Joanna Sinclair

Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector

BACKGROUND X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain (gamma(c)), resulting in disruption of development of T lymphocytes and natural-killer cells. B-lymphocyte function is also intrinsically compromised. Allogeneic bone-marrow transplantation is successful if HLA-matched family donors are available, but HLA-mismatched procedures are associated with substantial morbidity and mortality. We investigated the application of somatic gene therapy by use of a gibbon-ape-leukaemia-virus pseudotyped gammaretroviral vector. METHODS Four children with SCID-X1 were enrolled. Autologous CD34-positive haemopoietic bone-marrow stem cells were transduced ex vivo and returned to the patients without preceding cytoreductive chemotherapy. The patients were monitored for integration and expression of the gamma(c) vector and for functional immunological recovery. FINDINGS All patients have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two. No serious adverse events have been recorded. T cells responded normally to mitogenic and antigenic stimuli, and the T-cell-receptor (TCR) repertoire was highly diverse. Where assessable, humoral immunity, in terms of antibody production, was also restored and associated with increasing rates of somatic mutation in immunoglobulin genes. INTERPRETATION Gene therapy for SCID-X1 is a highly effective strategy for restoration of functional cellular and humoral immunity.

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+) T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

Capture and generation of adenovirus specific T cells for adoptive immunotherapy

Adenoviral infections represent a major cause of morbidity and mortality following haematopoietic stem cell transplantation. Current anti‐viral agents are virostatic and it is evident that elimination of adenovirus (ADV) infection is only achieved by recovery of cellular immunity. Using an interferon‐gamma (IFN‐γ) secretion and capture assay to isolate ADV‐specific T cells, followed by a 2 week expansion and restimulation protocol, we generated ADV T cells that may be used for cellular immunotherapy. In contrast to virus‐specific T cells for cytomegalovirus or Epstein‐Barr virus, the ADV response was dominated by CD4+ T cells and the majority of captured cells exhibited an effector/memory immunophenotype. Highly specific antigen responses were demonstrated by intracellular IFN‐γ expression and cytotoxicity assays when the expanded cells underwent restimulation with ADV‐pulsed target cells. Although T cells were initially generated in response to ADV species C, the expanded populations also showed strong activity against ADV species B, suggesting cross‐reactivity across ADV species; a finding that has important clinical consequences in the paediatric setting, where the majority of infections are caused by ADV type B and C. The protocols can be readily translated to generate ADV‐specific T cells suitable for clinical use and offer an effective immunotherapeutic strategy to control ADV infection.

Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitro

The transforming growth factor‐β‐related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta‐gonad‐mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34+ cells with different levels of c‐Kit expression and multipotency were identified. CD34+/c‐Kithigh cells express CD45 and are haematopoietic‐restricted progenitors. In contrast, CD34+/c‐Kitlow cells are Flk1+/CD45neg and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34+/c‐Kitlow cell pool within 48 h, via a combination of down modulation of the c‐Kit receptor in CD34+/c‐Kithigh cells and proliferation. In long‐term culture, BMP4 increased the growth/survival of CD34+/c‐Kithigh haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34+/c‐Kithigh progenitors cultured with BMP4 also generated adherent colonies typical of c‐Kitlow cells. These results suggest that BMP4 regulates c‐Kit expression and differentiation potential in CD34+ AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche.

Impaired neutrophil migration and phagocytosis in IRAK-4 deficiency

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