Delivery Of RNA Research Articles

Extracellular vesicle-mediated delivery of anti-miR-106b inhibits morphine-induced primary ciliogenesis in the brain

Repeated use of opioids such as morphine causes changes in theshape and signal transduction pathways of various braincells,including astrocytes and neurons, resulting in alterations in brain functioning and ultimately leading to opioid use disorder. We previously demonstrated that extracellular vesicle(EV)-induced primary ciliogenesis contributes to the development ofmorphine tolerance. Herein, we aimed to investigate theunderlying mechanisms and potential EV-mediated therapeutic approach to inhibit morphine-mediated primaryciliogenesis. We demonstrated that miRNA cargo inmorphine-stimulated-astrocyte-derived EVs (morphine-ADEVs) mediated morphine-induced primary ciliogenesis in astrocytes. CEP97 is a target of miR-106b and is a negative regulator of primary ciliogenesis. Intranasal delivery of ADEVs loaded with anti-miR-106b decreased the expression of miR-106b in astrocytes, inhibited primary ciliogenesis, and prevented the development of tolerance in morphine-administered mice. Furthermore, we confirmed primary ciliogenesis in the astrocytes of opioid abusers. miR-106b-5p in morphine-ADEVs induces primary ciliogenesis via targeting CEP97. Intranasal delivery of ADEVs loaded with anti-miR-106b ameliorates morphine-mediated primary ciliogenesis and prevents morphine tolerance. Our findings bring new insights into the mechanisms underlying primary cilium-mediated morphine tolerance and pave the way for developing ADEV-mediated small RNA delivery strategies for preventing substance use disorders.

Drug-delivery research and much more: interview with Dan Peer.

Drug-delivery research and much more: interview with Dan Peer.

Exosomal delivery of 7SK long non-coding RNA suppresses viability, proliferation, aggressiveness and tumorigenicity in triple negative breast cancer cells

AimsRN7SK (7SK), a highly conserved non-coding RNA, serves as a transcription regulatorvia interaction with a few proteins. Despite increasing evidences which support thecancer-promoting roles of 7SK-interacting proteins, limited reports address the direct link between 7SK and cancer. To test the hypothetic suppression of cancer by overexpression of 7SK, the effects of exosomal 7SK delivery on cancer phenotypes were studied. Materials and methodsExosomes derived from human mesenchymal stem cells were loadedwith 7SK (Exo-7SK). MDA-MB-231, triple negative breast cancer (TNBC), cell line was treated with Exo-7sk. Expression levels of 7SK were evaluated by qPCR. Cell viability was assessed via MTT and Annexin V/PI assays as well as qPCR assessment of apoptosis-regulating genes. Cell proliferation was evaluated by growth curve analysis, colony formation and cell cycle assays. Aggressiveness of TNBCs was evaluated via transwell migration and invasion assays and qPCR assessment of genes regulating epithelial to mesenchymal transition (EMT). Moreover, tumor formation ability was assessed using a nude mice xenograft model. Key findingsTreatment of MDA-MB-231 cells with Exo-7SK resulted in efficient overexpression of 7SK; reduced viability; altered transcription levels of apoptosis-regulating genes; reduced proliferation; reduced migration and invasion; altered transcription of EMT-regulating genes; and reduced in vivo tumor formation ability. Finally, Exo-7SK reduced mRNA levels of HMGA1, a 7SK interacting protein with master gene regulatory and cancer promoting roles, and its bioinformatically-selected cancer promoting target genes. SignificanceAltogether, as a proof of the concept, our findings suggest that exosomal delivery of 7SK may suppress cancer phenotypes via downregulation of HMGA1.

Auto- and paracrine rewiring of NIX-mediated mitophagy by insulin-like growth factor-binding protein 7 in septic AKI escalates inflammation-coupling tubular damage

AimsInflammation-coupling tubular damage (ICTD) contributes to pathogenesis of septic acute kidney injury (AKI), in which insulin-like growth factor-binding protein 7 (IGFBP-7) serves as a biomarker for risk stratification. The current study aims to discern how IGFBP-7 signalling influences ICTD, the mechanisms that underlie this process and whether blockade of the IGFBP-7-dependent ICTD might have therapeutic value for septic AKI. Materials and methodsIn vivo characterization was carried out in B6/JGpt-Igfbp7em1Cd1165/Gpt mice subjected to cecal ligation and puncture (CLP). Transmission electron microscopy, immunofluorescence, flow cytometry, immunoblotting, ELISA, RT-qPCR and dual-luciferase reporter assays were used to determine mitochondrial functions, cell apoptosis, cytokine secretion and gene transcription. Key findingsICTD augments the transcriptional activity and protein secretion of tubular IGFBP-7, which enables an auto- and paracrine signalling via deactivation of IGF-1 receptor (IGF-1R). Genetic knockout (KO) of IGFBP-7 provides renal protection, improves survival and resolves inflammation in murine models of cecal ligation and puncture (CLP), while administering recombinant IGFBP-7 aggravates ICTD and inflammatory invasion. IGFBP-7 perpetuates ICTD in a NIX/BNIP3-indispensable fashion through dampening mitophagy that restricts redox robustness and preserves mitochondrial clearance programs. Adeno-associated viral vector 9 (AAV9)-NIX short hairpin RNA (shRNA) delivery ameliorates the anti-septic AKI phenotypes of IGFBP-7 KO. Activation of BNIP3-mediated mitophagy by mitochonic acid-5 (MA-5) effectively attenuates the IGFBP-7-dependent ICTD and septic AKI in CLP mice. SignificanceOur findings identify IGFBP-7 is an auto- and paracrine manipulator of NIX-mediated mitophagy for ICTD escalation and propose that targeting the IGFBP-7-dependent ICTD represents a novel therapeutic strategy against septic AKI.

339 Mineral Coated Micropaarticles Delivering Chondroitinase ABC mRNA Improves Motor Function After Spinal Cord Injury in Rats

INTRODUCTION: Currently, there are no treatments to help regain a significant amount of the function that is lost after spinal cord injury (SCI). A primary reason for the lack in functional recovery is the glial scar, which forms after SCI and inhibits axonal regeneration. Chondroitinase ABC (ChABC), a bacterial enzyme, digests one of the main axonal inhibitors in the glial scar, chondroitin sulfate proteoglycans (CSPGs), and results in axonal regeneration and functional recovery. However, ChABC is not thermally stable and quickly becomes inactive at body temperature. Recently, messenger RNA (mRNA) delivery has emerged as an attractive strategy for non-virally producing proteins in vivo with higher activity than recombinant proteins and a more desirable safety profile than viral gene delivery. METHODS: ChABC mRNA synthesis was designed in Benchling Molecular Biology Suite and ordered as-synthesized plasmid (Genscript). Mineral coatings were grown on hydroxyapatite microparticles and lipoplexes with mRNA were bound to the coatings. Rat spinal cords were contused at the T10 level and the glial scar was allowed to form. Seven days post-injury MCMs with ChABC mRNA were injected into the epicenter of the injury. Rat Hindlimb function was assessed for seven weeks before the rats were harvested. Lastly, immunohistochemistry was used to assess CSPG digestion and axonal sprouting. RESULTS: MCMs delivering ChABC mRNA forced local overexpression of ChABC and significantly digested CSPGs after SCI, resulting in axonal sprouting and a significant improvement in hindlimb function. CONCLUSIONS: MCMs efficiently delivered ChABC mRNA to digest CSPGs in the glial scar after SCI.

Targeted delivery of RNAi to cancer cells using RNA-ligand displaying exosome.

Exosome is an excellent vesicle for invivo delivery of therapeutics, including RNAi and chemical drugs. The extremely high efficiency in cancer regression can partly be attributed to its fusion mechanism in delivering therapeutics to cytosol without endosome trapping. However, being composed of a lipid-bilayer membrane without specific recognition capacity for aimed-cells, the entry into nonspecific cells can lead to potential side-effects and toxicity. Applying engineering approaches for targeting-capacity to deliver therapeutics to specific cells is desirable. Techniques with chemical modification invitro and genetic engineering in cells have been reported to decorate exosomes with targeting ligands. RNA nanoparticles have been used to harbor tumor-specific ligands displayed on exosome surface. The negative charge reduces nonspecific binding to vital cells with negatively charged lipid-membrane due to the electrostatic repulsion, thus lowering the side-effect and toxicity. In this review, we focus on the uniqueness of RNA nanoparticles for exosome surface display of chemical ligands, small peptides or RNA aptamers, for specific cancer targeting to deliver anticancer therapeutics, highlighting recent advances in targeted delivery of siRNA and miRNA that overcomes the previous RNAi delivery roadblocks. Proper understanding of exosome engineering with RNA nanotechnology promises efficient therapies for a wide range of cancer subtypes.

(1098) Optimization and Safety of Lipoic Acid F127@pla Nanoparticles as New Therapeutic Vector for Rna Delivery in Cardiovascular Diseases

(1098) Optimization and Safety of Lipoic Acid F127@pla Nanoparticles as New Therapeutic Vector for Rna Delivery in Cardiovascular Diseases

Cation-free siRNA-cored nanocapsules for tumor-targeted RNAi therapy.

Cation-associated cytotoxicity limits the systemic administration of RNA delivery in vivo, demanding the development of non-cationic nanosystems. In this study, cation-free polymer-siRNA nanocapsules with disulfide-crosslinked interlayer, namely T-SS(-), were prepared via the following steps: 1) complexation of siRNA with a cationic block polymer cRGD-poly(ethylene glycol)-b-poly[(2-aminoethanethiol)aspartamide]-b-poly{N'-[N-(2-aminoethyl)-2-ethylimino-1-aminomethyl]aspartamide}, abbreviated as cRGD-PEG-PAsp(MEA)-PAsp(C=N-DETA), 2) interlayer crosslinking via disulfide bond in pH 7.4 solution, and 3) removal of cationic DETA pendant at pH 5.0 via breakage of imide bond. The cationic-free nanocapsules with siRNA cores not only showed great performance (such as efficient siRNA encapsulation, high stability in serum, cancer cell targeting via cRGD modification, and GSH-triggered siRNA release), but also achieved tumor-targeted gene silencing in vivo. Moreover, the nanocapsules loaded with siRNA against polo-like kinase 1 (siRNA-PLK1) significantly inhibited tumor growth without showing cation-associated toxicity side effects and remarkably improved the survival rate of PC-3 tumor-bearing mice. The cation-free nanocapsules could potentially serve as a safe and effective platform for siRNA delivery. STATEMENT OF SIGNIFICANCE: Cation-associated toxicity limits the clinical translation of cationic carriers for siRNA delivery. Recently, several non-cationic carriers, such as siRNA micelles, DNA-based nanogels, and bottlebrush-architectured poly(ethylene glycol), have been developed to deliver siRNA. However, in these designs, siRNA as a hydrophilic macromolecule was attached to the nanoparticle surface instead of being encapsulated. Thus, it was easily degraded by serum nuclease and often induced immunogenicity. Herein, we demonstrate a new type of cation-free siRNA-cored polymeric nanocapsules. The developed nanocapsules not only showed capacities including efficient siRNA encapsulation, high stability in serum, and cancer cell targeting via cRGD modification, but also achieved an efficient tumor-targeted gene silencing in vivo. Importantly, unlike cationic carriers, the nanocapsules exhibited no cation-associated side effects.

The tumor therapeutic potential of long non-coding RNA delivery and targeting.

Long non-coding RNAs (lncRNAs) is a type of RNA over 200nt long without any protein coding ability, which has been investigated relating to crucial biological function in cells. There are many key lncRNAs in tumor/normal cells that serve as a biological marker or a new target for tumor treatment. However, compared to some small non-coding RNA, lncRNA-based drugs are limited in clinical application. Different from other non-coding RNA, like microRNAs, most lncRNAs have a high molecular weight and conserved secondary structure, making the delivery of lncRNAs more complex than the small non-coding RNAs. Considering that lncRNAs constitute the most abundant part of the mammalian genome, it is critical to further explore lncRNA delivery and the subsequent functional studies for potential clinical application. In this review, we will discuss the function and mechanism of lncRNAs in diseases, especially cancer, and different approaches for lncRNA transfection using multiple biomaterials.

Rational design of a polysaccharide-based viral mimicry nanocomplex for potent gene silencing in inflammatory tissues

Rational design and fabrication of small interfering RNA (siRNA) delivery system with simple production scheme, specific targeting capability, responsiveness to endogenous stimuli and potential multi-functionalities remains technically challenging. Herein, we screen and design a virus-mimicking polysaccharide nanocomplex that shows specific gene delivery capability in a selective subset of leukocytes. A virus-inspired poly (alkyl methacrylate-co-methacrylic acid) fragment was conjugated on barley β-glucans (EEPG) to endow the nanocomplex with pH-dependent endosomal membrane destabilization capabilities, as confirmed both biologically and computationally. siRNA loaded EEPG nanocomplex is feasibly fabricated in a single-step manner, which exhibit efficient gene silencing efficacy towards Dectin-1+ monocytes/macrophages. The inherent targeting affinity and feasible gene silencing potency of EEPG nanocomplex are investigated in three independent murine inflammation models, including myocardial infarction, lung fibrosis and acute liver damage. Significant enhanced accumulation level of EEPG nanocomplex is observed in cardiac lesion site, indicating its exclusive targeting capability for ischemic heart diseases. As a proof of concept, siTGF-β based gene therapy is confirmed in murine model with heart fibrosis. Overall, our findings suggest the designed EEPG nanocomplex is favorable for siRNA delivery, which might have translational potential as a versatile platform in inflammation-related diseases.

Non-Viral Carriers for Nucleic Acids Delivery: Fundamentals and Current Applications.

Over the past decades, non-viral DNA and RNA delivery systems have been intensively studied as an alternative to viral vectors. Despite the most significant advantage over viruses, such as the lack of immunogenicity and cytotoxicity, the widespread use of non-viral carriers in clinical practice is still limited due to the insufficient efficacy associated with the difficulties of overcoming extracellular and intracellular barriers. Overcoming barriers by non-viral carriers is facilitated by their chemical structure, surface charge, as well as developed modifications. Currently, there are many different forms of non-viral carriers for various applications. This review aimed to summarize recent developments based on the essential requirements for non-viral carriers for gene therapy.

In Vivo RNA Delivery to Hematopoietic Stem and Progenitor Cells via Targeted Lipid Nanoparticles.

Ex vivo autologous hematopoietic stem cell (HSC) gene therapy has provided new therapies for the treatment of hematological disorders. However, these therapies have several limitations owing to the manufacturing complexities and toxicity resulting from required conditioning regimens. Here, we developed a c-kit (CD117) antibody-targeted lipid nanoparticle (LNP) that, following a single intravenous injection, can deliver RNA (both siRNA and mRNA) to HSCs in vivo in rodents. This targeted delivery system does not require stem cell harvest, culture, or mobilization of HSCs to facilitate delivery. We also show that delivery of Cre recombinase mRNA at a dose of 1 mg kg-1 can facilitate gene editing to almost all (∼90%) hematopoietic stem and progenitor cells (HSPCs) in vivo, and edited cells retain their stemness and functionality to generate high levels of edited mature immune cells.

Oral delivery of RNAi for cancer therapy.

Cancer is a major health concern worldwide and is still in a continuous surge of seeking for effective treatments. Since the discovery of RNAi and their mechanism of action, it has shown promises in targeted therapy for various diseases including cancer. The ability of RNAi to selectively silence the carcinogenic gene makes them ideal as cancer therapeutics. Oral delivery is the ideal route of administration of drug administration because of its patients' compliance and convenience. However, orally administered RNAi, for instance, siRNA, must cross various extracellular and intracellular biological barriers before it reaches the site of action. It is very challenging and important to keep the siRNA stable until they reach to the targeted site. Harsh pH, thick mucus layer, and nuclease enzyme prevent siRNA to diffuse through the intestinal wall and thereby induce a therapeutic effect. After entering the cell, siRNA is subjected to lysosomal degradation. Over the years, various approaches have been taken into consideration to overcome these challenges for oral RNAi delivery. Therefore, understanding the challenges and recent development is crucial to offer a novel and advanced approach for oral RNAi delivery. Herein, we have summarized the delivery strategies for oral delivery RNAi and recent advancement towards the preclinical stages.

STING Agonist-Derived LNP-mRNA Vaccine Enhances Protective Immunity Against SARS-CoV-2.

Lipid nanoparticle (LNP)-mediated delivery of messenger RNA (mRNA) COVID-19 vaccines has provided large-scale immune protection to the public. To elicit a robust immune response against SARS-CoV-2 infections, antigens produced by mRNAs encoding SARS-CoV-2 Spike glycoprotein need to be efficiently delivered and presented to antigen-presenting cells such as dendritic cells (DCs). As concurrent innate immune stimulation can facilitate the antigen presentation process, a library of non-nucleotide STING agonist-derived amino lipids (SALs) was synthesized and formulated into LNPs for mRNA delivery. SAL12 lipid nanoparticles (SAL12-LNPs) were identified as most potent in delivering mRNAs encoding the Spike glycoprotein (S) of SARS-CoV-2 while activating the STING pathway in DCs. Two doses of SAL12 S-LNPs by intramuscular immunization elicited potent neutralizing antibodies against SARS-CoV-2 in mice.

Hydrogels for RNA delivery.

RNA-based therapeutics have shown tremendous promise in disease intervention at the genetic level, and some have been approved for clinical use, including the recent COVID-19 messenger RNA vaccines. The clinical success of RNA therapy is largely dependent on the use of chemical modification, ligand conjugation or non-viral nanoparticles to improve RNA stability and facilitate intracellular delivery. Unlike molecular-level or nanoscale approaches, macroscopic hydrogels are soft, water-swollen three-dimensional structures that possess remarkable features such as biodegradability, tunable physiochemical properties and injectability, and recently they have attracted enormous attention for use in RNA therapy. Specifically, hydrogels can be engineered to exert precise spatiotemporal control over the release of RNA therapeutics, potentially minimizing systemic toxicity and enhancing in vivo efficacy. This Review provides a comprehensive overview of hydrogel loading of RNAs and hydrogel design for controlled release, highlights their biomedical applications and offers our perspectives on the opportunities and challenges in this exciting field of RNA delivery.

A report on the International Society for Cell & Gene Therapy 2022 Scientific Signature Series, “Therapeutic advances with native and engineered human extracellular vesicles”

The International Society for Cell & Gene Therapy Scientific Signature Series event “Therapeutic Advances With Native and Engineered Human EVs” took place as part of the International Society for Cell & Gene Therapy 2022 Annual Meeting, held from May 4 to 7, 2022, in San Francisco, California, USA. This was the first signature series event on extracellular vesicles (EVs) and a timely reflection of the growing interest in EVs, including both native and engineered human EVs, for therapeutic applications. The event successfully gathered academic and industrial key opinion leaders to discuss the current state of the art in developing and understanding native and engineered EVs and applying our knowledge toward advancing EV therapeutics. Latest advancements in understanding the mechanisms by which native and engineered EVs exert their therapeutic effects against different diseases in animal models were presented, with some diseases such as psoriasis and osteoarthritis already reaching clinical testing of EVs. The discussion also covered various aspects relevant to advancing the clinical translation of EV therapies, including EV preparation, manufacturing, consistency, site(s) of action, route(s) of administration, and luminal cargo delivery of RNA and other compounds.

Adeno-Associated Virus-Mediated Knockdown of Agmatinase Attenuates Inflammation and Tumorigenesis in a Mouse Model of Colitis-Associated Colorectal Cancer.

Agmatinase (AGMAT) is an enzyme that hydrolyzes agmatine to putrescine and urea. Here, we explored the functions of AGMAT in colorectal cancer (CRC). By performing gain-of-function and loss-of-function experiments, we investigated the roles of AGMAT in proliferation, cell cycle progression, and apoptosis of CRC cells. We also established a colitis-associated colorectal cancer (CAC) model by challenging mice with azoxymethane (AOM) and dextran sodium sulfate (DSS), and we subsequently silenced AGMAT expression in mice by adeno-associated virus 9 (AAV9)-mediated delivery of short hairpin RNA (shRNA). In vitro experiments showed that overexpression of AGAMT accelerated the proliferation and inhibited the apoptosis of CRC cells, and AGMAT knockdown exhibited the opposite effects. Interestingly, the oncogenic transcription factor c-Myc could bind to the AGMAT promoter and transcriptionally increase AGMAT expression in CRC cells. Additionally, c-Myc and AGMAT were upregulated in the colon of AOM/DSS-treated mice, and AGMAT silencing significantly mitigated colitis in AOM/DSS-treated mice, as evidenced by the increased colon length, attenuated crypt damage, and reduced levels of inflammatory indicators (myeloperoxidase, interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, and phosphorylated p65) in colon tissues. Notably, AGMAT silencing decreased both the number and size of tumors, reduced expression of proliferating cell nuclear antigen (PCNA), and inhibited the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in the colon of AOM/DSS-treated mice. Overall, we determined that AGMAT facilitates tumor progression in CRC. Our findings will be helpful in the search for potential therapeutic targets for CRC.

Functional restoration of a CFTR splicing mutation through RNA delivery of CRISPR adenine base editor

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal fortargeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities.

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis.

Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34+ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34+ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34+ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.

A narrow host-range and lack of persistence in two non-target insect species of a bacterial symbiont exploited to deliver insecticidal RNAi in Western Flower Thrips.

Insecticidal RNAi is a targeted pest insect population control measure. The specificity of insecticidal RNAi can theoretically be enhanced by using symbiotic bacteria with a narrow host range to deliver RNAi, an approach termed symbiont-mediated RNAi (SMR), a technology we have previously demonstrated in the globally-invasive pest species Western Flower Thrips (WFT). Here we examine distribution of the two predominant bacterial symbionts of WFT, BFo1 and BFo2, among genome-sequenced insects. Moreover, we have challenged two non-target insect species with both bacterial species, namely the pollinating European bumblebee, Bombus terrestris, and an insect predator of WFT, the pirate bug Orius laevigatus. Our data indicate a very limited distribution of either symbiont among insects other than WFT. Moreover, whereas BFo1 could establish itself in both bees and pirate bugs, albeit with no significant effects on insect fitness, BFo2 was unable to persist in either species. In terms of biosafety, these data, together with its more specific growth requirements, vindicate the choice of BFo2 for delivery of RNAi and precision pest management of WFT.