171 repair of spinal cord injury (SCI) using monocytes transfected with adenoviral vector expressing decorin in a rat SCI model

171 Repair of Spinal Cord Injury (SCI) Using Monocytes Transfected with Adenoviral Vector Expressing Decorin in a Rat SCI Model Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The Amer[.]

Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy

S68

NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY I

and different amounts of the complex injected into the subretinal

space of Balb/c mice Luciferase expression was quantifi ed up to

15 weeks post-injection Once the optimal amount of plasmid was

determined, PEGPOD was complexed with a GDNF-expressing

plasmid and injected into the subretinal space of Balb/c mice Mice

were subjected to 450-nm blue light at 14, 30, and 70 days

post-injection Electroretinograms (ERG) and thickness of outer nuclear

layer (ONL) were measured 7 days post-light treatment Results:

Reduced dose of the PEGPOD/DNA complex resulted in expression

of the luciferase up to 10 weeks post-injection When PEGPOD was

complexed with GDNF (PEGPOD/GDNF) and injected 30 days prior

to light treatment, signifi cant functional and anatomical rescue was

observed A- and B- wave amplitudes of mice injected 30 days prior

to light treatment were increased by 77% and 65%, respectively,

relative to control-injected mice While functional rescue of mice

injected with PEGPOD/GDNF 70 days prior to light treatment was

not signifi cant, ONL thickness of eyes injected with PEGPOD/

GDNF was signifi cantly increased by 25% relative to control-injected

eyes Conclusions: PEGPOD/GDNF complexes show biologically

signifi cant expression in retinas of a mouse model of light-induced

degeneration up to 70 days post-injection This study illustrates the

potential of PEGPOD/GDNF to mediate long-term rescue of retinal

diseases such as RP and AMD, and asserts the need for consideration

of this nonviral gene transfer system as a clinically viable approach

to treatment of these devastating diseases

169 Developing Engineered Zinc Finger

Repressors of Huntingtin as a Potential Therapy

for Huntington’s Disease

H Steve Zhang,1 David E Paschon,1 Josee Laganiere,1 Bryan

Zeitler,1 Matthew Mendel,1 Jennifer Cherone,1 Qi Yu,1 Jocelynn

Pearl,1 Joe Vu,1 Yolanda Santiago,1 Jeffrey C Miller,1 Edward J

Rebar,1 Lei Zhang,1 Philip D Gregory.1

1 Sangamo BioSciences Inc., Richmond, CA.

Huntington’s disease (HD) is an autosomal dominant

neurodegenerative disease caused by CAG-trinucleotide repeat

expansion in the fi rst exon of the Huntingtin (Htt) gene Repeat

lengths of 35 or fewer CAGs are normal and have no associated

pathophysiology, while those of 40 or more CAGs lead to HD with

100% penetrance, with longer repeats lengths correlating with earlier

disease onset The degeneration process primarily affects the basal

ganglia and cerebral cortex, and the disease is characterized by a

progressively worsening chorea, as well as cognitive and psychiatric

dysfunctions While neither the precise function of wild-type Htt nor

the mechanism by which mutant Htt (which contains an expanded

polyglutamine stretch)in HD pathogenesis if fully understood, results

from studying rodent models of HD demonstrate that reducing

both wild-type and mutant Htt levels, via strategies such as RNAi

or anti-sense oligonucleotides, can prevent or delay disease onset

Moreover, simultaneous partial down-regulation of both wild-type

and the expanded Htt allele (∼50%) appears to be tolerated in rodent

as well as non-human primates On the other hand, the complete

loss of wild-type Htt expression is embryonically lethal in mice and

numerous studies have identifi ed important functions of wild-type Htt

in various cellular processes Thus, strategies that selectively reduce

the expression of mutant and disease causing form of Htt represent

the ideal therapeutic approach Engineered zinc-finger protein

transcription factors (ZFP-TFs), which can be designed to target

virtually any gene, offer an attractive approach to the regulation of Htt

expression To this end, we designed a panel of ZFP transcriptional

repressors to bind the proximal promoter of Htt (which would regulate

both wild type and mutant Htt alleles) We show that such pan-allele

specifi c ZFP transcription factors were able to achieve 40-75%

down-regulation of Htt mRNA in transiently transfected HEK293

and Neuro2A cells, which was confi rmed at the Htt protein levels

To generate reagents capable of selectively repressing the mutant Htt allele – we designed ZFP DNA binding domains to selectively bind to the expanded CAG repeat, and investigated whether alleles carrying differing numbers of CAGs could be differentially regulated We show that ZFP repressors can be engineered such that they exhibit minimal repression of Htt alleles that contain normal CAG repeat lengths but greater than 50% repression of Ht alleles with expanded CAG repeat lengths These results demonstrate that both non-allele-specifi c and allele-specifi c repression of Htt can be achieved with engineered

ZFP repressors In vivo testing in HD models is therefore warranted

to determine as well as to compare the effi cacy and safety of these two approaches to the potential treatment of HD

170 Improved Neuroplasticity and Decreased Apoptosis by Intralesional Transplantation of Mesenchymal Stem Cells after Severe Spinal Cord Injury in Rats

Fátima M.C Caldeira,1 Fabíola B Fukushima,2 Mário S L Lavor,2

Carla M.O Silva,2 Isabel R Rosado,2 Paula M Costa,2 Karen M Oliveira,2 Jankerle N Boleoni,2 Rogéria Serakides,2 Alfredo M Góes,3 Eliane G Melo.2

1 Laboratório de Tecnologias para Terapia Gênica, FAV, Universidade de Brasília, Brasília, DF, Brazil; 2 Departamento

de Clínica e Cirurgia Veterinárias, EV, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; 3 Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

The present study aimed at evaluating the effect of mesenchymal stem cells (MSC), from bone marrow, in rats with spinal cord injury (SCI) Thus, 61 adult male Lewis rats were distributed in 3 groups:

sham (CN), placebo (PLA) and MSC (GCT) All animals underwent laminectomy of T12, whereas the animals in PLA and GGT underwent

SCI performed with a 70g rod for 5 minutes After 14 days, the surgical

site was exposed and for PLA and GCT, 2 μL PBS and 1.5 x 105 MSC diluted in 2 μL of PBS were administered at the injury epicenter, respectively The experimental design was completely randomized Neurological evaluation consisted of the Basso, Beatie, Bresnahan (BBB) test and a descriptive scale adapted method, performed every

4 days for 45 consecutive days In the second, third and fi fth weeks after inoculation, rats were euthanized, having their spinal cord collected for gene expression evaluation of BDNF, NT-3, Bcl-xL, Bax, and caspase-9 by RT-PCR In the fi fth week, the spinal cord was also assessed by immunohistochemical methods, using anti-NeuN, anti-eGFP, anti-GFAP, and anti-synaptophysin antibodies, as well

as by the TUNEL technique Neurological defi cits were observed

in the animals with injury, with difference between the medians of

groups PLA and GCT (p <0.05) There was no difference on neurons

identifi ed by NeuN antibody (p> 0.05) The higher synaptophysin immunostaining intensity and the lower formation of apoptotic bodies

for GCT was similar to CN (p> 0.05) PLA group showed a higher

expression of Bax and caspase-9 (p <0.05) Thus, we conclude that the MSC preserved synaptophysin and propitiously affected apoptosis

171 Repair of Spinal Cord Injury (SCI) Using Monocytes Transfected with Adenoviral Vector Expressing Decorin in a Rat SCI Model

Alireza Biglari,1 Reza Salarinia,1 Saeideh Mazloomzadeh,1 Iraj Jafari,1 Parisa Ranjzad,2 Paul A Kingston.2

1 Department of Molecular Medicine & Genetics, Zanjan University

of Medical Sciences, Zanjan, Islamic Republic of Iran; 2 Vascular Gene Therapy Unit, Core Technology Facility, The University of Manchester, Manchester, United Kingdom.

Background: Subsequent to spinal cord injury many pathological changes may occur that lead to inappropriate environment for

Molecular Therapy Volume 20, Supplement 1, May 2012

MUSCULO-SKELETAL GENE & CELL THERAPY I

repair, most important of which are cyst formation, glial scar and

ineffectiveness of monocytes Although monocytes have restorative

roles in tissue lesions, but with the incidence of spinal injury,

monocyte responses in the lesion site are inadequate and their numbers

are less than expected The glial scar formed as a result of SCI is rich in

Chondroitin Sulfate Proteoglycans (CSPG) that inhibit axon growth

Decorin is a small leucine rich proteoglycan that inhibits CSPGs

through various mechanisms Decorin directly promotes growth

of neurons as well as inhibiting glial scar formation In the current

study we attempted to improve treatment results using a combination

of monocyte and decorin features Methods: Rats were divided into

four groups of six at random Spinal cord injury was then performed

under general anaesthesia using the weight dropping method The cells

were injected at 4 days post-spinal cord injury Group one included

rats receiving normal saline, group two received monocytes, group

three received monocytes infected with adenoviruses encoding the

beta-galactosidase gene and group four received monocytes infected

with adenoviruses encoding the decorin gene A Basso, Beattie and

Bresnahan (BBB) score test was performed for four weeks Two weeks

before the end, biotin dextran amine was injected intra-cerebrally and

at the end of the fourth week tissue staining was performed Results

& Conclusion: Both BBB scores and axon count was signifi cantly

different between groups one and four However, no signifi cant

difference was detected between other groups It can be concluded that

the combined use of these methods (Decorin expressing Monocytes)

had better results in comparison with monocytes alone

172 Muscle Force Improvement by Long-Term

Systemic Expression of an AAV9 Minidystrophin

after Delivery in Young Adult GRMD Dogs without

Immune Suppression

Juan Li,1 Chunping Qiao,1 Dan Bogan,2 Ruhan Tang,1 Janet

Bogan,2 Tao Bian,1 Jianbin Li,1 Jennifer Dow,2 Zhenhua Yuan,1

Joshua C Grieger,2 Richard Jude Samulski,2 Joe Kornegay,2 Xiao

Xiao.1

1 Division of Molecular Pharmaceutics, Eshelman School of

Pharmacy, University of North Carolina at Chapel Hill, Chapel

Hill, NC; 2 Gene Therapy Center, School of Medicine, University of

North Carolina at Chapel Hill, Chapel Hill, NC.

Recently we reported effi cient and long-term transgene expression

without immune suppression in a young adult golden retriever

muscular dystrophy (GRMD) dog, by a single intravenous (i.v.)

injection of an AAV9 human minidystrophin vector containing a

muscle-specifi c (MS) promoter (ASGCT 2011 abstract) However,

we did not examine muscle functions in that dog To repeat the gene

expression results and also to examine immunology and muscle

force improvement, we carried out an experiment using 8 young

GRMD dogs from the same litter randomly divided into 2 groups,

2 males and 2 females per group for the gene therapy study In this

study,we fi rst pre-screened anti-AAV9 antibodies by an in vitro

infection assay and found no detectable neutralizing antibodies in all

the dogs At 12 weeks of age, four dogs (weight 5.8kg, 7.1kg, 7.5kg,

7.9 kg respectively) were injected with the AAV9-MS-human

Mini-dystrophin vector by a leg vein at the dose of 3.0x1013/kg The other

4 dogs were used as untreated controls Initial muscle biopsies at 4

weeks post injection showed detectable but low levels of minidys

expression by immunofl uorescent (IF) staining and western blot

The second muscle biopsies at four month post injection, however,

showed signifi cantly increased gene expression levels The results

of quantitative PCR of vector DNA were also consistent with the

IF staining and western blot data ELISPOT assays against minidys

peptide library at 7 weeks post AAV injection revealed no differences

in all 8 GRMD dogs and a normal control dog None of them had

readings above background level (50 spots/106 PBM cells) We next investigated if muscle forces could be improved after AAV9-MS-human Mini-dystrophin vector gene therapy Several phenotypic markers were evaluated in dogs at both 2 (pretreatment) and 6 months The principal outcome parameter was tetanic isometric tibiotarsal joint extension force, which showed consistent decline in GRMD dogs in our natural history studies Values for the gene therapy treated dogs increased from 1.75 N/kg ± 0.345 at 2 months to 2.05 N/kg ± 0.356 at 6 months, while those of untreated dogs decreased from 1.32 N/kg ± 0.11 to 1.09 ± 0.270 (p < 0.01) The muscle force data are

in general agreement with long-term expression of minidystrophin gene In addition, other therapeutic parameters such as muscle MRI and joint angle measurement are being investigated In conclusion, the above results further demonstrated that long-term minidystrophin gene expression could be achieved by a single i.v injection without immune suppression More importantly, muscle functions could

be signifi cantly improved as a result of AAV9-MS-minidystrophin gene therapy

173 RNA Interference Inhibits DUX4-Induced Muscle Toxicity In Vivo: Implications for a Targeted FSHD Therapy

Lindsay M Wallace,1,2 Sara E Garwick-Coppens,2 Jian Liu,2

Jacqueline Domire,2,3 Scott Q Harper.1,2,3,4

1 Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH; 2 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 3 Integrated Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH;

4 Department of Pediatrics, The Ohio State University, Columbus, OH.

Facioscapulohumeral muscular dystrophy (FSHD), one of the most common inherited muscle disorders, is characterized by asymmetric wasting and weakness in muscles of the face, scapula, and limbs The clinical features of FSHD were described over a century ago, and a chromosomal deletion that reduces the copy number of D4Z4 repeats on chromosome 4q35 was linked to the disease in 1992 Unfortunately, because these D4Z4 contractions do not completely remove any obvious genes, the pathogenic mechanisms underlying FSHD remained unresolved As a result, FSHD research for the last two decades has been primarily focused on understanding pathogenesis, while translational research for the disease has been largely lacking Today no targeted treatment for FSHD exists, but recent fi ndings suggest the barrier to translation may be lowering Several studies, including our work, support an FSHD pathogenesis model involving over-expression of the pro–myopathic gene, DUX4 Thus, DUX4 inhibition may be a promising therapeutic strategy for FSHD In this study, we tested a pre-clinical RNAi-based DUX4 gene silencing approach as a prospective treatment for FSHD We used adeno-associated viral (AAV) vector-delivered therapeutic microRNAs to correct DUX4-associated myopathy in vivo We found that our DUX4-targeted microRNAs (miDUX4) signifi cantly reduced DUX4 RNA and protein and protected mouse muscles from DUX4-induced damage Specifi cally, miDUX4-treated muscles showed minimal to no myofi ber degeneration, regeneration or activation of apoptotic pathways, while control-treated muscles had widespread damage associated with elevated DUX4 levels These histological improvements were also associated with a functional improvement measured by grip strength Our results provide proof–of–principle for RNAi therapy of FSHD through DUX4 inhibition, and importantly, this strategy can be modifi ed to target other genes that may be mis-expressed in FSHD