Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 103
Filter
1.
Chem Pharm Bull (Tokyo) ; 68(2): 129-132, 2020.
Article in English | MEDLINE | ID: mdl-32009079

ABSTRACT

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.


Subject(s)
DNA, Antisense/administration & dosage , RNA, Small Interfering/administration & dosage , Amines/chemistry , Animals , Cations/chemistry , DNA, Antisense/chemistry , DNA, Antisense/genetics , DNA, Antisense/pharmacokinetics , Disulfides/chemistry , Gene Silencing , HeLa Cells , Humans , Male , Mice, Inbred ICR , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacokinetics , Transfection/methods
2.
J Invest Dermatol ; 140(2): 435-444.e4, 2020 02.
Article in English | MEDLINE | ID: mdl-31421125

ABSTRACT

The activation of T helper 17 signaling plays a critical role in psoriasis pathogenesis, and systemically-administered IL-17 inhibitors are highly effective therapy for moderate-to-severe disease. We generated topically-delivered gene-regulating nanoconstructs, comprised of spherically-arrayed antisense DNA (liposomal spherical nucleic acids [L-SNAs]), which are able to penetrate human skin to knock down cutaneous gene targets. Topically-applied L-SNAs targeting the gene encoding the mouse IL-17A receptor (Il17ra) reversed the development of psoriasis clinically, histologically, and transcriptionally in imiquimod-treated psoriasis-like mouse skin. Il17ra L-SNAs reduced the modified PASI by 74% versus controls and decreased epidermal thickness by 56%. Il17ra L-SNA reduced Il17ra protein expression by 75% and significantly decreased the mRNA expression of psoriasis markers, including Defb4, Il17c, S100a7, Pi3, Krt16, and Tnfa versus scrambled spherical nucleic acid (Scr SNA) controls. A human IL17RA L-SNA penetrates 3-dimensional cultures and normal human explants to knock down IL17RA mRNA by 63% and 66%, respectively. After topical application to psoriatic 3-dimensional rafts, anti-human IL17RA L-SNAs reduced the expression of IL17RA (by 72%) and the IL-17-induced genes IL17C (by 85%), DEFB4 (by 83%), TNFA (by 77%), and PI3 (by 65%) versus scrambled L-SNA and vehicle controls (all P < 0.001). Taken together, these data suggest that targeted suppression of IL17RA is a promising new topical treatment strategy for psoriasis.


Subject(s)
DNA, Antisense/administration & dosage , Nanospheres/administration & dosage , Psoriasis/drug therapy , RNA, Messenger/drug effects , Receptors, Interleukin-17/antagonists & inhibitors , Administration, Cutaneous , Animals , Biomarkers/metabolism , Cells, Cultured , Disease Models, Animal , Gene Knockdown Techniques , Humans , Imiquimod/immunology , Keratinocytes , Liposomes , Mice , Primary Cell Culture , Psoriasis/chemically induced , Psoriasis/diagnosis , Psoriasis/immunology , RNA, Messenger/immunology , RNA, Messenger/metabolism , Receptors, Interleukin-17/genetics , Receptors, Interleukin-17/immunology , Severity of Illness Index , Skin/cytology , Skin/drug effects , Skin/immunology , Skin/pathology
3.
Cancer ; 124(19): 3881-3889, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30291796

ABSTRACT

BACKGROUND: Cetuximab combined with radiation therapy (RT) is an evidence-based treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, locoregional failure remains the primary cause of cancer-related death in this disease. Intratumoral injection of epidermal growth factor receptor (EGFR)-antisense plasmid DNA (EGFR-AS) is safe and has been associated with promising lesional responses in patients who have recurrent/metastatic HNSCC. For the current study, the authors investigated the antitumor effects of cetuximab and EGFR-AS in preclinical HNSCC models and reported their phase 1 experience adding intratumoral EGFR-AS to cetuximab RT. METHODS: Antitumor mechanisms were investigated in cell line and xenograft models. Phase 1 trial eligibility required stage IVA through IVC HNSCC and a measurable lesion accessible for repeat injections. Patients received standard cetuximab was for 9 weeks. EGFR-AS was injected weekly until they achieved a lesional complete response. RT was delivered by conventional fractionation for 7 weeks, starting at week 3. Research biopsies were obtained at baseline and week 2. RESULTS: When added to cetuximab, EGFR-AS decreased cell viability and xenograft growth compared with EGFR-sense control, partially mediated by reduced EGFR expression. Six patients were enrolled in the phase 1 cohort. No grade 2 or greater EGFR-AS-related adverse events occurred. The best lesional response was a complete response (4 patients), and 1 patient each had a partial response and disease progression. EGFR expression decreased in 4 patients who had available paired specimens. CONCLUSIONS: In preclinical models, dual EGFR inhibition with cetuximab and EGFR-AS enhanced antitumor effects. In a phase 1 cohort, intratumoral EGFR-AS injections, cetuximab, and RT were well tolerated. A phase 2 trial is needed to conduct an extended evaluation of safety and to establish efficacy.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cetuximab/administration & dosage , DNA, Antisense/administration & dosage , Head and Neck Neoplasms/therapy , Squamous Cell Carcinoma of Head and Neck/therapy , Aged , Aged, 80 and over , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Combined Modality Therapy , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Female , Genetic Therapy/methods , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/mortality , Head and Neck Neoplasms/pathology , Humans , Male , Mice , Mice, Nude , Middle Aged , Molecular Targeted Therapy/methods , Protein Kinase Inhibitors/administration & dosage , Radiotherapy, Adjuvant , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/mortality , Squamous Cell Carcinoma of Head and Neck/pathology , Xenograft Model Antitumor Assays
4.
Nano Lett ; 18(9): 5652-5659, 2018 09 12.
Article in English | MEDLINE | ID: mdl-30088771

ABSTRACT

One of the biggest obstacles for the use of antisense oligonucleotides as antibacterial therapeutics is their limited uptake by bacterial cells without a suitable carrier, especially in multi-drug-resistant bacteria with a drug efflux mechanism. Existing vectors, such as cell-penetrating peptides, are inefficient and nontargeting, and accordingly are not ideal carriers. A noncytotoxic tetrahedral DNA nanostructure (TDN) with a controllable conformation has been developed as a delivery vehicle for antisense oligonucleotides. In this study, antisense peptide nucleic acids (asPNAs) targeting a specific gene ( ftsZ) were efficiently transported into methicillin-resistant Staphylococcus aureus cells by TDNs, and the expression of ftsZ was successfully inhibited in an asPNA-concentration-dependent manner. The delivery system specifically targeted the intended gene. This novel delivery system provides a better platform for future applications of antisense antibacterial therapeutics and provides a basis for the development of a new type of antibacterial drug for multi-drug-resistant bacterial infections.


Subject(s)
Anti-Bacterial Agents/pharmacology , DNA, Antisense/pharmacology , Methicillin-Resistant Staphylococcus aureus/drug effects , Nanostructures/chemistry , Peptide Nucleic Acids/pharmacology , Staphylococcal Infections/drug therapy , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/chemistry , Bacterial Proteins/genetics , Cytoskeletal Proteins/genetics , DNA, Antisense/administration & dosage , DNA, Antisense/chemistry , Down-Regulation/drug effects , Drug Carriers/chemistry , Humans , Peptide Nucleic Acids/administration & dosage , Peptide Nucleic Acids/chemistry , Staphylococcal Infections/genetics
6.
Arq Neuropsiquiatr ; 76(4): 265-272, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29742241

ABSTRACT

Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.


Subject(s)
DNA, Antisense/administration & dosage , Genetic Therapy/methods , Muscular Atrophy, Spinal/therapy , Oligonucleotides/administration & dosage , Survival of Motor Neuron 1 Protein/administration & dosage , Humans , Injections, Spinal , Mutation , Phenotype
7.
Arq. neuropsiquiatr ; 76(4): 265-272, Apr. 2018. tab, graf
Article in English | LILACS | ID: biblio-888378

ABSTRACT

ABSTRACT Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.


RESUMO A atrofia muscular espinhal (AME) é uma grave doença dos neurônios motores, de grande variabilidade clínica e causada na maioria dos casos por mutação em homozigose no gene SMN1. Pelo menos quatro fenótipos clínicos distintos são reconhecidos com base na idade de início e no grau de envolvimento motor. Tal variabilidade clínica é em parte relacionada com o número de cópias do gene SMN2. Até recentemente, apenas terapias de suporte estavam disponíveis. Atualmente, terapias especificas estão sendo desenvolvidas com base em diferentes mecanismos para aumentar o nível de proteína SMN; em particular oligonucleotídeos antissenso por via intratecal e inserção de cópia do gene SMN1, via endovenosa, usando vetor viral. Nesta revisão, objetivamos discutir as mais recentes e promissoras estratégias terapêuticas, com consideração especial aos aspectos patogênicos da doença e aos mecanismos de ação de tais terapias.


Subject(s)
Humans , Oligonucleotides/administration & dosage , Muscular Atrophy, Spinal/therapy , Genetic Therapy/methods , DNA, Antisense/administration & dosage , Survival of Motor Neuron 1 Protein/administration & dosage , Phenotype , Injections, Spinal , Mutation
8.
Methods Mol Biol ; 1639: 127-138, 2017.
Article in English | MEDLINE | ID: mdl-28752452

ABSTRACT

Alpha-1 antitrypsin (AAT) is a serum protease inhibitor, mainly expressed in and secreted from hepatocytes, important for regulating neutrophil elastase activity among other proteases. Various mutations in AAT cause alpha-1 antitrypsin deficiency (AATD), a rare hereditary disorder that results in liver disease due to accumulation of AAT aggregates and lung disease from excessive neutrophil elastase activity. PiZ transgenic mice contain the human AAT genomic region harboring the most common AATD mutation, the Glu342Lys (Z) point mutation. These mice effectively recapitulate the liver disease exhibited in AATD patients, including AAT protein aggregates, hepatocyte death, and eventual liver fibrosis. Previously, we demonstrated that modified antisense oligonucleotides (ASOs) can dramatically reduce Z-AAT RNA and protein levels in PiZ mice enabling inhibition, prevention, and reversal of the associated liver disease. Here, we describe in detail usage of AAT-ASOs to knock down Z-AAT in PiZ mice with a focus on preparation and in vivo delivery of ASOs, as well as detailed workflows pertaining to the analysis of Z-AAT mRNA, plasma protein, and soluble/insoluble liver protein levels following ASO administration.


Subject(s)
Gene Knockdown Techniques/methods , Mutation/genetics , alpha 1-Antitrypsin/genetics , Animals , DNA, Antisense/administration & dosage , Disease Models, Animal , Humans , Liver/metabolism , Mice , Oligonucleotides, Antisense/administration & dosage , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Solubility , alpha 1-Antitrypsin/blood , alpha 1-Antitrypsin/metabolism
9.
Sci Rep ; 6: 21262, 2016 Feb 16.
Article in English | MEDLINE | ID: mdl-26880216

ABSTRACT

Chronic kidney disease is a progressive incurable pathology affecting millions of people. Intensive investigations aim to identify targets for therapy. We have previously demonstrated that abnormal expression of the Discoidin Domain Receptor 1 (DDR1) is a key factor of renal disease by promoting inflammation and fibrosis. The present study investigates whether blocking the expression of DDR1 after the initiation of renal disease can delay or arrest the progression of this pathology. Severe renal disease was induced by either injecting nephrotoxic serum (NTS) or performing unilateral ureteral obstruction in mice, and the expression of DDR1 was inhibited by administering antisense oligodeoxynucleotides either at 4 or 8 days after NTS (corresponding to early or more established phases of disease, respectively), or at day 2 after ligation. DDR1 antisense administration at day 4 stopped the increase of proteinuria and protected animals against the progression of glomeruloneprhitis, as evidenced by functional, structural and cellular indexes. Antisense administration at day 8 delayed progression -but to a smaller degree- of renal disease. Similar beneficial effects on renal structure and inflammation were observed with the antisense administration of DDR1 after ureteral ligation. Thus, targeting DDR1 can be a promising strategy in the treatment of chronic kidney disease.


Subject(s)
Discoidin Domain Receptor 1/genetics , Kidney Diseases/genetics , Animals , Cytokines/metabolism , DNA, Antisense/administration & dosage , Discoidin Domain Receptor 1/metabolism , Disease Models, Animal , Disease Progression , Fibrosis , Gene Expression , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Inflammation Mediators/metabolism , Kidney Diseases/metabolism , Kidney Diseases/pathology , Mice
10.
Genet Mol Res ; 14(3): 10087-95, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-26345946

ABSTRACT

We investigated the effects of hepatitis B virus (HBV) S/C double gene loci antisense locked nucleic acid on replication and expression of HBV in hepatitis transgenic mice. HBV mice (N = 30) were randomly divided into five groups of six mice: 5% glucose solution control, empty liposome control, single-target S, single-target C, and dual-target SC groups. An antisense locked nucleic acid fragment was injected into the mice. Serum HBsAg, serum HBV DNA, HBV C-mRNA expression in liver tissue, HbsAg and HbcAg expression in hepatocytes, serum albumin, alanine transaminase (ALT), urea nitrogen, and creatinine were detected. Liver and kidney sections were examined for the effects of antisense locked nucleic acid. The expression of HBsAg was markedly inhibited; the inhibition rates of the S, C, and SC target groups were 36.63, 31.50, and 54.87%, respectively; the replication of HBV DNA was also inhibited: 23.97, 21.13, and 35.83%, respectively. After injection at 1, 3, and 5 days, the corresponding rates for HBsAg inhibition were 14.40, 25.61, and 31.33%, and for HBV DNA inhibition they were 11.04, 19.24, and 24.13%. Compared with the control group, the differences in serum albumin, ALT, urea nitrogen, and creatinine in each group were not statistically significant, and the number of HbsAg- and HBcAg-positive cells in the mouse liver was significantly reduced. The liver and kidney tissues were normal. The gene therapy had significant inhibitory effects on the replication and expression of HBV in transgenic mice, and double-gene targeting was better than single-gene targeting.


Subject(s)
DNA, Antisense/genetics , Hepatitis B Core Antigens/genetics , Hepatitis B Surface Antigens/genetics , Hepatitis B/virology , Animals , DNA, Antisense/administration & dosage , DNA, Antisense/toxicity , Disease Models, Animal , Gene Expression Regulation, Viral , Hepatitis B/blood , Hepatitis B/pathology , Hepatitis B Surface Antigens/blood , Hepatitis B virus/genetics , Humans , Kidney Function Tests , Liver Function Tests , Mice , Mice, Transgenic , RNA, Messenger/genetics , Viral Load , Virus Replication/genetics
11.
Sci Rep ; 5: 12297, 2015 Jul 20.
Article in English | MEDLINE | ID: mdl-26189409

ABSTRACT

Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases.


Subject(s)
DNA, Antisense , Gold , Metal Nanoparticles , Theranostic Nanomedicine , Animals , Cell Line, Tumor , DNA, Antisense/administration & dosage , DNA, Antisense/chemistry , Disease Models, Animal , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Mice , Mice, Nude , Neoplasms/diagnosis , Neoplasms/genetics , Neoplasms/mortality , Neoplasms/therapy , Proto-Oncogene Proteins p21(ras)/genetics , Spectroscopy, Fourier Transform Infrared , Xenograft Model Antitumor Assays
12.
Biomaterials ; 62: 155-63, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26056726

ABSTRACT

Here, we designed biomimetic DNA nanoballs for delivery of multiple antisense oligonucleotides (ASOs). DNA templates with ASOs-complementary sequences were amplified by rolling circle amplification (RCA). RCA products were loaded with two types of ASOs by hybridization, condensed using adenovirus-derived Mu peptide, and coated with hyaluronic acid (HA) for delivery into CD44-overexpressing tumor cells. HA-coated, Mu peptide-condensed, dual ASO-loaded DNA nanoballs (HMA nanoballs) showed considerable cellular entry of Cy5-incorporated RCA product DNA and fluorescent ASOs, whereas Mu peptide-condensed, dual ASO-loaded DNA nanoballs (MA nanoballs) revealed limited uptake. Dual ASOs, Dz13 and OGX-427, delivered by HMA nanoballs could reduce the levels of protein targets and exert anticancer effects. Enhanced tumor distribution was observed for fluorescent HMA nanoballs than the corresponding MA nanoballs. Upon intravenous co-administration with doxorubicin, HMA nanoballs exerted the greatest anti-tumor effects among the groups. These results suggest HMA nanoballs as a nanoplatform for sequence-specific delivery of multiple ASOs and other functional oligonucleotides.


Subject(s)
DNA, Antisense/administration & dosage , DNA, Antisense/genetics , Nanospheres/chemistry , Neoplasms, Experimental/genetics , Neoplasms, Experimental/therapy , Transfection/methods , Biomimetic Materials/administration & dosage , Biomimetic Materials/chemical synthesis , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Diffusion , Genetic Therapy/methods , Humans , Nanocapsules/administration & dosage , Nanocapsules/chemistry , Nanocapsules/ultrastructure , Nanospheres/administration & dosage , Nanospheres/ultrastructure , Particle Size , Treatment Outcome
13.
Environ Toxicol Pharmacol ; 39(3): 1199-205, 2015 May.
Article in English | MEDLINE | ID: mdl-25982951

ABSTRACT

Tissue factor (TF) is a membranous glycoprotein that activates the coagulation system when blood vessels or tissues are damaged. TF was up-regulated in monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity model. The present study aimed to test the hypothesis that TF-dependent fibrin deposition occurs in liver toxicity induced by CCl4 in mice. Pericentral deposition of TF and fibrin is induced after CCl4-induced liver toxicity. The toxicity was evaluated by determination of serum activities of ALT, AST and ALP as well as GSH content and histopathological changes. The results showed that injection of mice with TF-antisense deoxyoligonucleotide (TF-AS) prevented the accumulation of TF and fibrin in the hepatic tissues. Furthermore, it significantly restored blood biochemical parameters, GSH content and distorted histopathological features caused by CCl4. The current study demonstrates that TF activation is associated with CCl4-induced liver injury. Furthermore, administration of TF-AS successfully prevented this type of liver injury.


Subject(s)
Carbon Tetrachloride/toxicity , Chemical and Drug Induced Liver Injury/pathology , Fibrin/metabolism , Thromboplastin/metabolism , Animals , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/metabolism , DNA, Antisense/administration & dosage , DNA, Antisense/pharmacology , Disease Models, Animal , Gene Expression Regulation, Enzymologic/drug effects , Glutathione/metabolism , Male , Mice , Thromboplastin/antagonists & inhibitors , Transaminases/blood
14.
Adv Drug Deliv Rev ; 87: 25-34, 2015 Jun 29.
Article in English | MEDLINE | ID: mdl-25912659

ABSTRACT

Synthetic, complementary DNA single strands and short interfering RNA double strands have been found to inhibit the expression of animal, plant, and viral genes in cells, animals, and patients, in a dose dependent and sequence specific manner. DNAs and RNAs, however, are readily digested in biological systems. Hence, chemists are obliged to design and synthesize nuclease-resistant analogs of normal DNA (Fig. 1).


Subject(s)
DNA, Antisense , Genetic Therapy , RNA, Antisense , Animals , DNA, Antisense/administration & dosage , DNA, Antisense/chemistry , DNA, Antisense/pharmacology , Humans , RNA Interference/drug effects , RNA, Antisense/administration & dosage , RNA, Antisense/chemistry , RNA, Antisense/pharmacology
15.
Cancer Chemother Pharmacol ; 74(1): 151-66, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24832107

ABSTRACT

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.


Subject(s)
Antineoplastic Agents/therapeutic use , DNA, Antisense/therapeutic use , DNA, Single-Stranded/therapeutic use , Gene Silencing/drug effects , Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , 5' Flanking Region/drug effects , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/metabolism , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , DNA, Antisense/administration & dosage , DNA, Antisense/pharmacokinetics , DNA, Antisense/pharmacology , DNA, Single-Stranded/administration & dosage , DNA, Single-Stranded/pharmacokinetics , DNA, Single-Stranded/pharmacology , Drug Compounding , Drug Stability , Female , Liposomes , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, SCID , Neoplasms/blood , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/pharmacokinetics , Oligodeoxyribonucleotides/pharmacology , Oligodeoxyribonucleotides/therapeutic use , Pharmaceutical Vehicles , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Random Allocation , Xenograft Model Antitumor Assays
16.
ACS Nano ; 7(4): 2948-59, 2013 Apr 23.
Article in English | MEDLINE | ID: mdl-23473419

ABSTRACT

The unique photophysical properties of noble metal nanoparticles contribute to their potential as photoactivated drug delivery vectors. Here we demonstrate the synthesis and characterization of 60-80 nm silver nanoparticles (SNPs) decorated with thiol-terminated photolabile DNA oligonucleotides. In vitro assays and fluorescent confocal microscopy of treated cell cultures show efficient UV-wavelength photoactivation of surface-tethered caged ISIS2302 antisense oligonucleotides possessing internal photocleavable linkers. As a demonstration of the advantages of these novel nanocarriers, we investigate properties including: enhanced stability to nucleases, increased hybridization activity upon photorelease, and efficient cellular uptake as compared to commercial transfection vectors. Their potential as multicomponent delivery agents for oligonucleotide therapeutics is shown through regulation of ICAM-1 (Intracellular Adhesion Molecule-1) silencing. Our results suggest a means to achieve light-triggered, spatiotemporally controlled gene silencing via nontoxic silver nanocarriers, which hold promise as tailorable platforms for nanomedicine, gene expression studies, and genetic therapies.


Subject(s)
DNA, Antisense/administration & dosage , DNA, Antisense/genetics , Gene Silencing , Intercellular Adhesion Molecule-1/genetics , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Nanocapsules/chemistry , Nanocapsules/radiation effects , Silver/chemistry , DNA, Antisense/chemistry , HeLa Cells , Humans , Materials Testing , Metal Nanoparticles/radiation effects , Nanocapsules/ultrastructure , Particle Size , Ultraviolet Rays
17.
Eur J Hum Genet ; 21(7): 774-8, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23211698

ABSTRACT

Recent development of next-generation DNA sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We applied NGS to study an A-T patient in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. The 160-kb ATM genomic region was divided into 31 partially overlapping fragments of 4-6 kb and amplified by long-range PCR in the patient and mother, who carried the same mutation by segregation. We identified six intronic variants that were shared by the two genomes and not reported in the dbSNP(132) database. Among these, c.1236-405C>T located in IVS11 was predicted to be pathogenic because it affected splicing. This mutation creates a cryptic novel donor (5') splice site (score 1.00) 405 bp upstream of the exon 12 acceptor (3') splice site. cDNA analysis showed the inclusion of a 212-bp non-coding 'pseudoexon' with a premature stop codon. We validated the functional effect of the splicing mutation using a minigene assay. Using antisense morpholino oligonucleotides, designed to mask the cryptic donor splice-site created by the c.1236-405C>T mutation, we abrogated the aberrant splicing product to a wild-type ATM transcript, and in vitro reverted the functional ATM kinase impairment of the patients' lymphoblasts. Resequencing is an effective strategy for identifying rare splicing mutations in patients for whom other mutation analyses have failed (DHPLC, MLPA, or cDNA sequencing). This is especially important because many of these patients will carry rare splicing variants that are amenable to antisense-based correction.


Subject(s)
Ataxia Telangiectasia/genetics , Ataxia Telangiectasia/therapy , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Morpholinos/administration & dosage , Protein Serine-Threonine Kinases/genetics , RNA Splice Sites/genetics , Tumor Suppressor Proteins/genetics , Ataxia Telangiectasia/pathology , Ataxia Telangiectasia Mutated Proteins , Base Sequence , Cell Line , DNA, Antisense/administration & dosage , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Introns/genetics , Mutation/genetics
18.
J Am Chem Soc ; 134(40): 16488-91, 2012 Oct 10.
Article in English | MEDLINE | ID: mdl-23020598

ABSTRACT

Spherical nucleic acid (SNA) constructs are promising new single entity gene regulation materials capable of both cellular transfection and gene knockdown, but thus far are promiscuous structures, exhibiting excellent genetic but little cellular selectivity. In this communication, we describe a strategy to impart targeting capabilities to these constructs through noncovalent functionalization with a complementary antibody-DNA conjugate. As a proof-of-concept, we designed HER2-targeting SNAs and demonstrated that such structures exhibit cell type selectivity in terms of their uptake, and significantly greater gene knockdown in cells overexpressing the target antigen as compared to the analogous antibody-free and off-target materials.


Subject(s)
Antibodies, Monoclonal/immunology , DNA, Antisense/administration & dosage , Immunoconjugates/immunology , Receptor, ErbB-2/immunology , Antibodies, Monoclonal/chemistry , Cell Line, Tumor , DNA, Antisense/chemistry , DNA, Antisense/genetics , DNA, Antisense/pharmacokinetics , Gene Knockdown Techniques , Humans , Immunoconjugates/chemistry , Models, Molecular , Receptor, ErbB-2/genetics
19.
Neurosci Res ; 72(3): 257-62, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22192464

ABSTRACT

The G protein-coupled receptor 39-b (GPR39-1b) is a splice variant of which is expressed in the central nervous and gastrointestinal systems. Previously, GPR39-1b was proposed to be the receptor for obestatin, but current evidence does not support this hypothesis. The purpose of the present work was to identify the role of GPR39-1b in anxiety and eating behaviors. Antisense oligonucleotides were infused at a constant rate into the cerebral lateral ventricles of rats and their effect on anxiety-like behavior and food intake was monitored. GPR39-1b antisense oligonucleotides produced anxiolytic-like effects in the elevated-plus maze test and in the black and white box test. Antisense oligonucleotides also decreased food intake. These results indicate that inhibition of GPR39-1b induces a decrease in anxiety-related behaviors and disturbs appetite.


Subject(s)
Anxiety/genetics , Behavior, Animal/drug effects , DNA, Antisense/administration & dosage , Eating/drug effects , Emotions/drug effects , Receptors, G-Protein-Coupled/genetics , Animals , Anxiety/metabolism , Appetite/drug effects , Appetite/genetics , Body Weight/drug effects , Body Weight/genetics , Dentate Gyrus/drug effects , Dentate Gyrus/metabolism , Eating/genetics , Male , Rats , Rats, Wistar , Receptors, G-Protein-Coupled/metabolism
20.
Cancer Lett ; 311(1): 77-84, 2011 Dec 01.
Article in English | MEDLINE | ID: mdl-21824723

ABSTRACT

Carbonyl reductase (CR) is an NADPH-dependent, mostly monomeric, cytosolic enzyme with broad substrate specificity for carbonyl compounds. CR appears to be involved in the regulation of tumour progression. However, molecular mechanisms of CR in tumour progression and clinical significance of CR status remain unclear in human uterine squamous cell carcinoma (SCC). Here, we investigated the clinical significance of CR using immunohistochemical analyses of human uterine cervical SCC tissues and how CR affects cancer cell behaviour in vitro. Paraffin sections from uterine cervical SCC tissues, FIGO stage Ib1-IIb (n = 67) were immunostained with anti-CR antibodies. Overall survival (OS) and progression-free survival (PFS) were analyzed by the Kaplan-Meier method. Sense and antisense CR cDNAs were transfected into a human uterine SCC cell line (SiHa) to investigate the role of CR in cancer cell invasion and metastasis. Immunohistochemical analyses showed that reduced CR expression patterns in primary cancer lesions were closely associated with a high incidence of pelvic lymph node metastasis, poor OS, and poor PFS. In an in vitro experiment, suppression of CR increased cancer cell invasion, secretion of MMP-2, -9 and cyclooxygenase-2 (COX-2) expression and decreased E-cadherin expression. On the other hand, over-expression of CR increased E-cadherin expression and decreased MMP-2, -9 secretion and COX-2 expression. The reduced CR expression pattern, as measured by immunohistochemistry, can be a useful predictor of lymph node metastasis and poor prognosis in patients with uterine SCC. This clinical result is supported by the in vitro data which show that suppression of CR expression promotes cancer cell invasion with decreased E-cadherin expression and increased MMP-2, -9 secretion.


Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Carcinoma, Squamous Cell/enzymology , Uterine Cervical Neoplasms/enzymology , Alcohol Oxidoreductases/biosynthesis , Alcohol Oxidoreductases/metabolism , Cadherins/biosynthesis , Cadherins/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2/genetics , Cyclooxygenase 2 Inhibitors/pharmacology , DNA, Antisense/administration & dosage , DNA, Antisense/genetics , Disease-Free Survival , Enzyme Precursors/biosynthesis , Enzyme Precursors/genetics , Female , Gelatinases/biosynthesis , Gelatinases/genetics , Humans , Immunohistochemistry , Lymphatic Metastasis , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/biosynthesis , Matrix Metalloproteinase 9/genetics , Neoplasm Invasiveness , Prognosis , Transfection , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology
SELECTION OF CITATIONS
SEARCH DETAIL
...