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

Transcription

1 Environmentally-responsive poly(aminoesters): Applications for the delivery of mra Dr. Timothy R. Blake Chem- Postdoc Retreat

2 Some problems require a multidisciplinary approach ew monomer and polymer synthesis ew cationic p sensitive materials In vivo imaging and quantification Alcohol oxidation catalysis Waymouth Lab Bioluminescent imaging Drug/gene delivery vehicles Wender Lab Contag Lab Functional biomaterials for gene delivery

3 Requirements for effective gene transfection Molecular Weight mra ca. 330 kda sira 13 kda Length ca nt 2 x 21 nt Secondary Structure Poorly defined Single strand Double stranded double helix Cationic ligomer 1 2 mra transient expression of any gene Size nm 2nm x 6 nm Effect Gene induction Gene silencing Limitations Readily degraded o protein induction 1 Pack and protect mra 2 3 Mediate cell entry Release cargo (escape endosome) Reach ribosome For translation Release is critical

4 sira delivery to acat cells In vitro gene delivery MTC-dodecyl MTC-guan R m C n 9a (m=4, n=4) 9d (m=7, n=7) % tdtomato knockdown This strategy is ineffective for mra delivery! Mean egfp Fluorescence (AU) cells Lipo2000 D:G 29:28 D:G 58:62 D:G 72:69 D:G 182:302 Design criteria for sira delivery vehicles do not apply to mra Successful expression of mra requires delivery and release D:G 43:44 (stat) PEG-D:G 19:14 PEG-D:G 40:33 PEG-G 27 TEG:D:G 25:24:22 D:G:D 6.5- D:G:D 96- x:9:x x:106:x Geihe, et al, PAS, 2012, 13171

5 ew developments provide another approach Pd Pd 2 2 Tf [(neocuproine)pd(ac)] 2 (Tf) 2 Boc Polymerizaion R Boc Tunable Mw arrow PDI Deprotection R rganocatalytic polymerization: designer materials 2 2 soluble stable>3days F 3 C CF 3 S Thiourea (TU) 1,8-Diazabicycloundec-7-ene (DBU) XR R [] Boc R 2 PBS p 7.4 Charge altering degradation Catalytic oxidation: monomer synthesis Functional, biocompatible smart materials mra delivery vehicles CARTs Blake and Waymouth, JACS, 2014 Chung, Blake, et al. JACS, 2013

6 Responsive poly(α-aminoesters) R Boc Deprotection R 2 p 7.4 *Fast and selective Cationic polymerà neutral small molecule First-order Plot 0 ln(%sm) y = x R² = t (min) T 1/2 p 7.4 <5 min T 1/2 p min T 1/2 p min *Wilson o *Andrey Rudenko

7 Proposed charge-canceling mechanism R 2 ac 3 (sat.) R R Base ative chemical ligation R R Base R R Base Fast and Selective R

8 mra delivery: cell culture R Using new amphipathic materials we can transfect mra and elicit the expression of reporter genes in multiple cell lines C GFP verlay % Transfection ela J774 EK-293 C epg2 Cells mra Alone 0% 0% 0 Cells mra Lipo 7 Flow cytometery: GFP expression Lipo 46.7% D:A 13: % Relative Viability Cells Alone 1 mra Complexes on-toxic GFP igher efficiency than commercial agent lipofectamine MTT cell viability assay McKinley, Blake, et al. PAS, 2017, E448-E456 8

9 Structure-activity relationships (L=Lipofectamine) (L=Lipofectamine) D:A 13:11 D:A 18:17 R R C C A B D:G 13:12 D:Pip 13:12 Cells L A B This polymerization strategy allows for rapid screening of new oligomers Block length matters on-degrading oligomers don t work Lipid is required R C C R 2 D 13 E R C D 2 McKinley, Blake, et al. PAS, 2017, E448-E456

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

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

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

13 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. ardy Dr. Masamitsu Kanada Dr. le Audun Werner aabeth Stanford University