Chandra Vargeese - Academia.edu (original) (raw)
Papers by Chandra Vargeese
Nature Biotechnology, 2005
The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to... more The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA 41.0 log 10 . The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.
Molecular Pharmaceutics, 2015
Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle med... more Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems.
Nucleic Acids Research, 1998
A new activator for the coupling of phosphoramidites to the 5′-hydroxyl group during oligonucleot... more A new activator for the coupling of phosphoramidites to the 5′-hydroxyl group during oligonucleotide synthesis is introduced. The observed time to complete coupling is twice as fast with 4,5-dicyanoimidazole (DCI) as the activator, compared with 1H-tetrazole. The effectiveness of DCI is thought to be based on its nucleophilicity. DCI is soluble in acetonitrile up to 1.1 M at room temperature and can be used as the sole coupling activator during routine automated solid phase synthesis of oligonucleotides. The addition of 0.1 M N-methylimidazole to 0.45 M 1H-tetrazole also results in higher product yields during oligonucleotide synthesis than observed with 1H-tetrazole alone.
Molecular Therapy, 2005
We then combined these validated shRNAs in a triple construct under the expression of a single pr... more We then combined these validated shRNAs in a triple construct under the expression of a single promoter and demonstrated functional activity of each of the individual shRNAs against their cognate targets. Results comparing these different strategies and their implication for shRNA-based drug development will be discussed.
Molecular Therapy, 2006
motif TFOs can induce gene modification in chromosomal DNA containing multiple copies of the muta... more motif TFOs can induce gene modification in chromosomal DNA containing multiple copies of the mutated gene via the effect of the triple helix itself. Gene modification with TFOs includes induced recombination between a DNA target and a donor DNA molecule, a process that allows a TFO to exert an effect at a distance from the third-strand binding site. In this work, we report the ability of a N3->P5 phosphoramidate-modified pyrimidine motif TFO to induce repair and recombination in the absence of any DNA-reactive conjugate. We establish the ability of this modified TFO when cotransfected with short, single-stranded donor DNAs to induce gene correction at a single-copy chromosomal locus in mammalian cells at frequencies up to 0.1%. We demonstrate sequence, target site, and dose-dependent effects of the TFO. Additionally, we show that the induction of recombination is independent of the binding motif and the orientation of the TFO (parallel versus anti-parallel), but is dependent on the donor fragment being homologous to the transcribed strand of the duplex. This work expands the number of targets available for triplex-mediated gene targeting and shows effective targeting at a chromosomal locus.
Journal of Viral Hepatitis, 2002
Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide a... more Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nucleaseresistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.
Gene Therapy, 2006
In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth... more In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mg of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mg of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential. Gene Therapy (2006) 13, 225-234.
![Research paper thumbnail of Biodistribution and Metabolism Studies of Lipid Nanoparticle-Formulated Internally [3H]-Labeled siRNA in Mice](https://attachments.academia-assets.com/53061440/thumbnails/1.jpg)
](Drug Metabolism and Disposition, 2014
Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA)... more Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [ 3 H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [ 3 H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [ 3 H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [ 3 H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [ 3 H]-SSB siRNA. dx.doi.org/10.1124/dmd.113.055434.
Bioorganic & Medicinal Chemistry Letters, 1998
Several 9-aralkyladenines have been prepared and their ADA inhibitory activity was determined. A ... more Several 9-aralkyladenines have been prepared and their ADA inhibitory activity was determined. A minimum of two carbon atoms separating the aromatic ring from the adenine-bearing carbon (C3') was found essential for potent activity. 0
AJP: Endocrinology and Metabolism, 2009
1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , Fe... more 1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , February 16, 2010; 107 : 3087-3092. PNAS Polisky B, Olefsky JM. Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes. The transcription factor TORC2 [transducer of regulated cAMP-responsive element-binding protein (CREB) activity 2] is a major regulator of hepatic gluconeogenesis and is increased in hyperglycemic rodent models. Because chronic hyperglycemia and increased hepatic glucose production, via increased gluconeogenesis, is a key feature of type 2 diabetes, an effective in vivo method to efficiently knock down TORC2 could provide a potential therapy for treating hyperglycemia and type 2 diabetes. To assess this, primary mouse hepatocytes, high-fat diet (HFD)-fed mice, and Zucker diabetic fatty (ZDF) rats were treated with a siRNA against TORC2 (siTORC2), which was delivered via a novel lipid nanoparticle system, or control siRNA (siCON). Compared with siCON, administration of siTORC2 resulted in highly efficient, sustained (1-3 wk) knockdown of TORC2 and its gluconeogenic target genes phosphoenolpyruvate carboxykinase and glucose-6-phophatase in primary mouse hepatocytes and in the livers of HFD-fed mice. In mice, this knockdown was specific to the liver and did not occur in kidney, skeletal muscle, or adipose tissue. In HFD-fed mice, siTORC2 reduced in vivo gluconeogenic capacity, fasting hepatic glucose production, and hyperglycemia, and led to improved hepatic and skeletal muscle insulin sensitivity. siTORC2 treatment also improved systemic hyperglycemia in ZDF rats. In conclusion, these results demonstrate the importance of TORC2 in modulating HGP in vivo and highlight a novel, liver-specific siRNA approach for the potential treatment of hyperglycemia and type 2 diabetes. transducer of regulated cAMP-responsive element-binding protein activity 2; diabetes; small interfering RNA Address for reprint requests and other correspondence: M. Saberi and D. Bjelica contributed equally to this work. Jerrold M. Olefsky,
Nature Biotechnology, 2005
The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to... more The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA 41.0 log 10 . The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.
Molecular Pharmaceutics, 2015
Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle med... more Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems.
Nucleic Acids Research, 1998
A new activator for the coupling of phosphoramidites to the 5′-hydroxyl group during oligonucleot... more A new activator for the coupling of phosphoramidites to the 5′-hydroxyl group during oligonucleotide synthesis is introduced. The observed time to complete coupling is twice as fast with 4,5-dicyanoimidazole (DCI) as the activator, compared with 1H-tetrazole. The effectiveness of DCI is thought to be based on its nucleophilicity. DCI is soluble in acetonitrile up to 1.1 M at room temperature and can be used as the sole coupling activator during routine automated solid phase synthesis of oligonucleotides. The addition of 0.1 M N-methylimidazole to 0.45 M 1H-tetrazole also results in higher product yields during oligonucleotide synthesis than observed with 1H-tetrazole alone.
Molecular Therapy, 2005
We then combined these validated shRNAs in a triple construct under the expression of a single pr... more We then combined these validated shRNAs in a triple construct under the expression of a single promoter and demonstrated functional activity of each of the individual shRNAs against their cognate targets. Results comparing these different strategies and their implication for shRNA-based drug development will be discussed.
Molecular Therapy, 2006
motif TFOs can induce gene modification in chromosomal DNA containing multiple copies of the muta... more motif TFOs can induce gene modification in chromosomal DNA containing multiple copies of the mutated gene via the effect of the triple helix itself. Gene modification with TFOs includes induced recombination between a DNA target and a donor DNA molecule, a process that allows a TFO to exert an effect at a distance from the third-strand binding site. In this work, we report the ability of a N3->P5 phosphoramidate-modified pyrimidine motif TFO to induce repair and recombination in the absence of any DNA-reactive conjugate. We establish the ability of this modified TFO when cotransfected with short, single-stranded donor DNAs to induce gene correction at a single-copy chromosomal locus in mammalian cells at frequencies up to 0.1%. We demonstrate sequence, target site, and dose-dependent effects of the TFO. Additionally, we show that the induction of recombination is independent of the binding motif and the orientation of the TFO (parallel versus anti-parallel), but is dependent on the donor fragment being homologous to the transcribed strand of the duplex. This work expands the number of targets available for triplex-mediated gene targeting and shows effective targeting at a chromosomal locus.
Journal of Viral Hepatitis, 2002
Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide a... more Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nucleaseresistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.
Gene Therapy, 2006
In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth... more In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mg of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mg of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential. Gene Therapy (2006) 13, 225-234.
Drug Metabolism and Disposition, 2014
Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA)... more Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [ 3 H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [ 3 H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [ 3 H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [ 3 H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [ 3 H]-SSB siRNA. dx.doi.org/10.1124/dmd.113.055434.
Bioorganic & Medicinal Chemistry Letters, 1998
Several 9-aralkyladenines have been prepared and their ADA inhibitory activity was determined. A ... more Several 9-aralkyladenines have been prepared and their ADA inhibitory activity was determined. A minimum of two carbon atoms separating the aromatic ring from the adenine-bearing carbon (C3') was found essential for potent activity. 0
AJP: Endocrinology and Metabolism, 2009
1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , Fe... more 1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , February 16, 2010; 107 : 3087-3092. PNAS Polisky B, Olefsky JM. Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes. The transcription factor TORC2 [transducer of regulated cAMP-responsive element-binding protein (CREB) activity 2] is a major regulator of hepatic gluconeogenesis and is increased in hyperglycemic rodent models. Because chronic hyperglycemia and increased hepatic glucose production, via increased gluconeogenesis, is a key feature of type 2 diabetes, an effective in vivo method to efficiently knock down TORC2 could provide a potential therapy for treating hyperglycemia and type 2 diabetes. To assess this, primary mouse hepatocytes, high-fat diet (HFD)-fed mice, and Zucker diabetic fatty (ZDF) rats were treated with a siRNA against TORC2 (siTORC2), which was delivered via a novel lipid nanoparticle system, or control siRNA (siCON). Compared with siCON, administration of siTORC2 resulted in highly efficient, sustained (1-3 wk) knockdown of TORC2 and its gluconeogenic target genes phosphoenolpyruvate carboxykinase and glucose-6-phophatase in primary mouse hepatocytes and in the livers of HFD-fed mice. In mice, this knockdown was specific to the liver and did not occur in kidney, skeletal muscle, or adipose tissue. In HFD-fed mice, siTORC2 reduced in vivo gluconeogenic capacity, fasting hepatic glucose production, and hyperglycemia, and led to improved hepatic and skeletal muscle insulin sensitivity. siTORC2 treatment also improved systemic hyperglycemia in ZDF rats. In conclusion, these results demonstrate the importance of TORC2 in modulating HGP in vivo and highlight a novel, liver-specific siRNA approach for the potential treatment of hyperglycemia and type 2 diabetes. transducer of regulated cAMP-responsive element-binding protein activity 2; diabetes; small interfering RNA Address for reprint requests and other correspondence: M. Saberi and D. Bjelica contributed equally to this work. Jerrold M. Olefsky,