bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2022–11–27
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. A cancer cell membrane coated nanoparticles-based gene delivery system for enhancing cancer therapy.
  2. Surface Design Options in Polymer- and Lipid-Based siRNA Nanoparticles Using Antibodies.
  3. Poly(aspartic acid)-Based Polymeric Nanoparticle for Local and Systemic mRNA Delivery.
  4. Substituting racemic ionizable lipids with stereopure ionizable lipids can increase mRNA delivery.
  5. Nanoemulsion Co-Loaded with XIAP siRNA and Gambogic Acid for Inhalation Therapy of Lung Cancer.
  6. Structural and biochemical characteristics of mRNA nanoparticles determine anti-SARS-CoV-2 humoral and cellular immune responses.
  7. SV-AUC as a stability-indicating method for the characterization of mRNA-LNPs.
  8. Electrospun fiber-mediated delivery of neurotrophin-3 mRNA for neural tissue engineering applications.
  9. Anticipating the Next Chess Move: Blocking SARS-CoV-2 Replication and Simultaneously Disarming Viral Escape Mechanisms.
  10. Rational Design and In Vivo Characterization of mRNA-Encoded Broadly Neutralizing Antibody Combinations against HIV-1.
  11. Processing of Lipid Nanodispersions into Solid Powders by Spray Drying.
  12. Nanostructured Lipid Carriers for Nose to Brain Delivery Targeting CNS: Diversified Role of Liquid Lipids for Synergistic Action.
  1. Int J Pharm. 2022 Nov 17. pii: S0378-5173(22)00970-X. [Epub ahead of print]629 122415
    A cancer cell membrane coated nanoparticles-based gene delivery system for enhancing cancer therapy.
    Zhou Fang, Min Zhang, Rui Kang, Mingxiao Cui, Mengdi Song, Kehai Liu.
      Gene therapy is a superior therapeutic means in cancer therapy. However, the instability of nucleic acid and the lack of suitable delivery carrier greatly restricts its further development and application. Herein, we coupled low molecular weight polyethyleneimine (LMW PEI) through disulfide bonds, then modified it with manganese dioxide (MnO2) nanosheets and nuclear localization signal peptide (NLS), as a p53 gene carrier, and finally coated it with B16F10 cell membrane to construct a novel gene-carrier system CM@MnO2-PEI-NLS-ss/p53 (M@MPNs/p53). Tumor cell membrane coating endows nanoparticles with homotypic targeting and immune escape capabilities, disulfide-crosslinked LMW-PEI has high transfection efficiency and low toxicity, and NLS peptides enhance nuclear delivery and improve p53 gene delivery efficiency; meanwhile, MnO2 nanosheets oxidize high intracellular concentration of glutathione (GSH), sensitizing p53 gene-mediated antitumor therapy. The results showed that the novel biofilm-camouflaged M@MPNs/p53 nanoparticles had a highly specific targeting effect on homologous cancer cells and could effectively inhibit tumor growth in vitro and in vivo. Besides, MnO2 loading improved p53-mediated tumor regression. This novel gene delivery platform is of great significance in improving gene delivery efficiency and enhancing anti-tumor therapy.
    Keywords:  Cancer cell membrane; Gene therapy; Homotypic targeting; Manganese dioxide nanosheets; Polyethyleneimine
    DOI:  https://doi.org/10.1016/j.ijpharm.2022.122415
  2. Int J Mol Sci. 2022 Nov 11. pii: 13929. [Epub ahead of print]23(22):
    Surface Design Options in Polymer- and Lipid-Based siRNA Nanoparticles Using Antibodies.
    Michael Gabel, Annkathrin Knauss, Dagmar Fischer, Markus F Neurath, Benno Weigmann.
      The mechanism of RNA interference (RNAi) could represent a breakthrough in the therapy of all diseases that arise from a gene defect or require the inhibition of a specific gene expression. In particular, small interfering RNA (siRNA) offers an attractive opportunity to achieve a new milestone in the therapy of human diseases. The limitations of siRNA, such as poor stability, inefficient cell uptake, and undesired immune activation, as well as the inability to specifically reach the target tissue in the body, can be overcome by further developments in the field of nanoparticulate drug delivery. Therefore, types of surface modified siRNA nanoparticles are presented and illustrate how a more efficient and safer distribution of siRNA at the target site is possible by modifying the surface properties of nanoparticles with antibodies. However, the development of such efficient and safe delivery strategies is currently still a major challenge. In consideration of that, this review article aims to demonstrate the function and targeted delivery of siRNA nanoparticles, focusing on the surface modification via antibodies, various lipid- and polymer-components, and the therapeutic effects of these delivery systems.
    Keywords:  antibody; gene delivery; lipid nanoparticle; polymer nanoparticle; siRNA; surface-functionalized nanoparticles; targeted drug delivery
    DOI:  https://doi.org/10.3390/ijms232213929
  3. Mol Pharm. 2022 Nov 21.
    Poly(aspartic acid)-Based Polymeric Nanoparticle for Local and Systemic mRNA Delivery.
    Youngrong Park, Abraham S Moses, Ananiya A Demessie, Prem Singh, Hyelim Lee, Tetiana Korzun, Olena R Taratula, Adam W G Alani, Oleh Taratula.
      Recently, therapeutics based on mRNA (mRNA) have attracted significant interest for vaccines, cancer immunotherapy, and gene editing. However, the lack of biocompatible vehicles capable of delivering mRNA to the target tissue and efficiently expressing the encoded proteins impedes the development of mRNA-based therapies for a variety of diseases. Herein, we report mRNA-loaded polymeric nanoparticles based on diethylenetriamine-substituted poly(aspartic acid) that induce protein expression in the lungs and muscles following intravenous and intramuscular injections, respectively. Animal studies revealed that the amount of polyethylene glycol (PEG) on the nanoparticle surface affects the translation of the delivered mRNA into the encoded protein in the target tissue. After systemic administration, only mRNA-loaded nanoparticles modified with PEG at a molar ratio of 1:1 (PEG/polymer) induce protein expression in the lungs. In contrast, protein expression was detected only following intramuscular injection of mRNA-loaded nanoparticles with a PEG/polymer ratio of 10:1. These findings suggest that the PEG density on the surface of poly(aspartic acid)-based nanoparticles should be optimized for different delivery routes depending on the purpose of the mRNA treatment.
    Keywords:  PEGylation; gene therapy; mRNA delivery; polymeric nanoparticle; systemic delivery
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.2c00738
  4. J Control Release. 2022 Nov 21. pii: S0168-3659(22)00782-9. [Epub ahead of print]
    Substituting racemic ionizable lipids with stereopure ionizable lipids can increase mRNA delivery.
    Alejandro J Da Silva Sanchez, Kun Zhao, Sebastian G Huayamares, Marine Z C Hatit, Melissa P Lokugamage, David Loughrey, Curtis Dobrowolski, Shuaishuai Wang, Hyejin Kim, Kalina Paunovska, Yanina Kuzminich, James E Dahlman.
      Lipid nanoparticles (LNPs) have delivered siRNA and mRNA drugs in humans, underscoring the potential impact of improving the therapeutic window of next-generation LNPs. To increase the LNP therapeutic window, we applied lessons from small-molecule chemistry to ionizable lipid design. Specifically, given that stereochemistry often influences small-molecule safety and pharmacokinetics, we hypothesized that the stereochemistry of lipids within an LNP would influence mRNA delivery. We tested this hypothesis in vivo using 128 novel LNPs that included stereopure derivatives of C12-200, an ionizable lipid that when formulated into LNPs delivers RNA in mice and non-human primates but is not used clinically due to its poor tolerability. We found that a novel C12-200-S LNP delivered up to 2.8-fold and 6.1-fold more mRNA in vivo than its racemic and C12-200-R controls, respectively. To identify the potential causes leading to increased delivery, we quantified LNP biophysical traits and concluded that these did not change with stereochemistry. Instead, we found that stereopure LNPs were better tolerated than racemic LNPs in vivo. These data suggest that LNP-mediated mRNA delivery can be improved by designing LNPs to include stereopure ionizable lipids.
    Keywords:  Lipid nanoparticles; Stereochemistry; mRNA
    DOI:  https://doi.org/10.1016/j.jconrel.2022.11.037
  5. Int J Mol Sci. 2022 Nov 18. pii: 14294. [Epub ahead of print]23(22):
    Nanoemulsion Co-Loaded with XIAP siRNA and Gambogic Acid for Inhalation Therapy of Lung Cancer.
    Minhao Xu, Lanfang Zhang, Yue Guo, Lu Bai, Yi Luo, Ben Wang, Meiyan Kuang, Xingyou Liu, Meng Sun, Chenhui Wang, Jing Xie.
      Lung cancer is a leading cause of cancer mortality worldwide, with a 5-year survival rate of less than 20%. Gambogic acid (GA) is a naturally occurring and potent anticancer agent that destroys tumor cells through multiple mechanisms. According to the literature, one of the most potent inhibitors of caspases and apoptosis currently known is the X-linked Inhibitor of Apoptosis Protein (XIAP). It is highly expressed in various malignancies but has little or no expression in normal cells, making it an attractive target for cancer treatment. Here we report the development of a chitosan (CS)-based cationic nanoemulsion-based pulmonary delivery (p.d.) system for the co-delivery of antineoplastic drugs (GA) and anti-XIAP small interfering RNA (siRNA). The results showed that the chitosan-modified cationic nanoemulsions could effectively encapsulate gambogic acid as well as protect siRNA against degradation. The apoptosis analysis confirmed that the cationic nanoemulsions could induce more apoptosis in the A549 cell line. In addition, most drugs and siRNAs have a long residence time in the lungs through pulmonary delivery and show greater therapeutic effects compared to systemic administration. In summary, this work demonstrates the applicability of cationic nanoemulsions for combined cancer therapy and as a promising approach for the treatment of lung cancer.
    Keywords:  RNAi; co-delivery; lung cancer; nanoemulsions; pulmonary delivery
    DOI:  https://doi.org/10.3390/ijms232214294
  6. Sci Adv. 2022 Nov 25. 8(47): eabo1827
    Structural and biochemical characteristics of mRNA nanoparticles determine anti-SARS-CoV-2 humoral and cellular immune responses.
    Yingying Shi, Jiaxin Huang, Yu Liu, Jing Liu, Xuemeng Guo, Jianhua Li, Liming Gong, Xin Zhou, Guofeng Cheng, Yunqing Qiu, Jian You, Yan Lou.
      The coronavirus disease 2019 (COVID-19) pandemic underlines the urgent need for effective mRNA vaccines. However, current understanding of the immunological outcomes of mRNA vaccines formulated under different nanoplatforms is insufficient. Here, severe acute respiratory syndrome coronavirus 2 receptor binding domain mRNA delivered via lipid nanoparticle (LNP), cationic nanoemulsion (CNE), and cationic liposome (Lipo) was constructed. Results demonstrated that the structural and biochemical characteristics of nanoparticles shaped their tissue dissemination, cellular uptake, and intracellular trafficking, which eventually determined the activation of antiviral humoral and cellular immunity. Specifically, LNP was mainly internalized by myocyte and subsequently circumvented lysosome degradation, giving rise to humoral-biased immune responses. Meanwhile, CNE and Lipo induced cellular-preferred immunity, which was respectively attributed to the better lysosomal escape in dendritic cells and the superior biodistribution in secondary lymphoid organs. Overall, this study may guide the design and clinical use of mRNA vaccines against COVID-19.
    DOI:  https://doi.org/10.1126/sciadv.abo1827
  7. Eur J Pharm Biopharm. 2022 Nov 18. pii: S0939-6411(22)00273-9. [Epub ahead of print]
    SV-AUC as a stability-indicating method for the characterization of mRNA-LNPs.
    Anian Thaller, Lukas Schmauder, Wolfgang Frieß, Gerhard Winter, Tim Menzen, Andrea Hawe, Klaus Richter.
      During the SARS-CoV2 pandemic mRNA vaccines in the form of lipid nanoparticles (LNPs) containing the mRNA, have set the stage for a new area of vaccines. Analytical methods to quantify changes in size and structure of LNPs are crucial, as changes in these parameters could have implications for potency. We investigated the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) as quantitative stability-indicating method to detect structural changes of mRNA-LNP vaccines upon relevant stress factors (freeze/thaw, heat and mechanical stress), in comparison to qualitative dynamic light scattering (DLS) analysis. DLS was capable to qualitatively determine size and homogeneity of mRNA-LNPs with sufficient precision. Stress factors, in particular freeze/thaw and mechanical stress, led to increased particle size and content of larger species in DLS and SV-AUC. Changes upon heat stress at 50 °C were only detected as increased flotation rates by SV-AUC. In addition, SV-AUC was able to observe changes in particle density, which cannot be detected by DLS. In conclusion, SV-AUC can be used as a highly valuable quantitative stability-indicating method for characterization of LNPs.
    Keywords:  Analytical Ultracentrifugation; Dynamic Light Scattering; Flotation; Lipid Nanoparticle; Nanoparticle Density; Particle Size Distribution; Vaccines
    DOI:  https://doi.org/10.1016/j.ejpb.2022.11.014
  8. Acta Biomater. 2022 Nov 21. pii: S1742-7061(22)00752-8. [Epub ahead of print]
    Electrospun fiber-mediated delivery of neurotrophin-3 mRNA for neural tissue engineering applications.
    Devan L Puhl, Jessica L Funnell, Tanner D Fink, Anuj Swaminathan, Martin Oudega, R Helen Zha, Ryan J Gilbert.
      Aligned electrospun fibers provide topographical cues and local therapeutic delivery to facilitate robust peripheral nerve regeneration. mRNA delivery enables transient expression of desired proteins that promote axonal regeneration. However, no prior work delivers mRNA from electrospun fibers for peripheral nerve regeneration applications. Here, we developed the first aligned electrospun fibers to deliver pseudouridine-modified (Ψ) neurotrophin-3 (NT-3) mRNA (ΨNT-3mRNA) to primary Schwann cells and assessed NT-3 secretion and bioactivity. We first electrospun aligned poly(L-lactic acid) (PLLA) fibers and coated them with the anionic substrates dextran sulfate sodium salt (DSS) or poly(3,4-dihydroxy-L-phenylalanine) (pDOPA). Cationic lipoplexes containing ΨNT-3mRNA complexed to JetMESSENGER® were then immobilized to the fibers, resulting in detectable ΨNT-3mRNA release for 28 days from all fiber groups investigated (PLLA+mRNA, 0.5DSS4h+mRNA, and 2pDOPA4h+mRNA). The 2pDOPA4h+mRNA group significantly increased Schwann cell secretion of NT-3 for 21 days compared to control PLLA fibers (p < 0.001-0.05) and, on average, increased Schwann cell secretion of NT-3 by ≥ 2-fold compared to bolus mRNA delivery from the 1µgBolus+mRNA and 3µgBolus+mRNA groups. The 2pDOPA4h+mRNA fibers supported Schwann cell secretion of NT-3 at levels that significantly increased dorsal root ganglia (DRG) neurite extension by 44% (p < 0.0001) and neurite area by 64% (p < 0.001) compared to control PLLA fibers. The data show that the 2pDOPA4h+mRNA fibers enhance the ability of Schwann cells to promote neurite growth from DRG, demonstrating this platform's potential capability to improve peripheral nerve regeneration. STATEMENT OF SIGNIFICANCE: Aligned electrospun fibers enhance axonal regeneration by providing structural support and guidance cues, but further therapeutic stimulation is necessary to improve functional outcomes. mRNA delivery enables the transient expression of therapeutic proteins, yet achieving local, sustained delivery remains challenging. Previous work shows that genetic material delivery from electrospun fibers improves regeneration; however, mRNA delivery has not been explored. Here, we examine mRNA delivery from aligned electrospun fibers to enhance neurite outgrowth. We show that immobilization of NT-3mRNA/JetMESSENGER® lipoplexes to aligned electrospun fibers functionalized with pDOPA enables local, sustained NT-3mRNA delivery to Schwann cells, increasing Schwann cell secretion of NT-3 and enhancing DRG neurite outgrowth. This study displays the potential benefits of electrospun fiber-mediated mRNA delivery platforms for neural tissue engineering.
    Keywords:  Biomaterials; Electrospinning; Gene delivery; Nerve regeneration; Neurotrophins; mRNA therapeutics
    DOI:  https://doi.org/10.1016/j.actbio.2022.11.025
  9. Genes (Basel). 2022 Nov 18. pii: 2147. [Epub ahead of print]13(11):
    Anticipating the Next Chess Move: Blocking SARS-CoV-2 Replication and Simultaneously Disarming Viral Escape Mechanisms.
    Samir Mansour Moraes Casseb, André Salim Khayat, Jorge Estefano Santana de Souza, Edivaldo Herculano Correa de Oliveira, Sidney Emanuel Batista Dos Santos, Pedro Fernando da Costa Vasconcelos, Paulo Pimentel de Assumpção.
      The COVID-19 pandemic initiated a race to determine the best measures to control the disease and to save as many people as possible. Efforts to implement social distancing, the use of masks, and massive vaccination programs turned out to be essential in reducing the devastating effects of the pandemic. Nevertheless, the high mutation rates of SARS-CoV-2 challenge the vaccination strategy and maintain the threat of new outbreaks due to the risk of infection surges and even lethal variations able to resist the effects of vaccines and upset the balance. Most of the new therapies tested against SARS-CoV-2 came from already available formulations developed to treat other diseases, so they were not specifically developed for SARS-CoV-2. In parallel, the knowledge produced regarding the molecular mechanisms involved in this disease was vast due to massive efforts worldwide. Taking advantage of such a vast molecular understanding of virus genomes and disease mechanisms, a targeted molecular therapy based on siRNA specifically developed to reach exclusive SARS-CoV-2 genomic sequences was tested in a non-transformed human cell model. Since coronavirus can escape from siRNA by producing siRNA inhibitors, a complex strategy to simultaneously strike both the viral infectious mechanism and the capability of evading siRNA therapy was developed. The combined administration of the chosen produced siRNA proved to be highly effective in successfully reducing viral load and keeping virus replication under control, even after many days of treatment, unlike the combinations of siRNAs lacking this anti-anti-siRNA capability. Additionally, the developed therapy did not harm the normal cells, which was demonstrated because, instead of testing the siRNA in nonhuman cells or in transformed human cells, a non-transformed human thyroid cell was specifically chosen for the experiment. The proposed siRNA combination could reduce the viral load and allow the cellular recovery, presenting a potential innovation for consideration as an additional strategy to counter or cope COVID-19.
    Keywords:  COVID-19; SARS-CoV-2; siRNA; treatment
    DOI:  https://doi.org/10.3390/genes13112147
  10. Antibodies (Basel). 2022 Oct 24. pii: 67. [Epub ahead of print]11(4):
    Rational Design and In Vivo Characterization of mRNA-Encoded Broadly Neutralizing Antibody Combinations against HIV-1.
    Elisabeth Narayanan, Samantha Falcone, Sayda M Elbashir, Husain Attarwala, Kimberly Hassett, Michael S Seaman, Andrea Carfi, Sunny Himansu.
      Monoclonal antibodies have been used successfully as recombinant protein therapy; however, for HIV, multiple broadly neutralizing antibodies may be necessary. We used the mRNA-LNP platform for in vivo co-expression of 3 broadly neutralizing antibodies, PGDM1400, PGT121, and N6, directed against the HIV-1 envelope protein. mRNA-encoded HIV-1 antibodies were engineered as single-chain Fc (scFv-Fc) to overcome heavy- and light-chain mismatch. In vitro neutralization breadth and potency of the constructs were compared to their parental IgG form. We assessed the ability of these scFv-Fcs to be expressed individually and in combination in vivo, and neutralization and pharmacokinetics were compared to the corresponding full-length IgGs. Single-chain PGDM1400 and PGT121 exhibited neutralization potency comparable to parental IgG, achieving peak systemic concentrations ≥ 30.81 μg/mL in mice; full-length N6 IgG achieved a peak concentration of 974 μg/mL, but did not tolerate single-chain conversion. The mRNA combination encoding full-length N6 IgG and single-chain PGDM1400 and PGT121 was efficiently expressed in mice, achieving high systemic concentration and desired neutralization potency. Analysis of mice sera demonstrated each antibody contributed towards neutralization of multiple HIV-1 pseudoviruses. Together, these data show that the mRNA-LNP platform provides a promising approach for antibody-based HIV treatment and is well-suited for development of combination therapeutics.
    Keywords:  HIV; antibodies; broadly neutralizing antibodies; mRNA
    DOI:  https://doi.org/10.3390/antib11040067
  11. Pharmaceutics. 2022 Nov 15. pii: 2464. [Epub ahead of print]14(11):
    Processing of Lipid Nanodispersions into Solid Powders by Spray Drying.
    Denise Steiner, Leonie V Schumann, Heike Bunjes.
      Spray drying is a promising technology for drying lipid nanodispersions. These formulations can serve as carrier systems for poorly water-soluble active pharmaceutical ingredients (APIs) that are loaded into the lipid matrix to improve their bioavailability. Once the API-loaded nanocarriers have been further processed into solid dosage forms, they could be administered orally, which is usually preferred by patients. Various solid lipids as well as oils were used in this study to prepare lipid nanodispersions, and it was shown that their nanoparticulate properties could be maintained when lactose in combination with SDS was used as matrix material in the spray-drying process. In addition, for lipid nanoemulsions loaded with fenofibrate, a good redispersibility with particle sizes below 300 nm at a lipid content of 26.8 wt.% in the powders was observed. More detailed investigations on the influence of the drying temperature yielded good results when the inlet temperature of the drying air was set at 110 °C or above, enabling the lactose to form an amorphous matrix around the embedded lipid particles. A tristearin suspension was developed as a probe to measure the temperature exposure of the lipid particles during the drying process. The results with this approach indicate that the actual temperature the particles were exposed to during the drying process could be higher than the outlet temperature.
    Keywords:  formulation technology; lipid nanoemulsions; nanotechnology; poorly water-soluble drug; solid lipid nanoparticles
    DOI:  https://doi.org/10.3390/pharmaceutics14112464
  12. Adv Pharm Bull. 2022 Aug;12(4): 763-771
    Nanostructured Lipid Carriers for Nose to Brain Delivery Targeting CNS: Diversified Role of Liquid Lipids for Synergistic Action.
    Avinash Ramrao Tekade, Pradeep Sadanand Mittha, Charushila Sopan Pisal.
      Neurological disorders such as Alzheimer's disease, Parkinson's disease, dementia, epilepsy, depression, migraine etc. are affecting more and more elderly people's day by day. Conventional route of administration to treat these diseases has to face a major hindrance that is blood brain and blood-cerebrospinal fluid (CSF) barrier to achieve desired concentration of drug at the site of action for therapeutic effect. Hence, intranasal route of delivery is considered as promising and alternative route to achieve desired goals. In last four decades, brain targeting strategies are widely studied and considered having great potential by researchers; especially intranasal delivery owing to its benefits. Various nano formulations such as nanoemulsions, nanosuspensions, hydrogels, in situ gels, dendrimers and lipidic formulations are studied widely. Lipid nano formulations especially second generation nanostructured lipid carriers offer greater advantages in terms of stability, fabrication techniques, scalability, drug loading and drug targeting. Nanostructured lipid carrier (NLCs) constitute of two major components viz solid lipid and liquid lipid in a specific ratio. In this review, authors have discussed about the possible synergistic actions of oils/liquid lipids with synthetic drugs resulting into great therapeutic benefits.
    Keywords:  Blood brain barrier; CNS targeting; Intranasal route; Liquid lipid; NLC; Synergistic action
    DOI:  https://doi.org/10.34172/apb.2022.078
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