bims-novged 2021-06-13 papers

on Non-viral vectors for gene delivery

Issue of 2021–06–13
thirteen papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Osteoporosis Remission and New Bone Formation with Mesoporous Silica Nanoparticles.
Low-Molecular-Weight Poly(ethylenimine) Nanogels Loaded with Ultrasmall Iron Oxide Nanoparticles for T1-Weighted MR Imaging-Guided Gene Therapy of Sarcoma.
Reactive oxygen species-responsive nanoplatforms for nucleic acid-based gene therapy of cancer and inflammatory diseases.
Ligand Conjugate Structure Activity Relationships and Enhanced Endosomal Escape with a Targeted Polymer Micelle in Non-Human Primates.
Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNP.
A Synergistic DNA-polydopamine-MnO2 Nanocomplex for Near-Infrared-Light-Powered DNAzyme-Mediated Gene Therapy.
High-throughput evaluation of polymeric nanoparticles for tissue-targeted gene expression using barcoded plasmid DNA.
In Vivo Editing of Macrophages through Systemic Delivery of CRISPR-Cas9-Ribonucleoprotein-Nanoparticle Nanoassemblies.
Salicylic acid-based nanomedicine with self-immunomodulatory activity facilitates microRNA therapy for metabolic skeletal disorders.
Highly Efficient MicroRNA Delivery Using Functionalized Carbon Dots for Enhanced Conversion of Fibroblasts to Cardiomyocytes.
Engineered materials for in vivo delivery of genome-editing machinery.
Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema.
Kidney-targeted delivery of PHD2 siRNA with nanoparticles alleviated renal ischemia/reperfusion injury.

Adv Sci (Weinh). 2021 Jun 06. e2101107

Osteoporosis Remission and New Bone Formation with Mesoporous Silica Nanoparticles.

Patricia Mora-Raimundo, Daniel Lozano, Manuel Benito, Francisca Mulero, Miguel Manzano, María Vallet-Regí.

  Nanotechnology changed the concept of treatment for a variety of diseases, producing a huge impact regarding drug and gene delivery. Among the different targeted diseases, osteoporosis has devastating clinical and economic consequences. Since current osteoporosis treatments present several side effects, new treatment approaches are needed. Recently, the application of small interfering RNA (siRNA) has become a promising alternative. Wnt/β-catenin signaling pathway controls bone development and formation. This pathway is negatively regulated by sclerostin, which knock-down through siRNA application would potentially promote bone formation. However, the major bottleneck for siRNA-based treatments is the necessity of a delivery vector, bringing nanotechnology as a potential solution. Among the available nanocarriers, mesoporous silica nanoparticles (MSNs) have attracted great attention for intracellular delivery of siRNAs. The mesoporous structure of MSNs permits the delivery of siRNAs together with another biomolecule, achieving a combination therapy. Here, the effectiveness of a new potential osteoporosis treatment based on MSNs is evaluated. The proposed system is effective in delivering SOST siRNA and osteostatin through systemic injection to bone tissue. The nanoparticle administration produced an increase expression of osteogenic related genes improving the bone microarchitecture. The treated osteoporotic mice recovered values of a healthy situation approaching to osteoporosis remission.

Keywords:  combination therapy; mesoporous silica nanoparticles; osteoporosis; osteostatin; small interfering RNAs

DOI:  https://doi.org/10.1002/advs.202101107
ACS Appl Mater Interfaces. 2021 Jun 09.

Low-Molecular-Weight Poly(ethylenimine) Nanogels Loaded with Ultrasmall Iron Oxide Nanoparticles for T1-Weighted MR Imaging-Guided Gene Therapy of Sarcoma.

Yucheng Peng, Yue Gao, Chao Yang, Rui Guo, Xiangyang Shi, Xueyan Cao.

  Cancer metastasis is still a major obstacle in clinical cancer therapy and a paramount cause of cancer deaths. Designing multifunctional nanoplatforms with an enhanced diagnostic sensitivity and anti-metastasis efficiency against tumors represents a major trend in current cancer management. Herein, we report the preparation of low-molecular-weight poly(ethylenimine) (PEI)-poly(ethylene glycol) (PEG) nanogels (NGs) loaded with transforming growth factor-β1 (TGF-β1) siRNA and ultrasmall iron oxide nanoparticles (Fe3O4 NPs) for gene therapy and T1-weighted magnetic resonance (MR) imaging of tumors and tumor metastasis in a mouse sarcoma model. In this work, ultrasmall Fe3O4 NPs stabilized by sodium citrate were first prepared and then mixed with PEI (800 Da) and PEG (400 Da)-diacrylate as a cross-linker to form Fe3O4/PEI-PEG NGs with an average size of 76.3 nm via an inverse microemulsion method. The developed hybrid NGs display good cytocompatibility and enhanced MR imaging performance (r1 relaxivity = 1.0346 mM-1 s-1). The Fe3O4/PEI-PEG NGs can be further used to compact TGF-β1 siRNA through electrostatic interaction and efficiently deliver siRNA to cancer cells and a tumor model to silence the TGF-β1 gene, which inhibits the growth and invasion of cancer cell in vitro significantly, as well as the growth of a subcutaneous sarcoma tumor model and lung metastasis in vivo. The designed hybrid NG-ultrasmall iron oxide NPs may be extended for the delivery of other drugs or genes for theranostics of different biological systems.

Keywords:  MR imaging; PEI; TGF-β1 siRNA; gene therapy; nanogels

DOI:  https://doi.org/10.1021/acsami.1c04081
Biomed Mater. 2021 Jun 11.

Reactive oxygen species-responsive nanoplatforms for nucleic acid-based gene therapy of cancer and inflammatory diseases.

Dandan Zhu, Wang Chen, Wenyi Lin, Ying Li, Xiaoxuan Liu.

  Nucleic acid-based gene therapy has recently made important progress toward clinical implementation, and holds tremendous promise for the treatment of some life-threatening diseases, such as cancer and inflammation. However, the on-demand delivery of nucleic acid therapeutics in target cells remains highly challenging. The development of delivery systems responsive to specific pathological cues of diseases is expected to offer promising alternatives for overcoming this problem. Among them, the reactive oxygen species (ROS)-responsive delivery systems, which in response to elevated ROS in cancer cells or activated inflammatory cells, can deliver nucleic acid therapeutics on-demand via ROS-induced structural and assembly behavior changes, constitute a promising approach for cancer and anti-inflammation therapies. In this short review, we briefly introduce the ROS-responsive chemical structures, ROS-induced release mechanisms and some representative examples to highlight the current progress in constructing ROS-responsive delivery systems. We aim to provide new insights into the rational design of on-demand gene delivery vectors.

Keywords:  cancer; inflammatory diseases; nucleic acids; on-demand delivery; reactive oxygen species (ROS)

DOI:  https://doi.org/10.1088/1748-605X/ac0a8f
Mol Ther. 2021 Jun 03. pii: S1525-0016(21)00308-7. [Epub ahead of print]

Ligand Conjugate Structure Activity Relationships and Enhanced Endosomal Escape with a Targeted Polymer Micelle in Non-Human Primates.

Richard James Holland, Kieu Lam, Xin Ye, Alan D Martin, Mark C Wood, Lorne Palmer, David Fraser, Kevin McClintock, Sara Majeski, Agnes Jarosz, Amy C H Lee, Emily P Thi, Adam Judge, James Heyes.

N-Acetylgalactosamine (GalNAc) conjugated small interfering RNA (siRNA) are a leading RNA interference (RNAi) platform allowing targeted inhibition of disease-causing genes in hepatocytes. More than a decade of development has recently resulted in the first approvals for this class of drugs. While substantial effort has been made to improve nucleic acid modification patterns for better payload stability and efficacy, relatively little attention has been given to the GalNAc targeting ligand. In addition, the lack of an intrinsic endosomal release mechanism has limited potency. Here we report a stepwise analysis of the structure activity relationships (SAR) of the components comprising these targeting ligands. We show that there is relatively little difference in biological performance between bi-, tri- and tetravalent ligand structures, while identifying other features that affect their biological activity more significantly. Further, we demonstrate that subcutaneous co-administration of a GalNAc-functionalized, pH responsive endosomal release agent markedly improved the activity and duration of effect for siRNA conjugates, without compromising tolerability, in non-human primates. These findings could address a significant bottleneck for future siRNA ligand conjugate development.

DOI: https://doi.org/10.1016/j.ymthe.2021.06.002
Mol Ther. 2021 Jun 03. pii: S1525-0016(21)00310-5. [Epub ahead of print]

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNP.

István Tombácz, Dorottya Laczkó, Hamna Shahnawaz, Hiromi Muramatsu, Ambika Natesan, Amir Yadegari, Tyler E Papp, Mohamad-Gabriel Alameh, Vladimir Shuvaev, Barbara L Mui, Ying K Tam, Vladimir Muzykantov, Norbert Pardi, Drew Weissman, Hamideh Parhiz.

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic, and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNP. Intravenous injection of CD4-targeted LNP loaded by Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNP in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

DOI: https://doi.org/10.1016/j.ymthe.2021.06.004
Nano Lett. 2021 Jun 08.

A Synergistic DNA-polydopamine-MnO2 Nanocomplex for Near-Infrared-Light-Powered DNAzyme-Mediated Gene Therapy.

Huaixin Zhao, Zhili Zhang, Duo Zuo, Linghui Li, Feng Li, Dayong Yang.

  DNAzyme is emerging for gene therapy. The administration of the in vivo catalytic activity of DNAzyme has proven important but challenging for clinical applications. Herein, we report a synergistic DNA-polydopamine-MnO2 nanocomplex, which enables near-infrared (NIR)-light-powered catalytic activity of DNAzyme in vivo. The nanocomplex has a hierarchical structure: a DNA nanoframework as the scaffold and polydopamine-MnO2 (PM) as the coating layer. The DNA nanoframework contains repeated DNAzyme sequences. PM assembles on the surface of the DNA nanoframework. When the nanocomplex accumulates at tumor sites, upon NIR-light radiation, polydopamine induces a temperature elevation at tumor sites via photothermal conversion; meanwhile, glutathione triggers decomposition of PM to release Mn2+ to activate DNAzyme in the cytoplasm for gene regulation. In vitro and in vivo experiments show that the PM-induced temperature elevation enhances the Egr-1 mRNA cleavage activity of DNAzyme, promoting downregulation of the Egr-1 protein in tumor cells. In addition, the temperature elevation induces heat stress, achieving a synergistic tumor ablation effect.

Keywords:  DNA nanotechnology; DNAzyme; Gene delivery; Gene therapy

DOI:  https://doi.org/10.1021/acs.nanolett.1c01727
J Control Release. 2021 Jun 04. pii: S0168-3659(21)00281-9. [Epub ahead of print]

High-throughput evaluation of polymeric nanoparticles for tissue-targeted gene expression using barcoded plasmid DNA.

Jayoung Kim, Hannah J Vaughan, Camila G Zamboni, Joel C Sunshine, Jordan J Green.

  Successful systemic gene delivery requires specific tissue targeting as well as efficient intracellular transfection. Increasingly, research laboratories are fabricating libraries of novel nanoparticles, engineering both new biomaterial structures and composition ratios of multicomponent systems. Yet, methods for screening gene delivery vehicles directly in vivo are often low-throughout, limiting the number of candidate nanoparticles that can be investigated. Here, we report a comprehensive, high-throughput method to evaluate a library of polymeric nanoparticles in vivo for tissue-specific gene delivery. The method involves pairing each nanoparticle formulation with a plasmid DNA (pDNA) that harbors a unique nucleotide sequence serving as the identifying "barcode". Using real time quantitative PCR (qPCR) for detection of the barcoded pDNA and quantitative reverse transcription PCR (RT-qPCR) for transcribed barcoded mRNA, we can quantify accumulation and transfection in tissues of interest. The barcode pDNA and primers were designed with sufficient sensitivity and specificity to evaluate multiple nanoparticle formulations per mouse, improving screening efficiency. Using this platform, we evaluated the biodistribution and transfection of 8 intravenously administered poly(beta-amino ester) (PBAE) nanoparticle formulations, each with a PBAE polymer of differential structure. Significant levels of nanoparticle accumulation and gene transfection were observed mainly in organs involved in clearance, including spleen, liver, and kidneys. Interestingly, higher levels of transfection of select organs did not necessarily correlate with higher levels of tissue accumulation, highlighting the importance of directly measuring in vivo transfection efficiency as the key barcoded parameter in gene delivery vector optimization. To validate this method, nanoparticle formulations were used individually for luciferase pDNA delivery in vivo. The distribution of luciferase expression in tissues matched the transfection analysis by the barcode qPCR method, confirming that this platform can be used to accurately evaluate systemic gene delivery.

Keywords:  Biodistribution; Gene delivery; High-throughput screening; Polymeric nanoparticle; Transfection

DOI:  https://doi.org/10.1016/j.jconrel.2021.05.047
Adv Ther (Weinh). 2019 Oct;pii: 1900041. [Epub ahead of print]2(10):

In Vivo Editing of Macrophages through Systemic Delivery of CRISPR-Cas9-Ribonucleoprotein-Nanoparticle Nanoassemblies.

Yi-Wei Lee, Rubul Mout, David C Luther, Yuanchang Liu, Laura Castellanos-García, Amy S Burnside, Moumita Ray, Gulen Yeşilbag Tonga, Joseph Hardie, Harini Nagaraj, Riddha Das, Erin L Phillips, Tristan Tay, Richard W Vachet, Vincent M Rotello.

  Macrophages are key effectors of host defense and metabolism, making them promising targets for transient genetic therapy. Gene editing through delivery of the Cas9-ribonucleoprotein (RNP) provides multiple advantages over gene delivery-based strategies for introducing CRISPR machinery to the cell. There are, however, significant physiological, cellular, and intracellular barriers to the effective delivery of the Cas9 protein and guide RNA (sgRNA) that have to date, restricted in vivo Cas9 protein-based approaches to local/topical delivery applications. Herein we describe a new nanoassembled platform featuring co-engineered nanoparticles and Cas9 protein that has been developed to provide efficient Cas9-sgRNA delivery and concomitant CRISPR editing through systemic tail-vein injection into mice, achieving >8% gene editing efficiency in macrophages of the liver and spleen.

Keywords:  CRISPR-Cas9 ribonucleoprotein delivery; gene editing; in vivo delivery; phagocyte targeting; protein delivery

DOI:  https://doi.org/10.1002/adtp.201900041
Acta Biomater. 2021 Jun 02. pii: S1742-7061(21)00334-2. [Epub ahead of print]

Salicylic acid-based nanomedicine with self-immunomodulatory activity facilitates microRNA therapy for metabolic skeletal disorders.

Yan Li, Bolei Cai, Zhaoyichun Zhang, Guanlin Qu, Lu Chen, Guojun Chen, Tingxizi Liang, Chi Yang, Ling Fan, Zhiyuan Zhang.

  Metabolic skeletal disorders remain a major clinical challenge. The complexity of this disease requires a strategy to address the net effects of both inflammation and impaired bone formation. microRNA-based gene therapy provides several therapeutic advantages to tackle these issues. Herein, we describe a microRNA-21 (miR-21) delivery system with an additional therapeutic effect from that of the delivery carrier itself. Poly (salicylic acid) (PSA) is, for the first time, synthesized via polycondensation of salicylic acid (SA), a bioactive ingredient widely used for anti-inflammation in medicine. PSA can self-assemble into nanoparticles (PSA-NPs) and can effectively deliver genes both in vitro and in vivo. The carrier was then attached to repetitive sequences of aspartate, serine, serine (DSS)6 for delivering miRNAs specifically to bone-formation surfaces. In vitro studies showed that miR-21@PSA-NP could effectively realize the intracellular delivery of miR-21 with low toxicity, while in vivo results indicated that the miR-21@PSA-NP-DSS6 prolonged blood circulation time, enhanced bone accumulation, and significantly improved the efficacy of miR-21-based bone anabolic therapy in osteoporotic mice. The constructed delivery system (miR-21@PSA-NP-DSS6) inherited the advantages of both SA and miR-21, which could ameliorate bone-inflamed niche and rescued the impaired bone formation ability. The synergy of anti-inflammatory and pro-osteogenic effects significantly improved trabecular bone microstructure in osteoporotic mice.

Keywords:  Drug delivery; metabolic skeletal disorders; nanomedicine; salicylic acid; self-immunomodulation

DOI:  https://doi.org/10.1016/j.actbio.2021.05.024
Int J Nanomedicine. 2021 ;16 3741-3754

Highly Efficient MicroRNA Delivery Using Functionalized Carbon Dots for Enhanced Conversion of Fibroblasts to Cardiomyocytes.

Lei Yang, Song Xue, Mingjun Du, Feng Lian.

   Introduction: The reprogramming of induced cardiomyocytes (iCMs) is of particular significance in regenerative medicine; however, it remains a great challenge to fabricate an efficient and safe gene delivery system to induce reprogramming of iCMs for therapeutic applications in heart injury. Here, we report branched polyethyleneimine (BP) coated nitrogen-enriched carbon dots (BP-NCDs) as highly efficient nanocarriers loaded with microRNAs-combo (BP-NCDs/MC) for cardiac reprogramming.
Methods: The BP-NCDs nanocarriers were prepared and characterized by several analytical techniques.
Results: The BP-NCDs nanocarriers showed good microRNAs-combo binding affinity, negligible cytotoxicity, and long-term microRNAs expression. Importantly, BP-NCDs/MC nanocomplexes led to the efficient direct reprogramming of fibroblasts into iCMs without genomic integration and resulting in effective recovery of cardiac function after myocardial infarction (MI).
Conclusion: This study offers a novel strategy to provide safe and effective microRNAs-delivery nanoplatforms based on carbon dots for promising cardiac regeneration and disease therapy.

Keywords:  branched polyethyleneimine; direct reprogramming; induced cardiomyocytes; microRNAs; nitrogen-enriched carbon dots

DOI:  https://doi.org/10.2147/IJN.S304873
Nat Rev Mater. 2019 Nov;4 726-737

Engineered materials for in vivo delivery of genome-editing machinery.

Sheng Tong, Buhle Moyo, Ciaran M Lee, Kam Leong, Gang Bao.

Genome editing technologies, such as CRISPR/Cas9, are promising for treating otherwise incurable genetic diseases. Great progress has been made for ex vivo genome editing; however, major bottlenecks exist in the development of efficient, safe, and targetable in vivo delivery systems, which are needed for the treatment of many diseases. To achieve high efficacy and safety in therapeutic in vivo genome editing, editing activities must be controlled spatially and temporally in the body, which requires novel materials, delivery strategies, and control mechanisms. Thus, there is currently a tremendous opportunity for the biomaterials research community to develop in vivo delivery systems that overcome the problems of low editing efficiency, off-targeting effect, safety, and cell and tissue specificity. In this Review, we summarize delivery approaches and provide perspectives on the challenges and possible solutions, aiming to stimulate further development of engineered materials for in vivo delivery of genome-editing machinery.

DOI: https://doi.org/10.1038/s41578-019-0145-9
Nat Commun. 2021 06 08. 12(1): 3460

Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema.

Dániel Szőke, Gábor Kovács, Éva Kemecsei, László Bálint, Kitti Szoták-Ajtay, Petra Aradi, Andrea Styevkóné Dinnyés, Barbara L Mui, Ying K Tam, Thomas D Madden, Katalin Karikó, Raghu P Kataru, Michael J Hope, Drew Weissman, Babak J Mehrara, Norbert Pardi, Zoltán Jakus.

Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.

DOI: https://doi.org/10.1038/s41467-021-23546-6
J Pharmacol Exp Ther. 2021 Jun 08. pii: JPET-AR-2021-000667. [Epub ahead of print]

Kidney-targeted delivery of PHD2 siRNA with nanoparticles alleviated renal ischemia/reperfusion injury.

Dengpiao Xie, Juan Wang, Gaizun Hu, Chaoling Chen, Hu Yang, Joseph K Ritter, Yun Qu, Ningjun Li.

  Background: Inhibition of HIF-prolyl hydroxylase (PHD) has been shown to protect against various kidney diseases. However, there are controversial reports on the effect of PHD inhibition in renoprotection. The present study determined whether delivery of PHD2 siRNA using a siRNA carrier, folic acid (FA)-decorated polyamidoamine dendrimer generation 5 (G5-FA), would mainly target kidneys and protect against renal ischemia/reperfusion injury (I/R). Methods: The renal I/R was generated by clipping the renal pedicle for 30 minutes in uninephrectomized mice. Mice were sacrificed 48 hours after I/R. Normal saline or G5-FA complexed with control or PHD2 siRNA was injected via tail vein 24 h before ischemia. Results: After the injection of near-infrared fluorescent dye-labeled G5-FA, the fluorescence was mainly detected in kidneys, but not in other organs. The reduction of PHD2 mRNA and protein was only observed in kidneys but not in other organs after injection of PHD2-siRNA- G5-FA complex. The injection of PHD2-siRNA-G5-FA significantly alleviated renal I/R injury, as shown by the inhibition of increases in serum creatinine and BUN, the blockade of increases in KIM-1 and NGAL and the improvement of histological damage compared with mice treated with control siRNA. Conclusion: PHD2 siRNA can be delivered specifically into kidneys using G5-FA and that local knockdown of PHD2 gene expression within the kidney alleviates renal I/R injury. Therefore, G5-FA is an efficient siRNA carrier to deliver siRNA into the kidney, and that local inhibition of PHD2 within the kidney may be a potential strategy for the management of acute I/R injury. Significance Statement Folic acid (FA)-decorated polyamidoamine dendrimer generation 5 (G5-FA) was demonstrated to be an effective carrier to deliver siRNA into kidneys. Delivery of PHD2 siRNA with G5-FA effectively protected the kidneys against the acute renal ischemia/reperfusion injury.

Keywords:  Fluorescence techniques; Hypoxia inducible factor (HIF1); kidney

DOI:  https://doi.org/10.1124/jpet.121.000667