bims-novged 2021-10-31 papers

on Non-viral vectors for gene delivery

Issue of 2021–10–31
ten papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Rational preparation and application of a mRNA delivery system with cytidinyl/cationic lipid.
Nanoparticle designs for delivery of nucleic acid therapeutics as brain cancer therapies.
Transferrin Receptor Targeted Polyplexes Completely Comprised of Sequence-defined Components.
Farnesylthiosalicylic acid-derivatized PEI-based nanocomplex for improved tumor vaccination.
Marked Increase in Tumor Transfection with a Truncated Branched Polymer.
Phenylboronic acid-functionalized polyaminoglycoside as an effective CRISPR/Cas9 delivery system.
Modulating expression of inhibitory and stimulatory immune 'checkpoints' using nanoparticulate-assisted nucleic acid delivery.
Confined growth of ZIF-8 in dendritic mesoporous organosilica nanoparticles as bioregulators for enhanced mRNA delivery in vivo.
Cetuximab-Modified Human Serum Albumin Nanoparticles Co-Loaded with Doxorubicin and MDR1 siRNA for the Treatment of Drug-Resistant Breast Tumors.
Intraperitoneal siRNA Nanoparticles for Augmentation of Gemcitabine Efficacy in the Treatment of Pancreatic Cancer.

J Control Release. 2021 Oct 23. pii: S0168-3659(21)00552-6. [Epub ahead of print]

Rational preparation and application of a mRNA delivery system with cytidinyl/cationic lipid.

Lei Li, Jinrong Long, Ye Sang, Xin Wang, Xinyang Zhou, Yufei Pan, Yiming Cao, Huiyuan Huang, Zhenjun Yang, Jing Yang, Shengqi Wang.

  The messenger RNA (mRNA)-based therapy, especially mRNA vaccines, has shown its superiorities in versatile design, rapid development and scale production, since the outbreak of coronavirus disease 2019 (COVID-19). Although the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines had been approved for application, unexpected adverse events were reported to be most likely associated with the mRNA delivery systems. Thus, the development of mRNA delivery system with good efficacy and safety remains a challenge. Here, for the first time, we report that the neutral cytidinyl lipid, 2-(4-amino-2-oxopyrimidin-1-yl)-N-(2,3-dioleoyl-oxypropyl) acetamide (DNCA), and the cationic lipid, dioleoyl-3,3'-disulfanediylbis-[2-(2,6-diaminohexanamido)] propanoate (CLD), could encapsulate and deliver the COVID-19 mRNA-1096 into the cytoplasm to induce robust adaptive immune response. In the formulation, the molar ratio of DNCA/CLD to a single nucleotide of COVID-19 mRNA-1096 was about 0.9: 0.5: 1 (the N/P ratio was about 7: 1). The DNCA/CLD-mRNA-1096 lipoplexes were rationally prepared by the combination of the lipids DNCA/CLD with the aqueous mRNA solution under mild sonication to stimulate multiple interactions, including H-bonding, π-stacking and electrostatic force between the lipids and the mRNA. After intramuscular applications of the DNCA/CLD-mRNA-1096 lipoplexes, robust neutralizing antibodies and long-lived Th1-biased SARS-CoV-2-specific cell immunity were detected in the immunized mice, thus suggesting the DNCA/CLD a promising mRNA delivery system. Moreover, our study might also inspire better ideas for developing mRNA delivery systems.

Keywords:  COVID-19; Lipids; Lipoplexes; mRNA delivery

DOI:  https://doi.org/10.1016/j.jconrel.2021.10.023
Adv Drug Deliv Rev. 2021 Oct 26. pii: S0169-409X(21)00392-6. [Epub ahead of print] 113999

Nanoparticle designs for delivery of nucleic acid therapeutics as brain cancer therapies.

Johan Karlsson, Kathryn M Luly, Stephany Y Tzeng, Jordan J Green.

  Glioblastoma (GBM) is an aggressive central nervous system cancer with a dismal prognosis. The standard of care involves surgical resection followed by radiotherapy and chemotherapy, but five-year survival is only 5.6% despite these measures. Novel therapeutic approaches, such as immunotherapies, targeted therapies, and gene therapies, have been explored to attempt to extend survival for patients. Nanoparticles have been receiving increasing attention as promising vehicles for non-viral nucleic acid delivery in the context of GBM, though delivery is often limited by low blood-brain barrier permeability, particle instability, and low trafficking to target brain structures and cells. In this review, nanoparticle design considerations and new advances to overcome nucleic acid delivery challenges to treat brain cancer are summarized and discussed.

Keywords:  BBB crossing; Biomaterials; Gene delivery; Glioblastoma; Intracellular delivery; Nanoparticles; RNA delivery; Targeted delivery

DOI:  https://doi.org/10.1016/j.addr.2021.113999
Macromol Rapid Commun. 2021 Oct 29. e2100602

Transferrin Receptor Targeted Polyplexes Completely Comprised of Sequence-defined Components.

Teoman Benli-Hoppe, Şurhan Göl-Öztürk, Özgür Öztürk, Simone Berger, Ernst Wagner, Mina Yazdi.

  Human transferrin protein (Tf) modified polyplexes have already displayed encouraging potential for receptor-mediated nucleic acid delivery into tumors. The use of a blood-derived targeting protein and polydisperse macromolecular cationic subunits however presents a practical challenge for pharmaceutical grade production. Here, Tf receptor (TfR) targeted siRNA polyplexes are designed that are completely composed of synthetic, monodisperse, and sequence-defined subunits generated by solid-phase supported synthesis. An optimized cationizable lipo-oligoaminoamide (lipo-OAA) is used for siRNA core polyplex formation, and a retro-enantio peptide (reTfR) attached via a monodisperse PEG spacer via click chemistry is applied for targeting. Improved gene silencing is demonstrated in TfR-expressing KB and DU145 cells. Analogous pDNA polyplexes are successfully used for receptor-mediated gene delivery in TfR-rich K562 cells and Neuro2A cells. Six lipo-OAAs differing in their lipidic domain and redox-sensitive attachment of lipid residues are tested in order to evaluate the impact of core polyplex stability on receptor- dependent gene transfer. This article is protected by copyright. All rights reserved.

Keywords:  gene silencing; gene transfer; nanoparticle; pDNA; siRNA; transferrin receptor

DOI:  https://doi.org/10.1002/marc.202100602
Mol Ther Nucleic Acids. 2021 Dec 03. 26 594-602

Farnesylthiosalicylic acid-derivatized PEI-based nanocomplex for improved tumor vaccination.

Yuang Chen, Yixian Huang, Haozhe Huang, Zhangyi Luo, Ziqian Zhang, Runzi Sun, Zhuoya Wan, Jingjing Sun, Binfeng Lu, Song Li.

  Cancer vaccines that make use of tumor antigens represent a promising therapeutic strategy by stimulating immune responses against tumors to generate long-term anti-tumor immunity. However, vaccines have shown limited clinical efficacy due to inefficient delivery. In this study, we focus on vaccine delivery assisted by nanocomplexes for cancer immunotherapy. Nanocomplex-mediated vaccination can efficiently deliver nucleic acids encoding neoantigens to lymphoid tissues and antigen-presenting cells. Polyethylenimine (PEI) was conjugated with farnesylthiosalicylic acid (FTS) to form micelles. Subsequent interaction with nucleic acids led to formation of polymer/nucleic acid nanocomplexes of well-controlled structure. Tumor transfection via FTS-PEI was much more effective than that by PEI, other PEI derivatives, or naked DNA. Significant numbers of transfected cells were also observed in draining lymph nodes (LNs). In vivo delivery of ovalbumin (OVA; a model antigen) expression plasmid (pOVA) by FTS-PEI led to a significant growth inhibition of the OVA-expressing B16 tumor through presentation of OVA epitopes as well as other epitopes via epitope spreading. Moreover, in vivo delivery of an endogenous melanoma neoantigen tyrosinase-related protein 2 (Trp2) also led to substantial tumor growth inhibition. FTS-PEI represents a promising transfection agent for effective gene delivery to tumors and LNs to mediate effective neoantigen vaccination.

Keywords:  farnesylthiosalicylic acid; immunotherapy; nanocomplex; neoantigen; polyethylenimine; vaccination

DOI:  https://doi.org/10.1016/j.omtn.2021.09.006
J Gene Med. 2021 Oct 29. e3396

Marked Increase in Tumor Transfection with a Truncated Branched Polymer.

Songhui Xu, Jiaxi He, Zuha Imtiyaz, Atul K Agrawal, Martin C Woodle, A James Mixson.

   BACKGROUND: We previously determined that polyplexes formed by linear H2K peptides were more effective in transfecting tumors in vivo than polyplexes formed by branched H2K4b-20 peptides. Based on trypsin digest and salt displacement studies, the linear H2K polyplexes were less stable than the branched H2K4b-20 polyplexes. Because binding and release of the polymer and DNA from the H2K4b-20 polyplex may account for the ineffectiveness, we investigated whether four-branched histidine-lysine (HK) peptides with varying numbers of amino acids in their branches would be more effective in their ability to increase gene expression in tumors in vivo.
METHODS: Linear and branched peptides with multiple -KHHK- motifs were synthesized by solid-phase synthesis. The branched H2K4b-20, -18, -14, and 12 peptides had 20, 18, 14, and 12 amino acids in their branches, respectively. These peptides were examined for their ability to carry luciferase-expressing plasmids to human breast cancer xenografts in a mouse model. With gel retardation and in vivo transfection, incorporating a targeting ligand and an endosomal lysis peptide into these polyplexes was also examined. A blocking antibody was pre-injected prior to the polyplexes to determine the role of neuropilin 1 in the uptake of these polyplexes by the tumor. The size of the polyplexes was measured by dynamic light scattering RESULTS: Of the four negative surface-charge polyplexes formed by the branched carriers, the H2K4b-14 polyplex was determined to be the most effective plasmid delivery platform to tumors. The incorporation of a targeting ligand and an endosomal lysis peptide into H2K4b-14 polyplexes further enhanced their ability to transfect tumors in vivo. Furthermore, after pre-injecting tumor-bearing mice with a blocking antibody to the neuropilin-1 receptor (NRP-1), there was a marked reduction of tumor gene expression with the modified H2K4b-14 polyplexes, suggesting that NRP-1 mediated their transport into the tumor.
CONCLUSION: This study established that branched peptides intermediate in length were very efficient in delivering plasmids to tumors in vivo.

Keywords:  cancer; gene therapy; neuropilin-1 receptor; non-viral; peptide; plasmid

DOI:  https://doi.org/10.1002/jgm.3396
Biomater Sci. 2021 Oct 26. 9(21): 7104-7114

Phenylboronic acid-functionalized polyaminoglycoside as an effective CRISPR/Cas9 delivery system.

Meiyu Shao, Yu Qi, Dandan Sui, Fu-Jian Xu.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a promising approach for cancer therapy, and its application practice urgently requires a safe and effective gene carrier. In this work, we focus on the design of a phenylboronic acid (PBA)-functionalized, disulfide bonded branched polyaminoglycoside (SS-HPT-P) as a robust delivery vector of the CRISPR-Cas9 system. SS-HPT-P showed great tumor-targeting performance, reduction-responsive degradability, and gene transfection ability. The typical pCas9-surv (one CRISPR-Cas9 plasmid that targets and knocks out the survivin gene) delivery mediated by SS-HPT-P exhibited gene editing performance in the A549 cell line, confirming the feasibility of SS-HPT-P to effectively deliver the CRISPR-Cas9 system. SS-HPT-P/pCas9-surv could effectively inhibit the proliferation of tumor cells both in vitro and in vivo, suggesting the potential of PBA-functionalized nanocarriers for cancer gene therapy. The present work provides a promising approach for the treatment of malignant tumors.

DOI: https://doi.org/10.1039/d1bm00185j
EBioMedicine. 2021 Oct 20. pii: S2352-3964(21)00417-5. [Epub ahead of print]73 103624

Modulating expression of inhibitory and stimulatory immune 'checkpoints' using nanoparticulate-assisted nucleic acid delivery.

Adam A Walters, Baljevan Dhadwar, Khuloud T Al-Jamal.

  Immune checkpoints are regulatory molecules responsible for determining the magnitude and nature of the immune response. The aim of immune checkpoint targeting immunotherapy is to manipulate these interactions, engaging the immune system in treatment of cancer. Clinically, the use of monoclonal antibodies to block immunosuppressive interactions has proven itself to be a highly effective immunotherapeutic intervention. Within the literature there are numerous candidates for next generation of immune checkpoint targeting strategies. One such example is the use of nucleic acid to alter expression levels of immune checkpoint molecules, either as antisense oligo nucleotides/siRNA, to downregulate inhibitory molecules, or mRNA/DNA, to express co-stimulatory molecules. A significant component of nucleic acid delivery is its formulation within a nanoparticulate system. In this review we discuss the progress of the preclinical application of nucleic acid-based immunotherapies to target a selection of co-inhibitory/co-stimulatory molecules. Furthermore, we identify the potential and current gaps within the literature which may form the basis of future work.

Keywords:  Immune checkpoint; Nanoparticle; RNA interference; siRNA

DOI:  https://doi.org/10.1016/j.ebiom.2021.103624
Natl Sci Rev. 2021 Aug;8(8): nwaa268

Confined growth of ZIF-8 in dendritic mesoporous organosilica nanoparticles as bioregulators for enhanced mRNA delivery in vivo.

Yue Wang, Hao Song, Chao Liu, Ye Zhang, Yueqi Kong, Jie Tang, Yannan Yang, Chengzhong Yu.

  Zeolitic imidazolate framework-8 (ZIF-8) and its composites have diverse applications. However, ZIF-8-based nanocomposites are mainly used as carriers in biomolecular delivery, with the functions of metal ions and ligands rarely used to modulate the biofunctions. In this work, dendritic mesoporous organosilica nanoparticles (DMONs) with tetrasulfide bond were used to confine ZIF-8 growth partially inside mesopores as a novel nanocomposite for mRNA delivery. Each component in the resultant DMONs-ZIF-8 contributed to mRNA delivery applications, including high loading benefitting from positively charged ZIF-8 and large mesopores of DMONs, endosomal escape promoted by the imidazole ring of ZIF-8, and long-term glutathione depletion mediated by both zinc ions and tetrasulfide bond. Combined together, DMONs-ZIF-8 demonstrated enhanced mRNA translation and better transfection efficiency than commercial products and toxic polymer-modified DMONs in vitro and in vivo.

Keywords:  bioregulator; cellular delivery; mRNA transfection; mesoporous organosilica nanoparticles; metal-organic framework

DOI:  https://doi.org/10.1093/nsr/nwaa268
Int J Nanomedicine. 2021 ;16 7051-7069

Cetuximab-Modified Human Serum Albumin Nanoparticles Co-Loaded with Doxorubicin and MDR1 siRNA for the Treatment of Drug-Resistant Breast Tumors.

Xin Yang, Yifan Wang, Si Chen, Shuang Zhang, Chunying Cui.

   Background: Breast cancer is the most prevalent cancer among women. Doxorubicin (DOX) is a common chemotherapeutic drug used to treat many different cancers. However, multidrug resistance limits the treatment of breast cancer. MDR1 siRNA (siMDR1) combinatorial therapy has attracted significant attention as a breakthrough therapy for multidrug resistance in tumors. However, naked siRNA is easily degraded by enzymatic hydrolysis requiring an siRNA carrier for its protection. Human serum albumin (HSA) was selected as the carrier due to its excellent biocompatibility, non-toxicity, and non-immunogenicity. Cetuximab was used to modify the HSA nanoparticles in order to target the tumor tissues.
Methods: This study used a central composite design response surface methodology (CCD-RSM) to investigate the optimal formula for HSA NPs preparation. Cex-HSA/DOX/MDR1 siRNA (C-H/D/M) was characterized by dynamic light scattering and transmission electron microscopy. The efficacy of C-H/D/M tumor growth inhibitory activity was investigated in vitro and in vivo using confocal imaging, MTT assay, and an MCF-7/ADR tumor-bearing mice model. RT-qPCR, ELISA analysis, and flow cytometry were used to investigate the in vitro antitumor mechanisms of C-H/D/M.
Results: The diameter and PDI of the C-H/D/M were 173.57 ± 1.30 nm and 0.027 ± 0.004, respectively. C-H/D/M promoted and maintained the sustained release and the uptake of DOX significantly. After transfection, the MDR1 mRNA and P-gp expression levels were down-regulated by 44.31 ± 3.6% (P < 0.01) and 38.08 ± 2.4% (P < 0.01) in an MCF-7/ADR cell line. The fluorescent images of the treated BALB/c nude mice revealed that C-H/D/M achieved targeted delivery of siMDR1 and DOX into the tumor tissue. The in vivo tumor inhibition results demonstrated that the tumor inhibition rate of the C-H/D/M treated group was 54.05% ± 1.25%. The biosafety results indicated that C-H/D/M did not induce significant damages to the main organs in vivo.
Conclusion: C-H/D/M can be used as an ideal non-viral tumor-targeting vector to overcome MDR and enhance the antitumor effect.

Keywords:  HSA; MDR; co-delivery carrier; gene silencing; human serum albumin; multidrug resistance; small interfere RNA

DOI:  https://doi.org/10.2147/IJN.S332830
Mol Pharm. 2021 Oct 26.

Intraperitoneal siRNA Nanoparticles for Augmentation of Gemcitabine Efficacy in the Treatment of Pancreatic Cancer.

Siyuan Tang, Yu Hang, Ling Ding, Weimin Tang, Ao Yu, Chuhan Zhang, Diptesh Sil, Ying Xie, David Oupický.

  Pancreatic ductal adenocarcinoma is a deadly disease with limited treatment options due to late diagnosis and resistance to conventional chemotherapy. Among emerging therapeutic targets, the CXCR4 chemokine receptor and polo-like kinase 1 (PLK1) play critical roles in the progression, metastasis, and chemoresistance of pancreatic cancer. Here, we tested the hypothesis that combining CXCR4 inhibition by a polymeric CXCR4 antagonist PAMD-CHOL with PLK1 knockdown by siRNA will enhance the therapeutic effect of gemcitabine (GEM) in the orthotopic model of metastatic pancreatic cancer. We formulated nanoparticles with cholesterol-modified PAMD and siPLK1 and found strong synergism when combined with GEM treatment in vitro in both murine and human pancreatic cancer cell lines. The biodistribution of the nanoparticles in orthotopic pancreatic cancer models revealed strong accumulation in primary and metastatic tumors, with limited hepatic disposition. The cholesterol-containing nanoparticles showed not only increased tumor accumulation than the cholesterol-lacking control but also deeper penetration into the tumors. In a therapeutic study in vivo, the triple combination of PAMD-CHOL/siPLK1 and GEM showed superior anticancer activity when compared with single and dual combination controls. In conclusion, PAMD-CHOL/siPLK1 nanoparticles synergistically enhance anticancer activity of GEM in pancreatic cancer and represent a promising addition to the treatment arsenal.

Keywords:  CXCR4 antagonism; PLK1; gemcitabine; intraperitoneal administration; pancreatic cancer; siRNA delivery

DOI:  https://doi.org/10.1021/acs.molpharmaceut.1c00653