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Background Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene ( RAG ) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor. Objective We sought to determine the efficacy of lentiviral vector (LV)–mediated GT in the murine model of OS (Rag2 R229Q/R229Q ) in correcting immunodeficiency and autoimmunity. Methods Lineage-negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild-type or OS-untransduced lineage-negative cells. Immunophenotyping, T-dependent and T-independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed. Results LV-mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild-type bone marrow (BM)−transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T-cell repertoire normalized, and gene-corrected mice responded properly to challenges in vivo . Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus. Conclusions Our data show that LV-mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.

Systemic delivery of lentiviral vector (LV) in immunocompetent mice leads to efficient in vivo cell transduction and expression of the encoded protein under the control of the ubiquitous promoter of human cytomegalovirus (CMV). However, antitransgene immune response results in clearance of transduced cells 4 weeks after injection. T regulatory cells (Tregs), which have been demonstrated to control immune responses in vivo, were tested for their ability to suppress antitransgene response leading to stable long-term expression. Adoptive transfer of natural CD4+CD25+ Tregs (nTregs) isolated from wild type (wt) mice or from transgene tolerant transgenic (tg) mice did not suppress the antitransgene immune response after LV delivery. These data demonstrate that neither increasing the endogenous pool of natural Tregs nor transferring nTregs selected in a transgene-expressing thymus can modulate the immune response and mediate sustained transgene expression. Conversely, adoptive transfer of antigen-presenting cells (APCs) isolated from transgene-tolerant tg mice efficiently reduced the immune response leading to stable LV-encoded protein expression in vivo. Reduction of CD8+ effector T cells was observed in LV-treated mice coinjected with transgene-expressing APCs compared with control mice. These data indicate that antitransgene immune response can be modulated by transgene-expressing APCs possibly through deletion of effector T cells.

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.