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1. The Interplay Between Viruses and RNAi Pathways in Insects

   https://doi.org/10.1146/annurev-ento-033020-090410  

   Bonning, Bryony C. ; Saleh, Maria-Carla ( January 2021 , Annual Review of Entomology)

   null (Ed.)

   As an overarching immune mechanism, RNA interference (RNAi) displays pathogen specificity and memory via different pathways. The small interfering RNA (siRNA) pathway is the primary antiviral defense mechanism against RNA viruses of insects and plays a lesser role in defense against DNA viruses. Reflecting the pivotal role of the siRNA pathway in virus selection, different virus families have independently evolved unique strategies to counter this host response, including protein-mediated, decoy RNA–based, and microRNA-based strategies. In this review, we outline the interplay between insect viruses and the different pathways of the RNAi antiviral response; describe practical application of these interactions for improved expression systems and for pest and disease management; and highlight research avenues for advancement of the field. 

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2. In vivo bone marrow microenvironment siRNA delivery using lipid–polymer nanoparticles for multiple myeloma therapy

   https://doi.org/10.1073/pnas.2215711120  

   Guimarães, Pedro_P G ; Figueroa-Espada, Christian G ; Riley, Rachel S ; Gong, Ningqiang ; Xue, Lulu ; Sewastianik, Tomasz ; Dennis, Peter S ; Loebel, Claudia ; Chung, Amanda ; Shepherd, Sarah J ; et al ( June 2023 , Proceedings of the National Academy of Sciences)

   Multiple myeloma (MM), a hematologic malignancy that preferentially colonizes the bone marrow, remains incurable with a survival rate of 3 to 6 mo for those with advanced disease despite great efforts to develop effective therapies. Thus, there is an urgent clinical need for innovative and more effective MM therapeutics. Insights suggest that endothelial cells within the bone marrow microenvironment play a critical role. Specifically, cyclophilin A (CyPA), a homing factor secreted by bone marrow endothelial cells (BMECs), is critical to MM homing, progression, survival, and chemotherapeutic resistance. Thus, inhibition of CyPA provides a potential strategy to simultaneously inhibit MM progression and sensitize MM to chemotherapeutics, improving therapeutic response. However, inhibiting factors from the bone marrow endothelium remains challenging due to delivery barriers. Here, we utilize both RNA interference (RNAi) and lipid–polymer nanoparticles to engineer a potential MM therapy, which targets CyPA within blood vessels of the bone marrow. We used combinatorial chemistry and high-throughput in vivo screening methods to engineer a nanoparticle platform for small interfering RNA (siRNA) delivery to bone marrow endothelium. We demonstrate that our strategy inhibits CyPA in BMECs, preventing MM cell extravasation in vitro. Finally, we show that siRNA-based silencing of CyPA in a murine xenograft model of MM, either alone or in combination with the Food and Drug Administration (FDA)-approved MM therapeutic bortezomib, reduces tumor burden and extends survival. This nanoparticle platform may provide a broadly enabling technology to deliver nucleic acid therapeutics to other malignancies that home to bone marrow.

    

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3. Ionizable Lipid Nanoparticles with Integrated Immune Checkpoint Inhibition for mRNA CAR T Cell Engineering

   https://doi.org/10.1002/adhm.202301515  

   Hamilton, Alex G ; Swingle, Kelsey L ; Joseph, Ryann A ; Mai, David ; Gong, Ningqiang ; Billingsley, Margaret M ; Alameh, Mohamad‐Gabriel ; Weissman, Drew ; Sheppard, Neil C ; June, Carl H ; et al ( December 2023 , Advanced Healthcare Materials)

   The programmed cell death protein 1 (PD‐1) signaling pathway is a major source of dampened T cell activity in the tumor microenvironment. While clinical approaches to inhibiting the PD‐1 pathway using antibody blockade have been broadly successful, these approaches lead to widespread PD‐1 suppression, increasing the risk of autoimmune reactions. This study reports the development of an ionizable lipid nanoparticle (LNP) platform for simultaneous therapeutic gene expression and RNA interference (RNAi)‐mediated transient gene knockdown in T cells. In developing this platform, interesting interactions are observed between the two RNA cargoes when co‐encapsulated, leading to improved expression and knockdown characteristics compared to delivering either cargo alone. This messenger RNA (mRNA)/small interfering RNA (siRNA) co‐delivery platform is adopted to deliver chimeric antigen receptor (CAR) mRNA and siRNA targeting PD‐1 to primary human T cells ex vivo and strong CAR expression and PD‐1 knockdown are observed without apparent changes to overall T cell activation state. This delivery platform shows great promise for transient immune gene modulation for a number of immunoengineering applications, including the development of improved cancer immunotherapies.

    

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4. Mechanisms, Applications, and Challenges of Insect RNA Interference

   https://doi.org/10.1146/annurev-ento-011019-025224  

   Zhu, Kun Yan ; Palli, Subba Reddy ( January 2020 , Annual Review of Entomology)

   The RNA interference (RNAi) triggered by short/small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been widely used as a research tool to study gene functions and has shown great potential for the development of novel pest management strategies. RNAi is highly efficient and systemic in coleopterans but highly variable or inefficient in many other insects. Differences in double-stranded RNA (dsRNA) degradation, cellular uptake, inter- and intracellular transports, processing of dsRNA to siRNA, and RNA-induced silencing complex formation influence RNAi efficiency. The basic dsRNA delivery methods include microinjection, feeding, and soaking. To improve dsRNA delivery, various new technologies, including cationic liposome–assisted, nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries, have been developed. Major challenges to widespread use of RNAi in insect pest management include variable RNAi efficiency among insects, lack of reliable dsRNA delivery methods, off-target and nontarget effects, and potential development of resistance in insect populations. 

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5. Systemic siRNA delivery to tumors by cell-penetrating α-helical polypeptide-based metastable nanoparticles

   https://doi.org/10.1039/C8NR03976C  

   Liu, Yang ; Song, Ziyuan ; Zheng, Nan ; Nagasaka, Kenya ; Yin, Lichen ; Cheng, Jianjun ( January 2018 , Nanoscale)

   Systemic, non-viral siRNA delivery for cancer treatment is mainly achieved via condensation by cationic materials ( e.g. , lipids and cationic polymers), which nevertheless, suffers from poor serum stability, non-specific tissue interaction, and unsatisfactory membrane activity against efficient in vivo gene knockdown. Here, we report the design of a metastable, cancer-targeting siRNA delivery system based on two functional polymers, PVBLG-8, a cationic, helical cell-penetrating polypeptide, and poly( l -glutamic acid) (PLG), an anionic random-coiled polypeptide. PVBLG-8 with rigid, linear structure showed weak siRNA condensation capability, and PLG with flexible chains was incorporated as a stabilizer which provided sufficient molecular entanglement with PVBLG-8 to encapsulate the siRNA within the polymeric network. The obtained PVBLG-8/siRNA/PLG nanoparticles (PSP NPs) with positive charges were sequentially coated with additional amount of PLG, which reversed the surface charge from positive to negative to yield the metastable PVBLG-8/siRNA/PLG@PLG (PSPP) NPs. The PSPP NPs featured desired serum stability during circulation to enhance tumor accumulation via the enhanced permeability and retention (EPR) effect. Upon acidification in the tumor extracellular microenvironment and intracellular endosomes, the partial protonation of PLG on PSPP NPs surface would lead to dissociation of PLG coating from NPs, exposure of the highly membrane-active PVBLG-8, and surface charge reversal from negative to positive, which subsequently promoted tumor penetration, selective cancer cell internalization, and efficient endolysosomal escape. When siRNA against epidermal growth factor receptor (EGFR) was encapsulated, the PSPP NPs showed excellent tumor penetration capability, tumor cell uptake level, EGFR silencing efficiency, and tumor growth inhibition efficacy in U-87 MG glioblastoma tumor spheroids in vitro and in xenograft tumor-bearing mice in vivo , outperforming the PSP NPs and several commercial reagents such as Lipofectamine 2000 and poly( l -lysine) (PLL). This study therefore demonstrates a facile and unique design approach of metastable and charge reversal NPs, which overcomes multiple biological barriers against systemic siRNA delivery toward anti-cancer treatment. 

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