531. Computational Pipeline for the Identification of Integration Sites and Novel Method for the Quantification of Clone Sizes in Clonal Tracking Studies

Gene-corrected cells in Gene Therapy (GT) treated patients can be tracked in vivo by means of vector integration site (IS) analysis, since each engineered clone becomes univocally and stably marked by an individual IS. As the proper IS identification and quantification is crucial to accurately perform clonal tracking studies, we designed a customizable and tailored pipeline to analyze LAM-PCR amplicons sequenced by Illumina MiSeq/HiSeq technology. The sequencing data are initially processed through a series of quality filters and cleaned from vector and Linker Cassette (LC) sequences with customizable settings. Demultiplexing is then performed according to the recognition of specific barcodes combination used upon library preparation and the sequences are aligned to the reference genome. Importantly, the human genome assembly Hg19 is composed of 93 contigs, among which the mitochondrial genome, unlocalized and unplaced contigs and some alternative haplotypes of chr6. While previous approaches aligned IS sequences only to the standard 24 human chromosomes, using the whole assembled genome allowed improving alignment accuracy and concomitantly increased the amount of detectable ISs. To date, we have processed 28 independent human sample sets retrieving 260,994 ISs from 189,270,566 sequencing reads. Although, sequencing read counts at each IS have been widely used to estimate the relative IS abundance, this method carries inherent accuracy constraints due to the rounds of exponential amplification required by LAM-PCR that might generate unbalances on the original clonal representation. More recently, a method based on genomic sonication has been proposed exploiting shear site counts to tag the number of original fragments belonging to each IS before PCR amplification. However, the number of cells composing a given clone could far exceed the number of fragments of different lengths that can be generated upon fragmentation in proximity of that given IS. This would rapidly saturate the available diversity of shear sites and progressively generate more and more same-site shearing on independent genomes. In order to overcome the described biases and reliably quantify ISs, we designed and tested a new LC encoding random barcodes. The new LC is composed of a known sequence of 29nt used as binding site for the primers upon amplification steps, a 6nt-random barcode, a fixed-anchor sequence of 6nt, a second 6nt-random barcode and a final known sequence of 22nt containing sticky ends for the three main restriction enzymes in use (MluI, HpyCH4IV and AciI). This peculiar design allowed increasing the accuracy of clonal diversity estimation since the fixed-anchor sequence acts as a control for sequencing reliability in the barcode area. The theoretical number of different available barcodes per clone (412=16,777,216) far exceeds the requirements for not saturating the original diversity of the analyzed sample (on average composed by around 50.000 cells). We validated this novel approach by performing assays on serial dilutions of individual clones carrying known ISs. The precision rate obtained was averagely around 99.3%, while the worst error rate reaches at most the 1.86%, confirming the reliability of IS quantification. We successfully applied the barcoded-LC system to the analysis of clinical samples from a Wiskott Aldrich Syndrome GT patient, collecting to date 50,215 barcoded ISs from 94,052,785 sequencing reads

Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.</p

Laboratory and beam test results of TOFFEE ASIC and ultra fast silicon detectors

In this report we present measurements performed on the full custom ASIC TOFFEE, designed to pre-amplify and discriminate signals of Ultra Fast Silicon Detectors. The ASIC has been characterized in laboratory with custom test boards, and with infrared laser light hitting the sensor emulating a minimum ionizing particle signal. Laser measurements show that a jitter term better than 50 (40) ps is achievable with a 10 (12) fC input charge.We also present some preliminary results on the TOFFEE performances, as obtained during recent beam tests with a 180 GeV/c pion beam, on the SPS-H8 beam line at CERN.Peer Reviewe

The future of small farms and small food businesses as actors in regional food security: A participatory scenario analysis from Europe and Africa

This research was supported by the `Small Farms, Small Food Businesses and Sustainable Food Security' (SALSA) project, which has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 677363. Funding for open access charge: CRUE-Universitat Politecnica de Valencia.Ortiz Miranda, D.; Moreno-Pérez, OM.; Arnalte-Mur, L.; Cerrada-Serra, P.; Martinez Gomez, VD.; Adolph, B.; Atela, J.... (2022). The future of small farms and small food businesses as actors in regional food security: A participatory scenario analysis from Europe and Africa. Journal of Rural Studies. 95:326-335. https://doi.org/10.1016/j.jrurstud.2022.09.0063263359

Online proton therapy monitoring: Clinical test of a Silicon-photodetector-based in-beam PET

Particle therapy exploits the energy deposition pattern of hadron beams. The narrow Bragg Peak at the end of range is a major advantage but range uncertainties can cause severe damage and require online verification to maximise the effectiveness in clinics. In-beam Positron Emission Tomography (PET) is a non-invasive, promising in-vivo technique, which consists in the measurement of the β+ activity induced by beam-tissue interactions during treatment, and presents the highest correlation of the measured activity distribution with the deposited dose, since it is not much influenced by biological washout. Here we report the first clinical results obtained with a state-of-the-art in-beam PET scanner, with on-the-fly reconstruction of the activity distribution during irradiation. An automated time-resolved quantitative analysis was tested on a lacrimal gland carcinoma case, monitored during two consecutive treatment sessions. The 3D activity map was reconstructed every 10 s, with an average delay between beam delivery and image availability of about 6 s. The correlation coefficient of 3D activity maps for the two sessions (above 0.9 after 120 s) and the range agreement (within 1 mm) prove the suitability of in-beam PET for online range verification during treatment, a crucial step towards adaptive strategies in particle therapy

Long-term and real-world safety and efficacy of retroviral gene therapy for adenosine deaminase deficiency

Adenosine deaminase (ADA) deficiency leads to severe combined immunodeficiency (SCID). Previous clinical trials showed that autologous CD34+ cell gene therapy (GT) following busulfan reduced-intensity conditioning is a promising therapeutic approach for ADA-SCID, but long-term data are warranted. Here we report an analysis on long-term safety and efficacy data of 43 patients with ADA-SCID who received retroviral ex vivo bone marrow-derived hematopoietic stem cell GT. Twenty-two individuals (median follow-up 15.4 years) were treated in the context of clinical development or named patient program. Nineteen patients were treated post-marketing authorization (median follow-up 3.2 years), and two additional patients received mobilized peripheral blood CD34+ cell GT. At data cutoff, all 43 patients were alive, with a median follow-up of 5.0 years (interquartile range 2.4-15.4) and 2 years intervention-free survival (no need for long-term enzyme replacement therapy or allogeneic hematopoietic stem cell transplantation) of 88% (95% confidence interval 78.7-98.4%). Most adverse events/reactions were related to disease background, busulfan conditioning or immune reconstitution; the safety profile of the real world experience was in line with premarketing cohort. One patient from the named patient program developed a T cell leukemia related to treatment 4.7 years after GT and is currently in remission. Long-term persistence of multilineage gene-corrected cells, metabolic detoxification, immune reconstitution and decreased infection rates were observed. Estimated mixed-effects models showed that higher dose of CD34+ cells infused and younger age at GT affected positively the plateau of CD3+ transduced cells, lymphocytes and CD4+ CD45RA+ naive T cells, whereas the cell dose positively influenced the final plateau of CD15+ transduced cells. These long-term data suggest that the risk-benefit of GT in ADA remains favorable and warrant for continuing long-term safety monitoring. Clinical trial registration: NCT00598481 , NCT034786