- Email: [email protected]
715. Targeted Delivery of Antisense Oligonucleotide and siRNA into Lung Cancer Cells
OLIGONUCLEOTIDE THERAPIES A system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors, or the accumulation of Ras, a cellular protein whose expression is regulated by endogenous miRNAs. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.
715. Targeted Delivery of Antisense Oligonucleotide and siRNA into Lung Cancer Cells Shyh-Dar Li,1 Leaf Huang.1 1 School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC. Selective gene inhibition by antisense oligonucleotide (AS-ODN) or siRNA therapeutics promises the treatment of diseases that can not be cured by conventional drugs. However, antisense therapy is hindered due to poor stability in physiological fluids and limited intracellular uptake of AS-ODN and siRNA. To address these problems, a ligand targeted and sterically stabilized nanoparticle formulation is developed in our lab. Human lung cancer cells overexpress the sigma receptor on their surface, thus can be targeted with a specific ligand such as anisamide. AS-ODN or siRNA against human survivin was mixed with a carrier DNA, calf thymus DNA, before complexed with protamine, a highly positively charged peptide. The resulting particles were coated with cationic liposomes consisting of DOTAP and cholesterol (1:1, molar ratio) to obtain stable LPD (liposome-protamine-DNA) nanoparticles. Ligand targeting and steric stabilization were then introduced by incubating preformed LPD nanoparticles with DSPE-PEG-anisamide, a PEGylated ligand lipid developed earlier in our lab, by the post insertion method. Non-targeted nanoparticles coated with DSPEPEG were also prepared as a control. Antisense activities of nanoparticles were determined by survivin mRNA down-regulation, survivin protein down-regulation, ability to trigger apoptosis in tumor cells, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to anticancer drugs. We found that tumor cell delivery and antisense activity of PEGylated nanoparticles were sequence dependent and rely on the presence of the anisamide ligand and could be competed by excess free ligand. Preliminary in vivo data showed that PEGylated LPD deliver siRNA predominantly into tumors after i.v. injection. Our encouraging results suggest that the ligand targeted and sterically stabilized nanoparticles can provide a selective delivery of AS-ODN and siRNA into lung cancer cells and perform antisense therapeutics. This research was supported by Industrial Technology Research Institute, Taiwan.
716. Differential Effects of Secondary Structure on Antisense Oligonucleotide (AON) and Short Interfering RNA (siRNA) Mediated mRNA Cleavage 1
2
Stephen I. Rudnick, Alan M. Gewirtz. Department of Pharmacology, University of Pennsylvania, Philadelphia, PA; 2Department of Medicine, University of Pennsylvania, Philadelphia, PA.
1
Post-transcriptional gene silencing (PTGS) impairs protein’s synthesis by targeting its messenger RNA (mRNA). The antisense oligonucleotide (AON) and RNA interference (RNAi) pathways can both accomplish PTGS by hybridization of a reverse complementary oligonucleotide and subsequent degradation of the mRNA by an RNaseH mechanism. Here we investigate the influence of specific mRNA secondary structure on targeting efficiency by S276
monitoring AON and siRNA mediated mRNA cleavage in Drosophila embryo whole cell lysates. Structure was induced within a short mRNA target by annealing 2’-O-methyl oligonucleotides (2’OMe ON) followed by in vitro cleavage assays. We demonstrate that annealing a single 2’OMe ON upstream or downstream of a 21 nucleotide (nt) target site enhances mRNA cleavage within both PTGS pathways by 7% to 30%. Inducing structure on both sides of the target site with two or more 2’OMe ON resulted in 23% to 76%, or 18% to 92% more product formation for the siRNA and AON pathways respectively when compared to controls without induced structure. When two 2’OMe ON were annealed incrementally closer to the center of the target site from either side, it was found that AON retained 80% or more of their activity as long as eight or more nucleotides were unblocked. When less than 8nt remained available, AON activity was reduced to half of its potential. In the same fashion, it was found that the siRNA retained 90% of its activity when 16nt or more were available. Otherwise, siRNA catalyzed cleavage was reduced to 52% to 61% as more of the target site was encroached upon. In order to better represent mRNA secondary structure, the 21nt target site was fully blocked by a single annealed 2’OMe ON. Bulges from 2 to 16nt in the induced double strand were introduced by incorporating non-complementary bases between two fully complementary 15nt regions. In all bulge model experiments, the siRNA pathway retained at least 48% of its activity. However, even with the largest bulge of 16nt, siRNA activity was limited to 75% of the control with no 2’OMe ON. In the presence of a 10nt or larger bulge, the AON pathway retained 74% of its activity, but was reduced to as little as 28% with smaller bulges. These data reveal a difference in the effect of mRNA secondary structure on siRNA and AON activity in vitro. The AON consistently degraded more mRNA than the siRNA, which may reflect a difference in the concentration of RNaseH and the siRNA incorporated in RISC. Any structure at the target site reduced siRNA activity, but it was never fully inhibited. AON could cleave the mRNA target if a minimal binding site of eight to ten adjacent nucleotides were accessible. If the target site was fully double stranded, the AON activity was affected to a greater extent than the siRNA. This suggests that there is an advantage to siRNA based target hybridization in comparison to AON when the target site is involved in complex secondary structure. In addition, the data supports the hypothesis that no processive scanning of the mRNA is involved in RNAi, but argues that hybridization occurs in more than a simple diffusion based model.
717. VCP Short Hairpin RNA Rescues ∆F508CFTR and Suppresses IL8 Levels: Therapeutic Implications in Cystic Fibrosis Neeraj Vij,1 Shengyun Fang,2 Pamela L. Zeitlin.1 Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD; 2Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD. 1
Background: Endoplasmic reticulum associated degradation (ERAD) is the major quality control pathway of the cell. The most common disease-causing protein folding mutation, ∆F508-CFTR, is destroyed by ERAD to cause cystic fibrosis (CF). Valosin containing protein (VCP) is an AAA ATPase that participates in retrograde translocation of misfolded proteins from the ER and degradation of polyubiquitinated protein by the proteasomes. We hypothesize that selective inhibition of VCP not only rescues ∆F508CFTR but also suppresses IL8 cytokine levels, the major inflammatory cytokine in CF airways. Methods: The VCP shRNA was PCR amplified from oligo/template containing the VCP hairpin using universal primers which contain XhoI (5’ prime) and EcoRI (3’ prime) flank. The PCR fragment was then cloned into the hairpin Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
715. Targeted Delivery of Antisense Oligonucleotide and siRNA into Lung Cancer Cells Shyh-Dar Li,1 Leaf Huang.1 1 School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC. Selective gene inhibition by antisense oligonucleotide (AS-ODN) or siRNA therapeutics promises the treatment of diseases that can not be cured by conventional drugs. However, antisense therapy is hindered due to poor stability in physiological fluids and limited intracellular uptake of AS-ODN and siRNA. To address these problems, a ligand targeted and sterically stabilized nanoparticle formulation is developed in our lab. Human lung cancer cells overexpress the sigma receptor on their surface, thus can be targeted with a specific ligand such as anisamide. AS-ODN or siRNA against human survivin was mixed with a carrier DNA, calf thymus DNA, before complexed with protamine, a highly positively charged peptide. The resulting particles were coated with cationic liposomes consisting of DOTAP and cholesterol (1:1, molar ratio) to obtain stable LPD (liposome-protamine-DNA) nanoparticles. Ligand targeting and steric stabilization were then introduced by incubating preformed LPD nanoparticles with DSPE-PEG-anisamide, a PEGylated ligand lipid developed earlier in our lab, by the post insertion method. Non-targeted nanoparticles coated with DSPEPEG were also prepared as a control. Antisense activities of nanoparticles were determined by survivin mRNA down-regulation, survivin protein down-regulation, ability to trigger apoptosis in tumor cells, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to anticancer drugs. We found that tumor cell delivery and antisense activity of PEGylated nanoparticles were sequence dependent and rely on the presence of the anisamide ligand and could be competed by excess free ligand. Preliminary in vivo data showed that PEGylated LPD deliver siRNA predominantly into tumors after i.v. injection. Our encouraging results suggest that the ligand targeted and sterically stabilized nanoparticles can provide a selective delivery of AS-ODN and siRNA into lung cancer cells and perform antisense therapeutics. This research was supported by Industrial Technology Research Institute, Taiwan.
716. Differential Effects of Secondary Structure on Antisense Oligonucleotide (AON) and Short Interfering RNA (siRNA) Mediated mRNA Cleavage 1
2
Stephen I. Rudnick, Alan M. Gewirtz. Department of Pharmacology, University of Pennsylvania, Philadelphia, PA; 2Department of Medicine, University of Pennsylvania, Philadelphia, PA.
1
Post-transcriptional gene silencing (PTGS) impairs protein’s synthesis by targeting its messenger RNA (mRNA). The antisense oligonucleotide (AON) and RNA interference (RNAi) pathways can both accomplish PTGS by hybridization of a reverse complementary oligonucleotide and subsequent degradation of the mRNA by an RNaseH mechanism. Here we investigate the influence of specific mRNA secondary structure on targeting efficiency by S276
monitoring AON and siRNA mediated mRNA cleavage in Drosophila embryo whole cell lysates. Structure was induced within a short mRNA target by annealing 2’-O-methyl oligonucleotides (2’OMe ON) followed by in vitro cleavage assays. We demonstrate that annealing a single 2’OMe ON upstream or downstream of a 21 nucleotide (nt) target site enhances mRNA cleavage within both PTGS pathways by 7% to 30%. Inducing structure on both sides of the target site with two or more 2’OMe ON resulted in 23% to 76%, or 18% to 92% more product formation for the siRNA and AON pathways respectively when compared to controls without induced structure. When two 2’OMe ON were annealed incrementally closer to the center of the target site from either side, it was found that AON retained 80% or more of their activity as long as eight or more nucleotides were unblocked. When less than 8nt remained available, AON activity was reduced to half of its potential. In the same fashion, it was found that the siRNA retained 90% of its activity when 16nt or more were available. Otherwise, siRNA catalyzed cleavage was reduced to 52% to 61% as more of the target site was encroached upon. In order to better represent mRNA secondary structure, the 21nt target site was fully blocked by a single annealed 2’OMe ON. Bulges from 2 to 16nt in the induced double strand were introduced by incorporating non-complementary bases between two fully complementary 15nt regions. In all bulge model experiments, the siRNA pathway retained at least 48% of its activity. However, even with the largest bulge of 16nt, siRNA activity was limited to 75% of the control with no 2’OMe ON. In the presence of a 10nt or larger bulge, the AON pathway retained 74% of its activity, but was reduced to as little as 28% with smaller bulges. These data reveal a difference in the effect of mRNA secondary structure on siRNA and AON activity in vitro. The AON consistently degraded more mRNA than the siRNA, which may reflect a difference in the concentration of RNaseH and the siRNA incorporated in RISC. Any structure at the target site reduced siRNA activity, but it was never fully inhibited. AON could cleave the mRNA target if a minimal binding site of eight to ten adjacent nucleotides were accessible. If the target site was fully double stranded, the AON activity was affected to a greater extent than the siRNA. This suggests that there is an advantage to siRNA based target hybridization in comparison to AON when the target site is involved in complex secondary structure. In addition, the data supports the hypothesis that no processive scanning of the mRNA is involved in RNAi, but argues that hybridization occurs in more than a simple diffusion based model.
717. VCP Short Hairpin RNA Rescues ∆F508CFTR and Suppresses IL8 Levels: Therapeutic Implications in Cystic Fibrosis Neeraj Vij,1 Shengyun Fang,2 Pamela L. Zeitlin.1 Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD; 2Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD. 1
Background: Endoplasmic reticulum associated degradation (ERAD) is the major quality control pathway of the cell. The most common disease-causing protein folding mutation, ∆F508-CFTR, is destroyed by ERAD to cause cystic fibrosis (CF). Valosin containing protein (VCP) is an AAA ATPase that participates in retrograde translocation of misfolded proteins from the ER and degradation of polyubiquitinated protein by the proteasomes. We hypothesize that selective inhibition of VCP not only rescues ∆F508CFTR but also suppresses IL8 cytokine levels, the major inflammatory cytokine in CF airways. Methods: The VCP shRNA was PCR amplified from oligo/template containing the VCP hairpin using universal primers which contain XhoI (5’ prime) and EcoRI (3’ prime) flank. The PCR fragment was then cloned into the hairpin Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy