Application of a Fas ligand encoding a recombinant adenovirus vector for prolongation of transgene expression

Abstract

An adenovirus vector encoding murine Fas ligand (mFasL) under an inducible control was derived. In vivo ectopic expression of mFasL in murine livers induced an inflammatory cellular infiltration. Furthermore, ectopic expression of mFasL by myocytes did not allow prolonged vector-mediated transgene expression. Thus, ectopic expression of functional mFasL in vector-transduced cells does not appear to confer, by itself, an immunoprivileged site sufficient to mitigate adenovirus vector immunogenicity.

FIG. 1

Apoptosis analysis of 293 cells expressing mFasL, performed as described in the text.

FIG. 2

Construction of a recombinant adenovirus vector encoding mFasL. (A) Map of the recombinant adenovirus vector. An expression cassette is inserted into the deleted E1A region. This cassette allows inducible expression of mFasL from the CAG promoter after excising of the stuffer Neo^r gene flanked by the Loxp sites. ITR, adenovirus inverted terminal repeats. Map units (m.u.) 0 to 100 are indicated. (B) Confirmation of identity of the adenovirus vector encoding mFasL, AdLoxpFasL. Adenovirus genome DNA was subjected to restriction endonuclease digestion with XhoI and analyzed by gel electrophoresis and Southern blotting. For Southern blotting, a ³²P-labeled mFasL probe was used. Lane 1, 1-kb DNA ladder; lane 2, XhoI digestion of AdLoxpFasL; lane 3, XhoI digestion of E1-deleted adenovirus vector lacking the mFasL gene; lane 4, Southern blot analysis as for lane 2; lane 5, Southern blot analysis as for lane 3.

FIG. 3

Analysis of gene expression characteristic of AdLoxpFasL by Northern blot analysis. Murine B6 lpr/lpr mouse macrophages were infected with either AdLoxpFasL plus AdCMVLacZ (lane 2) or AdLoxpFasL plus AxCANCre (lane 3). Lane 1 is an uninfected control. Twenty-four hours postinfection, total RNA was isolated and probed with mFasL and β-actin cDNAs.

FIG. 4

Characterization of the function of ectopic expression of mFasL in macrophages. The lpr/lpr macrophages were infected with either AdLoxpFasL plus AdCMVLacZ (A) or AdLoxpFasL plus AxCANCre (B) and mixed with ⁵¹Cr-labeled A20 cells at the indicated E/T ratios; after 6 h of incubation, the specific release of radioactive marker was determinated.

FIG. 5

Induction of autocrine suicide event by Fas-expressing target cells by AdLoxpFasL. HeLa cells were infected with AdLoxpFasL plus AdCMVLacZ or with AdLoxpFasL plus AxCANCre. After 24 h, cells were stained with trypan blue and analyzed in triplicate to determine the number of viable cells.

FIG. 6

Effect of ectopic expression of mFasL expression in the liver. Adult female C57BL/6 mice were injected intravenously with AdLoxpFasL plus AxCANCre (A), AdCMVLacZ (B), AdLoxpFasL plus AdCMVLacZ (C), or phosphate-buffered saline alone (D). Livers were harvested at 24 h postinjection, and sections were prepared for histological analysis staining with hematoxylin and eosin. Magnification, ×320.

FIG. 7

In situ detection of apoptotic cells in the liver. Adult female C57BL/6 mice were injected intravenously with AdLoxpFasL plus AxCANCre (A and B) or AdCMVLacZ (C and D). Livers were harvested at 24 h postinjection, and sections were prepared for histological analysis staining with hematoxylin and eosin (A and C) or with Hoechst 33258 reagent to detect apoptosis (B and D). Magnification, ×320.

FIG. 8

Effect of ectopic expression of mFasL expression on longevity of transgene expression in murine myocytes. Adult female BALB/c mice were injected intraglossally with the indicated vectors; control animals were not injected. At various times postinjection, luciferase activity was determined in harvested tissue. Each histogram represents the mean ± standard deviation for seven animals.