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
Alcohol oxidation catalysis
New cationic pH sensitive materials New monomer and polymer synthesis In vivo imaging and quantification
Drug/gene delivery vehicles Functional biomaterials for gene delivery Waymouth Lab Wender Lab Contag Lab Bioluminescent imaging
Cationic Oligomer
1 2 3 4
mRNA transient expression of “any” gene
Pack and protect mRNA Mediate cell entry Release cargo (escape endosome) Reach ribosome For translation
1 2 3 4
mRNA siRNA Molecular Weight
13 kDa Length
2 x 21 nt Secondary Structure Poorly defined Single strand Double stranded double helix Size 17-20 nm 2nm x 6 nm Effect Gene induction Gene silencing Limitations Readily degraded No protein induction
Release is critical
5000 10000 15000 20000
cells Lipo2000 D:G 29:28 D:G 58:62 D:G 72:69 D:G 182:302 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- x:9:x D:G:D 96- x:106:x
Mean eGFP Fluorescence (AU)
!
This strategy is ineffective for mRNA delivery
Design criteria for siRNA delivery vehicles do not apply to mRNA
85% tdTomato knockdown
siRNA delivery to HaCat cells
Geihe, et al, PNAS, 2012, 13171
O O O H O NH NH2 NH2 O O n MTC-guan O RO O O O C12H15 MTC-dodecyl m
9a (m=4, n=4) 9d (m=7, n=7)
TFA
Catalytic oxidation: monomer synthesis Organocatalytic polymerization: designer materials Functional, biocompatible “smart” materials
N N Pd O N N Pd O O O 2 2 OTf
[(neocuproine)Pd(OAc)]2(OTf)2 N N
1,8-Diazabicycloundec-7-ene (DBU)
N H N H S CF3 F3C
Thiourea (TU)
H2 N O H O TFA RO PBS pH 7.4 N N O HO O OH Charge altering degradation OH NR OH OH NH OH XR
[O]
O NBoc O
mRNA delivery vehicles “CARTs”
O NBoc O Boc N O H O RO Polymerizaion H2 N O H O TFA RO Tunable Mw Narrow PDI H2O soluble stable>3days Deprotection
Blake and Waymouth, JACS, 2014 Chung, Blake, et al. JACS, 2013
y = -0.0386x - 0.0676 R² = 0.99513
20 40 60 80
ln(%SM) t (min)
First-order Plot
T1/2 pH 7.4 <5 min T1/2 pH 6.4 11 min T1/2 pH 5.4 18 min
*Fast and selective Cationic polymerà neutral small molecule
*Wilson Ho *Andrey Rudenko
Deprotection Boc N O H O RO H2 N O H O TFA RO pH 7.4 N N O HO O OH
H2 N O H O TFA RO NaHCO3 (sat.) N N O HO O OH
RO H N O N O N O N O O O O O N O N O N O O O N O N O O H N O HO ROH O N O H H H H H H H H H H H H O N O OR H O H N O R O N O O H O H N N O HO O O OH
Fast and Selective
Base Base Base N N O O HO R R R
Native chemical ligation
GFP 0% 46.7% 99.5% mRNA Alone Lipo D:A 13:11 GFP Overlay Cells
1 1 0.2 0.4 0.6 0.8 1 1.2
Cells Alone mRNA Complexes
Relative Viability
Using new amphipathic materials we can transfect mRNA and elicit the expression of reporter genes in multiple cell lines
0% 8
100 80 60 40 20 % Transfection Cells mRNA Lipo 7 HeLa J774 HEK-293 CHO HepG2
Higher efficiency than commercial agent lipofectamine Non-toxic Flow cytometery: GFP expression MTT cell viability assay
O O O O O O C12H15
13
H3 N O H
11
RO TFA
McKinley, Blake, et al. PNAS, 2017, E448-E456
O O H2 N OH O RO O O O 18 17 TFA C12H15
O O O OH O RO O O O 13 12 O O C12H15 N H2 TFA
A B E D
O H N RO O H H 13
TFA
(L=Lipofectamine)
Block length matters Lipid is required
C
Non-degrading oligomers don’t work This polymerization strategy allows for rapid screening of new oligomers
D:A 13:11 D:A 18:17 D:G 13:12 D:Pip 13:12 D 13 Cells L A B
(L=Lipofectamine)
McKinley, Blake, et al. PNAS, 2017, E448-E456
O O O OH O RO O O O 13 12 O O TFA C12H15 H N NH2 NH2
O O O O O O C12H25
13
H2 N O H
11
RO
TFA
Confocal Microscopy
and cargo on a cell-by-cell basis Fluorophores
Conditions: HeLa cells, 10:1 +/- charge ratio, 4 hours following treatment D13:A11 D13:G12 Dansyl GFP Cy5 Merge
McKinley, Blake, et al. PNAS, 2017, E448-E456
O O O O O O C12H25
n
H2 N O H
m
O Dansyl O O O O O O H O O O O HN NH2 NH2
n m
O Dansyl C12H25
D13:A11 D13:G12
TFA TFA
Intramuscular
1 h 4 h 7 h 24 h 48 h 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Bioluminescent Intensity (p/s/cm^2/sr) 1 h 4 h 7 h 24 h 48 h
Intravenous (tail vein)
1 h 4 h 7 h 24 h 48 h 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Bioluminescent Counts (p/s) 1 h 4 h 7 h 24 h 48 h
Full circle=CART+mRNA Empty circle= mRNA alone
Localizes in spleen
7.5 ug mRNA 75uL PBS Luciferase mRNA administered at t=0 Luciferine administered at time of measurement
McKinley, Blake, et al. PNAS, 2017, E448-E456
Colin J. McKinley
Stanford University