Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions

Abstract

The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and alphav integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack alphav integrin binding, or lack both CAR and alphav integrin binding. These vectors have been used to examine the roles of CAR and alphav integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of alphav integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and alphav integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and alphav integrins can impact vector distribution in vivo. Disruption of both CAR and alphav integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

FIG. 1

Schematic diagram of Ad vectors with altered cell surface interactions. AdL.F* has a modification in the AB loop of fiber (see Materials and Methods) that abolishes CAR binding and has an HA epitope inserted in the fiber HI loop. In AdL.PB*, the penton base RGD motif has been replaced by an HA epitope. AdL.PB*F* combines the modifications found in AdL.PB* and AdL.F*.

FIG. 2

Specificity of transduction of AE25 (A) and AE25-HA (B) cells by the panel of vectors. Cells were incubated with vector (50 Pu/cell) or medium alone (−) for 60 min, washed to remove vector, and incubated for 18 h before being harvested. Competitors (fiber at 5 μg/ml, penton base at 100 μg/ml, or a combination of the two) were incubated with the cells for 60 min before addition of the vector. Cell lysates were assayed for luciferase expression. rlu, relative light units. Symbols: █ no competitor; , fiber; ░⃞, penton base; □, fiber plus penton base.

FIG. 3

Transduction of Ramos (A) and CHO (B) cells by a panel of vectors. Cells (10⁵) were incubated with vector at 50 Pu/cell (Ramos) or 1,000 Pu/cell (CHO) or with medium alone (−). Competitors and vectors were incubated with cells as described in the legend to Fig. 2, and cells were assayed for luciferase expression at 18 h. rlu, relative light units. Symbols are as in Fig. 2.

FIG. 4

Effects of fiber and penton base mutations in vivo on transduction of skeletal muscle. Luciferase activities were assayed in the gastrocnemius muscle 24 h after direct intramuscular injection of 10¹⁰ Pu of vector or buffer alone (mock) and were normalized to the protein content of the samples (relative light units [rlu] per microgram). Each column shows the average from four samples, and the error bars represent the standard deviations.

FIG. 5

Transduction of specific tissues following intravenous administrations with the panel of vectors. Animals received 10¹¹ Pu of vector intrajugularly. Liver, lung, heart, kidney, spleen, and muscle (gastrocnemius) were collected at 24 h postinjection and analyzed as described in the legend to Fig. 4. Four samples were used for each vector except for AdL, for which three were used. rlu, relative light units.

FIG. 6

Detection of vector DNA by Southern blot analysis, DNA isolated from liver (A), lung (B), heart (C), and spleen (D) was digested with KpnI and hybridized to a probe from the pol region of Ad5. The blots were exposed to X-ray film for 24 h (A) or 48 h (B to D). Quantitation of the bound probe using an InstantImager instrument is shown in the lower panels of A and D. The counts per minute reported were corrected by subtraction of background determined in adjacent regions of the blot. Samples from mock-treated animals were not above background.