Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb.

BACKGROUND: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. METHODS: In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. RESULTS: We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. CONCLUSION: This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.

Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.

Treatment of an infant with X-linked severe combined immunodeficiency (SCID-X1) by gene therapy in Australia.

OBJECTIVE: To report the outcome of gene therapy in an infant with X-linked severe combined immunodeficiency (SCID-X1), which typically causes a lack of T and natural killer (NK) cells. DESIGN AND SETTING: Ex-vivo culture and gene transfer procedures were performed at The Children's Hospital at Westmead, Sydney, NSW, in March 2002. Follow-up to March 2005 (36 months) is available. PATIENT: A 9-month-old male infant with confirmed SCID-X1 (including complete absence of T cells) with an NK+ phenotype (a less common variant of SCID-X1), and no HLA-identical sibling donor available for conventional bone marrow transplantation. PROCEDURE: CD34+ haemopoietic progenitor cells were isolated from harvested bone marrow and cultured with cytokines to stimulate cellular replication. Cells were then genetically modified by exposure to a retrovirus vector encoding human gamma c (the common gamma chain of several interleukin receptors; mutations affecting the gamma c gene cause SCID-X1). Gene-modified cells (equivalent to 1.3 x 10(6) CD34+/gamma c+ cells/kg) were returned to the infant via a central line. RESULTS: T cells were observed in peripheral blood 75 days after treatment, and levels increased rapidly to 0.46 x 10(9) CD3+ cells/L at 5 months. Within 2 weeks of the appearance of T cells, there was a distinct clinical improvement, with early weight gain and clearance of rotavirus from the gut. However, T-cell levels did not reach the reference range, and immune reconstitution remained incomplete. The infant failed to thrive and developed weakness, hypertonia and hyperreflexia in the legs, possibly the result of immune dysregulation. He went on to receive a bone marrow transplant from a matched unrelated donor 26 months after gene therapy. CONCLUSIONS: This is the first occasion that gene therapy has been used to treat a genetic disease in Australia. Only partial immunological reconstitution was achieved, most likely because of the relatively low dose of gene-corrected CD34+ cells re-infused, although viral infection during the early phase of T-cell reconstitution and the infant's NK+ phenotype may also have exerted an effect.

Differential subcellular localization of CD86 in human PBMC-derived macrophages and DCs, and ultrastructural characterization by immuno-electron microscopy.

We have previously reported the presence of a discrete reservoir of the costimulatory molecule CD86 in the cytoplasm of human monocytes freshly isolated from peripheral blood mononuclear cells (PBMC). In the current study, we have extended analysis of the subcellular localization of this molecule to in vitro PBMC-derived dendritic cells (DCs) and macrophages. In a sub-population of DCs, we observed by confocal microscopy an intracellular focal concentration of CD86 that bore striking similarities to that previously reported in monocytes. Further analyses revealed that this intracellular CD86 was not localized to the Golgi apparatus, MHC II compartments or endocytic structures, and required intact microtubules to retain structural integrity. A similar concentration of CD86 was not present in PBMC-derived macrophages. Electron microscopy revealed two distinct DC phenotypes containing either sparse or abundant cytoplasmic vesicles, and CD86 was found to be concentrated within the vesicular compartment of this latter phenotype. Collectively, these data not only identify and characterize a novel CD86-containing cytoplasmic compartment in human PBMC-derived DCs, but also define micro-structurally distinct DC subsets that differentially concentrate CD86 within cytoplasmic vesicles. Although the functional significance of these observations remains to be established, available evidence supports the conclusion that the focal concentration of CD86 is a storage reservoir that facilitates rapid deployment of this molecule to the DC surface when increased costimulatory capacity is required.

A novel splice-site mutation in the common gamma chain (gammac) gene IL2RG results in X-linked severe combined immunodeficiency with an atypical NK+ phenotype.

Mutations in the gene encoding the common gamma chain (gammac) of interleukin receptors 2, 4, 7, 9, 15 and 21 result in X-linked severe combined immunodeficiency (SCID-X1). Classically, this disease is characterised by an absence of T and NK cells, and near normal numbers of functionally deficient B cells (B(+), T(-), NK(-) phenotype). Atypical phenotypes have also been described, but relatively little is known about the mechanisms by which the underlying mutations impair gammac-dependent interleukin receptor signalling to produce these disease variants. Here we describe a novel splice-site mutation resulting in the presence of near normal numbers of functionally deficient NK cells (B(+), T(-), NK(+) phenotype), in a SCID-X1 infant who was subsequently treated by gene therapy. The mutation, c.468+3A>C affecting the third base of intron 3 in the IL2RG gene, was shown to result in the production of two aberrantly spliced gammac mRNA species and reduction of correctly spliced message to trace levels, consistent with failure to detect gammac on the surface of B and NK cells by FACS analysis. The infant's genotype-phenotype correlation supports the hypothesis that interleukin 15 receptor-mediated signalling is preferentially retained as the amount of cell surface gammac becomes limiting. The possible implications for immunological reconstitution following gene therapy are also discussed.

Promoter interference mediated by the U3 region in early-generation HIV-1-derived lentivirus vectors can influence detection of transgene expression in a cell-type and species-specific manner.

In a previous study using an early-generation VSV-G-pseudotyped lentivirus vector encoding enhanced green fluorescent protein (EGFP) under the transcriptional control of a human cytomegalovirus (CMV) immediate-early promoter, we examined transduction efficiency in dissociated dorsal root ganglia (DRG) cultures. In cultures of murine origin, transgene expression was observed solely in the sensory neurons with the stromal cell population failing to show evidence of transduction. In contrast, efficient and sustained transduction of both sensory neurons and the stromal cell population was observed in cultures of human origin. Given the widespread use of murine models in preclinical gene therapy studies, in the current study we investigated the basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures. The interspecies differences persisted at high multiplicities of infection, and irrespective of whether lentiviral vector stocks were packaged in the presence or absence of human immunodeficiency virus type 1 (HIV-1) accessory proteins. Cell-type specificity of CMV promoter expression, tropism of the VSV-G envelope, and blocks to molecular transduction were also precluded as possible mechanisms, thereby implicating transcriptional repression of the internal heterologous promoter. This promoter interference effect was found to be mediated by cis-acting sequences upstream of the core promoter elements located in the U3 region of the proviral long terminal repeats (LTRs). Deletion of this region, as in late-generation self-inactivating (SIN) lentivirus vectors, relieves this effect. This provides a basis for reevaluating data produced using early-generation U3-bearing lentivirus vectors and for reconciling these with results obtained using more contemporary SIN lentivirus vectors carrying a U3 deletion.

HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno-inhibitory phenotype through up-regulation of indoleamine 2,3-dioxygenase activity.

The mechanisms by which tumour cells escape recognition by the immune system or subvert antitumour effector responses remain poorly understood. In the course of investigating the potential of costimulatory signals in anticancer immunotherapy strategies, we have observed that HeLa cells (a human cervical carcinoma cell line) cocultured with peripheral blood lymphocytes (PBL) acquire the capacity to inhibit PBL proliferation in response to interleukin-2 (IL-2). This immuno-inhibitory phenotype was further shown to result from induction of the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO), by interferon-gamma (IFN-gamma) secreted from cocultured allo-reactive PBL. This enzyme has recently been shown to be a critically important modulator of immunological responses, most notably through the capacity to protect allogeneic concepti from alloreactive maternal lymphocytes. While the cytostatic consequences of IDO activity in tumour cells has received attention, the data presented in this report support the hypothesis that IDO activity may also act to impair antitumour immune responses.

The adenovirus E4 ORF6 and E1b 55 kDa proteins cooperate in a p53-independent manner to enhance transduction by recombinant adeno-associated virus vectors.

The observation that exposure of target cells to genotoxic stress or adenovirus infection enhances recombinant adeno-associated virus (rAAV) transduction is an important lead towards defining the rAAV transduction mechanism, and has significant implications for the exploitation of rAAV in gene therapy applications. The adenovirus-mediated enhancement of rAAV transduction has been mapped to the E4 ORF6 gene, and expression of E4 ORF6 alone has been considered necessary and sufficient to mediate this effect. Since p53 subserves an important function in the cellular response to genotoxic stress, and interacts with the E4 ORF6 gene product during adenovirus infection, we hypothesized that p53 function might be essential to the rAAV enhancement resulting from these cellular insults. In the current study, using the p53-null cell lines H1299 and Saos-2, we find that p53 is not essential to either genotoxic stress or adenovirus-mediated enhancement of rAAV transduction. We further demonstrate using HeLa, H1299 and Saos-2 cells that E4 ORF6 expression alone is not sufficient to enhance rAAV transduction and that coexpression of the adenovirus E1b 55 kDa protein is necessary. Together, these observations indicate that the mechanism by which adenovirus infection enhances rAAV transduction involves cooperative and interdependent functions of the E4 ORF6 and E1b 55 kDa proteins that are p53-independent.