Work Description

Date:
29 January, 2025

Dataset Title:
Anti-TNFα Antisense Oligonucleotide Conjugated PLG Nanoparticles Protect Transplanted Islets

Dataset Creators:
Elizabeth J. Bealer, Namit Padgaonkar, Kelly Crumley, Eiji Saito, Zoe Beekman, Alexa DeKorte, Thazha P Prakash, Alexey Revenko, and Lonnie D. Shea

Dataset Contact:
Elizabeth Bealer [email protected]

Funding:
Funding for this work was provided by NIH R01DK121462 and R01AI148076, and Breakthrough Type 1 Diabetes, and the Breakthrough Type 1 Diabetes Center of Excellence at the University of Michigan.

Key Points:
-PLG-aTNFα NPs benefit from synergy between ASO and NPs that results in broad knockdown of inflammation
-SC islets transplanted on microporous scaffolds to the intraperitoneal fat of streptozotocin-induced diabetic mice were protected after treatment with PLG-aTNFα NPs
-PLG-aTNFα NPs allow for ease of fabrication and lyophilization

Research overview:
One of the many challenges for islet transplantation as a treatment for type 1 diabetes is inflammation that contributes to islet de-differentiation and death. Innate immune cells such as monocytes and macrophages secrete TNFα, IL-1β, iNOS, and IL-6 which directly contribute to islet dysfunction. Attenuation of the early inflammatory response post-transplantation may protect cell survival and subsequent function. Herein, we investigate the development of anti-TNFα antisense oligonucleotide conjugated polylactide-co-glycolide nanoparticles (PLG-aTNFα NPs) as an anti-inflammatory therapy after stem cell-derived islet transplantation. PLG-aTNFα NPs are shelf stable and successfully reduce TNFα secretion and expression in inflammatory macrophages.

Synergy between the aTNFα antisense oligonucleotide and the polylactide-co-glycolide nanoparticles results in further knockdown of IL-1β, IL-6, iNOS, and IL-12 in vitro indicating PLG-aTNFα NPs may protect against the inflammatory cascade in vivo. In a diabetic mouse model, stem cell-derived islets transplanted to the peritoneal fat were protected after treatment with PLG-aTNFα NPs compared PLG NPs alone. TNFα and IL-1β expression was reduced in mice treated with PLG-aTNFα NPs indicating inflammation was reduced after transplant. PLG-aTNFα NPs reduce TNFα and protect islets, supporting their potential use a therapeutic in islet transplantation.

Methodology (brief - see full paper, 2025-ASONP-Manuscript-Final.docx, for detailed methods):

5’-/5AmMC12/CAAATCGGCTGACGGT-3’ antiTNFα-ASO was used in this work. Female C57BL/6 were used for bone marrow isolation and derivation of macrophages for in vitro testing of ASO NPs. PLG particles were fabricated using the oil in water single emulsion solvent evaporation method. Conjugation of ASO was done using EDC/NHS chemistry. Gene expression from macrophage cultures was done by lysing cells in Trizol followed by RNA isolation using the DirectZol RNA MicroPrep kit (Zymo) according to manufacturer’s instructions. Gene expression and fold change were quantified using the ΔΔCt method. Cytokines (TNFα, IL-12p40, IL-6, IL-1β) were measured in macrophage media supernatants by enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN) done by the University of Michigan Cancer Center Immunology Core.

PLG scaffolds were fabricated by compression molding PLG microspheres (75:25 mole ratio D,L-lactide to glycolide) and 250 to 425 µm salt crystals in a 1:30 ratio of PLG microspheres to salt. hSPC differentiations to stem cell islets (SC islets) were done based on previously published protocols. Reaggregated cell clusters were prepared for seeding onto microporous scaffolds and transplantation into mice. To seed scaffolds, cell clusters were distributed on both sides of the scaffold and incubated for 2 hours at 37°C. For these studies, 1 million SC islet cells were seeded on each scaffold. Scaffolds were transplanted to the epidydmal fat pads of diabetic male B6.Cg-Rag2tm1.1Cgn/J mice. ASO NPs were delivered to the tail vein of transplant recipients. Explanted scaffolds were sectioned and stained for C-peptide or processed to single cell suspensions to perform flow cytometry; analyzed by a Z5 cell analyzer (Bio-Rad). Statistical analyses were performed using GraphPad Software (La Jolla, CA) and graphed using Prism.

Files contained here:
Files include images from histology, raw data in excel, analyzed data in prism, and wsp files from Flowjo. Detailed description of figures - which are denoted in the file names - are in the 2024-ASONP-Manuscript-v2.docx.

- 2025-ASONP-Manuscript-Final.docx - Full paper.

- 2024-09-19_ASO-NPs_PaperFigures.pptx - All figures for manuscript, main and supplementary.

- 2025-ASOPaperSupp.pdf - Supplementary figures and figure descriptions for manuscript.

- 01-Mar-2023-Day7-Mono-Macros-compensation2.wsp - Flow data from Figure 1.

- 01-Mar-2023-DCDay7.wsp - Flow data from Figure 1.

- 03-Apr-2023-macrophageDay4.wsp - Flow data from Figure 1.

- 05-Apr-2023_Day4Monocytes.wsp - Flow data from Figure 1.

- 12-Jun-2023-NPExplantDay1_Final.wsp - Flow data from Figure 6.

- 21-Aug-2024-NP.wsp - Flow data from Figure 6.

- 22-Feb-2023-Day1.wsp - Flow data from Figure 6.

- 2024-ASO-NP_RawData.xlsx - PCR data from multiple figures.

- Fig6I-ASONP.jpg - Histology images from Figure 6.

- Fig6I-NoTreatment.jpg - Histology images from Figure 6.

- Fig6I-NP.jpg - Histology images from Figure 6.

- Fig6J-ASONP.jpg - Histology images from Figure 6.

- Fig6J-NoTreatment.jpg - Histology images from Figure 6.

- Fig6J-NP.jpg - Histology images from Figure 6.

- Figure1-Flow_Combined.prism - Analyzed flow data in Prism from Figure 1.

- Figure1.xlsx (and .csv) - Raw data from Figure 1.

- Figure2.1.prism - Analyzed PCR data in Prism from Figure 2.

- Figure2.prism - Analyzed PCR data in Prism from Figure 2.

- Figure2.xlsx (and .csv) and Figure2.2.xlsx (and .csv) - Raw PCR data in Excel from Figure 2.

- Figure3.prism - Analyzed PCR data in Prism from Figure 3.

- Figure4.prism - Analyzed PCR data in Prism from Figure 4.

- Figure3and4.xlsx - Raw ELISA data from Figures 3 and 4.

- Figure3.csv - Raw ELISA data from Figure 3.

- Figure4.csv - Raw ELISA data from Figure 4.

- Figure5.prism - Analyzed PCR data in Prism from Figure 5.

- Figure5.xlsx (and .csv) - Raw PCR and ELISA data from Figure 5.

- Figure6.1.prism - Analyzed PCR data in Prism from Figure 6.

- Figure6.2.prism - Analyzed PCR data in Prism from Figure 6.

- Figure6-PCR-imageanalysis.prism - Analyzed PCR data and histology quantification in Prism from Figure 6.

- Figure6_Day4ImageAnalysis.xlsx (and .csv) - Raw histology quantification values in Excel from Figure 6.

- Figure6_Day7ImageAnalysis.xlsx (and .csv) - Raw histology quantification values in Excel from Figure 6.

- Figure6_InflammationPCR.xlsx (and .csv) - Raw PCR data from Figure.

Use and Access:
This data set is made available under a Creative Commons Public Domain license (CC0 1.0).

To Cite this Work:
Bealer, E., Padgaonkar, N., Crumley, K., Saito, E., Beekman, Z., DeKorte, A., Prakash, T. P., Revenko, A., Shea, L. D. Anti-TNFα Antisense Oligonucleotide Conjugated PLG Nanoparticles Protect Transplanted Islets [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/dzq6-kk44