Adeno-associated virus vector-mediated delivery of pigment epithelium-derived factor restricts neuroblastoma angiogenesis and growth

Presented at the 35th Annual Meeting of the American Pediatric Surgical Association, Ponte Vedra, Florida, May 27-30, 2004.
https://doi.org/10.1016/j.jpedsurg.2004.09.049Get rights and content

Abstract

Purpose

The purpose of this study was to evaluate the ability of adeno-associated virus (AAV) vector-mediated delivery of pigment epithelium-derived factor (PEDF) to inhibit neuroblastoma (NB) xenograft growth. Pigment epithelium-derived factor was chosen for this study because, in addition to being a potent inhibitor of angiogenesis, it is capable of inducing neuronal differentiation.

Methods

Cohorts of mice received either recombinant AAV encoding human PEDF (rAAV-hPEDF) at a range of doses or control vector via tail vein. Subsequent hPEDF expression was measured by enzyme-linked immunoassay. After 6 weeks, the mice were given human NB cells by retroperitoneal injection and then killed 5 weeks later. Tumor weight, microvessel density, tumor differentiation, apoptosis, and levels of intratumoral vascular endothelial growth factor (VEGF) expression were determined at that time. In subsequent cohorts of mice, AAV-mediated murine PEDF expression was tested against both human NB xenografts and murine tumors.

Results

In a series of in vitro studies, PEDF was shown to inhibit endothelial cell activation and to stimulate differentiation of NB cell lines. After tail vein injection of rAAV-hPEDF, stable transgene expression was generated and correlated with levels of vector administration. Human NB xenograft growth was restricted by hPEDF in a dose-dependent fashion. Intratumoral VEGF expression and microvessel density were decreased, and tumor cell apoptosis was increased in PEDF-treated mice.

Conclusions

Treatment with PEDF had a significant impact on NB growth in mice when delivered continuously using a gene therapy–mediated approach. The activity of PEDF appears to be mediated in part by inhibition of tumor-induced angiogenesis through down-regulation of tumor-elaborated VEGF, with subsequent intratumoral apoptosis. Furthermore, hPEDF was able to induce NB differentiation in vitro and in vivo. In addition, antitumor efficacy was seen when mouse PEDF was used to treat syngeneic murine tumors. In our murine models, gene therapy–mediated delivery of PEDF appears promising for the treatment of neuroblastoma.

Section snippets

Cell lines

The human NB cell line NB-1691, provided by Dr P. Houghton (Memphis, Tenn), the murine NB cell line NXS2, provided by Dr R. Reisfeld (La Jolla, Calif), and the 293T cells (human embryonic kidney cells expressing SV40 large T antigen, American Type Culture Collection, Manassas, Va) were used in this study.

Inhibition of in vitro endothelial cell activation and tubule formation

Vascular endothelial growth factor (VEGF)–stimulated endothelial cell proliferation and basic fibroblast growth factor (bFGF)-stimulated endothelial cell migration and tubule formation assays

In vitro assessment of PEDF expression and function

Functional assessment of PEDF generated from the AAV PEDF vector was performed first. Conditioned medium from transfected cells was collected, and its ability to inhibit endothelial cell activation in vitro was assessed. Medium from cells expressing PEDF was able to inhibit bFGF-stimulated endothelial cell migration and VEGF-stimulated proliferation. As shown in Fig. 1A, a 34% decrease in endothelial cell migration and 35% decrease in endothelial cell proliferation were seen as compared with

Discussion

Adeno-associated virus vectors have several properties making them particularly promising vectors for gene therapy [7]. Importantly, rAAV has been shown to direct long-term transgene expression from nondividing cells in vivo [8], [26], [27]. Furthermore, AAV has an excellent safety profile, evokes little immune response because genes encoding wild-type protein are absent, is naturally replication-deficient, and is not known to be associated with any human disease. In recent studies, we have

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