Ad vectors modified to abolish native receptor interactions are depicted in Fig. . All express luciferase from a cytomegalovirus promoter in the E1 region. The fiber protein of AdL.F* carries amino acid substitutions within the AB loop which disable CAR binding, whereas AdL.PB* lacks the RGD motif within the penton base which binds αv integrins. An HA epitope was incorporated within the HI loop of the fiber protein of AdL.F* and in place of the penton base RGD in AdL.PB*. The fiber and penton base modifications, including the HA epitopes, were combined in AdL.PB*F*. Vector genomes were constructed by recombination in E. coli, and plasmids carrying the complete genomes of these vectors were transfected into 293 cells (AdL and AdL.PB*) or 293-HA cells which express a membrane-anchored single-chain antibody capable of binding the HA epitope (AdL.F* and AdL.PB*F*). Expansion of the vectors in production runs using 2 × 108 to 1 × 109 cells infected at a multiplicity of infection of 20 PU showed comparable yields for the ablated and unmodified vectors (Table ). The activities of AdL.F* and AdL.PB*F* were measured in focus-forming unit (FFU) assays on 293-HA cells. While AdL.F* exhibited PU/FFU ratios similar to those for AdL, AdL.PB*F* gave PU/FFU ratios about fourfold higher than those for AdL. This is consistent with the finding that luciferase expression from AdL.PB*F* is about 20% of the level detected from AdL following addition of equal particle numbers to 293-HA cells (data not shown).
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 (more ...)
Summary of vector yield and activity from multiple production runs.
The efficiency of gene transfer was analyzed by assaying luciferase expression in cells following incubation with Ad vectors. The effects of the vector modifications on transduction of AE25 cells are shown in Fig A. Whereas the addition of soluble fiber reduced AdL transduction by >70-fold, it had no effect on transduction by AdL.F*. This result is consistent with a model of transduction where the only role of fiber is to bind CAR. Interestingly, transduction by AdL.F* was reduced 12-fold in the presence of penton base protein, which had no effect on AdL transduction. Penton base affected AdL transduction only on combination with fiber, when a small additional reduction was seen. For AdL.PB*, only fiber inhibited transduction. The absence of additional penton base interactions for this vector is indicated by the lack of effect of penton base even when combined with fiber. Based on these results, a vector able to bind neither CAR nor αv integrins was constructed. This vector, designated AdL.PB*F* and also referred to as doubly ablated, transduced AE25 cells to a level nearly 3 orders of magnitude below what was seen with AdL. This residual transduction was less than 10-fold above the background measured in the absence of vector. Transduction by this vector was unaffected in the presence of either fiber or penton base or a combination of the two. Thus, it appeared that penton base inhibited transduction only when fiber was blocked from binding CAR and that this was mediated by the RGD motif. The significantly reduced transduction for the doubly ablated vector suggested that removal of the RGD motif could be critical for disabling the native transduction activity of Ad vectors in vivo.
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 (more ...)
On cells expressing a membrane-anchored single-chain antibody that binds to the HA peptide epitope incorporated in the coat proteins of the modified vectors, no significant differences were seen in transduction among the four vectors in the absence of competitors (Fig. B). Thus, the ability to bind cells via the anti-HA antibody overcomes the transduction deficiencies resulting from loss of CAR and αv integrins. Luciferase levels detected following infection with AdL in the presence or absence of competitors indicated no differences between AE25-HA and AE25 cells. On AE25-HA cells, unlike AE25 cells, AdL.F* exhibited no sensitivity to competition by penton base, presumably because the anti-HA–HA epitope interaction promotes binding, which masks this just as fiber-CAR binding does for AdL. AdL.PB*, on the other hand, remained very sensitive to competition by fiber protein on the AE25-HA cells. This suggested that the HA epitope inserted in place of the RGD motif in penton base was not effectively bound by the anti-HA protein. Transduction by AdL.PB*F* was dramatically increased on AE25-HA cells compared to that on AE25 cells. The results obtained with AE25-HA cells indicated that the CAR-ablated vector AdL.F* and the doubly ablated vector AdL.PB*F* efficiently transduced cells capable of binding the inserted HA epitope. Production of these vectors is dependent on this surrogate interaction, and this observation supports the idea that by incorporating appropriate ligands the modified vectors remain highly active in transduction.
The modified vectors were further evaluated on Ramos cells, which do not express αv integrins but do express CAR, and on CHO cells, which lack CAR but express αv integrins. In the presence of fiber protein, transduction of Ramos cells by AdL, at multiplicity of infection of 50 Pu/cell, dropped 3 orders of magnitude, virtually to background (Fig. A). Ramos cells were refractory to AdL.F*, so the additional modification of penton base gave no further reduction in transduction. CHO cells are poorly transduced by AdL, so all vectors were added at 1,000 Pu/cell. In contrast to the Ramos cells, CHO cells exhibited no effect of ablating CAR binding (AdL.F*) or competing with fiber (Fig. B). Transduction by AdL and by AdL.F*, however, were both inhibited about fivefold in the presence of penton base. The transduction phenotypes of the modified vectors on both Ramos and CHO cells further demonstrated the specificity of the modifications to fiber and penton base, which inhibit the interactions with CAR and αv integrins, respectively.
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 (more ...)
The activities of the panel of vectors in vivo were examined by direct intramuscular administration. Following injection into the gastrocnemius muscle, AdL.F* resulted in similar transduction to that due to AdL (Fig. ), indicating that CAR does not play a prominent role in transduction of skeletal muscle by Ad vectors. Transduction of muscle was decreased 3-fold for the penton-modified vector AdL.PB*, but AdL.PB*F* caused a dramatic reduction of nearly 100-fold. Thus, while knocking out CAR binding alone had no effect on expression following intramuscular injection, the combination of this modification with the penton base alteration resulted in a 25-fold drop relative to AdL.PB*.
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 (more ...)
Intravenous administration of Ad5-based vectors in mice results in preferential expression in the liver, which is also a prominent organ for CAR expression. Following intrajugular administration, the CAR-ablated vector AdL.F* caused reduced transduction of the liver compared to AdL (Fig. ), but luciferase expression remained nearly 3 orders of magnitude above that seen in livers from mock-injected animals. Deletion of the RGD motif from penton base (AdL.PB*) was as effective in reducing liver transduction as was disabling CAR binding. The vector disabled for both binding CAR and binding αv integrins (AdL.PB*F*) exhibited a drop in liver transduction of more than 700-fold compared to AdL. The reduced transduction by AdL.PB*F* was significantly lower than that by either AdL.F* (P < 0.03) or AdL.PB* (P < 0.03). The last two vectors exhibited decreases, respectively, of 10- and 20-fold versus AdL. Thus, the two modifications had a synergistic effect in reducing transduction of the liver. These results, together with the results from intramuscular injection, indicate that removing both the CAR and the αv integrin binding of Ad vectors is critical for reducing the native tropism of Ad vectors in vivo.
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 (more ...)
Additional tissues from these mice were analyzed for luciferase expression to examine the roles of CAR and αv integrin interactions. The level of transduction detected with AdL in all these tissues was ≤1% of that found in the liver. In the lung, heart, kidney, spleen, and muscle, abolishing CAR binding resulted in no reduction in transduction (Fig. ). In fact, lung, kidney, and muscle transduction appeared elevated. In contrast to ablating CAR binding, ablating αv integrin binding alone reduced transduction in the lung, heart, and kidney (P < 0.05, P < 0.01, and P < 0.01, respectively), although transduction of muscle was not significantly different from that by AdL. The doubly ablated vector exhibited reduced transduction relative to AdL in the spleen (P = 0.02) as well as the lung, heart, kidney, and muscle (P < 0.01 for all). With the exception of the spleen, ablating integrin binding was more effective than abolishing CAR binding for limiting transduction. In lung, heart, and muscle, AdL.PB*F* resulted in significantly less transduction than did AdL.PB* (P < 0.02, P < 0.05, and P < 0.04, respectively), and in all five tissues the transduction measured for AdL.PB*F* was not significantly above background. We detected no difference among the vectors in the low level of transduction of the superficial inguinal node or the diaphragm (data not shown). In all tissues where transduction by AdL was detected, AdL.PB*F* gave reduced luciferase expression.
Southern blot analyses were performed on DNA isolated from individual tissues to examine vector distribution independent of expression. Clear differences were seen among the panel of vectors in terms of genomes detected in the liver (Fig. A). Quantitation of bound probe showed a two- to threefold decrease for AdL.F* versus AdL, regardless of whether the weakest AdL.F* sample was included in calculating the means. A 9-fold decrease relative to AdL was seen for AdL.PB*, while the vector signal was reduced by 13-fold for AdL.PB*F*. Thus, in terms of vector particles localizing to the liver, αv integrin binding had a greater impact than CAR binding. The synergistic effect on transduction of combining the CAR and integrin binding modifications was not observed at the level of localization of particles to the liver.
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 (more ...)
AdL DNA was localized primarily in the liver at 24 h postadministration. In the lung and heart, the AdL DNA level was at or below the level of detection (Fig. B and C). As reported above, expression in these tissues was less than 1% of that detected in the liver. A similar reduction in vector genomes would give a signal barely above background in the Southern blot analysis. Vector DNA was more readily detected in the spleen (Fig. D). In addition, a different pattern was seen for the panel of vectors in this tissue. There was no difference between the amounts of AdL.F* and AdL, while the amount of AdL.PB* was reduced about twofold. By contrast, the amount of AdL.PB*F* was increased nearly threefold versus AdL. Quantitation of bound probe indicated that the amount of AdL detected per 5 μg of splenic DNA was 8% of that detected with an equal quantity of liver DNA. Since the normal mouse liver is about nine times the size of the normal spleen in terms of weight, the fraction of the AdL dose present in the spleen was quite small compared to that in the liver. While the AdL.PB*F* level was elevated in the spleen relative to that of AdL and was also nearly threefold higher than that of AdLPB*F* in the liver on a per-microgram-of-DNA basis, the size difference between the two organs indicates that the majority of this vector also localized to the liver.
We used real-time PCR analysis to further quantify differences in genome copies between the vectors within specific tissues. The results are presented as percentages of the numbers of copies detected for AdL in the respective tissue, since differences in PCR efficiency among organs limit a direct comparison of the number of vector copies detected in different tissues (Table ). The number of relative copies detected for each vector in the liver by PCR agreed well with the Southern blot results. Abolition of CAR binding resulted in two- to threefold drop in the number of vector genomes detected. For AdL.PB*, the reduction was 10-fold relative to AdL and significantly reduced relative to AdL.F* (P < 0.03). The level of the doubly ablated vector was reduced 15-fold relative to that of AdL and was not significantly lower than that of AdL.PB*. PCR detection of the different vectors in the spleen also matched the results of Southern blotting, with AdLPB*F* giving an elevation in the number of vector copies. We were able to detect vector DNA in the lung, heart, kidney, and diaphragm and found that loss of CAR binding did not result in a statistically significant reduction in the level of vector genomes in these tissues (Table ). In contrast, AdL.PB* levels were significantly reduced relative to AdL levels in the lung (P < 0.01) and heart (P < 0.01), as were the levels of AdL.PB*F* (P < 0.01 for both). The levels of AdL.PB*F* were also significantly reduced relative to those of AdL in the diaphragm (P < 0.02) and relative to those of AdL.F* in the kidney (P < 0.04). Thus, the penton base modification had a broader effect than the fiber modification in reducing the number of vector copies in these tissues.
Relative copies for the panel of vectors within specific tissues as detected by Taqman PCRa