- Email: [email protected]
667. Analysis and Validation of RNA Interference by 5’ RACE: Differential Effect between shRNA and siRNA
Oligonucleotide Therapies I endoplasmic reticulum (ER) stress, and protein mishandling have been associated with dopaminergic neuronal degeneration in these disorders. However, the mechanism(s) by which huSNCA or ER stress mediates dopaminergic cell death is not clearly understood. The transcription factor CHOP (C/EBP homologous protein) is induced by ER stress to promote apoptosis. Using lentiviral transduction and fluorescent activated cell sorting, we have generated inducible huSNCA expressing MN9D cell lines that stably express lentiviralH1-CHOP-shRNA-CMV-EGFP or lentiviral-H1-luciferase-shRNACMV-EGFP or control lentiviral-CMV-EGFP (devoid of shRNA). In addition to assessing chromatin condensation as a marker of cellular pyknosis, we have adapted the Alamar blue assay, which quantitatively measures the proliferation and viability of cells, to assess cell viability/cell death in inducible huSNCA expressing MN9D cells following doxycycline (dox) treatment. We have observed over 50% cell death following dox-induced huSNCA expression in these cells. More importantly, we demonstrate that while over-expression of human CHOP significantly increases cell death in unstressed MN9D cells, RNA interference-mediated silencing of CHOP expression, significantly protects MN9D cells from huSNCA-induced cell death. Together, these studies suggest a contributing pathogenic role of CHOP in mediating huSNCA-induced dopaminergic cell death and open a new investigational avenue for the development of therapeutics for Parkinson’s disease and related synucleinopathies.
667. Analysis and Validation of RNA Interference by 5’ RACE: Differential Effect between shRNA and siRNA
Yang Yu,1 Donald D. Rao,1 Padmasini Kumar,1 Zhaohui Wang,1 Neeta Mistry,1 Neil Senzer,1,2,3,4 Phillip B. Maples,1,2 Alex W. Tong,1,4 John J. Nemunaitis.1,2,3,4 1 Gradalis, Inc., Dallas, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX; 3Texas Oncology, P.A., Dallas, TX; 4Baylor Sammons Cancer Center, Dallas, TX. RNA interference (RNAi) has emerged as an extremely powerful tool in cancer research and holds strong potential for cancer treatment. RNAi down-regulates or silences the expression of a target gene, producing a stable “gene knockdown”. Small interference RNA (siRNA) mediated down-regulation of target mRNA was initially reported to be highly specific with sequence specific cleavage localizing at the middle of the target sequence. In support, several reports have demonstrated single nucleotide mismatches between siRNA and the target mRNA greatly decrease the rate of target mRNA cleavage. However, more recent studies have described siRNA induced off-target effects as a result of partial sequence matches between siRNA and non targeted mRNAs. Currently, we are developing miR30 based cleavage-dependent and –independent short hairpin RNA (shRNA) strategies for targeted cancer gene therapy to maximize the potential for tumor specific gene knockdown, permit sustained durability of knockdown effect, and to optimize therapeutic regulation. As an integral part of this project, we have initiated studies to address the on-target and off-target issues of our shRNA constructs. In order to do so, we adopted the 5’ Rapid Amplification of cDNA Ends (5’ RACE) method to assess the predictive accuracy and efficiency of shRNA induced target site cleavage. With gene specific primers, 5’ RACE allows us to amplify and examine cleavage products of target mRNA template as the result of either shRNA or siRNA treatment. Stathmin 1 over-expression has been identified as an overexpressed gene and protein duplex in a number of patients at the Mary Crowley Cancer Research Centers and in a spectrum of tumors in the literature. We developed shRNA constructs to knockdown stathmin 1 expression. The stathmin 1 specific 3’ primer was complementary to stathmin mRNA at 240 bp downstream from the presumptive targeted cleavage site. CCL-247 cells were treated with effective doses of either siRNA or shRNA (targeted to the same Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
sequence on stathmin mRNA). Total RNA was harvested 24hrs after transfection, the 5’ RACE PCR products were identified by gel electrophoresis and further confirmed by sequencing. Sequencing results revealed two siRNA induced cleavage products; one is the predicted product with cleavage at the middle of the targeted sequence and the second is 73 bp 3’ distal to the targeted mRNA cleavage site. Using the pSilencer/shRNA construct, only the predicted cleavage product at the target site was observed. No stathmn 1 specific cleavage products were observed in untreated cells or in scramble shRNA treated cells. This preliminary result reported here shows that both our siRNA & shRNA could effectively induce targeted mRNA cleavage; however, the cleavage site may differ between siRNA and shRNA. Studies are underway to further elucidate the differences between shRNA and siRNA treated cancer cells and the apparent differential off-target effects of the two therapeutics.
668. Restricted HIV-1 Escape with miR-Based shRNAs Targeting Mutliple Conserved Genomic Sequences
Jiyeon Son,1 Hongseok Ban,1 Premlata Shankar,2 Priti Kumar,2 Sang-Kyung Lee.1 1 Bioengineering, Hanyang University, Seoul, Republic of Korea; 2 Immune Disease Institute, Harvard Medical School, Boston. Lentiviral delivery of monospecific short hairpin RNAs (shRNA) can stably suppress HIV-1 infection in cell lines and primary cells. However, given the requirement of sequence homology for RNAimediated gene silencing, viral heterogeneity is a major hurdle for its therapeutic use against HIV-1 as even a single nucleotide substitution, especially in the central target region (regions 8-12) can completely abolish silencing. Therefore, simultaneously targeting multiple highly conserved viral sequences is thought to be the efficient way of safeguarding against viral propensity for escape from RNAi. As RNAi is known to induce the selection of escape variants with nucleotide changes in the target sequence, we screened conserved sequences in naturally occurring HIV isolates, since these are likely the ones with minimal loss in viral fitness. Of the 875 naturally occurring HIV isolates listed in the Los Alamos database, we selected two highly conserved 19 nucleotide sequences in the vif and tat genes with minimal/wobble nucleotide variations at positions 9, 10 and 11 of the target sequence to retain efficient siRNA binding even upon mutation. We tested efficacy of silencing the selected targets on HIV replication and analyzed whether combinatorial targeting of Vif and Tat would prevent viral escape. For this, we coexpressed these sequences as precursor microRNA (miRNA) stem-loop structures using a vector expressing pol II driven miR-155 miRNA backbone that allows efficient polycistronic expression of multiple miRNAs. We have confirmed siRNA expression by northern blots and successful inhibition of HIV by cotransfecting 293T cells with NL4-3, a molecular clone of HIV with the miR construct. Furthermore, no escape variants were detected upon culture. To test the effect of positional mutations in the target sequence on loss of silencing, we generated artificial mRNA targets expressing the target sequences in fusion with luciferase mRNA as an indicator. The mRNA targets were based on sequences from naturally occurring HIV variants reported in Los Alamos Database. Naturally variants with changes at the 9th position, either G9T in Tat or G9A in Vif were no longer responsive to silencing, but no natural variant has been reported with changes at both these positions. A variant of Vif, A11G was still amenable to inhibition, due to wobble base pairing of the variant residue with the siRNA strand as confirmed by the loss of inhibition observed with A11C that abolishes wobble binding. Interestingly, no mutations at position 10 were detected in any isolate. Taken together, our results suggest that bispecific miRNA targeting the highly conserved tat and vif sequences can completely inhibit all known isolates of HIV and S249
667. Analysis and Validation of RNA Interference by 5’ RACE: Differential Effect between shRNA and siRNA
Yang Yu,1 Donald D. Rao,1 Padmasini Kumar,1 Zhaohui Wang,1 Neeta Mistry,1 Neil Senzer,1,2,3,4 Phillip B. Maples,1,2 Alex W. Tong,1,4 John J. Nemunaitis.1,2,3,4 1 Gradalis, Inc., Dallas, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX; 3Texas Oncology, P.A., Dallas, TX; 4Baylor Sammons Cancer Center, Dallas, TX. RNA interference (RNAi) has emerged as an extremely powerful tool in cancer research and holds strong potential for cancer treatment. RNAi down-regulates or silences the expression of a target gene, producing a stable “gene knockdown”. Small interference RNA (siRNA) mediated down-regulation of target mRNA was initially reported to be highly specific with sequence specific cleavage localizing at the middle of the target sequence. In support, several reports have demonstrated single nucleotide mismatches between siRNA and the target mRNA greatly decrease the rate of target mRNA cleavage. However, more recent studies have described siRNA induced off-target effects as a result of partial sequence matches between siRNA and non targeted mRNAs. Currently, we are developing miR30 based cleavage-dependent and –independent short hairpin RNA (shRNA) strategies for targeted cancer gene therapy to maximize the potential for tumor specific gene knockdown, permit sustained durability of knockdown effect, and to optimize therapeutic regulation. As an integral part of this project, we have initiated studies to address the on-target and off-target issues of our shRNA constructs. In order to do so, we adopted the 5’ Rapid Amplification of cDNA Ends (5’ RACE) method to assess the predictive accuracy and efficiency of shRNA induced target site cleavage. With gene specific primers, 5’ RACE allows us to amplify and examine cleavage products of target mRNA template as the result of either shRNA or siRNA treatment. Stathmin 1 over-expression has been identified as an overexpressed gene and protein duplex in a number of patients at the Mary Crowley Cancer Research Centers and in a spectrum of tumors in the literature. We developed shRNA constructs to knockdown stathmin 1 expression. The stathmin 1 specific 3’ primer was complementary to stathmin mRNA at 240 bp downstream from the presumptive targeted cleavage site. CCL-247 cells were treated with effective doses of either siRNA or shRNA (targeted to the same Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
sequence on stathmin mRNA). Total RNA was harvested 24hrs after transfection, the 5’ RACE PCR products were identified by gel electrophoresis and further confirmed by sequencing. Sequencing results revealed two siRNA induced cleavage products; one is the predicted product with cleavage at the middle of the targeted sequence and the second is 73 bp 3’ distal to the targeted mRNA cleavage site. Using the pSilencer/shRNA construct, only the predicted cleavage product at the target site was observed. No stathmn 1 specific cleavage products were observed in untreated cells or in scramble shRNA treated cells. This preliminary result reported here shows that both our siRNA & shRNA could effectively induce targeted mRNA cleavage; however, the cleavage site may differ between siRNA and shRNA. Studies are underway to further elucidate the differences between shRNA and siRNA treated cancer cells and the apparent differential off-target effects of the two therapeutics.
668. Restricted HIV-1 Escape with miR-Based shRNAs Targeting Mutliple Conserved Genomic Sequences
Jiyeon Son,1 Hongseok Ban,1 Premlata Shankar,2 Priti Kumar,2 Sang-Kyung Lee.1 1 Bioengineering, Hanyang University, Seoul, Republic of Korea; 2 Immune Disease Institute, Harvard Medical School, Boston. Lentiviral delivery of monospecific short hairpin RNAs (shRNA) can stably suppress HIV-1 infection in cell lines and primary cells. However, given the requirement of sequence homology for RNAimediated gene silencing, viral heterogeneity is a major hurdle for its therapeutic use against HIV-1 as even a single nucleotide substitution, especially in the central target region (regions 8-12) can completely abolish silencing. Therefore, simultaneously targeting multiple highly conserved viral sequences is thought to be the efficient way of safeguarding against viral propensity for escape from RNAi. As RNAi is known to induce the selection of escape variants with nucleotide changes in the target sequence, we screened conserved sequences in naturally occurring HIV isolates, since these are likely the ones with minimal loss in viral fitness. Of the 875 naturally occurring HIV isolates listed in the Los Alamos database, we selected two highly conserved 19 nucleotide sequences in the vif and tat genes with minimal/wobble nucleotide variations at positions 9, 10 and 11 of the target sequence to retain efficient siRNA binding even upon mutation. We tested efficacy of silencing the selected targets on HIV replication and analyzed whether combinatorial targeting of Vif and Tat would prevent viral escape. For this, we coexpressed these sequences as precursor microRNA (miRNA) stem-loop structures using a vector expressing pol II driven miR-155 miRNA backbone that allows efficient polycistronic expression of multiple miRNAs. We have confirmed siRNA expression by northern blots and successful inhibition of HIV by cotransfecting 293T cells with NL4-3, a molecular clone of HIV with the miR construct. Furthermore, no escape variants were detected upon culture. To test the effect of positional mutations in the target sequence on loss of silencing, we generated artificial mRNA targets expressing the target sequences in fusion with luciferase mRNA as an indicator. The mRNA targets were based on sequences from naturally occurring HIV variants reported in Los Alamos Database. Naturally variants with changes at the 9th position, either G9T in Tat or G9A in Vif were no longer responsive to silencing, but no natural variant has been reported with changes at both these positions. A variant of Vif, A11G was still amenable to inhibition, due to wobble base pairing of the variant residue with the siRNA strand as confirmed by the loss of inhibition observed with A11C that abolishes wobble binding. Interestingly, no mutations at position 10 were detected in any isolate. Taken together, our results suggest that bispecific miRNA targeting the highly conserved tat and vif sequences can completely inhibit all known isolates of HIV and S249