bims-novged 2021-07-18 papers

on Non-viral vectors for gene delivery

Issue of 2021–07–18
nine papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Silica-coated calcium phosphate nanoparticles for gene silencing of NF-κB p65 by siRNA and their impact on cellular players of inflammation.
Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.
Synthesis of KUE-siRNA conjugates for prostate cancer cell-targeted gene silencing.
A folate-targeted PEGylated cyclodextrin-based nanoformulation achieves co-delivery of docetaxel and siRNA for colorectal cancer.
Substance P containing peptide gene delivery vectors for specifically transfecting glioma cells mediated by a neurokinin-1 receptor.
Ultrasound Image-guided Gene Delivery using Three-dimensional Diagnostic Ultrasound and Lipid-based Microbubbles.
Controlled CRISPR-Cas9 Ribonucleoprotein Delivery for Sensitized Photothermal Therapy.
Blockade of CD73 using siRNA loaded chitosan lactate nanoparticles functionalized with TAT-hyaluronate enhances doxorubicin mediated cytotoxicity in cancer cells both in vitro and in vivo.
Transfer of miRNA in tumor-derived exosomes suppresses breast tumor cell invasion and migration by inducing M1 polarization in macrophages.

Biomaterials. 2021 Jul 07. pii: S0142-9612(21)00369-0. [Epub ahead of print]276 121013

Silica-coated calcium phosphate nanoparticles for gene silencing of NF-κB p65 by siRNA and their impact on cellular players of inflammation.

Nataniel Białas, Elena K Müller, Matthias Epple, Ingrid Hilger.

  The transcription factor NF-κB and its signaling cascade both play key roles in all inflammatory processes. The most critical member of the NF-κB transcription factor family is p65. We investigated the role of cationic silica-coated calcium phosphate nanoparticles (spherical, diameter by SEM 50-60 nm; zeta potential about +26 mV; stabilized by polyethyleneimine) carrying encapsulated siRNA against NF-κB p65 and their influence on inflamed cells. The nanoparticles were taken up by cells of the blood compartment involved in the inflammatory response, particularly by monocytes, and to a lesser extent by endothelial cells and B-cells, but not by T-cells. The particles were found in endolysosomes where they were dissolved at low pH and released the siRNA into the cytoplasm. This was confirmed by dissolution experiments of model nanoparticles in simulated endolysosomal medium (pH 4.7) and by intracellular co-localization studies of double-labeled nanoparticles (using a negatively charged model peptide for siRNA). The encapsulated functional siRNA reverted the p65 gene and protein expression in inflamed monocytes, the main cells in immune response and surveillance, almost back to the non-inflammatory condition. Additionally, the nanoparticles suppressed the pro-inflammatory cytokine expression profiles (TNF-α, IL-6, IFN-β) in inflamed J774A.1 monocytes. Taken together, such nanoparticles can be applied for the treatment of inflammatory diseases.

Keywords:  Calcium phosphate; Gene silencing; Inflammation; NF-κB p65; Nanoparticles; Silica

DOI:  https://doi.org/10.1016/j.biomaterials.2021.121013
Nat Commun. 2021 07 09. 12(1): 4219

Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.

Moritz J Schmidt, Ashish Gupta, Christien Bednarski, Stefanie Gehrig-Giannini, Florian Richter, Christian Pitzler, Michael Gamalinda, Christina Galonska, Ryo Takeuchi, Kui Wang, Caroline Reiss, Kerstin Dehne, Michael J Lukason, Akiko Noma, Cindy Park-Windhol, Mariacarmela Allocca, Albena Kantardzhieva, Shailendra Sane, Karolina Kosakowska, Brian Cafferty, Jan Tebbe, Sarah J Spencer, Scott Munzer, Christopher J Cheng, Abraham Scaria, Andrew M Scharenberg, André Cohnen, Wayne M Coco.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

DOI: https://doi.org/10.1038/s41467-021-24454-5
Chembiochem. 2021 Jul 14.

Synthesis of KUE-siRNA conjugates for prostate cancer cell-targeted gene silencing.

Zhen Xi, Chao Yang, Dejun Ma, Liqing Lu, Xing Yang.

  The delivery of siRNAs to selectively target cells posed a great challenge in RNAi-based cancer therapy. The lack of suitable cell-targeting methods seriously restricted the advance in delivering siRNAs to extrahepatic tissues. Based on prostate-specific membrane antigen (PSMA)-targeting ligands, we here synthesized a series of KUE-siRNA conjugates and verified their effective cell uptake and gene silencing properties in prostate cancers. The results indicated that the KUE-siRNA conjugates could selectively enter PSMA + LNCaP cells, eventually down-regulating the STAT3 expression. Based on post-synthesis modification and receptor mediated endocytosis, this strategy of constructing ligand-siRNA conjugates might provide a general method of siRNA delivery for cell-targeted gene silencing.

Keywords:  Prostate cancer * PSMA-targeted * propargyl analog * KUE-siRNA conjugates * RNA interference

DOI:  https://doi.org/10.1002/cbic.202100243
Int J Pharm. 2021 Jul 13. pii: S0378-5173(21)00693-1. [Epub ahead of print] 120888

A folate-targeted PEGylated cyclodextrin-based nanoformulation achieves co-delivery of docetaxel and siRNA for colorectal cancer.

Yifang Zou, Fang Xiao, Liu Song, Bingxue Sun, Dandan Sun, Di Chu, Limei Wang, Shulan Han, Zhuo Yu, Caitriona M O'Driscoll, Jianfeng Guo.

  Docetaxel (DTX) is a chemotherapeutic agent used for a range of cancers, but it has little activity against colorectal cancer (CRC). However, combination therapy with other therapeutic agents is a potential strategy to enhance the efficacy of DTX in CRC treatment. The nuclear factor-κB (NF-κB) signaling pathway is implicated in a variety of malignancies (e.g., CRC), and the blockade of NF-κB may increase the sensitivity of cancer cells to chemotherapy. The application of small interference RNA (siRNA) to inhibit the translation of complementary mRNA has demonstrated the potential for cancer gene therapy. In this study, an amphiphilic cationic cyclodextrin (CD) nanoparticle modified with PEGylated folate (FA; a ligand to target folate receptor on CRC) has been developed for co-delivery of DTX and siRNA (against the RelA, a subunit of NF-κB) in the treatment of CRC. The resultant co-formulation (CD.DTX.siRelA.PEG-FA) achieved cell-specific uptake indicating the function of the folate targeting ligand. The CD.DTX.siRelA.PEG-FA nanoparticle enhanced the apoptotic effect of DTX with the downregulation of RelA expression, which significantly retarded the growth of CRC in mice, without causing significant toxicity. These results suggest that the FA-targeted PEGylated CD-based co-formulation provides a promising strategy for combining DTX and siRNA in treating CRC.

Keywords:  chemotherapy; colorectal cancer; combination therapy; gene therapy; nanoparticle

DOI:  https://doi.org/10.1016/j.ijpharm.2021.120888
J Mater Chem B. 2021 Jul 12.

Substance P containing peptide gene delivery vectors for specifically transfecting glioma cells mediated by a neurokinin-1 receptor.

Guihua Ding, Taoran Wang, Zhenbin Han, Long Tian, Qin Cheng, Longlong Luo, Baoquan Zhao, Chenhong Wang, Siliang Feng, Lianshuai Wang, Zhao Meng, Qingbin Meng.

Gene therapy provides a promising treatment for glioblastoma multiforme, which mainly depends on two key aspects, crossing the blood brain barrier (BBB) effectively and transfecting target cells selectively. In this work, we reported a series of peptide-based vectors for transfecting glioma cells specifically consisting of several functional segments including a cell-penetrating peptide, targeting segment substance P (SP), an endosomal escape segment, a PEG linker and a stearyl moiety. The conformations and DNA-loading capacities of peptide vectors and the self-assembly behaviors of peptide/pGL3 complexes were characterized. The in vitro gene transfection was evaluated in U87, 293T-NK1R, and normal 293T cell lines. The transfection efficiency ratio of P-02 (SP-PEG4-K(C18)-(LLHH)3-R9) to Lipo2000 in the U87 cell line was about 36% higher than that in the 293T cell line. The neurokinin-1 receptor (NK1R) in U87 cells mediated the transfection process via interactions with the ligand SP in peptide vectors. The mechanism of NK1R mediated transfection was demonstrated by the use of gene-modified 293T cells expressing NK1R, as well as the gene transfection in the presence of free SP. Besides, P-02 could promote the pGL3 plasmids to cross the BBB model in vitro and achieved the EGFP gene transfection in the brain of zebrafish successfully. The designed peptide vectors, owing to their specific transfection capacity in glioma cells, provide a potential approach for glioblastoma multiforme gene therapy.

DOI: https://doi.org/10.1039/d1tb00577d
J Drug Target. 2021 Jul 13. 1-26

Ultrasound Image-guided Gene Delivery using Three-dimensional Diagnostic Ultrasound and Lipid-based Microbubbles.

Daiki Omata, Lisa Munakata, Saori Kageyama, Yuno Suzuki, Tamotsu Maruyama, Tadamitsu Shima, Takumi Chikaarashi, Naoya Kajita, Kohji Masuda, Naoto Tsuchiya, Kazuo Maruyama, Ryo Suzuki.

  Gene therapy is a promising technology for genetic and intractable diseases. Drug delivery carriers or systems for genes and nucleic acids have been studied to improve transfection efficiency and achieve sufficient therapeutic effects. Ultrasound (US) and microbubbles have also been combined for use in gene delivery. To establish a clinically effective gene delivery system, exposing the target tissues to US is important. The three-dimensional (3D) diagnostic probe can three-dimensionally scan the tissue with mechanical regulation, and homogenous US exposure to the targeted tissue can be expected. However, the feasibility of therapeutically applying 3D probes has not been evaluated, especially gene delivery. In this study, we evaluated the characteristics of a 3D probe and lipid-based microbubbles (LB) for gene delivery and determined whether the 3D probe in the diagnostic US device could be used for efficient gene delivery to the targeted tissue using a mouse model. The 3D probe RSP6-16 with LB delivered pDNA to the kidney after systemic injection with luciferase activity similar to that of probes used in previously studies. No toxicity was observed after treatment and, therefore, the combined 3D probe and LB would deliver genes to targeted tissue safely and efficiently.

Keywords:  3D probe; diagnostic ultrasound device; gene delivery; microbubble; ultrasound

DOI:  https://doi.org/10.1080/1061186X.2021.1953510
Small. 2021 Jul 16. e2101155

Controlled CRISPR-Cas9 Ribonucleoprotein Delivery for Sensitized Photothermal Therapy.

Chao Chen, Yupei Ma, Shiyu Du, Yueyao Wu, Peiliang Shen, Tao Yan, Xueqing Li, Yujun Song, Zhengbao Zha, Xin Han.

  Manipulation of CRISPR delivery for stimuli-responsive gene editing is crucial for cancer therapeutics through maximizing efficacy and minimizing side-effects. However, realizing controlled gene editing for synergistic combination therapy remains a key challenge. Here, a near-infrared (NIR) light-triggered thermo-responsive copper sulfide (CuS) multifunctional nanotherapeutic platform is constructed to achieve controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and doxorubicin for tumor synergistic combination therapy involving in gene therapy, mild-photothermal therapy (PTT), and chemotherapy. The semiconductor CuS serves as a "photothermal converter" and can stably convert NIR light (808 nm) into local thermal effect to provide photothermal stimulation. The double-strand formed between CuS nanoparticle-linked DNA fragments and single-guide RNA is employed as a controlled element in response to photothermal stimulation for controlled gene editing and drug release. Hsp90α, one subunit of heat shock protein 90 (Hsp90), is targeted by Cas9 RNP to reduce tumor heat tolerance for enhanced mild-PTT effects (≈43 °C). Significant synergistic therapy efficacy can be observed by twice NIR light irradiation both in vitro and in vivo, compared to PTT alone. Overall, this exogenously controlled method provides a versatile strategy for controlled gene editing and drug release with potentially synergistic combination therapy.

Keywords:  CRISPR delivery; controlled release; mild-PTT; photothermal response; synergistic therapy

DOI:  https://doi.org/10.1002/smll.202101155
Int J Biol Macromol. 2021 Jul 07. pii: S0141-8130(21)01470-7. [Epub ahead of print]

Blockade of CD73 using siRNA loaded chitosan lactate nanoparticles functionalized with TAT-hyaluronate enhances doxorubicin mediated cytotoxicity in cancer cells both in vitro and in vivo.

Armin Mahmoud Salehi Khesht, Vahid Karpisheh, Parisa Sahami Gilan, Lyubov A Melnikova, Angelina Olegovna Zekiy, Mahdis Mohammadi, Mohammad Hojjat-Farsangi, Naime Majidi Zolbanin, Ata Mahmoodpoor, Hadi Hassannia, Leili Aghebati-Maleki, Reza Jafari, Farhad Jadidi-Niaragh.

  Chemotherapy drugs are still one of the first treatment options used in many cancers; however, problems such as cytotoxic side effects on normal cells after systemic administration and resistance to treatment have reduced the use of chemotherapeutics day by day. Targeted delivery of these drugs to the tumor site and sensitization of cancer cells to death induced by chemotherapy drugs are ways that can overcome the limitations of the use of these drugs. In this study, we designed and generated a novel nanocarrier composed of chitosan lactate nanoparticles (NPs) functionalized by HIV-1 derived TAT peptide (Transactivating transcriptional activator) and hyaluronate (HA) to deliver CD73 siRNA and doxorubicin to 4T1 and CT26 cancer cells, both in vivo and in vitro, as a novel combinatorial treatment strategy. The CD73 molecule plays a key role in many cancer cell behaviors such as proliferation, angiogenesis, metastasis, imunosuppression, and resistance to chemotherapy. Therefore, we decided to reduce the side effects of DOX by simultaneously transmitting CD73 siRNA and DOX by CL-TAT-HA NPs, increase the susceptibility of cancer cells to DOX-induced cell death, and stimulate anti-tumor immune responses, for the first time. These results indicated that simultaneous transfer of CD73 siRNA and DOX to cancer cells (4 T1 and CT26) increased cell death and inhibited the prolifration and spread of cancer cells. Also, the preferential aggregation of NPs in the tumor microenvironment reduced tumor growh, promoted the survival of tumor-bearing mice, and induced anti-tumor immune responses. These findings indicate that CL-TAT-HA NPs are a good candidate for targeted siRNA/drug delivery to cancer cells and the simultaneous transfer of CD73 siRNA and DOX to cancer cells using this nanocarrier can be used to treat cancer.

Keywords:  CD73; Cancer; Doxorubicin; Nanoparicle; siRNA

DOI:  https://doi.org/10.1016/j.ijbiomac.2021.07.034
Life Sci. 2021 Jul 07. pii: S0024-3205(21)00786-4. [Epub ahead of print] 119800

Transfer of miRNA in tumor-derived exosomes suppresses breast tumor cell invasion and migration by inducing M1 polarization in macrophages.

Maryam Moradi-Chaleshtori, Samaneh Shojaei, Samira Mohammadi-Yeganeh, Seyed Mahmoud Hashemi.

   AIMS: Macrophage repolarization from M1 to M2 phenotype is one of the hallmarks of malignancy. M2 macrophages are the most represented population in the tumor microenvironment and play an active role in tumor progression. In recent years, microRNAs (miRNAs) have been identified as a regulator of macrophage polarization.
MAIN METHODS: In this study, miR-130 was delivered to M2 macrophages using tumor-derived exosomes. Then, we evaluated the macrophage polarization status by assessment of specific markers and cytokines for M1 and M2 phenotype. The phagocytosis ability of macrophages was also investigated. Additionally, we performed migration and invasion assays to detect the effect of macrophage reprogramming on breast cancer cells migration and invasion.
KEY FINDINGS: The findings of the current study indicated that exosomes efficiently delivered miR-130 into macrophages. Delivery of miR-130 into macrophages resulted in upregulation of M1 specific markers and cytokines, including CD86, Irf5, Nos2, TNF-α, and IL-1β and downregulation of M2 specific markers and cytokines, including CD206, Ym1, Arg, TGF-β, and IL-10. The phagocytosis ability of macrophages also enhanced after treatment with miRNA-loaded exosomes. Furthermore, migration and invasion assays demonstrated reduced ability of 4T1 breast cancer cells for migration and invasion after macrophages reprogramming.
SIGNIFICANCE: These observations suggest that repolarization of M2 macrophages to M1 phenotype using miRNA-containing exosomes can be a therapeutic strategy against tumor invasion and metastasis in breast cancer.

Keywords:  Breast cancer; Exosome; Macrophage polarization; miRNA

DOI:  https://doi.org/10.1016/j.lfs.2021.119800