Projects

Gene editing

Cancer therapeutics

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of genome function. Using this system, DNA sequences within the endogenous genome and their functional outputs can be easily edited or modulated. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes . Recently, application of CRISPR/Cas9 system has become popular for therapeutic aims such as gene therapy.

We are using this platform to define a map of druggable genetic dependencies/essentialities across a number of selected cell lines; some of which are expected to be clinically actionable with currently available small molecules or other drugs. In parallel, we are seeking to understand molecular pathways that control expression of proteins involved in carcinogenesis and/or immune system evasion by tumours.

Cellular Therapeutics

Screening and gene editing technologies are used to screened for therapeutic modulation targets in human primary cells. CSHM scientists are looking for targets to control activation of immune responses in disease settings such as cancer. Primary cell screening, includes isolation of cells of interest of hematopoietic or non-hematopoietic origin and in vitro culturing and/or expansion. The ability to scale-up the expansion of cells is a major challenge in cell therapy. Scientists are establishing a variety of culturing protocols to support the expansion of various cell types. Currently, there is a focus on conventional T cells, regulatory T cells and NK cells.

Epigenetic modulation for the generation of innovative cellular immunotherapies against Graft versus Host Disease (EPICELL):

There is an urgent need for novel therapeutic strategies for graft versus host disease (GvHD) occurring after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whilst hypomethylating agents, such as Aza (5-azacitidine), have been shown to ex vivo generate immunoregulatory T cells, the major caveat in the use of these FOXP3+ regulatory T cells (Tregs) in clinical practice is the lack of specific surface markers for efficient purification. As indicated by our preliminary results, genes other than FOXP3 are responsible for the suppressor function of Aza -induced Tregs. We have showed that short in vitro treatment of T cells with Aza induces immunosuppressive HLA-G expressing T cells. The ultimate goal of the EPICELL consortium, in a research collaboration, is to develop a novel, easily generated and easily purified cellular immunotherapy against GvHD. Towards to this goal we plan to: a) Induce maximum and stable HLA-G expression and suppressor function in human T cells in vitro and test them under GvHD-like conditions, b) Generate clinical scale and clinical grade HLA-G+ Tregs, and c) Cryopreserve in vitro Aza-induced HLA-G+ suppressor cells, under GMP conditions, which will upon clinical trial approval be used for the in vivo investigation in acute GvHD. We anticipate that through EPICELL, we will ex vivo produce a new category of inducible regulatory T cells, the HA-induced HLA-G+ Tregs, with the perspective to enter clinical practice and be used as adoptive cellular therapy for GvHD and other T cell mediated diseases.

Genomics

The importance of genomic data in medicine has been ever increasing especially in the field of cancer biology and treatment decision for cancer patients. The use of next-generation sequencing (NGS) technologies has been a revolutionizing advancement in providing such information and at a timely manner for use in the clinic.

CSHM scientists are working to provide a high-throughput genomics platform for the biosciences community. This will include support and experimental design and advice, NGS library preparation methods, DNA extraction, long-read sequencing, long mate-pair library construction and exome capture, analysis from transcriptomics to exome sequencing, genome assembly, genome annotation and metagenomics.

NGS can often create large datasets that pose a computational challenge. The Center will provide a genome analytics solution by enabling high-performance computing support.

Retrospective Analsyis of Clonal Transformation (RETROCLONE):

The RETROCLONE proposal involves the analysis of the BCR repertoire and genetic aberrations of at least 50 patients with a B-LPDs and their comparison with a matched paired sample of the same patient, collected in an average 10 years prior to diagnosis (when the patient was registered as a volunteer bone marrow donor at Karaiskakio Foundation, PA1). Patients follow up samples at several time points will be also analysed and results will be compared with diagnosis and pre-diagnosis time point. Results will be also paralleled to paired samples from age matched normal controls and patients with MBL. The unique opportunity of CSHM / Karaiskakio Foundation to access samples of patients collected many years prior to their diagnosis and post diagnosis with a haematological malignancy provides a distinctive opportunity to carry out a Retrospective Analsyis of Clonal Transformation (RETROCLONE) events involved in leukogenesis. We hypothesize that one or more populations of pre-leukemic clones with distinct BCR sequences many years prior to diagnosis, evolve distinctively based on a sequence of specific mutation events and emerged at presentation as the predominant malignant clone. Our aims are a) to apply a novel NGS method coupled with network construction and population analysis of the BCR rearrangement sequences, in B-LPDs diagnostic, pre-diagnostic and post-diagnosis samples, b) test the Somatic Hypermutation (SHM) status and B-cell immunoglobulin stereotypy, c) Investigate gene mutation evolution in B-LPDs applying NGS sequencing for genes correlated with LPDs and d) assess the expression of crucial markers for LPDs prognosis in order to identify their association with pre-diagnostic clone characteristics. The outcome of this top quality high-impact project is expected to shed light on the mechanisms of disease evolution. This information will be of critical importance for improving patients’ monitoring and clinical outcome.

Bioinformatics

The development and widespread use of multi-omics has made imperative the use of bioinformatics. Bioinformatics allow scientists to gain a deeper and wider understanding of biological processes by analysing big datasets that are otherwise impossible to analyse. Major research efforts in the field include DNA/RNA/protein sequencing, sequence alignment, gene finding, genome assembly, drug design, drug discovery, protein structure alignment and prediction, prediction of gene expression and protein-protein interactions, genome-wide association studies, and the modelling of evolution. Equipped with advanced computing instruments and software, CSHM scientists will provide support to the broader research community of the region.

PDX models

In collaboration with local and international partners and taking advantage of its close association with its parent organisation Karaiskakio Foundation, the CSHM is working on establishing xenograft tumour models from patient-derived tumour tissue (PDTT). Patient-derived xenografts are believed to offer relevant predictive insights into clinical outcomes when evaluating the efficacy of novel therapies.