339 preservation of renal function in murine glycogen storage disease type i with a double stranded adeno associated virus vector

339 Preservation of Renal Function in Murine Glycogen Storage Disease Type I with a Double Stranded Adeno Associated Virus Vector Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The Am[.]

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Molecular Therapy Volume 19, Supplement 1, May 2011

GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II

Mucopolysaccharidosis IIIB in Adult Mice by

rAAV9 Trans-Blood-Brain-Barrier Gene Delivery

Haiyan Fu,1,2 Juliann DiRosario,1 Smruti Killedar,1,2 Kimberly

Zaraspe,1 Douglas M McCarty.1,2

1 Center for Gene Therapy, Research Institute at Nationwide

Children’s Hospital, Columbus, OH; 2 Department of Pediatrics,

The Ohio State University, Columbus.

The greatest challenge in developing therapies for

Mucopolysaccharidosis (MPS) IIIB is to achieve effi cient CNS

delivery across the blood-brain barrier (BBB) In this study, we used

the novel ability of AAV9 to cross the BBB from the vasculature to

achieve long-term global CNS, and widespread somatic restoration of

α-N-acetylglucosaminidase (NAGLU) activity A single intravenous

(IV) injection of rAAV9-CMV-hNAGLU, without extraneous

treatment to disrupt the BBB, restored NAGLU activity to normal or

above normal levels in adult MPS IIIB mice, leading to complete, or

near complete, correction of lysosomal storage pathology in the CNS

and periphery, and correction of astrocytosis and neurodegeneration

The IV-delivered rAAV9 vector also transduced abundant neurons

in the myenteric and submucosal plexus, suggesting peripheral

nervous system targeting While CNS entry did not depend on

osmotic disruption of the BBB, it was significantly enhanced

(≥2 fold) by pretreatment with an IV infusion of mannitol Most

important, we demonstrate that a single systemic rAAV9-NAGLU

gene delivery provides long-term neurological benefi ts in MPS IIIB

mice, resulting in signifi cant improvement in cognitive and motor

function, and extension of survival (18.8-20 months, ongoing) In

addition, much less vector is required to achieve functional benefi ts

by IV AAV9 delivery, in comparison to IV-mannitol-AAV2 regimen

These data suggest promising clinical potential using the

trans-BBB neurotropic rAAV9 vector for treating MPS IIIB and other

neurogenetic diseases

Disease by Increased Mannose-6-Phosphate

Receptor Expression in Target Tissues

Dwight D Koeberl,1 Baodong Sun,1 Jian Dai,1 Andrew Bird,1

Deeksha S Bali.1

1 Pediatrics, Duke University Medical Center, Durham, NC.

The underlying defi ciency of acid α-glucosidase (GAA) in Pompe

disease has been partially corrected by enzyme replacement (ERT)

However, skeletal muscle weakness persists in many patients on

ERT, and poor uptake of GAA by skeletal muscle has been linked

to low abundance of the cation-independent mannose-6-phosphate

receptor (CI-MPR) To further understand the role of CI-MPR in

Pompe disease, we crossed muscle-specifi c CI-MPR knockout (KO)

mice with GAA-KO (Pompe disease) mice We evaluated

adeno-associated virus (AAV) vector-mediated gene therapy in

CI-MPR-KO/GAA-KO (double KO) mice The essential role of CI-MPR was

emphasized by the lack of effi cacy from AAV vector administration,

as demonstrated by markedly reduced biochemical correction of

GAA defi ciency and of glycogen accumulations in double KO

mice, in comparison with administration of the same AAV vector in

GAA-KO mice We next attempted to increase CI-MPR expression

in skeletal muscle to demonstrate the dependence of biochemical

correction upon receptor-mediated uptake of GAA Therefore, the

AAV vector-transduced liver depot was enhanced by the addition of

a drug, clenbuterol, which was previously demonstrated to increase

the expression of CI-MPR in muscle The liver was transduced by

administering AAV-LSPhGAApA (2x1010 vector particles) to two

groups of 3 month-old male GAA-KO mice, and clenbuterol was

administered to one group of vector-treated mice The effect of

clenbuterol was evident, when Rotarod latency was increased by

75% following vector administration and clenbuterol treatment, in comparison with vector administration alone (p<2x10-5) The effi cacy from clenbuterol treatment was evaluated with regard to biochemical correction of GAA defi ciency and glycogen storage in striated muscles and the brain GAA activity was signifi cantly increased in the heart following vector administration and clenbuterol treatment,

in comparison with vector administration alone (p=0.03) Glycogen content was reduced in the diaphragm (p=0.04), soleus (p=0.0006), extensor digitorum longus (EDL; p=0.002), cerebrum (p<5x10-5), and cerebellum (p=0.03) following vector administration and clenbuterol treatment, in comparison with vector administration alone The basis for increased glycogen clearance during clenbuterol treatment was demonstrated by Western blotting detection of CI-MPR The signal for CI-MPR was increased in the EDL (p<0.05) and cerebrum (p<0.01) of clenbuterol-treated GAA-KO mice Furthermore, the effect of gene therapy with clenbuterol enhancement was evaluated

in 15 month-old female GAA-KO mice, because elderly, female mice with Pompe disease are particularly resistant to correction with AAV vector-mediated gene therapy Signifi cantly increased Rotarod latency (p=0.02) and weight (p=0.04) were demonstrated 12 weeks following the initiation of therapy, indicating that long-term effi cacy had been achieved in advanced Pompe disease In summary, enhanced CI-MPR expression might improve the effi cacy from gene therapy in Pompe disease through enhancing receptor-mediated uptake of GAA, and gene therapy could be similarly enhanced in other lysosomal storage disorders that primarily involve the brain

Glycogen Storage Disease Type I with a Double-Stranded Adeno-Associated Virus Vector

Xiaoyan Luo,1 Songtao Li,1 Andrew Bird,1 Michelle P Winn,2 Dwight D Koeberl.1

1 Pediatrics, Duke University Medical Center, Durham, NC;

2 Medicine, Duke University Medical Center, Durham, NC.

Glycogen storage disease type Ia (GSD-Ia) is caused by the defi ciency of glucose-6-phosphatase (G6Pase) in liver and kidney Long-term complications of GSD-Ia occur frequently, including life-threatening hypoglycemia and proteinuria progressing to renal failure To further develop gene therapy for GSD-Ia we cross-packaged a double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase with AAV2, AAV7, AAV8

and AAV9, and evaluated effi cacy in G6pase (-/-) mice The AAV2/8 vector previously prevented hypoglycemia during fasting in G6pase

(-/-) mice; however, the vector dose (1x1013vp/kg) exceeded the highest dose administered in clinical trials of AAV vector-mediated

gene therapy Therefore, a reduced dose was administered in G6pase

(-/-) mice (2x1012 vp/kg) for these 4 pseudotypes, and hypoglycemia during fasting was prevented for >6 months by the AAV2/8 and AAV2/9 vectors The AAV2/7 vector prevented hypoglycemia only

at the higher dose (1x1013 vp/kg), and the AAV2/2 vector did not prevent hypoglycemia Prolonged fasting for 8 hours revealed very signifi cant correction of hypoglycemia following AAV2/9 vector administration at the higher dose G6Pase activity was analyzed in

tissues of G6pase (-/-) mice following vector administration, and

G6Pase activity was signifi cantly increased in the kidney to 43% of normal activity following AAV2/9 vector administration at 7 months

of age Similarly, G6Pase activity was signifi cantly increased in the

liver of G6pase (-/-) mice following AAV vector administration, and

up to 48% of normal activity was demonstrated for the higher dose

of the AAV2/9 vector at 7 months of age Glycogen content was analyzed in tissues following vector administration to evaluate the biochemical activity of G6Pase expressed with each AAV vector The glycogen content of kidney was reduced by >70% in both the AAV2/7

and AAV2/9 vector-treated groups of G6pase (-/-) mice Glycogen

did not increase between 7 and 12 months of age in the kidney of the

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AAV2/9 vector-treated groups, and a dose-response was observed for

both AAV2/7 and AAV2/9 vectors The AAV2/2 vector-treated groups

had signifi cantly higher glycogen content in the kidney, in comparison

with the other 3 vectors Every vector treated group had signifi cantly

reduced glycogen content in the liver, in comparison with the liver of

untreated G6pase (-/-) mice G6Pase expression was demonstrated by

histochemical staining in the liver and kidney of G6pase (-/-) mice

that received the AAV2/9 vector In the kidney, G6Pase expression

was detected in many renal epithelial cells of G6pase (-/-) mice

treated with the higher dose of AAV2/9 at 7 and 12 months of age

Renal involvement in GSD-Ia progresses from microalbuminuria to

proteinuria, and therefore urinary microalbumin was quantifi ed for

vector-treated G6pase (-/-) mice Microalbuminuria was prevented by

vectors other than AAV2/2 Preservation of renal function in G6pase

(-/-) mice demonstrates the potential of AAV vectors for the correction

of inherited diseases of the kidney

Defi ciency in Adult Spfash Mice Using an

AAV2/8-Based Vector Expressing Codon-Optimized Human

OTC Gene

Lili Wang,1 Jianping Lin,1 Hiroki Morizono,2 Peter Bell,1 David

Jones,2 Deirdre McMenamin,1 Mark Batshaw,2 James M Wilson.1

1 Gene Therapy Program, Department of Pathology & Laboratory

Medicine, University of Pennsylvania, Philadelphia; 2 Center for

Genetic Medicine Research, Children’s National Medical Center,

Children’s Research Institute, Washington, DC.

Ornithine transcarbamylase defi ciency (OTCD) is an X-linked

recessive metabolic disease Severe OTC defi ciency (OTCD) can

result in hyperammonemic coma in the neonatal period which, if

left untreated, can rapidly become fatal Current therapies include

dialysis, the utilization of alternate nitrogen clearance pathways, and

liver transplantation for severely affected patients, however, there

is still an unacceptably high mortality rate AAV vector based gene

therapy would provide an alternative to current treatment options as

long as the vector delivers high level and stable transgene expression

in liver without substantial toxicity Previously, we have developed

an AAV2/8 based self-complementary vector expressing murine

OTC gene driven by a liver-specifi c TBG promoter A single tail

vein injection of 3x10e11 GC of vector in Spfash mice restored the

OTC enzyme activity in the livers of treated mice to a level similar

to wild-type In the current study, we replaced the murine OTC

gene with a codon-optimized human OTC gene Robust OTC gene

expression and correction of OTCD were achieved in Spfashmice

Three days after intravenous injection of vector at dose as low as

1x10e10 GC, urinary orotic acid in treated animals were corrected

to normal levels and sustained through the experiement Liver OTC

activity in Spfashmice treated with 3x10e10 GC of vector were about

2-fold of normal levels assayed one month after treatment Treated

mice had signifi cantly improved behavior scores following ammonia

challenge In contrary to previous conclusions drawn from studies

using adenovirus-based vectors, we demonstrated that human leader

peptides in OTC enzyme are fully functional in mouse Replacing the

human leader peptides with mouse leader peptides does not change

the enzyme activity The AAV2/8-human OTC vector we developed

has great potentials for treating OTCD in human

Long-Chain Acyl-CoA Deydrogenase Defi ciency by Recombinant Adeno-Associated Virus 9 Gene Therapy

Allison M Keeler,1 Glenn Walters,2 Thomas Conlon,2 Karin Green,1 Christian Mueller,1 Terence R Flotte.1

1 University of Massachusetts, Worcester, MA; 2 University of Florida, Gainesville, FL.

Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes is the rate-limiting step in mitochondrial beta-oxidation of fatty acids VLCAD defi ciency causes not only an energy defi cit but also an accumulation of long chain fatty acids VLCAD defi ciency presents

in three different phenotypes: the most severe form presents during infancy with cardiomyopathy and hepatopathy, a second form also occurs in infancy and is usually triggered by a preceding infection resulting also in hepatopathy, and fi nally the least severe presents in adolescents or young adults with muscle weakness In order for a therapy to comprehensively treat the heterogeneous phenotypes of this disease, it must correct the organs where pathogenesis occurs primarily in the liver, heart and skeletal muscle Recombinant adeno-associated virus serotype 9 (rAAV9) was chosen over other serotypes because of its broad-spectrum tissue tropism Therefore

we hypothesize that using intravenous rAAV9-expressing VLCAD will effectively target the liver, heart and skeletal muscle of VLCAD defi cient mice will in turn confer long-term correction of VLCAD defi ciency In this study, three cohorts of mice were injected with 1x1012vg of AAV9-VLCAD, AAV9-GFP or PBS Tissue transduction

by AAV9 was measured by a control virus expressing GFP; results showed GFP expression in the liver, heart, and to a lesser degree muscle Biochemical correction was seen in treated mice starting at two weeks post-injection, as characterized by a signifi cant drop in long chain accumulates in whole blood after an overnight fast This signifi cant reduction persisted until the fi rst cohort was terminated at twelve weeks Correction within the muscle was measured in vivo by Magnetic Resonance Spectrometry (MRS) at 14 weeks post injection

in the second cohort; preliminary scans have shown reduction of lipid peaks dropped within the muscle of treated VLCAD defi cient mice to the same levels as seen in wild-type animals In the future, MRS will be preformed on both the liver and the heart to see if reductions of lipids are also seen in those organs The third cohort

of mice underwent a fasting cold challenge six weeks post injection,

in which an overnight fast is followed by a challenge in a 4°C cold room During the cold-fast challenge, after 120 minutes all internal body temperatures of untreated VLCAD defi cient mice dropped below 20°C and became severely lethargic requiring euthanasia, whereas all of the VLCAD treated defi cient mice and the wild type controls maintained a body temperature averaging 28°C over the 150 minutes at 4°C In conclusion, AAV9 expressing VLCAD effectively targeted the liver, heart and skeletal muscles and conferred near total correction as seen by reduced fatty acid accumulates in the blood, and muscle as well as the improved the ability to thermoregulate in a cold environment These promising results implicate rAAV gene therapy

as a effective treatment for VLCAD defi ciency in humans

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