Jong Bum Lee

Technological development of structural DNA/RNA-based RNAi systems and their applications ☆

RNA interference (RNAi)-based gene therapy has drawn tremendous attention due to its highly specific gene regulation by selective degradation of any target mRNA. There have been multiple reports regarding the development of various cationic materials for efficient siRNA delivery, however, many studies still suffer from the conventional delivery problems such as suboptimal transfection performance, a lack of tissue specificity, and potential cytotoxicity. Despite the huge therapeutic potential of siRNAs, conventional gene carriers have failed to guarantee successful gene silencing in vivo, thus not warranting clinical trials. The relatively short double-stranded structure of siRNAs has resulted in uncompromising delivery formulations, as well as low transfection efficiency, compared with the conventional nucleic acid drugs such as plasmid DNAs. Recent developments in structural siRNA and RNAi nanotechnology have enabled more refined and reliable in vivo gene silencing with multiple advantages over naked siRNAs. This review focuses on recent progress in the development of structural DNA/RNA-based RNAi systems and their potential therapeutic applications. In addition, an extensive list of prior reports on various RNAi systems is provided and categorized by their distinctive molecular characters.

Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption

ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)‐associated protein‐9 nuclease (Cas9) can be used for the specific disruption of a target gene to permanently suppress the expression of the protein encoded by the target gene. Efficient delivery of the system to an intracellular target site should be achieved to utilize the tremendous potential of the genome‐editing tool in biomedical applications such as the knock‐out of disease‐related genes and the correction of defect genes. Here, we devise polymeric CRISPR/Cas9 system based on poly‐ribonucleoprotein (RNP) nanoparticles consisting of polymeric sgRNA, siRNA, and Cas9 endonuclease in order to improve the delivery efficiency. When delivered by cationic lipids, the RNP nanoparticles built with chimeric poly‐sgRNA/siRNA sequences generate multiple sgRNA‐Cas9 RNP complexes upon the Dicer‐mediated digestion of the siRNA parts, leading to more efficient disruption of the target gene in cells and animal models, compared with the monomeric sgRNA‐Cas9 RNP complex.

Bubbled RNA-Based Cargo for Boosting RNA Interference

As ribonucleic acid (RNA) nanotechnology has advanced, it has been applied widely in RNA‐based therapeutics. Among the range of approaches, enzymatically synthesized RNA structures for inducing RNA interference in cancer cells have potential for silencing genes in a target‐specific manner. On the other hand, the efficiency of gene silencing needs to be improved to utilize the RNA‐based system for RNAi therapeutics. This paper introduces a new approach for efficient generation of siRNA from bubbled RNA‐based cargo (BRC). The presence of bubbles in between to avoid nonfunctional short dsRNAs allows the RNA‐based cargoes to contain multiple Dicer‐cleavage sites to release the functional siRNAs when introduced to cells. BRCs can be synthesized easily in a one‐pot process and be purified by simple centrifugation. Furthermore, efficient target gene silencing by the bubbled structure is confirmed both in vitro and in vivo. Therefore, this bubbled RNA cargo system can be utilized for target‐specific RNAi therapeutics with high efficiency in the generation of functional siRNAs in the target cells.

Enzymatic Polymerization on DNA Modified Gold Nanowire for Label-Free Detection of Pathogen DNA.

This paper presents a label-free biosensor for the detection of single-stranded pathogen DNA through the target-enhanced gelation between gold nanowires (AuNW) and the primer DNAs branched on AuNW. The target DNA enables circularization of the linear DNA template, and the primer DNA is elongated continuously via rolling circle amplification. As a result, in the presence of the target DNA, a macroscopic hydrogel was fabricated by the entanglement of the elongated DNA with AuNWs as a scaffold fiber for effective gelation. In contrast, very small separate particles were generated in the absence of the target DNA. This label-free biosensor might be a promising tool for the detection of pathogen DNAs without any devices for further analysis. Moreover, the biosensor based on the weaving of AuNW and DNAs suggests a novel direction for the applications of AuNWs in biological engineering.

DNA aptamer-based carrier for loading proteins and enhancing the enzymatic activity

Here, we synthesized DNA microparticles comprised of thrombin binding aptamers via rolling circle amplification (RCA). These DNA aptamer particles could successfully load a number of thrombins and the complexes have shown improved thrombin activity. This new platform offers a protein loading and delivery system for medical applications.

RCA-Based Biosensor for Electrical and Colorimetric Detection of Pathogen DNA

For the diagnosis and prevention of diseases, a range of strategies for the detection of pathogens have been developed. In this study, we synthesized the rolling circle amplification (RCA)-based biosensor that enables detection of pathogen DNA in two analytical modes. Only in the presence of the target DNA, the template DNA can be continuously polymerized by simply carrying out RCA, which gives rise to a change of surface structure of Au electrodes and the gap between the electrodes. Electrical signal was generated after introducing hydrogen tetrachloroaurate (HAuCl4) to the DNA-coated biosensor for the improvement of the conductivity of DNA, which indicates that the presence of the pathogen DNA can be detected in an electrical approach. Furthermore, the existence of the target DNA was readily detected by the naked eyes through change in colors of the electrodes from bright yellow to orange-red after RCA reaction. The RCA-based biosensor offers a new platform for monitoring of pathogenic DNA with two different detection modes in one system.

Enzymatically Produced miR34a Nanoparticles for Enhanced Antiproliferation Activity

Among a range of functional forms of RNA, microRNAs (miRNAs) have held a great promise for their natural ability to influence cellular behavior by regulating gene expression. In particular, it is revealed that miR34a has a function as a tumor suppressor by regulating silent information regulator 1 expression, causing antiproliferation of tumor. To utilize functional miR34a as an effective cancer therapeutics, an enzymatic approach to synthesize miR34a nanoparticles is adapted. The enzymatically self‐assembled miR34a nanoparticles have a high cargo capacity and a suitable size for cellular uptake. Indeed, the miR34a nanoparticles are delivered successfully to PC3 cells, human prostate cancer cells, and suppress proliferation and migration of those cells. These results suggest that the enzymatically produced miR34a nanoparticles can function as an effective tumor suppressor. Furthermore, it is expected that the enzymatic synthesis of miRNA nanoparticles can be adapted universally for delivery of other functional miRNAs as well.

Library siRNA-generating RNA nanosponges for gene silencing by complementary rolling circle transcription

Since the discovery of RNA interference (RNAi), small interfering RNA (siRNA) has been powerful tools for gene downregulation in biomedical applications. Despite the outstanding efficacy of siRNA, the development of a therapeutic delivery system remains a challenge owing to the instability of RNA. In this study, we describe a new method for the design of siRNA-generating nanosponges by using complementary rolling circle transcription (cRCT), a technique that requires two complementary circular DNA. The sequences of one of the circular DNA are designed to have complete complementarity to the target mRNA resulting in double stranded RNA (dsRNA) that can be digested to siRNA by cellular Dicer activity. This siRNA design, called ‘library siRNA’, could be universally applied to fabricate RNA nanosponges targeting any known mRNA sequence.

A biomaterial approach to cell reprogramming and differentiation

Cell reprogramming of somatic cells into pluripotent states and subsequent differentiation into certain phenotypes has helped progress regenerative medicine research and other medical applications. Recent research has used viral vectors to induce this reprogramming; however, limitations include low efficiency and safety concerns. In this review, we discuss how biomaterial methods offer potential avenues for either increasing viability and downstream applicability of viral methods, or providing a safer alternative. The use of non-viral delivery systems, such as electroporation, micro/nanoparticles, nucleic acids and the modulation of culture substrate topography and stiffness have generated valuable insights regarding cell reprogramming.

CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells

As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used. For efficient induction of immune responses, current issues affecting the use of nucleic acid-based adjuvants, e.g. stability in physiological conditions, delivery to immune cells, and successful release within the phagolysosome, should be addressed. Here, we present CpG-based DNA microparticles (DNA-MPs) fabricated by complementary rolling circle amplification (cRCA) as adjuvants for enhancing immune response and production of selective antibody production. Using cRCA method, the sizes of CpG-based DNA-MPs were finely controlled (0.5 and 1 μm) with superior and provided mismatched single stranded form of CpG ODN region for specific cleavage site by DNase II within the phagolysosome. Fabricated CpG-based 1 μm DNA-MPs (DNA-MP-1.0) were successfully internalized into primary macrophages and macrophage cell line (RAW264.7 cells), and elicited superior cytokine production e.g. TNF-α and IL-6, compared to conventional CpG ODNs. After in vivo administration of DNA-MP-1.0 with model antigen ovalbumin (OVA), significantly elevated OVA-specific antibody production was observed. With this in mind, DNA-MP-1.0 could serve as a novel type of adjuvant for the activation of macrophages and the following production of selective antibodies without any noticeable toxicity in vitro and in vivo.