Figure 1.
Construction and characterization of scAAV vectors. (A) Structure of rAAV hFIX vectors. Each vector is shown schematically as it is packaged in the virion, with scAAV vectors shown as dimers. ssAAV-HCR-hAAT-FIX consists of the human apolipoprotein E/C-I gene locus control region (HCR) and the human α1 antitrypsin promoter (hAAT), a chicken β actin/rabbit β globin composite intron (IVS), 1.6-kb human FIX cDNA (hFIX), and a bovine growth hormone polyadenylation signal (BGpA) flanked by the AAV internal terminal repeats (ITRs shown as hairpin loop). Self-complementary scAAV-LP1-hFIXco vector containing the LP1 promoter consisting of core liver-specific elements from HCR (base pairs 134 to 442 of GenBank record HSU32510) and hAAT (base pairs 1747 to 2001 of GenBank record K02212), modified SV40 small t antigen intron (base pairs 4644 to 4552 of GenBank record J02400), codon-optimized hFIX (hFIXco), and SV40 late polyA (SV40 LpA; base pairs 2600 to 2733 of GenBank record J02400), and a deleted 3′ trs (Δtrs). scAAV-LP1-hFIX contains the wild-type human FIX cDNA from which the 3′ untranslated region has been deleted (ΔhFIX) instead of hFIXco. ssAAV-LP1-hFIXcoS has intact 5′ and 3′ ITR and in addition contains 760 bp noncoding stuffer sequence from the β-lactamase (Δbla). (B) Characterization of ss and scAAV2/8 viral particles. Viral particles were electrophoresed on a 1% neutral or alkaline agarose gel, Southern blotted, and hybridized with a vector-specific probe. The controls consisted of approximately 4.6- and 2.3-kbp fragments that were derived from ssAAV HCR-hAAT FIX and scAAV LP1-hFIXco plasmids cut with a double cutter, which cuts outside the ITR regions. The scAAV genome migrated with the 4.6-kb marker on the alkaline agarose gel and 2.3-kb marker on neutral gel, while the migration pattern of ssAAV vectors of differing size on these 2 gels did not change.

Construction and characterization of scAAV vectors. (A) Structure of rAAV hFIX vectors. Each vector is shown schematically as it is packaged in the virion, with scAAV vectors shown as dimers. ssAAV-HCR-hAAT-FIX consists of the human apolipoprotein E/C-I gene locus control region (HCR) and the human α1 antitrypsin promoter (hAAT), a chicken β actin/rabbit β globin composite intron (IVS), 1.6-kb human FIX cDNA (hFIX), and a bovine growth hormone polyadenylation signal (BGpA) flanked by the AAV internal terminal repeats (ITRs shown as hairpin loop). Self-complementary scAAV-LP1-hFIXco vector containing the LP1 promoter consisting of core liver-specific elements from HCR (base pairs 134 to 442 of GenBank record HSU32510) and hAAT (base pairs 1747 to 2001 of GenBank record K02212), modified SV40 small t antigen intron (base pairs 4644 to 4552 of GenBank record J02400), codon-optimized hFIX (hFIXco), and SV40 late polyA (SV40 LpA; base pairs 2600 to 2733 of GenBank record J02400), and a deleted 3′ trs (Δtrs). scAAV-LP1-hFIX contains the wild-type human FIX cDNA from which the 3′ untranslated region has been deleted (ΔhFIX) instead of hFIXco. ssAAV-LP1-hFIXcoS has intact 5′ and 3′ ITR and in addition contains 760 bp noncoding stuffer sequence from the β-lactamase (Δbla). (B) Characterization of ss and scAAV2/8 viral particles. Viral particles were electrophoresed on a 1% neutral or alkaline agarose gel, Southern blotted, and hybridized with a vector-specific probe. The controls consisted of approximately 4.6- and 2.3-kbp fragments that were derived from ssAAV HCR-hAAT FIX and scAAV LP1-hFIXco plasmids cut with a double cutter, which cuts outside the ITR regions. The scAAV genome migrated with the 4.6-kb marker on the alkaline agarose gel and 2.3-kb marker on neutral gel, while the migration pattern of ssAAV vectors of differing size on these 2 gels did not change.

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