A microRNA-regulated cell death-inducing gene therapy for T-cell Acute Lymphoblastic Leukemia

Project Information

miRToTALL

Grant agreement ID: 101069429

DOI

10.3030/101069429

Project closed

EC signature date 20 May 2022

Start date 1 June 2022

End date 31 May 2024

Funded under

• European Research Council (ERC)

Total cost

No data

EU contribution

€ 150 000,00

Coordinated by

FUNDACAO GIMM - GULBENKIAN INSTITUTE FOR MOLECULAR MEDICINE
Portugal

Periodic Reporting for period 1 - miRToTALL (A microRNA-regulated cell death-inducing gene therapy for T-cell Acute Lymphoblastic Leukemia)

Reporting period: 2022-06-01 to 2024-05-31

Summary of the context and overall objectives of the project

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. Although outcome has improved throughout the years with the use of combined chemotherapy, the aggressive regimens required for treatment efficacy often lead to significant short- and long-term side effects. Moreover, a significant fraction of T-ALL patients relapse, which have extremely poor prognosis.
The current project aligns with the core goal of our ERC Consolidator Grant IL7sigNETure (CoG-648455) to generate new targeted therapies for improved treatment of T-ALL.
We have envisioned a novel and ambitious gene therapy system that takes advantage of tumor specific microRNA (miR) expression profiles to selectively deliver a killing gene to tumor cells. MiRs are ~22 nucleotides small non-coding RNAs that regulate gene expression post-transcriptionally. Importantly, miR expression profiles have been shown to distinguish tumor from normal cells and tumors of different developmental origins and differentiation stages. Thus, we propose to develop a gene therapy that explores the differences between T-ALL and normal cells regarding their miR expression pattern, in order to: specifically identify leukemia cells and selectively induce the expression of a killing gene in the malignant cells. This microRNA-regulated cell death-inducing system has the transformative potential to overcome efficacy and side-effect limitations associated with current therapeutic strategies in T-ALL.
Overall, the scientific breakthrough of this project is the development of a gene therapy cell death-inducing system (miRTo) that uses cell-endogenous miRs to regulate the expression of a killing gene specifically in target cells. In addition to the stringent regulation by miRs specifically present and absent in target cells, the expression of the killing gene is further regulated by an inhibitor delivered simultaneously by the same gene therapy system and also regulated by cell-endogenous miRs. This dual layer of regulation, in one single vector, has not been applied before and will increase the specificity of this system.
Importantly, the way the technology is designed allows simple modifications to be introduced. The flexibility and adaptability of our technology makes it ideal for precision medicine, giving us the potential to generate personalized therapies and to treat a broad spectrum of diseases, in which patients attained of severe medical conditions and left without treatment alternatives may clinically benefit from the targeted delivery of a therapeutic gene. As such, the prospective clinical applications of this gene therapy are numerous, including, but not limited to, cancer and, in particular T-ALL, our main therapeutic focus.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The objectives of this project consisted of: 1) development of a gene therapy cell death-inducing system to treat T-ALL (miRToTALL), 2) its validation using functional in vitro and in vivo biologically-relevant validation models and, 3) building of the foundations for the long-term vision of miRToTALL translation into the clinic. The worked developed for the particular aims of each objective are described below.
Aim 1) Definition of a T-ALL-specific miR signature:
The identification of a T-ALL-specific miR profile is key to the success of our strategy. Prior to the start of this project, we have established T-ALL-specific miR expression profiles by bioinformatics analyses of publicly available miR expression datasets obtained from T-ALL and normal cells. Within this project we have evaluated the expression of these miRs by quantitative PCR, in T-ALL and non-T-ALL samples, including both primary cells and cell lines.
Overall, the quantitative analysis done evaluated the expression of 15 out of 16 predicted T-ALL-specific miRs in a total of 79 T-ALL and non-T-ALL samples, and allowed us to identify 13 miRs that are expressed as predicted.
Aim 2) Construction of miRToTALLs:
Using our miRTo prototype as starting material, we have built several miRToTALLs, based on different combinations of the miRs found to be expressed as predicted in T-ALL cells (Aim 1). Combinations of at least 2 different target sites (TS) and up to 5 TS regulating each one of the transgenes have been inserted into miRTo. The ability of these miRToTALLs to specifically and efficiently identify T-ALL cells has been tested in vitro (Aim3).
Upon discussion of our technology with a CDMO specialized in lentiviruses (LVs), we have developed improved miRTo backbone constructs. We are currently building miRToTALLs using these optimized versions of miRTo and the target sites of the miRs validated in Aim1 and used to build the initial miRToTALLs tested in vitro in Aim3.
Aim 3) In vitro testing of miRToTALLs:
To address the efficiency and specificity of the different miRToTALLs to selectively target leukemia cells in vitro, we have performed cultures of T-ALL and non-T-ALL cells transduced with miRToTALL (LVs). Flow cytometry analysis of the reporter gene expression allowed us determining which miRs are functional and targeting leukemia cells. These miR signatures are now being used to build miRToTALLs using as backbone the optimized versions of miRTo construct. These new constructs will to be next be then tested in vitro.
Once the most specific and efficient miRToTALL(s) have been identified, those will be used to test the killing gene.
Aim 4) In vivo testing of miRToTALLs:
Once the new set of miRToTALLs is developed and tested in vitro, we will be able to identify the miRToTALL(s) that selectively target T-ALL cells. These will then be tested in vivo.
Aim 5) miRToTALL project results dissemination and exploitation:
To support the future creation of a spin-off to exploit miRTo technology as a platform for cancer therapy we have developed a detailed business plan.
In addition, we have entered Regional phases for the existent PCT covering the technology platform patent and T-ALL as primary indication. Moreover, we have disseminated the project results through presentations at scientific conferences and relevant biotech and pharma industry trade shows.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our cell death-inducing gene therapy will address the lack of efficiency and specificity of current T-ALL therapies, and therefore the main beneficiaries will be T-ALL patients. However, since our technology can potentially be applied to any cancer type that presents a specific miR expression profile, its social impact may be much broader and significant. Besides the direct impact on patient health and recovery, the value of our therapy will benefit caregivers and health care providers, contributing to substantial reduction of the economic and social burden of cancer.
The successful proof of concept of miRToTALL, in the long-term, will result in a novel treatment for T-ALL. In this regard, the further development and testing of the miRToTALLs generated during this project will be key, for which we have already secured new funds.
Additionally, through the development of miRTo technology we further aim at establishing a cancer gene therapy platform technology with strong socioeconomic impact. As such, throughout this project we have developed a detailed business plan and actively engaged with different stakeholders such as CDMOs, CROs and Venture capitalist investors. Importantly, through these interactions, we have identified the key steps and milestones to support the creation of a spin-off to exploit miRTo technology.