bims-novged 2023-07-09 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–07–09
eight papers selected by
the Merkel lab, Ludwig-Maximilians University

A machine learning approach to predict cellular uptake of pBAE polyplexes.
In vitro and in vivo evaluation of clinically-approved ionizable cationic lipids shows divergent results between mRNA transfection and vaccine efficacy.
Nucleic-Acid-Templated Synthesis of Smart Polymer Vectors for Gene Delivery.
Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function.
Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA-Lipid Complexes.
Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.
PEG-Lipids: Quantitative Study of Unimers and Aggregates Thereof by the Methods of Molecular Hydrodynamics.
Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery.

Biomater Sci. 2023 Jul 04.

A machine learning approach to predict cellular uptake of pBAE polyplexes.

Aparna Loecher, Michael Bruyns-Haylett, Pedro J Ballester, Salvador Borros, Nuria Oliva.

The delivery of genetic material (DNA and RNA) to cells can cure a wide range of diseases but is limited by the delivery efficiency of the carrier system. Poly β-amino esters (pBAEs) are promising polymer-based vectors that form polyplexes with negatively charged oligonucleotides, enabling cell membrane uptake and gene delivery. pBAE backbone polymer chemistry, as well as terminal oligopeptide modifications, define cellular uptake and transfection efficiency in a given cell line, along with nanoparticle size and polydispersity. Moreover, uptake and transfection efficiency of a given polyplex formulation also vary from cell type to cell type. Therefore, finding the optimal formulation leading to high uptake in a new cell line is dictated by trial and error, and requires time and resources. Machine learning (ML) is an ideal in silico screening tool to learn the non-linearities of complex data sets, like the one presented herein, with the aim of predicting cellular internalisation of pBAE polyplexes. A library of pBAE nanoparticles was fabricated and the uptake studied in 4 different cell lines, on which various ML models were successfully trained. The best performing models were found to be gradient-boosted trees and neural networks. The gradient-boosted trees model was then analysed using SHapley Additive exPlanations, to interpret the model and gain an understanding into the important features and their impact on the predicted outcome.

DOI: https://doi.org/10.1039/d3bm00741c
Biomed Pharmacother. 2023 Jul 03. pii: S0753-3322(23)00856-9. [Epub ahead of print]165 115065

In vitro and in vivo evaluation of clinically-approved ionizable cationic lipids shows divergent results between mRNA transfection and vaccine efficacy.

Oscar Escalona-Rayo, Ye Zeng, Renzo A Knol, Thomas J F Kock, Dennis Aschmann, Bram Slütter, Alexander Kros.

  Ionizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their biological performance would extend the existing knowledge in the field. In the present study, we examined the effects of the three clinically-approved ICLs, Dlin-MC3-DMA, ALC-0315 and SM-102, as well as DODAP, on the in vitro and in vivo performance of LNPs for mRNA delivery and vaccine efficacy. mRNA-LNPs containing these lipids were successfully prepared, which were all found to be very similar in their physicochemical properties and mRNA encapsulation efficiencies. Furthermore, the results of the in vitro studies indicated that these mRNA-LNPs were efficiently taken up by immortalized and primary immune cells with comparable efficiency; however, SM-102-based LNPs were superior in inducing protein expression and antigen-specific T cell proliferation. In contrast, in vivo studies revealed that LNPs containing ALC-0315 and SM-102 yielded almost identical protein expression levels in zebrafish embryos, which were significantly higher than Dlin-MC3-DMA-based LNPs. Additionally, a mouse immunization study demonstrated that a single-dose subcutaneous administration of the mRNA-LNPs resulted in a high production of intracellular cytokines by antigen-specific T cells, but no significant differences among the three clinically-approved ICLs were observed, suggesting a weak correlation between in vitro and in vivo outcomes. This study provides strong evidence that ICLs modulate the performance of mRNA-LNPs and that in vitro data does not adequately predict their behavior in vivo.

Keywords:  Ionizable cationic lipids; Lipid nanoparticles; MRNA delivery; T cell response; Transfection

DOI:  https://doi.org/10.1016/j.biopha.2023.115065
Chembiochem. 2023 Jul 04. e202300333

Nucleic-Acid-Templated Synthesis of Smart Polymer Vectors for Gene Delivery.

José García Coll, Sebastien Ulrich.

  Nucleic acids are information-rich and readily available biomolecules which can be used to template the polymerization of synthetic macromolecules. Here, we highlight the control over the size, composition and sequence one can nowadays obtain using this methodology. We also showcase how templated processes exploiting dynamic covalent polymerization can, in return, result in therapeutic nucleic acids fabricating their own dynamic delivery vector - a biomimicking concept that can provide original solutions for gene therapies.

Keywords:  Dynamic covalent chemistry; Gene Delivery; Nucleic acid; Polymer; Template

DOI:  https://doi.org/10.1002/cbic.202300333
Nat Commun. 2023 Jul 06. 14(1): 4007

Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function.

Kai Liu, Ralf Nilsson, Elisa Lázaro-Ibáñez, Hanna Duàn, Tasso Miliotis, Marie Strimfors, Michael Lerche, Ana Rita Salgado Ribeiro, Johan Ulander, Daniel Lindén, Anna Salvati, Alan Sabirsh.

In terms of lipid nanoparticle (LNP) engineering, the relationship between particle composition, delivery efficacy, and the composition of the biocoronas that form around LNPs, is poorly understood. To explore this we analyze naturally efficacious biocorona compositions using an unbiased screening workflow. First, LNPs are complexed with plasma samples, from individual lean or obese male rats, and then functionally evaluated in vitro. Then, a fast, automated, and miniaturized method retrieves the LNPs with intact biocoronas, and multiomics analysis of the LNP-corona complexes reveals the particle corona content arising from each individual plasma sample. We find that the most efficacious LNP-corona complexes were enriched with high-density lipoprotein (HDL) and, compared to the commonly used corona-biomarker Apolipoprotein E, corona HDL content was a superior predictor of in-vivo activity. Using technically challenging and clinically relevant lipid nanoparticles, these methods reveal a previously unreported role for HDL as a source of ApoE and, form a framework for improving LNP therapeutic efficacy by controlling corona composition.

DOI: https://doi.org/10.1038/s41467-023-39768-9
ACS Nano. 2023 Jul 07.

Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA-Lipid Complexes.

János Szebeni, Bálint Kiss, Tamás Bozó, Keren Turjeman, Yael Levi-Kalisman, Yechezkel Barenholz, Miklós Kellermayer.

  Despite the worldwide success of mRNA-LNP Covid-19 vaccines, the nanoscale structures of these formulations are still poorly understood. To fill this gap, we used a combination of atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and the determination of the intra-LNP pH gradient to analyze the nanoparticles (NPs) in BNT162b2 (Comirnaty), comparing it with the well-characterized PEGylated liposomal doxorubicin (Doxil). Comirnaty NPs had similar size and envelope lipid composition to Doxil; however, unlike Doxil liposomes, wherein the stable ammonium and pH gradient enables accumulation of 14C-methylamine in the intraliposomal aqueous phase, Comirnaty LNPs lack such pH gradient in spite of the fact that the pH 4, at which LNPs are prepared, is raised to pH 7.2 after loading of the mRNA. Mechanical manipulation of Comirnaty NPs with AFM revealed soft, compliant structures. The sawtooth-like force transitions seen during cantilever retraction imply that molecular strands, corresponding to mRNA, can be pulled out of NPs, and the process is accompanied by stepwise rupture of mRNA-lipid bonds. Unlike Doxil, cryo-TEM of Comirnaty NPs revealed a granular, solid core enclosed by mono- and bilipid layers. Negative staining TEM shows 2-5 nm electron-dense spots in the LNP's interior that are aligned into strings, semicircles, or labyrinth-like networks, which may imply cross-link-stabilized RNA fragments. The neutral intra-LNP core questions the dominance of ionic interactions holding together this scaffold, raising the possibility of hydrogen bonding between mRNA and the lipids. Such interaction, described previously for another mRNA/lipid complex, is consistent with the steric structure of the ionizable lipid in Comirnaty, ALC-0315, displaying free ═O and -OH groups. It is hypothesized that the latter groups can get into steric positions that enable hydrogen bonding with the nitrogenous bases in the mRNA. These structural features of mRNA-LNP may be important for the vaccine's activities in vivo.

Keywords:  Doxil liposomes; SARS-CoV-2; atomic force microscopy; cryo-electron microscopy; dynamic light scattering; lipid nanoparticles; phospholipid membranes

DOI:  https://doi.org/10.1021/acsnano.2c11904
Res Sq. 2023 Jun 01. pii: rs.3.rs-2987323. [Epub ahead of print]

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.

Vignesh Narayan Hariharan, Jillian Caiazzi, Rachael Miller, Chantal Ferguson, Ellen Sapp, Hassan Fakih, Qi Tang, Nozomi Yamada, Raymond Furgal, Joseph Paquette, Brianna Bramato, Nicholas McHugh, Ashley Summers, Clemens Lochmann, Bruno Godinho, Samuel Hildebrand, Dimas Echeverria, Matthew Hassler, Julia Alterman, Marian DiFiglia, Neil Aronin, Anastasia Khvorova, Ken Yamada.

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ~ 32-fold over PS backbone and > 1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (~ 6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

DOI: https://doi.org/10.21203/rs.3.rs-2987323/v1
Anal Chem. 2023 Jul 07.

PEG-Lipids: Quantitative Study of Unimers and Aggregates Thereof by the Methods of Molecular Hydrodynamics.

Ilya Anufriev, Stephanie Hoeppener, Ivo Nischang.

Understanding the polymorphism of lipids in solution is the key to the development of intracellular delivery systems. Here, we study the dynamics of poly(ethylene glycol)-lipid (PEG-Lipid) conjugates aiming at a better understanding of their molecular properties and aggregation behavior in solution. Those PEG-Lipids are used as components of lipid nanoparticles (LNPs). LNPs are gaining increased popularity, e.g., by their utilization in modern vaccination strategies against SARS-CoV-2. Characterization of the systems is conducted by the classical methods of hydrodynamics in different solvents, such as ethanol and water, which are also commonly used for LNP formulation. We were able to elucidate the structurally associated hydrodynamic properties of isolated PEG-Lipids in ethanol, revealing the typically expected values of the hydrodynamic invariant for random coil polymers. By virtue of the same experimental setting, the PEG-Lipids' behavior in water was as well studied, which is a less good solvent than ethanol for the PEG-Lipids. Our experiments demonstrate that PEG-Lipids dissolved in water form well-defined micelles that can quantitatively be characterized in terms of their degree of aggregation of PEG-Lipid polymer unimers, their hydrodynamic size, and solvation, i.e., the quantitative determination of water contained or associated to the identified micelles. Quantitative results obtained from classical hydrodynamic analyses are fully supported by studies with standard dynamic light scattering (DLS). The obtained diffusion coefficients and hydrodynamic sizes are in excellent agreement with numerical results derived from analytical ultracentrifugation (AUC) data. Cryo-transmission electron microscopy (cryo-TEM) supports the structural insight from hydrodynamic studies, particularly, in terms of the observed spherical structure of the formed micelles. We demonstrate experimentally that the micelle systems can be considered as solvent-permeable, hydrated spheres.

DOI: https://doi.org/10.1021/acs.analchem.3c01999
J Am Chem Soc. 2023 Jul 03.

Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery.

Yi Lin, Xianjin Luo, Tobias Burghardt, Sarah Dorrer, Miriam Höhn, Ernst Wagner, Ulrich Lächelt.

The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.

DOI: https://doi.org/10.1021/jacs.3c01902