Correction of Sickle Cell Disease in Transgenic Mouse Models by Gene Therapy Robert Pawliuk, Karen A. Westerman, Mary E. Fabry, Emmanuel Payen, Robert Tighe, Eric E. Bouhassira, Seetharama A. Acharya, James Ellis, Irving M. London, Connie J. Eaves, R. Keith Humphries, Yves Beuzard, Ronald L. Nagel, and Philippe Leboulch

Supplemental Figure 1. (A) Hb^A-T87Q and HbF are potent inhibitors of HbS polymerization in vitro in contrast to HbA. The concentration of deoxygenated HbS in equilibrium with the polymer (CSAT) was determined using the p50 method (1) for various mixtures of HbS with test hemoglobins (1:1 ratio). p50 is the oxygen pressure at which half of the molecules of Hb are deoxygenated, as detected spectroscopically. Because the HbS polymer has a much lower oxygen affinity than HbS in solution, a sudden jump in p50 indicates the start of the formation of the polymer, and the concentration of Hb at which the jump occurs gives the CSAT value, expressed in g/dl. (B) Top: Southern blot analysis for proviral stability. DNA was digested with Afl II and probed with an exonic fragment of the human -globin gene. Lane 1, NIH 3T3 negative control; lanes 2-4, bone marrow, spleen and thymus DNA, respectively, from a representative C57Bl/6 recipient of ^A-T87Q-globin transduced bone marrow sacrificed 5 months post-transplantation; lanes 5 and 6, DNA from 2 day 12 spleen colonies generated using bone marrow from the primary C57Bl/6 recipient sacrificed 5 months post-transplantation. The expected 7.5Kb proviral band and a 3.2Kb endogenous band (EB) are marked in the left margin. Bottom: average proviral copy number in genomic DNA isolated from blood of ^A-T87Q-globin transduced C57Bl/6 mice 3 months post-transplantation (bar = SE). Quantification was performed by densitometry and comparison to NIH3T3 cells known to contain one copy of the provirus. (C) Diagram of the ^A-T87Q-globin provirus. HIV LTR, human immune deficiency type-1 virus long terminal repeat; + , packaging signal; cPPT/flap, central polypurine tract / DNA flap; RRE, Rev-responsive element; P, -globin promoter (from SnaB I to Cap site); ppt, polypurine tract. The 3´ -globin enhancer (up to downstream Avr II site), the 372 bp IVS2 deletion, the ^A-T87Q mutation (ACA Thr to CAG Gln) and DNase I hypersensitive sites (HS) 2 (Sma I to Xba I), HS3 (Sac I to Pvu II) and HS4 (Stu I to Spe I) of the -globin LCR are indicated. Southern blot analysis showed titers reaching 1.5 × 10⁹ infectious units per ml after pseudotyping with Vesicular Stomatitis Virus glycoprotein-G (VSV-G) and physical concentration.

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Supplemental Figure 2. Isoelectric focusing of RBC lysates from recipient mice 3 months post-transplantation showing the expected species of Hb. Lanes 1 and 2, blood deriving from SAD transplanted marrow; lanes 3 and 4, blood deriving from BERK transplanted marrow; lanes 1 and 3, mock transduction; lanes 2 and 4, transduction with ^A-T87Q-globin lentivirus. ^M, mouse -globin; ^H, human -globin; ^SIN, mouse single -globin; ^SAD, human SAD -globin; ^S, human sickle -globin; ^A-T87Q, human ^A-T87Q globin.

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Analysis of Position Effect Variegation. We asked whether the observed pancellular expression of human ^A-T87Q-globin protein in RBCs was the result of position-independent expression of the transferred ^A-T87Q-globin gene. To address this question, bone marrow from a representative primary C57Bl/6 recipient sacrificed 5 months post-transplantation was used to generate day 12 spleen colonies in secondary recipient mice. Southern blot analysis was performed on genomic DNA isolated from 20 individual spleen colonies following digestion with BamH I, which cuts only once within the integrated provirus. Ten distinct clones were observed with an average proviral copy number of 3.4 ± 0.3 (SE) (range 2-5). Primer extension analysis of mRNA from spleen colonies showed a wide variation in the amount of human ^A-T87Q-globin mRNA (data not shown). These results suggest that pancellular expression was the result of balanced expression from polyclonal stem cell reconstitution with multiple chromosomal integration sites rather than true position-independent expression.

II. Materials and Methods:

Whole-blood p50 analysis:

p50 measurements on whole blood were performed using a Hemoscan (Aminco, Silver Spring, MD).

Vector construction and virus production:

The human beta-globin gene including its promoter and 3´ enhancer sequence was cloned from BGT9 (2). A 374bp fragment was deleted from intron 2 and hypersensitive sites 2-4 were cloned from BGT33 (3). Virus stocks were generated by transient transfection of 293T cells with the recombinant lentiviral vector together with separate plasmids expressing HIV-1 Gag-Pol, Rev and Tat. DNA sequence of the vector will be provided upon request. One liter of virus was concentrated by ultracentrifugation at 25,000 RPM for 90 minutes at 4°C and the viral pellet resuspended in 300l of serum free medium (Life Technologies, Frederick, MD). The absence of replication competent retrovirus (RCR) was verified by mobilization assay as described (4). Viral titers were determined by Southern blot analysis.

Southern blot analysis:

Southern blot analysis was performed using standard methods (4). A ³²P-labeled exonic fragment of the human -globin gene was used as a probe. Quantification of vector copy number was achieved by densitometry using a phosphoimager with ImageQua^TM software (Molecular Dynamics, Sunnyvale, CA).

Bone marrow transduction and transplantation:

Donor mice were injected 4 days before bone marrow harvest with 150mg/kg of 5-fluorouracil (5-FU). Cells were prestimulated overnight in serum free medium (Life Technologies, Frederick, MD) supplemented with 200mM L-glutamine, 6ng/ml of murine Interleukin-3, 10ng/ml of human Interleukin-6, 10ng/ml of murine Interleukin-1 and 100ng/ml of murine Stem Cell Factor (Peprotech, Rocky Hill, NJ). Cells were exposed to concentrated viral supernatants on Retronectin^TM (Biowhittaker, East Rutherford, NJ) coated plates for 5-6 hours in the presence of 8g/ml of protamine sulfate (Sigma, St. Louis, MO). Following infection, cells were harvested and injected, without selection, into recipient mice given 1100cGy (¹²³Cs -rays) of total body irradiation (split dose of 550cGy over 3 hours).

FACS analysis:

RBCs were washed in PBS, fixed, permeabilized and stained with an FITC-labeled monoclonal antibody that specifically recognizes human HbA (PerkinElmer Wallac, Norton, OH). Samples were analyzed on a FACScan flow cytometer (Becton Dickinson, San Diego, CA).

Primer extension analysis.

Total RNA was extracted from 100-200l of blood using TRIzol reagent (LifeTechnologies, Frederick, MD). Primer extension was performed using the Primer Extension System-AMV Reverse Transcriptase kit (Promega, Madison, WI) according to the manufacturer's instructions. Primers for human ^A-globin [5´-CAGTAACGGCAGACTTCTCCTC-3´] and mouse ^single-globin [5´-TGATGTCTGTTTCTGGGGTTGTG-3´] generate extension products of 90bp and 53bp respectively. Extension products were radioactively labeled by including ³²P dCTP in the reaction mixture. Reactions were performed using 1g of RNA and run on a denaturing 7% polyacrylamide gel. Radioactive bands were quantified by phosphoimager analysis. Measurements were corrected for the number of dCTP residues in human (9) and mouse (6) extension products.

Protein analysis.

Quantification of -globin chains and Hb by HPLC was performed as described (5, 6).

RBC sickling:

Sickling of erythrocytes was studied as a function of PO2, from 0 to 150 mm Hg. Blood was diluted with PBS (340 mOsm) containing 5 mM glucose and 0.5 g/dl of bovine serum albumin. Cells were equilibrated with a mixture of air and nitrogen at the desired PO2, for 30 minutes at 37°C in a rotary shaker, and fixed by the addition of 4% formaldehyde equilibrated at the same PO2 and temperature. The reversal of sickling was determined after incubation of the fully deoxygenated cell suspension by reoxygenation with air at 0°C for one hour, prior to fixation with the formaldehyde solution equilibrated with air at 0°C. Proportions of sickle and non-sickle cells were determined by microscopy using Nomarski optics.

Hematology:

Red cell counts and total Hb were measured on a CBC analyzer (CBC Technologies, Oxford, CT). Reticulocytes were analyzed using the Sysmex SE 9000 system (Sysmex Corp of America, Long Grove, IL).

Urine concentrating ability:

Mice were deprived of water for 24 hours. At the end of this period, urine was collected onto Parafilm and the osmolarity measured after a 1:10 dilution with distilled water using a Microosmette (Precision Systems, Natic, MA).

RBC density gradients:

Density gradients were performed as previously described (7).

Delay time of HbS polymerization:

Polymer formation upon deoxygenation of purified Hb or membrane free hemolysates was studied by measuring the delay time of polymerization with the method described by Adachi and Asakura (8). In brief, after a temperature jump from 0°C to 30°C of the deoxygenated samples in a solution of 1.80 M potassium phosphate (pH 7.4) and 2mM of sodium dithionite, the turbidity induced by HbS polymerization was recorded at 700nm The probability factor for nucleation was derived from the measurement of the delay time performed at various Hb concentrations.

References for Supplementary Material:

1. R. E. Benesch et al., Analytical Biochem. 89, 162 (1978).
2. P. Pasceri, D. Pannell, X. Wu, J. Ellis, Blood 92, 653 (1998).
3. J. E. Rubin, P. Pasceri, X. Wu, P. Leboulch, J. Ellis, Blood 95, 3242 (2000).
4. R. Pawliuk, R. Kay, P. Lansdorp, R. K. Humphries, Blood 84, 2868 (1994).
5. M. Fabry et al., Blood 86, 2419 (1995).
6. C. Papadea and J. C. Cate, Clin. Chem. 42, 57 (1996).
7. M. Fabry et al., Blood 78, 217 (1991).
8. K. Adachi, T. Asakura, J. Mol. Biol. 144, 467 (1980).