Development of PET Radioisotope Copper-64-Labeled Theranostic Immunoliposomes for EGFR Overexpressing Cancer-Targeted Therapy and Imaging.

Combining standard surgical procedures with personalized chemotherapy and the continuous monitoring of cancer progression is necessary for effective NSCLC treatment. In this study, we developed liposomal nanoparticles as theranostic agents capable of simultaneous therapy for and imaging of target cancer cells. Copper-64 (64Cu), with a clinically practical half-life (t1/2 = 12.7 h) and decay properties, was selected as the radioisotope for molecular PET imaging. An anti-epidermal growth factor receptor (anti-EGFR) antibody was used to achieve target-specific delivery. Simultaneously, the chemotherapeutic agent doxorubicin (Dox) was encapsulated within the liposomes using a pH-gradient method. The conjugates of 64Cu-labeled and anti-EGFR antibody-conjugated micelles were inserted into the doxorubicin-encapsulating liposomes via a post-insertion procedure (64Cu-Dox-immunoliposomes). We evaluated the size and zeta-potential of the liposomes and analyzed target-specific cell binding and cytotoxicity in EGFR-positive cell lines. Then, we analyzed the specific therapeutic effect and PET imaging of the 64Cu-Dox-immunoliposomes with the A549 xenograft mouse model. In vivo therapeutic experiments on the mouse models demonstrated that the doxorubicin-containing 64Cu-immunoliposomes effectively inhibited tumor growth. Moreover, the 64Cu-immunoliposomes provided superior in vivo PET images of the tumors compared to the untargeted liposomes. We suggest that nanoparticles will be the potential platform for cancer treatment as a widely applicable theranostic system.

Novel Lipid Nanocomplex Co-Carrying Bcl2 siRNA and Quantum Dots for EGF Receptor-Targeted Anti-Cancer Theranosis.

Many different types of nanoparticles have been suggested for tumor-targeted theranosis. However, most systems were prepared through a series of complicated processes and could not even overcome the blood-immune barriers. For the accurate diagnosis and effective treatment of cancers, herein we suggested the lipid micellar structure capturing quantum dot (QD) for cancer theranosis. The QD/lipid micelles (QDMs) were prepared using a simple self-assembly procedure and then conjugated with anti-epidermal growth factor receptor (EGFR) antibodies for tumor targeting. As a therapeutic agent, Bcl2 siRNA-cholesterol conjugates were loaded on the surface of QDMs. The EGFR-directed QDMs containing Bcl2 siRNA, so-called immuno-QDM/siBcl2 (iQDM/siBcl2), exhibited the more effective delivery of QDs and siBcl2 to target human colorectal cancer cells in cultures as well as in mouse xenografts. The effective in vivo targeting of iQDM/siBcl2 resulted in a more enhanced therapeutic efficacy of siBcl2 to the target cancer in mice. Based on the results, anti-EGFR QDM capturing therapeutic siRNA could be suggested as an alternative modality for tumor-targeted theranosis.

Shaping Rolling Circle Amplification Products into DNA Nanoparticles by Incorporation of Modified Nucleotides and Their Application to In Vitro and In Vivo Delivery of a Photosensitizer.

Rolling circle amplification (RCA) is a robust way to generate DNA constructs, which are promising materials for biomedical applications including drug delivery because of their high biocompatibility. To be employed as a drug delivery platform, however, the DNA materials produced by RCA need to be shaped into nanoparticles that display both high cellular uptake efficiency and nuclease resistance. Here, we showed that the DNA nanoparticles (DNPs) can be prepared with RCA and modified nucleotides that have side-chains appended on the nucleobase are capable of interacting with the DNA strands of the resulting RCA products. The incorporation of the modified nucleotides improved cellular uptake efficiency and nuclease resistance of the DNPs. We also demonstrated that these DNPs could be employed as carriers for the delivery of a photosensitizer into cancer cells to achieve photodynamic therapy upon irradiation at both the in vitro and in vivo levels.

Sendai viroplexes for epidermal growth factor receptor-directed delivery of interleukin-12 and salmosin genes to cancer cells.

BACKGROUND: The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor-directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off-target effects of gene transfection. METHODS: The system consists of a well-verified cationic O,O'-dimyristyl-N-lysyl glutamate (DMKE), Sendai virus fusion (F) protein and hemagglutinin-neuraminidase (HN) protein, referred to as cationic Sendai F/HN virosomes. To achieve tumor-specific recognition, anti-epidermal growth factor (EGF) receptor antibody was coupled to the surface of the virosomes containing interleukin-12 (IL-12) and/or salmosin genes that have potent anti-angiogenetic functions. RESULTS: Among the virosomal formulations, the anti-EGF receptor (EGFR) viroplexes, prepared via complexation of plasmid DNA (pDNA) with cationic DMKE lipid, exhibited more efficient gene transfection to tumor cells over-expressing EGF receptors compared to the neutrally-charged anti-EGFR virosomes encapsulating pDNA. In addition, the anti-EGFR viroplexes with IL-12 and salmosin genes exhibited the most effective therapeutic efficacy in a mouse tumor model. Especially when combined with doxorubicin, transfection of the two genes via the anti-EGFR viroplexes exhibited an enhanced inhibitory effect on tumor growth and metastasis in lungs. CONCLUSIONS: The results of the present study suggest that anti-EGFR viroplexes can be utilized as an effective strategy for tumor-directed gene delivery. Copyright © 2016 John Wiley & Sons, Ltd.

Low-Power Phototransistor with Enhanced Visible-Light Photoresponse and Electrical Performances Using an IGZO/IZO Heterostructure.

In this study, we demonstrated the effective separation of charge carriers within the IGZO/IZO heterostructure by incorporating IZO. We have chosen IGZO for its high mobility and excellent on-off switching behavior in the front channel of our oxide-oxide heterostructure. Similarly, for an additional oxide layer, we have selected IZO due to its outstanding electrical properties. The optimized optoelectronic characteristics of the IGZO/IZO phototransistors were identified by adjusting the ratio of In:Zn in the IZO layer. As a result, the most remarkable traits were observed at the ratio of In:Zn = 8:2. Compared to the IGZO single-layer phototransistor, the IGZO/IZO(8:2) phototransistor showed improved photoresponse characteristics, with photosensitivity and photoresponsivity values of 1.00 × 107 and 89.1 AW-1, respectively, under visible light wavelength illumination. Moreover, the electrical characteristics of the IGZO/IZO(8:2) transistor, such as field effect mobility (µsat) and current on/off ratio (Ion/Ioff), were highly enhanced compared to the IGZO transistor. The µsat and Ion/Ioff were increased by about 2.1 times and 2.3 times, respectively, compared to the IGZO transistor. This work provides an approach for fabricating visible-light phototransistors with elevated optoelectronic properties and low power consumption based on an oxide-oxide heterostructure. The phototransistor with improved performance can be applied to applications such as color-selective visible-light image sensors and biometric sensors interacting with human-machine interfaces.

Enhancement of the Visible Light Photodetection of Inorganic Photodiodes via Additional Quantum Dots Layers.

Visible light photodetectors are extensively researched with transparent metal oxide holes/electron layers for various applications. Among the metal oxide transporting layers, nickel oxide (NiO) and zinc oxide (ZnO) are commonly adopted due to their wide band gap and high transparency. The objective of this study was to improve the visible light detection of NiO/ZnO photodiodes by introducing an additional quantum dot (QD) layer between the NiO and ZnO layers. Utilizing the unique property of QDs, we could select different sizes of QDs and responsive light wavelength ranges. The resulting red QDs utilized device that could detect light starting at 635 nm to UV (Ultra-violet) light wavelength and exhibited a photoresponsivity and external quantum efficiency (EQE) of 14.99 mA/W and 2.92% under 635 nm wavelength light illumination, respectively. Additionally, the green QDs, which utilized a device that could detect light starting at 520 nm, demonstrated photoresponsivity values of 8.34 mA/W and an EQE of 1.99% under 520 nm wavelength light illumination, respectively. In addition, we used X-ray photoelectron spectroscopy (XPS) and ultraviolet photoelectron spectroscopy (UPS) to investigate the origin of the photocurrents and the enhancement of the device's performance. This study suggests that incorporating QDs with metal oxide semiconductors is an effective approach for detecting visible light wavelengths in transparent optoelectronic devices.

Real-Time Ultraviolet Monitoring System with Low-Temperature Solution-Processed High-Transparent p-n Junction Photodiode with a Fast Responsive and High Rectification Ratio.

We introduce an enhanced performance organic-inorganic hybrid p-n junction photodiode, utilizing poly[bis(4-phenyl) (2,4,6-trimethylphenyl)amine] (PTAA) and ZnO, fabricated through a solution-based process at a low temperature under 100 °C. Improved interfacial electronic structure, characterized by shallower Gaussian standard deviation of the density-of-state distribution and a larger interface dipole, has resulted in a remarkable fold increase of ∼102 in signal-to-noise ratio for the device. This photodiode exhibits a high specific detectivity (2.32 × 1011 Jones, cm×Hz×W-1) and exceptional rectification ratio (5.47 × 104 at ±1 V). The primary light response, concentrated in the optimal thickness of the PTAA layer, contributes to response over the entire UVA region and rapid response speed, with rise and fall times of 0.24 and 0.64 ms, respectively. Furthermore, this work demonstrates immense potential of our device for health monitoring applications by enabling real-time and continuous measurements of UV intensity.

Highly Efficient ITO-Free Quantum-Dot Light Emitting Diodes via Solution-Processed PEDOT:PSS Semitransparent Electrode.

We present a study on the potential use of sulfuric acid-treated poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) as a viable alternative to indium tin oxide (ITO) electrodes in quantum dot light-emitting diodes (QLEDs). ITO, despite its high conductivity and transparency, is known for its disadvantages of being brittle, fragile, and expensive. Furthermore, due to the high hole injection barrier of quantum dots, the need for electrodes with a higher work function is becoming more significant. In this report, we present solution-processed, sulfuric acid-treated PEDOT:PSS electrodes for highly efficient QLEDs. The high work function of the PEDOT:PSS electrodes improved the performance of the QLEDs by facilitating hole injection. We demonstrated the recrystallization and conductivity enhancement of PEDOT:PSS upon sulfuric acid treatment using X-ray photoelectron spectroscopy and Hall measurement. Ultraviolet photoelectron spectroscopy (UPS) analysis of QLEDs showed that sulfuric acid-treated PEDOT:PSS exhibited a higher work function than ITO. The maximum current efficiency and external quantum efficiency based on the PEDOT:PSS electrode QLEDs were measured as 46.53 cd/A and 11.01%, which were three times greater than ITO electrode QLEDs. These findings suggest that PEDOT:PSS can serve as a promising replacement for ITO electrodes in the development of ITO-free QLED devices.

Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability.

Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).

Tumor-Targeted Erythrocyte Membrane Nanoparticles for Theranostics of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) cells do not contain various receptors for targeted treatment, a reason behind the poor prognosis of this disease. In this study, biocompatible theranostic erythrocyte-derived nanoparticles (EDNs) were developed and evaluated for effective early diagnosis and treatment of TNBC. The anti-cancer drug, doxorubicin (DOX), was encapsulated into the EDNs and diagnostic quantum dots (QDs) were incorporated into the lipid bilayers of EDNs for tumor bio-imaging. Then, anti-epidermal growth factor receptor (EGFR) antibody molecules were conjugated to the surface of EDNs for TNBC targeting (iEDNs). According to the confocal microscopic analyses and biodistribution assay, iEDNs showed a higher accumulation in EGFR-positive MDA-MB-231 cancers in vitro as well as in vivo, compared to untargeted EDNs. iEDNs containing doxorubicin (iEDNs-DOX) showed a stronger inhibition of target tumor growth than untargeted ones. The resulting anti-EGFR iEDNs exhibited strong biocompatibility, prolonged blood circulation, and efficient targeting of TNBC in mice. Therefore, iEDNs may be used as potential TNBC-targeted co-delivery systems for therapeutics and diagnostics.

Enhanced performance of flexible quantum dot light-emitting diodes using a low-temperature processed PTAA hole transport layer.

Low-temperature processing is important for improving the stability and performance of flexible quantum dot light-emitting diodes (QLEDs). In this study, QLEDs were fabricated using poly[bis(4-phenyl) (2,4,6-trimethylphenyl)amine] (PTAA) as a suitable hole transport layer (HTL) material owing to its low-temperature processability and vanadium oxide as the low-temperature solution-processable hole injection layer material. The maximum luminance and highest current efficiency of the QLEDs on a glass substrate with an optimal PTAA HTL was 8.9 × 104 Cd/m2 and 15.9 Cd/A, respectively, which was comparable to that of conventional devices. The QLEDs on a flexible substrate showed a maximum luminance of 5.4 × 104 Cd/m2 and highest current efficiency of 5.1 Cd/A. X-ray and ultraviolet photoelectron spectroscopies were used to investigate the chemical state and interfacial electronic structure according to the materials and the state changes of the HTL, respectively. The interfacial electronic structure showed that PTAA exhibited a better hole transport ability owing to its low hole injection barrier ([Formula: see text]). Moreover, QLEDs with a PTAA HTL could operate as photosensors under reverse bias conditions. These results indicate that the low-temperature-processed PTAA HTL is suitable for improving the performance of flexible QLEDs.

Checkpoint kinase 1/2 inhibition potentiates anti-tumoral immune response and sensitizes gliomas to immune checkpoint blockade.

Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cell mediated killing are less understood. Kinases are potentially druggable targets that drive tumor progression and might influence immune response. Here, we perform an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cell recognition. The screen reveals checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are currently being evaluated in clinical trials, in combination with PD-1 or PD-L1 blockade, lead to survival benefit in multiple preclinical glioma models. Mechanistically, loss of Chek2 enhances antigen presentation, STING pathway activation and PD-L1 expression in mouse gliomas. Analysis of human GBMs demonstrates that Chek2 expression is inversely associated with antigen presentation and T-cell activation. Collectively, these results support Chek2 as a promising target for enhancement of response to immune checkpoint blockade therapy in GBM.

EGF Receptor-Targeting Cancer Therapy Using CD47-Engineered Cell-Derived Nanoplatforms.

Introduction: Avoiding phagocytic cells and reducing off-target toxicity are the primary hurdles in the clinical application of nanoparticles containing therapeutics. For overcoming these errors, in this study, nanoparticles expressing CD47 proteins inhibiting the phagocytic attack of immune cells were prepared and then evaluated as an anti-cancer drug delivery vehicle. Methods: The CD47+ cell-derived nanoparticles (CDNs) were prepared from the plasma membranes of human embryonic kidney cells transfected with a plasmid encoding CD47. And the doxorubicin (DOX) was loaded into the CDNs, and anti-EGF receptor (EGFR) antibodies were conjugated to the surface of the CDNs to target tumors overexpressing EGFR. Results: The CD47+iCDNs-DOX was successfully synthesized having a stable structure. The CD47+CDNs were taken up less by RAW264.7 macrophages compared to control CDNs. Anti-EGFR CD47+CDNs (iCDNs) selectively recognized EGFR-positive MDA-MB-231 cells in vitro and accumulated more effectively in the target tumor xenografts in mice. Moreover, iCDNs encapsulating doxorubicin (iCDNs-DOX) exhibited the highest suppression of tumor growth in mice, presumably due to the enhanced DOX delivery to tumor tissues, compared to non-targeting CDNs or CD47- iCDNs. Discussion: These results suggest that the clinical application of biocompatible cell membrane-derived nanocarriers could be facilitated by functionalization with macrophage-avoiding CD47 and tumor-targeting antibodies.

Improving the Performance of Solution-Processed Quantum Dot Light-Emitting Diodes via a HfOx Interfacial Layer.

One of the major obstacles in the way of high-performance quantum dot light-emitting diodes (QLEDs) is the charge imbalance arising from more efficient electron injection into the emission layer than the hole injection. In previous studies, a balanced charge injection was often achieved by lowering the electron injection efficiency; however, high performance next-generation QLEDs require the hole injection efficiency to be enhanced to the level of electron injection efficiency. Here, we introduce a solution-processed HfOx layer for the enhanced hole injection efficiency. A large amount of oxygen vacancies in the HfOx films creates gap states that lower the hole injection barrier between the anode and the emission layer, resulting in enhanced light-emitting characteristics. The insertion of the HfOx layer increased the luminance of the device to 166,600 cd/m2, and the current efficiency and external quantum efficiency to 16.6 cd/A and 3.68%, respectively, compared with the values of 63,673 cd/m2, 7.37 cd/A, and 1.64% for the device without HfOx layer. The enhanced light-emitting characteristics of the device were elucidated by X-ray photoelectron, ultra-violet photoelectron, and UV-visible spectroscopy. Our results suggest that the insertion of the HfOx layer is a useful method for improving the light-emitting properties of QLEDs.

Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma.

PURPOSE: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. EXPERIMENTAL DESIGN: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. RESULTS: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. CONCLUSIONS: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

TOP2B Enzymatic Activity on Promoters and Introns Modulates Multiple Oncogenes in Human Gliomas.

PURPOSE: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. EXPERIMENTAL DESIGN: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. RESULTS: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. CONCLUSIONS: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma.

Only a subset of recurrent glioblastoma (rGBM) responds to anti-PD-1 immunotherapy. Previously, we reported enrichment of BRAF/PTPN11 mutations in 30% of rGBM that responded to PD-1 blockade. Given that BRAF and PTPN11 promote MAPK/ERK signaling, we investigated whether activation of this pathway is associated with response to PD-1 inhibitors in rGBM, including patients that do not harbor BRAF/PTPN11 mutations. Here we show that immunohistochemistry for ERK1/2 phosphorylation (p-ERK), a marker of MAPK/ERK pathway activation, is predictive of overall survival following adjuvant PD-1 blockade in two independent rGBM patient cohorts. Single-cell RNA-sequencing and multiplex immunofluorescence analyses revealed that p-ERK was mainly localized in tumor cells and that high-p-ERK GBMs contained tumor-infiltrating myeloid cells and microglia with elevated expression of MHC class II and associated genes. These findings indicate that ERK1/2 activation in rGBM is predictive of response to PD-1 blockade and is associated with a distinct myeloid cell phenotype.

A brain tumor-homing tetra-peptide delivers a nano-therapeutic for more effective treatment of a mouse model of glioblastoma.

Organ-specific cell-penetrating peptides (CPPs) are a class of molecules that can be highly effective at delivering therapeutic cargoes, and they are currently of great interest in cancer treatment strategies. Herein, we describe a new CPP (amino acid sequence serine-isoleucine-tyrosine-valine, or SIWV) that homes to glioblastoma multiforme (GBM) brain tumor tissues with remarkable specificity in vitro and in vivo. The SIWV sequence was identified from an isoform of annexin-A3 (AA3H), a membrane-interacting human protein. The mechanism of intracellular permeation is proposed to follow a caveolin-mediated endocytotic pathway, based on in vitro and in vivo receptor inhibition and genetic knockdown studies. Feasibility as a targeting agent for therapeutics is demonstrated in a GBM xenograft mouse model, where porous silicon nanoparticles (pSiNPs) containing the clinically relevant anticancer drug SN-38 are grafted with SIWV via a poly-(ethylene glycol) (PEG) linker. The formulation shows enhanced in vivo targeting ability relative to a formulation employing a scrambled control peptide, and significant (P < 0.05) therapeutic efficacy relative to free SN-38 in the GBM xenograft animal model.

Lung-targeted delivery of TGF-ß antisense oligonucleotides to treat pulmonary fibrosis.

Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-ß is a critical factor in the fibrotic process, downregulation of this cytokine has been considered a potential approach for disease treatment. Herein, we designed a new lung-targeted delivery technology based on the complexation of polymeric antisense oligonucleotides (pASO) and dimeric human ß-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-ß mRNA were polymerized by rolling circle amplification and complexed with DhBD23. After complexation with DhBD23, pASO showed improved serum stability and enhanced uptake by fibroblasts in vitro and lung-specific accumulation upon intravenous injection in vivo. The pASO/DhBD23 complex delivered into the lung downregulated target mRNA, and subsequently alleviated lung fibrosis in mice, as demonstrated by western blotting, quantitative reverse-transcriptase PCR (qRT-PCR), immunohistochemistry, and immunofluorescence imaging. Moreover, as the complex was prepared only with highly biocompatible materials such as DNA and human-derived peptides, no systemic toxicity was observed in major organs. Therefore, the pASO/DhBD23 complex is a promising gene therapy platform with lung-targeting ability to treat various pulmonary diseases, including pulmonary fibrosis, with low side effects.

Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity.

We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.