JP-2026076236-A - Gene vector regulation by cardiomyocyte-expressed microRNAs

Abstract

[Challenge] To provide gene vector control using microRNA expressed in cardiomyocytes. [Solution] This disclosure provides vectors and methods of using them for cell-type-specific repression of the expression of transgenes (e.g., cardiomyocyte reprogramming factors) using microRNA binding sites. The present invention generally relates to vectors and methods of using them for cell-type-specific repression of the expression of transgenes (e.g., cardiomyocyte reprogramming factors). This disclosure provides vectors comprising microRNA binding sites configured to promote specific repression of the expression of transgenes (e.g., cardiomyocyte reprogramming factors) in cardiomyocytes and cardiomyocyte progenitor cells compared to cardiac fibroblasts. [Selection Diagram] Figure 1

Inventors

• ローラ ロンバルディ
• エレナ シー． レオン
• タウニー ニール クレシ
• キャスリン エヌ． アイビー

Assignees

• テナヤ セラピューティクス， インコーポレイテッド

Dates

Publication Date

20260511

Application Date

20260120

Priority Date

20200302

Claims (1)

1. The invention described in the specification.

Description

Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62/984,183, filed on 2 March 2020, the contents of which are incorporated herein by reference in their entirety. Reference to the Sequence Listing This application was filed electronically via EFS-Web and includes a sequence listing submitted electronically in .txt format. The .txt file contains a sequence listing titled "TENA_017_01WO_SeqList_ST25.txt" created on February 25, 2021, with a size of 230 kilobytes. The sequence listing contained in this .txt file is part of this specification and is incorporated herein by reference in its entirety. This disclosure generally pertains to gene therapy vectors. Direct cardiac reprogramming is emerging as a strategy for creating new cardiomyocytes, leading to improved cardiac function in patients diagnosed with or at risk of cardiomyopathy, heart failure, or other heart diseases. Various combinations of genetic and chemical reprogramming factors have been shown to facilitate the reprogramming of other cells (e.g., fibroblasts) into cardiac cells (particularly cardiomyocytes). For example, a combination of three cardiac development transcription factors—GATA4, MEF2C, and TBX5 (GMT)—can be used to reprogram dermal or cardiac fibroblasts into induced cardiomyocyte (iCM)-like cells in mice. When GATA4, MEF2C, TBX5, MESP1, and MYOCD (GMTMM) are expressed together as a cocktail of factors, they alter the cell morphology from spindle-shaped to rod-shaped, causing the cells to exhibit spontaneous Ca2 + oscillations. HAND2, NKX2.5, the microRNAs miR-1 and miR-133, JAK, or TGF-β have been shown to promote such reprogramming. In humans, the addition of ETS2 and MESP1 to GMT induces heart-specific gene expression and sarcomere formation. Other combinations of factors for direct reprogramming have been described in the art, as outlined in Srivastava and DeWitt. Cell 166:1386-96 (2016). However, there remains a need in the art for alternative and improved reprogramming methods, such as vectors or vector systems, and means for carrying out such methods. This disclosure addresses this unmet need. Srivastava and DeWitt. Cell 166:1386-96 (2016) This invention generally relates to vectors for cell-type-specific repression of the expression of transgenes (e.g., cardiomyocyte reprogramming factors) and methods of using them. This disclosure provides vectors comprising microRNA binding sites configured to promote specific repression of transgene expression (e.g., cardiomyocyte reprogramming factors) in cardiomyocytes and cardiomyocyte progenitor cells compared to cardiac fibroblasts. In some embodiments, microRNAs are selected by treating cardiac fibroblasts with an effective amount of a composition that induces reprogramming of cardiac fibroblasts into cardiomyocytes, and measuring the expression of one or more microRNAs in cardiac fibroblasts against identified microRNAs that are expressed during the programming process and, optionally, selectively, expressed later in the reprogramming process. In some embodiments, the selected microRNAs are expressed in cardiac fibroblasts only after a predetermined time. In some embodiments, tissue- and cell-type-specific repression of expression allows expression in target cell types (e.g., cardiac fibroblasts) while repressing expression in non-target cell types (e.g., cardiomyocytes). In some embodiments, the microRNA binding sites allow expression in target cells for a sufficient time to cause effective conversion of target cells to non-target cells. A vector map according to one embodiment is shown.Four days after iPSC-CM transduction using AAV with an MOI of 160k, flow cytometry analysis revealed suppression of GFP transgene expression.Four days after hCF transduction with AAV at MOI 160k, flow cytometry analysis revealed a deletion in the suppression of GFP transgene expression.Four days after iPSC-CM transduction using AAV with an MOI of 500k, flow cytometry analysis revealed suppression of GFP transgene expression.The expression levels of microRNAs measured by qPCR after treatment with the My Δ3 A reprogramming cocktail are shown, compared to human iPSC-CMs.This shows the flow cytometry analysis of iPSC-CMs four days after transduction using AAV (MOI 500k).This shows the flow cytometry analysis of hCF two days after transduction using AAV (MOI 160k).This shows the immunofluorescence analysis of MOI 640k three weeks after AAV-mediated reprogramming.Throughout the research process, My Δ3 A with the miR-208 target cassette demonstrates its effectiveness against myocardial infarction, as measured by dynamic echocardiographic data of ejection fraction %.The echocardiographic data at the end of the in vivo trial, 8 weeks after injection, shows a significant increase in all groups compared to the negative control encoding GFP (n = 10–13 mice/group).This shows an analysis of fibrosis based on trichrome staining, in which muscle is sta