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Multi-step screening of DNA/lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression.

ABSTRACT: Lipid nanoparticles hold great potential as an effective non-viral vector for nucleic acid-based gene therapy. Plasmid DNA delivery can result in extended transgene expression compared to mRNA-based technologies, yet there is a lack of systematic investigation into lipid nanoparticle compositions for plasmid DNA delivery. Here, we report a multi-step screening platform to identify optimized plasmid DNA lipid nanoparticles for liver-targeted transgene expression. To achieve this, we analyze the role of different helper lipids and component ratios in plasmid DNA lipid nanoparticle-mediated gene delivery in vitro and in vivo. Compared to mRNA LNPs and in vivo-jetPEI/DNA nanoparticles, the identified plasmid DNA lipid nanoparticles successfully deliver transgenes and mediate prolonged expression in the liver following intravenous administration in mice. By addressing different physiological barriers in a stepwise manner, this screening platform can efficiently down select effective lipid nanoparticle candidates from a lipid nanoparticle library of over 1000 formulations. In addition, we substantially extend the duration of plasmid DNA nanoparticle-mediated transgene expression using a DNA/siRNA co-delivery approach that targets transcription factors regulating inflammatory response pathways. This lipid nanoparticle-based co-delivery strategy further highlights the unique advantages of an extended transgene expression profile using plasmid DNA delivery and offers new opportunities for DNA-based gene medicine applications.

SUBMITTER: Zhu Y

PROVIDER: S-EPMC9310361 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Multi-step screening of DNA/lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression.

Zhu Yining Y Shen Ruochen R Vuong Ivan I Reynolds Rebekah A RA Shears Melanie J MJ Yao Zhi-Cheng ZC Hu Yizong Y Cho Won June WJ Kong Jiayuan J Reddy Sashank K SK Murphy Sean C SC Mao Hai-Quan HQ

Nature communications 20220725 1

Lipid nanoparticles hold great potential as an effective non-viral vector for nucleic acid-based gene therapy. Plasmid DNA delivery can result in extended transgene expression compared to mRNA-based technologies, yet there is a lack of systematic investigation into lipid nanoparticle compositions for plasmid DNA delivery. Here, we report a multi-step screening platform to identify optimized plasmid DNA lipid nanoparticles for liver-targeted transgene expression. To achieve this, we analyze the r ...[more]

PMID: 35879315

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Project description:While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

Project description:Lipid nanoparticles (LNPs) have emerged as a viable, clinically-validated platform for the delivery of mRNA therapeutics. LNPs have been utilized as mRNA delivery systems for applications including vaccines, gene therapy, and cancer immunotherapy. However, LNPs, which are typically composed of ionizable lipids, cholesterol, helper lipids, and lipid-anchored polyethylene glycol, often traffic to the liver which limits the therapeutic potential of the platform. Several approaches have been proposed to resolve this tropism such as post-synthesis surface modification or the addition of synthetic cationic lipids. Methods: Here, we present a strategy for achieving extrahepatic delivery of mRNA involving the incorporation of bile acids, a naturally-occurring class of cholesterol analogs, during LNP synthesis. We synthesized a series of bile acid-containing C14-4 LNPs by replacing cholesterol with bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, or lithocholic acid) at various ratios. Results: Bile acid-containing LNPs (BA-LNPs) were able to reduce delivery to liver cells in vitro and improve delivery in a variety of other cell types, including T cells, B cells, and epithelial cells. Our subsequent in vivo screening of selected LNP candidates injected intraperitoneally or intravenously identified a highly spleen tropic BA-LNP: CA-100, a four-component LNP containing cholic acid and no cholesterol. These screens also identified BA-LNP candidates demonstrating promise for other mRNA therapeutic applications such as for gastrointestinal or immune cell delivery. We further found that the substitution of cholic acid for cholesterol in an LNP formulation utilizing a different ionizable lipid, C12-200, also shifted mRNA delivery from the liver to the spleen, suggesting that this cholic acid replacement strategy may be generalizable. Conclusion: These results demonstrate the potential of a four-component BA-LNP formulation, CA-100, for extrahepatic mRNA delivery that could potentially be utilized for a range of therapeutic and vaccine applications.

Project description:The use of siRNA therapeutics to treat cancer is a very promising approach. However, specific delivery of siRNAs to tumors remains a major challenge. The recent success of siRNA delivery to the liver has incentivized the development of biomaterials for siRNA delivery into tumors. Here, we report a new class of amino acid-modified lipids for siRNA delivery to cancer cells. Eight lipids were developed by headgroup modification with histidine and lysine. The lipids were screened in PC3-luciferase stable cells for gene silencing and cellular cytotoxicity study. The best lipid LHHK shows a pKa of 6.08, which is within the optimal pKa range of lipid nanoparticles (LNPs) for siRNA delivery. The LHHK LNP protects siRNA from serum degradation for up to 24 h and shows higher endosomal release and better cellular uptake compared to other lysine-modified lipids in PC3 cells. The LHHK LNP exhibits significant silencing activity of IKKα and IKBKE in prostate cancer and pancreatic cancer, respectively. Moreover, the LHHK LNP encapsulating IKBKE siRNA inhibits cell proliferation of pancreatic cancer cells and suppresses the tumor progression in a pancreatic cancer mouse model. STATEMENT OF SIGNIFICANCE: Lipid nanoparticle (LNP) is a promising platform for siRNA delivery. However, LNP is generally associated with high systemic toxicity. As a result, efficient and biodegradable lipids are highly needed for siRNA-based cancer therapy. Herein, we develop amino acid-modified biodegradable lipids. These lipids show very low cellular toxicity and high transfection efficiency. The best lipid LHHK shows a pKa of 6.08, which is within the optimal pKa range of LNPs for siRNA delivery. The LHHK LNP efficiently silences IKKα and IKBKE in prostate and pancreatic cancer, respectively. Moreover, the LHHK LNP encapsulating IKBKE siRNA inhibits cell proliferation and suppresses tumor growth of pancreatic cancer in vivo. These results suggest that amino acid-modified lipids possess a great potential for siRNA delivery in cancer therapy.

Dataset Information

Multi-step screening of DNA/lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression.

Publications

Multi-step screening of DNA/lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression.

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