148 the use of human iPSC derived cardiomyocytes for systematic comparison of cardiac transduction efficiency of AAV serotypes

148 The Use of Human iPSC Derived Cardiomyocytes for Systematic Comparison of Cardiac Transduction Efficiency of AAV Serotypes Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Ameri[.]

Molecular Therapy Volume 22, Supplement 1, May 2014

CELL AND GENE THERAPY FOR MYOGENIC DISORDERS

Phosphorodiamidate Morpholino Oligonucleotide

Improves Systemic Delivery and Dystrophin

Restoration in mdx Mice

Jason D Tucker,1 Mingxing Wang,1 Bo Wu,1 Pei Lu,1 Qi Lu.1

1 Neurology, Carolinas Healthcare System, Charlotte, NC.

The stability and low toxicity of phosphorodiamidate morpoholino

oligonucleotide (PMO), one of the most promising antisense

oligonucleotide chemistry currently available, with studies in animal

models and current clinical trials have demonstrated clear benefi ts

to subjects, a rare success in pharmaco-genetic interventions

However, low effi ciency, and non-specifi c delivery remain critical

barriers of oligonucleotide therapy in DMD and other diseases To

overcome these critical barriers, we have developed new polymers

toward more effective delivery systems without increased toxicity

Synthesized poly(ester amine)s (PEAs) through cross-linking low

molecular weight polyethylenimine and tris[2-(acryloyloxy)ethyl]

isocyanurate have been previously demonstrated as effective gene

and oligonucleotide delivery systems in vitro as well as in vivo

These polymers effectively condense with anionic cargo to particles

resistant to physiological conditions suffi cient to improve transfection

and reporter gene expression upon intramuscular administration as

compared to pDNA alone but also PEI 25k

The aim of this project has been to evaluate conjugation of these

new PEA polymers to directly impart cationic charge to otherwise

charge-neutral PMO toward more effective delivery while attempting

to address inherent toxicities of cationic oligonucleotide conjugates

Our results demonstrate the potential for synthetic polymer conjugates

on in vitro transfection, as well as intramuscular and systemic delivery

of antisense PMO for the restoration of dystrophin expression in the

mdx mouse

Parathyroid Hormone Type 1 Receptor Activation

Accelerate Muscle Differentiation and Muscle

Regeneration in Muscular Dystrophy

Shigemi Kimura,1 Kowasi Yoshioka.1

1 Department of Child Development, Kumamoto University

Graduate School, Kumamoto, Japan.

We have established genetically engineered embryonic stem cells

(ZHTc6-MyoD) that harbor a tetracycline-regulated expression vector

encoding myogenic transcriptional factor MyoD After removal of

tetracycline, almost all of the cells differentiated into myotubes, but a

few cells later re-formed colonies DNA microarray analysis showed

that the expression of parathyroid hormone type 1 receptor (PTH1R)

in the colonies that differentiated into muscle lineage cells is higher

in the colonies of undifferentiated ZHTc6-MyoD cells In addition to

the PTH1R required for the muscle differentiation of ZHTc6-MyoD

cells, parathyroid hormone (1-34) [PTH (1-34)] further accelerated

muscle differentiation In human and mouse skeletal muscle tissue,

most PTH1R-positive cells also expressed Pax7 and CD34, indicating

that these cells originated from satellite cells

The administration of 60 μg/kg per day of PTH (1-34) to mdx

mice (dystrophin-defi cient mice) was started at 4 weeks of age The

four limbs hanging wire test was performed on day 10 The mean

hang time of PTH-treated mdx mice (n=6) was 132±61.6 sec In

contrast, the mean hang time of saline-injected mdx mice (n=7) was

signifi cantly shorter, at 49.5±44.9 sec (P=0.03) The treadmill test was

performed on day 43 Mice were forced to run on a treadmill with

the belt parallel to the fl oor at a speed of 25 cm/sec for 100 min The

distance that they ran was recorded and compared All C57BL/10

mice (n=6) ran for 100 min, and the mean distance was 1486±7.4 m

PHT-treated mdx (n=6) mice ran a mean distance of 843.6±352 m,

although one of the PHT-treated mice did run for 100 min None of

the saline-injected mdx mice (n=7) ran for 100 min, and the mean

distance covered was 425±190 m The C57BL/10 mice (normal mice) ran signifi cantly longer than the saline-injected mdx mice (P<0.01), and the saline-injected mdx mice ran signifi cantly shorter than the PTH-treated mdx mice (P=0.03) The mice were sacrifi ced on day

60 for histological studies The diameters of myofi bres of C57BL/10 (n=5), saline-injected mdx (n=7), and PTH-treated mdx mice (n=6)

at the age of 13 weeks were 33.62±10.6, 25.41±10.4, and 31.16±12.9

μm, respectively The saline-injected mdx mice had smaller round

fi bres (regenerative fi bres) as compared to the PTH-treated mdx mice There were signifi cant differences (P<0.01) between the fi bre widths

of the groups The percentage of myofi bres with central nuclei in C57BL/10 (n=5), saline-injected mdx (n=7) and PTH-treated mdx mice (n=6) were 0.34±0.2, 93.2±2.0 and 83.01±4.0 % respectively There were also signifi cant differences (P<0.01) between the groups These results showed that there was improvement in the histologic

fi ndings in PTH-treated mdx mice We, therefore, concluded that PTH administration improved muscle weakness by enhancing muscle fi bre regeneration It appears that PTH administration may be effective

in the treatment of muscle diseases such as Duchenne muscular dystrophy This is the fi rst report that PTH1R and PTH accelerated the differentiation of muscle lineage cells

Cardiomyocytes for Systematic Comparison of Cardiac Transduction Effi ciency of AAV Serotypes

Zejing Wang,1,2 Xuan Guan,3 Véronique Blouin,4 Virginie Francois,4 Philippe Moullier,4 Martin K Childers.3,5

1 Clinical Research, Fred Hutchinson Cancer Research Center, Seattle; 2 Medicine, University of Washington, Seattle;

3 Rehabilitation Medicine, University of Washington, Seattle;

4 UMR1089 and Atlantic Gene Therapy Institute, Université de Nantes, Nantes Cedex 1, France; 5 Institute for Stem Cell and Regenerative Medicine, Seattle.

Adeno-associated viral (AAV) vectors-mediated gene replacement represents a promising therapeutic treatment in modern medicine

It has demonstrated effi cacy both in preclinical studies and clinical trials for a number of acquired and inherited diseases Several AAV serotypes, including AAV2, 5, 6, 8 and 9, have been tested for their ability in transducing cardiac muscle for their potential

of treating heart diseases Inconsistent fi ndings on their effi ciency have been reported due to the use of different model systems, vector administration routes and doses A direct comparison of these serotypes under the same condition is necessary to address this debate We have developed a system, in which human cardiomyocytes (CM) are differentiated from inducible pluripotent stem cells (iPSCs) derived from urine stem cells The iPSC-CM from healthy donors and a patient with Duchenne muscular dystrophy (DMD), in which cardiomyopathy is a leading cause of mortality, have been successfully characterized both phenotypically and physiologically In this current study, we set to systematically examine cardiac transduction effi ciency

of various AAV serotypes in a controlled manner in the iPSC-CM system Green fl uorescence protein (GFP) under the control of a CMV promoter is being used as a marker gene for all serotypes to be tested Immunofl uorecent staining and quantitative fl ow cytometry are being employed to detect expression of GFP and certain cardiomyocyte-specifi c marker genes Initial experiments used 2 serotypes, AAV6 and

8, and MOI ranging from 10 to 10,000 to determine an optimal titer in the DMD-iPSC-CM Our preliminary data showed gradual increase

in GFP expression from days 3 to 7 when robust GFP expression were observed for both vectors at MOI 1,000 (Figure 1) and above

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S56

CELL AND GENE THERAPY FOR MYOGENIC DISORDERS

Flow cytometry demonstrated 89% and 54% transduction

effi ciency of the defi ned cardiomyocytes by AAV6 and AAV8,

respectively, at MOI 10,000 We are currently repeating the same

set of studies using AAV9 and 10 to confi rm the titers for AAV6 and

8 Once an optimal titer is determined, a blinded experiment will be

conducted to compare transduction effi ciency of all major serotypes at

once in iPSC-CM from both normal and DMD patients: AAV1, 2, 5,

6, 8, 9 and 10 The knowledge gained in this study will provide much

needed information for clinical translation Further, this study will

demonstrate proof –of-principle the feasibility that drug-discovery

assays can be developed using iPSC-derived cardiomyocytes as a

biological reagent

Cargoes Delivery To Muscle

Gang Han,1 Xianjun Gao,1 Qingsong Wang,1 Limin Cao,1 Ben Gu,1

Haifang Yin.1

1 Research Center of Basic Medical Science, Tianjin Medical

University, Tianjin, China.

Duchenne muscular dystrophy (DMD) is one of the most common

and severe forms of muscular dystrophy, arising from mutations in

the dystrophin gene that preclude the synthesis of functional protein

Currently there is no treatment available in clinic Although antisense

oligonucleotides (AOs) – mediated exon skipping approach has shown

great promise for DMD patients from encouraging phase IIb clinical

trials, the lack of clear success of recent GSK phase III trials further

underline the importance of developing ways to further enhance the

systemic delivery effi ciency of current AOs

To address this issue, in our current study, we screened a number

of formulations, which have been extensively applied in clinic

with high safety profi les, in mdx mice to identify effective and

safe delivery systemis for AOs currently in clinical trials From

local intramuscular screening in mdx mice, we identifi ed four top

candidates which can increase the exon skipping efficiency of

phosphorodiamidate morpholino oligomer (PMO), with up to 6 fold

increase in dystrophin expression in tibialis anterior (TA) treated

with formulation F9 compared with that of saline Subsequent

systemic administration studies revealed that F9 can signifi cantly

enhance the exon skipping efficacy of PMOs across different

dosing regimens Notably, approximately 6.5 fold higher level of

dystrophin was restored with PMOs in the presence of formulation

F9 compared to saline at different tested dosing regimens Further

investigation indicated that the increased exon skipping effi ciency

of PMOs is attributed to the enhanced delivery of PMOs to muscle

conferred by F9 More importantly, F9 not only enhanced the delivery of PMOs but also applied to other nucleic acid cargoes e.g 2’-O-methylphosphorothioate RNA (2’OMe RNA) and siRNA Longer-term study further established the safety and effi cacy of F9

in enhancing different cargoes to muscle Our fi ndings demonstrate the potential of formulation F9 as an effective and safe formulation for cargoes in DMD and other muscle-related disorders

Muscular Dystrophy Using iPSCs from Duchenne Muscular Dystrophy Patients

Chantale Maltais,1 William-Édouard Gravel,1 Carl Lebel,1 Jacques

P Tremblay.1

1 Molecular Medecine, Laval University, Québec, Canada.

Duchenne Muscular Dystrophy is a hereditary myopathy characterized by a progressive muscular degeneration, due to the absence of dystrophin Among the possible therapies there is the autologous transplantation of myoblasts derived from induced pluripotent stem cells (hiPSCs) derived from the dystrophic patient himself Indeed, hiPSCs represents a renewable source of myogenic autologous cells and would thus not induce an immune response of the patient against the transplanted cells However, there is currently

no effective and reproducible protocol of differentiation of hiPSCs into myoblasts that results in considerable transplantation success We aim to induce myogenesis of hiPSCs with recombinant transcription factor proteins fused with a cell penetrating peptide, i.e., Tat These recombinant proteins were produced in bacteria and purifi ed Our results showed that these transcription factors were able to enter into mesenchymal-like cells The capacity of these recombinant proteins to induce a myogenic differentiation is currently under study When these therapeutic approaches will be established, they could be clinically applied to treat dystrophic patients

After Skeletal Muscle Injury in Hypertensive Spontaneously Rats

Roberta Sessa Stilhano,1 Leonardo Martins,1 Priscila Keiko Matsumoto,1 Sang Won Han.1

1 Biophysics, Federal University of Sao Paulo, Sao Paulo, Brazil.

Introduction: Fibrosis is a major acquired sequel after muscle injury, especially in more severe injuries in which healing occurs slowly, which prevents the proper muscle contraction and can lead

to chronic pain and muscle contractures Fibrosis is a necessary step for recovery of damaged tissue, but when this process is exacerbated,

as often occurs in cases of deeper lesions, causes imperfect tissue regeneration Hypertension may enhance fi brosis in cardiac muscle Aim: The main objective of this project is to prove that SHR (Spontaneously hypertensive rat) has more fi brosis than W (Wistar) after skeletal muscle injury

Methods: The laceration model of injury was performed using a scissor to cut partly the tibial muscle Animals were divided in the following groups: SHR_L (SHR with laceration), SHR_LS (SHR with laceration followed by suture), W_L (W with laceration) and W_LS (W with laceration followed by suture) After 30 days, animals were euthanized and the tibial muscles were removed for histological analysis The following parameters were analyzed: Injury’s area (Macro and microscopic), amount of adipocytes, fi brosis’s area and muscle’s weight

Results: L group present more injury’s area and adipocytes when compared with LS group There is no difference between L and LS in terms of fi brosis’s area The W_LS presented 1.3-fold more injury’s area than SHR_LS (p<0.05) However, SHR_LS presented 1.5-fold more fi brosis’s area than W_LS (p<0.05) The SHR_L presented 3-fold more adipocytes than W_L (p<0.001)