Retroviral Integrations in Gene Therapy Trials

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis

Penalized inference of the hematopoietic cell differentiation network via high-dimensional clonal tracking

Abstract Background During their lifespan, stem- or progenitor cells have the ability to differentiate into more committed cell lineages. Understanding this process can be key in treating certain diseases. However, up until now only limited information about the cell differentiation process is known. Aim The goal of this paper is to present a statistical framework able to describe the cell differentiation process at the single clone level and to provide a corresponding inferential procedure for parameters estimation and structure reconstruction of the differentiation network. Approach We propose a multidimensional, continuous-time Markov model with density-dependent transition probabilities linear in sub-population sizes and rates. The inferential procedure is based on an iterative calculation of approximated solutions for two systems of ordinary differential equations, describing process moments evolution over time, that are analytically derived from the process' master equation. Network sparsity is induced by adding a SCAD-based penalization term in the generalized least squares objective function. Results The methods proposed here have been tested by means of a simulation study and then applied to a data set derived from a gene therapy clinical trial, in order to investigate hematopoiesis in humans, in-vivo. The hematopoietic structure estimated contradicts the classical dichotomy theory of cell differentiation and supports a novel myeloid-based model recently proposed in the literature

Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT

First Case of Patient With Two Homozygous Mutations in MYD88 and CARD9 Genes Presenting With Pyogenic Bacterial Infections, Elevated IgE, and Persistent EBV Viremia

We described for the first time a female patient with the simultaneous presence of two homozygous mutations in MYD88 and CARD9 genes presenting with pyogenic bacterial infections, elevated IgE, and persistent EBV viremia. In addition to defective TLR/IL1R-signaling, we described novel functional alterations into the myeloid compartment. In particular, we demonstrated a defective production of reactive oxygen species exclusively in monocytes upon E. coli stimulation, the inability of immature mono-derived DCs (iDCs) to differentiate into mature DCs (mDCs) and the incapacity of mono-derived macrophages (MDMs) to resolve BCG infection in vitro. Our data do not provide any evidence for digenic inheritance in our patient, but rather for the association of two monogenic disorders. This case illustrates the importance of using next generation sequencing (NGS) to determine the most accurate and early diagnosis in atypical clinical and immunological phenotypes, and with particular concern in consanguineous families. Indeed, besides the increased susceptibility to recurrent invasive pyogenic bacterial infections due to MYD88 deficiency, the identification of CARD9 mutations underline the risk of developing invasive fungal infections emphasizing the careful monitoring for the occurrence of fungal infection and the opportunity of long-term antifungal prophylaxis

ALPS-Like Phenotype Caused by ADA2 Deficiency Rescued by Allogeneic Hematopoietic Stem Cell Transplantation

Adenosine deaminase 2 (ADA2) deficiency is an auto-inflammatory disease due to mutations in cat eye syndrome chromosome region candidate 1 (CECR1) gene, currently named ADA2. The disease has a wide clinical spectrum encompassing early-onset vasculopathy (targeting skin, gut and central nervous system), recurrent fever, immunodeficiency and bone marrow dysfunction. Different therapeutic options have been proposed in literature, but only steroids and anti-cytokine monoclonal antibodies (such as tumor necrosis factor inhibitor) proved to be effective. If a suitable donor is available, hematopoietic stem cell transplantation (HSCT) could be curative. Here we describe a case of ADA2 deficiency in a 4-year-old Caucasian girl. The patient was initially classified as autoimmune neutropenia and then she evolved toward an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype. The diagnosis of ALPS became uncertain due to atypical clinical features and normal FAS-induced apoptosis test. She was treated with G-CSF first and subsequently with immunosuppressive drugs without improvement. Only HSCT from a 9/10 HLA-matched unrelated donor, following myeloablative conditioning, completely solved the clinical signs related to ADA2 deficiency. Early diagnosis in cases presenting with hematological manifestations, rather than classical vasculopathy, allows the patients to promptly undergo HSCT and avoid more severe evolution. Finally, in similar cases highly suspicious for genetic disease, it is desirable to obtain molecular diagnosis before performing HSCT, since it can influence the transplant procedure. However, if HSCT has to be performed without delay for clinical indication, related donors should be excluded to avoid the risk of relapse or partial benefit due to a hereditary genetic defect

Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction

Wiskott–Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients

WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells

A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4+CD25+FOXP3+ natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS−/− mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS−/− nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS−/− nTreg cells failed to proliferate and to produce transforming growth factor β upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS−/− nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS−/− nTreg cells showed reduced in vitro suppressor activity on both WT and WAS−/− effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4+ effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients