business opportunities. In fact, several economic studies have already demonstrated a huge potential for job creation, innovation and growth . Through the NAVIGATOR project it is expected that

Prenatal diagnosis is currently performed using fetal genetic material that is sampled by invasive obstetric procedures with an inherent risk for fetal loss. The discovery of cell-free fetal DNA in maternal plasma opened up new avenues in the fetal diagnostics by facilitating the option of safer testing with maternal blood. The aim of the project is the development of a non-invasive method for early prenatal diagnosis of fetuses at-risk for beta-globinopathies, a group of severe hereditary anaemias of global significance. The project will develop a novel high-density SNP panel (HAPLONID) for the targeted next generation sequencing (NGS) of maternal plasma. A relative variant dosage (RVD) analysis will be developed for determining both the paternal and maternal haplotypes of the fetus. In parallel, droplet digital PCR technology will be used to develop a direct haplotype phasing approach of the maternal and paternal allele. The approach bypasses the need for mutation specific assays and is not dependent on the availability of other family members such as grandparents or other siblings. Therefore, the approach will be universally applicable to other countries and to other monogenic disorders. The implementation of NGS-based SNP detection will initially provide non-invasive prenatal diagnosis (NIPD) for beta-globinopathies. The overall approach will allow, for the first time, coverage of all pregnancies with exclusively non-invasive methods, with scope for IP protection and commercialisation of the HAPLONID. At the same time, the proposed project will provide a model and proof of concept for the development of NIPD assays for other inheritable single-gene diseases.

technologies for the protection of Cultural Heritage environment.

Clinical application of gene therapy for beta-thalassemia is currently based on gene disruption for reactivation of fetal hemoglobin (HbF) or on HBB gene addition, both of which have potential drawbacks for severe mutations. As alternative, specific correction of sufficiently common causative mutations is economically of interest, but is as yet not efficient enough for clinical application when based on homology-mediated precision repair. Employing both TALEN and CRISPR/Cas designer nucleases, we have established proof of concept for homology-independent mutation-specific repair of the exceedingly common beta-thalassemia mutation HBB{IVSI-110(G>A)}, which creates an aberrant HBB splice acceptor site. We have shown that in transgenic cell models and patient-derived primary cells, non-homologous-end-joining (NHEJ)-mediated disruption of the mutation or of its context sequences efficiently restores normal splicing, beta-globin levels and cell morphology. This project will perform preclinical assessment of our TALEN-, CRISPR/Cas- and BE-based mutation-specific therapeutics in primary cells in vitro and in chimeric mouse models, to validate efficacy, safety and long-term repopulation potential of modified cells, and suitability for clinical trials. Critically, the project will directly compare all three platforms with clinically applied disruption-based HbF reactivation (NCT03745287), in order to establish the relative merits and potential for commercialization of our HBB{IVSI-110(G>A)} mutation-specific therapeutics.

Thalassaemia is amongst the commonest single-gene disorders worldwide, and in Cyprus 9 out of 100 persons are carriers for one particular, severe beta-thalassaemia mutation, HBB-IVSI-110(G>A), with a relative carrier frequency of 76% in Cyprus and >20% in many EU countries. We have developed two highly efficient mutation-specific therapies for HBB-IVSI-110(G>A) thalassaemia. The first is based on genome editing by DNA-free delivery of TALE and CRISPR/Cas9 nucleases for specific DNA cleavage of the intronic mutation, so as to achieve destruction of the aberrant splice site and its sequence context, and to restore normal HBB splicing and expression. The second is based on short-hairpin-RNA (shRNA)-mediated knockdown of the aberrant HBB mRNA to boost protein expression from residual correct HBB mRNA, which unexpectedly outperforms gene addition by the GLOBE vector normal phenotype restoration. We have shown efficiency of both innovative approaches in cell lines and primary erythroid cells from HBB-IVSI-110(G>A)-homozygous patients, have submitted specific tools for consideration of patent protection, and with this project aim to refine and validate both approaches towards clinical application. For the genome editing approach, we will test shortlisted nucleases in additional, independent patient samples, will characterise off-target activity by genome-wide next-generation sequencing and established tagging technology, and will fine-tune the biosafety of nuclease delivery. For the shRNA approach, we will modify our lentiviral vectors to achieve cell-stage and tissue-specific shRNA expression and will use extant designer nucleases to direct the therapeutic transgene into the endogenous HBB or an inert (safe-harbour) genome site. Combining both lines of investigation draws synergistically on similar sample processing and analysis methods, allows direct comparison of both approaches and will provide and likely improve efficiency and biosafety data required for the submission of a clinical trial protocol.