586 choroid plexus directed viral gene therapy as a source of alpha n acetyl glucosaminidase enzyme replacement in cerebrospinal fluid for sanfilippo b syndrome

586 Choroid Plexus Directed Viral Gene Therapy as a Source of alpha N Acetyl Glucosaminidase Enzyme Replacement in Cerebrospinal Fluid for Sanfilippo B Syndrome Molecular Therapy Volume 21, Supplement[.]

Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

S224

PCC activity, survive to adulthood, and have signifi cant elevations

in propionyl-carnitine, methylcitrate, glycine, alanine, lysine,

ammonia, and markers associated with cardiomyopathy similar to

those in PA patients This adult model of PA allowed head to head

comparison of different gene therapy vectors for their effects on

PCCA expression, phenotype, and survival Liver-tropic adenovirus

serotype 5 (Ad5) vectors and adeno-associated virus 2/8 (AAV8)

vectors were engineered with identical codon-optimized human

PCCA cDNAs Ad5 and AAV8 vectors were injected intravenously

into adult mice at varied doses and the effects of the two vectors were

compared based on the number of PCCA genes that were delivered

Both vectors reduced propionyl-carnitine and methylcitrate levels,

but Ad5 mediated more rapid increases in PCCA protein and PCC

activity in the liver At equal or even 10-fold lower cDNA doses,

Ad5 mediated more robust PCCA protein expression in the mice As

expected, phenotypic correction was transient with fi rst generation

Ad5, whereas AAV8 mediated long-lasting effects Long-term head

to head comparisons of cDNA dose by AAV8, AAV-rh10, and

helper-dependent Ad5 and Ad6 vectors are underway and will be discussed

These data suggest that this PA model may be a useful platform for

optimizing PA therapeutics

584 Transcription Activator-Like Effector

Nuclease-Based Genome Editing for

Mucopolysaccharidosis Type I

Mark J Osborn,1 Colby G Starker,1 Anthony P DeFeo,1 Daniel F

Voytas,1 Bruce R Blazar,1 Jakub Tolar.1

1 University of Minnesota, Minneapolis, MN.

Transcription activator like effector nucleases (TALENs) are

genome-editing reagents that generate a DNA break at a user-defi ned

site in the genome and hold great promise as a tool for precision gene

repair Our goal is to apply TALENs for mucopolysaccharidosis Type

IH (MPS IH) Two TALEN candidates, specifi c for the 392X murine

IDUA gene mutation were constructed using the Golden Gate TALEN

assembly procedure and tested for their ability to mediate gene editing

by homologous recombination of a marker sequence The ability of

the TALEN candidates to mediate genomic modifi cation was assessed

in murine C1498 cells as well as fi broblasts derived from MPS mice

We observed activity consistent with TALEN cutting and DNA repair

by the two major repair pathways: non-homologous end joining

(NHEJ) and homology directed repair (HDR) We then employed the

TALEN in conjunction with an exogenous synthetic donor DNA that

contains the corrective base for the 392X mutation that allows for

selective outgrowth of corrected cells Following introduction of the

TALEN and donor into MPS fi broblasts we were able to document

permanent genotypic correction of the 392X mutation by direct

sequencing Conclusions: Our results to date show the applicability

of genome-editing strategies using engineered nucleases for MPS

This provides proof-of-concept for patient-specifi c gene-editing/

correction in primary human fi broblasts and serves as a platform for

translational ex vivo therapies

585 Gene Therapy for Mouse Model of Krabbe Disease

Hiroshi Kobayashi,1 Masamichi Ariga,1 Takahiro Fukuda,3 Takeo Iwamoto,4 Sayoko Izuka,1 Yota Shimada,1 Yoshikatsu Eto,2 Toya Ohashi.1,2

1 Dept of Gene Therapy, Institute of DNA Medicine, Jikei University School of Medicine, Tokyo, Japan; 2 Department of Genetic Disease and Genome Science, Jikei University School of Medicine, Tokoy, Japan; 3 Department of Neuropathology, Jikei University School of Medicine, Tokyo, Japan; 4 Division of Biochemistry, Research Core Facilities, Jikei University School of Medicine, Tokyo, Japan.

Krabbe disease is genetic neurodegenerative disease, due to genetic defi ciency of -galactocerebrosidase (GALC), results in the degeneration of myelin-forming cells (oligodendrocytes and Schwann cells) caused by an accumulation of galactosylsphingosine (psychosine), and the symptoms as the rapid progressive deterioration

of the nervous system (especially in infantile form) We tried gene therapy for mouse model of this disease We constructed 2nd generation lentiviral vector, with MCU3 promoter including GALC and GFP expression gene Firstly we checked this vector’s function

by transfection for some cell lines as 293A, Twi-2 (fi broblast from tail tip of Krabbe disease model mouse), detected dose dependent enzyme expression And we tried in vivo study with neonatal model mice We injected intravenously for facial vein of neonatal mouse, and detected pathological improvement in accumurative globoid cells in the sciatic nerve, effi cient decreasing psychosine accumulation in the brain (p=0.009), long-term expression of viral copy number in some organs including brain using real-time PCR, and improvement in body weight and twitching onset time point We studied the synergy effect

of gene therapy and substrate reduction therapy using L-Cycloserine,

an irreversible inhibitor of 3-ketodyhydrosphingosine synthase by continuous subcutaneous injection every other day from 5-6 days-old, and detected effi cient effect in body weight and life span (p=0.013) For more high viral titer and effective protein synthesis, we also tried gene therapy study using new lentiviral vector including GALC with codon-optimization As a conclusion, recombinant lentiviral vector mediated neonatal gene therapy for Krabbe mouse model should be effective, especially with sustrate reduction thrapy

586 Choroid Plexus-Directed Viral Gene Therapy as a Source of alpha-N-Acetyl-Glucosaminidase Enzyme Replacement in Cerebrospinal Fluid for Sanfi lippo B Syndrome

Patricia I Dickson,1 Shih-hsin Kan,1 Steven Q Le,1 Maria Reine Haddad,2 Eun-Young Choi,2 Anthony Donsante,2 Stephen G Kaler.2

1 Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CA; 2 Molecular Medicine Program, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo B) is an inherited lysosomal storage disorder with severe neurological deterioration caused by the deficiency of alpha-N-acetyl-glucosaminidase (NAGLU) and is characterized by the accumulation of glycosaminoglycan in the brain Enzyme replacement therapy for MPS IIIB is challenging, due to inadequate mannose 6-phosphorylation (M6P) which limits cellular uptake

of recombinantly produced human NAGLU Recently, a modifi ed recombinant human NAGLU fused to the receptor-binding motif

of the insulin-like growth factor 2 (rhNAGLU-IGF2) has shown enhanced ability to enter MPS IIIB fi broblasts via IGF2-mediated receptor-mediated endocytosis However, in order to reach the brain

as enzyme replacement therapy, rhNAGLU-IGF2 would require delivery by repeated injections into the cerebrospinal fl uid (CSF), an

Molecular Therapy Volume 21, Supplement 1, May 2013

Copyright © The American Society of Gene & Cell Therapy S225

NEUROLOGIC GENE & CELL THERAPY II

expensive and inconvenient treatment approach To begin to explore

a new potential therapeutic avenue for this disease, we constructed

a NAGLU-IGF2 cDNA, and documented robust expression and

NAGLU enzyme activity in transfected CHO and Hek293T cells

We prepared recombinant adeno-associated virus serotype 5 (rAAV5)

containing the NAGLU-IGF2 cDNA with the cytomegalovirus

enhancer/chicken -actin promoter to drive transgene expression

We are performing a pilot in vivo study in normal adult rats using

intracerebroventricular injections of 6×1010 rAAV5 viral particles,

under stereotaxic guidance The rAAV5 serotype is known to target

choroid plexus epithelia, specialized structures that project into

the brain’s ventricular cavities and show limited cellular turnover

Choroid plexuses produce CSF by transporting water and ions, and

regulate the biochemical and protein composition of CSF AAV vector

delivery to choroid plexus epithelia would provide a steady source of

human NAGLU-IGF2 in the CSF to treat the brain Testing in MPS

IIIB mice will evaluate the safety and effi cacy of this approach to

correct or prevent brain pathology by re-modeling choroid plexus

to continuously secrete NAGLU-IGF2 This unique therapeutic

approach seeks to combine the benefits of M6P-independent

endocytosis and viral gene therapy, and may enable effi cient

NAGLU-IGF2 distribution throughout central nervous system

587 Mitigation of DNA Damage Responses and

Oxidative Stress in Cells with Impaired Telomerase

Function

Larisa Pereboeva,1 Erik Westin,2,3 Mary Gannon,5 Lawrence

Lamb,1 Aloysius Klingelhutz,4 Frederick Goldman.2

1 Department of Medicine, Division of Hematology Oncology,

University of Alabama at Birmingham, Birmingham, AL;

2 Department of Pediatrics, Division of Hematology Oncology,

Children’s Hospital of Alabama, University of Alabama at

Birmingham, Birmingham, AL; 3 Department of Biochemistry

and Molecular Genetics, University of Alabama at Birmingham,

Birmingham, AL; 4 Department of Microbiology, University of

Iowa, Iowa City, IA; 5 Department of Genetics, University of

Alabama at Birmingham, Birmingham, AL.

The ‘telomeropathies’ are a newly described group of human

diseases with genetic mutations that decrease telomere length or

telomerase activity One such disease, Dyskeratosis congenita (DC) is

an inherited multisystem disorder characterized by premature aging,

cancer predisposition, bone marrow failure, and critically shortened

telomeres It is widely accepted that molecular pathology of DC cells

stems from abnormal signaling from shortened telomeres However,

exact mechanisms of this signaling are unknown and treatment

strategies are limited The requirement of telomerase activity is most

evident in highly proliferative cells, and those lacking this enzymatic

activity, either as a consequence of the natural aging process of

genetic mutation, demonstrate a growth disadvantage and features

of senescence We have recently shown that DC cells also have a

growth disadvantage, or stressed phenotype, that is associated with

increased basal levels of intracellular oxidative stress, apoptosis and

p53 expression We hypothesized that there is a causal relationship

between diminished telomerase function and imbalances in

steady-state levels of oxidative stress Therefore, restoration of telomerase

activity by transduction with exogenous telomerase may correct the

stressed phenotype of DC cells and normalize levels of ROS and p53

We have studied primary fi broblasts and lymphocytes derived from

DC patients as cell types representative of DC pathology Growth

disadvantage of both cell types in continuous cell culture compared

to corresponding normal controls was documented Studies on DC

fi broblasts revealed that heightened reactive oxygen species (ROS)

and DNA damage response (DDR) levels can be reduced in two

experimental conditions with a concomitant effect on cell growth

First, culturing DC fi broblasts in low oxygen conditions (5% O2)

partially restored their proliferative capacity In addition, we were able to ameliorate the proliferative disadvantage in DC fi broblasts

by introduction of exogenous TERT by way of transduction with a retroviral vector Importantly, expression of TERT reduced

steady-state levels of ROS in DC cells Recently we have also uncovered elevated basal levels of ROS and p53 expression in DC lymphocytes These levels, as well as replicative lifespan, can be partially normalized by treatment with antioxidant N-acetyl cysteine (NAC) These data suggest a regulatory role of telomeres or telomerase activity in maintaining homeostatic intracellular levels of ROS and p53

588 Abstract Withdrawn Neurologic Gene & Cell Therapy II

589 CNS Administration of an AAVrh.10 Gene Transfer Vector Expressing the Human ARSA cDNA to Nonhuman Primates Provides Widespread Activity across the CNS

Jonathan B Rosenberg,1 Dolan Sondhi,1 David G Rubin,1 Sébastien Monette,2 Alvin Chen,1 Sara A Cram,1 Benjamin Van de Graaf,1 Bishnu P De,1 Stephen M Kaminsky,1 Caroline Sevin,3,4 Patrick Aubourg,3,4 Ronald G Crystal.1

1 Weill Cornell Medical College, NY, NY; 2 Tri-Institutional Laboratory of Comparative Pathology, Weill Cornell Medical College and Memorial Sloan-Kettering Cancer Center, NY, NY;

3 INSERM U986, Université Paris-Descartes, Paris, France;

4 Bicêtre Hospital, Paris, France.

Metachromatic leukodystrophy (MLD), a fatal lysosomal lipid storage disorder caused by the defi ciency of the lysosomal enzyme, arylsulfatase A (ARSA), leads to an accumulation of sulfatides, causing a widespread demyelination in both the central (CNS) and peripheral (PNS) nervous systems MLD is a good candidate for gene therapy because the ARSA protein is secreted and is capable of cross correcting neighboring cells, i.e., it is not necessary to achieve gene transfer to every cell in the CNS Leveraging the knowledge that adeno-associated viruses (AAV) have demonstrated persistent and efficacious gene transfer in animal studies with excellent safety profi les in human clinical trials, and that the rhesus serotype AAVrh.10 expressing FLAG-tagged ARSA (AAVrh.10hARSA-FLAG) expresses ARSA throughout the CNS of rodents, we compared several routes of delivery with this vector in nonhuman primates to determine which would provide the broadest distribution of ARSA enzyme Five routes of vector delivery were tested using a total dose

of 1.5x1012 genome copies of AAVrh.10hARSA-FLAG for each route: (1) cortical delivery to the white matter (ventral to the cortex); (2) deep grey matter (putamen, thalamus, and caudate) plus the overlaying white matter; (3) convection enhanced delivery to same deep grey matter locations; (4) the lateral cerebral ventricle; and (5) intra-arterial delivery with hyperosmotic mannitol in the middle cerebral artery After 13 wk, the brains were analyzed for the distribution of ARSA protein and enzymatic activity The right hemisphere was subdivided into 1 cm3 cubes, homogenized and assayed for functional ARSA activity The left hemisphere was fi xed in formalin and sectioned for immunohistochemistry using the FLAG epitope The distribution

of ARSA activity in each of the 3 CNS (intracerebral) administered routes was signifi cantly higher than PBS-administered controls, while the intraventricular and intra-arterial routes failed to demonstrate measurable levels above controls Immunohistochemistry using anti-FLAG antibodies confi rmed these results with extensive ARSA expression from all 3 CNS (intracerebral) administered routes of delivery with staining in the cortex, white matter, deep grey matter

of the striatum, thalamus, choroid plexus and spinal cord dorsal root