Prerequisites for effective adenovirus mediated gene therapy of colorectal liver metastases in the rat using an intracellular neutralizing antibody fragment to p21-Ras

Abstract

Ras mutations are present in 40 -50% of colorectal cancers. Inactivating this oncogene may therefore reduce proliferation capacity. In order to target ras we studied the transduction efficacy and anti tumour activity of an adenoviral vector expressing an intracellular, neutralizing single chain antibody to p21-ras (Y28). In in vitro studies transfection levels of the K-ras mutated rat colon carcinoma cell line CC531 were studied using the LacZ marker gene. In our in vivo liver metastases model different routes of administration were evaluated to determine which regimen resulted in the best transfection levels and tumour responses: intravenous injection, intratumoural injection, isolated liver perfusion, or hepatic artery infusion. CC531 cells are readily transfected in vitro, resulting in significant inhibition of tumour cell proliferation by the Y28 construct. Intravenous injection did not result in any measurable transfection. Intratumoural injection resulted only in the transfection of tumour cells along the needle track. IHP as well as single HAI achieved low transfection levels of tumour tissue. Expression of Y28 was demonstrated in tumours after IT injection, HAI and IHP. Whereas, repeated HAI's clearly achieved expression in and around tumour associated vessels. Only five times repeated HAI's with Y28 resulted in a tumour response: in all animals tumour growth was inhibited, and in three rats out of eight a complete regression of the liver tumours was observed.

Gene Therapy in In Vivo Isolated Perfusion Models

“…When adenoviruses are used in IHP it has been demonstrated that high transduction percentages can be achieved (de Roos et al, 1997;Drazan et al, 1995;van Etten et al, 2004a;van Etten et al, 2002b). Regarding toxicity following IHP a maximum transient weight loss of 10% was observed, which could not be related to the viral vector as similar weight loss was observed upon sham IHP (van Etten et al , 2002b). Drazan et al, studied the influence of adenovirus mediate gene transfer on hepatic function in further detail by means histological, biochemical and functional studies (Drazan et al, 1995).…”

“…In an adenovirus based IHP model we are investigating the immune response upon IHP as well. Recently an impaired neutralizing antibody production was observed in rats (van Etten et al, 2004b). Manipulation of adenoviruses to reduce antigenicity is currently performed on a wide scale and this may in combination with local delivery be a future option for repeated delivery thereby offering possible clinical application options.…”

“…Only de Roos et al performed double infusion via the arterial as well as the portal vascularisation of the liver (de Roos et al, 1997). Our group currently performs IHP via the hepatic artery only (van Etten et al, 2003;van Etten et al, 2002b). Especially in liver metastases models the inflow site is of specific interest since the metastatic tumors are mainly vascularized by the hepatic artery (Breedis et al, 1954;Sigurdson et al, 1987).…”

“…We previously reported adenoviral IHP transferring a single chain antibody to p21 ras in to colon carcinoma cells (van Etten et al, 2002b), but with only 1-3 % transduced tumor cells a tumor response could not be induced. In this tumor model there was no bystander effect, and probably higher transduction rates were necessary.…”

Locoregional intravascular viral therapy of cancer: precision guidance for Paris's arrow?

“…Further proof of this hierarchy of administration methods was provided in an analysis of the administration of armed adenoviral vectors encoding LacZ or anti-p21ras antibody by intratumoural injection, IHP, HAI or intravenous infusion. 80 The model used was hepatic colorectal cancer metastases. The greatest level of infection was provided by intratumoural injection at 5%, with IHP providing 2–3% infection of target cells.…”