bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2022‒02‒06
ten papers selected by
Benjamin Winkeljann
Ludwig-Maximilians University

  1. ACS Appl Bio Mater. 2022 Feb 02.
      Small interfering RNA (siRNA) has been emerging as a highly selective and effective pharmaceutics for treating broad classes of diseases. However, the practical application of siRNA agent is often hampered by its poor crossing of the cellular membrane barrier and ineffective releasing from endosome to cytoplasm, leading to low gene silencing efficacy for clinical purposes. Thus far, cationic lipid and polymer-based vectors have been extensively explored for gene delivery. Yet condensing the rigid and highly negatively charged siRNA duplex to form a stable complex vehicle usually requires a large load of cationic carriers, prone to raising the toxicity issue for delivery. Herein, we develop a simple strategy that can efficiently condense the siRNAs into nanoparticle vehicles for target gene regulation. In this approach, we first employ a DNA-grafted polycaprolactone (DNA-g-PCL) brush as template to organize the small rigid siRNAs into a large brush-like structure (siRNA-brush) through nucleic acid hybridization. Then, the siRNA-brush assembly is condensed by an ionizable and biodegradable polymer (poly(β-amino ester), PBAE) under acidic buffer condition to form a stable nanoparticle for siRNA delivery. Compared to the free siRNAs with poor complexing capability with PBAE, the large brush-like siRNA assemblies with more complicated topological architecture significantly promotes their electrostatic interaction with PBAE, enabling the formation of complexed nanoparticles at low weight ratio of polymer to siRNA. Additionally, PBAE/siRNA-brush complexes exhibit good biocompatibility and stability under physiological condition, as well as enhanced cellular internalization. When equipped with functional siRNAs, the obtained delivery system demonstrates excellent downregulation of target genes both in vitro and in vivo, through which the progression of hypertrophic scars can be retarded with negligible adverse effects in an xenografted mouse model.
    Keywords:  RNA interference; hypertrophic scars; poly(β-amino ester); self-assembly; siRNA delivery
  2. Chem Commun (Camb). 2022 Jan 31.
      Small interfering RNA (siRNA)-based therapeutics represent a novel and compelling drug modality, provided that safe and competent vectors are available for their delivery. Here, we report a biodegradable amphiphilic poly(aminoester) dendrimer for effective siRNA delivery. This dendrimer is readily biodegradable upon enzyme action, and harnesses the delivery features of both lipid and polymer vectors thanks to its lipid/dendrimer hybrid structure. This study opens new perspectives for developing biodegradable and biocompatible vectors for siRNA therapeutics.
  3. Beilstein J Nanotechnol. 2022 ;13 82-95
      Polyethylenimine (PEI), which is frequently used for polyplex formation and effective gene transfection, is rarely recognized as a luminescent polymer. Therefore, it is usually tagged with an organic fluorophore to be optically tracked. Recently, we developed branched PEI (bPEI) superparamagnetic iron oxide nanoparticles (SPION@bPEI) with blue luminescence 1200 times stronger than that of bPEI without a traditional fluorophore, due to partial PEI oxidation during the synthesis. Here, we demonstrate in vitro dye-free optical imaging and successful gene transfection with luminescent SPION@bPEI, which was further modified for receptor-mediated delivery of the cargo selectively to cancer cell lines overexpressing the epidermal growth factor receptor (EGFR). Pro-apoptotic polyinosinic-polycytidylic acid sodium (PIC) was delivered to HeLa cells with SPION@bPEI and caused a dramatic reduction in the cell viability at otherwise non-toxic nanoparticle concentrations, proving that bPEI coating is still an effective component for the delivery of an anionic cargo. Besides, a strong intracellular optical signal supports the optically traceable nature of these nanoparticles. SPION@bPEI nanoparticles were further conjugated with Erbitux (Erb), which is an anti-EGFR antibody for targeting EGFR-overexpressing cancer cell lines. SPION@bPEI-Erb was used for the delivery of a GFP plasmid wherein the transfection was confirmed by the luminescence of the expressed gene within the transfected cells. Poor GFP expression in MCF7, a slightly better expression in HeLa, and a significant enhancement in the transfection of HCT116 cells proved a selective uptake and hence the targeting ability of Erb-tagged nanoparticles. Altogether, this study proves luminescent, cationic, and small SPION@bPEI nanoparticles as strong candidates for imaging and gene therapy.
    Keywords:  Erbitux; photoluminescence; polyethyleneimine; polyinosinic–polycytidylic acid sodium; superparamagnetic iron oxide nanoparticles
  4. ACS Appl Mater Interfaces. 2022 Feb 02.
      Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-κB (siNFκB) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air-liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NFκB gene silencing was achieved in LPS-stimulated 16HBE14o- cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.
    Keywords:  SAXS; cystic fibrosis; hybrid nanoparticles; lung mucus; siRNA delivery
  5. Front Cell Dev Biol. 2021 ;9 837777
      Enhancer RNAs (eRNAs) are noncoding RNAs that synthesized at active enhancers. eRNAs have important regulatory characteristics and appear to be significant for maintenance of cell identity and information processing. Series of functional eRNAs have been identified as potential therapeutic targets for multiple diseases. Nevertheless, the role of eRNAs on intervertebral disc degeneration (IDD) is still unknown yet. Herein, we utilized the nucleus pulposus samples of patients and identified a key eRNA (LINC02569) with the Arraystar eRNA Microarray. LINC02569 mostly locates in nucleus and plays an important role in the progress of IDD by activating nuclear factor kappa-B (NF-κB) signaling pathway. We used a cationic polymer brush coated carbon nanotube (oCNT-pb)-based siRNA delivery platform that we previously designed, to transport LINC02569 siRNA (si-02569) to nucleus pulposus cells. The siRNA loaded oCNT-pb accumulated in nucleus pulposus cells with lower toxicity and higher transfection efficiency, compared with the traditional siRNA delivery system. Moreover, the results showed that the delivery of si-02569 significantly alleviated the inflammatory response in the nucleus pulposus cells via inhibiting P65 phosphorylation and preventing its transfer into the nucleus, and meanwhile alleviated cell senescence by decreasing the expression of P21. Altogether, our results highlight that eRNA (LINC02569) plays important role in the progression of IDD and could be a potential therapeutic target for alleviation of IDD.
    Keywords:  Enhancer RNA; carbon nanotube; inflammation; intervertebral disc degeneration; senescence
  6. Nat Protoc. 2022 Feb 04.
      Macrophages in atherosclerotic lesions promote plaque progression and are an attractive therapeutic target in cardiovascular research. Here we present a protocol for synthesis of small interfering RNA (siRNA) nanoparticles (NP) that target lesional macrophages as a potential treatment for atherosclerosis. Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) activity in macrophages of advanced human and mouse atherosclerotic plaques drives necrosis by downregulating the expression of the efferocytosis receptor MerTK. Therefore, selective inhibition of CaMKIIγ in lesional macrophages holds great promise for the treatment of advanced atherosclerosis. We recently developed a siRNA NP platform that can selectively silence CaMKIIγ in macrophages, resulting in increased plaque stability. We provide a detailed protocol for the synthesis of NP components, the preparation and characterization (physicochemical and in vitro) of siRNA NPs, and the evaluation of in vivo therapeutic effects of siRNA NPs and their biocompatibility in atherosclerotic mice. Our siRNA-loaded polymer-lipid hybrid NPs are constructed via a robust self-assembly method, exhibiting excellent in vivo features for systemic siRNA delivery. Following this protocol, it takes 3-5 d to prepare the siRNA NPs, 8-10 d to characterize the NPs and 4-5 weeks to evaluate their therapeutic effects in established atherosclerotic mice. By changing the RNA molecules loaded in the NPs, lesional macrophages can be targeted for the exploration and validation of new targets/pathways in atherosclerosis.
  7. J Control Release. 2022 Jan 29. pii: S0168-3659(22)00052-9. [Epub ahead of print]343 175-186
      HMGB1 is an inflammatory factor produced by macrophages after liver injury, which plays a key role in promoting NASH progression and further developing into liver fibrosis and cirrhosis. In this study, a mannose-modified HMGB1-siRNA loaded stable nucleic acid lipid particle delivery system (mLNP-siHMGB1) was constructed to target liver macrophages with mannose receptor mediation, thereby silencing HMGB1 protein expression and treating NASH. We also examined the effect of co-administration with docosahexaenoic acid (DHA), a kind of unsaturated fatty acid, on NASH. The results showed that mLNP-siHMGB1 could target macrophages through mannose receptors, effectively silence HMGB1 gene, reduce the release of HMGB1 protein in the liver, regulate liver macrophages to be an anti-inflammatory M2 phenotype, effectively reduce hepatic lobular inflammation and bullous steatosis in the liver, and restore the liver function of NASH model mice to a normal level. After 8 weeks of combined treatment with mLNP-siHMGB1 and DHA, the liver function of NASH model mice recovered rapidly and the hepatic steatosis returned to normal level. In view of inflammation, a key factor in the progression of NASH, we provided an actively targeted siRNA delivery system in this study, and clarified the important role of the delivery system in phenotypic regulation of liver macrophages in NASH. In addition, we also demonstrated the effectiveness of DHA co-administration in NASH treatment. This study provided a useful idea and scientific basis for the development of therapeutic strategies for NASH in the future.
    Keywords:  HMGB1 siRNA; Macrophages; Mannose receptor; NASH; Stable nucleic acid lipid nanoparticles
  8. ACS Nano. 2022 Jan 31.
      The lack of safe and effective delivery across the blood-brain barrier and the profound immune suppressive microenvironment are two main hurdles to glioblastoma (GBM) therapies. Extracellular vesicles (EVs) have been used as therapeutic delivery vehicles to GBM but with limited efficacy. We hypothesized that EV delivery to GBM can be enhanced by (i) modifying the EV surface with a brain-tumor-targeting cyclic RGDyK peptide (RGD-EV) and (ii) using bursts of radiation for enhanced accumulation. In addition, EVs were loaded with small interfering RNA (siRNA) against programmed cell death ligand-1 (PD-L1) for immune checkpoint blockade. We show that this EV-based strategy dramatically enhanced the targeting efficiency of RGD-EV to murine GBM, while the loaded siRNA reversed radiation-stimulated PD-L1 expression on tumor cells and recruited tumor-associated myeloid cells, offering a synergistic effect. The combined therapy significantly increased CD8+ cytotoxic T cells activity, halting tumor growth and prolonging animal survival. The selected cell source for EVs isolation and the presented functionalization strategy are suitable for large-scale production. These results provide an EV-based therapeutic strategy for GBM immune checkpoint therapy which can be translated to clinical applications.
    Keywords:  extracellular vesicles; glioblastoma; immunotherapy; radiation therapy; targeted delivery
  9. Adv Mater. 2022 Feb 04. e2110618
      CRISPR-Cas9 may offer new therapeutics for genetic diseases through gene disruption via nonhomologous end joining (NHEJ) or gene correction via homology-directed repair (HDR). However, clinical transition of CRISPR technology is limited by the lack of safe and efficient delivery systems. Here, we report facilely fabricated pH-responsive polymer nanoparticles capable of safely and efficiently delivering Cas9 ribonucleoprotein alone (termed NHEJ-NP, diameter = 29.4 nm), or together with donor DNA (termed HDR-NP, diameter = 33.3 nm). Moreover, intravenously, intratracheally, and intramuscularly injected NHEJ-NP induced efficient gene editing in mouse liver, lung, and skeletal muscle, respectively. Intramuscularly injected HDR-NP also led to muscle strength recovery in a Duchenne muscular dystrophy mouse model. NHEJ-NP and HDR-NP possess many desirable properties including high payload loading content, small and uniform sizes, high editing efficiency, good biocompatibility, low immunogenicity, and ease of production, storage, and transport, making them great interest for various genome editing applications with clinical potentials. This article is protected by copyright. All rights reserved.
    Keywords:  CRISPR-Cas9; Genome Editing; Nanomedicine
  10. J Control Release. 2022 Jan 28. pii: S0168-3659(22)00055-4. [Epub ahead of print]
      Diabetes mellitus is a disease of metabolism, featuring persistent hyperglycaemia due to insufficient insulin secretion or insulin resistance. At present, the generation of new beta cells from autologous cells by ectopic expression of specific transcription factors is a promising treatment for diabetes. The application of this strategy urgently needs safe and effective gene delivery vectors. In this work, a therapeutic plasmid (pNPMN-PBase), combined multiple specific transcription factors Ngn3, Pdx1, Mafa and Neruod1 (NPMN), was firstly constructed. Then, phenylboronic acid (PBA)-functionalized branched polymers (SS-HPT-P) have been proposed to deliver pNPMN-PBasefor the promising treatment of diabetes. SS-HPT-P had good biocompatibility and low cytotoxicity, and could achieve liver-targeted delivery. SS-HPT-P/pNPMN-PBase system can effectively realize the liver delivery of exogenous therapeutic genes, induce the reprogramming of hepatocytes into beta-like cells, reestablish the endogenous insulin-expression system, and alleviate diabetes and its complications. The present study thus provides an effective strategy for the cell replacement therapy of diabetes.
    Keywords:  Diabetes; Gene therapy; Liver-targeted; Phenylboronic acid; Reprogramming; Vector