bims-novged 2023-01-15 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–01–15
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Liposome-polyethylenimine complexes for the effective delivery of HuR siRNA in the treatment of diabetic retinopathy.
Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.
Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.
Fine-Tuned Polymer Nanoassembly for Co-Delivery of Chemotherapeutic Drug and siRNA.
Acidification-Induced Structure Evolution of Lipid Nanoparticles Correlates with Their In Vitro Gene Transfections.
Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates.
In silico prediction of siRNA ionizable-lipid nanoparticles In vivo efficacy: Machine learning modeling based on formulation and molecular descriptors.
PEGylation and folic-acid functionalization of cationic lipoplexes-Improved nucleic acid transfer into cancer cells.
Recent progress and applications of poly(beta amino esters)-based biomaterials.
RNA and nanocarriers: next generation drug and delivery platform take center stage.

Drug Deliv Transl Res. 2023 Jan 11.

Liposome-polyethylenimine complexes for the effective delivery of HuR siRNA in the treatment of diabetic retinopathy.

Shibani Supe, Archana Upadhya, Santosh Tripathi, Vikas Dighe, Kavita Singh.

  Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)-induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents.

Keywords:  Diabetic retinopathy; HuR/ELAV; Lipopolyplexes; Polyethylenimine; VEGF-A; siRNA

DOI:  https://doi.org/10.1007/s13346-022-01281-9
J Control Release. 2023 Jan 09. pii: S0168-3659(23)00015-9. [Epub ahead of print]

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

Rima Kandil, Domizia Baldassi, Sebastian Böhlen, Joschka T Müller, David C Keul, Tonia Bargmann, Susann Dehmel, Yuran Xie, Aditi Mehta, Katherina Sewald, Olivia M Merkel.

  GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

Keywords:  Asthma; Cytokines; PCLS; Pulmonary delivery; T cell targeting; siRNA therapy

DOI:  https://doi.org/10.1016/j.jconrel.2023.01.014
Cells. 2022 Dec 30. pii: 156. [Epub ahead of print]12(1):

Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.

Kemao Xiu, Laura Saunders, Luan Wen, Jinxue Ruan, Ruonan Dong, Jun Song, Dongshan Yang, Jifeng Zhang, Jie Xu, Y Eugene Chen, Peter X Ma.

  Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor "BE4-max" pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.

Keywords:  CRISPR/Cas9; PEI; gene delivery; gene editing; nanoparticle; pDNA; polyplex

DOI:  https://doi.org/10.3390/cells12010156
Macromol Biosci. 2023 Jan 14. e2200529

Fine-Tuned Polymer Nanoassembly for Co-Delivery of Chemotherapeutic Drug and siRNA.

Yuanyuan Yuan, Yiyao Wang, Huiling Huang, Shuiliang Tao, Jinsheng Huang.

  Successful clinical application of siRNA to liver-associated diseases reinvigorates the RNAi therapeutics and delivery vectors, especially for anti-cancer combination therapy. Fine tuning of copolymer-based assembly configuration was highly important for a desirable synergistic cancer cell-killing effect via the co-delivery of chemotherapeutic drug and siRNA. Herein, an amphiphilic triblock copolymer methoxyl poly(ethylene glycol)-block-poly(L-lysine)-block-poly(2-(diisopropyl amino)ethyl methacrylate) (abbreviated as mPEG-PLys-PDPA or PLD) consisting of a hydrophilic diblock mPEG-PLys and a hydrophobic block PDPA was synthesized. Three distinct assemblies (i.e., nano-sized micelle, nano-sized polymersome, and microparticle) were acquired, along with the increase in PDPA block length. Furthermore, the as-obtained polymersome could efficiently co-deliver doxorubicin hydrochloride (DOX) as a hydrophilic chemotherapeutic model and siRNA against ADP-ribosylation factor 6 (siArf6) as an siRNA model into cancer cell via lysosomal pH-triggered payload release. PC-3 prostate cell was synergistically killed by the DOX- and siArf6-coloading polymersome (namely PLD@DOX/siArf6). PLD@DOX/siArf6 may serve as a robust nanomedicine for anti-cancer therapy. This article is protected by copyright. All rights reserved.

Keywords:  assembly; chemotherapy; drug delivery; polymeric nanovector; siRNA

DOI:  https://doi.org/10.1002/mabi.202200529
ACS Nano. 2023 Jan 06.

Acidification-Induced Structure Evolution of Lipid Nanoparticles Correlates with Their In Vitro Gene Transfections.

Zongyi Li, Jessica Carter, Luis Santos, Carl Webster, Christopher F van der Walle, Peixun Li, Sarah E Rogers, Jian Ren Lu.

  The rational design of lipid nanoparticles (LNPs) for enhanced gene delivery remains challenging because of incomplete knowledge of their formulation-structure relationship that impacts their intracellular behavior and consequent function. Small-angle neutron scattering has been used in this work to investigate the structure of LNPs encapsulating plasmid DNA upon their acidification (from pH 7.4 to 4.0), as would be encountered during endocytosis. The results revealed the acidification-induced structure evolution (AISE) of the LNPs on different dimension scales, involving protonation of the ionizable lipid, volume expansion and redistribution of aqueous and lipid components. A similarity analysis using an LNP's structural feature space showed a strong positive correlation between function (measured by intracellular luciferase expression) and the extent of AISE, which was further enhanced by the fraction of unsaturated helper lipid. Our findings reveal molecular and nanoscale changes occurring during AISE that underpin the LNPs' formulation-nanostructure-function relationship, aiding the rational design of application-directed gene delivery vehicles.

Keywords:  acidification-induced structure evolution (AISE); cellular expression; lipid nanoparticles; nonviral gene delivery; small-angle neutron scattering

DOI:  https://doi.org/10.1021/acsnano.2c06213
Sci Adv. 2023 Jan 13. 9(2): eadd4623

Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates.

Marco Herrera-Barrera, Renee C Ryals, Milan Gautam, Antony Jozic, Madeleine Landry, Tetiana Korzun, Mohit Gupta, Chris Acosta, Jonathan Stoddard, Rene Reynaga, Wayne Tschetter, Nick Jacomino, Oleh Taratula, Conroy Sun, Andreas K Lauer, Martha Neuringer, Gaurav Sahay.

Lipid nanoparticle (LNP)-based mRNA delivery holds promise for the treatment of inherited retinal degenerations. Currently, LNP-mediated mRNA delivery is restricted to the retinal pigment epithelium (RPE) and Müller glia. LNPs must overcome ocular barriers to transfect neuronal cells critical for visual phototransduction, the photoreceptors (PRs). We used a combinatorial M13 bacteriophage-based heptameric peptide phage display library for the mining of peptide ligands that target PRs. We identified the most promising peptide candidates resulting from in vivo biopanning. Dye-conjugated peptides showed rapid localization to the PRs. LNPs decorated with the top-performing peptide ligands delivered mRNA to the PRs, RPE, and Müller glia in mice. This distribution translated to the nonhuman primate eye, wherein robust protein expression was observed in the PRs, Müller glia, and RPE. Overall, we have developed peptide-conjugated LNPs that can enable mRNA delivery to the neural retina, expanding the utility of LNP-mRNA therapies for inherited blindness.

DOI: https://doi.org/10.1126/sciadv.add4623
Front Mol Biosci. 2022 ;9 1042720

In silico prediction of siRNA ionizable-lipid nanoparticles In vivo efficacy: Machine learning modeling based on formulation and molecular descriptors.

Abdelkader A Metwally, Amira A Nayel, Rania M Hathout.

  In silico prediction of the in vivo efficacy of siRNA ionizable-lipid nanoparticles is desirable as it can save time and resources dedicated to wet-lab experimentation. This study aims to computationally predict siRNA nanoparticles in vivo efficacy. A data set containing 120 entries was prepared by combining molecular descriptors of the ionizable lipids together with two nanoparticles formulation characteristics. Input descriptor combinations were selected by an evolutionary algorithm. Artificial neural networks, support vector machines and partial least squares regression were used for QSAR modeling. Depending on how the data set is split, two training sets and two external validation sets were prepared. Training and validation sets contained 90 and 30 entries respectively. The results showed the successful predictions of validation set log (siRNA dose) with Rval 2= 0.86-0.89 and 0.75-80 for validation sets one and two, respectively. Artificial neural networks resulted in the best Rval 2 for both validation sets. For predictions that have high bias, improvement of Rval 2 from 0.47 to 0.96 was achieved by selecting the training set lipids lying within the applicability domain. In conclusion, in vivo performance of siRNA nanoparticles was successfully predicted by combining cheminformatics with machine learning techniques.

Keywords:  QSAR; in vivo; ionizable lipids; machine learning; nanoparticles; siRNA

DOI:  https://doi.org/10.3389/fmolb.2022.1042720
Front Bioeng Biotechnol. 2022 ;10 1066887

PEGylation and folic-acid functionalization of cationic lipoplexes-Improved nucleic acid transfer into cancer cells.

Marco Hoffmann, Sven Gerlach, Christina Hoffmann, Nathalie Richter, Nils Hersch, Agnes Csiszár, Rudolf Merkel, Bernd Hoffmann.

  Efficient and reliable transfer of nucleic acids for therapy applications is a major challenge. Stabilization of lipo- and polyplexes has already been successfully achieved by PEGylation. This modification reduces the interaction with serum proteins and thus prevents the lipoplexes from being cleared by the reticuloendothelial system. Problematically, this stabilization of lipoplexes simultaneously leads to reduced transfer efficiencies compared to non-PEGylated complexes. However, this reduction in transfer efficiency can be used to advantage since additional modification of PEGylated lipoplexes with functional groups enables improved selective transfer into target cells. Cancer cells overexpress folate receptors because of a significantly increased need of folate due to high cell proliferation rates. Thus, additional folate functionalization of PEGylated lipoplexes improves uptake into cancer cells. We demonstrate herein that NHS coupling chemistries can be used to modify two commercially available transfection reagents (Fuse-It-DNA and Lipofectamine® 3000) with NHS-PEG-folate for increased uptake of nucleic acids into cancer cells. Lipoplex characterization and functional analysis in cultures of cancer- and healthy cells clearly demonstrate that functionalization of PEGylated lipoplexes offers a promising method to generate efficient, stable and selective nucleic acid transfer systems.

Keywords:  DNA-transfer; PEGylation; biocompatibility; functionalized lipoplexes; selective nucleic acid transfer

DOI:  https://doi.org/10.3389/fbioe.2022.1066887
J Control Release. 2023 Jan 06. pii: S0168-3659(23)00002-0. [Epub ahead of print]

Recent progress and applications of poly(beta amino esters)-based biomaterials.

Jingjing Wei, Linglin Zhu, Qiuyun Lu, Guicai Li, Youlang Zhou, Yumin Yang, Luzhong Zhang.

  Poly(beta-amino esters, PBAEs) are a promising class of cationic polymers synthesized from diacrylates and amines via Michael addition. Recently, PBAEs have been widely developed for drug delivery, immunotherapy, gene therapy, antibacterial, tissue engineering and other applications due to their convenient synthesis, good bio-compatibility and degradation properties. Herein, we mainly summarize the recent progress in the PBAEs synthesis and their applications. The amine groups of PBAEs could be protonated in low pH environment, exhibiting proton sponge and pH-sensitive abilities. Furthermore, the positive PBAEs can interact with negative genes via electrostatic interactions for efficient delivery of nucleic acids. Moreover, positive PBAEs could also directly kill bacteria by disrupting their membranes at high doses. Finally, PBAEs can augment the immune responses, and improve the bioactivity of hydrogels in tissue engineering.

Keywords:  And tissue engineering; Antibacterial application; Bio-orthogonal chemistry; Drug delivery; Gene therapy; Immunotherapy; Poly(beta-amino esters)

DOI:  https://doi.org/10.1016/j.jconrel.2023.01.002
Trends Biotechnol. 2023 Jan 04. pii: S0167-7799(22)00347-X. [Epub ahead of print]

RNA and nanocarriers: next generation drug and delivery platform take center stage.

Jessica Rouge.

The unprecedented rapid deployment of mRNA vaccines against COVID-19 can be traced back to the early studies of RNA nanocarriers, including the study by Zimmerman et al. which showcased the effectiveness of RNA nanocarriers in vivo. This study, among others, ultimately resulted in Onpattro, the first FDA-approved RNA formulation.

DOI: https://doi.org/10.1016/j.tibtech.2022.12.019