A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal

Abstract

Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells. We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals flanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells resulted in an approximately 10-fold increase in vector titer per passage. The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation. Large scale preparations of vector yielded semi-purified stocks of approximately 10(10) transducing virions/ml, with < 0.01% contamination by the E1-deleted helper virus. This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.

Figure 1

Generation of Ad-based vectors complemented by a helper virus containing a loxP-flanked packaging signal. Infection of Cre-expressing 293 cells with the helper virus results in excision of the viral packaging signal, rendering the helper virus DNA unpackagable. The helper virus will then provide all of the functions necessary in trans for replication and packaging of an Ad vector containing appropriate cis-acting elements [i.e., viral packaging signal (ψ⁺) and inverted terminal repeats]. Since all functions necessary for virion formation are provided by the helper virus, the majority of the Ad sequences contained in the vector can be replaced by a foreign gene and other non-Ad sequences (stuffer). The titer of the vector can be increased by serial passage in helper virus-infected 293Cre cells.

Figure 2

Structure of AdLC8 and analysis of Cre-mediated excision of the loxP flanked packaging signal (ψ) in 293Cre1 cells. (A) AdLC8 contains two loxP sites flanking the viral packaging signal and a neomycin gene (expanded), and was constructed as outlined in the Materials and Methods. The E1 and E3 deletions in AdLC8 remove sequences located between 339-3533 bp and 28,133–30,818 bp of the Ad genome, respectively. A PvuI restriction map of AdLC8, with fragment sizes indicated in kb, is shown above the schematic linear map, and PvuI restriction sites in the expanded regions are indicated (P). Fragments that hybridize to the pLC8-derived probe used during Southern blot analysis are indicated with an asterisk (∗). Cre-mediated recombination between the two loxP sites results in the disappearance of the 1.1- and 3.5-kb PvuI restriction fragments and formation of a novel 3.2-kb fragment. (B) 293 or 293Cre1 cells were infected with AdLC8 at a m.o.i. of 10 and DNA was extracted at times indicated, digested with PvuI, and separated on a 0.8% agarose gel. The 9.7-kb fragment is not shown. (C) DNA from the agarose gel shown in B was transferred to a nylon membrane and hybridized with a probe derived from pLC8 to monitor early Cre-mediated excision events. The 0–9-hr lanes were exposed to x-ray film for 1 hr, and the 12–48-hr lanes for 10 sec. Fragments of unrecombined AdLC8 are indicated by open arrows and recombination products by filled arrows. Molecular sizes (kb), are given on the left and right margins.

Figure 3

Structure of pRP1001 and vector amplification in AdLC8-infected 293Cre4 cells. (A) pRP1001 (shown in linear form), a derivative of pFG140 (35), contains sequences corresponding to the left end (3–5789 bp interrupted by the insertion of a bacterial plasmid [pMX2] at 1338 bp), and right end (29,792–35,924 bp) of the Ad genome, including the Ad packaging signal (ψ) and inverted terminal repeats. pRP1001 also contains the E. coli β-galactosidase gene (lacZ) between the murine CMV immediate early promoter and the simian virus 40 polyadenylyation signal, and an 8.3-kb insert of lambda DNA. In the presence of AdLC8 in 293 cells, pRP1001 is converted into linear molecules, replicated, and packaged into infectious virions (AdRP1001). (B) Serial passage of AdRP1001 (▪) in AdLC8-infected 293Cre4 cells. The titers on 293 cells (□) and A549 cells (░⃞) are also shown for each passage.

Figure 4

Structure of AdLC8cluc and amplification of AdRP1001 in AdLC8cluc-infected 293Cre1 cells. (A) AdLC8cluc is similar in structure to AdLC8 but lacks the neomycin gene and contains the firefly luciferase gene under regulation of the human CMV promoter and simian virus 40 polyadenylylation signal. (B) Serial passage of AdRP1001 (▪) in AdLC8cluc-infected 293Cre1 cells. The titer of AdLC8cluc at each passage is also shown (□).

Figure 5

Gradient profile of CsCl-purified virus from large scale preparation of AdRP1001 in AdLC8cluc-infected 293Cre1 cells. Fractions (≈40 μl) were collected through the two visible virus bands from the CsCl gradient, and each fraction was analyzed for the presence of AdRP1001 (bfu) and AdLC8cluc (pfu), as indicated in the Materials and Methods. An aliquot of each fraction was also used to infect 293 cells, and cell extracts prepared at 24 hr postinfection were assayed for luciferase activity. The luciferase values were compared with a standard curve of luciferase activity to determine the amount of luciferase protein produced per 10⁶ infected cells.