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3776 KiB

Open AccessArticle

Novel PEI/Poly-γ-Gutamic Acid Nanoparticles for High Efficient siRNA and Plasmid DNA Co-Delivery

by Shu-Fen Peng, Hung-Kun Hsu, Chun-Cheng Lin, Ya-Ming Cheng and Kuang-Hsing Hsu

Molecules 2017, 22(1), 86; https://doi.org/10.3390/molecules22010086 - 04 Jan 2017

Cited by 21 | Viewed by 10119

The efficient delivery of sufficient amounts of nucleic acids into target cells is critical for successful gene therapy and gene knockdown. The DNA/siRNA co-delivery system has been considered a promising approach for cancer therapy to simultaneously express and inhibit tumor suppressor genes and [...] Read more.

The efficient delivery of sufficient amounts of nucleic acids into target cells is critical for successful gene therapy and gene knockdown. The DNA/siRNA co-delivery system has been considered a promising approach for cancer therapy to simultaneously express and inhibit tumor suppressor genes and overexpressed oncogenes, respectively, triggering synergistic anti-cancer effects. Polyethylenimine (PEI) has been identified as an efficient non-viral vector for transgene expression. In this study, we created a very high efficient DNA/siRNA co-delivery system by incorporating a negatively-charged poly-γ-glutamic acid (γ-PGA) into PEI/nucleic acid complexes. Spherical nanoparticles with about 200 nm diameter were formed by mixing PEI/plasmid DNA/siRNA/γ-PGA (dual delivery nanoparticles; DDNPs) with specific ratio (N/P/C ratio) and the particles present positive surface charge under all manufacturing conditions. The gel retardation assay shows both nucleic acids were effectively condensed by PEI, even at low N/P ratios. The PEI-based DDNPs reveal excellent DNA/siRNA transfection efficiency in the human hepatoma cell line (Hep 3B) by simultaneously providing high transgene expression efficiency and high siRNA silencing effect. The results indicated that DDNP can be an effective tool for gene therapy against hepatoma. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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931 KiB

Open AccessArticle

Synthesis of a Morpholino Nucleic Acid (MNA)-Uridine Phosphoramidite, and Exon Skipping Using MNA/2′-O-Methyl Mixmer Antisense Oligonucleotide

by Suxiang Chen, Bao T. Le, Kamal Rahimizadeh, Khalil Shaikh, Narinder Mohal and Rakesh N. Veedu

Molecules 2016, 21(11), 1582; https://doi.org/10.3390/molecules21111582 - 22 Nov 2016

Cited by 21 | Viewed by 7650

Abstract

In this study, we synthesised a morpholino nucleoside-uridine (MNA-U) phosphoramidite and evaluated the potential of a MNA-modified antisense oligonucleotide (AO) sequences to induce exon 23 skipping in mdx mouse myotubes in vitro towards extending the applicability of morpholino chemistry with other nucleotide monomers. [...] Read more.

In this study, we synthesised a morpholino nucleoside-uridine (MNA-U) phosphoramidite and evaluated the potential of a MNA-modified antisense oligonucleotide (AO) sequences to induce exon 23 skipping in mdx mouse myotubes in vitro towards extending the applicability of morpholino chemistry with other nucleotide monomers. We designed, synthesised, and compared exon skipping efficiencies of 20 mer MNA-modified 2′-O-methyl RNA mixmer AO on a phosphorothioate backbone (MNA/2′-OMePS) to the corresponding fully modified 2′-O-methyl RNA AO (2′-OMePS) as a control. Our results showed that the MNA/2′-OMePS efficiently induced exon 23 skipping. As expected, the 2′-OMePS AO control yielded efficient exon 23 skipping. Under the applied conditions, both the AOs showed minor products corresponding to exon 22/23 dual exon skipping in low yield. As these are very preliminary data, more detailed studies are necessary; however, based on the preliminary results, MNA nucleotides might be useful in constructing antisense oligonucleotides. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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3450 KiB

Open AccessArticle

Synthesis of Polymer-Lipid Nanoparticles by Microfluidic Focusing for siRNA Delivery

by Yujing Li, Xueqin Huang, Robert J. Lee, Yuhang Qi, Kaikai Wang, Fei Hao, Yu Zhang, Jiahui Lu, Qingfan Meng, Shuai Li, Jing Xie and Lesheng Teng

Molecules 2016, 21(10), 1314; https://doi.org/10.3390/molecules21101314 - 17 Oct 2016

Cited by 19 | Viewed by 6334

Abstract

Polyethylenimine (PEI) as a cationic polymer is commonly used as a carrier for gene delivery. PEI-800 is less toxic than PEI-25K but it is also less efficient. A novel nanocarrier was developed by combining PEI-800 with a pH-sensitive lipid to form polymer-lipid hybrid [...] Read more.

Polyethylenimine (PEI) as a cationic polymer is commonly used as a carrier for gene delivery. PEI-800 is less toxic than PEI-25K but it is also less efficient. A novel nanocarrier was developed by combining PEI-800 with a pH-sensitive lipid to form polymer-lipid hybrid nanoparticles (P/LNPs). They were synthesized by microfluidic focusing (MF). Two microfluidic devices were used to synthesize P/LNPs loaded with VEGF siRNA. A series of P/LNPs with different particle sizes and distributions were obtained by altering the flow rate and geometry of microfluidic chips, and introducing sonication. Furthermore, the P/LNPs can be loaded with VEGF siRNA efficiently and were stable in serum for 12 h. Finally, P/LNPs produced by the microfluidic chip showed greater cellular uptake as well as down-regulation of VEGF protein level in both A549 and MCF-7 with reduced cellular toxicity. All in all, the P/LNPs produced by MF method were shown to be a safe and efficient carrier for VEGF siRNA, with potential application for siRNA therapeutics. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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2813 KiB

Open AccessArticle

Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine

by Yuran Xie, Bryan Killinger, Anna Moszczynska and Olivia M. Merkel

Molecules 2016, 21(10), 1334; https://doi.org/10.3390/molecules21101334 - 10 Oct 2016

Cited by 34 | Viewed by 8382

Abstract

The use of small interference RNA (siRNA) to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR) is overexpressed in many types of tumor cells and [...] Read more.

The use of small interference RNA (siRNA) to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR) is overexpressed in many types of tumor cells and therefore is a potential target for the selective delivery of siRNA to cancer cells. Here, we used the TfR binding peptide HAIYPRH (HAI peptide) conjugated to cationic polymer branched polyethylenimine (bPEI), optimized the coupling strategy, and the TfR selective delivery of siRNA was evaluated in cells with high (H1299) and low TfR expression (A549 and H460). The HAI-bPEI conjugate exhibited chemico-physical properties in terms of size, zeta-potential, and siRNA condensation efficiency similar to unmodified bPEI. Confocal microscopy and flow cytometry results revealed that HAI-bPEI selectively delivered siRNA to H1299 cells compared with A549 or H460 cells. Moreover, HAI-bPEI achieved more efficient glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene knockdown in H1299 cells compared with bPEI alone. However, despite optimization of the targeting peptide and coupling strategy, HAI-bPEI can only silence reporter gene enhanced green fluorescent protein (eGFP) at the protein level when chloroquine is present, indicating that further optimization of the conjugate is required. In conclusion, the HAI peptide may be useful to target TfR overexpressing tumors in targeted gene and siRNA delivery approaches. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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1767 KiB

Open AccessArticle

Development of an Innovative Intradermal siRNA Delivery System Using a Combination of a Functional Stearylated Cytoplasm-Responsive Peptide and a Tight Junction-Opening Peptide

by Hisako Ibaraki, Takanori Kanazawa, Yuuki Takashima, Hiroaki Okada and Yasuo Seta

Molecules 2016, 21(10), 1279; https://doi.org/10.3390/molecules21101279 - 24 Sep 2016

Cited by 20 | Viewed by 5858

Abstract

As a new category of therapeutics for skin diseases including atopic dermatitis (AD), nucleic acids are gaining importance in the clinical setting. Intradermal administration is noninvasive and improves patients′ quality of life. However, intradermal small interfering RNA (siRNA) delivery is difficult because of [...] Read more.

As a new category of therapeutics for skin diseases including atopic dermatitis (AD), nucleic acids are gaining importance in the clinical setting. Intradermal administration is noninvasive and improves patients′ quality of life. However, intradermal small interfering RNA (siRNA) delivery is difficult because of two barriers encountered in the skin: intercellular lipids in the stratum corneum and tight junctions in the stratum granulosum. Tight junctions are the major barrier in AD; therefore, we focused on functional peptides to devise an intradermal siRNA delivery system for topical skin application. In this study, we examined intradermal siRNA permeability in the tape-stripped (20 times) back skin of mice or AD-like skin of auricles treated with 6-carboxyfluorescein-aminohexyl phosphoramidite (FAM)-labeled siRNA, the tight junction modulator AT1002, and the functional cytoplasm-responsive stearylated peptide STR-CH₂R₄H₂C by using confocal laser microscopy. We found that strong fluorescence was observed deep and wide in the epidermis and dermis of back skin and AD-like ears after siRNA with STR-CH₂R₄H₂C and AT1002 treatment. After 10 h from administration, brightness of FAM-siRNA was significantly higher for STR-CH₂R₄H₂C + AT1002, compared to other groups. In addition, we confirmed the nontoxicity of STR-CH₂R₄H₂C as a siRNA carrier using PAM212 cells. Thus, our results demonstrate the applicability of the combination of STR-CH₂R₄H₂C and AT1002 for effective intradermal siRNA delivery. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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Review

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1915 KiB

Open AccessReview

Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease

by Valentina Sardone, Haiyan Zhou, Francesco Muntoni, Alessandra Ferlini and Maria Sofia Falzarano

Molecules 2017, 22(4), 563; https://doi.org/10.3390/molecules22040563 - 05 Apr 2017

Cited by 68 | Viewed by 13971

Abstract

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules [...] Read more.

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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872 KiB

Open AccessReview

What Is New in the miRNA World Regarding Osteosarcoma and Chondrosarcoma?

by Gaia Palmini, Francesca Marini and Maria Luisa Brandi

Molecules 2017, 22(3), 417; https://doi.org/10.3390/molecules22030417 - 07 Mar 2017

Cited by 60 | Viewed by 7699

Abstract

Despite the availability of multimodal and aggressive therapies, currently patients with skeletal sarcomas, including osteosarcoma and chondrosarcoma, often have a poor prognosis. In recent decades, advances in sequencing technology have revealed the presence of RNAs without coding potential known as non-coding RNAs (ncRNAs), [...] Read more.

Despite the availability of multimodal and aggressive therapies, currently patients with skeletal sarcomas, including osteosarcoma and chondrosarcoma, often have a poor prognosis. In recent decades, advances in sequencing technology have revealed the presence of RNAs without coding potential known as non-coding RNAs (ncRNAs), which provides evidence that protein-coding genes account for only a small percentage of the entire genome. This has suggested the influence of ncRNAs during development, apoptosis and cell proliferation. The discovery of microRNAs (miRNAs) in 1993 underscored the importance of these molecules in pathological diseases such as cancer. Increasing interest in this field has allowed researchers to study the role of miRNAs in cancer progression. Regarding skeletal sarcomas, the research surrounding which miRNAs are involved in the tumourigenesis of osteosarcoma and chondrosarcoma has rapidly gained traction, including the identification of which miRNAs act as tumour suppressors and which act as oncogenes. In this review, we will summarize what is new regarding the roles of miRNAs in chondrosarcoma as well as the latest discoveries of identified miRNAs in osteosarcoma. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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1657 KiB

Open AccessReview

Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases

by Wupeng Liao, Jinrui Dong, Hong Yong Peh, Lay Hong Tan, Kah Suan Lim, Li Li and Wai-Shiu Fred Wong

Molecules 2017, 22(1), 139; https://doi.org/10.3390/molecules22010139 - 17 Jan 2017

Cited by 31 | Viewed by 10791

Abstract

Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD) and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF). The advantage of direct accessibility for oligonucleotide molecules to the [...] Read more.

Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD) and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF). The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO), small interfering RNA (siRNA), and microRNA (miRNA) are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir) has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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1057 KiB

Open AccessReview

Delivery of RNAi Therapeutics to the Airways—From Bench to Bedside

by Yingshan Qiu, Jenny K. W. Lam, Susan W. S. Leung and Wanling Liang

Molecules 2016, 21(9), 1249; https://doi.org/10.3390/molecules21091249 - 20 Sep 2016

Cited by 54 | Viewed by 9711

Abstract

RNA interference (RNAi) is a potent and specific post-transcriptional gene silencing process. Since its discovery, tremendous efforts have been made to translate RNAi technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated [...] Read more.

RNA interference (RNAi) is a potent and specific post-transcriptional gene silencing process. Since its discovery, tremendous efforts have been made to translate RNAi technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). Similar to other nucleic acid-based therapeutics, the major hurdle of RNAi therapy is delivery. Pulmonary delivery is a promising approach of delivering RNAi therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. It is a non-invasive route of administration that is generally well accepted by patients. However, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. Understanding these barriers is essential for the development an effective RNA delivery system. In this review, the different barriers to pulmonary drug delivery are introduced. The potential of RNAi molecules as new class of therapeutics, and the latest preclinical and clinical studies of using RNAi therapeutics in different respiratory conditions are discussed in details. We hope this review can provide some useful insights for moving inhaled RNAi therapeutics from bench to bedside. Full article

(This article belongs to the Special Issue Nucleic Acid-based Drug)

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