Romito, M. | bioRxiv Science

bioRxiv2025

Modification and validation of a reference real-time RT-PCR method for the detection of a new African horse sickness virus variant

African horse sickness (AHS) is a disease affecting equids caused by the AHS virus (AHSV). The World Organization for Animal Health (WOAH) includes AHS as a notifiable disease and, upon detection within the European Union, immediate control and eradication measures are mandated. Thus, validated diagnostic methods for rapid AHSV detection are essential. The Aguero 2008 and Guthrie 2013 rRT-PCR methods have been widely validated for detection of any AHSV strain and are included as reference rRT-PCRs in the WOAH manual. However, the WOAH reference laboratory for AHS in the Republic of South Africa (RSA), reported an AHSV variant undetected by the Aguero 2008 rRT-PCR. Therefore, a set of modified primers and probe, containing degenerate positions to avoid mismatches with the sequence of the new RSA strain, was developed. The modified-Aguero method was validated by the WOAH reference laboratories in Spain and the UK employing a broad collection of AHSV strains, clinical samples as well as a synthetic RNA mimicking the target sequence of the new RSA AHSV variant (AHSV-sRNA-RSA). Comparative assessment of the modified-Aguero versus the WOAH reference rRT-PCRs, showed that the modified method exhibited a good diagnostic performance and enabled the detection of the new RSA AHSV variant nucleic acid.

microbiology↗

bioRxiv2023

CRISPR/Cas9-based disease modelling and functional correction of Interleukin 7 Receptor alpha Severe Combined Immunodeficiency in T-lymphocytes and hematopoietic stem cells

Interleukin 7 Receptor Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors proliferation and differentiation during lymphopoiesis resulting in absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukaemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modelling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knock-in strategy that targets IL7RA exon 1 and introduces via homology directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signalling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

molecular biology↗