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253. Optimization of mRNA for Therapeutic Delivery
PHYSICAL/NON-PHYSICAL METHODS OF DELIVERY Physical/Non-physical Methods of Delivery & Chemical/Molecular Conjugates 252. Engineering Exosomes as Therapeutic Delivery Vehicles Michelle E. Marcus,1 Joshua N. Leonard.1 1 Department of Chemical and Biological Engineering, Northwestern University, Evantson, IL.
Exosomes are biological nanovesicles that transfer proteins and nucleic acids between cells and have great potential as tunable therapeutic delivery vehicles; they are relatively easy to engineer, well-tolerated in vivo, and naturally capable of intracellular delivery of functional biomolecules. In order for this promise to be realized, robust and general methods for incorporating therapeutic cargo molecules into exosomes and for targeting therapeutic exosomes to specific destinations in vivo are required. Loading therapeutic RNA into exosomes poses several challenges. Transfer of RNA into isolated exosomes by electroporation can be inefficient and generates RNA precipitates. Alternatively, mass-actiondriven incorporation, in which the therapeutic RNA is overexpressed in exosome producing cells, is attractive but has not been as widely explored. In particular, it is not known how native RNA sorting and processing mechanisms and biophysical limitations may impact the efficacy of this approach for different RNA cargo molecules. Thus, to provide a robust and orthogonal strategy for exosome loading, we have developed a Targeted And Modular Exosome Loading (TAMEL) platform for directing the loading of a specific RNAs into exosomes. The TAMEL system comprises a packaging protein and a cargo RNA. The packaging protein is an RNA binding protein targeted to exosomes via fusion to an exosome-enriched protein. The cargo RNA is an RNA molecule displaying an RNA motif that is specifically bound by the packaging protein. Using this system, we have packaged small and large RNAs into exosomes and characterized the relationship between RNA size and packaging into exosomes by mass action alone or targeted means. This platform enables us to package mRNAs and RNAi-inducing RNA species into exosomes and to quantitatively evaluate delivery and functional modulation of gene expression in recipient cells. To enhance delivery of therapeutic exosomes to target recipient cells, we have also developed a technology for enhancing the display of targeting ligands on the exterior of exosomes. Previous attempts to target exosomes to specific recipient cells have been hindered by inconsistent targeting using different ligands or different exosomeproducing cell types. We determined that degradation of targeting peptides by endosomal proteases occurs during exosome biogenesis. To overcome this challenge, we have also developed a glycosylation strategy that protects peptides from degradation while maintaining their ability to bind target proteins. We applied this peptide targeting strategy to engineer enhanced delivery of exosomes to prostate cancer cells. The tools we have developed enable programming exosomes for targeted delivery of RNA cargos in a robust and straightforward manner that can be applied to a wide variety of therapeutic targets.
253. Optimization of mRNA for Therapeutic Delivery
Maggie L. Bobbin,1 Anton McCaffrey,2 John Burnett,1 John J. Rossi.1 1 City of Hope, Duarte, CA; 2Trilink Biotechnologies, La Jolla, CA. RNA-based therapeutics are more suitable than other gene-based therapies for applications requiring delivery with non-viral carriers, targeted delivery, transient expression, or the absence of integration into the genome. Dozens of small RNA-based therapeutics, such as siRNAs and cell-targeting RNA aptamers, have reached clinical testing, illustrating the practicability of non-viral RNA delivery. S96
Another potential therapeutic application is the delivery of mRNA encoding gene-modifying enzymes, as genome-editing technologies may offer new strategies to cure genetic or infectious diseases. However, delivery of protein-encoding mRNA presents new challenges, including mRNA stability, efficient protein translation, minimization of pro-inflammatory responses, and cellular uptake. Here we have investigated the effects of various chemical and structural modifications of mRNA to enhance the properties required for therapeutic use and delivery. To facilitate targeted delivery of mRNA, we have designed it to be annealed to any cell-specific RNA aptamer via Watson-Crick base pairing of complementary stick sequences located on both RNA. Stick location effect on mRNA translation was examined by adding stick sequence to the 5’UTR, 3’UTR or poly(A) tail of mRNA via in vitro transcription. To test functionality of the RNA, stick-modified mRNAs encoding eGFP, ffluc, and Cre recombinase were assayed for translational capability in cell culture. HEK293 and CHO cells were transfected with eGFP or ffluc mRNA and TransIT-mRNA (Mirus Bio) followed by protein expression analysis 12 to 96 hours later. For Cre mRNA, up to 500 ng of mRNA was transfected in HT1080 cells integrated with a loxP-reporter lentiviral vector. Cre recombinase protein production was analyzed indirectly using a color switch system in which Cre protein results in the excision of CFP and concurrent RFP expression, measured by flow cytometry. We found that adding stick sequence to the 3’UTR, 5’UTR and/or after the poly(A) tail has minimal effect on delivery with liposomes, but did affect translation. The functionality of stick-modified Cre mRNA was also tested in the mT/mG transgenic mouse model containing a similar Cre-inducible loxP-reporter system. We injected liposome complexes containing either the Cre mRNA with poly(A)-stick sequence or Cre mRNA with no stick sequence into the mT/mG mouse by IP injection. More efficient translation of the stick-containing mRNA in intestinal cells was observed. We hypothesized that poly(A)-stick sequences may protect mRNA from exonuclease degradation, and therefore measured stability of the mRNA with quantitative PCR (qPCR) and analyzed the duration of mRNA translation in cells. We are currently investigating the effects of sequence additions on stability and on in vivo inflammatory response in our mouse model by measuring levels of TNF-α and IFN-α by RT-PCR and ELISA. Our study revealed that such modifications may enhance mRNA stability and non-immunogenicity without compromising mRNA translational capacity. Moreover, our study continues to evaluate better, more targeted delivery systems relative to conventional modes of delivery, including lipid-based transfection, aptamers, PAMAM dendrimer, or electroporation approaches.
254. Next Generation SMART DRUGS: AptamerBased Cell-Targeted Cancer Therapies
Justin P. Dassie,1 Luiza I. Hernandez,1 Gregory S. Thomas,1 Matthew E. Long,3 William M. Rockey,2 Craig A. Howell,1 Frank J. Hernandez,1 Xiu Y. Liu,1 Lee A. Allen,1 Paloma H. Giangrande.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Radiation Oncology, University of Iowa, Iowa City, IA; 3Interdisciplinary Graduate Program in Molecular and Cellular Biology, University of Iowa, Iowa City, IA.
Cell-targeted drugs (aka smart drugs) deliver a therapeutic to a patient in a manner that increases the concentration of the medication in diseased areas of the body relative to others. The advantages of cell-targeted drugs are increased efficacy (reduction of the maximal effective dose) and safety (reduction of drug side-effects). While cell-targeted drugs can be developed to treat many diseases, such as cardiovascular disease and diabetes, perhaps the most sought-after application of cell-targeted drugs is the treatment of cancerous tumors. Notable examples of targeted cancer therapeutics (e.g., trastuzumab, Gleevec) demonstrate the utility of selectively controlling cancer cell proliferation/survival while limiting toxicity to normal cells. Despite Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Exosomes are biological nanovesicles that transfer proteins and nucleic acids between cells and have great potential as tunable therapeutic delivery vehicles; they are relatively easy to engineer, well-tolerated in vivo, and naturally capable of intracellular delivery of functional biomolecules. In order for this promise to be realized, robust and general methods for incorporating therapeutic cargo molecules into exosomes and for targeting therapeutic exosomes to specific destinations in vivo are required. Loading therapeutic RNA into exosomes poses several challenges. Transfer of RNA into isolated exosomes by electroporation can be inefficient and generates RNA precipitates. Alternatively, mass-actiondriven incorporation, in which the therapeutic RNA is overexpressed in exosome producing cells, is attractive but has not been as widely explored. In particular, it is not known how native RNA sorting and processing mechanisms and biophysical limitations may impact the efficacy of this approach for different RNA cargo molecules. Thus, to provide a robust and orthogonal strategy for exosome loading, we have developed a Targeted And Modular Exosome Loading (TAMEL) platform for directing the loading of a specific RNAs into exosomes. The TAMEL system comprises a packaging protein and a cargo RNA. The packaging protein is an RNA binding protein targeted to exosomes via fusion to an exosome-enriched protein. The cargo RNA is an RNA molecule displaying an RNA motif that is specifically bound by the packaging protein. Using this system, we have packaged small and large RNAs into exosomes and characterized the relationship between RNA size and packaging into exosomes by mass action alone or targeted means. This platform enables us to package mRNAs and RNAi-inducing RNA species into exosomes and to quantitatively evaluate delivery and functional modulation of gene expression in recipient cells. To enhance delivery of therapeutic exosomes to target recipient cells, we have also developed a technology for enhancing the display of targeting ligands on the exterior of exosomes. Previous attempts to target exosomes to specific recipient cells have been hindered by inconsistent targeting using different ligands or different exosomeproducing cell types. We determined that degradation of targeting peptides by endosomal proteases occurs during exosome biogenesis. To overcome this challenge, we have also developed a glycosylation strategy that protects peptides from degradation while maintaining their ability to bind target proteins. We applied this peptide targeting strategy to engineer enhanced delivery of exosomes to prostate cancer cells. The tools we have developed enable programming exosomes for targeted delivery of RNA cargos in a robust and straightforward manner that can be applied to a wide variety of therapeutic targets.
253. Optimization of mRNA for Therapeutic Delivery
Maggie L. Bobbin,1 Anton McCaffrey,2 John Burnett,1 John J. Rossi.1 1 City of Hope, Duarte, CA; 2Trilink Biotechnologies, La Jolla, CA. RNA-based therapeutics are more suitable than other gene-based therapies for applications requiring delivery with non-viral carriers, targeted delivery, transient expression, or the absence of integration into the genome. Dozens of small RNA-based therapeutics, such as siRNAs and cell-targeting RNA aptamers, have reached clinical testing, illustrating the practicability of non-viral RNA delivery. S96
Another potential therapeutic application is the delivery of mRNA encoding gene-modifying enzymes, as genome-editing technologies may offer new strategies to cure genetic or infectious diseases. However, delivery of protein-encoding mRNA presents new challenges, including mRNA stability, efficient protein translation, minimization of pro-inflammatory responses, and cellular uptake. Here we have investigated the effects of various chemical and structural modifications of mRNA to enhance the properties required for therapeutic use and delivery. To facilitate targeted delivery of mRNA, we have designed it to be annealed to any cell-specific RNA aptamer via Watson-Crick base pairing of complementary stick sequences located on both RNA. Stick location effect on mRNA translation was examined by adding stick sequence to the 5’UTR, 3’UTR or poly(A) tail of mRNA via in vitro transcription. To test functionality of the RNA, stick-modified mRNAs encoding eGFP, ffluc, and Cre recombinase were assayed for translational capability in cell culture. HEK293 and CHO cells were transfected with eGFP or ffluc mRNA and TransIT-mRNA (Mirus Bio) followed by protein expression analysis 12 to 96 hours later. For Cre mRNA, up to 500 ng of mRNA was transfected in HT1080 cells integrated with a loxP-reporter lentiviral vector. Cre recombinase protein production was analyzed indirectly using a color switch system in which Cre protein results in the excision of CFP and concurrent RFP expression, measured by flow cytometry. We found that adding stick sequence to the 3’UTR, 5’UTR and/or after the poly(A) tail has minimal effect on delivery with liposomes, but did affect translation. The functionality of stick-modified Cre mRNA was also tested in the mT/mG transgenic mouse model containing a similar Cre-inducible loxP-reporter system. We injected liposome complexes containing either the Cre mRNA with poly(A)-stick sequence or Cre mRNA with no stick sequence into the mT/mG mouse by IP injection. More efficient translation of the stick-containing mRNA in intestinal cells was observed. We hypothesized that poly(A)-stick sequences may protect mRNA from exonuclease degradation, and therefore measured stability of the mRNA with quantitative PCR (qPCR) and analyzed the duration of mRNA translation in cells. We are currently investigating the effects of sequence additions on stability and on in vivo inflammatory response in our mouse model by measuring levels of TNF-α and IFN-α by RT-PCR and ELISA. Our study revealed that such modifications may enhance mRNA stability and non-immunogenicity without compromising mRNA translational capacity. Moreover, our study continues to evaluate better, more targeted delivery systems relative to conventional modes of delivery, including lipid-based transfection, aptamers, PAMAM dendrimer, or electroporation approaches.
254. Next Generation SMART DRUGS: AptamerBased Cell-Targeted Cancer Therapies
Justin P. Dassie,1 Luiza I. Hernandez,1 Gregory S. Thomas,1 Matthew E. Long,3 William M. Rockey,2 Craig A. Howell,1 Frank J. Hernandez,1 Xiu Y. Liu,1 Lee A. Allen,1 Paloma H. Giangrande.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Radiation Oncology, University of Iowa, Iowa City, IA; 3Interdisciplinary Graduate Program in Molecular and Cellular Biology, University of Iowa, Iowa City, IA.
Cell-targeted drugs (aka smart drugs) deliver a therapeutic to a patient in a manner that increases the concentration of the medication in diseased areas of the body relative to others. The advantages of cell-targeted drugs are increased efficacy (reduction of the maximal effective dose) and safety (reduction of drug side-effects). While cell-targeted drugs can be developed to treat many diseases, such as cardiovascular disease and diabetes, perhaps the most sought-after application of cell-targeted drugs is the treatment of cancerous tumors. Notable examples of targeted cancer therapeutics (e.g., trastuzumab, Gleevec) demonstrate the utility of selectively controlling cancer cell proliferation/survival while limiting toxicity to normal cells. Despite Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy