Volume 139, Issue 3, Pages 999-1007.e2 (September 2010) Lentiviral Vectors That Express UGT1A1 in Liver and Contain miR-142 Target Sequences Normalize Hyperbilirubinemia in Gunn Rats  Françoise Schmitt, Séverine Remy, Anne Dariel, Maude Flageul, Virginie Pichard, Sébastien Boni, Claire Usal, Anne Myara, Sophie Laplanche, Ignacio Anegon, Philippe Labrune, Guillaume Podevin, Nicolas Ferry, Tuan Huy Nguyen  Gastroenterology  Volume 139, Issue 3, Pages 999-1007.e2 (September 2010) DOI: 10.1053/j.gastro.2010.05.008 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Time course of serum bilirubin levels in treated animals. Gunn rats received lentiviral vector encoding hUGT1A1 under the control of the mTTR liver-specific promoter carrying or not miR-142 target sequences at a dose of 1.5 × 107 TU/g per animal. Each value plotted represents the mean ± SD for uninjected rat controls (n = 4, closed circles), mTTR.hUGT1A1-injected rats (n = 3, closed squares), and mTTR.hUGT1A1.142T rats (n = 5, open squares). Control rats developed severe hyperbilirubinemia. *P < .05 injected vs control animals. Gastroenterology 2010 139, 999-1007.e2DOI: (10.1053/j.gastro.2010.05.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Detection of hUGT1A1 antibodies by enzyme-linked immunosorbent assay. Antibodies against hUGT1A1 were detected by enzyme-linked immunosorbent assay in serum of (A) mTTR.hUGT1A1-injected and (B) mTTR.hUGT1A1.142T-injected rats (closed squares). Titrations of sera from a Gunn rat injected with oncoretroviral vector encoding hUGT1A1 were used as a positive control (open squares). Each plot represents an individual animal. Results are expressed as arbitrary OD values with a significance threshold of 0.5 at dilutions greater than 1:400. Administration of mTTR.hUGT1A1.142T vector did not lead to formation of hUGT1A1 antibodies except in one animal (#138). Gastroenterology 2010 139, 999-1007.e2DOI: (10.1053/j.gastro.2010.05.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Effect of addition of target sequences to miR-142 in vector backbone on transgene expression level in rat hematopoietic cells. Rat hematopoietic cells were transduced with PGK.GFP.142T (PGK-mirT) and PGK.GFP (PGK) lentiviral vectors. (A) Gunn rat bone marrow and (B) NR8383 cells were transduced at an MOI of 1 or 10. The percentage of GFP-positive cells is indicated in the lower right corner of each plot. Control infections were conducted with medium only (mock). (C) Gunn rat dendritic cells were transduced at an MOI of 20 (left panel). The percentage of transduction (mean ± SD, n = 3) and mean intensity of fluorescence (each plot represents an individual animal) are shown in the middle and right panels, respectively. (D) T-cell response to GFP in rats injected with PGK.GFP.142T or PGK.GFP vectors. Splenocytes were isolated from naive and vector-injected rats and cultured in the presence of recombinant GFP for 4 days following restimulation or not with CD3/CD28 antibodies before FACS analysis to determine the proportion of activated CD8+ and CD4+ T cells. Each plot corresponds to one experimental animal. Gastroenterology 2010 139, 999-1007.e2DOI: (10.1053/j.gastro.2010.05.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Follow-up of vector genome content in the liver after hUGT1A1 gene transfer in cured Gunn rats. Genomic DNA was isolated from liver samples at 1 and 6 months after administration of mTTR.hUGT1A1.miR-142T vectors. The number of vector copies per haploid genome (C/G) was determined by qPCR. Each plot represents an individual animal. Gastroenterology 2010 139, 999-1007.e2DOI: (10.1053/j.gastro.2010.05.008) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Transduction efficacy in the rat liver after intraportal administration of lentiviral vectors. Gunn rats received lentiviral vector encoding GFP under the control of the mTTR promoter at a dose of 1.5 × 107 TU/g (n = 3) and were killed at day 9 postinjection. (A) The presence of GFP-positive hepatocytes in the liver was detected by immunohistochemistry. Hematoxylin counterstained. Original magnification 220×. (B) The number of vector copies/haploid genome (C/G) was determined by qPCR. The percentage of GFP-positive hepatocytes is also indicated for each animal. Gastroenterology 2010 139, 999-1007.e2DOI: (10.1053/j.gastro.2010.05.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Off-target transgene expression from a liver-specific promoter in rat hematopioietic cells. (A) NR8383 rat macrophage cells and (B) Gunn rat dendritic cells were transduced with lentiviral vectors encoding GFP under the control of the mTTR promoter at a MOI of 1 or 10 and 20, respectively before FACS analysis. The percentage of GFP-positive cells and mean intensity of fluorescence are indicated in the lower right corner of each plot. Control infections were done with medium only (mock). Significant expression of GFP was observed from the liver-specific lentiviral vector in transduced rat hematopoietic cells. Gastroenterology 2010 139, 999-1007.e2DOI: (10.1053/j.gastro.2010.05.008) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Hematoxylin and eosin stainings of paraffin-embedded liver sections of non-injected Gunn rat (left) and Gunn rat that has been injected with mTTR.hUGT1A1 vectors (right) at month 6 post-vector delivery. Gastroenterology 2010 139, 999-1007.e2DOI: (10.1053/j.gastro.2010.05.008) Copyright © 2010 AGA Institute Terms and Conditions