34. Retroviral Vector-Mediated Neonatal Gene Therapy Results in Long-Term and Therapeutic Levels of Canine Factor VIII Expression in Hemophilia A Mice and Dogs
Authors: Lingfei Xu, Timothy C. Nichols, Rita Sarkar, Stephanie McCorquodale, Dwight Bellinger, Katherine P. Ponder
Journal: Molecular Therapy (2004)

Hemophilia A is a common severe bleeding disorder due to a deficiency of clotting Factor VIII (FVIII). Affected individuals are at risk for spontaneous bleeding, which can be life-threatening. It has been difficult to achieve fully therapeutic levels of expression of FVIII by gene therapy in large animal models. The hypothesis of this study is that neonatal gene therapy with a retroviral vector (RV) could produce a sustained correction of the hemophilic coagulopathy in hemophilia A, and might prevent the development of inhibitory antibodies. A Moloney murine leukemia (MLV)-based RV that contains the liver-specific human 1-antitrypsin promoter, the canine B-domain deleted Factor VIII (cFVIII) cDNA, and the Woodchuck hepatitis virus post-transcriptional regulatory element was generated. Neonatal hemophilia A mice were injected with 11010transducing units (TU)/kg of RV via the temporal vein at birth (N=10). Plasma cFVIII activity was determined by chromogenic substrate assay (COATEST FVIII) using normal canine plasma as the standards. In RV-treated hemophilia A mice, cFVIII activity was 92.5+/-20.1% of normal at 1 month after transduction, and remained high at 109.4+/-10.8% of normal at 11 months thus far. The cFVIII was also functional in an aPTT assay with 23.5% of normal cFVIII levels, and allowed all treated mice to survive after tail-clip. A previous study showed that 50% of mice produced inhibitors after neonatal transduction of a vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped RV expressing human FVIII. However, in our study, none of 12 mice that were transduced as neonates produced cFVIII inhibitors, which was determined by Bethesda assay. To verify the capability of making inhibitors in hemophilia A mice, a single dose of RV was given IV into 6-week old mice at 3.3109 TU/kg (N=3). All three mice made cFVIII inhibitors at 2-3 weeks after transduction. We conclude that completely therapeutic levels of cFVIII can be achieved in mice after neonatal transduction, and neonatal gene therapy with an amphotropic RV expressing cFVIII is not immunogenic. Based upon the above results, we performed neonatal RV transduction in two Chapel Hill hemophilia A dogs. RV was given IV at 3 days after birth at 9109 TU/kg and 7109 TU/kg, respectively. The whole blood clotting time (WBCT) in both dogs has been normalized since day 4 after RV transduction, and remained in the normal range for 4 months thus far. The plasma cFVIII activity in one RV-treated dog was 417% of normal by COATEST assay and 430% of normal by aPTT assay, and the aPTT time was normalized in this dog. The plasma cFVIII activity in another dog was 353% of normal by COATEST assay and 145% of normal by aPTT assay, and the aPTT time was shortened, but not normalized, in this dog. The plasma cFVIII activities have remained stable in both dogs for 4 months thus far. No cFVIII inhibitors were detected. No bleeding has occurred. We conclude that stable expression of therapeutic levels of cFVIII have been achieved in hemophilia A mice and dogs with neonatal transduction of an MLV-based RV.