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dc.contributor.advisorAnastasia Khvorova
dc.contributor.authorHaraszti, Reka A
dc.date2022-08-11T08:08:46.000
dc.date.accessioned2022-08-23T16:07:59Z
dc.date.available2022-08-23T16:07:59Z
dc.date.issued2018-05-15
dc.date.submitted2018-05-15
dc.identifier.doi10.13028/M2Z68X
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32358
dc.description.abstractExtracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data. We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5'-phosphate with 5'-E-vinylphosphonate and chemical stabilization of all nucleotides with 2'-O-methyl and 2'-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol. Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated via differential ultracentrifugation from two-dimensional cultures of the same cells. In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both in vitro and in vivo. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectExosomes
dc.subjectEngineered Exosomes
dc.subjectsiRNA
dc.subjectLipidomics
dc.subjectProteomics
dc.subjectLipid Nanoparticles
dc.subjectLiposomes
dc.subjectDelivery
dc.subjectNeurons
dc.subjectBrain
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectChemical and Pharmacologic Phenomena
dc.subjectComplex Mixtures
dc.subjectLipids
dc.subjectMedical Biochemistry
dc.subjectMedical Biotechnology
dc.subjectMedical Cell Biology
dc.subjectMedical Molecular Biology
dc.subjectMedicinal and Pharmaceutical Chemistry
dc.subjectNanomedicine
dc.subjectNervous System Diseases
dc.subjectNeurosciences
dc.subjectNucleic Acids, Nucleotides, and Nucleosides
dc.subjectOther Analytical, Diagnostic and Therapeutic Techniques and Equipment
dc.subjectPharmaceutics and Drug Design
dc.subjectTherapeutics
dc.titleEngineered Exosomes for Delivery of Therapeutic siRNAs to Neurons
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1977&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/971
dc.legacy.embargo2018-11-14T00:00:00-08:00
dc.identifier.contextkey12128478
refterms.dateFOA2022-08-25T04:31:44Z
html.description.abstract<p>Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data.</p> <p>We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5'-phosphate with 5'-<em>E</em>-vinylphosphonate and chemical stabilization of all nucleotides with 2'-<em>O</em>-methyl and 2'-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol.</p> <p>Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated <em>via</em> differential ultracentrifugation from two-dimensional cultures of the same cells.</p> <p>In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both <em>in vitro</em> and <em>in vivo</em>. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field.</p>
dc.identifier.submissionpathgsbs_diss/971
dc.contributor.departmentRNA Therapeutics Institute
dc.description.thesisprogramTranslational Science
dc.identifier.orcid0000-0001-9925-6337


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