Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver.

Human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type I diabetes. However, limits to the efficiency of rAAV serotype 2 (rAAV2)-mediated gene transfer have been reported for both of these cell targets. Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting. In order to test this, we generated rAAV particles that display a ligand (ApoE) that targets the low-density lipoprotein receptor, which is present on both of these cell types. The rAAV/ApoE viruses greatly enhanced the efficiency of transduction of both islet cells ex vivo and murine hepatocytes in vivo when compared to native rAAV2 serotype (220- and four-fold, respectively). The use of receptor-targeted rAAV particles may circumvent the lower abundance of receptors on certain nonpermissive cell types.

Targeting tumor blood vessels: an adjuvant strategy for radiation therapy.

BACKGROUND AND PURPOSE: The neovascularization of tumor cells is a prerequisite if a clinically relevant tumor size is to be reached. A continuously expanding vessel network supplying nutritional requirements and removing waste products is essential for continued tumor development, growth and survival. RESULTS: In many tumors, the growing endothelium is unable to fully support the demands of the neoplastic cell population. As a consequence of the inadequacies of the resulting aberrant vasculature, microenvironmental conditions develop in tumors which are not only detrimental to the response of tumors to conventional anticancer treatments, but may lead to or predispose cells to genetic modifications resulting in more aggressive phenotypes and higher metastatic potential. Yet the utter dependence of the tumor on its induced vessel formation for growth, survival and spread has also created a great deal of enthusiasm for developing therapeutic approaches to specifically targeting the tumor microcirculation. CONCLUSIONS: The application of such strategies as adjuvants to conventional radiation treatments offers unique opportunities to develop more effective cancer therapies.

Developing VDEPT for DT-diaphorase (NQO1) using an AAV vector plasmid.

PURPOSE: One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background. METHODS: The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol. RESULTS: The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to approximately 200 nmol/min/mg, and the NQO1 activity in KHT/iv increased approximately 10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis. CONCLUSIONS: Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.