A modular platform for targeted RNAi therapeutics

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1 SUPPLEMENTARY INFORMATION Letters In the format provided by the authors and unedited. A modular platform for targeted RNAi therapeutics Ranit Kedmi 1, Nuphar Veiga 1, Srinivas Ramishetti 1, Meir Goldsmith 1, Daniel Rosenblum 1, Niels Dammes 1, Inbal HazanHalevy 1, Limor Nahary 2, Shani LeviatanBenArye 1, Michael Harlev 3, Mark Behlke 4, Itai Benhar 2, Judy Lieberman 5 and Dan Peer 1 * 1 Laboratory of Precision NanoMedicine, School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Department of Materials Sciences and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Center for Nanoscience and Nanotechnology, Cancer Biology Research Center, Tel Aviv University, Tel Aviv, Israel. 2 School of Molecular Cell Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. 3 Veterinary Service Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 4 Integrated DNA Technologies Inc., Coralville, IA, USA. 5 Program in Cellular and Molecular Medicine, Boston Children s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA. R.K. and N.V. contributed equally to this work. * peer@tauex.tau.ac.il Nature Nanotechnology Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 A modular platform for targeted RNAi therapeutics Ranit Kedmi 1, Nuphar Veiga 1, Srinivas Ramishetti 1, Meir Goldsmith 1, Daniel Rosenblum 1, Niels Dammes 1, Inbal HazanHalevy 1, Limor Nahary 2, Shani LeviatanBenArye, Michael Harlev, Mark Behlke, Itai Benhar, Judy Lieberman 5, and Dan Peer 1* Affiliations: 1 Laboratory of Precision NanoMedicine, School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Department of Materials Sciences and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Center for Nanoscience and Nanotechnology, Cancer Biology Research Center, Tel Aviv University, Tel Aviv, 69978, Israel 2 School of Molecular Cell Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. 3 Veterinary Service Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel 4 Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA 5 Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA. R.K. and N.V. contributed equally to this work. * Correspondence: Dan Peer, peer@tauex.tau.ac.il Supplementary Figures

3 a b V H L EVQLSVRLNXEAWGFSEDSCKASGYTFTDYNMDWV KQSHEKSLEWIGYINPYSGDTIYNHKFKDKATLTVDK SSNIAYMELRSLTSEDTAVYYCARGGYDYGDHWGQ GTTLTVSS GSAGGGGSGGGGSGGGGS i ii iii iv v vi V L DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQ QKPGKSPKTLIYRANRLVXGVPSRFSGSGSGQDYSL TISSLEYEDMGIYYCLQDDEFPRTFGGGTKLEIK 37 c unstain RIg scfv RIg scfv RIg scfv Counts αhis Supplementary Figure 1: Generation of antirat Ig scfv. (a) Amino acid sequence of secondary (mouse antirat) scfv, cloned from RG7/1.3 hybridoma. (b) Polyacrylamide/SDS gel electrophoresis of samples from ASSET production process, uninduced culture (ii), induced culture (iii), insoluble fraction of induced culture (iv), soluble fraction of induced culture (v) and purified ASSET (vi). Stained with Coomassie blue. Red box indicates scfv. Data is representative of 5 independent experiments. (c) Representative histograms of TK1 cell line, preincubated or not with αitgb7 RIg (red), assayed for binding of antirat Ig scfv. Antirat Ig scfv binding in the absence of RIg or in the absence of antirat Ig scfv (grey) served as controls. Representative histograms of 3 independent experiments. Numbers indicate cell count in each gate.

4 a b LNPs ASSET 100nm Free sirna 0.5% Triton c Time in plasma (min) Free sirna TsiLNPs 1% Triton Relative densitometry analysis Supplementary Figure 2: TsiLNP characterization. (a) Transmission electron microscopy images of LNP before (top) and after (bottom) ASSET incorporation. (b) Agarose gel electrophoresis to detect sirnas in medium released from Tritonpermeabilized or intact LNP, before and after ASSET incorporation. (c) Analysis of TsiLNPs integrity and sirna stability in plasma over 240 minutes. A cropped image of agarose gel electrophoresis to detect sirna released from TsiLNP by Triton or intact TsiLNP. Lanes were cropped from the same gel. Data in (ac) are representative of 3 independent experiments.

5 a b LNPs ASSET RIg Standard curve µg IgG: RIg (cct) ASSET RIg (T) ASSET RIg Counts d Counts c Mock LNPs Mock RIg TsiLNPs ASSET LNPs RIg silnps TsiLNPs Cy5 mcherry e GFP GFP silnps GFP GFP GFP TsiLNPs GFP cctsilnps Supplementary Figure 3: ASSET incorporation into LNPs. (a) RIg binding to LNP depends on ASSET. Protein composition of purified TsiLNP was assessed by immunoblot probed with αhis (ASSET) or antirat IgG. Cropped blot is presented. (b) Gel quantification by Coomassie blue staining of RIg incorporation into TsiLNP (T) and cctsilnp (cct), compared to RIg standard curve. Cropped blot from the same original gel is presented to each sample. (c) Binding of Cy5siRNAloaded αlfa1coated (blue) or uncoated (grey) TsiLNP to TK1 cells, compared to untreated cells (black), assessed by flow cytometry analysis of Cy5 fluorescence. (d) Representative histograms of ASSET mcherry fluorescence after TK1 cells were incubated with αlfa1 TsiLNP (red). Controls (in indicated gray scale) were RIg alone (αlfa1), ASSET LNP lacking RIg, and silnp lacking ASSET but incubated with RIg. Shown is a representative of 3

6 replicate experiments. (e) Experimental design testing Cy5siRNAloaded αcd34 cctsilnp, TsiLNP and silnp interaction with rat Fc receptor, transiently express in HEK293T cells along with GFP reporter. Data in (ad) are representative of 3 independent experiments.

7 a Mononuclear, CD4 Mononuclear CD25 CD4 CD3 CD19 CD11b Mock Itgb7 TsiLNPs CD3 TsiLNPs CD4 TsiLNPs CD25 TsiLNPs Cy5 b CD4 Counts CD8 CD3 Supplementary Figure 4: In vivo selective uptake of TsiLNP. (a) Blood leukocytes isolated 1.5 hours after intravenous injection of Cy5siRNAloaded TsiLNP assembled with indicated RIgs (αitgb7, αcd3, αcd4 and αcd25) were costained for CD4, CD3, CD8, CD19, CD11b or CD25 and analyzed by

8 flow cytometry. Shown are representative contour plots of gated CD4 live mononuclear cells (left column only) or of ungated live mononuclear cells. (b) Uptake of αcd3 TsiLNP caused CD3 receptor downmodulation, but did not affect CD4 or CD8 staining. Blood cells from mice that were mocktreated or treated with αcd3 TsiLNP were stained for CD4, CD8 and CD3. Histograms below show CD3 staining of gated CD4 or CD8 cells (gating in dot blots above). Data in (ab) are representative of 3 independent experiments (as biological replicates).

9 a Relative TNFα Levels **** Mock Luc TNF b Mock Iso TsiLNPs Ly6C TsiLNPs c 3 **** Ly6C Relative Cy5 MFI 2 1 Cy5 0 RIg: Iso Ly6C Supplementary Figure 5: TNF gene knockdown invitro and Ly6C TsiLNP selective uptake (a) TNF gene knockdown, relative to GAPDH, by qrtpcr in RAW cells incubated with TNF or luciferase sirnaloaded TsiLNP. Data are mean ± SD, n = 3, ****p < (twosided Student s ttest.). (b, c) Uptake of Cy5siRNAloaded, αly6c or isotype control TsiLNP into Ly6C blood cells harvested 1 h after intravenous injection, evaluated by flow cytometry Shown are representative contour blots of live mononuclear cells (b) and relative Cy5 fluorescence of Ly6C vs LyC6 cells (c). Data are mean ± SD, n = 3, ****p < (twosided student s T test). Data in (ac) are representative of 3 independent experiments (as biological replicates).

10 a b Mock Luc PLK1 Relative PLK1 Levels Mock Luc PLK1 Counts FL2a c Percent Survival Untreated Iso PLK1 CD29 PLK1 Time (days) Supplementary Figure 6: TsiLNPmediated therapeutic gene silencing in a MCL (a) PLK1 gene knockdown, relative to GAPDH, by qrtpcr in Granta519 cells incubated with PLK1 or luciferase sirnaloaded TsiLNP. Data are mean ± SD, n = 3, ***p = (twosided Student s ttest.). (b) Representative cell cycle distribution, 60h post treatments with αcd29lnpssiluc or αcd29lnpssiplk1, analyzed by flow cytometry. The data is representative of 3 independent experiments. (c) Survival curves of MCLbearing mice. Corresponding treatments (2 mg sirna/kg body) were administrated at seven time points as listed in the experimental section via retroorbital route. n = 10 animals per group, ***P = P values and significance were determined by logrank Mantel Cox test with Bonferroni correction.

11 Supplementary Tables S14 Supplementary table 1: Characterization of LNP by Dynamic Light Scattering and ζpotential. Data presented are mean ± SD of six independent preparations. LNP ASSET LNP Hydrodynamic diameter (d, nm) 66 ± ± 0.75 ζpotential (mv) 4.28 ± ± 0.5 Supplementary table 2: Comparison of αcd34 TsiLNP and cctsilnp. Data are presented as mean ± SD of three independent preparations. LNP yield IgG incorporation RIg K d (nm) tested by ELISA cctsilnp 23.5% ± % ± ± TsiLNP 94.9% ± % ± ± Supplementary table 3: Kinetic measurements of masset binding affinity as analyzed by Surface Plasmon Resonance (SPR). Data is representative of 3 independent experiments. Concentration RI (RU) Drift Req (RU) kobs (1/s) (nm) (RU/s) E E E E

12 Supplementary table 4: masset's kinetic constants as analyzed using Biacore evaluation software 4.1. Data is representative of 3 independent experiments. ka (1/Ms) kd (1/s) Rmax KA (1/M) KD (M) Chi2 (RU) 3.85E E E E

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