196 dosage thresholds and vector serotype define AAV targeting of rod and cone photoreceptors in non human primates

196 Dosage Thresholds and Vector Serotype Define AAV Targeting of Rod and Cone Photoreceptors in Non Human Primates Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society[.]

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

S76

Neurologic & Ophthalmic Gene & Cell Therapy I

Lysosomal Storage Disease by Intracranial AAV

Gene Therapy

Victoria J Jones,1 Allison M Bradbury,1 Misako Hwang,1 Stanley

G Leroy,2 Stacy Maitland,2 Aime K Johnson,3 Miguel

Sena-Esteves,2 Douglas R Martin.1

1 Scott-Ritchey Res Ctr/Dept Anat, Phys, Pharm, Auburn University

College of Vet Med, Auburn, AL; 2 Gene Therapy Center, University

of Massachusetts Medical School, Worcester, MA; 3 Dept Clinical

Sciences, Auburn University College of Vet Med, Auburn, AL.

Defi ciency of lysosomal β-galactosidase (βgal) causes storage of

GM1 ganglioside, resulting in progressive neurological deterioration

and death, often by 5 years of age AAV gene therapy has been

extraordinarily successful in the GM1 mouse model, (Mol Ther,

15:30, 2007; PLoS One, 5:e13468, 2010), resulting in enhanced

survival and complete clearance of storage in the brains of GM1

mice Because the mouse brain is ∼1000 times smaller and much

less complex than the human brain, it is important to test AAV

gene therapy in an animal model whose brain size and complexity

more closely resemble humans First reported ∼40 years ago, the

feline GM1 model presents an unparalleled opportunity to evaluate

AAV gene therapy in a non-rodent, ‘large animal’ prior to initiating

human clinical trials In the current study, AAV2/1 or AAV2/rh8

vectors expressing a feline βgal cDNA (3.1-12.0e12 g.c total) were

injected bilaterally into the thalamus and deep cerebellar nuclei of

2-month old GM1 cats (disease onset ∼3.5 months) In treated brains

collected 4-16 weeks post-injection, βgal was distributed throughout

the entire anterior-posterior axis of the cerebrum and cerebellum at

levels up to 4 times normal Cervical and lumbar spinal cord regions

demonstrated βgal activity 0.5 -1 times normal, and fi lipin staining

demonstrated extensive clearance of storage material Little evidence

of an infl ammatory cellular infi ltrate was observed in H&E-stained

brain sections, though serum antibody titers to the AAV vectors

were pronounced (∼1:65,000) Long-term therapeutic experiments

in 2-month old GM1 cats were conducted using the same vector dose

and route of delivery Currently, AAV-treated GM1 cats are 16, 14 and

12 months of age, with no evidence of clincal neurological disease

(untreated humane endpoint, 7.7 ± 0.8 months, n=9) Treated GM1

cats demonstrate normalization of MRI brain lesions and absence

of gait abnormalities typical of untreated cats Other than serum

antibody titers, no evidence of vector toxicity has been documented,

and no βgal activity was detected in peripheral blood mononuclear

cells Further encouraging results come from treatments performed

nearer to clinical disease onset (3.0 months of age, 2 weeks prior to

disease onset), in which 2 GM1 cats remain neurologically normal

at 8.3 months of age These translational studies provide strong

support for the initiation of AAV-based clinical trials for human

GM1 gangliosidosis

Leukodystrophy: Optimized Clinical Protocol

Caroline Sevin,1,2 Françoise Piguet,1 Dolan Sondhi,3 Marie-Anne Colle,4 Thomas Roujeau,1,5 Sylvie Raoul,6 Jack-Yves Deschamps,7 Celine Bouquet,1 Ornella Ahouansou,1 Marie Vanier,8 Philippe Moullier,9 Yan Cherel,4 Neil R Hackett,3 Michel Zerah,1,5 Ronald

G Crystal,3 Patrick Aubourg,1,2 Nathalie Cartier.1,2

1 INSERM U745, Paris, France; 2 Pediatric Neurology and Endocrinology, St Vincent de Paul Hospital, Paris, France; 3 Weill Medical College of Cornell University, New York; 4 UMR INRA

703, Nantes, France; 5 Pediatric Neurosurgery, Necker-Enfants Malades Hospital, Paris, France; 6 Neurosurgery, CHU Nord, Nantes, France; 7 National School of Veterinary Medicine, Nantes, France; 8 Inserm U820, Lyon, France; 9 Inserm U649, Nantes, France.

Metachromatic Leukodystrophy (MLD) is a lethal neurodegenerative disease caused by defi ciency of Arylsulfatase A (ARSA) The most severe late-infantile form starts around 1-2 years, leading to death within a few years, without available treatment Among potential new therapeutic interventions, brain gene therapy could ensure rapid and sustained delivery of ARSA enzyme in the brain, a prerequisite to arrest the neurodegenerative process in due time We have demonstrated effi ciency and safety of intracerebral delivery of adeno-associated-vector serotype 5 (AAV5) encoding human ARSA

in MLD mice and non-human primates We recently demonstrated that AAVrh.10 encoding ARSA improves more rapidly MLD mice that AAV5-ARSA and, importantly, allows normalization of sulfatide isoforms that accumulate specifi cally in oligodendrocytes

In a clinical perspective, we optimized in non-human primates the neurosurgical procedure to allow simultaneous infusion of vector at

12 different brain sites in less than 2 1/2 hours We also developed

a brain imaging protocol using MRI to select the injection sites and evaluate the tolerance of the surgical procedure Following the same protocol planed to be used in patients, we demonstrated in non-human primates that intracerebral injection of AAVrh.10/ARSA vector (1.1.10exp11 viral particles per hemisphere in selected areas of the white matter) is well tolerated and results in the diffusion of the vector

in 66 to 90% of the injected hemisphere We also documented up to 31% increased activity of ARSA, reaching foreseeable therapeutic levels As compared to our previous results with AAV5/ARSA, the use of a 20-fold lower dose of AAVrh.10 vector injection led to higher amounts of vector and increased level of ARSA activity in the brain Toxicological studies are in progress towards phase I/II tolerance and effi ciency clinical trial in late 2011 This trial will enroll fi ve patients with rapidly progressing MLD AAVrh.10/ARSA vector will be administrated to 12 locations in the CNS, guided by brain imaging Safety and effi ciency parameters will be evaluated up to 2 years, a period that will be suffi cient enough to assess the potential therapeutic effi ciency of this brain gene therapy strategy in rapidly progressing forms of MLD

Defi ne AAV Targeting of Rod and Cone Photoreceptors in Non-Human Primates

Luk H Vandenberghe,1 Peter Bell,1 Albert M Maguire,2 Ru Xiao,1 Rebecca Grant,1 Jean Bennett,2 James M Wilson.1

1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2 F M Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA.

Gene therapy is emerging as a therapeutic modality for treatment

of retinal disorders Rod and/or cone photoreceptors are the primary cell types in many inherited retinal degenerations and successful gene therapy for those diseases will therefore require identifi cation

Molecular Therapy Volume 19, Supplement 1, May 2011

of effi cient and safe targeting vectors Previously, we injected

non-human primate eyes with 6 AAV serotypes via a subretinal route in

order to determine relative effi ciencies of transduction of the retinal

pigment epithelium (RPE) and characterize the histological and

immunological consequences of vector administration at various

doses Here, we further examined the injected eyes qualitatively and

quantitatively for photoreceptor transduction Non-human primates

are the only animal models with a macula and therefore we also

sought to determine the expression profi le in the macula, including

the fovea Based on an interesting transduction profi le in the murine

retina, AAV2, 7, 8, 9 and the engineered capsid isolates rh64R1 and

rh8R were subretinally injected into cynomolgus macaque eyes at

doses ranging from 10E8 to 10E11 GC per eye of a GFP expressing

AAV Eyes were sectioned and analyzed histologically 2 to 6 months

following injection Morphometry for transduction effi ciency and

intensity highlighted substantial differences between vector serotypes

AAV8 was the most effi cient serotype in transducing photoreceptors

at the moderate dose of 10E9 GC At a dose of 10E10 GC however,

all serotypes with the exception of AAV2, demonstrated very

effi cient and roughly equivalent levels of GFP expression in the

photoreceptor cell layer Detailed histological analysis of 3 macular

regions, including the fovea, and a fourth non-macular site illustrated

striking differences in the relative targeting effi ciency of cones and

rods Whereas all vectors transduced rods quite readily, AAV9 and

rh8R emerged as substantially more effi cient in cone transduction both

within and outside of the fovea At higher doses, the effi ciency of cone

transduction of the other vectors can be boosted, but nevertheless,

the differential permissivity of cones versus rods for each vector

remains apparent

with Bone Marrow Transplantation in the Treatment

of Infantile Neuronal Ceroid Lipofuscinosis

Shannon L Macauley,1 Marie S Roberts,1 Mark S Sands.1

1 Internal Medicine, Washington University School of Medicine,

Saint Louis, MO.

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited,

neurodegenerative lysosomal storage disease (LSD) affecting the

central nervous system (CNS) during infancy INCL is caused by

mutations in the CLN1 gene leading to a defi ciency in the lysosomal

enzyme, palmitoyl protein thioesterase 1 (PPT1) Pathological

changes in the CNS include brain atrophy, cortical thinning,

autofl uorescent accumulation, neurodegeneration, and glial activation

The clinical signs include seizures, blindness, motor dysfunction,

mental retardation and premature death by 6 years of age The

PPT1-/- mouse accurately mimics the human disease PPT1-PPT1-/- mice have

a decreased life span, visual defects, cognitive defi cits, epilepsy,

and motor dysfunction PPT1-/- brains show similar pathological

changes to INCL patients including autofl uorescent accumulation,

neurodegeneration, glial activation as well as brain atrophy The

similarities between PPT1-/- mice and children with INCL make

this an excellent tool to investigate therapeutic strategies for the

treatment of INCL Currently, gene and stem cell therapies are in

clinical trials for the treatment of INCL and late infantile neuronal

ceroid lipofuscinosis (LINCL) Although both therapies demonstrated

partial improvements on biochemical and histological parameters,

the behavioral improvements were modest and no improvement in

lifespan was seen Furthermore, another clinical study utilizing bone

marrow transplantation (BMT) as a potential therapy for INCL or

LINCL proved unsuccessful Thus, given the complexity of INCL

and the inadequate therapeutic benefi t from any singular therapy, we

hypothesized that combining therapeutic modalities must be used to

be effi cacious In this study, we combined intracranially (IC) delivered

AAV2/5 expressing PPT1 with bone marrow transplantation (BMT)

At birth, PPT1-/- mice received six injections of AAV2/5-PPT1

delivered bilaterally to the anterior cortex, thalamus/hippocampus, and cerebellum The following day the treated PPT1-/- mice were irradiated with a myeloreductive-conditioning regimen (400 rads) followed by BMT via a temporal vein injection of 1x106 bone marrow derived stem cells BMT alone provided no enzyme activity

to the brain and did not improve lifespan or motor function, similar

to clinical trials Surprisingly, neonatal BMT synergized with IC delivery of AAV2/5-PPT1 to dramatically increase the median lifespan of PPT1-/- mice (untreated PPT1-/- ∼ 8mo, AAV2/5-PPT1-only ∼ 13mo, AAV2/5-PPT1/BMT ∼ 17mo) The combination also resulted in sustained improvements in motor function (constant speed and rocking rotorod) On the constant speed rotorod paradigm, the AAV2/5-PPT1+BMT mice performed similar to wildtype mice until

13 months of age and then their performance steadily decreased through 17 months The combination therapy also demonstrated improvements on the rocking test when compared to untreated mice through 11 months of age and then steadily decreased thereafter This combination of CNS-directed gene therapy and BMT demonstrates clinical improvements unparalleled by any previous pre-clinical study

or clinical trial done in INCL

Type 1

Megan K Keiser,3 Ryan L Boudreau,1 Beverly L Davidson.1,2,3

1 Internal Medicine, University of Iowa, Iowa City, IA; 2 Neurology, University of Iowa, Iowa City, IA; 3 Graduate Program of Neuroscience, University of Iowa, Iowa City, IA.

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant late onset neurodegenerative disease SCA1 toxicity is caused by an expanded polyglutamine tract in ataxin-1 The disease affects cerebellar and brainstem neurons, including Purkinje cells Motor symptoms include gait, balance, and motor coordination defi cits Currently, there are no curative therapies for SCA1, however, evidence supports that reducing mutant ataxin-1 expression may provide therapeutic benefi t RNA interference (RNAi) is a naturally occurring process which mediates gene silencing and is currently being investigated as therapy for dominant diseases such as SCA1 Previous work from our laboratory has shown that RNAi-mediated silencing of a mutant human ataxin-1 transgene improves neuropathological and behavioral phenotypes in a transgenic mouse model of SCA1 The results from this proof-of-concept study highlight the potential of RNAi therapy for treating SCA1 patients Subsequently, we have initiated studies

to test this therapeutic strategy in the knock-in mouse model of SCA1, which more closely genocopies human patients We have designed several artifi cial microRNAs (miRNAs) targeting conserved sequences in both the mouse and human ataxin-1 This approach may facilitate the transition of therapeutic sequences from mouse studies

to human We screened the artifi cial miRNAs in vitro against both transgenic and endogenously expressed ataxin-1 Two therapeutic candidates (siSCA1.1, siSCA1.2) showed effective silencing of ataxin-1 in both human and mouse cell lines siSCA1.1 and siSCA1.2 were placed into adeno-associated viral (AAV) vectors for in vivo delivery Pilot studies showed expression in cerebellar Purkinje cells, knock down of target, and reduction of protein expression At 30 weeks of age (25 weeks after therapy) the SCA1 mutant mice treated with siSCA1.1 trend toward improved motor phenotypes relative to controls Additional tests of therapeutic effi cacy are underway This data provides the fi rst evidence of effective RNAi against ataxin-1

in the knock-in model of SCA1