bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023–02–19
ten papers selected by
the Merkel lab, Ludwig-Maximilians University



  1. Chembiochem. 2023 Feb 13. e202200801
      Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, the low stability, immunogenicity, and impermeability of mRNA to the cell membrane greatly limit their potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA-based vaccines to prevent COVID-19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue-targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing using LNPs. Finally, we provide a perspective on the future of LNP-based mRNA delivery for CRISPR/Cas9 genome editing and treatment of genetic disorders.
    Keywords:  CRISPR/Cas9; cell-selective; genome editing; lipid nanoparticles; mRNA delivery
    DOI:  https://doi.org/10.1002/cbic.202200801
  2. ACS Appl Mater Interfaces. 2023 Feb 17.
      Genome editing mediated by the CRISPR-Cas system holds great promise for the treatment of genetic diseases. However, safe and efficient in vivo delivery of CRISPR genome editing machinery remains a challenge. Here, we report a lipopeptide-based nanoparticle (LNP) that can efficiently deliver the CRISPR Cas9/sgRNA ribonucleoprotein (RNP) and enable efficient genome editing both in vitro and in vivo. An artificial lipopeptide, GD-LP, was constructed by linking a hydrophilic guanidinium-rich head to an oleic acid-based hydrophobic tail via a disulfide bond. LNP formed by the self-assembly of GD-LP can easily form a complex with RNP with a loading content of up to 20 wt %. The resulting RNP-LNP nanocomplex led to 72.6% gene editing efficiency in GFP-HEK cells with negligible cytotoxicity. The LNP also showed significantly higher transfection efficiencies than Lipofectamine 2000 for the delivery of mRNA in NIH 3T3 and RAW 264.7 and the delivery of plasmid DNA in B78 cells. In vivo studies showed that intramuscular injection of the RNP-LNP nanocomplex in Ai14 mice induced efficient gene editing in muscular tissues. Moreover, the delivery of Cas9 RNP and donor DNA by LNP (i.e., RNP/ssODN-LNP nanocomplex) restored dystrophin expression, reduced skeletal muscle fibrosis, and significantly improved muscle strength in a Duchenne muscular dystrophy (DMD) mouse model.
    Keywords:  CRISPR; Cas9/sgRNA ribonucleoprotein (RNP); delivery of genome editor; genome editing; guanidinium; lipopeptide-based nanoparticle; muscle editing
    DOI:  https://doi.org/10.1021/acsami.2c21683
  3. J Control Release. 2023 Feb 10. pii: S0168-3659(23)00102-5. [Epub ahead of print]355 406-416
      The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system is a technology that is used to perform site-specific gene disruption, repair, and the modification of genomic DNA via DNA repair mechanisms, and is expected to be a fundamental therapeutic strategy for the treatment of infectious diseases and genetic disorders. For clinical applications, the non-viral vector-based delivery of the CRISPR/Cas ribonucleoprotein (RNP) is important, but the poor efficiency of delivery and the lack of a practical method for its manufacture remains as an issue. We report herein on the development of a lipid nanoparticle (LNP)-based Cas RNP delivery system based on optimally designed single stranded oligonucleotides (ssODNs) that allow efficient in vivo genome editing. The formation of sequence-specific RNP-ssODN complexes was found to be important for the functional delivery of RNP. Furthermore, the melting temperature (Tm) between sgRNA and ssODN had a significant effect on in vivo gene knockout efficiency. An ssODN with a high Tm resulted in limited knockout (KO) activity while that at near room temperature showed the highest KO activity, indicating the importance of the cytosolic release of RNPs. Two consecutive intravenous injections of the Tm optimized formulation achieved approximately 70% and 80% transthyretin KO at the DNA and protein level, respectively, without any obvious toxicity. These findings represent a significant contribution to the development of safe in vivo CRISPR/Cas RNP delivery technology and its practical application in genome editing therapies.
    Keywords:  Genome editing; Lipid nanoparticles; Liver; Melting temperature; Ribonucleoprotein; Single stranded oligonucleotides
    DOI:  https://doi.org/10.1016/j.jconrel.2023.02.008
  4. J Mater Chem B. 2023 Feb 16.
      Messenger RNA (mRNA) has emerged as a new and efficient agent for the treatment of various diseases. The success of lipid nanoparticle-mRNA against the novel coronavirus (SARS-CoV-2) pneumonia epidemic has proved the clinical potential of nanoparticle-mRNA formulations. However, the deficiency in the effective biological distribution, high transfection efficiency and good biosafety are still the major challenges in clinical translation of nanomedicine for mRNA delivery. To date, a variety of promising nanoparticles have been constructed and then gradually optimized to facilitate the effective biodistribution of carriers and efficient mRNA delivery. In this review, we describe the design of nanoparticles with an emphasis on lipid nanoparticles, and discuss the manipulation strategies for nanoparticle-biology (nano-bio) interactions for mRNA delivery to overcome the biological barriers and improve the delivery efficiency, because the specific nano-bio interaction of nanoparticles usually remoulds the biomedical and physiological properties of the nanoparticles especially the biodistribution, mechanism of cellular internalization and immune response. Finally, we give a perspective for the future applications of this promising technology. We believe that the regulation of nano-bio interactions would be a significant breakthrough to improve the mRNA delivery efficiency and cross biological barriers. This review may provide a new direction for the design of nanoparticle-mediated mRNA delivery systems.
    DOI:  https://doi.org/10.1039/d2tb02455a
  5. Methods Mol Biol. 2023 ;2622 127-137
      To provide long circulating nanoparticles able to carry a gene to tumor cells, we have designed anionic pegylated lipoplexes which are pH sensitive. The reduction of positive charges in nucleic acid carriers allows reducing the elimination rate, increasing circulation time in the blood, leading to improved tumor accumulation of lipid nanoparticles. Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. The neutralization of the particle surface charge as a function of the pH was monitored by dynamic light scattering in order to determine the ratio between anionic and cationic lipids that would give pH-sensitive complexes. This ratio has been optimized to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes was checked by DNA accessibility to Picogreen. The transfection efficiency and pH-sensitive property of these formulations were shown in vitro using bafilomycin, a vacuolar H+-ATPase inhibitor.
    Keywords:  Anionic cholesterol; Anionic lipoplexes; Gene delivery to tumor; Pegylated lipoplexes; pH-sensitive lipoplexes
    DOI:  https://doi.org/10.1007/978-1-0716-2954-3_11
  6. Nanoscale Res Lett. 2023 Feb 17. 18(1): 18
      Recent years have witnessed an increased interest in the development of nanoparticles (NPs) owing to their potential use in a wide variety of biomedical applications, including drug delivery, imaging agents, gene therapy, and vaccines, where recently, lipid nanoparticle mRNA-based vaccines were developed to prevent SARS-CoV-2 causing COVID-19. NPs typically fall into two broad categories: organic and inorganic. Organic NPs mainly include lipid-based and polymer-based nanoparticles, such as liposomes, solid lipid nanoparticles, polymersomes, dendrimers, and polymer micelles. Gold and silver NPs, iron oxide NPs, quantum dots, and carbon and silica-based nanomaterials make up the bulk of the inorganic NPs. These NPs are prepared using a variety of top-down and bottom-up approaches. Microfluidics provide an attractive synthesis alternative and is advantageous compared to the conventional bulk methods. The microfluidic mixing-based production methods offer better control in achieving the desired size, morphology, shape, size distribution, and surface properties of the synthesized NPs. The technology also exhibits excellent process repeatability, fast handling, less sample usage, and yields greater encapsulation efficiencies. In this article, we provide a comprehensive review of the microfluidic-based passive and active mixing techniques for NP synthesis, and their latest developments. Additionally, a summary of microfluidic devices used for NP production is presented. Nonetheless, despite significant advancements in the experimental procedures, complete details of a nanoparticle-based system cannot be deduced from the experiments alone, and thus, multiscale computer simulations are utilized to perform systematic investigations. The work also details the most common multiscale simulation methods and their advancements in unveiling critical mechanisms involved in nanoparticle synthesis and the interaction of nanoparticles with other entities, especially in biomedical and therapeutic systems. Finally, an analysis is provided on the challenges in microfluidics related to nanoparticle synthesis and applications, and the future perspectives, such as large-scale NP synthesis, and hybrid formulations and devices.
    DOI:  https://doi.org/10.1186/s11671-023-03792-x
  7. Int J Biol Macromol. 2023 Feb 11. pii: S0141-8130(23)00556-1. [Epub ahead of print] 123663
      Drug resistance in cancer chemotherapy is a major confounding factor affecting the effectiveness of chemotherapeutic agents, thereby leading to poor clinical outcomes. Most chemotherapeutic drugs can induce protective autophagy and increase the resistance of tumors to chemotherapeutic drugs and reduce effective drug delivery to tumor cells. In this study, a tri-drug nanocomposite (NP) delivery system was devised using carboxymethyl β-dextran (CMD) and protamine sulfate (PS), two natural materials with good bio-compatibility. They were designed to carry the chemotherapeutic drug docetaxel (DTX), the autophagy inhibitor chloroquine (CQ), and Atg5 siRNA to cancer cells. The CQ + DTX + Atg5 siRNA NPs was driven by electrostatic interaction and self-assembly methods. The breast cancer cell line MDA-MB-231 was used for both cell culture and establishing mouse xenograft model. Our findings demonstrated that CQ and Atg5 siRNA encapsulated in NPs could enhance the sensitivity of tumor cells to DTX. The NPs exhibited remarkable considerable therapeutic effects for treating triple-negative breast cancer (TNBC) and good biosafety. Therefore, we established a novel multifunctional nanoplatform based on CMD and PS that enhances chemotherapeutic drug sensitivity through an autophagy inhibition strategy, providing new opportunities to overcome conventional drug resistance and enhance therapeutic efficiency against TNBC.
    Keywords:  Autophagy inhibition; Carboxymethyl β-dextran,Protamine; Combination antitumor therapy; Nanocomposites; Triple negative breast cancer
    DOI:  https://doi.org/10.1016/j.ijbiomac.2023.123663
  8. bioRxiv. 2023 Feb 03. pii: 2023.02.01.526706. [Epub ahead of print]
      Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in a model of viral pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus developing worse lung pathology and more consistently losing body weight relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. We conclude that anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.
    DOI:  https://doi.org/10.1101/2023.02.01.526706
  9. Int J Pharm. 2023 Feb 11. pii: S0378-5173(23)00137-0. [Epub ahead of print]635 122717
      In this work, the effect of cryoprotectant type and concentration and freeze-drying process parameters were evaluated to determine an optimal freeze-drying process for celecoxib-loaded solid lipid nanoparticles. Different cryoprotectants were tested at different weight ratios (cryoprotectant:lipid). Trehalose, maltose, and sucrose at a 1:1 wt ratio were selected for further use in optimizing the freeze-drying process through experimental designs to accurately define the freezing, primary, and secondary drying conditions of the freeze-drying process. The optimal freeze-dried solid lipid nanoparticles were subjected to a 6-month stability study at either 4 °C or 25 °C/60% RH, resulting in significant growth when the nanoparticles were stored at 25 °C/60% RH. The best results were obtained with trehalose as a cryoprotectant and storage at 4 °C. Furthermore, the in vitro release data showed a significantly different release profile before and after optimization of the freeze-drying process, suggesting that the optimization of the freeze-drying process affected the quality of the freeze-dried cake. In conclusion, a successful lyophilization process was obtained due to rational cooperation between a good formulation and optimal conditions in the freezing and drying steps. This yielded an acceptable non-collapsed freeze-dried cake with good redispersibility, minimal changes in physicochemical properties, and long-term stability at 4 °C.
    Keywords:  Celecoxib; Design of experiments; Freeze-drying optimization; Solid lipid nanoparticles
    DOI:  https://doi.org/10.1016/j.ijpharm.2023.122717