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Blood, Vol. 114, Issue 3, 526-534, July 16, 2009
Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector
Blood Ramezani and Hawley
114: 526
Supplemental materials for: Ramezani and Hawley
Determination of vector copy number by real-time quantitative PCR
Genomic DNA was extracted using the GenElute Kit (Sigma-Aldrich, St. Louis, MO). Real-time quantitative PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) using the Power SYBR Green PCR Master Mix kit (Applied Biosystems) as described by the manufacturer. The 20-µL reaction volume, which included 5 µL of DNA template, 10 µL of SYBR Green PCR Master Mix, and 300 nM of each primer DNA (300 ng), was amplified in duplicate with human fVIII-specific primers (forward, 5′-GAAAGTCACAGGAGTAACT-3′; reverse, 5′-TCCCTGAAAAACCTTTACT-3′). All fVIII values were normalized with respect to the endogenous murine Gapdh gene, amplified with Gapdh-specific primers (forward, 5′-ATCCCAGAGCTGAACG-3′; reverse, 5′-TGTCTCCTGCGACTTC-3′). A stock of plasmid DNA containing the fVIII cDNA was used to prepare standards of known copy number for the standard curve. DNA extracted from NIH3T3 cells transduced with a single copy of the MSGV-sfVIIIΔB vector was used as an internal reference standard. DNA from naive BAL-fVIIIKO mice was used as a negative control. Reactions consisted of the following thermal cycling conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The plasmid standard dilution series was used to plot a regression curve of Ct against copies of fVIII cDNA. The predicted number of vector copies per cell of each sample was calculated from the regression equation and normalized to the Gapdh reference standard. A conversion factor of 6 pg/diploid genome equivalent was used to calculate the copy number on a per cell basis. Detection of fVIII and anti-fVIII antibodies
fVIII activity was determined by a chromogenic functional assay (COATEST VIII:C/4; DiaPharma Group, Inc., West Chester, OH) as described previously.1 All samples were assayed in triplicate and the means calculated. Reconstituted, normal pooled human plasma (Calibration plasma; DiaPharma Group, Inc., West Chester, OH, USA), which has 1000 mIU or 100% activity, equivalent to 200 ng/ml, was used to generate the standard curve. For determination of in vivo fVIII expression, fVIII activity was assayed in citrated murine plasma. Frozen plasma samples were thawed in a 37°C water bath and immediately assayed by COATEST. The negative control was pooled plasma from BAL-fVIIIKO mice. To measure anti-fVIII antibodies, a control group which included 5 age-matched naive BAL-fVIIIKO mice received 4 intravenous injections of 10 IU plasma/albumin-free recombinant full-length human fVIII (ADVATE, Baxter Healthcare Corp., Westlake Village, CA) in PBS at weekly intervals. PB samples were obtained from the transplanted and the control mice for plasma collection to assess total anti-fVIII antibodies. Antibodies against fVIII were measured as described previously.2 The antibody concentration was reported as the average of at least two readings within the linear portion of the standard curve. The standard curve was generated by serial dilutions of a monoclonal anti-human fVIII light chain antibody (ESH4; American Diagnostica, Inc., Stamford, CT) in PBS/1% BSA/0.05% Tween 20. The sensitivity of the ELISA was ~10 ng/ml. REFERENCES
1. Moayeri M, Ramezani A, Morgan RA, Hawley TS, Hawley RG. Sustained phenotypic correction of hemophilia A mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells. Mol. Ther. 2004;10:892–902.
2. Moayeri M, Hawley TS, Hawley RG. Correction of murine hemophilia A by hematopoietic stem cell gene therapy. Mol. Ther. 2005;12:1034–1042.
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