Progress in RNA-targeting and Gene Editing Therapies for Transthyretin Amyloidosis Cardiomyopathy / 罕见病研究

Transthyretin(TTR) protein is a tetramer protein, synthesized mainly by the liver. TTR can be misfolded and deposited as amyloid fibrilae and deposited in the myocardial interstroma leading to transthyroxin amyloidosis cardiomyopathy (ATTR-CM). ATTR-CM was included in China's First List of Rare Diseases. Therapeutic strategies for ATTR-CM include blocking TTR synthesis in the liver, stabilizing TTR tetramers and destroying TTR fibra. Small molecule drugs such as tafamidis and diflunisal offer new treatment options for patients. Chlorobenzolic acid became the first drug approved by the U.S. Food and Drug Administration for the treatment of ATTR-CM. Small interfering RNA(siRNA)patisiran and antisense oligonucleotide (ASO)inotersen block TTR expression in the liver and have been approved for the treatment of ATTR variant polyneuropathy (ATTRv-PN)and are in phase Ⅲ trials for the treatment of ATTR-CM. Other siRNA drugs, vutrisiran, and ASO, eplontersen, are being evaluated for clinical efficacy. This article reviews the development of RNA-targeted therapeutics and gene-editing drugs using CRISPR-Cas9.

Therapeutics in paediatric genetic diseases: Current and future landscape

There are more than 7,000 paediatric genetic diseases (PGDs) but less than 5% have treatment options. Treatment strategies targeting different levels of the biological process of the disease have led to optimal health outcomes in a subset of patients with PGDs, where treatment is available. In the past 3 decades, there has been rapid advancement in the development of novel therapies, including gene therapy, for many PGDs. The therapeutic success of treatment relies heavily on knowledge of the genetic basis and the disease mechanism. Specifically, gene therapy has been shown to be effective in various clinical trials, and indeed, these trials have led to regulatory approvals, paving the way for gene therapies for other types of PGDs. In this review, we provide an overview of the treatment strategies and focus on some of the recent advancements in therapeutics for PGDs.

Research progress of RNA drugs delivery / 药学学报

Ribonucleic acid (RNA) medicines have strong therapeutic potential for numerous rare genetic illnesses and malignancies because of its exact programmability based on Watson-Crick base pairing principle and unique ability to regulate gene expression. However, RNA medicines still have limitations in many areas, including stability, half-life time, immunogenicity, organ selectivity, cellular uptake and endosomal escape efficiency despite their great therapeutic potentials. This review briefly introduced numerous RNA medications [mostly messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and antisense oligonucleotide (ASO)] that have intrigued of researchers in recent years, as well as their action mechanism in vivo. A number of delivery techniques, such as chemical modification, ligands coupling and nanocarriers have been proposed. The manufacture and applications of lipid nanoparticle, polymer nanoparticle and exosomes were discussed in depth. The goal of this work is to give a theoretical foundation and design concepts for the development of effective and safe RNA delivery technology, as well as to facilitate RNA therapeutic clinical translation.

A Comprehensive Review of Duchenne Muscular Dystrophy: Genetics, Clinical Presentation, Diagnosis, and Treatment

Duchenne Muscular Dystrophy (DMD) is a genetic disorder involving progressive muscle deterioration leading to loss of mobility, cardiomyopathy, and respiratory complications leading to an early death by the fourth decade of life. Males are affected more often as DMD results from a mutation in the dystrophin gene residing on the X chromosome. The DMD genetic mutation results in a complete functional lack of dystrophin, which culminates as an inadequate connection between the intracellular actin filaments and the extracellular skeleton of muscle. Boys affected by DMD clinically present with muscle weakness before age five, are often wheelchair-bound by age 12, and rarely survive beyond the third decade of life. Traditional treatment strategies have focused primarily on quality-of-life improvement and have included the use of glucocorticoids and physical therapy. No cure currently exists, however many novel treatments for DMD are currently being explored. Some of these involve gene therapy, exon skipping, stop codon skipping, CRISPR technology interventions, and the use of a retinal dystrophin isoform. In this comprehensive review, we recapitulate the literature findings to summarize the history, epidemiology, genetics, clinical presentation, diagnosis, and current and future strategies for the treatment of Duchenne Muscular Dystrophy.

Preparation of folate-targeted anti-miR- 221 anionic liposome and its in vitro anti-hepatocellular carcinoma effect / 中国药房

OBJECTIVE To prep are folate-targeted miR- 221 antisense oligonucleotide （anti-miR-221）delivery system ，and to preliminarily evaluate its in vitro anti-cancer effect on hepatocellular carcinoma. METHODS Folate-targeted anti-miR- 221 liposomes（FRL）were prepared by thin-film dispersion method ；the particle size ，Zeta potential and encapsulation efficiency were determined. The delivery efficiency of folate-targeted anionic liposome in human hepatoma HepG 2 cells was determined by in vitro cellular uptake experiment using calcein as the model drug. Flow cytometry was used to detect the effects of FRL on the apoptosis and cell cycle of HepG 2 cells. RESULTS The particle size of prepared FRL was （172.70±3.76）nm，Zeta potential was （－1.16± 0.15）mV and encapsulation efficiency was （83.53±1.85）％. In vitro cellular uptake experiments showed that folate-targeted anionic liposome successfully delivered calcein to HepG 2 cells，and the delivery efficiency in targeted group was higher than that of non-targeted liposome group （P＜0.01）. Apoptosis experiment results showed that the apoptotic rate of HepG 2 cells treated with FRL was significantly higher than that of non-targeted liposome （P＜0.01）. In cell cycle experiment ，FRL could shorten the S phase fraction of HepG 2 cells and induced arrest in the G 0/G1 and G 2/M phases. CONCLUSIONS FRL can encapsulate anti-miR-221 well and deliver it to hepatocellular carcinoma HepG 2 cells successfully ，and has a good in vitro anti-hepatoma effect in inducing apoptosis and cell cycle regulation.

Managing intrathecal administration of nusinersen in adolescents and adults with 5q-spinal muscular atrophy and previous spinal surgery / Manejo da administração intratecal de nusinersena em adolescentes e adultos com atrofia muscular espinhal 5q e cirurgia prévia de coluna

ABSTRACT Background: Spinal muscular atrophy (SMA) is a neurodegenerative disease of lower motor neurons associated with frequent occurrence of spinal deformity. Nusinersen is an antisense oligonucleotide that increases SMN protein level and is administrated by frequent intrathecal lumbar injections. Thus, spinal deformities and previous spinal surgery are important challenges for drug delivery in SMA. Objective: To report imaging methods used for Nusinersen injection in SMA patients. Methods: Nusinersen injection procedures in SMA types 2 and 3 patients who had previous spinal surgery were analyzed retrospectively to describe the imaging and puncture procedures, as well as the occurrence of complications. Results: Nine SMA patients (14 to 50 years old) underwent 57 lumbar punctures for nusinersen injection. Six patients had no interlaminar space available; in five of them, a transforaminal approach was used, and another one underwent a surgery to open a posterior bone window for the injections. Transforaminal puncture was performed using CT scan in three cases and fluoroscopy in the other two, with a similar success rate. One patient in the transforaminal group had post-procedure radiculitis, and another one had vagal reaction (hypotension). In three cases, with preserved interlaminar space, injections were performed by posterior interlaminar puncture, and only one adverse event was reported (post-puncture headache). Conclusion: In SMA patients with previous spinal surgery, the use of imaging-guided intervention is necessary for administering intrathecal nusinersen. Transforaminal technique is indicated in patients for whom the interlaminar space is not available, and injections should always be guided by either CT or fluoroscopy.

RESUMO Introdução: A atrofia muscular espinal (AME) é uma desordem neurodegenerativa dos motoneurônios inferiores frequentemente associada à ocorrência de deformidade da coluna vertebral. Nusinersena é um oligonucleotídeo antisense que aumenta os níveis da proteína SMN, sendo administrado através de injeções lombares intratecais frequentes. Assim, deformidades da coluna vertebral e abordagem cirúrgica prévia são desafios importantes para a administração de medicamentos na AME. Objetivo: descrever os métodos de imagens utilizados para administração do Nusinersena nos pacientes com AME. Métodos: Os procedimentos de administração de nusinersena em pacientes com AME dos tipos 2 e 3 submetidos à cirurgia prévia da coluna foram analisados retrospectivamente para descrever os métodos de imagem e punção, e a ocorrência de complicações. Resultados: Nove pacientes com AME (14 a 50 anos) foram submetidos a 57 punções lombares para administração de nusinersena. Seis pacientes tinham enxerto ósseo ou nenhum espaço interlaminar disponível; em cinco deles foi utilizada uma abordagem transforaminal, e outra paciente foi submetida à abertura cirúrgica de janela óssea para as injeções. A punção transforaminal foi realizada usando tomografia computadorizada (TC) em três casos e fluoroscopia nos outros dois, com taxa de sucesso semelhante. Um paciente no grupo de abordagem transforaminal apresentou radiculite pós-procedimento e outro apresentou reação vagal (hipotensão). Em três casos, com espaço interlaminar preservado, foram realizadas técnica de punção interlaminar posterior e apenas um evento adverso foi relatado (cefaleia pós-punção). Conclusão: Em pacientes com AME e cirurgia prévia, o uso de intervenção guiada por imagem é necessário para a administração de nusinersena. A técnica transforaminal é indicada nos casos onde o espaço interlaminar não está disponível, devendo ser guiada por TC ou técnicas de imagem fluoroscópica.

Long non-coding RNAs: From disease code to drug role

Enormous studies have corroborated that long non-coding RNAs (lncRNAs) extensively participate in crucial physiological processes such as metabolism and immunity, and are closely related to the occurrence and development of tumors, cardiovascular diseases, nervous system disorders, nephropathy, and other diseases. The application of lncRNAs as biomarkers or intervention targets can provide new insights into the diagnosis and treatment of diseases. This paper has focused on the emerging research into lncRNAs as pharmacological targets and has reviewed the transition of lncRNAs from the role of disease coding to acting as drug candidates, including the current status and progress in preclinical research. Cutting-edge strategies for lncRNA modulation have been summarized, including the sources of lncRNA-related drugs, such as genetic technology and small-molecule compounds, and related delivery methods. The current progress of clinical trials of lncRNA-targeting drugs is also discussed. This information will form a latest updated reference for research and development of lncRNA-based drugs.

Cortical Axonal Secretion of BDNF in the Striatum Is Disrupted in the Mutant-huntingtin Knock-in Mouse Model of Huntington's Disease

Deficient BDNF signaling is known to be involved in neurodegenerative diseases such as Huntington's disease (HD). Mutant huntingtin (mhtt)-mediated disruption of either BDNF transcription or transport is thought to be a factor contributing to striatal atrophy in the HD brain. Whether and how activity-dependent BDNF secretion is affected by the mhtt remains unclear. In the present study, I provide evidence for differential effects of the mhtt on cortical BDNF secretion in the striatum during HD progression. By two-photon imaging of fluorescent BDNF sensor (BDNF-pHluorin and -EGFP) in acute striatal slices of HD knock-in model mice, I found deficient cortical BDNF secretion regardless of the HD onset, but antisense oligonucleotide (ASO)-mediated reduction of htts only rescues BDNF secretion in the early HD brain before the disease onset. Although secretion modes of individual BDNF-containing vesicle were not altered in the pre-symptomatic brain, the full-fusion and partial-fusion modes of BDNF-containing vesicles were significantly altered after the onset of HD symptoms. Thus, besides abnormal BDNF transcription and transport, our results suggest that mhtt-mediated alteration in activity-dependent BDNF secretion at corticostriatal synapses also contributes to the development of HD.

Preparation and evaluation of new cyanoacrylate nanosphere with alcoxyle side group for gene delivery / 国际药学研究杂志

Objective To prepare a new(alcoxyle cyanoacrylate)-based nanosphere for brain targeting gene delivery and evaluate its physicochemical properties,capability of delivery of transforming growth factor beta 2(TGF-β2)antisense oligonucle?otides(ASON),and its potential use on tumor cell suppression in vitro.Methods The cationic nanospheres(NS)were prepared by emulsion polymerization method with DEAE-dextran as cationic stabilizer.The ASON were adsorbed by charge interaction,and poly?sorbate-80 was used as brain-targeting modification.The morphology was observed by transmission electron microscopy(TEM).The average particle size and Zeta potential were determined by dynamic light scattering(DLS). The ultraviolet spectrophotometry was used to determine the entrapment efficiency and drug loading.Agarose gel electrophoresis was used to analyze the optimal loading ratio of ASON-NS,and also the protection of ASON in DNaseⅠand serum containing environment.The release rate of ASON was deter?mined by dialysis.The cytotoxicity on L929 cells and the anti-tumor activity on A172 cells were evaluated by MTS.Results The TEM showed a typical round nanospheres morphology,and no adhesion was detected.The particle size was(79.04±4.33)nm,the disper?sion coefficient was 0.04 ± 0.03,the Zeta potential was(33.60 ± 0.60)mV. The encapsulation efficiency of ASON-NS was(83.14 ± 1.90)%,and the drug loading of ASON-NS was(11.59±0.56)%.The NS provided ASON protection against the Dnase I and serum containing environment. The NS-ASON could effectively deliver ASON into A172 cells and show anti-tumor activity. Besides,little L929 cytotoxicity was detected.Conclusion A new cyanoacrylate nanosphere with alcoxyle side group for brain targeting gene deliv?ery was prepared successfully. It had good ASON loading and delivery capability,providing new carrier materials for nucleic acid drugs.

Microbubbles with HIF-1α Antisense Oligonucleotide Enhanced Ultrasound on Inhibition of Tumor Growth / 中国医学影像学杂志

Purpose After the angiotripsy treatment of tumor,tumor blood vessels have different degrees of regeneration,leading to incomplete tumor necrosis,which may be related to the high expression of HIF-lα.To explore the feasibility of microbubble with HIF-1α antisense oligonucleotide combined with ultrasound inhabit tumor growth.Materials and Methods Twenty-four rabbits with VX2 tumor were randomly assigned to three groups:microbubbles with HIF-1α antisense oligonucleotide plus ultrasound (HMB+US) (n=8),microbubbles plus ultrasound (MB+US) (n=8),therapeutic ultrasound alone without microbubbles (US) (n=8).Pulsed focused ultrasound was delivered directly to the tumor surface for 10 minutes during intravenous infusion of microbubbles with HIF-1α antisense oligonucleotide in the experimental group.The control groups were applied only microbubbles or saline injection.The procedure was repeated every 72 hours until the 30th day.Contrast enhanced US and grey scale ultrasonography were acquired after every treatment to get the tumor perfusion images,size and volume measurements.Results The average maximum diameter of the HMB+US,MB+US,and the US groups grows from (1.13±0.19) cm,(1.17±0.21) cm,(1.22±0.17) cm to (1.60±0.45) cm,(2.11 ±0.57) cm,(3.43 ± 0.71) cm within a 30d-experimental period,respectively.No statistical difference in average diameter was observed among three groups before treatment (P＞0.05).The average maximum diameter of the HMB+US group was significantly smaller than that of the two control groups at the end of experiment (P＜0.05),and the average maximum diameter of the MB+US group was significantly smaller than that of the US group (P＜0.05).Conclusion Microbubbles with HIF-1α antisense oligonucleotide can enhance vascular damage effect ofangiotripsy,which can be a novel method for tumor treatment.

Antisense oligonucleotide in the treatment of brain diseases:research advances / 国际药学研究杂志

Antisense oligonucleotide(ASON)has attracted a growing attention with the rapid development of biological tech?nology. ASON can be designed to target mRNA in a sequence-specific manner to stop,alter,or induce particular disease-related gene functions. Thus,its drugs have emerged as a very promising new class of therapeutics at the molecular genetic level with high specifici?ty and low toxicity. ASON has shown good application prospects in the treatment of brain diseases,by means of brain-targeted delivery and chemical modification. In this review,we summarize the latest development of ASON drugs and their applications on the treatment of Alzheimer disease,Parkinson′s disease and brain tumors.

The influence of silencing miRNA-155 on proliferation and apoptosis of human cutaneous squamous cell carcinoma cell line A431 / 天津医药

Objective To investigate the effects of antisense oligonucleotide against miRNA-155 (AS-miRNA-155) on proliferation,apoptosis and invasion and migration abilities of human cutaneous squamous cell carcinoma cell line A431. Methods AS-miRNA-155 was transfected into human cutaneous squamous cell carcinoma A431 cells by using LipofectamineTM 2000. Blank control without transfection and transfected with non-sense sequence were used as non-sense sequence control. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miRNA-155 in A431 cells. Cell proliferation was analyzed using dimethyl thiazolyldiphenyl tetrazolium (MTT) assay. Cell cycle arrest and apoptosis were studied by flow cytometry (FCM). Invasion and migration were measured by Transwell chamber assays. Results The relative expression of miRNA-155 mRNA was lower in the transfection group than that in the blank control group and the negative control group (F=634.57, P<0.01), but there was no significant difference between the blank control group and the negative control group. After 72 h transfection, the survival rate was significantly lower in the transfection group than that of the blank control group and the negative control group, and the transfection rate decreased significantly by 120 h (P<0.05). Cells of G0/G1 phase increased, Cells of S phase reduced, the overall PI value decreased in transfection group, and the apoptosis rate of A431 cells, migration and invasion of cells increased (P<0.05). There was no significant difference in G2/M cycle between transfection group, blank control group and negative control group. There were no significant differences in A431 cell apoptosis rate, cell migration and invasive ability between blank control group and negative control group. Conclusion Antisense oligonucleotide against miRNA-155 can inhibit the expression of miRNA-155, the proliferation and promote the apoptosis of human cutaneous squamous cell carcinoma A431 cells, which indicates that miRNA-155 may become a new target for the regulation of gene expression in cutaneous squamous cell carcinoma.

A preliminary study of AS1411 mediated STAT3 antisense oligonucleotide targeting tumor cells / 复旦学报（医学版）

Objective To observe the effect of AS1411-mediated signal transduction and activator of transcription 3 (STAT3) antisense oligonucleotide (ASO) targeting tumor cells.Methods RNA was used as coupling molecules to link the targeting molecules AS1411 and effector molecules ASO.Prediction and analysis of the secondary structure of the pre-synthesis of chimeric molecules by RNA Structure software.Agarose gel electrophoresis was used to test the stability of chimeric molecules in serum and cell lysis solution.Using flow cytometry and confocal fluorescence microscope were used to estimate the internalization of AS1411-mediated STAT3 ASO.Inhibitory effect of ASO on the growth of tumor cells was detected by CCK-8 kit.RT-PCR and Western blot was used to measure the expression levels of tumor related genes.Results STAT3 ASOs mediated by AS1411 can enter tumor cells efficiently to inhibit the transcription and translation of C-myc,Cyclin D1,Bcl-xl and PD-L1 gene,and also can inhibit the growth of Du145 cells.Conclusions AS1411-mediated STAT3 ASO can enter tumor cells and act as anti-tumor medicine.

Synthesis and antisense properties of 2'β-F-/2'α-F-2'-deoxy-uridines modified oligonucleotides with 4'-C-(2-methoxyethoxy) substituent / 药学学报

Chemical modification is critical for the therapeutic applications of antisense oligonucleotides. Novel 4'-C-MOE and 2'-fluoro-modified monomer 2'-F-4'-C-MOE-araU and its epimeric 2'-F-4'-C-MOE-rU were synthesized from 2'-fluorinated arabinourine (2'-F-araU) and 2'-fluorouridine (2'-F-rU), respectively. Their phosphoramidites were synthesized and successfully incorporated into oligodeoxynucleotides. The mismatch discrimination ability of these unnatural monomers and their effect on thermal stability were evaluated in the context of dsDNA and DNA-RNA chimeras. The thermal denaturation studies showed that the incorporation of 2'-F-4'-C-MOE-araU led to enhanced binding affinity to complementary RNA strand and almost equivalent binding ability to complementary DNA, when compared with 2'-F-4'-C-MOE-rU and 2'-F-araU modified duplexes.Especially a C-H…F-C pseudohydrogen bond was supposed to contribute more binding affinity at uridine-purine steps, meanwhile, 2'-F-4'-C-MOE-araU had almost the same base discriminatory ability as uridine in dsDNA and DNA-RNA chimeras, while 2'-F-4'-C-MOE-rU was found to have only moderate RNA hybridization ability. However, 2'-F-4'-C-MOE-araU at 3'-end of oligonucleotide could not led to more nuclease hydrolytic stability than that with 2'-F-4'-C-MOE-rU modification. These results demonstrated the feasibility of C4'-MOE modification on 2'-F-ANA and the dramatic effects of the 2'-F substituent, which provides a new approach fo r further chemical modification of antisense drugs.

Establishment of Mouse Models with Kidney-positive Deficiency with Antisense Oligonucleotide-loaded Bio-degradable Polylactic Acid Microspheres / 中国药房

OBJECTIVE:To investigate the feasibility of mouse models establishment with kidney-positive deficiency with anti-sense oligonucleotide-loaded biodegradable polylactic acid microspheres. METHODS:Three-week old mice were randomized into groups A,B,C,D and E,with 6 mice in each group,where the mice in group A were given subcutaneous injection 0.2 ml nor-mal saline,and those in groups B-E were given subcutaneous injection 0.1,0.2,0.4 and 0.8 mg each mice of antisense oligonucle-otide-loaded biodegradable polylactic acid microspheres. After the administration,the weights and activities of mice in all groups were observed;and one month later,their livers,adrenal glands and brains were collected to detect the expression of glucocorti-coid receptor (GR) by real-time quantitative polymerase chain reaction (RT-PCR). RESULTS:All mice except those in group A were found to have kidney-positive deficiency symptoms such as developmental delay,light weight,less activities and gathering to-gether. Compared to group A,other groups showed lower GR expressions in liver,adrenal gland and brain. There was statistically difference (P<0.05). CONCLUSIONS:Antisense oligonucleotide-loaded biodegradable polylactic acid microspheres can inhibit GR expression,and the mice whose GR expression is inhibited show symptoms of kidney-positive deficiency,which provides a re-ference for the establishment of mouse models with kidney-positive deficiency.

Effects of FKBP51 acting on Caspase-3 and rat hippocampal CA1 area neuronal necrosis in cerebral ischemia reperfusion injury / 中国基层医药

Objective To explore the effect of FKBP51 acting on Caspase-3 and hippocampal CA1 area neu-ronal necrosis in cerebral ischemia reperfusion injury of rat.Methods SD rats were randomly divided into Sham group,ischemia reperfusion group ( I/R group ) , TE buffer group ( TE group ) , FKBP51 antisense oligonucleotide group (FKBP51 ASODN group) and FKBP51 missense oligonucleotide group (FKBP51 MSODN group).Transient global cerebral ischemia rats models were made by four-vessel method.We used Western blot to detect the expression of FKBP51,the effect of FKBP51 ASODN to FKBP51 expression and Caspase-3 activity;while we used HE staining technique to detect FKBP51 ASODN effect to rat hippocampal CA1 area neuronal necrosis.Results (1) In Sham group and I/R group (0min,15min,30min,1h,3h,6h,1d,3d),FKBP51 expressed,and the difference among the groups was no statistical significance (F=0.64,P>0.05).(2)The expression of FKBP51 in FKBP51 ASODN group was obviously reduced, and the difference was statistically significant compared with Sham group ( t =8.21, P <0.05).(3)The expression of Cleaved-Caspase-3 in Sham group obviously declined than the other groups,the differ-ence between them was statistically significant (F=12.31,P<0.05);The expression of FKBP51 in FKBP51 ASODN group was decreasing compared with FKBP51 MSODN group,and the difference was statistical significance(t=9.71, P<0.05).(4)HE staining showed:the number of Sham group (186.3 ±2.5) hippocampal CA1 pyramidal cells was most.The cells arranged densely,and nucleoli were large and round,the difference was statistically significant com-pared with the other groups (χ2 =81.91,P<0.05);The hippocampal CA1 pyramidal cells of I/R group (15.4 ± 2.6),TE group (18.5 ±2.2) and FKBP51 MSODN group (17.5 ±1.8) were almost completely disappeared,only left a few residual cells,a great quantity of denaturated cells which presented karyopykosis,tinctorialed endochylema, ruptured of membrane and released cell content;the hippocampal CA1 pyramidal cells FKBP51 ASODN group (92.8 ±2.6) survival increased significantly compared with other group,the difference was statistically significant (χ2 =52.36,P<0.05).Conclusion In cerebral ischemia reperfusion injury,FKBP51 can enhance the activation of Caspase-3 (Cleaved-Caspase-3) expression and inhibit the survival of the neurons.

Effect of PDGFR-αASODN/lip2000 complex implantation into vitreous cavity for proliferative vitreoretinopathy in rabbit / 实用医学杂志

Objective To examine the effect of PDGF-αreceptor on proliferative vitreoretinopathy (PVR) in rabbits. Methods Different concentrations of PDGFR-α ASODN were mixed with lip 2000, and the final proportion of PDGFR-α ASODN/lip2000 complex was 1∶1、1∶2.5 and 1∶5 respectively. All the complexes were incubated with cultured human retinal pigment epithelium for 24 hours before transfection. The rabbits were divided into group A (RPE cells)、group B and C (1.0、2.0 μmol/L PDGFR-αASODN lipofectin transfection of RPE cells) with 8 eyes each. The level of PVR were estimated by indirect ophthalmoscope examination; the fundus changes were estimated by histopathology; and the expression of PDGFA was detected by immunohistochemistry. Results The highest transduction efficiency was PDGFR-α ASODN/ lip2000 ratio to 1∶2.5. The degree of proliferative vitreoretinopath , the fundus changes and the density of PDGFA in group B and group C were significantly lower than that in group A, while group C more lower than group B. Conclusion PDGF-αreceptor antisense oligonucleotides can inhibit the development of experimental proliferative vitreoretinopathy.

Effects of Apollon antisense oligonucleotide on proliferation and apoptosis of K562 cells / 中华实用儿科临床杂志

Objective To investigate the effects of Apollon antisense oligonucleotide (ASODN) on proliferation,apoptosis and drug resistance of human leukemia(K562) cells.Methods Specific phosphorothioate ASODN and missense oligonucleotide (MSODN) of Apollon mRNA were synthesized and transfected into K562 cells following cationic liposome.The proliferation inhibition of K562 cells was assessed by MTT.The apoptosis rate was detected by Annexin V-FITC.The sensitivity of K562 cells to etoposide and vincristine was detected by MTT.Results Apollon antisense oligonucleotide inhibition of K562 cells with the concentration and time increased.ASODN at a final concentration of 600 nmol/L could significantly inhibit the K562 cell proliferation.The apoptosis rate was apparently increased (P ＜ 0.01).Conclusions Apollon ASODN may decrease Apollon gene expression,suppress K562 cells proliferation effectively,and induce significant apoptosis of K562 cells.Apollon ASODN is able to reverse the drug resistance via inhibition of Apollon expression and inducement of apoptosis.

Cholesterol conjugated spermine as a delivery modality of antisense oligonucleotide

The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.

Effect of suppression of platelet-derived growth factor-α receptor expression with antisense oligonucleotide on proliferation and apoptosis of retinal pigment epithelium cell / 中华实验眼科杂志

BackgroundRetinal pigment epithelial(RPE) cells can secrete platelet-derived growth factor (PDGF) and PDGF receptor(PDGFR).Studies have shown that PDGF plays a key role in the formation of proliferative vitreous retinopathy(PVR). ObjectiveThis study was to investigate the proliferation and apoptosis changes of RPE after blockage of the PDGFR-α expression by antisense oligonucleotide ( ASODN ) in vitro. Methods Human RPE cells strain was cultured in low glucose DMEM with 10％ fetal bovine serum.Logarithmic phase cells were collected and incubated in 96-well plate at the density of 5 × 105 cells/hole.PDGFR-α ASODN was transfected into RPE cells at different concentrations for 48 hours.The cells of the blank control group were regularly cultured without any transfection.The changes of PDGFR-α expression were detected by reverse transcription-polymerase chain reaction(RT-PCR),and the proliferation of RPE was detected by MTT as the A490 value.Hoechst 33258 fluorescence staining was used to determine the apoptosis of RPE.Flow cytometry method (FCM) was applied to detect the change of cell cycle and apoptosis rate of RPE cells. ResultsThe A490 values of RPE cells were 1.45±0.12,1.07±0.06,0.65±0.05 in blank control group,1.0 μmol/L Lipo-ASODN group and 2.0 μmol/L Lipo-ASODN group with the significant difference(P=0.00 ),and that of 1.0 μmol/L Lipo-ASODN group and 2.0 μ mol/L Lipo-ASODN group were significantly lower than the blank control group ( P =0.00,0.00).Hoechst 33258 staining showed that the apoptosis cells were obviously more in Lipo-ASODN group compared with blank control group.PDGFR-α ASODN transfection induced an increase of percentage of RPE cells in G0/G1 phase( F =206.70,P =0.00),and the apoptosis rates in 1.0 μmol/L Lipo-ASODN group and 2.0 μmol/L Lipo-ASODN group were significantly enhanced in comparison with blank control group ( 37.8 ± 1.3 vs 10.5 ± 0.1,61.2 ± 1.9 vs 10.5 ± 0.1 ) ( F =1808.90,P =0.00 ).Expression intensity of PDGFR-α mRNA in RPE cells in Lipo-ASODN groups was lower. ConclusionsBlocking the PDGFR-α expression with ASODN technology can suppress proliferation and induce apoptosis of RPE cells.Intensity of PDGFR-α mRNA expression in RPE cells is ASODN dose-dependent.ASODN targeted to PDGFR-α offers an experimental basis of the gene therapy for PVR.