Lysosomes before reaching Even after successful cellular internalization, inefficient endosomal escape remains a major bottleneck in RNAi therapy

The gap

Evidence profile

Sourced from the future work and limitations of the source papers, classified as general, drawn from work published between 2023 and 2026, spanning 3 journals. Those papers have been cited 194 times in total.

Supporting evidence — 3 representative gaps

• Engineered and Mimicked Extracellular Nanovesicles for Therapeutic Delivery (2024) · Nanomaterials · cited 36× · doi

  Since exosomes were identified quite early as high-potential drug-delivery vehicles, this review primarily focuses on recent nanovesicular loading strategies for nucleic acid, protein drugs, and small molecules. However, the last decade demonstrated that exo- some-mediated drug-delivery methods suffer from low mass production, delivery rates, and high cost. One supplementary important issue remains that the specific functions of exosomes are not fully understood, making it challenging to ensure their long-term safety and efficacy. Moreover, one must remember that exosomes have different membrane and cargo compositions and biological functions, depending on source cells and environmen- tal conditions, inducing reproducibility issues. For all these reasons, they cannot be con- sidered as simply acting like empty carriers, especially when they are derived from circu- lating cancer or infected cells. Therefore, the potential of exosome-mimes or analogues was studied more recently. Nevertheless, considering exosomes or exosome-like vesicles, the loading of exogen ther- apeutic agents is almost necessary. Unfortunately, gentle co-incubation is inefficient, and Nanomaterials 2024, 14, 639 26 of 35 the alternative techniques partly damage the vesicles. Therefore, it is essential to ensure consistency of exosomes and drugs structure and function. We especially focused on the recent developments of exosomes-mimes, because they open new lines considering the important issues that are required to be addressed before exosomes can be approved for clinical use. Although exosome-based therapeutics have succeeded in numerous studies, several hurdles also remain. We will announce them hereafter, to put into perspective the path that remains to be taken before their clinical application Enhancing EV secretion is one of the challenges, and several approaches have been developed, such as culturing under turbulent fluids, under acidic conditions, under salty stress, etc. However, it is important to note that exosome composition is dependent on the environmental conditions. This questions the conservation of the attempted function un- der these near-to-lethal stresses. Even if exosomes have intrinsic addressing capacity thanks to surface components and autologous recognition, important research energy was dedicated to further modify- ing their surface with supplementary targeting ability or extended circulating half-life. Such approaches are developed for EVs, but also for the exosome-mimicking particles. This time, it must be noticed that such approaches can potentially induce immune reac- tions, especially if these peptides can be systemically released. Consequently, enhancing the anchoring of such targeting peptides on exosomes is a real issue. Another line of work would be to develop the embedding of exosomes and exosome- like structures in biocompatible hydrogels to control th

  generalfuture work
  Keywords: exosomes exosome important delivery conditions like especially approaches high potential drug recent loading drugs supplementary
• Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem? (2023) · RNA · cited 158× · doi

  Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem? STEVEN F. DOWDY Department of Cellular and Molecular Medicine, UCSD School of Medicine, La Jolla, California 92093, USA ABSTRACT With over 15 FDA approved drugs on the market and numerous ongoing clinical trials, RNA therapeutics, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), have shown great potential to treat human disease. Their mechanism of action is based entirely on the sequence of validated disease-causing genes without the prerequisite knowledge of protein structure, activity or cellular location. In contrast to small molecule therapeutics that passively diffuse across the cell membrane’s lipid bilayer, RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and instead are taken up into cells by endocytosis. However, endosomes are also com- posed of a lipid bilayer barrier that results in endosomal capture and retention of 99% of RNA therapeutics with 1% or less entering the cytoplasm. Although this very low level of endosomal escape has proven sufficient for liver and some CNS disorders, it is insufficient for the vast majority of extra-hepatic diseases. Unfortunately, there are currently no acceptable solutions to the endosomal escape problem. Consequently, before RNA therapeutics can be used to treat widespread hu- man disease, the rate-limiting delivery problem of endosomal escape must be solved in a nontoxic manner. Keywords: ASOs; RNA therapeutics; delivery; endosomal escape; siRNAs INTRODUCTION During the first year of our laboratory some 29 years ago, we indirectly stumbled into the endosomal escape prob- lem (Ezhevsky et al. 1997), and we have been working directly on the problem for the last 15-plus years (Wadia et al. 2004; Lönn et al. 2016). Given the magnitude of impact that solving the endosomal escape problem would have on the entire RNA therapeutics field (and perhaps others), if it was easy, it would have already been solved. Unfortunately, endosomal escape has remained a highly In fact, the recalcitrant problem (Dowdy et al. 2022). more we work on it, the greater the appreciation I have for how difficult it will be to successfully overcome while maintaining a low level of cytotoxicity. My guess is that it will take years of significant effort from multiple groups be- fore we devise a clinically acceptable approach to endoso- mal escape. Built on ∼50 years of oligonucleotide chemistry that has resulted in an increased on-target activity and metabolic stability, while decreasing off-target activity and immuno- genicity (Dowdy 2017; Khvorova and Watts 2017; Crooke et al. 2021), there are currently more than 15 FDA ap- proved combined siRNAs, phosphorothioate backbone ASOs and neutral phosphorodiamidate morpholino oligo- mer (PMO) RNA therapeutics targeting disease-causing genes in the liver, muscle and CNS (Hammond et al. 2021; Corey et al. 2022). As one example, inclisiran, a GalNAc–siRNA conjugate targeting the PSCK9 gene in liv- er to treat hypercholesterolemia, has a single-dose 6 mo duration of response (Fitzgerald et al. 2017). Not surpris- ingly, due to these clinical successes, there has been sig- nificant interest and investment in RNA therapeutics by biotechs and large pharmaceutical companies resulting in numerous ongoing early- and late-stage clinical trials. The mechanism of action of oligonucleotide RNA thera- peutics is based entirely on targeting the mRNA sequence of validated disease-causing genes and does not a priori require the structure of the protein product, knowing the protein activity and/or its cellular location (Dowdy 2017). In comparison to small molecule drugs that, based on an extracellular concentration gradient, are capable of pas- sively diffusing across the cell membrane (Lipinski 2004), RNA therapeutics are too charged, too large and/or too hydrophilic to diffuse across the cell membrane and in- stead are taken up by endocytosis into cells (Fig. 1). Corresponding author: [email protected] Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna .079507.122. Freely available online through the RNA Open Access option. © 2023 Dowdy This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/ by-nc/4.0/. 396 RNA (2023) 29:396–401; Published by Cold Spring Harbor Laboratory Press for the RNA Society

  generalfuture work
  Keywords: therapeutics endosomal escape problem dowdy disease cellular activity across membrane years clinical small sirnas asos
• RNAi-Loaded Nanocarriers Targeting the IL-6/JAK-STAT3-NF-κB Cytokine Axis in Rheumatoid Arthritis (2026) · Journal of Drug Delivery and Therapeutics · doi

  lysosomes before reaching Even after successful cellular internalization, inefficient endosomal escape remains a major bottleneck in RNAi therapy. A substantial fraction of delivered siRNA is degraded within the cytoplasm, preventing incorporation into the RNAinduced silencing complex (RISC).74 This intracellular trafficking challenge necessitates advanced carrier designs capable of membrane destabilization or fusion to release siRNA into the cytosol. However, enhancing endosomal escape must be carefully balanced against cytotoxicity, as excessive membrane disruption may induce cellular stress or apoptosis in non-target tissues. 10.3 Off-Target Effects and Immune Activation Advances in transcriptomics and single-cell sequencing have revealed substantial heterogeneity among RA patients, with distinct molecular endotypes defined by differential activation of IL-6/STAT3, TNF/NF-κB, or therapeutics can be interferon pathways. RNAi customized to selectively silence genes most relevant to an individual’s inflammatory profile, paving the way for precision medicine approaches.71 Such adaptability distinguishes RNAi nanomedicine from fixed-target biologics and suggests a future in which therapeutic regimens are guided by molecular diagnostics to achieve optimized disease control. RNAi therapeutics may inadvertently silence unintended transcripts due to partial sequence complementarity, leading to off-target gene modulation. Such effects can disrupt physiological pathways and complicate safety assessments.75 Furthermore, double-stranded RNA molecules may activate innate immune receptors such as TLR3, TLR7, and RIG-I, resulting in interferon infection.75,76 Although responses that mimic viral chemical modification of siRNA (e.g., 2′-O-methyl or ISSN: 2250-1177 CODEN (USA): JDDTAO Ahirwar et al. Journal of Drug Delivery & Therapeutics. 2026; 16(3):328-355 substitutions) phosphorothioate reduce immunogenicity, achieving an optimal balance between stability, specificity, and biological activity remains a central design challenge.

  generallimitationsevidence 5/5
  Keywords: rnai target sirna therapeutics cellular endosomal escape remains substantial challenge membrane effects immune activation molecular

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