bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023‒07‒23
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University

  1. ACS Appl Mater Interfaces. 2023 Jul 21.
      Gene therapy has emerged as a significant advancement in medicine in recent years. However, the development of effective gene delivery vectors, particularly polymer vectors, remains a significant challenge. Limited understanding of the internal structure of polymer vectors has hindered efforts to enhance their efficiency. This work focuses on investigating the impact of polymer structure on gene delivery, using the well-known polymeric vector poly(β-amino ester) (PAE) as a case study. For the first time, we revealed the distinct characteristics of individual polymer components and their synergistic effects-the appropriate combination of different components within a polymer (high MW and low MW components) on gene delivery. Additionally, artificial intelligence (AI) analysis was employed to decipher the relationship between the polymer component distribution (PCD) and gene transfection performance. Guided by this analysis, a series of highly efficient polymer vectors that outperform current commercial reagents such as jetPEI and Lipo3000 were developed, among which the transfection efficiency of the PAE-B1-based polyplex was approximately 1.5 times that of Lipo3000 and 2 times that of jetPEI in U251 cells.
    Keywords:  artificial intelligence; gene delivery vectors; machine learning; pDNA delivery; poly(β-amino ester); polymer component distribution (PCD)
  2. ACS Nano. 2023 Jul 17.
      Delivery of mRNA-based therapeutics to the perinatal brain holds great potential in treating congenital brain diseases. However, nonviral delivery platforms that facilitate nucleic acid delivery in this environment have yet to be rigorously studied. Here, we screen a diverse library of ionizable lipid nanoparticles (LNPs) via intracerebroventricular (ICV) injection in both fetal and neonatal mice and identify an LNP formulation with greater functional mRNA delivery in the perinatal brain than an FDA-approved industry standard LNP. Following in vitro optimization of the top-performing LNP (C3 LNP) for codelivery of an adenine base editing platform, we improve the biochemical phenotype of a lysosomal storage disease in the neonatal mouse brain, exhibit proof-of-principle mRNA brain transfection in vivo in a fetal nonhuman primate model, and demonstrate the translational potential of C3 LNPs ex vivo in human patient-derived brain tissues. These LNPs may provide a clinically translatable platform for in utero and postnatal mRNA therapies including gene editing in the brain.
    Keywords:  congenital brain disease; fetal gene therapy; gene editing; ionizable lipid nanoparticles; mRNA delivery
  3. Macromol Biosci. 2023 Jul 19. e2300177
      The present study expands the versatility of cationic poly(2-oxazoline) (POx) copolymers as a PEG-free platform for gene delivery to immune cells, such as monocytes and macrophages. Several block copolymers are developed by varying non-ionic hydrophilic blocks (poly(2-methyl-2-oxazoline) (pMeOx) or poly(2-ethyl-2-oxazoline) (pEtOx), cationic blocks, and an optional hydrophobic block (poly(2-isopropyl-2-oxazoline) (iPrOx). The cationic blocks are produced by side chain modification of 2-methoxy-carboxyethyl-2-oxazoline (MestOx) block precursor with diethylenetriamine (DET) or tris(2-aminoethyl)amine (TREN). For the attachment of a targeting ligand, mannose, we employed azide-alkyne cycloaddition click chemistry methods. Of the two cationic side chains, polyplexes made with DET-containing copolymers transfect macrophages significantly better than those made with TREN-based copolymer. Likewise, non-targeted pEtOx-based diblock copolymer is more active in cell transfection than pMeOx-based copolymer. The triblock copolymer with hydrophobic block iPrOx performs poorly compared to the diblock copolymer which lacks this additional block. Surprisingly, attachment of a mannose ligand to either of these copolymers is inhibitory for transfection. Despite similarities in size and design, mannosylated polyplexes result in lower cell internalization compared to non-mannosylated polyplexes. Thus, PEG-free, non-targeted DET- and pEtOx-based diblock copolymer outperforms other studied structures in the transfection of macrophages and displays transfection levels comparable to GeneJuice, a commercial non-lipid transfection reagent. This article is protected by copyright. All rights reserved.
    Keywords:  macrophages; mannose targeting; poly(2-oxazolines); transfection
  4. Arch Insect Biochem Physiol. 2023 Jul 15. e22036
      There has been limited success in the usage of exogenous small interference RNA (siRNA) or small hairpin RNA (shRNA) to trigger RNA interference (RNAi) in insects. Instead, long double-stranded RNAs (dsRNA) are used to induce knockdown of target genes in insects. Here, we compared the potency of si/sh RNAs and dsRNA in Colorado potato beetle (CPB) cells. CPB cells showed highly efficient RNAi response to dsRNA. However, si/sh RNAs were inefficient in triggering RNAi in CPB cells. Confocal microscopy observations of Cy3 labeled-si/sh RNA cellular uptake revealed reduced si/sh RNA uptake compared to dsRNA. si/sh RNAs were stable in the conditioned media of CPB cells. Although in a small amount, when internalized by CPB cells, the si/sh RNAs were processed by the Dicer enzyme. Lipid-mediated transfection and chimeric dsRNA approaches were used to improve the delivery of si/sh RNAs. Our results suggest that the uptake of si/sh RNAs is inefficient in CPB cells, resulting in ineffective RNAi response. However, with the help of effective delivery methods, si/sh RNA could be a useful option for developing target-specific RNAi-mediated biopesticides.
    Keywords:  RNAi; cellular uptake; shRNA; siRNA
  5. Mol Pharm. 2023 Jul 18.
      The recent clinical and commercial success of lipid nanoparticles (LNPs) for nucleic acid delivery has incentivized the development of new technologies to manufacture LNPs. As new technologies emerge, researchers must determine which technologies to assess and how to perform comparative evaluations. In this article, we use a quality-by-design approach to systematically investigate how the mixer technology used to form LNPs influences LNPstructure. Specifically, a coaxial turbulent jet mixer and a staggered herringbone microfluidic mixer were systematically compared via matched formulation and process conditions. A full-factorial design-of-experiments study with three factors and three levels was executed for each mixer to compare process robustness in the production of antisense oligonucleotide (ASO) LNPs. ASO-LNPs generated with the coaxial turbulent jet mixer were consistently smaller, had a narrower particle size distribution, and had a higher ASO encapsulation as compared to the microfluidic mixer, but had a greater variation in internal structure with less ordered cores. A subset of the study was replicated for mRNA-LNPs with comparable trends in particle size and encapsulation, but more frequent bleb features for LNPs produced by the coaxial turbulent jet mixer. The study design used here provides a road map for how researchers may compare different mixer technologies (or process changes more broadly) and how such studies can inform process robustness and manufacturing control strategies.
    Keywords:  antisense oligonucleotide; lipid nanoparticle; messenger RNA; microfluidic mixer; nucleic acid; turbulent jet mixer
  6. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2023 Jun 25. pii: 1008-9292(2023)03-0306-12. [Epub ahead of print]52(3): 306-317
      OBJECTIVES: To investigate the effect and mechanism of lipid nanoparticle (LNP) delivery of small interfering RNA (siRNA) targeting Cyp2e1 gene on subacute alcoholic liver injury in mice.METHODS: siRNA targeting Cyp2e1 gene was encapsulated in LNP (si-Cyp2e1 LNP) by microfluidic technique and the resulting LNPs were characterized. The optimal dose of si-Cyp2e1 LNP administration was screened. Forty female C57BL/6N mice were randomly divided into blank control group, model control group, si-Cyp2e1 LNP group, LNP control group and metadoxine group. The subacute alcoholic liver injury mouse model was induced by ethanol feeding for 10 d plus ethanol gavage for the last 3 d. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and the superoxide dismutase (SOD) activity as well as malondialdehyde, reactive oxygen species, glutathione, triacylglycerol, total cholesterol contents in liver tissue were measured in each group, and liver index was calculated. The expression of genes related to oxidative stress, lipid synthesis and inflammation in each group of mice were measured by realtime RT-PCR.
    RESULTS: Compared with the model control group, the levels of liver index, serum ALT, AST activities, malondialdehyde, reactive oxygen species, triacylglycerol, total cholesterol contents in liver tissue decreased, but the SOD activity as well as glutathione increased in the si-Cyp2e1 LNP group (all P<0.01). Hematoxylin-eosin staining result showed disorganized hepatocytes with sparse cytoplasm and a large number of fat vacuoles and necrosis in the model control group, while the si-Cyp2e1 LNP group had uniformly sized and arranged hepatocytes with normal liver tissue morphology and structure. Oil red O staining result showed si-Cyp2e1 LNP group had lower fat content of the liver compared to the model control group (P<0.01), and no fat droplets accumulated. Anti-F4/80 monoclonal antibody fluorescence immunohistochemistry showed that the si-Cyp2e1 LNP group had lower cumulative optical density values compared to the model control group (P<0.01) and no significant inflammatory reaction. Compared with the model control group, the expression of catalytic genes P47phox, P67phox and Gp91phox were reduced (all P<0.01), while the expression of the antioxidant enzyme genes Sod1, Gsh-rd and Gsh-px were increased (all P<0.01). The mRNA expression of the lipid metabolism genes Pgc-1α and Cpt1 were increased (all P<0.01) and the lipid synthesis-related genes Srebp1c, Acc and Fasn were decreased (all P<0.01); the expression of liver inflammation-related genes Tgf-β, Tnf-α and Il-6 were decreased (all P<0.01).
    CONCLUSIONS: The si-Cyp2e1 LNP may attenuate subacute alcoholic liver injury in mice mainly by reducing reactive oxygen levels, increasing antioxidant activity, blocking oxidative stress pathways and reducing ethanol-induced steatosis and inflammation.
    Keywords:  Inflammation; Lipid metabolic synthesis; Lipid nanoparticles; Mice; Oxidative stress; Small interfering RNA; Subacute alcoholic liver injury
  7. Chembiochem. 2023 Jul 17. e202300449
      Lipids are key constituents of numerous biomedical drug delivery technologies. Here, we present the design, synthesis, and biophysical characterizations of a library of cationic lipids containing an acetal residue in their linker region. These cationic acetal lipids (CALs) could be conveniently prepared through a trans-acetalization protocol from commercially available precursors. NMR studies highlighted the conformational rigidity at the acetal residue and the high hydrolytic stability of these CALs. Fluorescence anisotropy studies revealed that the CAL with pyridinium headgroup (CAL1) formed highly cohesive vesicular aggregates in water. These structural and self-assembly features of the CAL1 allowed up to 196% w/w loading of Curcumin (Cur) as a representative hydrophobic drug. A reconstitutable formulation of Cur was obtained as a result, which could deliver the drug inside mammalian cells with very high efficiency. The hemocompatibility and cytocompatibility of CAL1 was significantly enhanced by creating a coating of polydopamine (PDA) onto its vesicular assemblies to produce hybrid lipid-polymer nanocapsules. This work demonstrates rapid access to the useful synthetic lipid formulations with high potential in drug and gene delivery applications.
    Keywords:  Acetal; Cationic lipids; Cytocompatible; drug delivery; self-assembly
  8. Bull Exp Biol Med. 2023 Jul 19.
      The presence of anti-polyethylene glycol (PEG) antibodies can limit the clinical efficacy of PEGylated drugs and cause anaphylactic reactions in patients. Monocytes/macrophages are effector cells involved in IgG-mediated passive systemic anaphylaxis. We studied the influence of human blood serum on the efficiency of uptake of PEGylated nanoparticles by human blood monocytes. It has been shown that magnetic nanoparticles modified with PEG-3000 and solid lipid nanoparticles containing PEG-2000 are avidly internalized by human blood monocytes in vitro, the uptake efficiency depends on the features (composition) of donor blood serum, but does not correlate with the level of the IgG antibody against PEG.
    Keywords:  anti-PEG IgG antibody; magnetic nanoparticles; monocytes; polyethylene glycol; solid lipid nanoparticles
  9. J Pharm Sci. 2023 Jul 19. pii: S0022-3549(23)00292-7. [Epub ahead of print]
      Inhalation-based drug delivery systems have gained attention as potential therapeutic options for various respiratory diseases. Among these systems, nanoparticles are being explored as drug carriers because of their ability to deliver therapeutic agents directly to the lungs. It is essential to accurately evaluate the intrapulmonary behavior of nanoparticles to optimize drug delivery and achieve selective targeting of lung lesions. Prior research used the Förster resonance energy transfer (FRET) phenomenon to study the in vivo behavior of nanoparticles as drug carriers. In this study, image reconstruction involving bleed-through compensation was used to quantitatively assess the behavior of FRET nanoparticles in the lungs. When the nanoparticles for FRET fluorescence imaging, which employed 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) as the donor and as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide (DiR) the acceptor, were administered to mouse lungs, whole-body in vivo imaging could not compensate for the influence of respiration and heartbeat. However, ex vivo imaging of excised lungs enabled the quantitative evaluation of the time-concentration profiles and distribution of nanoparticles within the lungs. This imaging technique is particularly useful for the development of inhalable nanoparticles that specifically target the lesions and exhibit controlled-release capabilities within the lungs.
    Keywords:  Near-infrared FRET imaging; Pulmonary drug delivery system; intrapulmonary distribution; nanoparticle dissociation; polymeric nanoparticles
  10. Int J Biol Macromol. 2023 Jul 18. pii: S0141-8130(23)02777-0. [Epub ahead of print] 125882
      In this study, a targeted pH-sensitive polymersome incorporating doxorubicin (DOX) was manufactured implementing diblock copolymer of hyaluronic acid-b-pPoly (β-amino ester) (HA-PBAE). The hydrophilic DOX was loaded into the aqueous compartment of HA-PBAE polymersomal structure during nanoprecipitation process with 60 % ± 3.0 entrapment efficiency (EE%) and 5.3 % ± 0.2 loading content (LC%) while demonstrating spherical morphology with size of 196 ± 3.8 nm and PDI of 0.3. The prepared platform (DOX-HA-PBAE) illustrated accelerated DOX release in acidic pH 5.4, and showed significantly higher cytotoxicity and cellular internalization in comparison with free DOX against 4T1 cell line (CD44 positive cell). In contrast, no significant growth inhibition was observed in CHO cell line (CD44 negative cell). Furthermore, DOX-HA-PBAE platform displayed higher therapeutic efficacy, favorable tumor accumulation and lower systemic toxicity in comparison with free DOX based on obtained experimental data in ectopic 4T1 tumor model in BALB/c Female mice in terms of tumor growth rate, survival rate, body weight loss, ex vivo biodistribution and pathological evaluations. The obtained results demonstrated that DOX-HA-PBAE polymersomes have potential to be used in metastatic breast cancer therapy with promising characteristics in terms of tumor growth suppression and safety profile.
    Keywords:  Doxorubicin; Hyaluronic acid; Metastatic breast cancer; Poly (β-amino ester); Targeted drug delivery; pH sensitive
  11. Antiviral Res. 2023 Jul 19. pii: S0166-3542(23)00155-9. [Epub ahead of print] 105677
      Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a respiratory virus that causes COVID-19 disease, with an estimated global mortality of approximately 2%. While global response strategies, which are predominantly reliant on regular vaccinations, have shifted from zero COVID to living with COVID, there is a distinct lack of broad-spectrum direct acting antiviral therapies that maintain efficacy across evolving SARS-CoV-2 variants of concern. This is of most concern for immunocompromised and immunosuppressed individuals who lack robust immune responses following vaccination, and others at risk for severe COVID and long-COVID. RNA interference (RNAi) therapeutics induced by short interfering RNAs (siRNAs) offer a promising antiviral treatment option, with broad-spectrum antiviral capabilities unparalleled by current antiviral therapeutics and a high genetic barrier to antiviral escape. Here we describe novel siRNAs, targeting highly conserved regions of the SARS-CoV-1 and 2 genome of both human and animal species, with multi-variant antiviral potency against eight SARS-CoV-2 lineages - Ancestral VIC01, Alpha, Beta, Gamma, Delta, Zeta, Kappa and Omicron. Treatment with our siRNA resulted in significant protection against virus-mediated cell death in vitro, with >97% cell survival (P < 0.0001), and corresponding reductions of viral nucleocapsid RNA of up to 99.9% (P < 0.0001). When compared to antivirals; Sotrovimab and Remdesivir, the siRNAs demonstrated a more potent antiviral effect and similarly, when multiplexing siRNAs to target different viral regions simultaneously, an increased antiviral effect was observed compared to individual siRNA treatments (P < 0.0001). These results demonstrate the potential for a highly effective broad-spectrum direct acting antiviral against multiple SARS-CoV-2 variants, including variants resistant to antivirals and vaccine generated neutralizing antibodies.
    Keywords:  Broad spectrum antiviral; RNAi; SARS-CoV-2; Variants of concern; siRNA therapeutic