bims-novged 2022-01-30 papers

on Non-viral vectors for gene delivery

Issue of 2022–01–30
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Highly effective gene delivery based on cyclodextrin multivalent assembly in target cancer cells.
Linear-branched poly(β-amino esters)/DNA nano-polyplexes for effective gene transfection and neural stem cell differentiation.
Direct Comparison of Chol-siRNA Polyplexes and Chol-DsiRNA Polyplexes Targeting STAT3 in a Syngeneic Murine Model of TNBC.
Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles.
Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size.
Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery.
Principles for optimization and validation of mRNA lipid nanoparticle vaccines against COVID-19 using 3D bioprinting.
In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift.
Glutathione targeted tragacanthic acid-chitosan as a non-viral vector for brain delivery of miRNA-219a-5P: An in vitro/in vivo study.
Targeted Nanoparticle for Co-delivery of HER2 siRNA and a Taxane to Mirror the Standard Treatment of HER2+ Breast Cancer: Efficacy in Breast Tumor and Brain Metastasis.
A new liposomal nanocarrier for co-delivery of gedunin and p-glycoprotein siRNA to target breast cancer stem cells.

J Mater Chem B. 2022 Jan 24.

Highly effective gene delivery based on cyclodextrin multivalent assembly in target cancer cells.

Yao-Hua Liu, Yu Liu.

Nucleic acid condensation and controlled release remain significant challenges of gene therapy in the fields of chemical biology and nanotechnology. In this work, we have reported a polysaccharide supramolecular assembly constructed using upconversion nanoparticles encapsulated by β-cyclodextrin-grafted hyaluronic acid (HACD-UCNPs) and spermine modified with arylazopyrazoles (AAPS). Through UV-Vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), gel electrophoresis, confocal laser imaging and combination experiments, such an assembly can achieve not only nucleic acid condensation but also targeted cells delivery and controlled release. Furthermore, we investigated the ability of the system to deliver siRNA under hypoxic conditions, and the subsequent NIR irradiation regulation achieved the two-step release of RNA, obtaining the best effect. This strategy provides a new approach for nucleic acid condensation and targeted delivery, which may bring broad potential in gene therapy.

DOI: https://doi.org/10.1039/d1tb02585f
Biomed Mater. 2022 Jan 24.

Linear-branched poly(β-amino esters)/DNA nano-polyplexes for effective gene transfection and neural stem cell differentiation.

Yuheng Cao, Haobo He, Kaili Cao, Yongyang Liu, Dehua Huang, Tunwei Li, Guangcun Chen.

  Controllable regulation of stem cell differentiation is a critical concern in stem cell-based regenerative medicine. In particular, there are still great challenges in controlling the directional differentiation of neural stem cells (NSCs) into neurons. Herein, we developed a novel linear-branched poly(β-amino esters) (S4-TMPTA-BDA-DT, STBD) through a two-step reaction. The synthesized STBD linear branched polymers possess multiple positively charged amine terminus and degradable intermolecular ester bonds, thus endowing them with excellent properties such as high gene load, efficient gene delivery, and effective gene release and transcription in cells. In the mCherry transfection test, a high transfection efficiency of approximately 70% was achieved in primary NSCs after a single transfection. Moreover, STBD also showed high biocompatibility to NSCs without disturbing their viability and neural differentiation. With the high gene delivery property, STBD is capable of delivering siRNA (shSOX9) expression plasmid into NSCs to significantly interfere with the expression of SOX9, thus enhancing the neuronal differentiation and maturation of NSCs. The STBD/DNA nano-polyplex represents a powerful non-viral approach of gene delivery for manipulating the differentiation of stem cells, showing broad application prospects in NSC-based regenerative therapy for treating neurodegenerative diseases.

Keywords:  neural stem cells; neuronal differentiation; non-viral gene delivery; β-amino ester

DOI:  https://doi.org/10.1088/1748-605X/ac4e64
Noncoding RNA. 2022 Jan 13. pii: 8. [Epub ahead of print]8(1):

Direct Comparison of Chol-siRNA Polyplexes and Chol-DsiRNA Polyplexes Targeting STAT3 in a Syngeneic Murine Model of TNBC.

Zhen Ye, Mai Mohamed Abdelmoaty, Stephen M Curran, Shetty Ravi Dyavar, Devendra Kumar, Yazen Alnouti, Don W Coulter, Anthony T Podany, Rakesh K Singh, Joseph A Vetro.

  RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after intravenous (IV) administration. We previously found that polymer complexes (polyplexes) formed between 3'-cholesterol-modified siRNA (Chol-siRNA) or DsiRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase RNAi potency against stably expressed LUC mRNA in primary syngeneic murine breast tumors after daily IV dosing. Chol-DsiRNA polyplexes, however, maintain LUC mRNA suppression for ~48 h longer after the final dose than Chol-siRNA polyplexes, which suggests that they are the better candidate formulation. Here, we directly compared the activities of Chol-siRNA polyplexes and Chol-DsiRNA polyplexes in primary murine 4T1 breast tumors against STAT3, a therapeutically relevant target gene that is overexpressed in many solid tumors, including breast cancer. We found that Chol-siSTAT3 polyplexes suppressed STAT3 mRNA in 4T1 tumors with similar potency (half-maximal ED50 0.3 mg/kg) and kinetics (over 96 h) as Chol-DsiSTAT3 polyplexes, but with slightly lower activity against total Stat3 protein (29% vs. 42% suppression) and tumor growth (11.5% vs. 8.6% rate-based T/C ratio) after repeated IV administration of equimolar, tumor-saturating doses every other day. Thus, both Chol-siRNA polyplexes and Chol-DsiRNA polyplexes may be suitable clinical candidates for the RNAi therapy of breast cancer and other solid tumors.

Keywords:  Chol-DsiRNA polymer micelles; Chol-siRNA polymer micelles; DsiRNA delivery; RNAi; RNAi delivery; drug delivery; siRNA delivery

DOI:  https://doi.org/10.3390/ncrna8010008
J Control Release. 2022 Jan 22. pii: S0168-3659(22)00041-4. [Epub ahead of print]

Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles.

Martijn J W Evers, Wenjuan Du, Qiangbing Yang, Sander A A Kooijmans, Arjan Vink, Mies van Steenbergen, Pieter Vader, Saskia C A de Jager, Sabine A Fuchs, Enrico Mastrobattista, Joost P G Sluijter, Zhiyong Lei, Raymond Schiffelers.

  Lipid Nanoparticles (LNPs) are a promising drug delivery vehicle for clinical siRNA delivery. Modified mRNA (modRNA) has recently gained great attention as a therapeutic molecule in cardiac regeneration. However, for mRNA to be functional, it must first reach the diseased myocardium, enter the target cell, escape from the endosomal compartment into the cytosol and be translated into a functional protein. However, it is unknown if LNPs can effectively deliver mRNA, which is much larger than siRNA, to the ischemic myocardium. Here, we evaluated the ability of LNPs to deliver mRNA to the myocardium upon ischemia-reperfusion injury functionally. By exploring the bio-distribution of fluorescently labeled LNPs, we observed that, upon reperfusion, LNPs accumulated in the infarct area of the heart. Subsequently, the functional delivery of modRNA was evaluated by the administration of firefly luciferase encoding modRNA. Concomitantly, a significant increase in firefly luciferase expression was observed in the heart upon myocardial reperfusion when compared to sham-operated animals. To characterize the targeted cells within the myocardium, we injected LNPs loaded with Cre modRNA into Cre-reporter mice. Upon LNP infusion, Tdtomato+ cells, derived from Cre mediated recombination, were observed in the infarct region as well as the epicardial layer upon LNP infusion. Within the infarct area, most targeted cells were cardiac fibroblasts but also some cardiomyocytes and macrophages were found. Although the expression levels were low compared to LNP-modRNA delivery into the liver, our data show the ability of LNPs to functionally deliver modRNA therapeutics to the damaged myocardium, which holds great promise for modRNA-based cardiac therapies.

Keywords:  Lipid nanoparticles; Modified modRNA; Myocardial infarction; Reperfusion injury; Systemic delivery

DOI:  https://doi.org/10.1016/j.jconrel.2022.01.027
Pharm Res. 2022 Jan 26.

Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size.

Jiaxing Di, Zhili Du, Kangzeng Wu, Shanshan Jin, Xun Wang, Tonglei Li, Yuhong Xu.

   PURPOSE: Lipid nanoparticles (LNPs) are widely utilized as means to deliver mRNA molecules. However, metric connections between biodistribution and pharmacokinetics (PK) of the nanoparticle carrier and transgene expression dynamics remain largely unknown.
METHODS: LNPs containing mRNAs encoding the firefly luciferase gene were prepared with varying sizes. Biodistributions of injected LNPs in mice were measured by fluorescence bioimaging or liquid chromatography with tandem mass spectrometry. In addition, luciferase expression levels were determined by bioluminescence imaging and enzyme activity assays.
RESULTS: Some intramuscularly injected LNPs were found circulating in the system, resulting in accumulation in the liver and spleen, especially when the LNP sizes were relatively small. Bigger LNPs were more likely to remain at the injection site. Transgene expression in the liver was found most prominent compared with other organs and tissues.
CONCLUSIONS: Biomolecules such as mRNAs encapsulated in locally injected LNPs can reach other organs and tissues via systemic circulation. Gene expression levels are affected by the LNP biodistribution and pharmacokinetics (PK), which are further influenced by the particle size and injection route. As transfection efficiency varies in different organs, the LNP exposure and mRNA expression are not linearly correlated.

Keywords:  distribution; lipids nanoparticle; mRNA; transfection luciferase

DOI:  https://doi.org/10.1007/s11095-022-03166-5
Drug Deliv. 2022 Dec;29(1): 386-398

Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery.

Lidan Wang, Jingping Geng, Linlin Chen, Xiangli Guo, Tao Wang, Yanfen Fang, Bonn Belingon, Jiao Wu, Manman Li, Ying Zhan, Wendou Shang, Yingying Wan, Xuemei Feng, Xianghui Li, Hu Wang.

  The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency. We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro. After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo.

Keywords:  36 + GFP; Cell-permeable peptides (CPPs); Dot1l; plasmid DNA delivery; supercharged protein

DOI:  https://doi.org/10.1080/10717544.2022.2030430
Nano Today. 2022 Jan 21. 101403

Principles for optimization and validation of mRNA lipid nanoparticle vaccines against COVID-19 using 3D bioprinting.

Massimiliano Papi, Daniela Pozzi, Valentina Palmieri, Giulio Caracciolo.

  BioNTech/Pfizer's Comirnaty and Moderna's SpikeVax vaccines consist in mRNA encapsulated in lipid nanoparticles (LNPs). The modularity of the delivery platform and the manufacturing possibilities provided by microfluidics let them look like an instant success, but they are the product of decades of intense research. There is a multitude of considerations to be made when designing an optimal mRNA-LNP vaccine. Herein, we provide a brief overview of what is presently known and what still requires investigation to optimize mRNA LNP vaccines. Lastly, we give our perspective on the engineering of 3D bioprinted validation systems that will allow faster, cheaper, and more predictive vaccine testing in the future compared with animal models.

Keywords:  COVID-19; administration; bioprinting; lipid nanoparticles; mRNA; vaccines

DOI:  https://doi.org/10.1016/j.nantod.2022.101403
Biomaterials. 2021 Dec 29. pii: S0142-9612(21)00695-5. [Epub ahead of print]281 121339

In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift.

Azadeh Kheirolomoom, Aris J Kare, Elizabeth S Ingham, Ramasamy Paulmurugan, Elise R Robinson, Mo Baikoghli, Mohammed Inayathullah, Jai W Seo, James Wang, Brett Z Fite, Bo Wu, Spencer K Tumbale, Marina N Raie, R Holland Cheng, Lisa Nichols, Alexander D Borowsky, Katherine W Ferrara.

  Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Keywords:  Lipid nanoparticle; Reporter gene; T-cell activation; T-cell transfection; mRNA

DOI:  https://doi.org/10.1016/j.biomaterials.2021.121339
Int J Biol Macromol. 2022 Jan 20. pii: S0141-8130(22)00115-5. [Epub ahead of print]200 543-556

Glutathione targeted tragacanthic acid-chitosan as a non-viral vector for brain delivery of miRNA-219a-5P: An in vitro/in vivo study.

Nahal Shamaeizadeh, Jaleh Varshosaz, Mina Mirian, Mehdi Aliomrani.

  Multiple sclerosis (MS) is a progressive chronic demyelinating and neurodegenerative disease. The symptoms could only be diminished through stimulated remyelination. Although administration of microRNA-219a-5P (miR-219) seems to recover the damages, it is hampered by the challenging delivery of genes to the central nervous system across the blood-brain barrier. To enhance the CNS delivery of miR-219, a novel non-viral targeted vector was appraised by conjugating chitosan (Ch) to tragacanthic acid (TA) and glutathione (Glu). The nanoparticles were characterized and injected into the cuprizone model of MS mice to investigate the in vivo features of the resulting polyplex. Transmission electron microscopy, luxol fast blue staining, and proteolipid protein 1 (Plp1) overexpression confirmed more compact myelin sheaths following the administration of the targeted miR-219 nanoparticles and positron emission tomography (PET) scan also demonstrated the reduced inflammation and higher cell regeneration in the brain. Fluorescence microscopy and in vivo imaging were employed to identify miR-219 accumulation patterns in mice. The polyplex led to miR-219 overexpression, crystallin alpha B upregulation, and apolipoprotein E downregulation. It was concluded that glutathione targeted Ch/TA nanoparticles could be exploited as a feasible non-viral vector for miR-219 specific targeting to the brain, miR-219 overexpression and inflammation abatement in MS.

Keywords:  Multiple sclerosis; Tragacanthic acid; miR-219

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.01.100
Small. 2022 Jan 27. e2107550

Targeted Nanoparticle for Co-delivery of HER2 siRNA and a Taxane to Mirror the Standard Treatment of HER2+ Breast Cancer: Efficacy in Breast Tumor and Brain Metastasis.

Worapol Ngamcherdtrakul, Daniel S Bejan, William Cruz-Muñoz, Moataz Reda, Husam Y Zaidan, Natnaree Siriwon, Suphalak Marshall, Ruijie Wang, Molly A Nelson, Justin P C Rehwaldt, Joe W Gray, Kullervo Hynynen, Wassana Yantasee.

  The first-line treatment of advanced and metastatic human epidermal growth factor receptor type 2 (HER2+) breast cancer requires two HER2-targeting antibodies (trastuzumab and pertuzumab) and a taxane (docetaxel or paclitaxel). The three-drug regimen costs over $320,000 per treatment course, requires a 4 h infusion time, and has many adverse side effects, while achieving only 18 months of progression-free survival. To replace this regimen, reduce infusion time, and enhance efficacy, a single therapeutic is developed based on trastuzumab-conjugated nanoparticles for co-delivering docetaxel and siRNA against HER2 (siHER2). The optimal nanoconstruct has a hydrodynamic size of 100 nm and specifically treats HER2+ breast cancer cells over organ-derived normal cells. In a drug-resistant orthotopic HER2+ HCC1954 tumor mouse model, the nanoconstruct inhibits tumor growth more effectively than the docetaxel and trastuzumab combination. When coupled with microbubble-assisted focused ultrasound that transiently disrupts the blood brain barrier, the nanoconstruct inhibits the growth of trastuzumab-resistant HER2+ BT474 tumors residing in the brains of mice. The nanoconstruct has a favorable safety profile in cells and in mice. Combination therapies have become the cornerstone of cancer treatment and this versatile nanoparticle platform can co-deliver multiple therapeutic types to ensure that they reach the target cells at the same time to realize their synergy.

Keywords:  HER2+; breast cancer; nanoparticles; siRNAs; taxane

DOI:  https://doi.org/10.1002/smll.202107550
Nat Prod Res. 2022 Jan 27. 1-4

A new liposomal nanocarrier for co-delivery of gedunin and p-glycoprotein siRNA to target breast cancer stem cells.

Mohan Singh Rana, Meran Keshawa Ediriweera, Umapriyatharshini Rajagopalan, Desiree Nedra Karunaratne, Kamani Hemamala Tennekoon, Sameera Ranganath Samarakoon.

  Gedunin is a secondary metabolite found in neem tree. Since the first discovery of this compound, its bio-active properties have been continuously evaluated. However, the low hydrophobicity of gedunin decreases its bioavailability and pharmacokinetic profile. In the present investigation, a new liposomal nanocarrier for co-delivery of gedunin and P-glycoprotein (P-gp) siRNA [siRNA coated liposomal gedunin (Lipo-Ged-siRNA)] was developed to improve the anti-proliferative activity of gedunin. Characteristics of prepared Lipo-Ged-siRNA demonstrated promising effects. Lipo-Ged-siRNA showed greater anti-proliferative effects (IC50-8.5 µg/mL) followed by pure gedunin (IC50- 40.2 µg/mL) in breast cancer stem cells (bCSCs). Immunofluorescence analysis demonstrated reduced expression of P-gp following exposure to Lipo-Ged-siRNA. Furthermore, Lipo-Ged-siRNA affected the expression of ABCB1, Cyclin D1, Bax, p53, and surviving genes in bCSCs.

Keywords:  Gedunin; P-glycoprotein; breast cancer stem cells; co-delivery; liposomal nanocarrier

DOI:  https://doi.org/10.1080/14786419.2022.2032048