Projects / Programmes
Development of anti-inflammatory regulatory T cells (Treg) for therapy of autoimmune diabetes using designed transcriptional factors and CRISPR/Cas9 system
| Code |
Science |
Field |
Subfield |
| 3.07.00 |
Medical sciences |
Metabolic and hormonal disorders |
|
| Code |
Science |
Field |
| B000 |
Biomedical sciences |
|
| Code |
Science |
Field |
| 3.02 |
Medical and Health Sciences |
Clinical medicine |
diabetes type 1, reprograming of regulatory T cells, designed transcriptional regulators, CRISPR/Cas9; autoimmunity
methods,
Researchers (16)
Organisations (3)
Abstract
Diabetes mellitus type 1 (T1D) is a chronic metabolic and immune disease, characterized by hyperglycaemia, resulting from impaired insulin secretion by the destruction of beta cells of the pancreas, sensitivity to insulin action or both. Diabetes and its complications have become a major public health problem in all countries causing physical morbidity, premature mortality and a heavy burden on health services. Although insulin administration is lifesaving for T1D patients, this therapy does not cure the disease. Therefore, research aimed at developing novel approaches to therapies for curing the disease are well justified. One such direction of therapy that is already in clinical trials phase I is aimed at shifting the balance from pro-inflammatory conventional T-cells (Tconv) to increased pool of immuno-suppressing regulatory T-cells (Tregs). However, no such approach or trial has yet resulted in clinical remission and few have had significant impact on halting the progression of T1D.
Here we propose an innovative strategy towards development of a therapy for T1D combining novel technological advances of synthetic biology and state of the art knowledge of immunology and clinical diabetology by the members of this project consortium. Recent emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases provides extremely powerful and precise tools for genome engineering but also offers another safer use by regulation of the endogenous gene expression. In contrast to gene therapy methods, with potential risks side effects of the genome editing, our proposal is based on the design of transcriptional regulators based on fusion of potent transcriptional effector domains (e.g. VPR transcriptional activation domain) to catalytically inactivated Cas9 (dCas9) domain. They will be designed to induce or suppress the transcription of the target genes without risks of genome editing. We propose to reprogram Tconv into immunosuppressing Tregs by the use of dCas9-based designed transcription regulators to upregulate key endogenous transcription factors that underlay stable, therapeutically relevant Treg phenotype. Project will include three main stage: i) design, selection and validation of the sgRNAs to upregulate transcription of genes for Treg reprogramming, ii) identification of the effective combination of reprogramming factors to induce switch Tconv towards Treg-specific phenotype and iii) isolation and reprogramming of Tconv from blood of patients with T1D and their reprogramming into therapeutic Tregs. SgRNAs will be optimized for increased transcription in combination with cCas9-VPR for each of the up to 10 reprogramming transcription factors identified by bioinformatics methods. After optimization of the upregulation of transcription of each of the TFs the optimal combination will be identified to obtain the transcriptional profile and maintain the stable Treg phenotype, which will be finally tested on real patient cells isolated from blood.
The project group has expertise and strong track record in designed transcription factor regulators (Gaber et al., Nature Chem. Biol. 2014, Lebar et al., Nature Commun. 2014). We improved the degree of transcriptional activation by more than 1000 fold (Lebar et al., ACS SynBio 2016 and patent pending). We have already demonstrated up- regulation of gene expression of some Treg reprogramming genes (e.g. FoxP3) as well as dCas9 protein transduction and human cell delivery. The ultimate goal of the project is very ambitions but feasible, based on the expertise of the project team in immunology, metabolism, bioinformatics and synthetic biology and encouraging preliminary results. This proposal is a proof of principle study to identify key regulators to reprogram Tconv into Treg population in cell lines and primary cells from T1D patients to provide a platform for clinical trials in this and other autoimmune diseases. Our proposed approach can also be applied
Significance for science
Advancement of Basic Research
The major idea behind the proposed study is to identify target genes and their combinations to develop T-reg inducing proteins using CRISPR/Cas9 system linked with effector activators of transcription. Final objective is to use this newly developed basic knowledge and tools to re-programme conventional T-cells isolated from diabetic patient’s PBMC into therapeutic regulatory T-cells. Published studies on Treg differentiation have already identified several genes and agents that induce Treg-like propagation and development in vitro or in animal models but the definitive list of essential master genes for stable trans-differentiation of conventional T cells into Tregs has not yet been identified. In this respect we can contribute new basic scientific knowledge to the field. Our experiments should also provide advancement of knowledge on how immune system and inflammatory components are intertwined in the Treg trans-differentiation process and how this impacts development or halting of autoimmune diabetes.
Development of new genetic resources.
One important deliverable of this project is development of novel genetic resources. Under objectives 1-3 we will develop DNA constructs targeting several genes involved in Treg differentiation process. These genetic resources will all be done for human genes. They will not only be useful for the study proposed herein but also for future clinical work in our or other groups. In vitro and ex vivo resources developed will be valuable for various metabolic-immunity type studies connected to autoimmune diabetes and possibly for other autoimmune disease where certain genes that we will target also play an important role (e.g. TGF-beta, IL2…).
Development of New Technologies.
Although TALE and CRISPR/Cas9 studies have dominated the field of genome engineering in recent years, most studies used this technology to modify the genome by deleting, inserting or replacing gene elements. We will focus entirely on developing new tools using CRISPR/Cas9 systems to regulate endogenous gene expression by activating or repressing them. This approach may provide novel and an optimized solution for efficient regulation of multiple endogenous genes with a potential for different clinical applications.
Significance for the country
We expect that our study will provide results directly relevant to clinic to introduce novel approach in treating autoimmune diabetes, which is now recognised as one of the most common chronic diseases of childhood. This could lead to early and more targeted development of therapies with a consequence to increase of the quality of life of patients that currently rely on daily administration of insulin. Our ex vivo studies on blood cells from diabetic patients should yield important new insights into the possibility of increasing the pool of therapeutic Treg cells with possible implications for the development of new treatments in the future. Our study could provide a basis for collaboration with some pharmaceutical companies to develop a scale up protocols to reprogram patients’ conventional T cells into stable and long-lived therapeutic Treg population.
Most important scientific results
Final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
