SCID MPS I Mice Using the Sleeping Beauty Transposon System

SCID MPS I Mice Using the Sleeping Beauty Transposon System

Inborn Errors of Metabolism I administered, is costly, and has not resulted in significant benefit to bone or neurological complications. Murine macro...

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Inborn Errors of Metabolism I administered, is costly, and has not resulted in significant benefit to bone or neurological complications. Murine macrophages treated in-vitro with GP-huGBA DNA formulations efficiently phagocytosed the GP formulations and expressed human glucocerebrosidase. Administration of the GP-GBA DNA formulations to Gaucher mice resulted in extensive particle uptake and increased glucocerebrosidase expression in target tissues. Compared to untreated Gaucher mice, oral administration of GP-GBA DNA formulations to Gaucher mice produced an increase in liver GBA activity and a decrease in tissue Gaucher cells. Preliminary findings from a small pilot study also suggest that this therapy sufficiently corrects tissue GBA activity to ameliorate symptoms in treated, compared to untreated, severely affected Gaucher mice. The potential of this ingestible macrophage targeted strategy to improve delivery and restoration of huGBA to tissues, suggests that this approach could achieve significant reversal of tissue pathology, including bone. In addition to enabling a safer, more efficient and cost effective treatment for Gaucher disease, this macrophage targeted therapeutic strategy could be useful for a wide range of other medical conditions, including low bone density and inflammatory diseases.

122. Long-Term Expression of α-L-Iduronidase and Correction of Lysosomal Pathology in NOD/ SCID MPS I Mice Using the Sleeping Beauty Transposon System

Elena L. Aronovich,1 Jason B. Bell,1 Shaukat A. Khan,2 Lalitha R. Belur,1 Roland Gunther,3 Brenda L. Koniar,3 Patricia A. Schachern,4 Pankaj Gupta,2 Cathy S. Carlson,5 Chester B. Whitley,6 R. Scott McIvor,1 Perry B. Hackett.1 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2Medicine, University of Minnesota, Minneapolis, MN; 3Research Animal Resources, University of Minnesota, Minneapolis, MN; 4Otolaryngology, University of Minnesota, Minneapolis, MN; 5Veterinary Population Medicine, University of Minnesota, St. Paul, MN; 6Pediatrics, University of Minnesota, Minneapolis, MN. MPS I is an autosomal recessive lysosomal storage disorder caused by α-L-iduronidase (IDUA) deficiency. We tested the efficacy of the Sleeping Beauty (SB) transposon system as a gene therapy vector in a mouse model. MPS I mouse exhibits many of the clinical manifestations of the respective human disease, including elevated glycosaminoglycan (GAG) levels in tissues, organomegaly, skeletal dysplasia, and neurologic deficits. To avoid interference of immune responses, NOD/SCID MPS I mouse was used. SB transposon plasmids were constructed for high-level expression of human IDUA. The expression cassette for SB transposase was inserted outside the transposon. Plasmids for transposition control transposon were constructed not to express SB transposase. 25 µg of plasmids were hydrodynamically injected into 4-6-wk old mice, after which about 99% of transgene expression is in the liver. All treated mice showed long-term, high levels of IDUA in plasma. However, females (F) treated with the complete SB system (transposon plus the SB transposase) maintained higher IDUA levels than those treated with the transposon plasmid alone, in which IDUA activity declined to <10-fold WT. In males (M) IDUA remained stable in both groups. Curiously, as of 2 wks post-injection (p.i.) and on, IDUA activities in M compared to F were higher. This gender-related difference was especially striking when the injected DNA dose was reduced from 25 to 5 µg. In mice injected with 25 µg DNA five months p.i. the ranges of IDUA activities in organs of 11 females and 7 males that stably expressed IDUA in treated (T) and untreated (U) mice were:

Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

Liver Spleen Lung Heart Aorta Kidney Sm Intestine Lg Intestine

Females(T) 106-1180 14-152 1-10 1-8 3-8 1-5 0.4-6 0.4-2

Males(T) 727-4733 58-324 4-29 4-14 8-52 2-65 4-14 1-11

Females&Males(U) 6±2 29±14 8±4 4±1 6±1 2±1 5±3 5±1

GAG levels in these tissues and the liver/body weight ratio in treated animals were within normal range compared to untreated NOD/SCID MPS I mice, likely a result of partial to complete restoration of deficient IDUA activity. IDUA activity significantly above background could be also measured in the brain, although the mechanism by which IDUA activity enters the brain is not known. Using faxitron radiography, we obtained images that suggested correction of long-bone pathology that is associated with IDUA deficiency. Our results demonstrate that in the absence of host immune responses, SB-mediated delivery of IDUA to the liver confers longterm (>4 mo), stable transgene expression in mice that can ‘cure’ phenotypic disorders associated with MPS I disease.

123. Humanized Hybrid Human/Porcine Factor VIII Displays High Expression Properties Required for Lentiviral Gene Therapy of Hemophilia A

Christopher B. Doering,1 Gabriela Denning,2 Elisabeth Javazon,1 Bagirath Gangadharan, David McCarty, H. Trent Spencer. 1 Aflac Cancer Center and Blood Disorders Service, Emory University School of Medicine, Atlanta, GA; 2Expression Therapeutics, LLC, Atlanta, GA.

Human coagulation factor VIII (fVIII) has proven difficult to express at therapeutic levels in patients with hemophilia A using clinical gene-transfer technologies. Recently, we showed that the fVIII expression barrier can be overcome in vitro and in vivo using the orthologous porcine fVIII transgene. Furthermore, we demonstrated that 1) the increased expression of fVIII transgenes containing high expression porcine sequences results from an enhanced rate of secretion and 2) the responsible sequence determinants reside in two non-contiguous regions. We now have initiated gene-transfer studies using humanized, high-expression, hybrid human/porcine (HP) fVIII transgenes delivered by self-inactivating (SIN) human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV)-based vectors. These studies were performed using an optimized HP-fVIII construct, designated ET-3, which is 90% identical to the common B-domain-deleted (BDD) human fVIII constructs used in most pre-clinical and clinical studies. VSV-G pseudotyped, recombinant HIV-1-based virus containing an EF1-α promoter, the ET-3 transgene, the woodchuck posttranscriptional regulatory element and a deletion in the U3 region of the 3’ LTR was used to transduce HEK-293 cells at MOIs of 0.3, 0.9 and 2.7. A positive correlation was observed between ET-3 expression and MOI with peak fVIII production of 28 units/106 cells/24 hr from a polyclonal population containing means of of.6 proviral genomes and 5,900 fVIII RNA transcripts per cell. Subsequent clonal analysis revealed similar RNA transcript and fVIII production levels indicating stable gene transfer. For comparison, ET-3 expression was 6-fold greater than was observed following lentiviral transfer using an identical vector encoding a BDD human fVIII construct. Although HIV-ET-3 efficiently transduced HEK-293-T cells, it was ineffective at transducing murine cells. Therefore, murine experiments were performed using a recombinant SIV-based vector containing the MSCV-LTR driving ET-3 expression. Stem cell antigen-1 + hematopoietic stem and progenitor cells were transduced ex vivo prior to transplantation into lethally-irradiated hemophilia A mice. Recipient mice had mean plasma fVIII activity levels of ~0.3 units/ ml (26% normal human level) and 0.1 units/ml at 2 and 12 weeks post-transplantation, respectively, demonstrating long-term fVIII S47