiran | TRPV1 | Dry eye disease | – | Topical | Completed |
Cosdosiran | Caspase 2 | Primary angle-closure glaucoma | – | IV | Terminated |
Teprasiran | p53 | Cardiac surgery–associated AKI | – | IV | Completed |
Nedosiran | Hepatic LDH | Primary hyperoxaluria | GalNAc | SC | Enrolling by invitation |
Fitusiran (ALN-AT3SC) | SERPINC1 (antithrombin) | Hemophilia A/B | GalNAc | SC | Active, not recruiting |
siRNA has many advantages compared with traditional small molecules and antibodies. These include abundant disease targets, high development success rate, short development time, robust and long-lasting efficacy, and outstanding attributes of platform-based modalities. Despite the broad application prospects of siRNA drugs in clinical practice, their development faces pivotal challenges, including targeted accumulation and cellular uptake (entry), endolysosomal escape (escape), and in vivo pharmaceutical performance (efficacy).
Fig 5. Challenges that limit the clinical translation and application of siRNA. Images adapted from Trends in Molecular Medicine. 2024 Jan 1;30(1):13-24.
3. siRNA-LNPs vs. siRNA-GalNAc Conjugates
The choice between LNP delivery and GalNAc conjugation for siRNA therapeutics depends on specific therapeutic requirements and target profiles[8][9]. GalNAc conjugates offer several advantages: simplified synthesis on solid-state oligonucleotide synthesizers; chemically defined composition amenable to mass spectrometry characterization; subcutaneous administration; and reduced immunogenicity. These advantages have led to GalNAc technology largely replacing LNPs for liver-targeted siRNA therapeutics[10].
However, LNPs maintain distinct advantages including potential for extrahepatic targeting and protection from degradation in harsh endolysosomal environments. Direct comparison studies show that LNP delivery provides more rapid onset of action with shorter duration, while GalNAc conjugates exhibit delayed onset but prolonged effect due to slow release from endosomal compartments[8]. The metabolic stability of siRNA in acidic subcellular compartments is critical for GalNAc conjugates but less important for LNP-delivered siRNA[8].
4. LNP Manufacturing and Microfluidic Production
The manufacturing of siRNA-LNPs has evolved significantly, with microfluidic mixing emerging as the gold standard for production[11][12]. This process involves rapid mixing of lipid solutions in ethanol with siRNA in mildly acidic aqueous buffer, achieving near-perfect encapsulation efficiency (>90%) and precise particle size control[13].
Fig 6. siRNA-LNP synthesis with microfluidic mixers.
Recent advances have introduced parallelized microfluidic devices capable of producing LNPs at scales exceeding 18.4 L/h, representing a 100-fold increase compared to single-channel devices[14]. The parallelized microfluidic device (PMD) incorporates ladder geometry with flow resistors to ensure uniform distribution across multiple mixing channels, enabling scalable production without compromising particle quality or biological activity.
5. Applications of siRNA-LNPs
5.1 Cancer Therapy
siRNA-LNPs have demonstrated significant potential in oncology applications, with multiple clinical trials investigating various targets. ALN-VSP, an LNP formulation targeting both VEGF and kinesin spindle protein (KSP), completed a first-in-humans Phase I study in patients with advanced solid tumors[18]. The study demonstrated detectable drug levels in both hepatic and extrahepatic tumor biopsies, evidence of siRNA-mediated mRNA cleavage. Additionally, it showcased antitumor activity, including complete regression of liver metastases in endometrial cancer.
Fig 7. Clinical activity of ALN-VSP in patients with cancer. (A) complete response in endometrial cancer patient 021 with multiple liver metastases. (B) decrease in blood flow (Ktrans) in liver tumors of patients treated with ALN-VSP. (C) DCE-MRI images from patient 012 with metastatic PNET treated at 0.7 mg/kg. (D), decrease in spleen volume in patients treated with ALN-VSP. Image adapted from Cancer discovery. 2013 Apr 1;3(4):406-17.
STP707 by Sirnaomics represents another promising cancer therapeutic, targeting both TGF-β1 and COX-2 mRNA simultaneously using polymeric nanoparticles. Phase I trials showed that 74% of evaluable patients achieved stable disease, with pancreatic cancer patients demonstrating particularly encouraging results with an average of 92 days of stable disease[19]. However, no siRNA anticancer therapeutics have progressed to Phase III trials as of 2025, reflecting the ongoing challenges in delivering siRNA effectively to solid tumors[20].
5.2 Selective Organ Targeting (SORT) Technology
A major breakthrough in LNP technology is the development of Selective Organ Targeting (SORT) LNPs, which incorporate a fifth lipid component to alter biodistribution patterns beyond the liver[21][22]. SORT technology leverages an endogenous targeting mechanism where different SORT molecules recruit specific serum proteins that interact with cognate receptors in target organs[22].
The mechanism involves three sequential steps: desorption of PEG lipids from the LNP surface exposing underlying SORT molecules, binding of distinct serum proteins to the exposed SORT molecules, and subsequent interactions between surface-bound proteins and tissue-specific receptors. Lung-targeting SORT LNPs using permanently cationic lipids, spleen-targeting formulations with permanently anionic lipids, and liver-enhanced targeting with ionizable amino lipids have all demonstrated successful tissue-specific delivery in preclinical studies[23].
Fig 8. SORT nanoparticles for tissue-specific mRNA delivery have unique biodistribution and ionization behavior. Image adapted from Proceedings of the National Academy of Sciences. 2021 Dec 28;118(52):e2109256118.
5.3 Safety and Toxicity Considerations
siRNA-LNP safety profiles have been extensively characterized through clinical experience and preclinical studies. The primary safety concerns relate to ionizable lipid toxicity, particularly at high doses where LNPs can accumulate in liver sinusoidal endothelial cells (LSECs) and activate Toll-like receptors (TLRs). This in turn triggers cytokine release, leading to neutrophilic inflammation[24]. This toxicity mechanism has been successfully addressed through hepatocyte-specific targeting using GalNAc modification, which dramatically reduces LSEC uptake and associated inflammatory responses.
Aside from ionizable lipids, PEGylated lipids also present concerns for repeated dosing due to potential anti-PEG antibody formation. This can cause accelerated blood clearance and reduced therapeutic efficacy. Recent innovations include replacing PEGylated lipids with polysarcosine (pSar) to reduce complement activation and replacing ionizable lipids with biodegradable trehalose glycolipids to minimize toxicity[25].
6. Future of siRNA-LNPs
6.1 Endosomal Escape
A fundamental challenge in siRNA-LNP therapeutics is achieving efficient endosomal escape, with studies showing that less than 2% of internalized siRNA successfully reaches the cytoplasm[15]. The endosomal escape process relies on the pH-sensitive properties of ionizable lipids, which become positively charged in acidic endosomes. This forms destabilizing hexagonal (HII) phase structures that facilitate membrane disruption[16].
Recent research using super-resolution microscopy has revealed that endosomal escape occurs preferentially from early endocytic/recycling compartments rather than late endosomes[17]. The study identified that Rab11-positive recycling endosomes have the highest probability for mRNA escape, with escape events captured from endosomal recycling tubules. This mechanistic understanding suggests that the high positive curvature of recycling tubules may create membrane instabilities conducive to RNA release.
Fig 9. Schematic representation of the Proton Sponge Effect: Due to the buffering ability of ionizable lipids, there is a huge influx of protons by activation of the proton pump in endolysosomal compartments. To neutralize the membrane potential, an inflow of chloride ion is triggered creating an osmotic imbalance which is followed by water intake. This leads to endolysosomal compartment swelling and eventually burst, which releases the cargo. Image adapted from Proceedings of the National Academy of Sciences. 2024 Mar 12;121(11):e2307800120.
6.2 Next-Generation Ionizable Lipids
The development of advanced ionizable lipids continues to drive improvements in siRNA-LNP efficacy and safety. Recent work has focused on structure-activity relationships, identifying four necessary and sufficient criteria for effective siRNA delivery: specific structural characteristics; optimal pKa values (typically 6.2-6.5); appropriate lipid tail saturation; and suitable headgroup properties[26][27].
Artificial intelligence-driven approaches are accelerating ionizable lipid discovery, with machine learning models predicting critical properties such as apparent pKa and delivery efficiency[27]. Recent advances include unsaturated thioether ionizable lipids with modified headgroups that demonstrate more than 200-fold improvement in in vivo mRNA delivery compared to earlier generations[28]. These next-generation lipids achieve optimal physicochemical properties while maintaining low toxicity and reduced hemolytic activity.
Fig 10. Overview of AI-driven rational design of ionizable lipids for mRNA lipid nanoparticles. Image adapted from Nature Communications. 2024 Dec 30;15(1):10804.
Recent advances in automation technology have led to the development of sophisticated platforms capable of preparing and characterizing hundreds of LNP formulations in a single experimental run. The precision and reproducibility afforded by these robotic systems ensure direct head-to-head comparisons between formulations, providing researchers with reliable data to guide further development decisions.
Fig 11. NanoGenerator Flex-S Plus: A high throughput automatic workstation from Precigenome
Fig 12. Workflow with the NanoGenerator Flex-S Plus containing library prep, LNP synthesis and ethanol removal
6.3 Clinical Pipeline and Future Directions
The siRNA therapeutic landscape demonstrates unprecedented growth with over 150 industry-sponsored clinical trials conducted between 2020-2024, representing a compound annual growth rate of 79.5%[29]. Current late-stage development includes multiple Phase III candidates across diverse therapeutic areas including cardiovascular disease (olpasiran for Lp(a) reduction), hematology (fitusiran for hemophilia), and acute care medicine (teprasiran for acute kidney injury).
Future directions for siRNA-LNP technology include expansion beyond liver targeting through SORT and other advanced targeting strategies, combination therapies incorporating siRNA with mRNA or gene editing systems, and personalized medicine approaches using patient-specific siRNA sequences[30]. The global market for small interfering RNA (siRNA) therapeutics is experiencing rapid growth driven by expanding clinical approvals, novel delivery platforms, and increasing R&D investment. Market size is estimated at USD 2.55 billion in 2024, with projections reaching USD 12.38 billion by 2033 at a CAGR of 17.4%[31].
7. Conclusion
siRNA-LNP therapeutics have achieved clinical validation through FDA approvals and demonstrated therapeutic potential across multiple disease areas. While current applications predominantly focus on hepatic diseases, technological advances in SORT targeting, next-generation ionizable lipids, and manufacturing scalability are expanding the therapeutic scope. The successful integration of AI-driven design, advanced manufacturing processes, and mechanistic understanding of endosomal escape positions siRNA-LNPs as a transformative platform for precision medicine, offering unprecedented opportunities to target previously "undruggable" proteins and pathways across diverse therapeutic applications.
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