1. An Adenoviral Expression System for AAV Rep78 Using Homologous Recombination 
Cheryl A. Carlson, Dmitry M. Shayakhmetov, André Lieber 
Molecular Therapy 
Volume 6, Issue 1, Pages (July 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Reconstitution of the AAV rep78 gene by homologous recombination between two first-generation Ad vectors. (A) Schematic of the recombination principle. Two first-generation parental Ad vectors were created such that one, Ad.5‘rep78, contained a non-functional 5'portion of the rep78 gene (5‘rep78) under constitutive control of an ApoE AAT promoter [26] in place of the normal Ad El region. We insulated the promoter from upstream viral regulatory elements by the addition of the chicken β-globin HS-4 domain (HS4) [49]. The other parental vector, Ad.3‘rep78, contained an SV40 polyadenylation signal (SV40pA) followed by a non-functional 3’ portion of the rep78 gene (3‘rep78) in place of the El region which overlapped with the 5’ portion by 661 bp. (Note that for convenience, we depicted the Ad.3’ rep78 vector in the opposite orientation from the Ad.5‘rep78 vector.) Upon co-infection of permissive cells and viral DNA replication, homologous recombination occurs between the parental vectors creating a progeny genome, ΔAd.rep78, devoid of all viral genes with an ApoEhAAT driven functional rep78 gene. The numbers shown for the rep78 gene correspond to base pair numbers from the wild-type AAV2 genome where the translation start site is base pair 321. The rep78 gene used in this study was modified [20] to not express any of the other Rep proteins. Ψ, Ad packaging signal; Ad ITR, Ad inverted terminal repeat; Ad E2, E3, E4, Ad E2 region, Ad E3 region except base pairs 28597–28602 and 30005–30750 (based upon pJM17 [50]), and the E4 region. (B) Southern blot analysis of 6 μg of total DNA extracted from 293 cells 48 hours after infection with Ad.5‘rep78 (10 PFU/cell), Ad.3‘rep78 (10 PFU/cell), or Ad.5‘rep78 (5 PFU/cell) plus Ad.3‘rep78 (5 PFU/cell) (Combined). The expected sizes are 35.9 kb for Ad.5‘rep78, 34.6 kb for Ad.3‘rep78, and 5.5 kb for ΔAd.rep78. The entire rep78 gene was used as a probe.
Molecular Therapy 2002 6, 91-98DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Schematics of the rescue substrates used to assess Rep78 functionality. (A) We employed two different plasmids containing AAV ITRs flanking a transgene cassette as rescue substrates. Rep78 can excise or “rescue” the AAV ITR flanked transgene cassettes. One plasmid consisted of 10.3 kb that yielded a 3.5-kb monomeric rescue product; the other consisted of 6.7 kb giving a 4.9-kb monomeric rescue product. (B) A second type of rescue substrate was created by homologous recombination as depicted. We designed a parental first-generation Ad vector with a transgene cassette and inserted it into the Ad E1 region flanked by 1.2-kb inverted repeat sequences (IRs) having an AAV ITR located at the 5’ end. Upon viral DNA replication, the inverted repeats mediate homologous recombination generating the progeny genome ΔAd.AAVITR with duplicated AAV ITRs. ΔAd.AAVITR genomes are suitable substrates for rescue by Rep78. Ψ, Ad packaging signal; Ad ITR, Ad inverted terminal repeat; Ad E2, E3, E4, Ad E2 region, Ad E3 region except base pairs 28597–28602 and 30005–30750 (based upon pJM17 [50]), and the E4 region.
Molecular Therapy 2002 6, 91-98DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Demonstration of the functionality of the reconstituted rep78 gene by rescue assay. (A) Rep78 rescue assay in 293 cells. The left panel employed the 10.3-kb plasmid as a rescue substrate that yields a 3.5-kb monomeric rescue. The right panel used the ΔAd.AAVITR recombination system (Fig. 2B) to provide a substrate for generating a 5.0-kb monomeric rescue product. We infected 293 cells as indicated with Ad.5‘rep78, Ad.3‘rep78, or Ad.AAVITR at 5 PFU/cell. (An irrelevant control vector was used to bring the total MOI up to 15 PFU/cell in each case.) Two hours post-infection, we transfected the cells by calcium phosphate co-precipitation for six hours as shown with 5 μg of plasmid rescue substrate, 5 μg of a plasmid expressing Rep78 from a CMV promotor (pCMVrep78) as a positive control, or an irrelevant plasmid to bring the total transfected DNA up to 10 μg in each case. Thirty hours after infection, total DNA was extracted, and 6 μg of each sample was analyzed by Southern blot with a rescue substrate specific probe located internal to the two AAV ITRs. Arrows indicate co-migration of viral and chromosomal DNA (v/c), nicked circular rescue plasmid substrate (ncs), linear plasmid substrate (ls), supercoiled plasmid substrate (scs), ΔAd.AAVITR monomers of rescue substrate (ms), ΔAd.AAVITR dimers of rescue substrate (ds), respective monomers of rescued product (mrp), and respective dimers of rescued product (drp). (B) Rep78 rescue assay in Hep 3B cells. We employed the ΔAd.AAVITR recombination system as the rescue substrate. Hep 3B cells were infected as indicated with Ad.5‘rep78, Ad.3‘rep78, Ad.AAVITR, or wild-type Ad5 (wt Ad5). Wild-type Ad5 was applied at 10 PFU/cell. The other first-generation vectors were applied at 30 PFU/cell with an irrelevant vector to bring the total MOI up to 100 PFU/cell when wild-type Ad5 was present or 83 PFU/cell in the absence of wild-type Ad5. Two and a half hours post-infection with wild-type Ad5 (complete cytopathic effect) or 72 hours without wild-type Ad5, we extracted total DNA and analyzed 10 μg of each sample by Southern blot with a rescue substrate specific probe located internal to the two AAV ITRs. Arrows depict the same gel migrations as in the right panel of (A).
Molecular Therapy 2002 6, 91-98DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Delivery of functional reconstituted transgenes by purified ΔAd.rep78 vectors. Rep78 mediated rescue activity was assessed in 293 cells by employing the 6.7-kb plasmid that yields a 4.9-kb monomeric rescue product as the rescue substrate. First, we transfected the 293 cells by calcium phosphate co-precipitation for 6 hours with 5 μg of rescue plasmid template and 5 μg of pCMVrep78 (positive control) or an irrelevant plasmid (to bring the total transfected DNA up to 10 μg in each case) as shown. Next, we infected the cells for three hours with ΔAd.rep78, Ad.5‘rep78, Ad.3‘rep78, or an irrelevant first-generation control vector (Ad.control), or both at the indicated number of genomes per cell where “X” equals 100 genomes per cell (note: 100 genomes of a first-generation Ad vector roughly corresponds to 5 PFU). After 3 hours, remaining virus was removed and one set of infections was treated with 10 mM HU (right panels). We had to reverse the order for transfection and infection in this rescue assay because HU treatment adversely affects transduction efficiency and must be initiated before Ad vectors begin to replicate. Total DNA was extracted 48 hours post-infection. We analyzed 6 μg of each sample by Southern blot analysis with a rescue substrate specific probe (upper panels) and a rep78 specific probe (lower panels). We exposed the right panels longer than the left ones for demonstrative purposes (note the relative intensities of pCMVrep78 and the plasmid rescue substrate). The following gel migrations are indicated by arrows: 1) co-migration of viral and chromosomal DNA (v/c); 2) nicked circular rescue plasmid substrate (ncs); 3) linear plasmid substrate (ls); 4) supercoiled plasmid substrate (scs); 5) rescue product monomer (mrp); 6) nicked circular pCMVrep78 (ncr); 7) linear pCMVrep78 (lr); 8) supercoiled pCMVrep78 (scr); and 9) ΔAd.rep78 (Δ).
Molecular Therapy 2002 6, 91-98DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions