bims-novged 2021-10-24 papers

on Non-viral vectors for gene delivery

Issue of 2021–10–24
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Prospects of Cationic Carbosilane Dendronized Gold Nanoparticles as Non-viral Vectors for Delivery of Anticancer siRNAs siBCL-xL and siMCL-1.
Optimization of Synthesis of the Amino Lipid ECO for Effective Delivery of Nucleic Acids.
Development of a T cell-targeted siRNA delivery system against HIV-1 using modified superparamagnetic iron oxide nanoparticles: An in vitro study.
Orthogonal Design of Experiments for Optimization of Lipid Nanoparticles for mRNA Engineering of CAR T Cells.
Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers.
Ultrasonic particles: An approach for targeted gene delivery.
Designed Antitumor Peptide for Targeted siRNA Delivery into Cancer Spheroids.
Stimulus-Responsive Smart Nanoparticles-Based CRISPR-Cas Delivery for Therapeutic Genome Editing.
Asymmetric Silica Nanoparticles with Tailored Spiky Coverage Derived from Silica-Polymer Cooperative Assembly for Enhanced Hemocompatibility and Gene Delivery.
Ultrasound-targeted nucleic acid delivery for solid tumor therapy.
Enhanced cancer therapeutic efficiency of NO combined with siRNA by caspase-3 responsive polymers.
Nanoparticle-Mediated In Situ Molecular Reprogramming of Immune Checkpoint Interactions for Cancer Immunotherapy.

Pharmaceutics. 2021 Sep 24. pii: 1549. [Epub ahead of print]13(10):

Prospects of Cationic Carbosilane Dendronized Gold Nanoparticles as Non-viral Vectors for Delivery of Anticancer siRNAs siBCL-xL and siMCL-1.

Viktar Abashkin, Elżbieta Pędziwiatr-Werbicka, Rafael Gómez, Francisco Javier de la Mata, Volha Dzmitruk, Dzmitry Shcharbin, Maria Bryszewska.

  Cancer is one of the most important problems of modern medicine. At the present time, gene therapy has been developed against cancer, which includes the delivery of anticancer small interfering RNAs (siRNAs) directed at cancer proteins. The prospect of creating drugs based on RNA interference implies the use of delivery systems. Metal nanoparticles are the most studied objects for medicine, including their application as non-viral vectors. We have synthesized gold nanoparticles (AuNPs) modified with cationic carbosilane dendrons of 1-3 generations, with a positive charge on the surface, gold nanoparticles can effectively bind small interfering RNAs. Using a photometric viability test and flow cytometry, we assessed the ability of dendronized gold nanoparticles in delivering siRNAs to tumor cells. The efficiency of the complexes in initiating apoptosis was measured and, also, the overall effect of proapoptotic siRNA on cells. AuNP15 has both the highest efficacy and toxicity. The delivery efficiency in suspension cell lines was 50-60%. Complexes with targeted siRNA decreased cell viability by 20% compared to control and initiated apoptosis.

Keywords:  carbosilane dendrons; gene therapy; gold nanoparticles; siRNA

DOI:  https://doi.org/10.3390/pharmaceutics13101549
Pharmaceuticals (Basel). 2021 Oct 02. pii: 1016. [Epub ahead of print]14(10):

Optimization of Synthesis of the Amino Lipid ECO for Effective Delivery of Nucleic Acids.

Andrew L Schilb, Josef H Scheidt, Amita M Vaidya, Zhanhu Sun, Da Sun, Sangjoon Lee, Zheng-Rong Lu.

  Nucleic acids are promising for a variety of therapies, such as cancer therapy and the gene therapy of genetic disorders. The therapeutic efficacy of nucleic acids is reliant on the ability of their efficient delivery to the cytosol of the target cells. Amino lipids have been developed to aid in the cytosolic delivery of nucleic acids. This work reports a new and efficient synthetic pathway for the lipid carrier, (1-aminoethyl) iminobis [N-(oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO). The previous synthesis of the ECO was inefficient and presented poor product quality control. A solution-phase synthesis of the ECO was explored, and each intermediate product was characterized with better quality control. The ECO was synthesized with a relatively high yield and high purity. The formulations of the ECO nanoparticles were made with siRNA, miRNA, or plasmid DNA, and characterized. The transfection efficiency of the nanoparticles was evaluated in vitro over a range of N/P ratios. The nanoparticles were consistent in size with previous formulations and had primarily a positive zeta potential. The ECO/siLuc nanoparticles resulted in potent luciferase silencing with minimal cytotoxicity. The ECO/miR-200c nanoparticles mediated the efficient delivery of miR-200c into the target cells. The ECO/pCMV-GFP nanoparticles resulted in substantial GFP expression upon transfection. These results demonstrate that the solution-phase synthetic pathway produced pure ECO for the efficient intracellular delivery of nucleic acids without size limitation.

Keywords:  ECO; RNA interference; gene therapy; miRNA; plasmid; siRNA; synthesis

DOI:  https://doi.org/10.3390/ph14101016
J Pharm Sci. 2021 Oct 18. pii: S0022-3549(21)00559-1. [Epub ahead of print]

Development of a T cell-targeted siRNA delivery system against HIV-1 using modified superparamagnetic iron oxide nanoparticles: An in vitro study.

Sara Kamalzare, Vahid Iranpur Mobarakeh, Farnaz Sadat Mirzazadeh Tekie, Maliheh Hajiramezanali, Farhad Riazi-Rad, Sepideh Yoosefi, Zahra Normohammadi, Shiva Irani, Mohamadreza Tavakoli, Pooneh Rahimi, Fatemeh Atyabi.

  In spite of the promising properties of small interfering RNAs (siRNAs) in the treatment of infectious diseases, safe and efficient siRNA delivery to target cells is still a challenge. In this research, an effective siRNA delivery approach (against HIV-1) has been reported using targeted modified superparamagnetic iron oxide nanoparticles (SPIONs). Trimethyl chitosan-coated SPION (TMC-SPION) containing siRNA was synthesized and chemically conjugated to a CD4-specific monoclonal antibody (as a targeting moiety). The prepared nanoparticles exhibited a high siRNA loading efficiency with a diameter of about 85 nm and a zeta potential of +28 mV. The results of the cell viability assay revealed the low cytotoxicity of the optimized nanoparticles. The cellular delivery of the targeted nanoparticles (into T cells) and the gene silencing efficiency of the nanoparticles (containing anti-nef siRNA) were dramatically improved compared to those of nontargeted nanoparticles. In conclusion, this study offers a promising targeted delivery platform to induce gene silencing in target cells. Our approach may find potential use in the design of effective vehicles for many therapeutic applications, particularly for HIV treatment.

Keywords:  CD4; HIV-1 nef; SPION; siRNA delivery

DOI:  https://doi.org/10.1016/j.xphs.2021.10.018
Nano Lett. 2021 Oct 20.

Orthogonal Design of Experiments for Optimization of Lipid Nanoparticles for mRNA Engineering of CAR T Cells.

Margaret M Billingsley, Alex G Hamilton, David Mai, Savan K Patel, Kelsey L Swingle, Neil C Sheppard, Carl H June, Michael J Mitchell.

  Viral engineered chimeric antigen receptor (CAR) T cell therapies are potent, targeted cancer immunotherapies, but their permanent CAR expression can lead to severe adverse effects. Nonviral messenger RNA (mRNA) CAR T cells are being explored to overcome these drawbacks, but electroporation, the most common T cell transfection method, is limited by cytotoxicity. As a potentially safer nonviral delivery strategy, here, sequential libraries of ionizable lipid nanoparticle (LNP) formulations with varied excipient compositions were screened in comparison to a standard formulation for improved mRNA delivery to T cells with low cytotoxicity, revealing B10 as the top formulation with a 3-fold increase in mRNA delivery. When compared to electroporation in primary human T cells, B10 LNPs induced comparable CAR expression with reduced cytotoxicity while demonstrating potent cancer cell killing. These results demonstrate the impact of excipient optimization on LNP performance and support B10 LNPs as a potent mRNA delivery platform for T cell engineering.

Keywords:  CAR T; T cell engineering; lipid nanoparticles; mRNA delivery

DOI:  https://doi.org/10.1021/acs.nanolett.1c02503
J Am Chem Soc. 2021 Oct 21.

Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers.

Dapeng Zhang, Elena N Atochina-Vasserman, Devendra S Maurya, Matthew Liu, Qi Xiao, Juncheng Lu, George Lauri, Nathan Ona, Erin K Reagan, Houping Ni, Drew Weissman, Virgil Percec.

Targeted and efficient delivery of nucleic acids with viral and synthetic vectors is the key step of genetic nanomedicine. The four-component lipid nanoparticle synthetic delivery systems consisting of ionizable lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled by microfluidic or T-tube technology, have been extraordinarily successful for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA relying on amphiphilic Janus dendrimers and glycodendrimers developed in our laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple injection in acetate buffer, rather than by microfluidic devices, and provides a very efficient system for delivery of mRNA to lung. Here we report the replacement of most of the hydrophilic fragment of the dendron from IAJDs, maintaining only its ionizable amine, while changing its interconnecting group to the hydrophobic dendron from amide to ester. The resulting IAJDs demonstrated that protonated ionizable amines play dual roles of hydrophilic fragment and binding ligand for mRNA, changing delivery from lung to spleen and/or liver. Replacing the interconnecting ester with the amide switched the delivery back to lung. Delivery predominantly to liver is favored by pairs of odd and even alkyl groups in the hydrophobic dendron. This simple structural change transformed the targeted delivery of mRNA mediated with IAJDs, from lung to liver and spleen, and expands the utility of DNPs from therapeutics to vaccines.

DOI: https://doi.org/10.1021/jacs.1c09585
Adv Drug Deliv Rev. 2021 Oct 15. pii: S0169-409X(21)00391-4. [Epub ahead of print] 113998

Ultrasonic particles: An approach for targeted gene delivery.

Aidan P G Walsh, Henry N Gordon, Karlheinz Peter, Xiaowei Wang.

  Gene therapy has been widely investigated for the treatment of genetic, acquired, and infectious diseases. Pioneering work utilized viral vectors; however, these are suspected of causing serious adverse events, resulting in the termination of several clinical trials. Non-viral vectors, such as lipid nanoparticles, have attracted significant interest, mainly due to their successful use in vaccines in the current COVID-19 pandemic. Although they allow safe delivery, they come with the disadvantage of off-target delivery. The application of ultrasound to ultrasound-sensitive particles allows for a direct, site-specific transfer of genetic materials into the organ/site of interest. This process, termed ultrasound-targeted gene delivery (UTGD), also increases cell membrane permeability and enhances gene uptake. This review focuses on the advances in ultrasound and the development of ultrasonic particles for UTGD across a range of diseases. Furthermore, we discuss the limitations and future perspectives of UTGD.

Keywords:  gene transfer; microbubble; nucleic acid; sonoporation; targeted therapy; ultrasonic irradiation

DOI:  https://doi.org/10.1016/j.addr.2021.113998
ACS Appl Mater Interfaces. 2021 Oct 17.

Designed Antitumor Peptide for Targeted siRNA Delivery into Cancer Spheroids.

Silvia Cirillo, Mhd Anas Tomeh, Robert N Wilkinson, Chris Hill, Stephen Brown, Xiubo Zhao.

  Antimicrobial/anticancer peptides (AMPs/ACPs) have shown promising results as new therapeutic agents in cancer thearpy. Among them, the designed amphiphilic α-helical peptide G(IIKK)3I-NH2 (G3) displayed great affinity and specificity in targeting cancer cells. Here, we report new insights on how G3 penetrates cancer cells. G3 showed high specificity to HCT-116 colon cancer cells compared to the HDFs (human neonatal primary dermal fibroblasts) control. With high concentrations of peptide, a clear cancer cell membrane disruption was observed through SEM. Gene knockdown of the endocytic pathways demonstrated that an energy-dependent endocytic pathway is required for the uptake of the peptide. In addition, G3 can protect and selectively deliver siRNAs into cancer cells and successfully modulated their gene expression. Gene delivery was also tested in 3D cancer spheroids and showed deep penetration delivery into the cancer spheroids. Finally, the in vivo toxicity of G3 was evaluated on zebrafish embryos, showing an increasing toxicity effect with concentration. However, the toxicity of the peptide was attenuated when complexed with siRNA. In addition, negligible toxicity was observed at the concentration range for efficient gene delivery. The current results demonstrate that G3 is promising as an excellent agent for cancer therapy.

Keywords:  RNAi screening; antimicrobial/anticancer peptides; endocytic pathway; gene knockdown; siRNA delivery

DOI:  https://doi.org/10.1021/acsami.1c14761
Int J Mol Sci. 2021 Oct 19. pii: 11300. [Epub ahead of print]22(20):

Stimulus-Responsive Smart Nanoparticles-Based CRISPR-Cas Delivery for Therapeutic Genome Editing.

Muhammad Naeem, Mubasher Zahir Hoque, Muhammad Ovais, Chanbasha Basheer, Irshad Ahmad.

  The innovative research in genome editing domains such as CRISPR-Cas technology has enabled genetic engineers to manipulate the genomes of living organisms effectively in order to develop the next generation of therapeutic tools. This technique has started the new era of "genome surgery". Despite these advances, the barriers of CRISPR-Cas9 techniques in clinical applications include efficient delivery of CRISPR/Cas9 and risk of off-target effects. Various types of viral and non-viral vectors are designed to deliver the CRISPR/Cas9 machinery into the desired cell. These methods still suffer difficulties such as immune response, lack of specificity, and efficiency. The extracellular and intracellular environments of cells and tissues differ in pH, redox species, enzyme activity, and light sensitivity. Recently, smart nanoparticles have been synthesized for CRISPR/Cas9 delivery to cells based on endogenous (pH, enzyme, redox specie, ATP) and exogenous (magnetic, ultrasound, temperature, light) stimulus signals. These methodologies can leverage genome editing through biological signals found within disease cells with less off-target effects. Here, we review the recent advances in stimulus-based smart nanoparticles to deliver the CRISPR/Cas9 machinery into the desired cell. This review article will provide extensive information to cautiously utilize smart nanoparticles for basic biomedical applications and therapeutic genome editing.

Keywords:  CRISPR/Cas9 delivery; smart nanoparticles; stimulus CRISPR delivery; therapeutic genome editing

DOI:  https://doi.org/10.3390/ijms222011300
ACS Appl Mater Interfaces. 2021 Oct 19.

Asymmetric Silica Nanoparticles with Tailored Spiky Coverage Derived from Silica-Polymer Cooperative Assembly for Enhanced Hemocompatibility and Gene Delivery.

Xiuzhen Lin, Weixi Wu, Jianye Fu, Yannan Yang, Bing Guo, Chengzhong Yu, Hao Song.

  Asymmetric mesoporous silica nanoparticles (AMSNs) with one side featuring a spiky nanotopography, while the other side is smooth and solid, were synthesized via an ethylenediamine (EDA)-directed silica-polymer cooperative assembly approach. By simply varying the EDA amount (x), AMSNs-x samples with adjustable spiky surface coverage were obtained. It is demonstrated that a spiky coverage higher than 50% improved the hemocompatibility of AMSN-x, possibly due to the reduced contact area of the smooth side exposed to the red blood cell (RBC) membrane. Moreover, AMSNs-175 and AMSNs-200 with high spiky coverage enhanced their plasmid DNA (pDNA) loading and binding capability, as well as cellular uptake into HEK-293T cells, thus resulting in high transfection performance. The good hemocompatibility and high performance in pDNA delivery of AMSNs-x with high spiky coverage allow them to serve as promising nonviral vectors for potential applications in gene therapies and DNA vaccines.

Keywords:  asymmetric; gene delivery; hemolysis; mesoporous silica nanoparticles; spiky nanostructure

DOI:  https://doi.org/10.1021/acsami.1c13517
J Control Release. 2021 Oct 13. pii: S0168-3659(21)00539-3. [Epub ahead of print]

Ultrasound-targeted nucleic acid delivery for solid tumor therapy.

Mark R Schwartz, Anna C Debski, Richard J Price.

  Depending upon multiple factors, malignant solid tumors are conventionally treated by some combination of surgical resection, radiation, chemotherapy, and immunotherapy. Despite decades of research, therapeutic responses remain poor for many cancer indications. Further, many current therapies in our armamentarium are either invasive or accompanied by toxic side effects. In lieu of traditional pharmaceutics and invasive therapeutic interventions, gene therapies offer more flexible and potentially more durable approaches for new anti-cancer therapies. Nonetheless, many current gene delivery approaches suffer from low transfection efficiency due to physiological barriers limiting extravasation and uptake of genetic material. Additionally, systemically administered gene therapies may lack target-specificity, which can lead to off-target effects. To overcome these challenges, many preclinical studies have shown the utility of focused ultrasound (FUS) to increase macromolecule uptake in cells and tissue under image guidance, demonstrating promise for improved delivery of therapeutics to solid tumors. As FUS-based drug delivery is now being tested in several clinical trials around the world, FUS-targeted gene therapy for solid tumor therapy may not be far behind. In this review, we comprehensively cover the literature pertaining to preclinical attempts to more efficiently deliver therapeutic genetic material with FUS and offer perspectives for future studies and clinical translation.

Keywords:  Focused ultrasound; Gene delivery; Ultrasound; cancer

DOI:  https://doi.org/10.1016/j.jconrel.2021.10.010
J Control Release. 2021 Oct 13. pii: S0168-3659(21)00541-1. [Epub ahead of print]

Enhanced cancer therapeutic efficiency of NO combined with siRNA by caspase-3 responsive polymers.

Yi Yan, Cheng-Han Wang, Shi-He Cui, Lin Zhai, Jing Sun, Xiao-Yu Liu, Xin Chen, Yi Sun, Hong-Gang Qian, Xiang Gao, Yi-Da Tang, Yuan-Jun Zhu, Yu-Jie Shi, Qiang Zhang, Jian-Cheng Wang.

  The combination of nitric oxide (NO) and siRNA is highly desirable for cancer therapy. Here, the furoxans-grafted PEI polymer (FDP) with caspase-3 responsive cleavable DEVD linker was synthesized, and used to bind siRNAs via electrostatic interaction and self-assembled into FDP/siRNA nanoplexes by hydrophobic force. After cellular uptake and lysosomal escape, the FDP/siRNA nanoplexes could achieve GSH-triggered NO release, and then increase the activity of caspase-3. The activated caspase-3 could specifically cleave the DEVD peptide sequence and enhance cell apoptosis. With the cleavage of DEVD peptide sequence, the disassembly of FDP/siRNA nanoplexes was further promoted, thereby resulting in increased siRNAs of ~40% were released at 48 h compared with the caspase-3 non-responsive FDnP/siRNA nanoplexes. By this way, cell apoptosis promotion and cell proliferation inhibition was achieved by siRNA-based downregulation of EGFR protein and the upregulated activity of caspase-3, followed by the enhanced cascade release of NO from FDP/siRNA nanoplexes. Furthermore, in vivo results demonstrated the improved anti-cancer efficiency of FDP/siEGFR nanoplexes without any detectable side effects. Therefore, it is believed that the caspase-3 responsive cleavable furoxans-grafted PEI polymers could provide a potential and efficient enhancement for cancer therapeutic efficiency by the co-delivery of nitric oxide and siRNA.

Keywords:  Cancer therapy; Caspase-3 responsive; DEVD peptide; Nitric oxide; siRNA

DOI:  https://doi.org/10.1016/j.jconrel.2021.10.012
ACS Nano. 2021 Oct 22.

Nanoparticle-Mediated In Situ Molecular Reprogramming of Immune Checkpoint Interactions for Cancer Immunotherapy.

Adam A Walters, Gemma Santacana-Font, Jin Li, Nadia Routabi, Yue Qin, Nathalie Claes, Sara Bals, Julie Tzu-Wen Wang, Khuloud T Al-Jamal.

  Immune checkpoint blockade involves targeting immune regulatory molecules with antibodies. Preclinically, complex multiantibody regimes of both inhibitory and stimulatory targets are a promising candidate for the next generation of immunotherapy. However, in this setting, the antibody platform may be limited due to excessive toxicity caused by off target effects as a result of systemic administration. RNA can be used as an alternate to antibodies as it can both downregulate immunosuppressive checkpoints (siRNA) or induce expression of immunostimulatory checkpoints (mRNA). In this study, we demonstrate that the combination of both siRNA and mRNA in a single formulation can simultaneously knockdown and induce expression of immune checkpoint targets, thereby reprogramming the tumor microenvironment from immunosuppressive to immunostimulatory phenotype. To achieve this, RNA constructs were synthesized and formulated into stable nucleic acid lipid nanoparticles (SNALPs); the SNALPs produced were 140-150 nm in size with >80% loading efficiency. SNALPs could transfect macrophages and B16F10 cells in vitro resulting in 75% knockdown of inhibitory checkpoint (PDL1) expression and simultaneously express high levels of stimulatory checkpoint (OX40L) with minimal toxicity. Intratumoral treatment with the proposed formulation resulted in statistically reduced tumor growth, a greater density of CD4+ and CD8+ infiltrates in the tumor, and immune activation within tumor-draining lymph nodes. These data suggest that a single RNA-based formulation can successfully reprogram multiple immune checkpoint interactions on a cellular level. Such a candidate may be able to replace future immune checkpoint therapeutic regimes composed of both stimulatory- and inhibitory-receptor-targeting antibodies.

Keywords:  SNALP; immune checkpoint; immunotherapy; mRNA; siRNA

DOI:  https://doi.org/10.1021/acsnano.1c04456