bims-novged 2022-03-06 papers

on Non-viral vectors for gene delivery

Issue of 2022–03–06
nine papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Rationally designed cationic amphiphilic peptides for selective gene delivery to cancer cells.
Tumour-derived extracellular vesicle membrane hybrid lipid nanovesicles enhance siRNA delivery by tumour-homing and intracellular freeway transportation.
Bioengineered Protein Nanocage by Small Heat Shock Proteins Delivering mTERT siRNA for Enhanced Colorectal Cancer Suppression.
Effective Transfection and Gene Silencing of Primary Murine Macrophages with Small Interfering RNA.
Designing siRNA/chitosan-methacrylate complex nanolipogel for prolonged gene silencing effects.
Self-immolative polyplexes for DNA delivery.
High-throughput synthesis and characterization of next-generation lipid nanoparticles for enhanced in vivo performance.
Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.
Targeted siRNA nanocarrier: a platform technology for cancer treatment.

Int J Pharm. 2022 Feb 23. pii: S0378-5173(22)00174-0. [Epub ahead of print]617 121619

Rationally designed cationic amphiphilic peptides for selective gene delivery to cancer cells.

Roja Hadianamrei, Jiqian Wang, Stephen Brown, Xiubo Zhao.

  Gene therapy has gained increasing attention as an alternative to pharmacotherapy for treatment of various diseases. The extracellular and intracellular barriers to gene delivery necessitate the use of gene vectors which has led to the development of myriads of gene delivery systems. However, many of these gene delivery systems have pitfalls such as low biocompatibility, low loading efficiency, low transfection efficiency, lack of tissue selectivity and high production costs. Herein, we report the development of a new series of short cationic amphiphilic peptides with anticancer activity for selective delivery of small interfering RNA (siRNA) and antisense oligodeoxynucleotides (ODNs) to cancer cells. The peptides consist of alternating dyads of hydrophobic (isoleucine (I) or leucine (L)) and hydrophilic (arginine (R) or lysine (L)) amino acids. The peptides exhibited higher preference for transfection of HCT 116 colorectal cancer cells compared to human dermal fibroblasts (HDFs) and induced higher level of gene silencing in the cancer cells. The nucleic acid complexation and transfection efficiency of the peptides was a function of their secondary structure, their hydrophobicity and their C-terminal amino acid. The peptides containing L in their hydrophobic domain formed stronger complexes with siRNA and successfully delivered it to the cancer cells but were unable to release their cargo inside the cells and therefore could not induce any gene silencing. On the contrary, the peptides containing I in their hydrophobic domain were able to release their associated siRNA and induce considerable gene silencing in cancer cells. The peptides exhibited higher selectivity for colorectal cancer cells and induced less gene silencing in fibroblasts compared to the lipid-based commercial transfection reagent DharmaFECT™ 1. The results from this study can serve as a tool for rational design of new peptide-based gene vectors for high selective gene delivery to cancer cells.

Keywords:  Cancer cell selectivity; Cationic amphiphilic peptide; Gene delivery; Nonviral vector; siRNA

DOI:  https://doi.org/10.1016/j.ijpharm.2022.121619
J Extracell Vesicles. 2022 Mar;11(3): e12198

Tumour-derived extracellular vesicle membrane hybrid lipid nanovesicles enhance siRNA delivery by tumour-homing and intracellular freeway transportation.

Xin Zhou, Yunqiu Miao, Ying Wang, Shufang He, Linmiao Guo, Junsong Mao, Mingshu Chen, Yuting Yang, Xinxin Zhang, Yong Gan.

  Extracellular vesicles (EVs) have been proved a promising small interfering RNA (siRNA) delivery vehicle to mediate gene-silencing. Tumour-derived extracellular vesicles (TDEVs) as genetic exchange vectors in the tumour microenvironment, enable intercellular communication for a wide range of endogenous cargo molecules, such as RNAs and proteins. However, the oncogenic cargo of TDEVs limits their application in siRNA delivery for cancer therapy. Herein, we isolated TDEVs from hepatocellular carcinoma (HCC) cells and derived TDEV membranes by abandoning their content. Innovative TDEV membrane hybrid lipid nanovesicles (LEVs) were then fabricated by fusion of TDEV membranes and phospholipids to realize precise delivery to tumours and highly efficient transfection of siRNA. The TDEV membranes endow LEVs with 'homing' targeting ability, facilitating specific internalisation into parent HCC cells primarily through heparan sulfate proteoglycan-mediated pathways. Unlike conventional lipid-based nanovesicles, LEVs can bypass the endosomal degradation pathway, boost the delivery of siRNA through the Golgi and endoplasmic reticulum (ER) intracellular 'freeway' transportation, achieving a 1.7-fold improvement in siRNA transfection efficiency compared with liposomes. Additionally, siRNA loaded LEVs were demonstrated to enhance the antitumour efficacy in HCC bearing mice through effective gene silencing in the tumour sites. Our results highlight the potential application of the TDEV membrane-derived nanovesicles as an advanced siRNA delivery strategy for cancer therapy.

Keywords:  hepatocellular carcinoma; hybrid lipid nanovesicles; siRNA delivery; tumour homing; tumour-derived extracellular vesicles

DOI:  https://doi.org/10.1002/jev2.12198
ACS Appl Bio Mater. 2022 Mar 02.

Bioengineered Protein Nanocage by Small Heat Shock Proteins Delivering mTERT siRNA for Enhanced Colorectal Cancer Suppression.

Hao Wang, Ning Liu, Fuxu Yang, Nannan Hu, Mingyue Wang, Meiying Cui, Nico Bruns, Xingang Guan.

  The efficient delivery of small interfering RNA (siRNA) for target gene silencing holds great promise for cancer therapy. Protein nanocages have attracted considerable attention as ideal drug delivery systems because of their material-derived advantages and unique structural properties. However, most studies about siRNA delivery have not indicated the real role of protein nanocages in inhibiting tumor growth in vivo. Herein, we fabricated an efficient siRNA delivery system using a small heat shock protein (Hsp) nanocage decorated with Arg-Gly-Asp (RGD) and the transactivator of transcription (Tat) peptide. Hsp-Tat-RGD NC showed good cellular uptake and lysosomal escape in colorectal cancer cells. In addition, the nanocage could efficiently transfect siRNA into the cytoplasmic area of CT26 cells. Hsp-Tat-RGD NC delivering telomerase reverse transcriptase (TERT)-targeting siRNA could significantly downregulate TERT protein expression and trigger tumor cell apoptosis in vitro. More importantly, Hsp-Tat-RGD/siTERT complexes nearly completely inhibited the tumor growth after five times of treatment in mice bearing CT26 xenograft. Our results demonstrate the great potential of the Tat/RGD-decorated Hsp nanocage as a promising siRNA delivery platform for cancer therapy.

Keywords:  RGD; TERT; cancer therapy; protein nanocage; siRNA delivery

DOI:  https://doi.org/10.1021/acsabm.1c01221
Methods Mol Biol. 2022 ;2419 125-132

Effective Transfection and Gene Silencing of Primary Murine Macrophages with Small Interfering RNA.

Kim van Kuijk, Jan Nagenborg, Judith C Sluimer.

  Transfection of murine primary macrophages to silence genes can be a challenging procedure because this cell type has developed mechanisms to evade cellular intrusion. The introduction of small interfering RNA (siRNA) encapsulated in liposomes to the cell to decrease gene expression is one of the methods that can be used to achieve gene silencing. There are different commercially available compounds to introduce siRNA into the cell, including Lipofectamine RNAiMAX and HiPerfect. The chapter will describe a method for gene silencing in mouse primary macrophages using liposome-based transfection of siRNA.

Keywords:  Macrophages; Primary cells; RNA interference; Transfection; siRNA

DOI:  https://doi.org/10.1007/978-1-0716-1924-7_8
Sci Rep. 2022 Mar 03. 12(1): 3527

Designing siRNA/chitosan-methacrylate complex nanolipogel for prolonged gene silencing effects.

Ye Cao, Yang Fei Tan, Yee Shan Wong, Muhammad Aminuddin, Bhuthalingam Ramya, Melvin Wen Jie Liew, Jiaxin Liu, Subbu S Venkatraman.

Despite immense revolutionary therapeutics potential, sustaining release of active small interfering RNA (siRNA) remains an arduous challenge. The development of nanoparticles with siRNA sustained release capabilities provides an avenue to enhance the therapeutic efficacy of gene-based therapy. Herein, we present a new system based on the encapsulation of siRNA/chitosan-methacrylate (CMA) complexes into liposomes to form UV crosslinkable Nanolipogels (NLGs) with sustained siRNA-release properties in vitro. We demonstrated that the CMA nanogel in NLGs can enhance the encapsulation efficiency of siRNA and provide sustained release of siRNA up to 28 days. To understand the particle mechanism of cellular entry, multiple endocytic inhibitors have been used to investigate its endocytosis pathways. The study saw positively charged NLGs entering cells via multiple endocytosis pathways, facilitating endosomal escape and slowly releasing siRNA into the cytoplasm. Transfection experiments confirmed that the crosslinked NLG delivery system provides effective transfection and prolonged silencing effect up to 14 days in cell cultures. We expect that this sustained-release siRNA NLG platform would be of interest in both fundamental biological studies and in clinical applications to extend the use of siRNA-based therapies.

DOI: https://doi.org/10.1038/s41598-022-07554-0
Biomater Sci. 2022 Feb 28.

Self-immolative polyplexes for DNA delivery.

Quinton E A Sirianni, TianDuo Wang, Aneta Borecki, Zhengyu Deng, John A Ronald, Elizabeth R Gillies.

Nucleic acids have immense potential for the treatment and prevention of a wide range of diseases, but delivery vehicles are needed to assist with their entry into cells. Polycations can reversibly complex with nucleic acids via ionic interactions to form polyplexes and transport them into cells, but they are still hindered by the need to balance cytotoxicity and delivery effectiveness. In this work, we describe a new self-immolative polyglyoxylamide (PGAm) platform designed to address these challenges by complexing nucleic acids via multivalent interactions in the polymeric form and releasing them upon depolymerization. Nine PGAms were synthesized and characterized, with different end-caps and variable cationic pendent groups. The PGAms underwent depolymerization under mildly acidic conditions, with rates dependent on their pendent groups and end-caps. They complexed plasmid DNA, forming cationic nanoparticles, and released it upon depolymerization. Cytotoxicity assays of the PGAms and polyplexes in HEK 293T cells showed a decrease in toxicity following depolymerization, and all samples exhibited much lower toxicity than a commercial non-degradable linear polyethyleneimine (jetPEI) transfection agent. Transfection assays revealed that selected PGAms provided similar levels of reporter gene expression to jetPEI in vitro with a PGAm analogue of poly[2-(dimethylamino)ethyl methacrylate] having particularly interesting activity that was dependent on depolymerization, along with low cytotoxicity. Overall, these results indicate that end-to-end depolymerization of self-immolative polymers can provide a new and promising tool for nucleic acid delivery.

DOI: https://doi.org/10.1039/d1bm01684a
Nanomedicine (Lond). 2022 Mar 03.

High-throughput synthesis and characterization of next-generation lipid nanoparticles for enhanced in vivo performance.

Bryant C Nelson, Sven Even Borgos.



Keywords:  DNA barcodes; antisense oligonucleotides; combinatorial synthesis; drug delivery; high-throughput screening; lipid nanoparticles; mRNA barcodes; mRNA-LNP; nanomedicines; nucleic acid delivery

DOI:  https://doi.org/10.2217/nnm-2022-0024
Mol Ther. 2022 Feb 28. pii: S1525-0016(22)00154-X. [Epub ahead of print]

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.

Rkia El-Kharrag, Kurt E Berckmueller, Ravishankar Madhu, Margaret Cui, Gabriela Campoy, Heather M Mack, Carl B Wolf, Anai M Perez, Olivier Humbert, Hans-Peter Kiem, Stefan Radtke.

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires GMP facilities similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NP), which could also be used for in vivo administration, for the delivery of mRNA and nucleases to human GCSF-mobilized CD34+ cells. NP-mediated ex vivo delivery showed no toxicity, and the efficiency directly correlated with the charge of nanoparticles. In a side-by-side comparison with electroporation, NP-mediated gene editing allowed for a 3-fold reduction in the amount of reagents with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.

DOI: https://doi.org/10.1016/j.ymthe.2022.02.026
Oncogene. 2022 Feb 26.

Targeted siRNA nanocarrier: a platform technology for cancer treatment.

Nicole Bäumer, Jessica Tiemann, Annika Scheller, Theresa Meyer, Lisa Wittmann, Matias Ezequiel Gutierrez Suburu, Lilo Greune, Matthias Peipp, Neele Kellmann, Annika Gumnior, Caroline Brand, Wolfgang Hartmann, Claudia Rossig, Carsten Müller-Tidow, Dario Neri, Cristian A Strassert, Christian Rüter, Petra Dersch, Georg Lenz, H Phillip Koeffler, Wolfgang E Berdel, Sebastian Bäumer.

The small arginine-rich protein protamine condenses complete genomic DNA into the sperm head. Here, we applied its high RNA binding capacity for spontaneous electrostatic assembly of therapeutic nanoparticles decorated with tumour-cell-specific antibodies for efficiently targeting siRNA. Fluorescence microscopy and DLS measurements of these nanocarriers revealed the formation of a vesicular architecture that requires presence of antibody-protamine, defined excess of free SMCC-protamine, and anionic siRNA to form. Only these complex nanoparticles were efficient in the treatment of non-small-cell lung cancer (NSCLC) xenograft models, when the oncogene KRAS was targeted via EGFR-mediated delivery. To show general applicability, we used the modular platform for IGF1R-positive Ewing sarcomas. Anti-IGR1R-antibodies were integrated into an antibody-protamine nanoparticle with an siRNA specifically against the oncogenic translocation product EWS/FLI1. Using these nanoparticles, EWS/FLI1 knockdown blocked in vitro and in vivo growth of Ewing sarcoma cells. We conclude that these antibody-protamine-siRNA nanocarriers provide a novel platform technology to specifically target different cell types and yet undruggable targets in cancer therapy by RNAi.

DOI: https://doi.org/10.1038/s41388-022-02241-w