Figure 1.
AAV Single-Strand Split gfp Vector System.
A) Two AAV genomes are depicted, vectors A and B, that have the following genetic arrangement: i) vector A contains the CMV promoter upstream of a partial gfp coding sequence (cds) followed by a splicedonor site, and ii) vector B contains intron elements, the remainder of the gfp cds followed by a poly-adenylation signal. The inverted terminal repeats (ITRs) of the distinct genomes are labeled 1–4 and yellow highlights the intron region. B) The functional concatemer: a single DNA molecule for the ITR 2–3 linkage which results in functional GFP after splice removal of the intron. C) DNA 80-mers are depicted individually having 40 nt of homology to the color matched positions on vectors A and B. The crooked lines represent the splice donor and acceptor sequences and the intron is highlighted in yellow.
Figure 2.
Oligonucleotides Direct AAV2 Concatemerization.
A) Human embryonic kidney cells were co-infected with the split gfp vectors (A and B; Fig. 1) at a multiplicity of infection of 200 for each, vector A and B. Then, DNA oligonucleotides (80 nM) displaying 40 nucleotides of different sequence homology to vector A and B genomes were transfected in (see Fig. 1, non-homologous = NH). Three days post-treatment, GFP+ cells were quantitated by flow cytometry. An 80-mer without vector A and B homology was used as the non-homologous control (NH). The tested oligos are described (Fig. 1 and in the text) and opposite polarities were also investigated (Fwd = sense = non-template strand, Rev = anti-sense). Results are presented as %GFP + cells. B) Fluorescent microscopy of cells treated as in A) with a NH oligo or the H-Ib Fwd oligo used for transfection. C) Cells were treated as described in A). Additionally a single vector with the identical split gfp reporter as the split vectors (a CMV promoter upstream of the gfp coding sequence interrupted by the hcg intron) was investigated (single vector Gfp). Cells treated as in A) with the split vectors, or the single vector, followed by oligo transfection were harvested 3 days post-treatment. Western blotting was then performed to quantitate Gfp protein (NH 2X = twice the total protein loaded compared to all other lanes). Using densitometry, the relative levels of Gfp abundance was determined and the acquired values are also depicted.
Figure 3.
Characterization of Oligo-Assisted AAV Genome Concatemerization.
A) Human embryonic kidney (HEK) cells were co-infected by AAV2 single strand split gfp vectors at a multiplicity of infection (MOI) of 200 for each vector. Then, either H-Ib Fwd or the non-homologous (NH) oligos were used for transfection (80 nM). GFP positive cells were quantitated by flow cytometry at the indicated times. B) HEK cells were co-transduced with the indicated MOI of each split gfp vector and subsequently transfected with the H-Ib Fwd oligo or a NH control (80 nM). Three days later the number of GFP+ cells was determined by flow cytometry. C) HEK cells were co-transduced with the AAV2 split gfp vectors at a MOI of 200 and the indicated concentrations of H-Ib Fwd was used for transfection. GFP+ cells were determined on day 3. The presented results were not normalized to the efficiencies of co-infection and oligo transfection. D) Oligo size analysis for stimulation of OAGR. Cells were co-infected with the split vector system as above and then either the H-Ib Fwd oligo or derivatives of that sequence were then used for transfection. %GFP positive cells were determined on day 3 by flow cytomtery. nt = nucleotide.
Figure 4.
Junction Characterization of Functional Concatemers.
A) This cartoon depicts our AAV split gfp vector system and the position of primers (block arrows) used for functional concatemer amplification. B. Evaluation of amplification products by southern blotting (gfp probe) showed a unique band formed only from DNA isolated for H-I treated cells. This band was directly cloned and sequenced.
Figure 5.
Oligo-Assisted AAV Genome Concatemerization of Self-Complementary Split gfp Vectors.
A) Four split gfp systems are depicted for self-complimentary (sc) AAV2 genomes. The genomes differ in the orientation of their respective cassettes (see text for details) in relation to the open or closed ends of the molecule. A broken box encases the type of junction necessary for functional GFP. B) The pairs of split gfp vectors depicted in A were used for co-infection of human embryonic kidney cells at a multiplicity of infection (MOI) of 200 for each particle type. Then, either H-Ib Fwd or a non-homologous (NH) oligo was used for transfection. Three days later, GFP+ cells were determined by flow cytometry. The results are presented as fold-induction which is the number of GFP+ cells given the H-Ib oligo divided by the number of GFP+ cells given the NH oligo.
Figure 6.
Oligo-Assisted AAV Genome Recombination of Circularization Dependent AAV.
A) The cartoon depicts single vector split gfp systems that are capable of GFP production after either intra- or inter-molecular dimerization. The relevant system features are depicted; the arrow represents the CMV promoter followed by the 5′ portion of the gfp coding sequence (cds) and an splice donor sequence. On the far left side is the hcg intron and the remaining gfp cds followed by a poly-adenylation sequence. The entire intron is highlighted in yellow. Both the single-strand (ss) and self-complementary (sc) versions are shown. B) AAV2 capsids packaged with either genome depicted in A were used to separately infect human embryonic kidney cells at the indicated viral genomes per cell. Then, either the H-Ib Fwd or a non-homologous (NH) oligo was used for transfection. Three days later, GFP+ cells were determined by flow cytometry. In all cases the single-strand construct demonstrated a significant (unpaired student t test) increase, yet less than 2-fold, with the H-Ib Fwd oligo compared to the NH control. No significant changes were note for the sc construct.
Figure 7.
A Model for Oligo-Assisted AAV Genome Recombination.
Following the capsid release of the 4.7 kb AAV genomes, presumably in the nucleus, oligos with homology to each vector anneal to their single-strand (ss) target, displacing the remaining terminal ends of the genome. This annealing event promotes the desired genome dimer orientation and viral genome replication using the inverted terminal repeats for initiation. Extension of the 3′ oligo end is also a possibility. Exploitation of host repair enzymes for intermolecular recombination follows resulting in a double strand product with the vector junction sequence corresponding to that of the oligo. In the case of the H-Ib oligo this recombination event results in an approximate 400 bp deletion of the ITR junction. The recombined dimer is near double the length of the independent genomes. Although it was not conclusively demonstrated, this model favors physical incorporation of the transfected oligo into the oligo-directed concatemer (described in discussion). Also depicted are perhaps the naturally favored products initially following transduction, circular monomers, which do not produce GFP.