- Email: [email protected]
514. Robust RNAi Enhancement Via Human Argonaute-2 Overexpression from Plasmids, Viral Vectors or Cell Lines
OLIGONUCLEOTIDE & RNAI THERAPEUTICS In summary, the CaP/PLGA-EPI composite nanoparticle system efficiently delivers siRNAs into cancer cells in a robust and sustained manner both in vitro and in vivo.
515. Ultra-Stable Trivalent RNA Nanoparticles for Targeting to Cancers and HIV-1
Kato Shum,1 Peixuan Guo,2 John J. Rossi.1 Molecular and Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA; 2College of Pharmacy, University of Kentucky, Lexington, KY. 1
514. Robust RNAi Enhancement Via Human Argonaute-2 Overexpression from Plasmids, Viral Vectors or Cell Lines
Kathleen Boerner,1 Dominik Niopek,2 Daniel Gilbert,3 Konrad Streetz,4 Hans-Georg Kraeusslich,5 Dirk Grimm.5 1 Infectious Diseases/Virology, Heidelberg University Hospital, Heidelberg, Germany; 2Bioinformatics and Functional Genomics, German Cancer Research Center, Heidelberg, Germany; 3Medical Biotechnology, University of Erlangen-Nuernberg, Erlangen, Germany; 4Medicine III, University Hospital Aachen, Aachen, Germany; 5Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany. Argonaute-2 (Ago2) is the only human Ago protein capable of cleaving small RNA-targeted mRNAs, making it a unique and vital player in RNAi applications, including therapeutic gene silencing. Recently, we and others have found that cellular Ago2 levels limit the efficiency of si or shRNAs, as transient Ago2 overexpression from a plasmid increased the potency of co-transfected shRNAs in cultured cells. We also obtained first data that co-delivery of AAV vectors expressing Ago2 can boost the efficiency and persistence of AAV-encoded shRNAs in murine livers and concomitantly alleviate shRNA toxicity. While this suggested numerous benefits from Ago2 overexpression, our strategies remained limited by their need for Ago2 co-delivery by an extra plasmid or AAV, in turn hampering further (pre-)clinical development. Besides, none of our constructs was readily applicable in siRNA screens which usually require distinct DNA/RNA delivery methods. Here, we have overcome these restrictions by engineering a new generation of all-in-one plasmids and AAV vectors carrying two integrated Ago2 and shRNA expression cassettes. Moreover, we stably inserted our Ago2 expression cassette into a series of human cell lines and obtained several clones with up to 25-fold higher Ago2 levels but otherwise identical phenotypes as their parental cells (growth, morphology, cDNA/miRNA profiles). Using various endo- and exogenous targets, including clinically relevant p53 and hepatitis B virus, we then demonstrated that all three strategies increased si/shRNA knockdown efficiencies in cell culture by up to ten-fold, especially for inherently poor si/shRNAs. When we tested our new stable Ago2 cells in an siRNA screen of 800 genes implied in the human kinome, 1) more siRNAs reached the hit criteria, 2) RNAi phenotypes were frequently enhanced and 3) interassay variability was largely reduced. Together with our additional findings that Ago2 overexpression permits the use of lower siRNA doses and facilitates combinatorial RNAi with multiple si/shRNAs, these data illustrate the vast potential of our new cell lines and tools for in vitro RNAi studies. Importantly, we also confirmed the benefits from concurrent shRNA and Ago2 overexpression from a single AAV in vivo. In livers of mice transgenic for human alpha-1-antitrypsin (hAAT), a vector co-endoding Ago2 and anti-hAAT shRNA induced 85% knockdown which lasted for at least 6 months, whereas a YFP/ shRNA control merely gave 50% and only for 2 months. Notably, long-term Ago2 overexpression caused no liver or gross toxicity. In summary, we report a new set of widely applicable and highly versatile tools which enhance custom si/shRNAs via transient or stable Ago2 co-expression, and which should improve future RNAi applications in cells, animals and, hopefully, eventually also humans.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
One of the advantages of nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. The field of RNA nanotechnology is emerging. Our group has successfully designed a bivalent pRNAbased nanoparticle as a cell type specific delivery vehicle for both efficient siRNA delivery and cell specific targeting. However, controlled assembly of stable RNA nanoparticles with multiple functionalities, which retain their original role, remains challenging due to refolding after fusion. Herein, we report a novel design of thermodynamically stable T-shaped RNA nanoparticles to carry three therapeutic RNA motifs by self-assembly of re-engineered small RNA fragments. We achieved a significant progress in construction of pRNA-based nanoparticles containing a new CD4 aptamer and two dicer substrate siRNAs. The CD4 aptamer acts as a targeting vehicle to facilitate selective intracellular access of nanoparticles into CD4+ T cells when CD4 is shuttled from the plasma membrane to the cytoplasm. We isolated new CD4 aptamers from a combination of the protein-SELEX and the cell-SELEX and proved that the aptamer bound tightly and specifically internalized into cytoplasm of CD4+ expressing cells but not cell lines without CD4 expression. The aptamer was further assembled with a phi 29 RNA backbone and two siRNAs to construct nanoparticles, which were was extremely stable and highly resistant to urea denaturation. More importantly, gene silencing effects were observed when the nanoparticles are transfected into the cell. We are poised to test these nanoparticles in vivo in a humanized mouse model for the treatment of HIV-1 and T cell leukemia.
516. Directed Evolution-Based Dissection of the Four Human Argonaute Proteins
Nina Schuermann,1 Leonardo Trabucco,2 Christian Bender,3 Rob Russell,2 Dirk Grimm.1 1 Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany; 2 Heidelberg University, Cluster of Excellence CellNetworks, Heidelberg, Germany; 3Translational Oncology, University of Mainz, Medical Center, Mainz, Germany.
In humans, four different Argonaute proteins, Ago1-4, act at the core of the RNA-induced silencing complex and thereby play essential roles in numerous physiological or pathological processes as well as during RNAi applications. A paramount goal in current RNAi research is hence to fully decipher the function of these proteins in order to complete our understanding of natural RNAi mechanisms. Concurrently, a precise knowledge of human Ago biology is imperative for the success and safety of future clinical RNAi therapies considering disturbing findings in small animals that shRNA-induced Ago/RNAi perturbation can accelerate cancer, cause organ failure, or trigger morbidity and lethality. In the past, a combination of point/domain swapping mutagenesis with biochemical assays has resulted in the identification and initial annotation of the four signature Ago protein domains, N, PAZ, MID and PIWI. Here, we introduce an alternative potent technology into the RNAi field which complements the existing methods, i.e., DNA family shuffling (DFS). Its hallmark is the molecular evolution of chimeric DNA/proteins through fragmentation of multiple related parental genes followed by reassembly based on partial sequence homologies, yielding large libraries of hybrid molecules which can be screened for phenotypes of interest. To provide proof-of-concept for the potential of DFS to functionally dissect Ago proteins, we focused on the curiosity that S199
515. Ultra-Stable Trivalent RNA Nanoparticles for Targeting to Cancers and HIV-1
Kato Shum,1 Peixuan Guo,2 John J. Rossi.1 Molecular and Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA; 2College of Pharmacy, University of Kentucky, Lexington, KY. 1
514. Robust RNAi Enhancement Via Human Argonaute-2 Overexpression from Plasmids, Viral Vectors or Cell Lines
Kathleen Boerner,1 Dominik Niopek,2 Daniel Gilbert,3 Konrad Streetz,4 Hans-Georg Kraeusslich,5 Dirk Grimm.5 1 Infectious Diseases/Virology, Heidelberg University Hospital, Heidelberg, Germany; 2Bioinformatics and Functional Genomics, German Cancer Research Center, Heidelberg, Germany; 3Medical Biotechnology, University of Erlangen-Nuernberg, Erlangen, Germany; 4Medicine III, University Hospital Aachen, Aachen, Germany; 5Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany. Argonaute-2 (Ago2) is the only human Ago protein capable of cleaving small RNA-targeted mRNAs, making it a unique and vital player in RNAi applications, including therapeutic gene silencing. Recently, we and others have found that cellular Ago2 levels limit the efficiency of si or shRNAs, as transient Ago2 overexpression from a plasmid increased the potency of co-transfected shRNAs in cultured cells. We also obtained first data that co-delivery of AAV vectors expressing Ago2 can boost the efficiency and persistence of AAV-encoded shRNAs in murine livers and concomitantly alleviate shRNA toxicity. While this suggested numerous benefits from Ago2 overexpression, our strategies remained limited by their need for Ago2 co-delivery by an extra plasmid or AAV, in turn hampering further (pre-)clinical development. Besides, none of our constructs was readily applicable in siRNA screens which usually require distinct DNA/RNA delivery methods. Here, we have overcome these restrictions by engineering a new generation of all-in-one plasmids and AAV vectors carrying two integrated Ago2 and shRNA expression cassettes. Moreover, we stably inserted our Ago2 expression cassette into a series of human cell lines and obtained several clones with up to 25-fold higher Ago2 levels but otherwise identical phenotypes as their parental cells (growth, morphology, cDNA/miRNA profiles). Using various endo- and exogenous targets, including clinically relevant p53 and hepatitis B virus, we then demonstrated that all three strategies increased si/shRNA knockdown efficiencies in cell culture by up to ten-fold, especially for inherently poor si/shRNAs. When we tested our new stable Ago2 cells in an siRNA screen of 800 genes implied in the human kinome, 1) more siRNAs reached the hit criteria, 2) RNAi phenotypes were frequently enhanced and 3) interassay variability was largely reduced. Together with our additional findings that Ago2 overexpression permits the use of lower siRNA doses and facilitates combinatorial RNAi with multiple si/shRNAs, these data illustrate the vast potential of our new cell lines and tools for in vitro RNAi studies. Importantly, we also confirmed the benefits from concurrent shRNA and Ago2 overexpression from a single AAV in vivo. In livers of mice transgenic for human alpha-1-antitrypsin (hAAT), a vector co-endoding Ago2 and anti-hAAT shRNA induced 85% knockdown which lasted for at least 6 months, whereas a YFP/ shRNA control merely gave 50% and only for 2 months. Notably, long-term Ago2 overexpression caused no liver or gross toxicity. In summary, we report a new set of widely applicable and highly versatile tools which enhance custom si/shRNAs via transient or stable Ago2 co-expression, and which should improve future RNAi applications in cells, animals and, hopefully, eventually also humans.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
One of the advantages of nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. The field of RNA nanotechnology is emerging. Our group has successfully designed a bivalent pRNAbased nanoparticle as a cell type specific delivery vehicle for both efficient siRNA delivery and cell specific targeting. However, controlled assembly of stable RNA nanoparticles with multiple functionalities, which retain their original role, remains challenging due to refolding after fusion. Herein, we report a novel design of thermodynamically stable T-shaped RNA nanoparticles to carry three therapeutic RNA motifs by self-assembly of re-engineered small RNA fragments. We achieved a significant progress in construction of pRNA-based nanoparticles containing a new CD4 aptamer and two dicer substrate siRNAs. The CD4 aptamer acts as a targeting vehicle to facilitate selective intracellular access of nanoparticles into CD4+ T cells when CD4 is shuttled from the plasma membrane to the cytoplasm. We isolated new CD4 aptamers from a combination of the protein-SELEX and the cell-SELEX and proved that the aptamer bound tightly and specifically internalized into cytoplasm of CD4+ expressing cells but not cell lines without CD4 expression. The aptamer was further assembled with a phi 29 RNA backbone and two siRNAs to construct nanoparticles, which were was extremely stable and highly resistant to urea denaturation. More importantly, gene silencing effects were observed when the nanoparticles are transfected into the cell. We are poised to test these nanoparticles in vivo in a humanized mouse model for the treatment of HIV-1 and T cell leukemia.
516. Directed Evolution-Based Dissection of the Four Human Argonaute Proteins
Nina Schuermann,1 Leonardo Trabucco,2 Christian Bender,3 Rob Russell,2 Dirk Grimm.1 1 Infectious Diseases/Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, Germany; 2 Heidelberg University, Cluster of Excellence CellNetworks, Heidelberg, Germany; 3Translational Oncology, University of Mainz, Medical Center, Mainz, Germany.
In humans, four different Argonaute proteins, Ago1-4, act at the core of the RNA-induced silencing complex and thereby play essential roles in numerous physiological or pathological processes as well as during RNAi applications. A paramount goal in current RNAi research is hence to fully decipher the function of these proteins in order to complete our understanding of natural RNAi mechanisms. Concurrently, a precise knowledge of human Ago biology is imperative for the success and safety of future clinical RNAi therapies considering disturbing findings in small animals that shRNA-induced Ago/RNAi perturbation can accelerate cancer, cause organ failure, or trigger morbidity and lethality. In the past, a combination of point/domain swapping mutagenesis with biochemical assays has resulted in the identification and initial annotation of the four signature Ago protein domains, N, PAZ, MID and PIWI. Here, we introduce an alternative potent technology into the RNAi field which complements the existing methods, i.e., DNA family shuffling (DFS). Its hallmark is the molecular evolution of chimeric DNA/proteins through fragmentation of multiple related parental genes followed by reassembly based on partial sequence homologies, yielding large libraries of hybrid molecules which can be screened for phenotypes of interest. To provide proof-of-concept for the potential of DFS to functionally dissect Ago proteins, we focused on the curiosity that S199