Adeno-Associated Virus Vector Gene Delivery Elevates Factor I Levels and Downregulates the Complement Alternative Pathway In Vivo.

The complement system is a key component of innate immunity, but impaired regulation influences disease susceptibility, including age-related macular degeneration and some kidney diseases. While complete complement inhibition has been used successfully to treat acute kidney disease, key unresolved challenges include strategies to modulate rather than completely inhibit the system and to deliver therapy potentially over decades. Elevating concentrations of complement factor I (CFI) restricts complement activation in vitro and this approach was extended in the current study to modulate complement activation in vivo. Sustained increases in CFI levels were achieved using an adeno-associated virus (AAV) vector to target the liver, inducing a 4- to 5-fold increase in circulating CFI levels. This led to decreased activity of the alternative pathway as demonstrated by a reduction in the rate of inactive C3b (iC3b) deposition and more rapid formation of C3 degradation products. In addition, vector application in a mouse model of systemic lupus erythematosus (NZBWF1), where tissue injury is, in part, complement dependent, resulted in reduced complement C3 and IgG renal deposition. Collectively, these data demonstrate that sustained elevation of CFI reduces complement activation in vivo providing proof-of-principle support for the therapeutic application of AAV gene delivery to modulate complement activation.

MatCol: a tool to measure fluorescence signal colocalisation in biological systems.

Protein colocalisation is often studied using pixel intensity-based coefficients such as Pearson, Manders, Li or Costes. However, these methods cannot be used to study object-based colocalisations in biological systems. Therefore, a novel method is required to automatically identify regions of fluorescent signal in two channels, identify the co-located parts of these regions, and calculate the statistical significance of the colocalisation. We have developed MatCol to address these needs. MatCol can be used to visualise protein and/or DNA colocalisations and fine tune user-defined parameters for the colocalisation analysis, including the application of median or Wiener filtering to improve the signal to noise ratio. Command-line execution allows batch processing of multiple images. Users can also calculate the statistical significance of the observed object colocalisations compared to overlap by random chance using Student's t-test. We validated MatCol in a biological setting. The colocalisations of telomeric DNA and TRF2 protein or TRF2 and PML proteins in >350 nuclei derived from three different cell lines revealed a highly significant correlation between manual and MatCol identification of colocalisations (linear regression R2 = 0.81, P < 0.0001). MatCol has the ability to replace manual colocalisation counting, and the potential to be applied to a wide range of biological areas.

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.

Limiting {gamma}c expression differentially affects signaling via the interleukin (IL)-7 and IL-15 receptors.

X-linked severe combined immunodeficiency (SCID-X1) results from mutations in the IL2RG gene, which encodes the common gamma chain (gammac) of the receptors for interleukin (IL)-2, 4, 7, 9, 15, and 21. Affected infants typically lack T and natural killer (NK) cells as a consequence of loss of signaling via the IL-7 receptor (IL-7R) and the IL-15R, respectively. In some infants, however, autologous NK cells are observed despite failure of T-cell ontogeny. The mechanisms by which mutations in gammac differentially impact T- and NK-cell ontogeny remain incompletely understood. We used SCID-X1 patient-derived EBV-transformed B cells to test the hypothesis that the IL-15R-mediated signaling is preferentially retained as gammac expression becomes limiting. Signal transduction via the IL-15R was readily detected in control EBV-transformed B cells, and via the IL-7R when modified to express IL-7Ralpha. Under the same experimental conditions, patient-derived EBV-transformed B cells expressing trace amounts of gammac proved incapable of signal transduction via the IL-7R while retaining the capacity for signal transduction via the IL-15R. An equivalent result was obtained in ED-7R cells modified to express varying levels of gammac. Collectively, these results confirm that signal transduction via the IL-15R, and hence NK ontogeny, is preferentially retained relative to the IL-7R as gammac expression becomes limiting.

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.

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.