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2' fana modified foxp3 antisense oligonucleotides and methods of use thereof

An antisense oligonucleotide and nucleotide technology, applied in the field of hybrid chimera antisense oligonucleotides, can solve the problems of poor target cell delivery, poor stability, and off-target effects

Pending Publication Date: 2021-06-15
AUM LIFETECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although conventional AONs have been effective in discovery and preclinical studies, their transfer to the clinic faces many challenges, including target accessibility, off-target effects, poor stability, and poor delivery to target cells
[0011] There is currently an unmet need for new therapeutics utilizing next-generation AON chemicals to reduce Treg immunosuppression and enhance anti-tumor immunity, especially in lung cancer

Method used

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  • 2' fana modified foxp3 antisense oligonucleotides and methods of use thereof
  • 2' fana modified foxp3 antisense oligonucleotides and methods of use thereof
  • 2' fana modified foxp3 antisense oligonucleotides and methods of use thereof

Examples

Experimental program
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example 1

[0175] Materials and methods

[0176] Antibodies and flow cytometry. Flow cytometry (BDPharmingen) was performed using commercially available conjugated monoclonal antibodies (mAbs). Anti-Foxp3 mAb was FJK-16s (eBioscience), and β-actin antibody was rabbit mAb (CellSignaling). Flow cytometry was performed on a Cyan flow cytometer (Beckman Coulter) and data were analyzed using FlowJo 8 software (Tree-Star). CD4 was sorted from age- and sex-matched Foxp3YFP-cre mice using a FACS Aria cell sorter (BD Bioscience, UPenn Cell Sorting Facility) + YFP + (Foxp3 + ) and CD4 + YFP - (Foxp3 - )cell.

[0177] Spleens and surrounding lymph nodes were harvested and processed for single cell suspension of lymphoblasts. Conventional T cells (Tconv, CD4 + CD25 - ) and Treg (CD4 + CD25 + )cell. For cell sorting, from Foxp3 cre Lymphocytes were isolated from YFP mice and purified based on CD4 expression as above. Then, the CD4 + YFP + (Foxp3 + ) and CD4 + YFP - Cells are so...

example 2

[0186] FOXP3 FANA vs. FOXP3 expressing cells Assessment of the role of the quantity

[0187] The effect of Foxp3 FANA on the number of Foxp3 expressing cells was assessed by flow cytometry.

[0188] Splenocytes were treated with CD3 mAb and different FANA sequences for 3 days in vitro. Such as figure 1 As shown in , the scrambled control FANA indicated that when the FANA concentration was 2.5 μM, there were 14.8% Foxp3-expressing cells in the cell population isolated from the spleen, and when the concentration was 5 μM, there were 9.57% Foxp3-expressing cells. Treatment with Foxp3 FANA sequences reduces the number of Foxp3 expressing cells. For example, with AUM-FANA-6, the percentage of cells decreased to 4.63% at 2.5 μM and 2.97% at 5 μM, respectively.

[0189] Purified Treg cell populations were independently treated in vitro with CD3 / CD28 magnetic beads for three days in the presence of 10 U / ml IL-2 and several FANA sequences. Such as figure 2 As shown in , the scra...

example 3

[0193] Assessment of cellular uptake of FANA oligonucleotides

[0194] In vivo uptake of FANA was assessed 24 hours after injection of 10 mg / kg fluorescent (APC) labeled scrambled oligonucleotides into mice. Cells were harvested from spleen, lymph nodes and blood and analyzed by flow cytometry.

[0195] CD8 was analyzed by tracking the expression of CD8 and APC in cells + CD8 - cell. Such as Figure 4 As shown in , FANA signal was significantly detected in CD8 cells at all three locations, indicating that CD8 cells were successfully transfected in vivo and that CD8 + In vivo uptake of FANA by cells.

[0196] Non-Treg cells as YFP using a mouse model expressing Foxp3 tagged with yellow fluorescent protein (YFP) - (Foxp3 - ) cells were analyzed and uptake of labeled FANA by non-Treg cells was assessed. Such as Figure 5 As shown in , FANA signal was significantly detected in cells from all three locations (spleen, lymph node, and blood) that did not express Foxp3, ind...

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Abstract

The present invention is directed to hybrid chimera antisense oligonucleotides including deoxyribonucleotide and 2'-deoxy-2'-fluoro-beta-D-arabinonucleotide which binds to a Foxp3 mRNA, and to methods of use thereof. The methods include the use for reducing expression level of Foxp3 gene, increasing anti-tumor activity, and treating cancer in a subject.

Description

[0001] Cross References to Related Applications [0002] Pursuant to 35 U.S.C. §119(e), this application claims priority to U.S. Serial Nos. 62 / 737,061, filed September 26, 2018, and U.S. Serial Nos. 62 / 739,001, filed September 28, 2018, the entire contents of which are incorporated by reference Incorporated into this article as a whole. [0003] Incorporation of Sequence Listings [0004] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The attached sequence listing text file is named AUM1190_2WO_Sequence_Listing.txt, it was created in _______, and its size is ____kb. Said files can be accessed using Microsoft Word on a computer using Windows OS. [0005] governmental support [0006] This invention was made with government support under grants 5R01CA177852 and 5R01AI123241 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0007] The presen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34A61P35/00C12N15/113
CPCC12N15/113A61P35/00C12N2310/11C12N2310/322C12N2310/315C12N2310/32C12N2310/3533A61K31/7088A61K45/06A61N5/10
Inventor V·艾西瓦亚W·W·汉考克
Owner AUM LIFETECH INC