bims-novged 2021-11-14 papers

on Non-viral vectors for gene delivery

Issue of 2021–11–14
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

AMP functionalized curdlan nanoparticles as a siRNA carrier: Synthesis, characterization and targeted delivery via adenosine A2B receptor.
Modulating intracellular pathways to improve non-viral delivery of RNA therapeutics.
Efficient and safe small RNA delivery to macrophage using peptide-based nanocomplex.
Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery.
Cation-Free siRNA Micelles as Effective Drug Delivery Platform and Potent RNAi Nanomedicines for Glioblastoma Therapy.
Core-shell lipoplexes inducing active macropinocytosis promote intranasal delivery of c-Myc siRNA for treatment of glioblastoma.
Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations.
Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors.
Possibility for double optimization of siRNA intracellular delivery efficiency and antibacterial activity: Structure screening of pH-sensitive triblock amphiphilic polycation micelles.
Co-delivery of Dexamethasone and a MicroRNA-155 Inhibitor Using Dendrimer-Entrapped Gold Nanoparticles for Acute Lung Injury Therapy.
Role of Ionizable Lipids in SARS-CoV-2 Vaccines As Revealed by Molecular Dynamics Simulations: From Membrane Structure to Interaction with mRNA Fragments.

Int J Biol Macromol. 2021 Oct 29. pii: S0141-8130(21)02296-0. [Epub ahead of print]193(Pt A): 866-873

AMP functionalized curdlan nanoparticles as a siRNA carrier: Synthesis, characterization and targeted delivery via adenosine A2B receptor.

Qingming Bao, Tsogzolmaa Ganbold, Qiburi Qiburi, Mingming Bao, Shuqin Han, Huricha Baigude.

  Receptor-mediated endocytosis has been used for tissue targeted delivery of short interfering RNA (siRNA) drugs. Herein, we investigated adenosine receptor (AR) as a candidate for receptor-mediated siRNA internalization. We synthesized adenosine functionalized cationic curdlan derivatives (denote CuAMP polymers). One of these polymers, CuAMP4, efficiently delivered siRNA to breast cancer cells expressing high level of A2B receptor. The internalization of siRNA loaded CuAMP4 by cancer cells was inhibited by free AMP as well as endocytosis inhibitors. Moreover, knockdown of A2BR by siRNA, or pre-treatment of the cells with anti-A2BR antibody, strongly inhibited the cellular uptake of CuAMP4. Our findings confirmed that A2BR can be utilized for cell type specific siRNA delivery, and CuAMP4 NP may be a promising delivery system for cancer cell targeted delivery of therapeutic siRNAs.

Keywords:  Curdlan; Receptor-mediated endocytosis; siRNA delivery

DOI:  https://doi.org/10.1016/j.ijbiomac.2021.10.138
Adv Drug Deliv Rev. 2021 Nov 08. pii: S0169-409X(21)00434-8. [Epub ahead of print] 114041

Modulating intracellular pathways to improve non-viral delivery of RNA therapeutics.

Thijs Van de Vyver, Stefaan C De Smedt, Koen Raemdonck.

  RNA therapeutics (e.g. siRNA, oligonucleotides, mRNA, etc.) show great potential for the treatment of a myriad of diseases. However, to reach their site of action in the cytosol or nucleus of target cells, multiple intra- and extracellular barriers have to be surmounted. Several non-viral delivery systems, such as nanoparticles and conjugates, have been successfully developed to meet this requirement. Unfortunately, despite these clear advances, state-of-the-art delivery agents still suffer from relatively low intracellular delivery efficiencies. Notably, our current understanding of the intracellular delivery process is largely oversimplified. Gaining mechanistic insight into how RNA formulations are processed by cells will fuel rational design of the next generation of delivery carriers. In addition, identifying which intracellular pathways contribute to productive RNA delivery could provide opportunities to boost the delivery performance of existing nanoformulations. In this review, we discuss both established as well as emerging techniques that can be used to assess the impact of different intracellular barriers on RNA transfection performance. Next, we highlight how several modulators, including small molecules but also genetic perturbation technologies, can boost RNA delivery by intervening at differing stages of the intracellular delivery process, such as cellular uptake, intracellular trafficking, endosomal escape, autophagy and exocytosis.

Keywords:  cellular delivery; cellular uptake; conjugates; endosomal escape; intracellular barriers; intracellular trafficking; nanoparticles; nucleic acid therapeutics

DOI:  https://doi.org/10.1016/j.addr.2021.114041
Biotechnol Bioeng. 2021 Nov 11.

Efficient and safe small RNA delivery to macrophage using peptide-based nanocomplex.

Yeong Chae Ryu, Yoo Eun Lee, Byeong Hee Hwang.

  As one of the gene therapies, RNA interference (RNAi) effectively suppresses only specific genes, targeting various diseases in which they are involved. For the successful process of RNAi, efficient and safe delivery of small RNAs, including siRNA and shRNA, is essential. Herein, an S-R11 fusion peptide, SPACE peptide conjugated with poly-arginine, was introduced to deliver small RNAs into immune cells that are difficult to transfect. This S-R11 peptide stably formed a spontaneous self-assembling nanocomplex through electrostatic attraction and hydrogen bonding with small RNAs. The nanocomplex showed about 5.3-fold better permeation efficiency than the conventional LipofectamineTM 2000 for RAW 264.7 macrophage cells. Moreover, it induced about 66.2% silencing effect of the target gene in the cells activated with polyinosinic:polycytidylic acid (poly (I:C)). In addition, the cell viability of fusion peptide was ensured even in a concentration range exceeding the concentration used in the nanocomplex. Based on these results, it is expected that the nanocomplex in this study can be used as a new gene delivery system that can overcome the challenge of gene therapies to immune cells. This article is protected by copyright. All rights reserved.

Keywords:  Gene silencing; nanocomplex; peptide; self-assembly; small RNA

DOI:  https://doi.org/10.1002/bit.27988
J Control Release. 2021 Nov 03. pii: S0168-3659(21)00584-8. [Epub ahead of print]

Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery.

Kelsey L Swingle, Margaret M Billingsley, Sourav K Bose, Brandon White, Rohan Palanki, Apeksha Dave, Savan K Patel, Ningqiang Gong, Alex G Hamilton, Mohamad-Gabriel Alameh, Drew Weissman, William H Peranteau, Michael J Mitchell.

  Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is the in utero delivery of messenger RNA (mRNA). mRNA is a gene therapeutic that has previously been investigated for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to concerns of immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects ex utero stability in amniotic fluid, and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen ex vivo LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in vitro in fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.

Keywords:  Gene therapy; In utero; Nucleic acid therapeutics; lipid nanoparticles; mRNA

DOI:  https://doi.org/10.1016/j.jconrel.2021.10.031
Adv Mater. 2021 Nov;33(45): e2104779

Cation-Free siRNA Micelles as Effective Drug Delivery Platform and Potent RNAi Nanomedicines for Glioblastoma Therapy.

Tong Jiang, Yonghan Qiao, Weimin Ruan, Dongya Zhang, Qingshan Yang, Guoying Wang, Qunzhi Chen, Fengping Zhu, Jinlong Yin, Yan Zou, Rongjun Qian, Meng Zheng, Bingyang Shi.

  Nanoparticle-based small interfering RNA (siRNA) therapy shows great promise for glioblastoma (GBM). However, charge associated toxicity and limited blood-brain-barrier (BBB) penetration remain significant challenges for siRNA delivery for GBM therapy. Herein, novel cation-free siRNA micelles, prepared by the self-assembly of siRNA-disulfide-poly(N-isopropylacrylamide) (siRNA-SS-PNIPAM) diblock copolymers, are prepared. The siRNA micelles not only display enhanced blood circulation time, superior cell take-up, and effective at-site siRNA release, but also achieve potent BBB penetration. Moreover, due to being non-cationic, these siRNA micelles exert no charge-associated toxicity. Notably, these desirable properties of this novel RNA interfering (RNAi) nanomedicine result in outstanding growth inhibition of orthotopic U87MG xenografts without causing adverse effects, achieving remarkably improved survival benefits. Moreover, as a novel type of polymeric micelle, the siRNA micelle displays effective drug loading ability. When utilizing temozolomide (TMZ) as a model loading drug, the siRNA micelle realizes effective synergistic therapy effect via targeting the key gene (signal transducers and activators of transcription 3, STAT3) in TMZ drug resistant pathways. The authors' results show that this siRNA micelle nanoparticle can serve as a robust and versatile drug codelivery platform, and RNAi nanomedicine and for effective GBM treatment.

Keywords:  cation-free; glioblastoma; micelle; siRNA; spherical nucleic acid

DOI:  https://doi.org/10.1002/adma.202104779
Acta Biomater. 2021 Oct 29. pii: S1742-7061(21)00711-X. [Epub ahead of print]

Core-shell lipoplexes inducing active macropinocytosis promote intranasal delivery of c-Myc siRNA for treatment of glioblastoma.

Yang Hu, Kuan Jiang, Dongli Wang, Shengyu Yao, Linwei Lu, Huan Wang, Jie Song, Jianfen Zhou, Xingyan Fan, Yong Wang, Weiyue Lu, Jian Wang, Gang Wei.

  Glioblastoma is the most common and aggressive primary brain tumor, whose malignancy is closely correlated with elevated proto-oncogene c-myc. Intranasal administration emerges as a potential approach to deliver gene into the brain and interfere c-Myc expression. However, powerful permeability in nasal mucosa, selective delivery to glioma and avoidance of premature release during remote transport are imperative to ensure the therapeutic effectiveness. To address the above concerns, herein we constructed a lipoplex based on pre-compression of c-Myc-targeting siRNA (sic-Myc) by octaarginine and subsequent encapsulation by liposome modified with a selected peptide derived from penetratin, named 89WP. It was found that the lipoplex exhibited a stable core-shell structure and could be preferentially internalized along with cell debris by glioma cells via active macropinocytosis. Through this cellular uptake pathway, the lipoplex avoided being entrapped by lysosome and released siRNA in cytoplasm within 4 h, inducing substantial downregulation of c-Myc mRNA and protein expression of glioma cells. Furthermore, due to significantly enhanced permeability in tumor spheroids and nasal mucosa, the lipoplex was competent to deliver more siRNA to orthotopic glioma after intranasal administration, and therefore prolonged the survival time of glioma-bearing mice by inducing apoptosis. STATEMENT OF SIGNIFICANCE: In the present work, a lipoplex was designed to address the unmet demands on intranasal siRNA delivery to the brain for treatment of glioma. First, a powerful peptide was selected to enable the lipoplex to penetrate nasal mucosa. Second, we found the lipoplex could be selectively internalized along with cell debris by glioma cells via active macropinocytosis, and recorded the entire process. This cellular uptake pathway not only prevented the lipoplex being entrapped by lysosome, but also increased distribution of the lipoplex in orthotopic glioma. Third, this lipoplex provided additional protection for siRNA to avoid premature release during transport from nasal to brain. Overall, this lipoplex improved the gene delivery efficiency of intranasal administration and was promising in the perspective of selectively silencing disease-related genes in intracranial tumor.

Keywords:  Glioblastoma; Intranasal drug delivery; Macropinocytosis; Penetratin; siRNA

DOI:  https://doi.org/10.1016/j.actbio.2021.10.042
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Nov 01. pii: S1570-0232(21)00496-7. [Epub ahead of print]1186 123015

Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations.

Xiujuan Jia, Yong Liu, Angela M Wagner, Michelle Chen, Yuejie Zhao, Katelyn J Smith, Dan Some, Andreas M Abend, Justin Pennington.

  The potential of lipid nanoparticles (LNPs) as nucleic acid delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of LNP-based mRNA SARS-CoV-2 vaccines in late 2020. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. This work presents the first description of a facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from multi-angle light scattering, ultraviolet and refractive index detectors following separation of the LNPs by size-exclusion chromatography. The analysis was validated by size-based fractionation of the LNPs with subsequent offline analysis of the fractions. Four LNPs formulated with different PEG-lipids and different lipid compositions were tested. Good agreement was observed between the online and offline size-based RNA distributions among all four LNPs, demonstrating the utility of the online method for LNP-encapsulated RNA in general, and suggesting a means for simplified biophysical quantitation of a dosing-related critical quality attribute.

Keywords:  Lipid nanoparticle; Multi-angle light scattering; Online analysis; RNA; Size exclusion chromatography; Size-dependent

DOI:  https://doi.org/10.1016/j.jchromb.2021.123015
Nat Commun. 2021 Nov 08. 12(1): 6445

Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors.

Aji Alex M Raynold, Danyang Li, Lan Chang, Julien E Gautrot.

In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.

DOI: https://doi.org/10.1038/s41467-021-26695-w
Colloids Surf B Biointerfaces. 2021 Oct 26. pii: S0927-7765(21)00622-6. [Epub ahead of print]209(Pt 2): 112178

Possibility for double optimization of siRNA intracellular delivery efficiency and antibacterial activity: Structure screening of pH-sensitive triblock amphiphilic polycation micelles.

Yufeng Zhang, Yanliang Dong, Junhui Zhou, Wenjun Huang, Yidi Wu, Shuyue Zhao, Yongli Shi, Suping Bai, Chunhui Li, Yuanyu Huang, Anjie Dong.

  Optimal combination of hydrophobic-hydrophilic balance, proton buffering and electrostatic interaction is the key issue for designing polycations as efficient gene vectors and antibacterial agents. Herein, we screened a series of pH-sensitive quaternary ammonium-based amphiphilic triblock copolymers, mPEG2k-P(DPAa/DMAb)-PQAc (TDDE-x), which had different pKa values and proton buffering capacities. Significantly, we found that both the highest siRNA intracellular delivery efficiency and the strongest antibacterial capacity occurred on TDDE-3 micelles with the segment structure of mPEG2k-P(DPA50/DMA56)-PQA55. The TDDE-3/siRNA complex achieved 67% silencing efficiency on H9C2 cells (N/P = 5, 50 nM siRNA), higher than the advanced commercial transfection reagents RNAiMAX (58%) and Lipo2000 (30%). Moreover, TDDE-3 micelles showed quite low MICs of 32 μg/mL and 8 μg/mL against E. coli and S. aureus, respectively. Further studies on the structure-function relationship indicated that TDDE-3 micelles could mediate robust endosome escape and siRNA cytosolic release, and strong bacterial cell membrane-destabilizing function. Undoubtedly, this work reveals the possibility for double optimization of siRNA intracellular delivery efficiency and antibacterial activity of amphiphilic polycations by reasonable structure design, which is significant for low-cost development and clinical translation of efficient multifunctional polycations.

Keywords:  Amphiphilic triblock polycations; Antibacterial activity; Hydrophobic-hydrophilic balance; Proton buffering capacity; SiRNA delivery

DOI:  https://doi.org/10.1016/j.colsurfb.2021.112178
Biomacromolecules. 2021 Nov 10.

Co-delivery of Dexamethasone and a MicroRNA-155 Inhibitor Using Dendrimer-Entrapped Gold Nanoparticles for Acute Lung Injury Therapy.

Changsheng Li, Jin Li, Yu Fan, Dayuan Wang, Mengsi Zhan, Mingwu Shen, Xiangyang Shi.

Development of nanomedicines for effective therapy of acute lung injury (ALI), a common critical respiratory failure syndrome, remains to be challenging. We report here a unique design of a functional nanoplatform based on generation 5 (G5) poly(amidoamine) dendrimer-entrapped gold nanoparticles (Au DENPs) to co-deliver dexamethasone (Dex) and a microRNA-155 inhibitor (miR-155i) for combination chemotherapy and gene therapy of ALI. In this study, we synthesized Au DENPs with 10 Dex moieties attached per G5 dendrimer and an Au core diameter of 2.1 nm and used them to compress miR-155i. The generated polyplexes own a positive zeta potential (16-26 mV) and a small hydrodynamic diameter (175-230 nm) and display desired cytocompatibility and efficient miR-155i delivery to lipopolysaccharide (LPS)-activated alveolar macrophages, thus upregulating the suppressor of cytokine signaling 1 and IL-10 expression and downregulating the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Likewise, as a synthetic glucocorticoid with a potent anti-inflammatory property, the attached Dex on the surface of Au DENPs could inhibit pro-inflammatory cytokine secretion by down-regulating cyclooxygenase-2 expression in the LPS-activated alveolar macrophages. The integration of Dex and miR-155i within one nanoformulation enables superior downregulation of pro-inflammatory cytokines for successful repair of damaged lung tissues in an ALI model, as demonstrated by histological examinations and pro-inflammatory cytokine downregulation in ALI lesion at the gene and protein levels. Such a combined chemotherapy and gene therapy strategy enabled by dendrimer nanotechnology may hold great promise to treat other types of inflammatory diseases.

DOI: https://doi.org/10.1021/acs.biomac.1c01081
J Phys Chem Lett. 2021 Nov 11. 11199-11205

Role of Ionizable Lipids in SARS-CoV-2 Vaccines As Revealed by Molecular Dynamics Simulations: From Membrane Structure to Interaction with mRNA Fragments.

Markéta Paloncýová, Petra Čechová, Martin Šrejber, Petra Kührová, Michal Otyepka.

Recent advances in RNA-based medicine have provided new opportunities for the global current challenge, i.e., the COVID-19 pandemic. Novel vaccines are based on a messenger RNA (mRNA) motif with a lipid nanoparticle (LNP) vector, consisting of high content of unique pH-sensitive ionizable lipids (ILs). Here we provide molecular insights into the role of the ILs and lipid mixtures used in current mRNA vaccines. We observed that the lipid mixtures adopted a nonlamellar organization, with ILs separating into a very disordered, pH-sensitive phase. We describe structural differences of the two ILs leading to their different congregation, with implications for the vaccine stability. Finally, as RNA interacts preferentially with IL-rich phases located at the regions with high curvature of lipid phase, local changes in RNA flexibility and base pairing are induced by lipids. A proper atomistic understanding of RNA-lipid interactions may enable rational tailoring of LNP composition for efficient RNA delivery.

DOI: https://doi.org/10.1021/acs.jpclett.1c03109