bims-novged 2021-12-05 papers

on Non-viral vectors for gene delivery

Issue of 2021–12–05
eight papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Design of a new cell penetrating peptide for DNA, siRNA and mRNA delivery.
Investigating histidinylated highly branched poly(lysine) for siRNA delivery.
A scalable and robust cationic lipid/polymer hybrid nanoparticle platform for mRNA delivery.
Influence of Aib-Containing Amphipathic Helical Chain Length in MAP(Aib)-cRGD as Carrier for siRNA Delivery.
Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy.
Engineered ε-decalactone lipomers bypass the liver to selectively in vivo deliver mRNA to the lungs without targeting ligands.
Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.
Polymers Strive for Accuracy: From Sequence-Defined Polymers to mRNA Vaccines against COVID-19 and Polymers in Nucleic Acid Therapeutics.

J Gene Med. 2021 Dec 02. e3401

Design of a new cell penetrating peptide for DNA, siRNA and mRNA delivery.

Salif Ali, Candice Dussouillez, Beatriz Padilla, Benoît Frisch, A James Mason, Antoine Kichler.

   BACKGROUND: Delivery systems, including peptide-based ones, that destabilize endosomes in a pH-dependent manner are increasingly used to deliver cargoes of therapeutic interest such as nucleic acids and proteins into mammalian cells.
METHODS: The negatively charged amphipathic alpha-helicoidal forming peptide named HELP is a derivative from the bee venom melittin and was shown to have a pH-dependent activity with the highest lytic activity at pH 5.0 while becoming inactive when the pH is increased. In the present work, we asked whether replacement in the HELP peptide of the glutamic acid residues by histidines - whose protonation state is sensitive to the pH changes that occur during endosomal acidification - can transform this fusogenic peptide into a carrier able to deliver different nucleic acids into mammalian cells.
RESULTS: Our results show that the resulting HELP-4H peptide displays high plasmid DNA, siRNA and mRNA delivery capabilities. Importantly, in contrast to other cationic peptides its transfection activity was only marginally affected by the presence of serum. Using circular dichroism, we found that acidic pH did not induce significant conformational changes for HELP-4H.
CONCLUSIONS: Taken together, we were able to develop a new cationic histidine rich peptide able to efficiently deliver various nucleic acids into cells.

Keywords:  DNA transfection; cationic peptide; cell penetrating peptide; histidine residues; mRNA vectorization; siRNA delivery

DOI:  https://doi.org/10.1002/jgm.3401
J Mater Chem B. 2021 Dec 01.

Investigating histidinylated highly branched poly(lysine) for siRNA delivery.

Ali Alazzo, Nurcan Gumus, Pratik Gurnani, Snjezana Stolnik, Ruman Rahman, Keith Spriggs, Cameron Alexander.

The temporary silencing of disease-associated genes utilising short interfering RNA (siRNA) is a potent and selective route for addressing a wide range of life limiting disorders. However, the few clinically approved siRNA therapies rely on lipid based formulations, which although potent, provide limited chemical space to tune the stability, efficacy and tissue selectivity. In this study, we investigated the role of molar mass and histidinylation for poly(lysine) based non-viral vectors, synthesised through a fully aqueous thermal condensation polymerisation. Formulation and in vitro studies revealed that higher molar mass derivatives yielded smaller polyplexes attributed to a greater affinity for siRNA at lower N/P ratios yielding greater transfection efficiency, albeit with some cytotoxicity. Histidinylation had a negligible effect on formulation size, yet imparted a moderate improvement in biocompatibility, but did not provide any meaningful improvement over silencing efficiency compared to non-histidinylated derivatives. This was attributed to a greater degree of cellular internalisation for non-histidinylated analogues, which was enhanced with the higher molar mass material.

DOI: https://doi.org/10.1039/d1tb01793d
Int J Pharm. 2021 Nov 25. pii: S0378-5173(21)01120-0. [Epub ahead of print] 121314

A scalable and robust cationic lipid/polymer hybrid nanoparticle platform for mRNA delivery.

Randall A Meyer, G Patrick Hussmann, Norman C Peterson, Jose Luis Santos, Anthony D Tuesca.

mRNA based gene therapies hold the potential to treat multiple diseases with significant advantages over DNA based therapies, including rapid protein expression and minimized risk of mutagenesis. However, successful delivery of mRNA remains challenging, and clinical translation of mRNA therapeutics has been limited. This study investigated the use of a lipid/polymer hybrid (LPH) nanocarrier for mRNA, designed to address key delivery challenges and shuttle mRNA to targeted tissues. LPH nanocarriers were synthesized using a scalable microfluidic process with a variety of material compositions and mRNA loading strategies. Results show that a combination of permanently ionized and transiently, pH-dependent ionizable cationic lipids had a synergistic effect upon on mRNA gene translation, when compared to each lipid independently. Upon intravenous administration, particles with adsorbed mRNA outperformed particles with encapsulated mRNA for protein expression in the lungs and the spleen despite significant LPH nanoparticle localization to the liver. In contrast, encapsulated particles had higher localized expression when injected intramuscularly with protein expression detectable out to 12 days post injection. Intramuscular administration of particles with OVA mRNA resulted in robust humoral immune response with encapsulated outperforming adsorbed particles in terms of antibody titers at 28 days. These results demonstrate LPH nanocarriers have great potential as a vehicle for mRNA delivery and expression in tissues and that tissue expression and longevity can be influenced by LPH composition and route of administration.

DOI: https://doi.org/10.1016/j.ijpharm.2021.121314
Chem Biodivers. 2021 Nov 29.

Influence of Aib-Containing Amphipathic Helical Chain Length in MAP(Aib)-cRGD as Carrier for siRNA Delivery.

Shun-Ichi Wada, Akira Shibaike, Junsuke Hayashi, Hidehito Urata.

  MAP(Aib)-cRGD, which is a conjugate of an α-aminoisobutyric acid (Aib)-containing amphipathic helical peptide [MAP(Aib)] with a α v b 3 integrin binding ligand, cRGD, at the C-terminus of the helical peptide, has been developed for siRNA delivery into cells. In this work, we synthesized three peptides containing 19 ( PI ), 18 ( PII ), and 17 ( PIII ) amino acid residues in the helical peptide, which lack Aib, Leu-Aib, and Lys-Leu-Aib residues present in the C-terminus of the helical peptide of the parent MAP(Aib)-cRGD, respectively. MAP(Aib)-cRGD showed the siRNA delivery into cells and the RNAi effect both in the presence and in the absence of serum in reaction media. In contrast, PI delivered siRNA into cells, and this was followed by the RNAi effect in only serum-free reaction media. On the other hand, siRNA delivery was abolished by the further reduction of the number of residues ( PII and PIII ) in the C-terminus. Our data indicate that the Aib-containing helical part requires 20 residues in the conjugation of the helical peptide with cRGD for the construction of carrier for siRNA delivery into cells.

Keywords:  amphipathic helical peptide, α-aminoisobutyric acid (Aib), RGD, siRNA, MAP(Aib)-cRGD

DOI:  https://doi.org/10.1002/cbdv.202100728
Bioact Mater. 2022 Mar;9 590-601

Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy.

Chunhui Li, Tongren Yang, Yuhua Weng, Mengjie Zhang, Deyao Zhao, Shuai Guo, Bo Hu, Wanxuan Shao, Xiaoxia Wang, Abid Hussain, Xing-Jie Liang, Yuanyu Huang.

  CRISPR/Cas9-based gene editing has emerged as a powerful biotechnological tool, that relies on Cas9 protein and single guided RNA (sgRNA) to edit target DNA. However, the lack of safe and efficient delivery carrier is one of the crucial factors restricting its clinical transformation. Here, we report an ionizable lipid nanoparticle (iLP181, pKa = 6.43) based on iLY1809 lipid enabling robust gene editing in vitro and in vivo. The iLP181 effectively encapsulate psgPLK1, the best-performing plasmid expressing for both Cas9 protein and sgRNA targeting Polo-like kinase 1 (PLK1). The iLP181/psgPLK1 nanoformulation showed uniformity in size, regular nanostructure and nearly neutral zeta potential at pH 7.4. The nanoformulation effectively triggered editing of PLK1 gene with more than 30% efficiency in HepG2-Luc cells. iLP181/psgPLK1 significantly accumulated in the tumor for more than 5 days after a single intravenous injection. In addition, it also achieved excellent tumor growth suppression compared to other nucleic acid modalities such as siRNA, without inducing adverse effects to the main organs including the liver and kidneys. This study not only provides a clinically-applicable lipid nanocarrier for delivering CRISPR/Cas system (even other bioactive molecules), but also constitutes a potential cancer treatment regimen base on DNA editing of oncogenes.

Keywords:  CRISPR/Cas; Cancer therapy; Gene editing; Lipid nanoparticle; PLK1

DOI:  https://doi.org/10.1016/j.bioactmat.2021.05.051
Mater Horiz. 2021 Aug 01. 8(8): 2251-2259

Engineered ε-decalactone lipomers bypass the liver to selectively in vivo deliver mRNA to the lungs without targeting ligands.

Mahmoud M Abd Elwakil, Tianle Gao, Takuya Isono, Yusuke Sato, Yaser H A Elewa, Toshifumi Satoh, Hideyoshi Harashima.

RNA drugs hold real potential for tackling devastating diseases that are currently resistant to small molecule drugs or monoclonal antibodies. However, since these drugs are unstable in vivo and unable to pass through cellular membranes their clinical realization is limited by their successful delivery to target sites. Herein we report on the design of a combinatorial library of polyester lipomers based on the renewable monomer, ε-decalactone (ε-DL), via organocatalytic ring-opening polymerization for mRNA delivery. The ε-DL lipomers showed a surprisingly efficient ability to target the lungs upon intravenous administration. Interestingly, most of the lipomers achieved functional EGFP expression in the lungs, while minimally transfecting hepatocytes and splenic cells. This simple approach for the design of biodegradable materials has the potential for the clinical translation of genetic medicines for the treatment of lung diseases.

DOI: https://doi.org/10.1039/d1mh00185j
ACS Nano. 2021 Nov 29.

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.

Albert Kamanzi, Yifei Gu, Radin Tahvildari, Zachary Friedenberger, Xingqi Zhu, Romain Berti, Marty Kurylowicz, Dominik Witzigmann, Jayesh A Kulkarni, Jerry Leung, John Andersson, Andreas Dahlin, Fredrik Höök, Mark Sutton, Pieter R Cullis, Sabrina Leslie.

  Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

Keywords:  drug-delivery; genetic medicines; lipid nanoparticles; microscopy; nanomedicines; single-molecule; vaccines

DOI:  https://doi.org/10.1021/acsnano.1c04862
J Am Chem Soc. 2021 Nov 28.

Polymers Strive for Accuracy: From Sequence-Defined Polymers to mRNA Vaccines against COVID-19 and Polymers in Nucleic Acid Therapeutics.

Aman Ishaqat, Andreas Herrmann.

Unquestionably, polymers have influenced the world over the past 100 years. They are now more crucial than ever since the COVID-19 pandemic outbreak. The pandemic paved the way for certain polymers to be in the spotlight, namely sequence-defined polymers such as messenger ribonucleic acid (mRNA), which was the first type of vaccine to be authorized in the U.S. and Europe to protect against the SARS-CoV-2 virus. This rise of mRNA will probably influence scientific research concerning nucleic acids in general and RNA therapeutics in specific. In this Perspective, we highlight the recent trends in sequence-controlled and sequence-defined polymers. Then we discuss mRNA vaccines as an example to illustrate the need of ultimate sequence control to achieve complex functions such as specific activation of the immune system. We briefly present how mRNA vaccines are produced, the importance of modified nucleotides, the characteristic features, and the advantages and challenges associated with this class of vaccines. Finally, we discuss the chances and opportunities for polymer chemistry to provide solutions and contribute to the future progress of RNA-based therapeutics. We highlight two particular roles of polymers in this context. One represents conjugation of polymers to nucleic acids to form biohybrids. The other is concerned with advanced polymer-based carrier systems for nucleic acids. We believe that polymers can help to address present problems of RNA-based therapeutic technologies and impact the field beyond the COVID-19 pandemic.

DOI: https://doi.org/10.1021/jacs.1c08484