Volume 127, Issue 4, Pages 1222-1232 (October 2004) Gene therapy for human α1-antitrypsin deficiency in an animal model using SV40- Derived vectors  Yu–You Duan, Jian Wu, Jian–Liang Zhu, Shu–Ling Liu, Iwata Ozaki, David S. Strayer, Mark A. Zern  Gastroenterology  Volume 127, Issue 4, Pages 1222-1232 (October 2004) DOI: 10.1053/j.gastro.2004.07.058 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Schematic illustration of rSV40-derived vectors: (A) pSV(mAT) and (B) pSV(AT589T). (A) Tag and tag genes were excised from the wild-type SV40 genome, and a polylinker was replaced at the downstream site of the SV40 early promoter (SV40-P), which overlaps with the SV40 origin of replication. The modified SV40 viral genome was recombined with a portion of pT7Blue plasmid to generate a new SV40 construct, pT7SVP, as described in Materials and Methods. Full-length modified cDNA of human α1-AT was cloned into pT7SVP, and the resulting vector is named as pSV(mAT), including an additional copy of SV40-P head-to-tail with the existing viral promoter. (B) Ribozyme 589T cassette was inserted into pT7SV(Δ) and resulted in pSV(AT589T), in which ribozyme 589T is under the control of the tRNA promoter (tRNA-P) in this cassette containing PolIII termination signals. Both rSV40s were produced by removing pT7 plasmid carrier with PmeI, recirculating it, and transfecting it into COS-7 cells. The resulting rSV40s packaged and amplified by COS-7 cells were then band purified by ultracentrifugation. AT589T, human α1-AT ribozyme; hm-alpha AT, the human modified α1-AT cDNA; SV40-PA, SV40 polyA; PmeI, restriction nuclease. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Effects of SV40-derived vectors on human α1-AT mRNA levels in human hepatoma cells. (A) Quantitative RT-PCR analysis of human α1-AT expression in HLE and HLF cells after transduction with SV(mAT), containing human α1-AT–modified cDNA. RNA was extracted from the transduced HLE or HLF cells 72 hours after transduction, and α1-AT mRNA levels were evaluated by quantitative RT-PCR using human glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene. The data were summarized from 2 independent experiments and expressed as fold increase compared with untransduced cells. (B) Quantitative RT-PCR analysis of human α1-AT mRNA levels in PLC/PRF/5 cells after transduction with SV(AT589T), containing α1-AT ribozyme. Human α1-AT mRNA levels in the transduced cells were determined 72 hours after the transduction by quantitative RT-PCR using human glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene and expressed as relative levels of gene expression based on untransduced controls. The data were summarized from 5 independent experiments. ***P < 0.001 compared with controls. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 In situ RT-PCR of a liver section after injection with SV(mAT) via the portal vein. Mice were injected 8 times over 4 days through an indwelling catheter in the portal vein. Each injection consisted of 0.3 mL of high-titer virus (∼1 × 1012 IU/mL). For the amplification of RNA sequences for SV40, reverse transcription was performed for the specific sequences using antisense primers as described in Materials and Methods. Brown color is indicative of SV40 RNA within the cells. (A) Injected mouse liver. The mouse was killed 2 days after the last injection. (B) Untransduced mouse liver. (C) Histology of the liver from a mouse after 2 inoculations a day for 4 days with SV(mAT), containing modified α1-AT cDNA (H&E staining). The mouse was killed 4 days after the last injection. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Northern blot and Western blot analyses of human α1-AT mRNA and protein in ICR mice. (A) Northern blot hybridization analysis of human α1-AT mRNA transcripts in mouse liver after intravenous injection of SV(mAT), containing modified α1-AT cDNA. Mice were injected 8 times over 4 days with 0.3 mL of high-titer virus (∼1 × 1012 IU/mL). Fourteen days after beginning inoculations with the recombinant virus, the mice were killed. RNA was extracted from 2 individual mice (1 and 2) that were infected with SV(mAT). (B) Western blot analysis of human α1-AT protein in the mouse livers. The liver protein was extracted 65 weeks after administration of 8 injections over 4 days with 0.3 mL of high-titer virus (3.5 × 1012 IU/mL). (Upper panel) (for detection of human α1-AT): lane 1, transgenic mice carrying human PiZ allele, 9.0 μg; lane 2, mouse B in Figure 5, 60 μg; lane 3, mouse C in Figure 5, 60 μg; lane 4, control ICR mouse. The dilution of the primary antibody was 1:100, and detection antibody was diluted at 1:1000. (Lower panel) for detection of mouse albumin, 15 ng protein was loaded in each lane; the primary antibodies were diluted at 1:500 and the second antibodies at 1:2000. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Human α1-AT levels in 3 ICR mice transduced with SV40-derived modified human α1-AT cDNA vector. Blood samples were collected by tail incision after the injection of the construct through an indwelling catheter in the portal vein. Mice were injected 8 times over 4 days of 0.3 mL of high-titer virus (3.5 × 1012 IU/mL). Human serum α1-AT levels were determined by ELISA using specific anti-human α1-AT antibodies, and the value for each mouse is shown separately. No human α1-AT protein was detectable in untreated ICR mice at all time points. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Inhibition of human α1-AT in transgenic mice by SV40-derived ribozyme construct. (A) Northern blot analysis of PiZ transcripts in livers of transgenic mice that were treated with SV(AT589T), containing a specific hammerhead ribozyme against human α1-AT. Mice were injected 8 times over 4 days with 0.3 mL of high-titer virus (1.3 × 1012 IU/mL). Fourteen days after the injection through an indwelling catheter in the portal vein, the mice were killed. RNA was extracted from the liver, and Northern blot analysis was performed. Lane 1, Hep G2 cells; lane 2, ICR mouse; lane 3, untransduced mouse transgenic for PiZ allele; lane 4, transgenic mouse inoculated once a day for 4 days; lane 5, transgenic mouse inoculated twice a day for 4 days. (B) Western blot analysis of the same experiment shown in A. A transgenic mouse was injected twice a day for 4 days and then killed 14 days after infection (the same mouse as shown in lane 5 in A). Proteins from HepG2 cells and untransduced transgenic (Tg) mouse liver were used as positive controls. HepG2 cells, 5 μg; mouse liver samples (20 μg each sample). The primary antibodies were diluted at 1:400 and the second antibodies at 1:2000. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 Serum human α1-AT levels in transgenic mice after the injection of SV40-derived ribozyme construct. Ribozyme-inoculated mice were assayed at intervals, shown in the graph, after the inoculations. Mice were injected 8 times over 4 days with 0.3 mL of high-titer virus (4.4 × 1012 IU/mL). Serum α1-AT levels were measured by ELISA as described in Materials and Methods. (A) Three mice had relatively high basal levels of human α1-AT. (B) Two additional transgenic mice with relatively low basal levels of human α1-AT before the inoculation. Serum human α1-AT levels in 7 control transgenic mice were unchanged during the experiments. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 8 Sustained inhibition of human α1-AT synthesis in the livers of transgenic mice by SV40-derived ribozyme construct. (A) Western blot analysis of human α1-AT protein in livers of transgenic mice that were injected with SV(AT589T)-containing ribozyme. Mice were injected 8 times over 4 days with 0.3 mL of high-titer virus (4.4 × 1012 IU/mL). (Upper panel) Western blot analysis of human α1-AT protein in the transgenic mice. Lane 1, transgenic (Tg) control mouse 1 carrying human PiZ allele without ribozyme treatment (serum human α1-AT level, 854 μg/mL); lane 2, the molecular weight standard; lane 3, the Tg control mouse 2 without ribozyme treatment (serum human α1-AT level, 747 μg/mL); lane 4, mouse D in Figure 7 was killed 6 weeks after injection (α1-AT levels before ribozyme, 175 μg/mL); lane 5, mouse A in Figure 7 was killed 12 weeks after injection (α1-AT level before ribozyme, 1830 μg/mL); lane 6, mouse B in Figure 7 was killed 12 weeks after injection (α1-AT level before ribozyme, 1306 μg/mL); lane 7, mouse C in Figure 7 was killed 16 weeks after injection (α1-AT level before ribozyme, 2439 μg/mL); lane 8, control ICR mouse (CM, not transgenic). Twenty micrograms of protein was loaded in each lane; the primary antibodies were diluted at 1:400 and the second antibodies at 1:2000. (Lower panel) Western blot analysis of mouse albumin levels in the corresponding mouse liver. Fifteen nanograms of protein was loaded in each lane; the primary antibodies were diluted at 1:400 and the second antibodies at 1:2000. (B) Quantitative analysis for Western blots by densitometry. Relative density was calculated based on transgenic control mouse 2. The labeling is the same as in A. Gastroenterology 2004 127, 1222-1232DOI: (10.1053/j.gastro.2004.07.058) Copyright © 2004 American Gastroenterological Association Terms and Conditions