Volume 18, Issue 8, Pages 1553-1558 (August 2010) Ex Vivo Transduction and Transplantation of Bone Marrow Cells for Liver Gene Delivery of α1-Antitrypsin  Hong Li, Yuanqing Lu, Rafal P Witek, Lung-Ji Chang, Martha Campbell-Thompson, Marda Jorgensen, Bryon Petersen, Sihong Song  Molecular Therapy  Volume 18, Issue 8, Pages 1553-1558 (August 2010) DOI: 10.1038/mt.2010.116 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Ex vivo transduction of bone marrow (BM) cells. Mouse BM cells were seeded in a 24-well plate (1 × 104 cells/well; n = 3) and infected with the Lenti-CB-hAAT vector at 100 multiplicity of infection (MOI), rAAV1-CB-hAAT, rAAV8-CB-hAAT at 104 MOI, and phosphate-buffered saline (PBS), respectively. The cumulative hAAT in the culture medium was measured by enzyme-linked immunosorbent assay. Circles, Lenti-CB-hAAT; triangles, rAAV1-CB-hAAT; squares, rAAV8-CB-hAAT; dashes, lower limit of quantification (LLOQ). hAAT level of PBS group (negative control) was below LLOQ. hAAT, human α1-antitrypsin. Molecular Therapy 2010 18, 1553-1558DOI: (10.1038/mt.2010.116) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Transplantation of BM cells into mouse liver. (a) Outline of the experiment. The recipients (female C57BL/6) were i.p. injected twice (2-week interval) with 50 mg/kg of monocrotaline (MCT) and received partial hepatectomy (PHx) to remove 70% of liver mass before transplantation. BM cells were isolated from the femurs and tibias of male C57BL/6 mice. The newly purified BM cells were infected with Lenti-CB-hAAT, rAAV1-CB-hAAT, rAAV8-CB-hAAT vector, respectively, for 2 hours, washed, and transplanted into the recipient liver by portal vein injection or intrasplenic injection. Serum hAAT levels were monitored by human AAT-specific enzyme-linked immunosorbent assay. Recipients were killed at 14 weeks after transplantation. Liver repopulation was measured by immunostaining. (b–i) Detection of expression of human α1-antitrypsin (hAAT) in recipient liver after transplantation of viral vector–infected BM cells by immunostaining. (b) Liver section from C57BL/6 mouse transplanted with Lenti-CB-hAAT-infected BM cells (brown). (c) Image b view at larger magnification. (d) Liver section from C57BL/6 mouse transplanted with rAA1-CB-hAAT-infected BM cells. (e) Image d view at larger magnification. (f) Liver section from C57BL/6 mouse transplanted with rAAV8-CB-hAAT-infected BM cells. (g) Image e view at larger magnification. (h) Human liver section served as positive control. (i) Liver section from untransplanted C57BL/6 mouse served as negative control. (j) Quantification of hAAT-positive cells in rAAV8-hAAT (n = 3), Lenti-hAAT (n = 3), and rAAV1-hAAT (n = 4) groups. BM, bone marrow. Molecular Therapy 2010 18, 1553-1558DOI: (10.1038/mt.2010.116) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Detection of donor cells in recipient liver. (a–c) Double fluorescence immunostaining for human α1-antitypsin (hAAT) and green fluorescent protein (GFP). (a) Liver section from C57BL/6 mouse transplanted with rAAV8-CB-hAAT-infected bone marrow cells showing hAAT expression (red). (b) Liver section same as in a stained for GFP with anti-GFP antibody (green). (c) Merge image of a and b. Representative slides were viewed at ×100 magnification. (d–f) Fluorescence in situ hybridization (FISH) for Y chromosome in the recipient liver. Green signal shows X chromosome (X); red signal shows Y chromosome (Y). Molecular Therapy 2010 18, 1553-1558DOI: (10.1038/mt.2010.116) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Hepatic differentiation of bone marrow (BM) cells. Coexpression of human α1-antitypsin (hAAT) and mouse albumin detected by immunostaining. (a,b) Liver section from C57BL/6 mouse transplanted with rAAV8-CB-hAAT-infected BM cells stained for hAAT (brown). (c,d) Liver section adjacent to the section in a and b, respectively, stained for albumin (red). Black arrows point to both AAT-positive and albumin-positive cells. Asterisks: location indicator. Representative slides were viewed at ×20 magnification. Molecular Therapy 2010 18, 1553-1558DOI: (10.1038/mt.2010.116) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Multiorgan homing of transplanted bone marrow (BM) cells. (a) Spleen section subjected to fluorescence in situ hybridization for detecting Y chromosome. (b) Quantification of GFP+ cells by immunostaining. Representative images of immunostaining for GFP (brown) in spleen (c,d), in bone (e,f), and in lung (g,h). (a,c,e,g) Tissue sections were from female C57BL/6 mouse at 8 weeks after transplantation with rAAV8-CB-GFP vector–infected male BM cells. (d,f,h) Tissue sections from untransplanted C57BL/6 mouse served as negative control. Black arrowheads indicate the observed GFP staining. Representative slides were viewed at ×20 magnification. GFP, green fluorescent protein. Molecular Therapy 2010 18, 1553-1558DOI: (10.1038/mt.2010.116) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Detection of expression of human α1-antitrypsin (hAAT) in the recipient serum. Bone marrow cells from C57BL/6 mice were infected with ssAAV8-CB-hAAT vector at 1 × 104 particles/cells for 2 hours and transplanted into the liver of partially hepatectomized C57BL/6 recipient (2 × 107 cells/mouse; n = 4) by splenic injection. The transgene expression was monitored by measuring the serum level of hAAT. Squares are the serum from the treatment group; dashes are the lower limit of quantification (LLOQ). The serum level of hAAT from untransplanted C57BL/6 mouse (negative control) was below the LLOQ. Molecular Therapy 2010 18, 1553-1558DOI: (10.1038/mt.2010.116) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions