Placement of viral regulatory genes under the control of the hTERT promoter, which is active in a majority of human cancers (Kim et al.
), is increasingly regarded as an ideal way to restrict the replication of oncolytic adenoviruses to malignant tissues. Several tumor cell-replicating, hTERT-driven adenoviruses have been described (Wirth et al.
); however, they differ most notably from Adeno-hTERT-E1A, the oncolytic adenovirus described in the present study, insofar as they contain either one or both E1B region genes: E1B-19kDa and E1B-55kDa. Several of these hTERT promoter-regulated viruses include upstream hTERT regulatory sequences, which may contain repressor sequences absent from the hTERT core promoter. The core hTERT promoter fragment presently used to regulate E1A expression in Adeno-hTERT-E1A was chosen to maximize activity in cancer cell lines based on earlier promoter deletion analysis (Horikawa et al.
; Poole et al.
). Evaluation of the hTERT-E1A expression cassette demonstrated functionality, and the hTERT-luciferase reporter cassette showed a cell line-specific pattern of reporter activity that was qualitatively similar to the endogenous hTERT activity. Thus, the hTERT core promoter sequence appears to confer high levels of expression in a cell line-dependent manner, with the truncated promoter behaving qualitatively similar to the endogenous full-length promoter.
kDa-deleted, oncolytic adenovirus ONYX-015 has been evaluated in multiple phase II and phase III clinical trials (Crompton and Kirn, 2007
kDa protein is known to bind, sequester, and facilitate the degradation of p53 (Steegenga et al.
; Grand et al.
), and as such, ONYX-015 was first believed to replicate only in p53-deficient cells, imparting tumor cell specificity to viral replication (Bischoff et al.
). However, ONYX-015 was later shown to replicate in certain cells bearing a wild-type p53 gene (Dix et al.
), suggesting the possible risk of permissive replication in normal tissues. E1B-55
kDa protein also plays a role in host cell protein synthesis shutoff and in late adenoviral mRNA export, thus restricting replication to tumor cells, as well as normal cells, where this transport is phenocopied by other factors (O'Shea et al.
). These observations indicate a need to improve the safety of E1B-55
kDa-deleted oncolytic adenoviruses. The second E1B region gene product, E1B-19
kDa, functions like the antiapoptotic mitochondrial protein Bcl-2: it binds to and represses the proapoptotic transcription repressor Btf (Imazu et al.
; Kasof et al.
), and it inactivates the proapoptotic Bcl2 family member Bax (Han et al.
). Both E1B genes delay adenovirus-induced cell death long enough for completion of viral replication and repackaging. In many cancer cells, where apoptosis is already suppressed, these antiapoptotic genes are not required for viral replication (Rao et al.
), enabling these genes to be deleted in the construction of oncolytic adenoviruses (Duque et al.
; Harrison et al.
). The present deletion of both E1B genes in the context of high hTERT promoter-regulated E1A protein expression yielded a highly oncolytic virus that retains high cancer cell selectivity with greatly reduced replication in primary hepatocytes.
The high levels of E1A protein achieved in Adeno-hTERT-E1A-infected cells also likely contributed to the increased oncolytic activity seen with Adeno-hTERT-E1A as compared with ONYX-015 in all tumor cell lines tested except for A549. E1A, an immediate-early viral protein, is typically expressed within 6
hr of infection and is crucial for the onset of viral replication and subsequent expression of further downstream early and late viral genes (Nevins, 1981
). As such, Adeno-hTERT-E1A infection led to oncolysis that was more rapid and more complete than after ONYX-015 infection in the case of MCF-7/4HC (breast), U251 (brain), and MDA-MB-231 (breast) cancer cells. E1A itself has substantial intrinsic cytotoxic activity (Hale and Braithwaite, 1999
; Zhou et al.
; Rao et al.
) and its high-level expression in the absence of both anti-apoptotic E1B proteins in Adeno-hTERT-E1A-infected cells resulted in early and high levels of apoptosis, which most likely also contributed to cell death. In our oncolysis and hexon-staining studies, cells infected with ONYX-015 did not appear to go through apoptotic cell death as evidenced by lack of PARP cleavage and TUNEL staining (). They appear to have two discrete morphologies, one of a healthy cell bearing normal cell-line specific characteristics and one of a rounded-up cell about to lyse, whereas those infected with Adeno-hTERT-E1A typically appear as sickly, rounded-up cells, which were shown to undergo apoptosis.
Adeno-hTERT-E1A and ONYX-015 differ with respect to five adenoviral gene products, in addition to their distinct E1A promoter regulatory elements, making it difficult to definitively identity the factors responsible for the unique properties of each virus. However, ONYX-015 was initially chosen to assess the oncolytic potential of Adeno-hTERT-E1A while comparing it with a clinically evaluated adenovirus. ONYX-015, but not Adeno-hTERT-E1A, encodes the anti-apoptotic factor E1B-19
kDa, as well as the E3 region adenoviral death protein (ADP), which facilitates late-stage lysis and release of mature viral progeny from infected A549 cells (Doronin et al.
). The similar oncolytic profiles of both viruses in A549 cells at earlier time points might be explained by efficient viral lysis in the ONYX-015-infected cells as compared with the early apoptotic death seen in Adeno-hTERT-E1A-infected cells. We considered the possibility that the viral ADP protein might explain why the long-term lysis and spread of ONYX-015 in A549 cells is superior, outpacing Adeno-hTERT-E1A in its ability to kill the cells, in particular at low MOIs. However, it does not appear that the ADP protein is responsible for the increased lysis and spread of ONYX-015 compared with Adeno-hTERT-E1A in A549 cells, as indicated by the much higher levels of functional virus seen in lysates of ONYX-015-infected cells as compared with Adeno-hTERT-E1A-infected cells. Indeed, in other studies, E1B-19
kDa-deleted adenoviruses were equally able to produce viral plaques and lyse A549 cells, independent of whether ADP was present (Subramanian et al.
). Plaque formation and disruption of cell lysis were impeded only in the presence of E1B-19
kDa, which stabilizes the nuclear lamin structure, and only in the absence of ADP, which counteracts this added nuclear stability late in the viral life cycle to instigate efficient cell lysis (Subramanian et al.
). Accordingly, in the case of Adeno-hTERT-E1A, in which both proteins in the antagonist pair, E1B-19
kDa and ADP, are absent, viral release per se is not the problem. The more extensive lysis of A549 cells by ONYX-015 as compared with Adeno-hTERT-E1A must therefore result from other factors unique to ONYX-015 that confer its ability to efficiently replicate and produce high functional viral titers in A549 cells, in spite of the lower levels of E1A. One possibility is that the high levels of E1A protein produced in A549 cells infected with Adeno-hTERT-E1A kill many of the host cells prematurely via apoptosis, thereby decreasing the efficiency of successive rounds of viral replication and reinfection.
E1A can be used as a therapeutic gene to induce antitumoral responses, including inhibition of angiogenesis (Zhou et al.
). E1A also can sensitize tumor cells to chemotherapy and radiation (Sanchez-Prieto et al.
; Martin-Duque et al.
). Therefore, the high level of E1A expression achieved in Adeno-hTERT-E1A-infected tumor cells may not only provide for tumor-specific regulation of viral replication but may induce other E1A-dependent antitumor mechanisms. Differences in apoptosis induction between Adeno-hTERT-E1A and ONYX-015 may reflect the absence of both antiapoptotic genes (E1B-55
kDa and E1B-19
kDa) in Adeno-hTERT-E1A, as compared with ONYX-015, which retains the anti-apoptotic Bcl-2 analog E1B-19
kDa, as well as the elevated expression of E1A, which can induce p53 and stimulate apoptosis (Hale and Braithwaite, 1999
; Kasof et al.
). Indeed, high E1A levels correlated with a high apoptotic index in U251, A549, MDA-MB-231, and MCF-7 cells infected with Adeno-hTERT-E1A (data not shown). The similar A549 cell oncolytic profiles exhibited by both viruses at higher MOIs at earlier but not later time points might be explained by the delivered dose of Adeno-hTERT-E1A initially inducing high levels of cell death via E1A-dependent apoptosis. ADP-containing replication-competent adenoviruses, such as ONYX-015, induce cell death via a necrosis-like pathway that is apoptotic machinery independent (Abou El Hassan et al.
), which is not the case for Adeno-hTERT-E1A. As such, the cell death induced by Adeno-hTERT-E1A may best be described as telomerase-regulated E1A suicide gene therapy using a replication-conditional virus.
Adeno-hTERT-E1A displayed superior antitumor activity compared with ONYX-015 against breast tumor MDA-MB-231 xenografts, with no apparent increase in host toxicity. Moreover, the genetic modifications introduced into Adeno-hTERT-E1A greatly decreased its replication competence in primary human hepatocytes. This can be explained by the absence of hTERT expression in human hepatocytes, which precludes E1A expression and all downstream events in the adenoviral replication cycle. The use of hTERT promoter sequences to regulate Adeno-hTERT-E1A replication likely contributes to its substantially decreased replication in hepatocytes compared with ONYX-015, where E1A induction, genome replication, and viral protein production were readily detected, as were viral completion/repackaging and capsid assembly, as indicated by hexon staining. Adenoviral E1A protein has been linked to acute hepatotoxicity via induction of tumor necrosis factor-α and transaminitis in mice (Engler et al.
). Although hTERT RNA was not detectable in hepatocytes, we could still detect E1A and hexon protein in a few cells infected with Adeno-hTERT-E1A. These cells may be hepatocytes undergoing abortive replication, cells with a mutated hTERT and an apoptosis status that reflects oncogenic potential, or perhaps the presence of other liver-associated cell types in the human hepatocyte preparations. The absence of both antiapoptotic proteins in Adeno-hTERT-E1A should allow primary host cells to apoptose and thereby abort any instances of viral infection that could otherwise lead to viral replication. The increased oncolytic response associated with complete deletion of the E1B region therefore does not infringe on the replication specificity of Adeno-hTERT-E1A and its safety profile against primary tissues. These findings were confirmed in primary hepatocytes isolated from three independent donors, highlighting the improved safety and replication specificity of Adeno-hTERT-E1A, and further substantiating the use of the hTERT core promoter to simultaneously improve cancer selectivity and minimize host toxicity.
While this study was in progress, Bilsland and coworkers described the use of an hTERT promoter-regulated E1A cassette in an oncolytic adenovirus lacking the entire E1B gene region, and reported a phenotype similar to that of our Adeno-hTERT-E1A virus, in particular elevated E1A expression and a modified oncolytic profile (Bilsland et al.
). Their results support our finding that deletion of the E1B region enhances early oncolysis, which may diminish replication completion and virion maturation. The present study extends those findings to include an investigation of the effects of Adeno-hTERT-E1A on apoptosis induction, viral release, hepatocyte safety, and antitumor activity in vivo.
In conclusion, we have engineered an oncolytic adenovirus, Adeno-hTERT-E1A, which displays an improved oncolytic profile in many cancer cell lines and substantially reduced replication in human hepatocytes compared with ONYX-015, an oncolytic virus that has undergone extensive testing in clinical trials. The diminished propensity of Adeno-hTERT-E1A to replicate in a primary organ such as the liver, which is highly susceptible to adenoviral infection (Wood et al.
), may allow for an increase in virus dose to improve therapeutic activity without increasing hepatic toxicity. Future studies will examine potential combination therapies in which Adeno-hTERT-E1A will be used with anticancer prodrug-activating enzymes, such as cytochrome P-
450 2B11 (Jounaidi et al.
), to further enhance therapeutic potential.