Cre/loxP-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging

Abstract

Previous analyses have demonstrated that packaging of the adenovirus type 5 (Ad5) genome is dependent on at least seven cis-acting elements, called AI to AVII, which are located in the left-end region of the genome. These elements have different packaging efficiencies, and without AI through AV, viral DNA cannot be packaged. Here we report the identification of the cis-acting Ad5 packaging domain in vivo by using the Cre/loxP system. We found that an adenoviral DNA fragment (nt 192 to nt 358), which includes elements AI to AV, is excised by Cre recombinase and packaged into capsids. Furthermore, this mutant adenovirus replicated so efficiently by repetitive propagation that its purification by CsCI equilibrium gradient was possible. This study clarified that the region from nt 358 to nt 454 on the viral genome is sufficient for packaging. Recently, the helper-dependent adenoviral vector (HDAd) construction system has been developed for the purpose of gene therapy. This system uses a helper virus with two parallel loxP sites flanking the packaging signal. This region is eliminated by Cre-mediated excision, which prevents helper virus packaging. Our data provide useful information regarding factors affecting efficient elimination.

Influence of loxP insertion upstream of the cis‐acting packaging domain on adenovirus packaging efficiency

“…3a, columns 1, 5, and 9), which are nearly equal to the expected initial ratio, namely, 1 / (1 + 0.3) ≈ 0.8 (dotted line, columns 1-12). The GFP ratios were approximately 0.2, 0.2 and 0.2 (columns 13, 17 and 21, respectively), which were also nearly equal to the expected initial ratio: 0.3 / (1 + 0.3) ≈ 0.2 (dotted line, columns [13][14][15][16][17][18][19][20][21][22][23][24].…”

“…AdLC8cluc, which is the most popular helper virus used for the construction of HDAdV, contains a set of loxP at 191 nt in SgrAI and at 358 nt (9 (24). Thus, the 454-nt position appears to be better than the 358-nt position for the downstream insertion of loxP in the helper virus because all seven A-repeats present between 194 nt and 380 nt (19) are removed by Cre/loxP deletion.…”

“…The virus titers of helper viruses containing loxP at 143 nt in the AflIII cleavage site or at 192 nt in the BsrGI site have been studied (24,26) (Fig. 1a).…”

Self-inactivating helper virus for the production of high-capacity adenoviral vectors

“…Interestingly, infective particles can be detected, but PCR and Southern blot analysis revealed that virtually all of them are devoid of C. Encapsidation of viral genomes in the absence of all the core elements that integrate the packaging signal is a very inefficient event 21,22 that probably requires the forced conditions established here, and generates inactive particles. 23 Alternatively, viral genomes can attach to the capsids and become internalized in cells without being really encapsidated. 24 This suggests that further reduction of HV contamination, if necessary, would require additional mechanisms different from the selective cleavage of C. Other methods based on delayed HV encapsidation 11 or genome size restrictions 25 can be complemented with the strategy described here.…”

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper‐dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

“…The following cell lines were used: human embryonic kidney cells (HEK293), monkey kidney cells COS7 (American Type Culture Collection, Manassas, VA, USA) and HEK293 cells that stably express the Cre recombinase (Cre-293) [12]. These cells were maintained as monolayers in Dulbecco's modified Eagle's medium (D-MEM) (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 50 U/ml penicillin and 50 U/ml streptomycin (Sigma, St. Louis MO, USA).…”