Reinstatement of Retroviral Infectivity via Non-Covalent Attachment of DOTAP, DOPE and Cholesterol to Murine Leukemia Virus-Like Particles Hybrid Viral/Lipid Gene Delivery Vectors Authors Rahul K. Keswani, Mihael Lazebnik & Daniel W. Pack Laboratory of Advanced Drug and Gene Delivery Department of Chemical and Biomolecular Engineering University of Illinois, Urbana-Champaign AIChE Annual Meeting 2011 October 18, 2011 Session: Nucleic Acid Delivery II 1
Overview Introduction & Background Why hybrid vectors? What are RVLPs? Results Transfections Uptake Sizes Morphology Conclusions and Future Work 2
Gene Therapy ASGCT defines Gene Therapy as The treatment of disease by either replacing damaged or abnormal genes with normal ones, or by providing new genetic instructions to help fight disease. Retrovirus Adenovirus Adeno-associated Virus Plasmid Polymer Liposomes Polymers Polyethylenimine (PEI) Poly-L-Lysine (PLL) Polyamidoamine (PAMAM) Chitosan Lipids Cationic transfection lipids (DOTAP;DC-Chol) Neutral Helper lipids (DOPE;Cholesterol) Sources: http://4.bp.blogspot.com/_9bbho-qfjms/thwfbs0hdqi/aaaaaaaaaea/smn7nutvdha/s1600/virus+diagram.gif http://biomarker.cdc.go.kr/biomarker/pathogenimg/adenovirus_en.gif http://www.ucl.ac.uk/ioo/research/web%20ready%20images%20apr%202007/8%20untitled-1-aav.jpg 3
Comparison Why do we need a hybrid design? Retrovirus Synthetic Vector Transfection Efficiency Excellent Poor-excellent Size Small Small-medium Gene Expression Stable Transient Toxicity No Yes/No Flexible Targeting No Yes Pathogenicity Yes Unknown/No Immunogenicity Yes Little Production Expensive Inexpensive Stability Poor Robust 4
Retrovirus Structure Protease Lipid bilayer Envelope Protein Reverse Transcriptase Integrase Icosahedral capsid shell RNA (2 copies) 5
Retrovirus Envelope Protein needs a suitable alternative Envelope protein provides good efficiency but Immunogenicity Pathogenicity Poor Stability Poor Processing Single Tropism Retrovirus-Like Particle (RVLP) 6
Brief Protocol Synthetic envelopes are added non-covalently to RVLPs GP293Luc Transfect with plasmid encoding for envelope protein Vortex and Incubate for 2-4 h Quantify via Real-Time PCR assay 7
Synthetic Envelopes This study improves the feasibility of the hybrid vector design Generation 1 LF2000 PEI PLL Generation 2 Chitosan DOTAP/DOPE/Cholesterol Scale bar = 100 nm Ramsey et al, 2010, Journal of Controlled Release 8
Synthetic Envelopes Lipids (φ) are efficient non-viral gene delivery vectors Cationic Lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) Helper Lipids (Neutral) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) Cholesterol 9
φ-rvlps Transfection Efficiency is dependent on the lipid composition 10
φ-rvlps Transfection Efficiency is dependent on the lipid composition 11
φ-rvlps Transfection Efficiency is dependent on the lipid composition 12
φ-rvlps Transfection Efficiency is dependent on the lipid composition 13
φ-rvlps Transfection Efficiency is dependent on the lipid composition (DOTAP-heavy) and (DOTAP-light,DOPE-heavy) lipid compositions performed worse DOPE and Cholesterol, if present in approximately equal proportions, was optimal On repeat trials, DOTAP:DOPE:Cholesterol = 5:8:7 (w) best composition for consistently good transfection efficiency 14
Measuring Cellular Uptake in HEK293 cells RVLPs labeled with DiD prior to formation of hybrid vectors + Lipophilic fluorescent dye 15
φ-rvlps Cationic charge on lipids improves uptake but has no effect on transfection 5 μg lipid/10 9 RVLPs 16
φ-rvlps Cationic charge on lipids improves uptake but has no effect on transfection 10 μg lipid/10 9 RVLPs 17
φ-rvlps Cationic charge on lipids improves uptake but has no effect on transfection 20 μg lipid/10 9 RVLPs 18
Normalized Metabolic Activity Lipid Toxicity 505 was toxic 10 µg/ml to HEK293 cells 1.600 1.400 1.200 1.000 0.800 0.600 0.400 0.200 0.000 Lipo550 Lipo587 Lipo505 0.5 5 50 500 Lipids (µg/ml) 19
φ-rvlps TEM of hybrid vectors shows enveloping of RVLP 20
Size Reduction for φ-rvlps Extrusion of lipids led to smaller sizes of hybrid vectors Simple Hydration Type Only Lipid 5* 10* Lipo587 269 ± 8 278 ± 10 697 ± 60 Lipo505 353 ± 5 443 ± 6 601 ± 7 Lipo550 294 ± 48 493 ± 114 1190 ± 184 Sizes in nm; * - µg/10 9 RVLPs After Extrusion Type Only Lipid 5* 10* Lipo587 176 ± 3 334 ± 5 374 ± 10 Lipo505 172 ± 3 235 ± 5 244 ± 5 Lipo550 134 ± 2 202 ± 2 397 ± 8 Sizes in nm; * - µg/10 9 RVLPs 21
Size Reduction for φ-rvlps TEM scans of hybrid vectors formed by extrusion method 587 alone 22
Size Reduction for φ-rvlps TEM scans of hybrid vectors formed by extrusion method Lipo587-10 µg/10 9 RVLPs 23
Normalized RLU/mg total protein Size Reduction for φ-rvlps Normalized RLU/mg total protein Normalized RLU/mg total protein 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Extruded lipid hybrid vectors had mixed performance in transfections 4.5 Lipo550 4.0 Lipo505 Lipo550e 3.5 Lipo505e 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Lipid (μg)/10 9 RVLPs Lipid (μg)/10 9 RVLPs 9.0 8.0 Lipo587 7.0 Lipo587e 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 5 10 15 20 25 30 Lipid (μg)/10 9 RVLPs 24
Conclusions and Future Work Optimized lipid composition for lipid-based hybrid vectors in HEK293 cells Uptake has no correlation with subsequent transfection within the cells Size reduction of hybrid vector aggregates successfully achieved via lipid extrusion Formation of smaller liposomes led to smaller hybrid vectors Synthetic envelope decides the intracellular navigation route? - Amphotropic Virus = Lipo-RVLPs > Polymer-RVLPs (10-fold difference) Future Work Compare the intracellular trafficking of lipid-based hybrid vectors with chitosan-based hybrid vectors and amphotropic virus Evaluate receptor-targeted gene delivery and serum performance via PEGylated hybrid vectors 25
Acknowledgements Professor Daniel W. Pack (Advisor) Colleagues: Joshua, Neel, Steve, Nate, Dave, Lily, Ti-An, Shy Chyi, Kalena, Kara, Victor, Mark, Yujie, Mihael, Noel Undergrads Mikhil, Andreina, Janelle, Ian, Hao Facilities Institute of Genomic Biology (Real-Time PCR, Confocal Microscopy) Beckman Institute (Confocal Microscopy) Roy J. Carver Biotechnology Center (Real-Time PCR, Flow Cytometry) Center for Microscopic Imaging Lou Ann Miller (Transmission Electron Microscopy) Frederick Seitz Materials Research Laboratory (Transmission Electron Microscopy) Cell Media Facility (Sandy McMasters) Funding National Science Foundation National Institutes of Health Hanratty Travel Award Graduate College Travel Award Biotechnology Center, UIUC (Catherine Connor Outstanding Dissertation Award) 26
Comparison Why do we need a hybrid design? Retrovirus Hybrid Vector Synthetic Vector Transfection Efficiency Excellent Poor-Excellent Poor-excellent Size Small Medium-Large Small-medium Gene Expression Stable Stable 1 Transient Toxicity No Yes/No Yes/No Flexible Targeting No Yes 2 Yes Pathogenicity Yes Unknown Unknown/No Immunogenicity Yes Little Little Production Expensive Cheaper Inexpensive Stability Poor Robust Robust 1 Depends on synthetic envelope 2 Untested 27
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Real-Time PCR Assay Protocol RNA Isolation Reverse Transcription Real-Time PCR 29
Real-Time PCR Assay Quantification 30
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