Environmentally-responsive poly(aminoesters): Applications for the - PowerPoint PPT Presentation

Environmentally-responsive poly(aminoesters): Applications for the delivery of mRNA Dr. Timothy R. Blake Chem-H Postdoc Retreat 05-02-17

Some problems require a multi- disciplinary approach New cationic pH sensitive materials New monomer and In vivo imaging polymer synthesis and quantification Alcohol oxidation catalysis Waymouth Lab Bioluminescent imaging Contag Lab Drug/gene delivery vehicles Wender Lab Functional biomaterials for gene delivery

Requirements for effective gene transfection mRNA siRNA Molecular ca. 330 kDa 13 kDa Weight Length ca. 1000 nt 2 x 21 nt Secondary Poorly defined Double Structure Single strand stranded double helix Size 17-20 nm 2nm x 6 nm mRNA Effect Gene induction Gene transient expression of Cationic Oligomer silencing “ any ” gene Limitations Readily No protein degraded induction - - -- -- - - -- -- - - -- -- - - -- -- - - -- -- - - -- -- - - -- -- - - -- -- 2 1 Pack and protect 1 mRNA 2 Mediate cell entry - - -- -- 3 4 - - -- -- - - -- -- - - -- -- - - -- -- - - -- -- Release cargo - - -- -- - - -- -- 3 - - -- -- (escape endosome) - - -- -- 4 Reach ribosome Release is critical For translation

In vitro gene delivery siRNA delivery to HaCat cells MTC-dodecyl MTC-guan O O O H RO O O O m n O O O O C 12 H 15 9a (m=4, n=4) NH NH 2 9d (m=7, n=7) 85% tdTomato NH 2 knockdown TFA ! This strategy is ineffective for mRNA delivery Mean eGFP Fluorescence (AU) 20000 Design criteria for siRNA delivery vehicles 15000 do not apply to mRNA 10000 • Successful expression of mRNA requires delivery and release 5000 0 cells Lipo2000 D:G 29:28 D:G 58:62 D:G 72:69 D:G D:G 43:44 PEG-D:G PEG-D:G PEG-G 27 TEG:D:G D:G:D 6.5- D:G:D 96- 182:302 (stat) 19:14 40:33 25:24:22 x:9:x x:106:x Geihe, et al, PNAS , 2012, 13171

New developments provide another approach CF 3 2 Polymerizaion Deprotection O S O O TFA Boc H 2 F 3 C N N 2 OTf O N N N N N O H H H N O H RO RO Pd NBoc Thiourea (TU) Pd O O O O H 2 O soluble Tunable Mw N Narrow PDI stable>3days N 1,8-Diazabicycloundec-7-ene Organocatalytic polymerization: [(neocuproine)Pd(OAc)] 2 (OTf) 2 (DBU) designer materials OH OH O PBS pH 7.4 [O] HO O XR O N TFA H 2 OH OH O N O H N RO O OH NH NR NBoc Charge altering degradation Catalytic oxidation: Functional, biocompatible monomer synthesis “smart” materials mRNA delivery vehicles “CARTs” Blake and Waymouth, JACS, 2014 Chung, Blake, et al. JACS, 2013

Responsive poly( α -aminoesters) Deprotection pH 7.4 HO O O O TFA Boc H 2 N N O H N O H N RO RO O OH *Fast and selective Cationic polymer à neutral small molecule First-order Plot 0 -0.5 -1 T 1/2 pH 7.4 <5 min ln(%SM) -1.5 T 1/2 pH 6.4 11 min -2 T 1/2 pH 5.4 18 min -2.5 y = -0.0386x - 0.0676 R² = 0.99513 -3 -3.5 0 20 40 60 80 *Wilson Ho t (min) *Andrey Rudenko

Proposed charge-canceling mechanism NaHCO 3 (sat.) HO O O N H 2 TFA O O O O H H H H H H H N O H N R RO N N N N O OH RO O O O O O Base N O O H H H H H Native chemical ligation R O N N N O O O H OR O Base O HO N O O H H H ROH R N N N O O O O Base O HO O N N H R O N N O H O OH O O O N Fast and Selective N HO O

mRNA delivery: cell culture O O H 3 Using new amphipathic materials we can transfect mRNA and N O H RO O O elicit the expression of reporter genes in multiple cell lines 13 11 TFA O O C 12 H 15 GFP Overlay 100 HeLa 0% J774 80 Cells % Transfection HEK-293 CHO 60 HepG2 mRNA 40 0% Alone 20 0 Cells mRNA Lipo 7 46.7% Lipo Flow cytometery: GFP expression 99.5% D:A 13:11 1.2 1 1 1 Relative Viability 0.8 GFP 0.6 Non-toxic 0.4 0.2 Higher efficiency than 0 Cells Alone mRNA commercial agent lipofectamine Complexes MTT cell viability assay 8 McKinley, Blake, et al. PNAS , 2017, E448-E456

Structure-activity relationships ( L =Lipofectamine) ( L =Lipofectamine) D:A 18:17 D:A 13:11 O O O O H 2 H 2 N O H N RO O O RO O O OH TFA 13 11 TFA 17 18 O O O O C 12 H 25 C 12 H 15 A B D:G 13:12 D:Pip 13:12 O O O O RO O O O OH RO O O O OH Cells L A B 12 12 13 13 O O O O O O O O NH 2 H C 12 H 15 N C 12 H 15 TFA This polymerization strategy allows for rapid NH 2 C D TFA screening of new oligomers N H 2 D 13 Block length matters O H H N O H Non-degrading oligomers don’t work RO 13 TFA Lipid is required E McKinley, Blake, et al. PNAS , 2017, E448-E456

To express mRNA, mRNA must escape endosome Confocal Microscopy - Allows for independent imaging of transporter D13:G12 D13:A11 and cargo on a cell-by-cell basis O O O O H 2 O H N O H Fluorophores Dansyl O O O O Dansyl O O O TFA n m - Dansyl : attached to transporter n m O O O O O O - GFP : indicates expression has occurred TFA C 12 H 25 C 12 H 25 - Cy5 : attached to mRNA HN NH 2 NH 2 Dansyl GFP Cy5 Merge D 13 :G 12 D 13 :A 11 Conditions: HeLa cells, 10:1 +/- charge ratio, 4 hours following treatment McKinley, Blake, et al. PNAS , 2017, E448-E456

mRNA expression via multiple routes of administration in vivo Intramuscular Intravenous (tail vein) 1 h 4 h 7 h 24 h 48 h 1 h 4 h 7 h 24 h 48 h 1.00E+06 1.00E+06 Full circle=CART+mRNA Empty circle= mRNA alone Bioluminescent Counts (p/s) Bioluminescent Intensity 1.00E+05 1.00E+05 (p/s/cm^2/sr) 1.00E+04 1.00E+04 1.00E+03 1.00E+03 1 h 4 h 7 h 24 h 48 h 1 h 4 h 7 h 24 h 48 h Luciferase mRNA administered at t=0 Localizes in spleen 7.5 ug mRNA Luciferine administered at time of measurement 75uL PBS McKinley, Blake, et al. PNAS , 2017, E448-E456

Conclusions • Our oligomerization strategy is a great platform for rapid synthesis and screening of new delivery vehicles • Rapidly degrading biocompatible oligomers allow for mRNA delivery and robust protein expression • Release of mRNA from the CART polyplex is required for protein expression

Acknowledgements Prof. Robert M. Waymouth Prof. Paul A. Wender Prof. Chris T. Contag Prof. Ronald Levy Colin J. McKinley Dr. Jessica R. Vargas Dr. Jonathon M. Hardy Dr. Masamitsu Kanada Dr. Ole Audun Werner Haabeth Stanford University