bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2023‒09‒17
eight papers selected by
the Merkel lab, Ludwig-Maximilians University

  1. Nanoscale Horiz. 2023 Sep 11.
      The design of nanomaterial-based nucleic acid formulations is one of the biggest endeavours in the search for clinically applicable gene delivery systems. Biopolymers represent a promising subclass of gene carriers due to their physicochemical properties, biodegradability and biocompatibility. By modifying melanin-like polydopamine nanoparticles with poly-L-arginine and poly-L-histidine blends, we obtained a novel catch-and-release gene delivery system for efficient trafficking of pDNA to human cells. A synergistic interplay of nanoparticle-bound poly-L-arginine and poly-L-histidine was observed and evaluated for pDNA binding affinity, cell viability, gene release and transfection. Although the functionalisation with poly-L-arginine was crucial for pDNA binding, the resulting nanocarriers failed to release pDNA intracellularly, resulting in limited protein expression. However, optimal pDNA release was achieved through the co-formulation with poly-L-histidine, essential for pDNA release. This effect enabled the design of gene delivery systems, which were comparable to Lipofectamine in terms of transfection efficacy and the catch-and-release surface modification strategy can be translated to other nanocarriers and surfaces.
  2. Int J Biol Macromol. 2023 Sep 11. pii: S0141-8130(23)03742-X. [Epub ahead of print] 126845
      Aminated curdlan derivatives are highly effective nucleic acid carriers. Previously, we proved that the ligand-functionalized curdlan derivatives have greatly enhanced cell type specificity induced by receptor-mediated internalization in vitro. In this study, to improve biocompatibility and enhance tumor-targeting efficacy of the curdlan derivative, we pegylated the adenosine functionalized amino curdlan derivative (denoted by pAVC polymer). We confirmed that the uptake of pAVC polymer carrying siRNA by tumor cells was adenosine receptor (AR)-dependent and was specifically inhibited by AMP but not by GMP. The pAVC polymers not only preserved the receptor recognition and exhibited significantly decreased cytotoxicity but also showed remarkable tumor targeting efficiency in vivo. The nanoparticles formulated from siRNA (against STAT3) and pAVC4 polymer, which bears the highest degree of PEG substitution, delivered siRNA highly specifically to tumor tissue, knocked down STAT3, and inhibited tumor growth. The pAVC polymers may be a promising carrier for tumor specific delivery of nucleic acid drugs.
    Keywords:  Adenosine receptor; Curdlan nanoparticles; Melanoma; STAT3; siRNA delivery
  3. Natl Sci Rev. 2023 Oct;10(10): nwad214
      Messenger RNA (mRNA) vaccine is revolutionizing the methodology of immunization in cancer. However, mRNA immunization is drastically limited by multistage biological barriers including poor lymphatic transport, rapid clearance, catalytic hydrolysis, insufficient cellular entry and endosome entrapment. Herein, we design a mRNA nanovaccine based on intelligent design to overcome these obstacles. Highly efficient nanovaccines are carried out with machine learning techniques from datasets of various nanocarriers, ensuring successful delivery of mRNA antigen and cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) to targets. It activates stimulator of interferon genes (STING), promotes mRNA-encoded antigen presentation and boosts antitumour immunity in vivo, thus inhibiting tumour growth and ensuring long-term survival of tumour-bearing mice. This work provides a feasible and safe strategy to facilitate STING agonist-synergized mRNA immunization, with great translational potential for enhancing cancer immunotherapy.
    Keywords:  STING; cancer immunotherapy; mRNA; nanoparticles; vaccine
  4. Bioeng Transl Med. 2023 Sep;8(5): e10392
      Induction of potent immune responses toward tumors remains challenging in cancer immunotherapy, in which it only showed benefits in a minority of patients with "hot" tumors, which possess pre-existing effector immune cells within the tumor. In this study, we proposed a nanoparticle-based strategy to fire up the "cold" tumor by upregulating the components associated with T and NK cell recruitment and activation and suppressing TGF-β1 secretion by tumor cells. Specifically, LTX-315, a first-in-class oncolytic cationic peptide, and TGF-β1 siRNA were co-entrapped in a polymer-lipid hybrid nanoparticle comprising PLGA, DSPE-mPEG, and DSPE-PEG-conjugated with cRGD peptide (LTX/siR-NPs). The LTX/siR-NPs showed significant inhibition of TGF-β1 expression, induction of type I interferon release, and triggering immunogenic cell death (ICD) in treated tumor cells, indicated via the increased levels of danger molecules, an in vitro setting. The in vivo data showed that the LTX/siR-NPs could effectively protect the LTX-315 peptide from degradation in serum, which highly accumulated in tumor tissue. Consequently, the LTX/siR-NPs robustly suppressed TGF-β1 production by tumor cells and created an immunologically active tumor with high infiltration of antitumor effector immune cells. As a result, the combination of LTX/siR-NP treatment with NKG2A checkpoint inhibitor therapy remarkably increased numbers of CD8+NKG2D+ and NK1.1+NKG2D+ within tumor masses, and importantly, inhibited the tumor growth and prolonged survival rate of treated mice. Taken together, this study suggests the potential of the LTX/siR-NPs for inflaming the "cold" tumor for potentiating the efficacy of cancer immunotherapy.
    Keywords:  LTX‐315; cancer immunotherapy; hybrid nanoparticle; tumor microenvironment
  5. Mol Pharm. 2023 Sep 12.
      mRNA vaccines encoding a single spike protein effectively prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the emergence of SARS-CoV-2 variants leads to a wide range of immune evasion. Herein, a unique trivalent mRNA vaccine based on ancestral SARS-CoV-2, Delta, and Omicron variant spike receptor-binding domain (RBD) mRNAs was developed to tackle the immune evasion of the variants. First, three RBD mRNAs of SARS-CoV-2, Delta, and Omicron were coencapsulated into lipid nanoparticles (LNPs) by using microfluidic technology. After that, the physicochemical properties and time-dependent storage stability of the trivalent mRNA vaccine nanoformulation were tested by using dynamic light scattering (DLS). In vitro, the trivalent mRNA vaccine exhibited better lysosomal escape ability, transfection efficiency, and biocompatibility than did the commercial transfection reagent Lipo3000. In addition, Western blot analyses confirmed that the three RBD proteins can be detected in cells transfected with the trivalent mRNA vaccine. Furthermore, ex vivo imaging analysis indicated that the livers of BALB/c mice had the strongest protein expression levels after intramuscular (IM) injection. Using a prime-boost strategy, this trivalent vaccine elicited robust humoral and T-cell immune responses in both the high-dose and low-dose groups and showed no toxicity in BALB/c mice. Three specific IgG antibodies in the high-dose group against SARS-CoV-2, Delta, and Omicron variants approached ∼1/1,833,333, ∼1/1,866,667, and ∼1/925,000, respectively. Taken together, two doses of inoculation with the trivalent mRNA vaccine may provide broad and effective immunization responses against SARS-CoV-2 and variants.
    Keywords:  Lipid nanoparticle; SARS-CoV-2; immune evasion; trivalent mRNA vaccine; variant
  6. Bioconjug Chem. 2023 Sep 14.
      The interaction between lipid nanoparticles (LNPs) and serum proteins, giving rise to a unique identification in the form of the protein corona, has been shown to be associated with novel recognition by cell receptors. The presence of the corona enveloping the nanoparticle strongly affects the interplay with immune cells. The immune responses mediated by protein corona can affect nanoparticle toxicity and targeting capabilities. But the intracellular signaling of LNPs after corona formation resulting in the change of nanoparticles' ability to provoke immune responses remains unclear. Therefore, a more systematic and delineated approach must be considered to present the correlation between corona complexes and the shift in nanoparticle immunogenicity. Here, we studied and reported the inhibiting effect of the absorbed proteins on the LNPs on the NLRP3 inflammasome activation, a key intracellular protein complex that modulates several inflammatory responses. Ionizable lipid as a component of LNP was observed to play an important role in modulating the activation of NLRP3 inflammasome in serum-free conditions. However, in the presence of serum proteins, the corona layer on LNPs caused a significant reduction in the inflammasome activation. Reduction in the lysosomal rupture after treatment with corona-LNPs significantly reduced inflammasome activation. Furthermore, a strong reduction of cellular uptake in macrophages after the corona formation was observed. On inspecting the uptake mechanisms in macrophages using transport inhibitors, lipid formulation was found to play a critical role in determining the endocytic pathways for the LNPs in macrophages. This study highlights the need to critically analyze the protein interactions with nanomaterials and their concomitant adaptability with immune cells to evaluate nano-bio surfaces and successfully design nanomaterials for biological applications.
  7. Biosens Bioelectron. 2023 Aug 25. pii: S0956-5663(23)00576-6. [Epub ahead of print]241 115634
      Spatially resolved transfection, intracellular delivery of proteins and nucleic acids, has the potential to drastically speed up the discovery of biologically active cargos, for instance for the development of cell therapies or new genome engineering tools. We recently demonstrated the use of a high-density microelectrode array for the targeted electrotransfection of cells grown on its surface, a process called High-Definition Electroporation (HD-EP). We also developed a framework based on Design of Experiments to quickly establish optimized electroporation conditions across five different electrical pulse parameters. Here, we used this framework to optimize the transfection efficiency of primary fibroblasts with a mCherry-encoding mRNA, resulting in 98% of the cells expressing the desired fluorescent protein without any sign of cell death. That transfection yield is the highest reported so far for electroporation. Moreover, varying the pulse number was shown to modulate the fluorescence intensity of cells, indicating the dosage-controlled delivery of mRNA and protein expression. Finally, exploiting the single-electrode addressability of the microelectrode array, we demonstrated spatially resolved, high efficiency, sequential transfection of cells with three distinct mRNAs. Since the chip can be easily redesigned to feature a much large number of electrodes, we anticipate that this methodology will enable the development of dedicated screening platforms for analysis of mRNA variants at scale.
    Keywords:  Adherent cells; CMOS microelectrode arrays; Design of experiments; RNA delivery; Spatially resolved electroporation; Transfection
  8. Sci Transl Med. 2023 Sep 13. 15(713): eadf4100
      With the success of messenger RNA (mRNA) vaccines against coronavirus disease 2019, strategies can now focus on improving vaccine potency, breadth, and stability. We designed and evaluated domain-based mRNA vaccines encoding the wild-type spike protein receptor binding domain (RBD) or N-terminal domain (NTD) alone or in combination. An NTD-RBD-linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2° to 8°C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In BALB/c mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses from viral challenge were observed against wild-type, beta, delta, or omicron (BA.1) viruses compared with mRNA-1273-immunized mice, especially at lower vaccine dosages. K18-hACE2 mice immunized with mRNA-1283 or mRNA-1273 as a primary series demonstrated similar degrees of protection from challenge with SARS-CoV-2 Delta and Omicron variants at all vaccine dosages. These results support clinical assessment of mRNA-1283, which has now entered clinical trials (NCT05137236).