Novel Human Gene Transfer Vectors: Evaluation of Wild-Type and Recombinant Animal Adenoviruses in Human-Derived Cells

Abstract

Major disadvantages of human adenovirus (hAd) vectors in gene therapy include preexisting or induced immune responses, and possible coreplication of recombinant hAd in the presence of wild-type hAds. These disadvantages may be overcome by using nonhuman, animal adenoviruses (aAds). We evaluated four different aAds for their potential use as viral vectors. The canine adenovirus type 2 (CAV2) and bovine adenovirus type 3 (BAV3) appeared to be suitable systems, as they infect human cells. CAV2, but not BAV3, caused cytotoxicity, and only limited (CAV2) or no (BAV3) production of infectious virus particles was observed after infection of human cell lines. CAV2 showed higher expression of endogenous genes than did BAV3 in the tested human cells. No interference between hAd and CAV2 or BAV3, such as recombination of DNA or cross-activation of virus replication, was observed in up to five passages in double-infected human cells. Transfection of cloned genomic CAV2 or BAV3 DNA into appropriate permissive cell lines rescued infectious virus. Furthermore, we produced a recombinant E1-deleted BAV3, and showed that it could infect and express a reporter gene in various human cell types. The goal was to construct and evaluate recombinant (E1-deleted) animal adenoviruses (aAds) as new vector systems for human gene therapy. The rationale for developing aAds for human use is the potential higher safety and efficiency, as compared with human adenoviruses (hAds). Coreplication and recombination with preexisting hAds should not be possible owing to lack of homology, and preexisting immunity in the general population should be limited. Of the four aAds we evaluated, BAV3 appeared to be the best candidate. It infects human cells without showing growth or cytotoxic effects, viral gene expression was barely detectable, and no trans-activation of either virus was detected in coinfections with hAd5. Rescue of virus in permissive cells, from plasmids containing the CAV2 or BAV3 genome, confirmed our approach. Furthermore, an E1-deleted recombinant BAV3 was constructed and shown to transduce and express the lacZ reporter gene in human cells.

Biology of Ovine Adenovirus Infection of Nonpermissive Cells

“…The realization that preexisting immunity to human adenoviruses in the majority of the population poses a major problem for the use of these vectors prompted us and others (18,28,29,32,34,38,47) to explore adenoviruses of nonhuman origin. Many of the results are promising and suggest that nonhuman adenoviruses are a useful alternative and addition to the currently used human adenovirus-derived vectors.…”

CAR chasing: canine adenovirus vectors–all bite and no bark?

“…Nonetheless, Ad5 propagates in vitro in porcine cells [144], and in vivo in super-infected cotton rats and rabbits [58], while bovine serotype 3 (BAV-3) also propagates in cotton rats [94]. Although others reported that CAV-2 may replicate in some human cells [115], we found that CAV-2 does not propagate (make new progeny) [67]. Furthermore, we did not consider the low level of CAV-2 replication a significant drawback when choosing a nonhuman Ad.…”

Development and Characterization of Bovine × Human Hybrid Cell Lines That Efficiently Support the Replication of both Wild-Type Bovine and Human Adenoviruses and Those with E1 Deleted

“…On the basis of the observations that (i) 293 cells express HAV5 E1 proteins but do not support the replication of wildtype (wt) BAV3 (43), (ii) Madin-Darby bovine kidney (MDBK) cell line is an excellent host for growing and titrating wt BAV3, and (iii) there is efficient functional trans-complementation between BAV3 E1A and HAV5 E1A, we hypothesized that the concept of somatic cell hybrids could be easily tested by generating hybrids that support replication of BAV3⌬E1AE3. The rationale for generating bovine ϫ human hybrid (BHH) cell lines is presented in Fig.…”