bims-novged 2022-10-09 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–10–09
twelve papers selected by
the Merkel lab, Ludwig-Maximilians University

RGD peptide-based lipids for targeted mRNA delivery and gene editing applications.
Development of Amino Acid-Modified Biodegradable Lipid Nanoparticles for siRNA Delivery.
Core-shell Structured Chitosan-polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency.
Transferrin-modified chitosan nanoparticles for targeted nose-to-brain delivery of proteins.
Machine Learning Guided Structure Function Predictions Enable In Silico Nanoparticle Screening for Polymeric Gene Delivery.
Phosphatidylserine Lipid Nanoparticles Promote Systemic RNA Delivery to Secondary Lymphoid Organs.
Nanoparticle-based CRISPR/Cas delivery: An emerging tactic for cancer therapy.
Synthesis of Double hydrophilic block copolymers poly(2-isopropyl-2-oxazoline-b-ethylenimine) and their DNA transfection efficiency.
Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates.
CRISPR Spherical Nucleic Acids.
The Mucoadhesive Nanoparticle-Based Delivery System in the Development of Mucosal Vaccines.
Current therapeutic strategies and possible effective drug delivery strategies against COVID-19.

RSC Adv. 2022 Sep 05. 12(39): 25397-25404

RGD peptide-based lipids for targeted mRNA delivery and gene editing applications.

Jingya Qin, Lulu Xue, Ningqiang Gong, Hanwen Zhang, Sarah J Shepherd, Rebecca M Haley, Kelsey L Swingle, Michael J Mitchell.

mRNA therapeutics are promising platforms for protein replacement therapies and gene editing technologies. When delivered via non-viral gene delivery systems, such as lipid nanoparticles (LNPs), mRNA therapeutics are easy to produce and show low toxicity and immunogenicity. However, LNPs show limited delivery efficiency and tissue specificity in certain applications. To overcome this, we designed RGD peptide (Arg-Gly-Asp) based ionizable lipids, which can be formulated into LNPs for integrin binding on cells and targeted mRNA delivery. RGD-LNPs were formulated using microfluidic devices and screened in vitro for size, mRNA encapsulation efficiency, transfection efficiency, and cell viability. A lead candidate, 1A RGD-based hybrid LNP, showed effective mRNA encapsulation and transfection, and was selected for further testing, including the co-delivery of Cas9 mRNA and sgRNA for gene editing applications. In vitro, 1A RGD-based hybrid LNP outperformed a non-targeted control LNP and showed GFP knockout efficiencies up to 90%. Further, the improved cellular uptake was reversed in the presence of soluble RGD, supporting the hypothesis that this improved uptake is RGD-dependent. In vivo, 1A RGD-based hybrid LNPs showed comparable mRNA delivery to the liver and spleen, when compared to a non-targeted control, and had increased expression in the whole body. Overall, this RGD-based hybrid LNP system is a promising platform for targeted mRNA delivery, which may allow for mRNA-based protein replacement and gene editing in a more efficient and specific manner with reduced off-target effects.

DOI: https://doi.org/10.1039/d2ra02771b
Acta Biomater. 2022 Sep 30. pii: S1742-7061(22)00637-7. [Epub ahead of print]

Development of Amino Acid-Modified Biodegradable Lipid Nanoparticles for siRNA Delivery.

Pratikkumar Patel, John Fetse, Chien-Yu Lin, Yuhan Guo, Md Reaid Hasan, Maryam Nakhjiri, Zhen Zhao, Akshay Jain, Kun Cheng.

  The use of siRNA therapeutics to treat cancer is a very promising approach. However, specific delivery of siRNAs to tumors remains a major challenge. The recent success of siRNA delivery to the liver has incentivized the development of biomaterials for siRNA delivery into tumors. Here, we report a new class of amino acid-modified lipids for siRNA delivery to cancer cells. Eight lipids were developed by headgroup modification with histidine and lysine. The lipids were screened in PC3-luciferase stable cells for gene silencing and cellular cytotoxicity study. The best lipid LHHK shows a pKa of 6.08, which is within the optimal pKa range of lipid nanoparticles (LNPs) for siRNA delivery. The LHHK LNP protects siRNA from serum degradation for up to 24 hours and shows higher endosomal release and better cellular uptake compared to other lysine-modified lipids in PC3 cells. The LHHK LNP exhibits significant silencing activity of IKKα and IKBKE in prostate cancer and pancreatic cancer, respectively. Moreover, the LHHK LNP encapsulating IKBKE siRNA inhibits cell proliferation of pancreatic cancer cells and suppress the tumor progression in a pancreatic cancer mouse model. These results suggest that amino acid-modified lipids possess a great potential for siRNA delivery in cancer therapy. STATEMENT OF SIGNIFICANCE: Lipid nanoparticle (LNP) is a promising platform for siRNA delivery. However, LNP is generally associated with high systemic toxicity. As a result, efficient and biodegradable lipids are highly needed for siRNA-based cancer therapy. Herein, we develop amino acid-modified biodegradable lipids. These lipids show very low cellular toxicity and high transfection efficiency. The best lipid LHHK shows a pKa of 6.08, which is within the optimal pKa range of LNPs for siRNA delivery. The LHHK LNP efficiently silences IKKα and IKBKE in prostate and pancreatic cancer, respectively. Moreover, the LHHK LNP encapsulating IKBKE siRNA inhibits cell proliferation and suppresses tumor growth of pancreatic cancer in vivo. These results suggest that amino acid-modified lipids possess a great potential for siRNA delivery in cancer therapy.

Keywords:  Amino acid; lipid nanoparticle; pKa; pancreatic cancer; prostate cancer; siRNA

DOI:  https://doi.org/10.1016/j.actbio.2022.09.065
Macromol Biosci. 2022 Oct 06. e2200314

Core-shell Structured Chitosan-polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency.

Jens Casper, Laura Nicolle, Melanie Willimann, Evrim Ümit Kuzucu, Alan Tran, Perrine Robin, Pascal Detampel, Hiu Man Grisch-Chan, Beat Thöny, Jörg Huwyler, Sandrine Gerber-Lemaire.

  Gene therapy emerged as a promising treatment option for acquired and inherited diseases. The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially non-viral gene delivery systems are of increasing interest. However, accomplishing therapeutic transgene expression levels and limited tolerability of these systems remain a challenge. Therefore, we investigate in the improvement of nucleic acid delivery using depolymerized chitosan - polyethylenimine DNA complexes (dCS-PEI/DNA). These core complexes are further entrapped into chitosan-based shells, functionalized with polyethylene glycol and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physico-chemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-LPEI/DNA complex with a shell consisting of dCS-PEG-COOH, which resulted in improved nucleic acid encapsulation, cellular uptake, transfection efficiency, and transfection potency in human hepatoma HuH-7 cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induce a high reporter gene signal for three days. We conclude that ternary core-shell structured particles comprising functionalized chitosan are a promising gene delivery technology for both in vitro as well as in vivo applications. This article is protected by copyright. All rights reserved.

Keywords:  Chitosan; Gene delivery; Nanoparticles; Polyethylenimine; Ternary complex

DOI:  https://doi.org/10.1002/mabi.202200314
Drug Deliv Transl Res. 2022 Oct 07.

Transferrin-modified chitosan nanoparticles for targeted nose-to-brain delivery of proteins.

Bettina Gabold, Friederike Adams, Sophie Brameyer, Kirsten Jung, Christian L Ried, Thomas Merdan, Olivia M Merkel.

  Nose-to-brain delivery presents a promising alternative route compared to classical blood-brain barrier passage, especially for the delivery of high molecular weight drugs. In general, macromolecules are rapidly degraded in physiological environment. Therefore, nanoparticulate systems can be used to protect biomolecules from premature degradation. Furthermore, targeting ligands on the surface of nanoparticles are able to improve bioavailability by enhancing cellular uptake due to specific binding and longer residence time. In this work, transferrin-decorated chitosan nanoparticles are used to evaluate the passage of a model protein through the nasal epithelial barrier in vitro. It was demonstrated that strain-promoted azide-alkyne cycloaddition reaction can be utilized to attach a functional group to both transferrin and chitosan enabling a rapid covalent surface-conjugation under mild reaction conditions after chitosan nanoparticle preparation. The intactness of transferrin and its binding efficiency were confirmed via SDS-PAGE and SPR measurements. Resulting transferrin-decorated nanoparticles exhibited a size of about 110-150 nm with a positive surface potential. Nanoparticles with the highest amount of surface bound targeting ligand also displayed the highest cellular uptake into a human nasal epithelial cell line (RPMI 2650). In an air-liquid interface co-culture model with glioblastoma cells (U87), transferrin-decorated nanoparticles showed a faster passage through the epithelial cell layer as well as increased cellular uptake into glioblastoma cells. These findings demonstrate the beneficial characteristics of a specific targeting ligand. With this chemical and technological formulation concept, a variety of targeting ligands can be attached to the surface after nanoparticle formation while maintaining cargo integrity.

Keywords:  Brain delivery; Chitosan nanoparticles; Glioblastoma; Nose-to-brain; Transferrin receptor

DOI:  https://doi.org/10.1007/s13346-022-01245-z
Acta Biomater. 2022 Oct 04. pii: S1742-7061(22)00644-4. [Epub ahead of print]

Machine Learning Guided Structure Function Predictions Enable In Silico Nanoparticle Screening for Polymeric Gene Delivery.

Dennis Gong, Elana Ben-Akiva, Arshdeep Singh, Hannah Yamagata, Savannah Est-Witte, Julie K Shade, Natalia A Trayanova, Jordan J Green.

  Developing highly efficient non-viral gene delivery reagents is still difficult for many hard-to-transfect cell types and, to date, has mostly been conducted via brute force screening routines. High throughput in silico methods of evaluating biomaterials can enable accelerated optimization and development of devices or therapeutics by exploring large chemical design spaces quickly and at low cost. This work reports application of state-of-the-art machine learning algorithms to a dataset of synthetic biodegradable polymers, poly(beta-amino ester)s (PBAEs), which have shown exciting promise for therapeutic gene delivery in vitro and in vivo. The data set includes polymer properties as inputs as well as polymeric nanoparticle transfection performance and nanoparticle toxicity in a range of cells as outputs. This data was used to train and evaluate several state-of-the-art machine learning algorithms for their ability to predict transfection and understand structure-function relationships. By developing an encoding scheme for vectorizing the structure of a PBAE polymer in a machine-readable format, we demonstrate that a random forest model can satisfactorily predict DNA transfection in vitro based on the chemical structure of the constituent PBAE polymer in a cell line dependent manner. Based on the model, we synthesized PBAE polymers and used them to form polymeric gene delivery nanoparticles that were predicted in silico to be successful. We validated the computational predictions in two cell lines in vitro, RAW 264.7 macrophages and Hep3B liver cancer cells, and found that the Spearman's R correlation between predicted and experimental transfection was 0.57 and 0.66 respectively. Thus, a computational approach that encoded chemical descriptors of polymers was able to demonstrate that in silico computational screening of polymeric nanomedicine compositions had utility in predicting de novo biological experiments. STATEMENT OF SIGNIFICANCE: : Developing highly efficient non-viral gene delivery reagents is difficult for many hard-to-transfect cell types and, to date, has mostly been explored via brute force screening routines. High throughput in silico methods of evaluating biomaterials can enable accelerated optimization and development for therapeutic or biomanufacturing purposes by exploring large chemical design spaces quickly and at low cost. This work reports application of state-of-the-art machine learning algorithms to a large compiled PBAE DNA gene delivery nanoparticle dataset across many cell types to develop predictive models for transfection and nanoparticle cytotoxicity. We develop a novel computational pipeline to encode PBAE nanoparticles with chemical descriptors and demonstrate utility in a de novo experimental context.

Keywords:  computational; gene delivery; library; machine learning; nanoparticle; poly(beta-amino ester); polymer

DOI:  https://doi.org/10.1016/j.actbio.2022.09.072
Nano Lett. 2022 Oct 04.

Phosphatidylserine Lipid Nanoparticles Promote Systemic RNA Delivery to Secondary Lymphoid Organs.

Sijin Luozhong, Zhefan Yuan, Tara Sarmiento, Yu Chen, Wenchao Gu, Caleb McCurdy, Wenting Gao, Ruoxin Li, Stephan Wilkens, Shaoyi Jiang.

  Secondary lymphoid organs (SLOs) are an important target for mRNA delivery in various applications. While the current delivery method relies on the drainage of nanoparticles to lymph nodes by intramuscular (IM) or subcutaneous (SC) injections, an efficient mRNA delivery carrier for SLOs-targeting delivery by systemic administration (IV) is highly desirable but yet to be available. In this study, we developed an efficient SLOs-targeting carrier using phosphatidylserine (PS), a well-known signaling molecule that promotes the endocytic activity of phagocytes and cellular entry of enveloped viruses. We adopted these biomimetic strategies and added PS into the standard four-component MC3-based LNP formulation (PS-LNP) to facilitate the cellular uptake of immune cells beyond the charge-driven targeting principle commonly used today. As a result, PS-LNP performed efficient protein expression in both lymph nodes and the spleen after IV administration. In vitro and in vivo characterizations on PS-LNP demonstrated a monocyte/macrophage-mediated SLOs-targeting delivery mechanism.

Keywords:  cell-targeting delivery; lipid nanoparticles; mRNA delivery; secondary lymphoid organs

DOI:  https://doi.org/10.1021/acs.nanolett.2c03234
Curr Med Chem. 2022 Oct 06.

Nanoparticle-based CRISPR/Cas delivery: An emerging tactic for cancer therapy.

Fahima Dilnawaz, Sarbari Acharya.

  Genome editing arose as a new promising approach for the treatment of innumerable intricate ailments including cancer. Over the past couple of decades, delivery technologies that have serendipitously been developed using viral vectors are successful to some extent in protein and nucleic acid delivery but their effectiveness still lags due to their efficiency, tissue targeting capabilities, and toxicity which must be further improved. With the infiltration of nanotechnology into every sphere of life, nano-vehicles can be implemented as an ideal modality that can overcome challenges, also can be introspective as new genome editing tools for cancer therapy owing to the safety and efficiency in clinical settings. Such projected substitution can help in developing highly efficacious therapy regimes which are successful in clinical settings. This emerging approach of incorporation of genome editors (CRISPR/Cas) in different nano vehicles and their utility in targeting various aspects of cancer therapy like treatment, diagnostics, modelling has been comprehensively done in this review.

Keywords:  CRISPER; Cas9; cancer; gene editing; nanoparticles; nanotechnology

DOI:  https://doi.org/10.2174/0929867329666221006112615
Macromol Biosci. 2022 Oct 03. e2200296

Synthesis of Double hydrophilic block copolymers poly(2-isopropyl-2-oxazoline-b-ethylenimine) and their DNA transfection efficiency.

Gwendoline Delecourt, Laetitia Plet, Yann Le Guen, Ozgul Tezgel, Guillaume Tresset, Patrick Midoux, Tristan Montier, Véronique Bennevault, Philippe Guégan.

  Gene delivery is now a part of our therapeutic arsenal for vaccination and treatments of inherited or acquired diseases. Polymers represent an opportunity to develop new synthetic vectors for gene transfer, with a prerequisite of improved delivery and reduced toxicity compared to existing polymers. Here, we report the synthesis in a two-step's procedure of linear poly(ethylenimine-b-2-isopropyl-2-oxazoline) block copolymers with the linear polyethylenimine (lPEI) block of various molar masses; the molar mass of the poly(2-isopropyl-2-oxazoline) (PiPrOx) block been set to 7 kg.mol-1 . Plasmid DNA condensation is successfully achieved, and in vitro transfection efficiency of the copolymers is at least comparable to that obtained with the lPEI of same molar mass. lPEI-b-PiPrOx block copolymers are however less cytotoxic than their linear counterparts. PiPrOx could be a good alternative to PEG which is often used in drug delivery systems. The grafting of histidine moieties on the lPEI block of lPEI-b-PiPrOx does not provide any real improvement of the transfection efficiency. A weak DNA condensation is observed, due to increased steric hindrance along the lPEI backbone. The low cytotoxicity of lPEI-b-PiPrOx makes of this family a good candidate for future gene delivery developments. This article is protected by copyright. All rights reserved.

Keywords:  Oxazoline; PEGylation alternative; PEI; gene therapy

DOI:  https://doi.org/10.1002/mabi.202200296
Mol Ther Nucleic Acids. 2022 Sep 24.

Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates.

Yuta Suzuki, Takayuki Miyazaki, Hiroki Muto, Kenji Kubara, Yohei Mukai, Ryuji Watari, Shinya Sato, Keita Kondo, Shin-Ichi Tsukumo, Koji Yasutomo, Masashi Ito, Kappei Tsukahara.

mRNA and lipid nanoparticles have emerged as powerful systems for the preparation of vaccines against SARS-CoV-2 infection. The emergence of novel variants or the necessity of cold chain logistics for approved mRNA vaccines undermines the investigation of next-generation systems that could preserve both potency and stability. However, the correlation between lipid nanoparticle composition and activity is not fully explored. Here, we screened a panel of ionizable lipids in vivo and identified lead lipid nanoparticles with a branched-tail lipid structure. Buffer optimization allowed the determination of lyophilization conditions, where lipid nanoparticle-encapsulated mRNA encoding SARS-CoV-2 spike protein could induce robust immunogenicity in mice after one month of storage at 5°C and 25°C. Intramuscularly injected lipid nanoparticles distributed in conventional dendritic cells in mouse lymph nodes induced balanced Th1/Th2 responses against SARS-CoV-2 spike protein. In nonhuman primates, two doses of 10 or 100 μg mRNA induced higher spike-specific binding geometric mean titers than those from a panel of SARS-CoV-2-convalescent human sera. Immunized sera broadly inhibited the viral entry receptor ACE2 from binding to the spike protein in all six strains tested, including variants of concern. These results could provide useful information for designing next-generation mRNA vaccines.

DOI: https://doi.org/10.1016/j.omtn.2022.09.017
J Am Chem Soc. 2022 Oct 06.

CRISPR Spherical Nucleic Acids.

Chi Huang, Zhenyu Han, Michael Evangelopoulos, Chad A Mirkin.

The use of CRISPR/Cas9 systems in genome editing has been limited by the inability to efficiently deliver the key editing components to and across tissues and cell membranes, respectively. Spherical nucleic acids (SNAs) are nanostructures that provide privileged access to both but have yet to be explored as a means of facilitating gene editing. Herein, a new class of CRISPR SNAs are designed and evaluated in the context of genome editing. Specifically, Cas9 ProSNAs comprised of Cas9 cores densely modified with DNA on their exteriors and preloaded with single-guide RNA were synthesized and evaluated for their genome editing capabilities in the context of multiple cell lines. The radial orientation of the DNA on the Cas9 protein surface enhances cellular uptake, without the need for electroporation or transfection agents. In addition, the Cas9 proteins defining the cores of the ProSNAs were fused with GALA peptides on their N-termini and nuclear localization signals on their C-termini to facilitate endosomal escape and maximize nuclear localization and editing efficiency, respectively. These constructs were stable against protease digestion under conditions that fully degrade the Cas9 protein, when not transformed into an SNA, and used to achieve genome editing efficiency between 32 and 47%. Taken together, these novel constructs and advances point toward a way of significantly broadening the scope of use and impact of CRISPR-Cas9 genome editing systems.

DOI: https://doi.org/10.1021/jacs.2c07913
Int J Nanomedicine. 2022 ;17 4579-4598

The Mucoadhesive Nanoparticle-Based Delivery System in the Development of Mucosal Vaccines.

Kai Zhao, Yinzhuo Xie, Xuezheng Lin, Wei Xu.

  Mucosal tissue constitutes the largest interface between the body and the external environment, regulating the entry of pathogens, particles, and molecules. Mucosal immunization is the most effective way to trigger a protective mucosal immune response. However, the majority of the currently licensed vaccines are recommended to be administered by intramuscular injection, which has obvious shortcomings, such as high production costs, low patient compliance, and lack of mucosal immune response. Strategies for eliciting mucosal and systemic immune responses are being developed, including appropriate vaccine adjuvant, delivery system, and bacterial or viral vectors. Biodegradable mucoadhesive nanoparticles (NPs) are the most promising candidate for vaccine delivery systems due to their inherent immune adjuvant property and the ability to protect the antigen from degradation, sustain the release of loaded antigen, and increase the residence time of antigen at the administration site. The current review outlined the complex structure of mucosa, the mechanism of interaction between NPs and mucosa, factors affecting the mucoadhesion of NPs, and the application of the delivery system based on mucoadhesive NPs in the field of vaccines. Moreover, this review demonstrated that the biodegradable and mucoadhesive NP-based delivery system has the potential for mucosal administration of vaccines.

Keywords:  delivery system; mucosal adhesion; mucosal immunization; mucosal vaccine; nanoparticles

DOI:  https://doi.org/10.2147/IJN.S359118
Curr Drug Deliv. 2022 Oct 04.

Current therapeutic strategies and possible effective drug delivery strategies against COVID-19.

Debabrata Ghosh Dastidar, Dipanjan Ghosh, Swarnarsree Ghosh, Gopal Chakrabarti.

  COVID-19 pandemic is the biggest global crisis. The frequent mutations in coronavirus to generate new mutants is of major concern. The pathophysiology of SARS-CoV-2 infection has been well studied to find out suitable molecular targets and candidate drugs for effective treatment. FDA-recommended etiotropic therapies are currently followed along with mass vaccination. The drug delivery system and the route of administration have a great role to enhance the efficacy of therapeutic agents and vaccines. Since COVID-19 primarily infects the lungs in the affected individuals, pulmonary administration may be the best possible route for the treatment of COVID-19. Liposomes, solid lipid nanoparticles, polymeric nanoparticles, porous microsphere, dendrimers, and nanoparticles encapsulated microparticles are the most suitable drug delivery systems for targeted drug delivery. The solubility, permeability, chemical stability, and biodegradability of drug molecules are the key factors for the right selection of suitable nanocarriers. The application of nanotechnology has been instrumental in the successful development of mRNA, DNA and subunit vaccines, as well as the delivery of COVID-19 therapeutic agents.

Keywords:  COVID-19; SARS-CoV-2; monoclonal antibody; plasma therapy; pulmonary administration; targeted drug delivery; vaccine.

DOI:  https://doi.org/10.2174/1567201819666221004094509