bims-novged 2022-03-13 papers

on Non-viral vectors for gene delivery

Issue of 2022–03–13
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Polyvinylamine with moderate binding affinity as a highly effective vehicle for RNA delivery.
Nanoparticles-mediated CRISPR/Cas gene editing delivery system.
Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.
Size and Charge Characterization of Lipid Nanoparticles for mRNA Vaccines.
Optimization of cationic polymer-mediated transfection for RNA interference.
Combinatorial Polycation Synthesis and Causal Machine Learning Reveal Divergent Polymer Design Rules for Effective pDNA and Ribonucleoprotein Delivery.
Visualization of the process of a nanocarrier-mediated gene delivery: stabilization, endocytosis and endosomal escape of genes for intracellular spreading.
Design and Development of Branched Poly(ß-aminoester) Nanoparticles for Interleukin-10 Gene Delivery in a Mouse Model of Atherosclerosis.
The Unexpected Importance of the Primary Structure of the Hydrophobic Part of One-Component Ionizable Amphiphilic Janus Dendrimers in Targeted mRNA Delivery Activity.
Metformin Bicarbonate-Mediated Efficient RNAi for Precise Targeting of TP53 Deficiency in Colon and Rectal Cancers.
Structural Fingerprinting of Short Interfering RNA Therapeutics by Solution Nuclear Magnetic Resonance Spectroscopy.
Development of a fibrin-mediated gene delivery system for the treatment of cystinosis via design of experiment.

J Control Release. 2022 Mar 03. pii: S0168-3659(22)00113-4. [Epub ahead of print]345 20-37

Polyvinylamine with moderate binding affinity as a highly effective vehicle for RNA delivery.

Ye Tian, Yipu Zhao, Chong Yin, Shenxing Tan, Xue Wang, Chaofei Yang, Tuo-Di Zhang, Xi Zhang, Fei Ye, Jing Xu, Xianglong Wu, Li Ding, Jie Zhang, Jiawei Pei, Xue-Ting Wang, Rui Xue Zhang, Jianrong Xu, Weisi Wang, Carlos D M Filipe, Todd Hoare, Da-Chuan Yin, Airong Qian, Xudong Deng.

  Polymeric carriers for RNA therapy offer potential advantages in terms of low immunogenicity, promoting modifiability and accelerating intracellular transport. However, balancing high transfection efficacy with low toxicity remains challenging with polymer-based vehicles; indeed, polyethyleneimine (PEI) remains the "gold standard" polymer for this purpose despite its significant toxicity limitations. Herein, we demonstrate the potential of polyvinylamine (PVAm), a commodity high-charge cationic polymer used in the papermaking industry and has similar structure with PEI, as an alternative carrier for RNA delivery. High levels of transfection of normal, tumor, and stem cells with a variety of RNA cargoes including small interfering RNA (siRNA), microRNA (miRNA), and recombinant RNA can be achieved in vitro under the proper complex conditions. While, both the anti-tumor effect achieved in a xenograft osteosarcoma model and lipid-lowering activity observed in a hyperlipidemia mice indicate the potential for highly effective in vivo activity. Of note, both the transfection efficiency and the cytotoxicity of PVAm compare more favorably with those of PEI, with PVAm offering the additional advantages of simpler purification and significantly lower cost. In addition, the mechanism for the difference in transfection efficiency between PVAm and PEI is explored by molecular docking as well as analyzing the process of association and dissociation between polymers (PVAm and PEI) and nucleic acids. Our research provides a novel, non-toxic, and cost-effective carrier candidate for next generation RNA therapy, and elucidates the potential mechanism of PVAm for its efficient delivery of RNA.

Keywords:  Binding affinity; Gene therapy; Polymeric nanocomplex; Polyvinylamine; RNA delivery; RNA-polymer interaction

DOI:  https://doi.org/10.1016/j.jconrel.2022.03.003
ChemMedChem. 2022 Mar 08.

Nanoparticles-mediated CRISPR/Cas gene editing delivery system.

Phei Er Saw, Guo-Hui Cui, Xiaoding Xu.

  The CRISPR/Cas gene editing system utilizes CRISPR RNA to guide the endonuclease in specifically breaking target gene, and then repairs genomic DNA by the means of homology directed repair (HDR) and non-homologous end joining (NHEJ). The gene editing system can only play its role in gene editing when it enters the nucleus. This crucial step in the process of gene editing is the major hurdle to gene therapy as it is still a huge challenge to efficiently deliver the CRISPR/Cas system to target tissues and cells. The low delivery efficiency hinders the clinical transformation of this technology. At present, delivery systems mainly include physical methods, viral vectors, and non-viral vectors. Due to the advantages of nanomaterial, it is currently being used rapidly in developing non-viral delivery systems. This review focuses on the mechanism of CRISPR/Cas and the delivery of gene editing system, following the research progress of nanoparticle-mediated gene editing.

Keywords:  CRISPR/Cas; gene editing; gene therapy; nanoparticle

DOI:  https://doi.org/10.1002/cmdc.202100777
J Control Release. 2022 Mar 02. pii: S0168-3659(22)00109-2. [Epub ahead of print]

Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing.

Se-Youl Chae, Euihwan Jeong, Seounghun Kang, Yeajee Yim, Jin-Soo Kim, Dal-Hee Min.

  Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.

Keywords:  Cas9 ribonucleoprotein; Gene knockout; Genome editing; Myostatin; Porous nanoparticle

DOI:  https://doi.org/10.1016/j.jconrel.2022.02.035
Anal Chem. 2022 Mar 07.

Size and Charge Characterization of Lipid Nanoparticles for mRNA Vaccines.

Camille Malburet, Laurent Leclercq, Jean-François Cotte, Jérôme Thiebaud, Emilie Bazin, Marie Garinot, Hervé Cottet.

Messenger RNA vaccines have come into the spotlight as a promising and adaptive alternative to conventional vaccine approaches. The efficacy of mRNA vaccines relies on the ability of mRNA to reach the cytoplasm of cells, where it can be translated into proteins of interest, allowing it to trigger the immune response. However, unprotected mRNA is unstable and susceptible to degradation by exo- and endonucleases, and its negative charges are electrostatically repulsed by the anionic cell membranes. Therefore, mRNA needs a delivery system that protects the nucleic acid from degradation and allows it to enter into the cells. Lipid nanoparticles (LNPs) represent a nonviral leading vector for mRNA delivery. Physicochemical parameters of LNPs, including their size and their charge, directly impact their in vivo behavior and, therefore, their cellular internalization. In this work, Taylor dispersion analysis (TDA) was used as a new methodology for the characterization of the size and polydispersity of LNPs, and capillary electrophoresis (CE) was used for the determination of LNP global charge. The results obtained were compared with those obtained by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE).

DOI: https://doi.org/10.1021/acs.analchem.1c04778
Genet Mol Biol. 2022 ;pii: S1415-47572022000200701. [Epub ahead of print]45(2): e20210237

Optimization of cationic polymer-mediated transfection for RNA interference.

Xiaojie Fan, Jingnan Yang, Guangyao Wu, Meiyi Wang, Xiaoxia Cheng, Chang Liu, Qian Liu, Yanan Wen, Shuangshuang Meng, Zhenxing Wang, Xuhong Lin, Lei An.

Transfection efficiency was estimated to optimize the conditions for RNA interference (RNAi), including transfection time, validity, and nucleic acid concentration and type, using the EZ Trans Cell Reagent, a cationic polymer. An shRNA against GFP was designed and transfected into cells using the EZ transfection reagent. The shRNA significantly decreased the expression of GFP. In addition, pre-diluted transfection reagent at room temperature and small nucleic acids increased the transfection efficiency, which peaked at 24 h. Compared with circular nucleic acids, linear nucleic acids showed higher transfection efficiency and a higher genome integration rate. We optimized cationic polymer-mediated RNAi conditions, and our data will be useful for future RNAi studies.

DOI: https://doi.org/10.1590/1678-4685-GMB-2021-0237
JACS Au. 2022 Feb 28. 2(2): 428-442

Combinatorial Polycation Synthesis and Causal Machine Learning Reveal Divergent Polymer Design Rules for Effective pDNA and Ribonucleoprotein Delivery.

Ramya Kumar, Ngoc Le, Felipe Oviedo, Mary E Brown, Theresa M Reineke.

The development of polymers that can replace engineered viral vectors in clinical gene therapy has proven elusive despite the vast portfolios of multifunctional polymers generated by advances in polymer synthesis. Functional delivery of payloads such as plasmids (pDNA) and ribonucleoproteins (RNP) to various cellular populations and tissue types requires design precision. Herein, we systematically screen a combinatorially designed library of 43 well-defined polymers, ultimately identifying a lead polycationic vehicle (P38) for efficient pDNA delivery. Further, we demonstrate the versatility of P38 in codelivering spCas9 RNP and pDNA payloads to mediate homology-directed repair as well as in facilitating efficient pDNA delivery in ARPE-19 cells. P38 achieves nuclear import of pDNA and eludes lysosomal processing far more effectively than a structural analogue that does not deliver pDNA as efficiently. To reveal the physicochemical drivers of P38's gene delivery performance, SHapley Additive exPlanations (SHAP) are computed for nine polyplex features, and a causal model is applied to evaluate the average treatment effect of the most important features selected by SHAP. Our machine learning interpretability and causal inference approach derives structure-function relationships underlying delivery efficiency, polyplex uptake, and cellular viability and probes the overlap in polymer design criteria between RNP and pDNA payloads. Together, combinatorial polymer synthesis, parallelized biological screening, and machine learning establish that pDNA delivery demands careful tuning of polycation protonation equilibria while RNP payloads are delivered most efficaciously by polymers that deprotonate cooperatively via hydrophobic interactions. These payload-specific design guidelines will inform further design of bespoke polymers for specific therapeutic contexts.

DOI: https://doi.org/10.1021/jacsau.1c00467
J Nanobiotechnology. 2022 Mar 09. 20(1): 124

Visualization of the process of a nanocarrier-mediated gene delivery: stabilization, endocytosis and endosomal escape of genes for intracellular spreading.

Zhongzheng Ma, Yang Zheng, Zijian Chao, Hongtao Chen, Yunhui Zhang, Meizhen Yin, Jie Shen, Shuo Yan.

  Nanoparticles have been widely applied as gene carrier for improving RNA interference (RNAi) efficiency in medical and agricultural fields. However, the mechanism and delivery process of nanoparticle-mediated RNAi is not directly visualized and elucidated. Here we synthesized a star polymer (SPc) consisted of a hydrophilic shell with positively-charged tertiary amine in the side chain, which was taken as an example to investigate the mechanism in gene delivery. The SPc could assemble with dsRNA spontaneously through electrostatic force, hydrogen bond and van der Waals force. Interestingly, the SPc could protect dsRNA from degradation by RNase A and insect hemolymph, thus remarkably increasing the stability of dsRNA. Meanwhile, the SPc could efficiently promote the cellular uptake and endosomal escape for intracellular spreading of dsRNA. Transcriptome analysis revealed that the SPc could up-regulate some key genes such as Chc, AP2S1 and Arf1 for activating clathrin-mediated endocytosis. Furthermore, the suppression of endocytosis hindered the cellular uptake of SPc-delivered dsRNA in vitro, and the subsequent RNAi effect was also disappeared in vivo. To our knowledge, our study is the first direct visualization of the detailed cellular delivery process and mechanism of nanocarrier-mediated gene delivery. Above mechanism supports the application of nanocarrier-based RNAi in gene therapy and pest management.

Keywords:  Cellular uptake; Clathrin; Endocytosis; Nanocarrier; RNA interference; dsRNA

DOI:  https://doi.org/10.1186/s12951-022-01336-6
Acta Biomater. 2022 Mar 04. pii: S1742-7061(22)00131-3. [Epub ahead of print]

Design and Development of Branched Poly(ß-aminoester) Nanoparticles for Interleukin-10 Gene Delivery in a Mouse Model of Atherosclerosis.

Nicholas Distasio, France Dierick, Talin Ebrahimian, Maryam Tabrizian, Stephanie Lehoux.

  Atherosclerosis progression is a result of chronic and non-resolving inflammation, effective treatments for which still remain to be developed. We designed and developed branched poly(ß-amino ester) nanoparticles (NPs) containing plasmid DNA encoding IL-10, a potent anti-inflammatory cytokine to atherosclerosis. The NPs (NP-VHPK) are functionalized with a targeting peptide (VHPK) specific for VCAM-1, which is overexpressed by endothelial cells at sites of atherosclerotic plaque. The anionic coating affords NP-VHPK with significantly lower toxicity than uncoated NPs in both endothelial cells and red blood cells (RBCs). Following injection of NP-VHPK in ApoE-/- mice, Cy5-labelled IL-10 significantly accumulates in both whole aortas and aortic sinus sections containing plaque compared to injection with a non-targeted control. Furthermore, IL-10 gene delivery results in an attenuation of inflammation locally at the plaque site. NP-VHPK may thus have the potential to reduce the inflammatory component of atherosclerosis in a safe and effective manner. STATEMENT OF SIGNIFICANCE: Atherosclerosis is a chronic inflammatory disease that results in the formation of lipid-laden plaques within vascular walls. Although treatments using drugs and antibodies are now beginning to address the inflammation in atherosclerosis, neither is sufficient for long-term therapy. In this paper, we introduce a strategy to deliver genes encoding the anti-inflammatory protein interleukin-10 (IL-10) in vivo. We showed that Branched Poly(ß-aminoester) carrying the IL-10 gene are able to localize specifically at the plaque via surface-functionalized targeting moieties against inflamed VCAM-1 and/or ICAM-1 and to facilitate gene transcription by ECs to increase the local concentration of the IL-10 within the plaque. To date, there is no report involving non-viral nanotechnology to provide gene-based therapies for atherosclerosis.

Keywords:  VCAM-1; anti-inflammatory cytokine; atherosclerosis; gene therapy; interleukin-10; poly(ß-amino ester); targeting; toxicity

DOI:  https://doi.org/10.1016/j.actbio.2022.02.043
J Am Chem Soc. 2022 Mar 09.

The Unexpected Importance of the Primary Structure of the Hydrophobic Part of One-Component Ionizable Amphiphilic Janus Dendrimers in Targeted mRNA Delivery Activity.

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

Viral and synthetic vectors for delivery of nucleic acids impacted genetic nanomedicine by aiding the rapid development of the extraordinarily efficient Covid-19 vaccines. Access to targeted delivery of nucleic acids is expected to expand the field of nanomedicine beyond most expectations. Both viral and synthetic vectors have advantages and disadvantages. The major advantage of the synthetic vectors is their unlimited synthetic capability. The four-component lipid nanoparticles (LNPs) are the leading nonviral vector for mRNA used by Pfizer and Moderna in Covid-19 vaccines. Their synthetic capacity inspired us to develop a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA. The first experiments on IAJDs provided, through a rational-library design combined with orthogonal-modular accelerated synthesis and sequence control in their hydrophilic part, some of the most active synthetic vectors for the delivery of mRNA to lung. The second experiments employed a similar strategy, generating, by a less complex hydrophilic structure, a library of IAJDs targeting spleen, liver, and lung. Here, we report preliminary studies designing the hydrophobic region of IAJDs by using dissimilar alkyl lengths and demonstrate the unexpectedly important role of the primary structure of the hydrophobic part of IAJDs by increasing up to 90.2-fold the activity of targeted delivery of mRNA to spleen, lymph nodes, liver, and lung. The principles of the design strategy reported here and in previous publications indicate that IAJDs could have a profound impact on the future of genetic nanomedicine.

DOI: https://doi.org/10.1021/jacs.2c00273
Nano Today. 2022 Apr;pii: 101406. [Epub ahead of print]43

Metformin Bicarbonate-Mediated Efficient RNAi for Precise Targeting of TP53 Deficiency in Colon and Rectal Cancers.

Jiangsheng Xu, Yunhua Liu, Sheng Liu, Wenquan Ou, Alisa White, Samantha Stewart, Katherine H R Tkaczuk, Lee M Ellis, Jun Wan, Xiongbin Lu, Xiaoming He.

  Colon and rectal cancers are the leading causes of cancer-related deaths in the United States and effective targeted therapies are in need for treating them. Our genomic analyses show hemizygous deletion of TP53, an important tumor suppressor gene, is highly frequent in both cancers, and the 5-year survival of patients with the more prevalent colon cancer is significantly reduced in the patients with the cancer harboring such deletion, although such reduction is not observed for rectal cancer. Unfortunately, direct targeting TP53 has been unsuccessful for cancer therapy. Interestingly, POLR2A, a gene essential for cell survival and proliferation, is almost always deleted together with TP53 in colon and rectal cancers. Therefore, RNA interference (RNAi) with small interfering RNAs (siRNAs) to precisely target/inhibit POLR2A may be an effective strategy for selectively killing cancer cells with TP53 deficiency. However, the difficulty of delivering siRNAs specifically into the cytosol where they perform their function, is a major barrier for siRNA-based therapies. Here, metformin bicarbonate (MetC) is synthesized to develop pH-responsive MetC-nanoparticles with a unique "bomb" for effective cytosolic delivery of POLR2A siRNA, which greatly facilitates its endo/lysosomal escape into the cytosol and augments its therapeutic efficacy of cancer harboring TP53 deficiency. Moreover, the MetC-based nanoparticles without functional siRNA show notable therapeutic effect with no evident toxicity or immunogenicity.

Keywords:  POLR2A; endosomal escape; gene delivery; metformin; targeted therapy

DOI:  https://doi.org/10.1016/j.nantod.2022.101406
Nucleic Acid Ther. 2022 Mar 09.

Structural Fingerprinting of Short Interfering RNA Therapeutics by Solution Nuclear Magnetic Resonance Spectroscopy.

Owen B Becette, Anh Tran, Jace W Jones, John P Marino, Robert G Brinson.

  Nucleic acids are an increasingly popular platform for the development of biotherapeutics to treat a wide variety of illnesses, including diseases where traditional drug development efforts have failed. To date, there are 14 short oligonucleotide therapeutics and 2 messenger RNA (mRNA) vaccines approved by the U.S. Food and Drug Administration (FDA), which demonstrates the potential of nucleic acids as a platform for the development of safe and effective medicines and vaccines. Despite the increasing popularity of nucleic acid-based drugs, there has been a paucity of high-resolution structural techniques applied to rigorously characterize these molecules during drug development. Here, we present application of nuclear magnetic resonance (NMR) methods to structurally "fingerprint" short oligonucleotide therapeutics at natural isotope abundance under full formulation conditions. The NMR methods described herein leverage signals arising from the native structural features of nucleic acids, including imino, aromatic, and ribose resonances, in addition to non-native chemistries, such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and phosphorothioate (PS) modifications, introduced during drug development. We demonstrate the utility of the NMR methods to structurally "fingerprint" a model short interfering RNA (siRNA) and a sample that simulated the drug product Givosiran. We anticipate broad applicability of the NMR methods to other nucleic acid-based therapeutics due to the generalized nature of the approach and ability to monitor many quality attributes simultaneously.

Keywords:  NMR fingerprinting; drug development; oligonucleotide therapeutics; quality attributes; siRNA; structure

DOI:  https://doi.org/10.1089/nat.2021.0098
Sci Rep. 2022 Mar 08. 12(1): 3752

Development of a fibrin-mediated gene delivery system for the treatment of cystinosis via design of experiment.

Valeria Graceffa.

Cystinosis is a rare disease, caused by a mutation in the gene cystinosin and characterised by the accumulation of cystine crystals. Advantages of biomaterial-mediated gene delivery include reduced safety concerns and the possibility to cure organs that are difficult to treat using systemic gene transfer methods. This study developed novel fibrin hydrogels for controlled, localised gene delivery, for the treatment of cystinosis. In the first part, fabrication parameters (i.e., DNA, thrombin, and aprotinin concentrations) were optimised, using a Design of Experiment (DOE) methodology. DOE is a statistical engineering approach to process optimisation, which increases experimental efficiency, reduces the number of experiments, takes into consideration interactions between different parameters, and allows the creation of predictive models. This study demonstrated the utility of DOE to the development of gene delivery constructs. In the second part of the study, primary fibroblasts from a patient with cystinosis were seeded on the biomaterials. Seeded cells expressed the recombinant CTNS and showed a decrease in cystine content. Furthermore, conditioned media contained functional copies of the recombinant CTNS. These were taken up by monolayer cultures of non-transfected cells. This study described a methodology to develop gene delivery constructs by using a DOE approach and ultimately provided new insights into the treatment of cystinosis.

DOI: https://doi.org/10.1038/s41598-022-07750-y