954. Toward Hemophilia A Gene Therapy: Safety-Modified Gammaretroviral Vector Expressing a Secretion-Enhanced FVIII Molecule with Reduced Immunogenicity

that it may be possible to express high levels of a secreted protein from HSC-derived erythroid cells engraftcd in minimally ablated subjects. The prospect ofcombining reduced intensity conditioning, a reduced risk of insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increases the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.

954. Toward Hemophilia A Gene Therapy: Safety-Modified Gammaretroviral Vector Expressing a Secretion-Enhanced FVIII Molecule with Reduced Immunogenicity Ali Ramezani , Teresa S. Hawley, Robert G. Hawley. J Department ofAnatomy and Cell Biology. and the Flow Cytometry Core Facility, The George Washington University Medical Center; Washington. DC. HemophiliaA is an X-linked recessive genetic bleeding disorder caused by a deficiency or functional defect in coagulation factor VIII (FVIII). We have previously demonstrated the potential of hematopoietic stem cell-directed retroviral-mediated FVIII gene transfer as a curative therapeutic strategy for hemophilia A (Moayeri et al., Mol. Ther. 12:I034-1042, 2005). However, the emergence of leukemia in gene therapy patients with X-linked severe eombined immunodeficiency disease due to retroviral vector insertional mutagenesis has prompted reevaluation ofthe safety profile ofretrovira1mediated gene delivery, We recently constructed and evaluated a self-inactivating retroviral vector (MSinSB) which not only mediates persistent high level transgene expression in both murine and human hematopoietic stem/progenitor cells, but unlike the parental MSCV vector, remains transcriptionally active when transduced murine embryonic stem cells are differentiated into hematopoietic cells in vitro (Ramezani et al., Mol. Ther. 14:245-254,2006). Based on the outcome of this study, we have selected the MSinSB vector for further safety modifications which are anticipated to reduce the risks of oncogene activation during rctroviral genomic integration. MSinSB was first modified to generate the RMSinSB vector in which the U3 enhancer/promoter region of the 5' LTR was replaced with that of the Rous sarcoma virus promoter and the U5 region of the 3' LTR was deleted, further minimizing vector sequence homology. In an attempt to reduce the risk of insertional activation of neighboring oncogenes, the enhancer-blocking components of the chicken ~-globin 5'HS4 insulator (a single copy of the 42-bp FII element) and a homologous region from the human 'l-ccll receptor alB BEAD-A insulator were inserted into the U3 region of the SIN 3' L:I'R of RMSinSB, creating RMSinEB . Furthermore, in order to prevent vector RNA read-through into adjacent genomic sequences, the safety-modified Woodchuck hepatitis virus posttranscriptional regulatory element (OPRE) was incorporated into the 3' untranslated region, giving rise to RMSinOEB. Using a new flow cytometrybased assay that we developed, the FlIIBEAD-A combination - which is <100 bp in length - was shown to be as effective as the prototypical 1.2-kb 5'HS4 insulator in enhancer blocking activity. Insertion of the OPRE caused a 3-fold increase in both the titer and expression levels ofthe RMSinOEB vector. In parallel , a B domaindeleted FVllI transgene was optimized for more efficient glycosylation-facilitated secretion by incorporating amino acids 778-996 ofthe B-domain and concomitantly rendered less immunogenic by introducing theA2-domain mutations R484A1R489A1P492A. This bioengineered enhanced FVIII (cFVIII) transgene was subcloned into the RMSinOEB vector. Experiments to evaluate the genotoxic potential of the RMSin-eFVllI-OEB vector in hematopoietic stem cell mutagenesis assays and to assess the in vivo therapeutic efficacy and inhibitory antibody response to eFVIII in hemophilia A mice are currently under investigation, and the findings obtained wiII be presented. S364

955. An Essential Role of Factor VIII Light Chain in Facilitating Heavy Chain Secretion

Lingxia Chen,' Fuxiang Zhu,' Juan Li,3 Hui Lu,' Haiyan Jiang,' Shangzhen Zhou, I Rita Sarkar,' Valder R. Arruda, 1 Jinhui Wang, I Chuma J. Chike-Obi,' Hua Zhu,' Glenn F. Pierce,' Steven W. Pipe/ Xiang-Qin Liu,2 Xiao Xiao,' Rodney M. Camire; Weidong Xiao.' JDepartment ofPediatrics, University ofPennsylvania Medical Center and The Children s Hospital ofPhiladelphia. Philadelphia, PA; 2Department ofBiochemistry and Molecular Biology, Dalhousie University, Halifax. NS. Canada; 3Molecular Pharmaceutics. School of Pharmacy; University ofNorth Carolina . Chapel Hill. NC.."Preclinical Development. Bayer HealthCare Pharmaceuticals. Berkeley. CA.. SDepartment ofPediatrics. University ofMichigan, Ann Arbor; MI. Coagulation Factor VllI (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. However, FVIII heavy chain itself is secreted 10-100 fold less efficiently than the light chain. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIIl heavy chain secretion, which suggested a role of factor VllI LC in regulating HC secretion. To further delineate the mechanism of FVIII secretion, we designed an intein strategy to study the effects of the addition of light chain to the heavy chain on the secretion of FVllI molecules. The accurate and efficient intein-mediated trans-splicing reaction allowed us to compare the secretion of heavy chain alone and heavy chain joined with light chain in the ER or the Golgi . We compared FVIII heavy chain secretion with or without light chain via posttranslational protein trans-splicing. As demonstrated in vitro, ligation of the light chain to the heavy chain significantly increased heavy chain secretion. Such heavy chain secretion increases were also confirmed in vivo by hydrodynamic injection of FVllI intein plasmids into hemophilia A mice. The control groups injected with pLC-INT alone or pHC-INT alone did not show any clotting activity. However, when these two plasm ids were injected together, they produced a vel)' high level of FVllI clotting activity, reaching 50% of positive control group receiving BDD-FVIll expressing plasmid. In addition, such enhancement of He secretion can also observed when the light chain is supplied in trans, which is probably caused by the spontaneously association of heavy chain and light chain in the secretion pathway. To further characterize how the FVlII light chain might affect the FVlII heavy chain secretion, we constructed FVIlI expressing plasmid, pFVlII-LC-d) and pFVlII-LC-d2, which include deletions in the light chain region. Only )-2% ofthe secreted protein level ofpBDD-FVlII was detected by ELISA . In contrast, intracellular levels of FVlII protein for these defective mutants were approximately twice as much as that BDD-FVlII expressing plasmid. It suggested that the main effects caused by such deletions were in the process ofsecretion . Collectively, FVlIIlight chain may facilitate FVlII secretion through promoting correct heavy chain folding . Factor VIII heavy chain alone or defects in the light chain region seriously impair its secretion.

956. A High Specific Activity Factor VIII Variant and Its Application to AAV-Mediated Gene Transfer for Hemophilia A

Vi-Lin Liu,' Raffaella Toso,' Heesoon Chang; Rodney Camirc.P Katherine High.P JHematology. Children s Hospital ofPhiladelphia. Philadelphia, PA; 2University ofPennsylvania. Philadelphia. PA.

Hemophilia A (HA) is an inherited X-linked bleeding disorder caused by absence or low level of functional factor VIII (FVlII). Molecular 'Therapy Volume 15. Supplement I• .\1.,)' 2007 C () p y ri~ ht © "1l1CAmerican Society of Gene Therapy