bims-novged 2021-08-22 papers

on Non-viral vectors for gene delivery

Issue of 2021–08–22
ten papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Novel Ellipsoid Chitosan-Phthalate Lecithin Nanoparticles for siRNA Delivery.
Effect of using amino acids in the freeze-drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection.
Lipid nanoparticles for mRNA delivery.
Biomimetic cell membrane-coated DNA nanoparticles for gene delivery to glioblastoma.
Preparation of siRNA-PLGA/Fab'-PLGA mixed micellar system with target cell-specific recognition.
Zn2+-Coordination-Driven RNA Assembly with Retained Integrity and Biological Functions.
Smart Polymeric Nanoparticles with pH-Responsive and PEG-Detachable Properties (II): Co-Delivery of Paclitaxel and VEGF siRNA for Synergistic Breast Cancer Therapy in Mice.
Minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) model of GalNAc-conjugated siRNA disposition and gene silencing in preclinical species and humans.
Amelioration of post-traumatic osteoarthritis via nanoparticle depots delivering small interfering RNA to damaged cartilage.
JNK2 Gene Silencing for Elastic Matrix Regenerative Repair.

Front Bioeng Biotechnol. 2021 ;9 695371

Novel Ellipsoid Chitosan-Phthalate Lecithin Nanoparticles for siRNA Delivery.

Ramzi Mukred Saeed, Mohammed Abdullah, Mamoun Ahram, Mutasem Omar Taha.

  Small interfering RNA (siRNA) has received increased interest as a gene therapeutic agent. However, instability and lack of safe, affordable, and effective carrier systems limit siRNA's widespread clinical use. To tackle this issue, synthetic vectors such as liposomes and polymeric nanoparticles have recently been extensively investigated. In this study, we exploited the advantages of reduced cytotoxicity and enhanced cellular penetration of chitosan-phthalate (CSP) together with the merits of lecithin (LC)-based nanoparticles (NPs) to create novel, ellipsoid, non-cytotoxic, tripolyphosphate (TPP)-crosslinked NPs capable of delivering siRNA efficiently. The resulting NPs were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and were found to be ellipsoid in the shape of ca. 180 nm in size, exhibiting novel double-layer shells, with excellent stability at physiological pH and in serum solutions. MTT assay and confocal fluorescence microscopy showed that CSP-LC-TPP NPs are non-cytotoxic and efficiently penetrate cancer cells in vitro. They achieved 44% silencing against SLUG protein in MDA-MB-453 cancer cells and were significantly superior to a commercial liposome-based transfection agent that achieved only 30% silencing under comparable conditions. Moreover, the NPs protected their siRNA cargos in 50% serum and from being displaced by variable concentrations of heparin. In fact, CSP-LC-TPP NPs achieved 26% transfection efficiency in serum containing cell culture media. Real-time wide-field fluorescence microscopy showed siRNA-loaded CSP-LC-TPP NPs to successfully release their cargo intracellularly. We found that the amphoteric nature of chitosan-phthalate polymer promotes the endosomal escape of siRNA and improves the silencing efficiency.

Keywords:  Chitosan nanoparticles; ellipsoid nanoparticles; gene delivery; non-viral vectors; siRNA delivery

DOI:  https://doi.org/10.3389/fbioe.2021.695371
Biomed Rep. 2021 Sep;15(3): 72

Effect of using amino acids in the freeze-drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection.

Min Tang, Yoshiyuki Hattori.

  Recently, small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) have become a crucial research tool for studying gene function. Easy and reliable siRNA transfection with a large set of siRNAs is required for the successful screening of gene function. Reverse (Rev)-transfection with freeze-dried siRNA lipoplexes is validated for siRNA transfection with a large set of siRNAs in a multi-well plate. In our previous study, it was shown that Rev-transfection with siRNA lipoplexes freeze-dried in disaccharides or trisaccharides resulted in long-term stability with a high level of gene-knockdown activity. In the present study, the effects of amino acids used as cryoprotectants in the freeze-drying of siRNA lipoplexes on gene knockdown via Rev-transfection were assessed. A total of 15 types of amino acids were used to prepare freeze-dried siRNA lipoplexes, and it was found that the freeze-drying of siRNA lipoplexes with amino acid concentrations >100 mM strongly suppressed targeted gene expression regardless of the amino acid type; however, some amino acids strongly upregulated or downregulated gene expression in the cells transfected with negative control siRNA. Amongst the amino acids tested, the presence of asparagine showed specific gene-knockdown activity, forming large cakes after freeze-drying and retaining a favorable siRNA lipoplex size after rehydration. These findings provide valuable information regarding amino acids as cryoprotectants for Rev-transfection using freeze-dried siRNA lipoplexes for the efficient delivery of siRNA into cells.

Keywords:  amino acids; cationic liposome; freeze-drying; reverse transfection; small interfering RNA

DOI:  https://doi.org/10.3892/br.2021.1448
Nat Rev Mater. 2021 Aug 10. 1-17

Lipid nanoparticles for mRNA delivery.

Xucheng Hou, Tal Zaks, Robert Langer, Yizhou Dong.

  Messenger RNA (mRNA) has emerged as a new category of therapeutic agent to prevent and treat various diseases. To function in vivo, mRNA requires safe, effective and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and mRNA release. Lipid nanoparticles have successfully entered the clinic for the delivery of mRNA; in particular, lipid nanoparticle-mRNA vaccines are now in clinical use against coronavirus disease 2019 (COVID-19), which marks a milestone for mRNA therapeutics. In this Review, we discuss the design of lipid nanoparticles for mRNA delivery and examine physiological barriers and possible administration routes for lipid nanoparticle-mRNA systems. We then consider key points for the clinical translation of lipid nanoparticle-mRNA formulations, including good manufacturing practice, stability, storage and safety, and highlight preclinical and clinical studies of lipid nanoparticle-mRNA therapeutics for infectious diseases, cancer and genetic disorders. Finally, we give an outlook to future possibilities and remaining challenges for this promising technology.

Keywords:  Drug delivery; Drug development

DOI:  https://doi.org/10.1038/s41578-021-00358-0
J Control Release. 2021 Aug 13. pii: S0168-3659(21)00427-2. [Epub ahead of print]338 22-32

Biomimetic cell membrane-coated DNA nanoparticles for gene delivery to glioblastoma.

Sangrok Han, Youngki Lee, Minhyung Lee.

  Gene therapy has been introduced as an alternative to radiation and chemical therapy for glioblastoma. Biomimetic nanoparticles coated with cell membranes (CM) have advantages such as high biocompatibility and prolong half-life. To apply CM coated nanoparticles to gene delivery, the polyethylenimine (PEI25k)/plasmid DNA (pDNA) complexes were coated with CM from C6 rat glioblastoma cells. With the CM covering, the PEI25k/pDNA complexes formed stable nanoparticles with negative surface charge. The PEI25k/pDNA/CM nanoparticles had high colloidal stability and could be stored for approximately 20 days without aggregation. The transfection efficiency of the PEI25k/pDNA/CM nanoparticles was higher than that of the PEI25k/pDNA complex in serum-containing medium. This suggests that serum does not interfere with transfection efficiency of the nanoparticles. Moreover, the PEI25k/pDNA/CM nanoparticles had lower toxicity than the PEI25k/DNA complex in vitro and in vivo. The PEI25k/pDNA/CM nanoparticles prepared with CMs of different types of cells were transfected into cells. The results showed that the PEI25k/pDNA/CM nanoparticles with the C6 CM had the highest transfection efficiency to C6 cells, suggesting the homotypic targeting effect. The therapeutic effects of the nanoparticles were evaluated in intracranial C6 transplanted glioblastoma animal models. The PEI25k/pDNA/CM nanoparticles were prepared with herpes simplex virus thymidine kinase plasmid (pHSVtk) and injected into the tumor locally. The results showed that the PEI25k/pHSVtk/CM nanoparticles induced higher HSVtk expression compared with the PEI25k/pHSVtk complex. Furthermore, tumor size was reduced more efficiently by the PEI25k/pHSVtk/CM nanoparticles than by the PEI25k/pHSVtk complex. Overall results indicate that PEI25k/pDNA/CM nanoparticles are suitable for pDNA delivery to glioblastoma.

Keywords:  Cell membrane; Gene carrier; Gene therapy; Glioblastoma; Ternary complex

DOI:  https://doi.org/10.1016/j.jconrel.2021.08.021
Sci Rep. 2021 Aug 18. 11(1): 16789

Preparation of siRNA-PLGA/Fab'-PLGA mixed micellar system with target cell-specific recognition.

Mai Hazekawa, Takuya Nishinakagawa, Takeshi Mori, Miyako Yoshida, Takahiro Uchida, Daisuke Ishibashi.

Small interfering RNAs (siRNAs) are susceptible to nucleases and degrade quickly in vivo. Moreover, siRNAs demonstrate poor cellular uptake and cannot cross the cell membrane because of its polyanionic characteristics. To overcome these challenges, an intelligent gene delivery system that protects siRNAs from nucleases and facilitates siRNA cellular uptake is required. We previously reported the potential of siRNA-poly(D,L-lactic-co-glycolic acid; PLGA) micelles as an effective siRNA delivery tool in a murine peritoneal dissemination model by local injection. However, there was no effective formulation for siRNA delivery to target cells via intravenous injection. This study aimed to prepare siRNA-PLGA/Fab'-PLGA mixed micelles for siRNA delivery to target floating cells and evaluate its formulation in vitro. As the target siRNA protein in CEMx174, CyclinB1 levels were significantly reduced when siRNA-PLGA/Fab'-PLGA mixed micelles were added to cells compared with siRNA-PLGA micelles. siRNA-PLGA/Fab'-PLGA mixed micelles have high cell permeability and high target cell accumulation by endocytosis because flow cytometry detected labeling micelles in target cells. This study supports siRNA-PLGA/Fab'-PLGA mixed micelles as an effective siRNA delivery tool. This formulation can be administered systemically in dosage form against target cells, including cancer metastasis or blood cancer.

DOI: https://doi.org/10.1038/s41598-021-96245-3
Angew Chem Int Ed Engl. 2021 Aug 17.

Zn2+-Coordination-Driven RNA Assembly with Retained Integrity and Biological Functions.

Ronghua Yang, Zhen Zou, Libei He, Xiangxi Deng, Huangxiang Wang, Ziyun Huang, Qian Xue, Zhihe Qing, Yanli Lei, Juewen Liu.

  Metal coordination-directed biomolecule crosslinking in nature has been used for synthetic various biopolymers, including DNA, peptides, proteins, and polysaccharides. However, biopolymer of RNA has been avoided as yet due to the poor stability of the RNA molecules, the formation of biopolymer may alter biological function of the biological molecules. Herein, for the first time, we report Zn2+-driven RNA self-assembly forming spherical nanoparticles, and with retaining the integrity and biological functions of RNA. Various functional RNAs of different compositions, shapes, and lengths from 20 to nearly 1000-nucleotides were packaged, highlighting the versatility of this approach. The assembled nanospheres possess superior RNA loading efficiency, pharmacokinetics, and bioavailability. In vitro and in vivo evaluation demonstrated mRNA delivery for expressing GFP proteins, and microRNA delivery to triple negative breast cancer. This coordination-directed self-assembly behavior amplifies the horizons of RNA coordination chemistry and application scope of RNA-based therapeutic.

Keywords:  Gene delivery; RNA self-assembly; metal-organic coordination; nanobiotechnology; nanomedicine

DOI:  https://doi.org/10.1002/anie.202110404
Int J Nanomedicine. 2021 ;16 5479-5494

Smart Polymeric Nanoparticles with pH-Responsive and PEG-Detachable Properties (II): Co-Delivery of Paclitaxel and VEGF siRNA for Synergistic Breast Cancer Therapy in Mice.

Mingji Jin, Yan Hou, Xiuquan Quan, Liqing Chen, Zhonggao Gao, Wei Huang.

   Background: The dual-loaded nano-delivery system can realize chemotherapeutic drug and small interfering RNA (siRNA) co-loading as well as enhance the therapeutic effect of drugs on tumors through a synergistic effect, while reducing their toxic and side effects on normal tissues.
Methods: Previously, we developed layered smart nanoparticles (NPs) to co-deliver survivin siRNA as well as small molecule drugs for lung cancer. In this study, we used such smart NPs to co-deliver paclitaxel (PTX) and siRNA against vascular endothelial growth factor (VEGF) gene for breast cancer therapy in mice models. For the prepared NPs, characterizations such as particle size, zeta potential, gel electrophoresis imaging and in vitro stability were investigated. Then, 4T1 cells were used to evaluate the in vitro VEGF silencing capacity, tumor cell inhibitory and anti-apoptotic abilities. Finally, an orthotopic model of mouse breast cancer was established to evaluate the in vivo antitumor effects and safety properties of PTX-siRNAVEGF-NPs.
Results: We prepared PTX-siRNAVEGF-NPs with particle size of 85.25 nm, PDI of 0.261, and zeta potential of 5.25 mV. The NPs with VEGF siRNA effectively knocked down the expression of VEGF mRNA. Cell counting kit-8 (CCK-8) and apoptosis assays revealed that the PTX-siRNAVEGF-NPs exhibited antiproliferation effect of PTX on 4T1 cells. The in vivo anti-tumor study indicated that PTX-siRNAVEGF-NPs could exert an antitumor effect by inhibiting the formation and development of new blood vessels in tumor tissues, thereby cutting off nutrient and blood supplies required for tumor tissue growth. Both the anti-tumor efficacy and in vivo safety of the PTX-siRNAVEGF-NPs group were better than that of the PTX-NPs and siRNAVEGF-NPs groups.
Conclusion: The combination of PTX and VEGF siRNA exerts good antitumor effect on 4T1 tumor cells. This study provides a theoretical and practical basis for breast cancer therapy.

Keywords:  VEGF siRNA; breast cancer; co-delivery; paclitaxel

DOI:  https://doi.org/10.2147/IJN.S313339
J Pharmacol Exp Ther. 2021 Aug 19. pii: JPET-AR-2021-000805. [Epub ahead of print]

Minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) model of GalNAc-conjugated siRNA disposition and gene silencing in preclinical species and humans.

Vivaswath S Ayyar, Dawei Song, Songmao Zheng, Thomas Carpenter, Donald L Heald.

  Conjugation of small interfering ribonucleic acid (siRNA) to tris-N-acetylgalactosamine (tris-GalNAc) can enable highly selective, potent, and durable knockdown of targeted proteins in the liver. However, potential knowledge gaps between in vitro experiments, preclinical species, and clinical scenarios remain. A minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) model for GalNAc-conjugated siRNA (GalNAc-siRNA) was developed using published data for fitusiran (ALN-AT3), an investigational compound targeting liver antithrombin (AT), to delineate putative determinants governing the whole body-to-cellular PK-PD of GalNAc-siRNA and facilitate preclinical-to-clinical translation. The model mathematically linked relevant mechanisms: i) hepatic biodistribution, ii) tris-GalNAc binding to asialoglycoprotein receptors (ASGPRs) on hepatocytes, iii) ASGPR endocytosis and recycling, iv) endosomal transport and escape of siRNA, v) cytoplasmic RNA-induced silencing complex (RISC) loading, vi) degradation of target mRNA by bound RISC, and vii) knockdown of protein. Physiological values for 36 out of 48 model parameters were obtained from the literature. Kinetic parameters governing (GalNAc)3-ASGPR binding and internalization were derived from published studies of uptake in hepatocytes. The proposed model well characterized reported PK, RISC dynamics, and knockdown of AT mRNA and protein by ALN-AT3 in mice. The model bridged multiple PK-PD datasets in preclinical species (mice, rat, monkey) and successfully captured reported plasma PK and AT knockdown in a Phase-I ascending dose study. Estimates of in vivo potency (SC50 ) were similar (~2-fold) across species. Subcutaneous absorption and serum AT degradation rate constants scaled across species by body weight with allometric exponents of -0.29 and -0.22. The proposed mechanistic modeling framework characterizes the unique PK-PD properties of GalNAc-siRNA. Significance Statement (GalNAc)3-conjugated siRNA therapeutics enable liver-targeted gene therapy and precision medicine. Using a translational and systems-based minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) modeling approach, drug- and system-specific determinants influencing GalNAc-siRNA functionality in preclinical species (mice, rats, monkeys) and humans were investigated. The developed model successfully integrated and characterized relevant published in vitro-derived biomeasures, mechanistic PK-PD profiles in animals, and observed clinical PK-PD responses for an investigational GalNAc-siRNA (fitusiran). This modeling effort delineates the whole body-to-cellular disposition and liver-targeted pharmacodynamics of GalNAc-siRNA.

Keywords:  RNA/siRNA; liver/hepatic; pharmacodynamics; pharmacokinetic/pharmacodynamic modeling/PKPD; pharmacokinetics; systems pharmacology

DOI:  https://doi.org/10.1124/jpet.121.000805
Nat Biomed Eng. 2021 Aug 19.

Amelioration of post-traumatic osteoarthritis via nanoparticle depots delivering small interfering RNA to damaged cartilage.

Sean K Bedingfield, Juan M Colazo, Fang Yu, Danielle D Liu, Meredith A Jackson, Lauren E Himmel, Hongsik Cho, Leslie J Crofford, Karen A Hasty, Craig L Duvall.

The progression of osteoarthritis is associated with inflammation triggered by the enzymatic degradation of extracellular matrix in injured cartilage. Here we show that a locally injected depot of nanoparticles functionalized with an antibody targeting type II collagen and carrying small interfering RNA targeting the matrix metalloproteinase 13 gene (Mmp13), which breaks down type II collagen, substantially reduced the expression of MMP13 and protected cartilage integrity and overall joint structure in acute and severe mouse models of post-traumatic osteoarthritis. MMP13 inhibition suppressed clusters of genes associated with tissue restructuring, angiogenesis, innate immune responses and proteolysis. We also show that intra-articular injections of the nanoparticles led to greater reductions in disease progression than either a single injection or weekly injections of the steroid methylprednisolone. Sustained drug retention by targeting collagen in the damaged extracellular matrix of osteoarthritic cartilage may also be an effective strategy for the treatment of osteoarthritis with other disease-modifying drugs.

DOI: https://doi.org/10.1038/s41551-021-00780-3
Tissue Eng Part A. 2021 Aug 19.

JNK2 Gene Silencing for Elastic Matrix Regenerative Repair.

Sarah Carney, Thomas Broekelmann, Robert Mecham, Anand Ramamurthi.

Elastic fibers do not naturally regenerate in many proteolytic disorders, such as in abdominal aortic aneurysms (AAAs), and prevent restoration of tissue homeostasis. We have shown drug-based attenuation of the stress-activated protein kinase, JNK-2 to stimulate elastic matrix neoassembly and to attenuate cellular proteolytic activity. We now investigate if JNK2 gene knockdown with siRNA provides greater specificity of action and improved regenerative/anti-proteolytic outcomes in a proteolytic injury culture model of rat aneurysmal smooth muscle cell (EaRASMCs). An siRNA dose of 12.5nM delivered with a transfection reagent significantly enhanced downstream elastic fiber assembly and maturation vs untreated EaRASMC cultures. The optimal siRNA dose was also delivered as a complex with a polymeric transfection vector, polyethyleneimine (PEI) in preparation for future in vivo delivery. Linear 25kDa PEI-siRNA (5:1 molar ratio of amine to phosphate) and linear 40kDa PEI-siRNA (2.5:1 ratio) were effective in downregulating the JNK2 gene, and significantly increasing expression of elastic fiber assembly proteins, and decreases in elastolytic matrix metalloprotease (MMP)-2 vs treatment controls to significantly increase mature elastic fiber assembly. The current work has identified siRNA dosing and siRNA-PEI complexing conditions that are safe and efficient in stimulating processes contributing to improved elastic matrix neoassembly via JNK2 gene knockdown. The results represent a mechanistic basis of a broader therapeutic approach to reverse elastic matrix pathophysiology in tissue disorders involving aberrations of elastic matrix homeostasis, such as in aortic aneurysms.

DOI: https://doi.org/10.1089/ten.TEA.2020.0221