455 neonatal delivery of scAAV9 SMN: impact of disease development in two models of spinal muscular atrophy

455 Neonatal Delivery of scAAV9 SMN Impact of Disease Development in Two Models of Spinal Muscular Atrophy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene[.]

Molecular Therapy Volume 19, Supplement 1, May 2011

Copyright © The American Society of Gene & Cell Therapy S175

GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II

potentially lethal metabolic decompensations, hyperammonemia,

neurological complications, and a potentially lethal cardiomyopathy

A murine model of PA, created by knocking out a portion of the Pcca

gene, exhibits elevated levels of metabolites and neonatal lethality

within the fi rst 48 hours We have previously demonstrated the

ability of rAAV8 gene delivery to rescue the murine model of PA

from lethality and decrease the levels of disease related metabolites

In light of the cardiomyopathy observed in patients with PA, the

cardiac histology of the Pcca-/- mice was examined to determine

whether cardiac abnormalities are present Histology revealed

gross abnormalities of the cardiac tissue However, Pcca-/- mice

treated by rAAV8 gene delivery of the PCCA gene did not exhibit

histopathological changes of the cardiac tissue compared to

wild-type mice These results demonstrate the potential effi cacy of rAAV8

gene delivery as an alternative treatment to transplantation for the

potentially lethal cardiomyopathy observed in PA

Muscle Results in Sustained Reduction of

Hyperbilirubinemia in an Animal Model of

Crigler-Najjar Syndrome Type 1

Nunzia Pastore,1 Rosa Maria Sepe,1 Francesco Vetrini,1 Edoardo

Nusco,1 Alberto Auricchio,1,2 Nicola Brunetti-Pierri.1,2

1 Telethon Institute of Genetics and Medicine, Naples, Italy;

2 Pediatrics, Federico II University, Naples, Italy.

Crigler-Najjar syndrome is an autosomal recessive disorder

characterized by severe unconjugated hyperbilirubinemia due to

defi ciency of the liver-specifi c uridine diphospho-glucuronosyl

transferase 1 A1 (UGT1A1) Current therapy relies on phototherapy

to prevent life-threatening elevations of serum bilirubin levels but

liver transplantation is the only permanent treatment Given the

mortality and morbidity related to the transplant procedures, there is

high motivation at developing gene therapy for this disorder Although

correction of the defi cient enzymatic activity in the affected organ,

i.e the liver, would be most straightforward, expression within an

ectopic tissue to clear toxic metabolites from the circulation is very

attractive The muscle is the preferred tissue for this goal because

of its simple and safe access through intramuscular (IM) injections

Moreover, the IM route has been investigated extensively for gene

therapy of various diseases and in human clinical trials as well In

this study, we have investigated the effi cacy of muscle-directed gene

therapy for Crigler-Najjar syndrome type 1 using Adeno Associated

Viral (AAV) vectors A serotype 1 AAV vector expressing the

UGT1A1 under the control of the muscle-specifi c creatine kinase

(MCK) promoter was injected at the dose of 3x10e12 genome

copies/kg into the muscles of one month-old Gunn rats, the animal

model of Crigler-Najjar syndrome type 1 IM injections of AAV

vectors resulted in the expression of functionally active UGT1A1

enzyme in the muscle as demonstrated by Western blot analysis and

enzyme assay on muscle tissues AAV-injected Gunn rats showed

an approximately 50% reduction of baseline serum bilirubin levels

by 3 weeks post-injection which were sustained for at least 1 year

post-injection Taken together, these data show that clinically relevant

and sustained reduction of serum bilirubin levels can be achieved by

simple and safe IM injections in the Gunn rats AAV-mediated muscle

directed gene therapy has potential for the treatment of patients with

Crigler-Najjar syndrome type 1

of Disease Development in Two Models of Spinal Muscular Atrophy

Jacqueline Glascock,1 Monir Shababi,1 Christian Lorson.1

1 Molecular Microbiology and Immunology and Veterinary Pathobiology, University of Missouri, Columbia, MO.

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1) Despite its ubiquitous expression, low levels

of full length SMN protein result in the preferential loss of lower motor neurons It has been shown that replacement of SMN1 using viral vectors is a viable option for the rescue of motor neurons and the treatment of SMA in the delta 7 mouse model of SMA In this study, we used self-complementary adeno-associated virus (scAAV) carrying SMN1 to compare two different routes of viral delivery in the delta 7 model scAAV9-SMN1 was delivered to postnatal day 1 mice via intravenous (IV) or intracerebroventricular (ICV) injection Both routes of scAAV9-SMN1 delivery resulted in a signifi cant increase in lifespan and weight compared to untreated mice with a subpopulation of mice surviving more than 200 days However, mice given ICV injections gained signifi cantly more weight than their IV treated counterparts Survival analysis shows that ICV treated mice displayed fewer early deaths than IV treated animals

Preliminary results have shown that SMN protein levels in the brain and spinal cord of ICV treated animals are higher than SMN protein levels in IV treated animals This study demonstrates that route of delivery is a crucial component of gene therapy treatment for SMA These results prompted a study of this vector’s (scAAV9-SMN1) ability to rescue a more severe model of disease We now show that while lifespan is signifi cantly improved in this more severe model, the inability to fully rescue suggests that either prenatal delivery is required or that the model is too severe to be fully rescued

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

S176

GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II

Model Mice by a Single Intramuscular Injection of

AAV Type 8 Vector Expressing Tissue-Nonspecifi c

Alkaline Phosphatase

Tae Matsumoto,1,2 Koichi Miyake,1 Noriko Miyake,1 Hideo

Orimo,3 Sonoko Narisawa,4 José Millán,4 Yoshitaka Fukunaga,2

Takashi Shimada.1

1 Department of Biochemistry and Molecular Biology, Division

of Gene Therapy Research Center for Advanced Medical

Technology, Nippon Medical School, Tokyo, Japan; 2 Department

of Pediatrics, Nippon Medical School, Tokyo, Japan; 3 Division of

Structural Biology and Metabolism, Department of Biochemistry

and Molecular Biology, Nippon Medical School, Tokyo, Japan;

4 Sanford Children’s Health Research Center, Sanford-Burnham

Medical Research Institute, La Jolla, CA.

Hypophosphatasia (HPP) is an inherited systemic skeletal

disease characterized by a defi ciency in tissue-nonspecifi c alkaline

phosphatase (TNALP), which leads to abnormal mineralization of

skeletal and dental tissues Patients with the infantile form may appear

normal at birth, however, the prognosis of this form is poor Although

enzyme replacement therapy or bone marrow transplantation was tried

in these 30 years, there is no curative treatment for hypophosphatasia

Recently, it was demonstrated that TNALP knockout (AKP2-/-) mice,

which are good models for infantile form hypophosphatasia, could

be treated by injection of a bone-targeted form of human TNALP

in which a deca-aspartate sequence is linked to the C terminal end

of soluble TNALP (TNALP-D10) We also reported that a single

intravenous systemic injection of lentiviral or adeno-associated virus

type 8 (AAV8) vectors expressing TNALP-D10 rescued neonatal

knockout mice However, the safety of a systemic injection of either

lentiviral or AAV into neonates has not be established In this study,

to develop a more practical gene therapy for HPP, we examined the

feasibility of AAV8 vector-mediated muscle directed gene therapy

for neonatal HPP Neonatal AKP2-/- mice (n=6) were treated with

a single intramuscular injection of AAV8-TNALP-D10 (1x1012 vg/

body) High level of plasma ALP activity (14.6 ± 4.5 U/ml) was then

detected for more than one year, thereafter X-ray analysis showed

mature bone mineralization was detected in treated AKP2-/- mice

but not control AAV8 vector expressing GFP injected mice Treated

knockout mice lived for more than 1 year with normal physical

activity and healthy appearance, while control mice died within

3 weeks Ectopic calcifi cation and abnormal calcium metabolism

were not detected in treated mice These fi ndings suggest that AAV

mediated intramuscular neonatal gene therapy is a highly practical

option to cure the severe infantile form of HPP

of the Small Molecules ManNAc and Sialic Acid

To Rescue Hyposialylation in a Mouse Model of Hereditary Inclusion Body Myopathy (HIBM)

Tal Yardeni,1,4 Carla Ciccone,1 Shelley Hoogstraten-Miller,2 Daniel Darvish,3 Yair Anikster,4 John Nemunaitis,5,6 Phil Maples,5 Chris

M Jay,5 William A Gahl,1 Marjan Huizing.1

1 MGB, NHGRI/NIH, Bethesda, MD; 2 OLAM, NHGRI/NIH, Bethesda, MD; 3 HIBM Research Group, Encino, CA; 4 Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel; 5 Gradalis Inc., Dallas, TX; 6 Mary Crowley Cancer Research Centers, Dallas, TX.

HIBM is an adult-onset, progressive neuromuscular disorder, caused by mutations in the ubiquitously expressed, key enzyme

of sialic acid (SA) synthesis, UDP-GlcNAc 2-epimerase/ManNAc

kinase, encoded by the GNE gene We created an HIBM mouse

model, mimicking the Persian-Jewish founder mutation Mutant mice (-/-) unexpectedly died before day 3 of life (P3) from severe glomerulopathy due to hyposialylation Renal fi ndings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin Oral administration

of the SA precursor, N-Acetyl-D-mannosamine (ManNAc), yielded survival beyond P3 in 43% of the mutant pups Then we assessed the effi ciency and effi cacy of ManNAc/SA delivery in liposomes

(ManNAc-Lipoplex,SA-Lipoplex), and human GNE gene delivery

in liposomes (hGNE-Lipoplex) in our HIBM mouse model Newborn

pups (P1) were retro-orbitally injected with ManNAc-Lipoplex,

SA-Lipoplex or hGNE-Lipoplex Mice were watched for clinical

signs and survival beyond P3, and tissues were tested at P5 for

sialylation, histology, glomerular disease and hGNE expression hGNE-Lipoplex injections yielded no surviving -/- pups beyond P3, however, WT survivors showed hGNE expression in tested tissues

at P5, indicating no toxicity of hGNE-Lipoplex and effi cient gene

delivery to tissues Interestingly, -/- pups that died before P3 also

showed hGNE expression in their tissues (as early as P2); treatment at

P1 may not allow enough time for suffi cient protein translation and SA production In contrast, ManNAc-Lipoplex injections at P1 yielded survival beyond P3 in >90% of -/- pups SA-Lipoplex yielded survival beyond P3 in >80% of -/- pups in contrast to SA in drinking water that didn’t yielded any -/- pups surviving beyond P3.The surviving mutant pups showed improved glomerular histology and improved sialylation of glomerular sialoproteins at P5 ManNAc-Lipoplex treated -/- mice continued to live beyond weaning; the oldest mice are now 8 months The development of a muscular phenotype in these mice, similar to the symptoms of HIBM, and the effects of ManNAc treatment on these symptoms can now be assessed Our studies demonstrate: 1)retro-orbital injection in newborn mice is an effi cient method for systemic delivery of compounds; 2)small molecules can

be effi ciently delivered in Lipoplex; 3)systemic delivery of a gene in Lipoplex yields gene expression in tissues after one day; 4)ManNAc-Lipoplex/SA-Lipoplex can be applied to increase sialic acid levels and systemic or intramuscular ManNAc-Lipoplex therapy should be considered for the treatment of patients with HIBM, and may also be considered for other disorders of hyposialylation (i.e.,certain cancers, certain renal disorders)