IS-Seq: a bioinformatics pipeline for integration sites analysis with comprehensive abundance quantification methods.

BACKGROUND: Integration site (IS) analysis is a fundamental analytical platform for evaluating the safety and efficacy of viral vector based preclinical and clinical Gene Therapy (GT). A handful of groups have developed standardized bioinformatics pipelines to process IS sequencing data, to generate reports, and/or to perform comparative studies across different GT trials. Keeping up with the technological advances in the field of IS analysis, different computational pipelines have been published over the past decade. These pipelines focus on identifying IS from single-read sequencing or paired-end sequencing data either using read-based or using sonication fragment-based methods, but there is a lack of a bioinformatics tool that automatically includes unique molecular identifiers (UMI) for IS abundance estimations and allows comparing multiple quantification methods in one integrated pipeline. RESULTS: Here we present IS-Seq a bioinformatics pipeline that can process data from paired-end sequencing of both old restriction sites-based IS collection methods and new sonication-based IS retrieval systems while allowing the selection of different abundance estimation methods, including read-based, Fragment-based and UMI-based systems. CONCLUSIONS: We validated the performance of IS-Seq by testing it against the most popular  analytical workflow available in the literature (INSPIIRED) and using different scenarios. Lastly, by performing extensive simulation studies and a comprehensive wet-lab assessment of our IS-Seq pipeline we could show that in clinically relevant scenarios, UMI quantification provides better accuracy than the currently most widely used sonication fragment counts as a method for IS abundance estimation.

High-throughput analysis of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing.

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) has shown clear neurological benefit in rare diseases, which is achieved through the engraftment of genetically modified microglia-like cells (MLCs) in the brain. Still, the engraftment dynamics and the nature of engineered MLCs, as well as their potential use in common neurogenerative diseases, have remained largely unexplored. Here, we comprehensively characterized how different routes of administration affect the biodistribution of genetically engineered MLCs and other HSPC derivatives in mice. We generated a high-resolution single-cell transcriptional map of MLCs and discovered that they could clearly be distinguished from macrophages as well as from resident microglia by the expression of a specific gene signature that is reflective of their HSPC ontogeny and irrespective of their long-term engraftment history. Lastly, using murine models of Parkinson's disease and frontotemporal dementia, we demonstrated that MLCs can deliver therapeutically relevant levels of transgenic protein to the brain, thereby opening avenues for the clinical translation of HSPC-GT to the treatment of major neurological diseases.

Interferon gene therapy reprograms the leukemia microenvironment inducing protective immunity to multiple tumor antigens.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing.

Convergent mutations and kinase fusions lead to oncogenic STAT3 activation in anaplastic large cell lymphoma.

A systematic characterization of the genetic alterations driving ALCLs has not been performed. By integrating massive sequencing strategies, we provide a comprehensive characterization of driver genetic alterations (somatic point mutations, copy number alterations, and gene fusions) in ALK(-) ALCLs. We identified activating mutations of JAK1 and/or STAT3 genes in ∼20% of 88 [corrected] ALK(-) ALCLs and demonstrated that 38% of systemic ALK(-) ALCLs displayed double lesions. Recurrent chimeras combining a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were also discovered in WT JAK1/STAT3 ALK(-) ALCL. All these aberrations lead to the constitutive activation of the JAK/STAT3 pathway, which was proved oncogenic. Consistently, JAK/STAT3 pathway inhibition impaired cell growth in vitro and in vivo.