403. Long-Term, High alpha-L-iduronidase Expression in MPS I Dogs Following Neonatal, Intravenous, Retroviral Vector Gene Therapy

METABOLIC DISORDERS & LYSOSOMAL STORAGE DISEASES 401. Quantitative Bioluminescence Imaging of Transgene Expression In Vivo Garrett R. Rettig,1 Marie McAnuff,1 Dijie Liu,1 Ji-Seon Kim,1 Kevin G. Rice.1 1 College of Pharmacy, Medicinal Chemistry, The University of Iowa, Iowa City, IA. Bioluminescent imaging (BLI) is a widely used in vivo method to determine the location and relative intensity of luciferase expression in mice. In the present study, we have rigorously validated BLI to establish its utility as a quantitative measurement of luciferase expression for comparing the efficiency of non-viral gene transfer vectors in vivo. Precise quantities of plasmid DNA encoding the luciferase gene were hydrodynamically dosed in mice and luciferase expression was measured at different time intervals following an i.p. dose of D-luciferin. BLI provided a linear response when dosing 100 pg to 5 mg of plasmid DNA and integrating the light units measured in liver, resulting in a dynamic range that spanned 5orders of magnitude. The level of luciferase expression was found to be a direct function of D-luciferin dose. BLI was used to serially sample mice to determine the time course of luciferase expression and to establish the influence of multi-dosing of substrate. BLI was found to be just as sensitive as a standard luciferase assay applied to homogenized mouse liver. The results establish a limit-of-detection of 20 pg of luciferase/liver following a hydrodynamic dose of 100 pg of plasmid DNA. BLI was also utilized to quantify the efficiency of knockdown of gene expression following siRNA dosing. The results demonstrate that BLI is both sensitive and linear and allows for the direct comparison of the efficiency of gene transfer vectors that target the liver.

402. Intracellular Trafficking of QD-FRET Nanoparticles for Gene Delivery Hunter H. Chen,1 Yi-Ping Ho,2 Tza-Huei Wang,2 Kam W. Leong.3 1 Biomedical Engineering, Johns Hopkins University, Baltimore, MD; 2Mechanical Engineering, Johns Hopkins University, Baltimore, MD; 3Biomedical Engineering, Duke University, Durham, NC. Development of a safe and effective nonviral gene vector remains a significant challenge in the field of gene therapy. Rational design of more efficient gene carriers will be possible only with mechanistic insights of the rate-limiting steps in the nonviral gene transfer process. In order to examine the intracellular transport of DNA nanoparticles and their dissociated individual components, we have labeled nanoparticles with quantum dots (QD) and fluorescent organic dyes as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). QDs have emerged as efficient FRET donors due to their broad absorption and narrow emission spectra, and high quantum yield, thus minimizing cross-talk between the donor and acceptor. Chitosan and plasmid DNA (pEGFP-C1) were individually labeled with a fluorescent organic dye and CdSe-ZnS quantum dots, respectively. Nanoparticles were subsequently formed by complex coacervation. Size (100-400 nm) and zeta-potential (+14 mV) of the nanoparticles, as determined by photon correlation spectroscopy and laser Doppler anemometry, were not affected. Emission at 670 nm due to FRET from intact chitosan-DNA nanoparticles was confirmed by single molecule detection, fluorescence microscopy, and spectrofluorometry (Fig. 1A). TEM images showed the encapsulation of the QD-labeled plasmids within nanoparticles (Fig. 1B). When the nanoparticles were disrupted by addition of heparin and/or chitosanase, FRET was reduced or abrogated (Fig. 1A), indicating the high sensitivity of FRET to detect subtle changes in nanoparticle state. The intracellular distribution and unpacking of QD-FRET chitosan-DNA nanoparticles in different cell types were S154

followed by confocal microscopy. Intact and decomplexed nanoparticles were observed in acidic vesicles within the cell and in the perinuclear space within 4 hours after transfection. By 48 hours post-transfection, GFP-positive cells containing released plasmid DNA were also detected. This study represents a first attempt to evaluate the trafficking and unpacking behaviors of chitosan-DNA nanoparticles intracellularly, which are significant transfection barriers for this type of nanoparticles.

METABOLIC DISORDERS & LYSOSOMAL STORAGE DISEASES 403. Long-Term, High alpha-L-iduronidase Expression in MPS I Dogs Following Neonatal, Intravenous, Retroviral Vector Gene Therapy Anne Traas,1 Ping Wang,1 Xiucui Ma,2 Patricia O’Donnell,1 Meg Sleeper,1 Gus Aguirre,1 Mark Haskins,1 Katherine P. Ponder.2 1 Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; 2Internal Medicine, Washington University, St. Louis, MO. Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficient activity of alpha-L-iduronidase (IDUA). Manifestations include bone and joint disease, heart disease, and neurological dysfunction. The MPS I dog has a G to A transition in the donor splice site of intron 1 creating a premature termination codon at the exon-intron junction. The disease in the dog resembles the clinical manifestation in humans. We treated six MPS I dogs at three days of age intravenously with 2.5 to 10.1 X 10E9 (mean 3.6+/-2.9 SD) transducing units (TU)/kg of a retroviral vector (RV) expressing canine IDUA from the human 1-antitrypsin promoter (hAAT-cIDUA-WPRE). The group had 22-749 nmol/ml/hr of serum IDUA activity (mean=337+/-334; normal=14.6 +/-2.4, N=7) (Fig. 1). The 3 oldest dogs at more than 1 year of age have stable activity at 1.3x, 2.1x, and 41x normal. The serum activity was not proportional to the dose. Clinically in the dog, early manifestations of MPS I are much less severe than those of MPS VII previously reported. Early signs include facial dysmorphia, prolapse of the 3rd eyelid, corneal clouding, mitral valve insufficiency, and joint disease. The 3 RVtreated dogs at 1 year of age showed improvement in corneal clouding, facial dysmorphia, 3rd eyelid prolapse, and valvular heart disease. Corneal clouding scores were 0/3,1/3, and 0/3 for the RV-treated dogs (serum IDUA activity of 35, 22, and 684 nmol/ml/h, respectively) compared with scores of 2/3 for four 6 month-old untreated MPS I dogs. Scores for facial dysmorphia were 0/4 for all RV-treated dogs compared to 2-3/4 for 3 untreated dogs. No treated MPS I dog exhibited 3rd eyelid prolapse, a sign seen in all untreated dogs. At 1 year of age, none of the 3 RV-treated dogs had a cardiac murmur, whereas two of the three oldest untreated MPS I dogs had a grade 2/6 murmur of mitral insufficiency. Two untreated dogs were euthanized at 1 year of age due to signs of severe cervical spinal cord disease, no signs of which are present in any of the treated dogs, the oldest of which is 17 months. One dog was treated Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

METABOLIC DISORDERS & LYSOSOMAL STORAGE DISEASES with 1.3X10E9 TU/kg at 7 weeks of age and at 6 weeks posttreatment has 2.9 nmol/ml/hr (17% of normal) IDUA activity in serum (I-173, Fig 1), which has been stable since 4 days posttransduction. These data show that in contrast to previous treatment modalities, neonatal, intravenous, RV gene therapy was successful in producing stable serum IDUA levels in MPS I dogs, improving early clinical disease.

combination of two AAV2/1 vectors expressing individually PAH and GTPCH-PTPS were co-injected into the same hind leg muscles. As a control, an AAV2/1-vector expressing only PAH with GTPCH was not therapeutic. This non-invasive application is the basis to develop an efficient therapy for PKU using a triple-cistronic gene transfer into skeletal muscle.

405. Correction of the Murine Model of Hereditary Tyrosinemia Type I Using Messenger RNA as a Source of Transposase for Sleeping Beauty Mediated Integration of the FAH Gene

404. Therapeutic Correction of PKU in a Mouse Model by Ectopic Expression of PAH and Its BH4Cofactor Genes in Skeletal Muscle by a Recombinant Triple-Cistronic AAV2-Based Pseudotype 1 Vector Zhaobing Ding,1 Cary O. Harding,2 Alexandre Rebuffat,1 Lina Elzaouk,1 Jon A. Wolff,3 Beat Thony.1 1 Department of Pediatrics, University of Zurich, Zurich, Switzerland; 2Department of Pediatrics and Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR; 3Department of Pediatrics and Department of Medical Genetics, University of Wisconsin, Madison, WI. Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism with a deficiency of the hepatic phenylalanine hydroxylase (PAH) leading to toxic accumulation of circulating phenylalanine (Phe) in blood, resulting in growth failure, microcephaly, seizures, and mental retardation. PAH catalyzes the hydroxylation of Phe to tyrosine and needs oxygen and tetrahydrobiopterin (BH4) as cofactor. BH4 biosynthesis requires the consecutive action of the enzymes GTPCH and PTPS, with dihydroneopterin triphosphate as the first intermediate. Here we aimed at expressing the PAH system in skeletal muscle to degrade serum Phe in PKU patients, as this tissue is abundant, easy accessible, and persistent due to its post-mitotic nuclei. However, BH4 is abundant in liver but scarce in skeletal muscle, as the cofactorsynthesizing enzyme GTPCH is absent in muscle tissue, and PTPS is expressed at low levels. We first demonstrated that transgenic PKU mice that had no liver PAH and expressed coordinately PAH along with GTPCH in skeletal muscle tissue accumulated dihydroneopterin triphosphate and remained hyperphenylalaninemic unless synthetic BH4-cofactor was supplied by intraperitoneal injections. Thus, PTPS activity is definitely limiting in skeletal muscle to synthesize sufficient BH4 and to support Phe hydroxylation. A recombinant triple-cistronic AAV2based pseudotype 1 vector expressing PAH along with the two cDNA-genes for BH4 biosynthesis, GTPCH and PTPS, was then generated. Upon single injections of at least 3.5x10e12 recombinant triple-cistronic AAV2/1 vector particles into each of the gastrocnemius muscles of the hind legs of the PKU mouse model Pah-enu2 resulted in long-term clearance of blood Phe, including complete phenotypic reversion. A similar therapeutic effect was achieved when a Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

Andrew Wilber,1,2,3 Kirk J. Wangensteen,1,4 Yixin Chen,7 Lijuan Zhou,7 Joel L. Frandsen,1,2,3 Jason Bell,1 Zongyu Chen,6 Stephen C. Ekker,1,3,4 R. Scott McIvor,1,2,3,6 Xin Wang.5,6,7 1 The Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, Minneapolis, MN; 2Gene Therapy Program, Institutue of Human Genetics, University of Minnesota, Minneapolis, MN; 3Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 4Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN; 5Stem Cell Institute, University of Minnesota, Minneapolis, MN; 6Cancer Center, University of Minnesota, Minneapolis, MN; 7Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN. Sleeping Beauty (SB) is a DNA transposon capable of mediating chromosomal integration and stable expression in vertebrate cells when co-delivered with a source of transposase. In all pre-clinical reports where SB-mediated gene insertion in somatic cells has been used to correct mouse models of human disease, the transposase component has been provided as a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to remobilization and reintegration of transposons. Such continued expression of transposase is a key safety concern in development of the SB transposon system for clinical applications. As an alternate source of transposase, we have previously shown that in vitro transcribed transposase-encoding messenger RNA (mRNA) can effectively mediate transposon insertion both in vitro and in mouse liver (Wilber et al., Molecular Therapy, 2005, in press). Here, we test the use of transposaseencoding mRNA plus transposon DNA for gene therapy of hereditary tyrosinemia type I by first evaluating several parameters for systemic delivery and expression of mRNA in mice. We also introduce a method to quantitatively track repopulating liver cells by in vivo bioluminescence imaging after co-delivery of a DNA or RNA source of transposase with a bi-functional transposon encoding both mouse fumaryl acetoacetate hydrolase (FAH) and firefly luciferase (luc) genes in FAH deficient mice by rapid, high-volume injection into the tail vein. Liver repopulation was quantitatively monitored over time by increasing luc activity, measured as light emitted from the liver. Using this method, we determined that supplying SB transposase in the form of mRNA results in selective repopulation of corrected hepatocytes with stable co-expression of both FAH and luc. Plasma taken from animals 5 months after co-infusion with transposase mRNA contained levels of succinylacetone (the clinical determinant of tyrosinemia) that were nearly normalized. Amino acid levels were also normalized, suggesting normal liver metabolism of catabolized protein products (including urea and glucose). We further demonstrated the stability of integration by transplanting hepatocytes (250,000) into FAH deficient recipient mice. All transplanted animals survived NTBC withdrawal and gained weight consistently over a period of 90 days and demonstrated stable expression of luc. In summary, we demonstrate for the first time S155