Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells by Dana N. Levasseur, Thomas M. Ryan, Kevin M. Pawlik, and Tim M. Townes Blood Volume 102(13):4312-4319 December 15, 2003 ©2003 by American Society of Hematology

Insertion of a potent antisickling human β-globin gene into a lentiviral vector. Insertion of a potent antisickling human β-globin gene into a lentiviral vector. (A) Schematic of the human β-globin LCR and downstream genes. HS cores are indicated by arrows. Genes are illustrated by filled boxes and are represented according to scale. (B) The lentiviral/antisickling β-globin expression construct is shown. HS2 (1203 bp), HS3 (1213 bp), and HS4 (954 bp) sequences, the 3′ globin enhancer, the 266-bp β-globin promoter (βp) and the βAS3 globin gene are drawn to scale. The HIV-1 LTR is shown with a 3′ SIN deletion; ψ indicates packaging signal; SD and SA, splice donor and acceptor sites, respectively; RRE, Rev-responsive element; cPPT/CTS, central polypurine tract or DNA flap/central termination sequence; and WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (C) Lentiviral vector copy number determination. Lane 1 is the PCR product obtained from bone marrow of a KO-TG sickle mouse after amplification, labeled primer extension, and Bsu36I digestion; lanes 2, 3, and 4 are products of representative lenti/βAS3-transduced mice; and lane 5 is a knockout-transgenic animal expressing exclusively human HbAS3. Dana N. Levasseur et al. Blood 2003;102:4312-4319 ©2003 by American Society of Hematology

Isolation of highly purified Sca-1+c-Kit+Lin- HSCs from sickle mice Isolation of highly purified Sca-1+c-Kit+Lin- HSCs from sickle mice.WBM was sorted on a MACS column to obtain Sca-1-enriched cells. Isolation of highly purified Sca-1+c-Kit+Lin- HSCs from sickle mice.WBM was sorted on a MACS column to obtain Sca-1-enriched cells. Three-color preparative FACS was then performed to isolate Sca-1+, c-Kit+, Lin-/low HSCs. Purified KLS cells satisfy gating requirements for R1 (A), R2 (D), and R3 (C). R4 (B) indicates the c-Kit+ and Lin-/low cell population. Dana N. Levasseur et al. Blood 2003;102:4312-4319 ©2003 by American Society of Hematology

Therapeutic βAS3-globin expression levels in murine erythrocytes following lentiviral gene delivery. Therapeutic βAS3-globin expression levels in murine erythrocytes following lentiviral gene delivery. (A) IEF of hemolysates from recipient mice at 5 to 7 months after transplantation. The first 4 lanes are human HbS, murine hemoglobin, human HbAS3, and mock-transduced controls. The next 5 lanes are 5 primary lenti/βAS3 mice reconstituted with more than 99% donor cells. The final lane is a representative secondary transplant at 4 months after transplantation. (B) Single-cell expression analysis of lenti/βAS3 reticulocytes. Following thiazole orange staining, reticulocytes were sorted by flow cytometry. mRNA was isolated from individual lysed cells, reverse transcribed, PCR amplified, radioactively labeled by primer extension, and digested with Bsu36I, which digests βAS3 but not βS. Data are from 13 representative reticulocytes. Population controls of 100 cells from the same transplant (TP), an HbAS3 knockout-transgenic (AS3), or a donor knockout-transgenic sickle mouse (S) are also shown. (C) Lentiviral vector copy number determination in bone marrow G/M, erythroid progenitors, and WBM from secondary recipients at 7 months after transplantation. G/M is granulocyte/macrophage (Gr1+ and CD11b/Mac1+), T is erythroid progenitor (Ter119+), and WBM is whole bone marrow. Dana N. Levasseur et al. Blood 2003;102:4312-4319 ©2003 by American Society of Hematology

Correction of abnormal RBC morphology in lenti/βAS3 rescued transplants. Correction of abnormal RBC morphology in lenti/βAS3rescued transplants. (A) Blood smear from a mock-transplanted animal with characteristic sickled erythrocytes and a pronounced reticulocytosis. (B-C) Two representative primary transplant recipients of βAS3-transduced stem cells. No sickled cells were observed in any fields examined. (D) Wild-type C57Bl6 control. Original magnification × 120. Blood smears were stained with Wright-Giemsa. Dana N. Levasseur et al. Blood 2003;102:4312-4319 ©2003 by American Society of Hematology

Amelioration of spleen, liver, and kidney pathology in lenti/βAS3 mice Amelioration of spleen, liver, and kidney pathology in lenti/βAS3 mice.(A) Spleen, liver, and kidney sections were analyzed at low (original magnifications are × 10 for spleen and kidney and × 40 for liver; top 3 rows and bottom row) and high magnification ... Amelioration of spleen, liver, and kidney pathology in lenti/βAS3mice.(A) Spleen, liver, and kidney sections were analyzed at low (original magnifications are × 10 for spleen and kidney and × 40 for liver; top 3 rows and bottom row) and high magnification (original magnification, × 100; bottom rows for spleen and liver and the middle row for kidney). In lenti/βAS3-transduced mice, normal splenic red and white pulp is observed and virtually no pools of sickle erythrocytes or infarcts are evident. In livers of lenti/βAS3 animals, focal areas of necrosis and aggregation of sickled erythrocytes are not observed; also, extramedullary hematopoiesis and hemosiderin deposition are absent. Kidneys of lenti/βAS3 mice appear normal and free of the disruptive vascular RBC pooling and hemosiderin deposits observed in mock-treated animals. (B) Correction of splenomegaly in lenti/βAS3-transduced mice. All sections except the bottom panel were stained with hematoxylin-eosin; the bottom panel was stained with Gomori iron. Dana N. Levasseur et al. Blood 2003;102:4312-4319 ©2003 by American Society of Hematology