חידושים בהחלפת הגן הפגום בניוון שרירים דושן ובקר

State of the Art of

Gene Transfer in DMD Kevin Flanigan, MD Center for Gene Therapy Nationwide Children’s Hospital Columbus, Ohio

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Emerging viral gene therapy approaches • Gene replacement: lessons learned – Alpha-sarcoglycan – Microdystrophin

• Myostatin inhibition (follistatin) • Expression of other genes (Galgt2)

Dystrophinopathies: Clinical diagnosis Duchenne muscular dystrophy (DMD): Onset age 3-5 Pelvic girdle weakness Tight heel cords CK 50-100X normal Loss of ambulation by age 12 (range 7-12) Death by age 20 (historically)

Becker muscular dystrophy (BMD): Classic definition: loss of ambulation > age 12 Alternatively: “intermediate muscular dystrophy� for loss of ambulation ages 12 through15 BMD for loss of ambulation >age 15

Limb-girdle syndromes in adulthood Muscle aches (myalgias) Isolated cardiomyopathy

X Roberts, Genome Biology, 2001

Roberts, Genome Biology, 2001

Dystrophin Mutations 

Dystrophin gene (Xp21.1) is huge:  

2.4 million base pairs 79 exons and 8 promoters

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Large deletions (≥ 1 exon) account for ~65% of DMD/BMD patients

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~5% have duplications ~15% of boys have nonsense mutations Remainder are frameshifting insertions/deletions, splice site mutations, missense mutations

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Dystrophin mutations: Duchenne vs Becker   

Size of deletion does not correlate well with phenotype Best correlation is whether the deletion is “in-frame” or “out-of-frame” In-frame deletions are more likely to result in translation of a protein with partial function  (i.e.,

out-of-frame deletions are DMD ~90% of the time)

van Deutekom et al, N Engl J Med. 2007 Dec 27;357(26):2677-86

Non-Viral: Plasmids

• Circular DNA replicated in bacteria • Does not generate immune responses to the vector (can be re-administered) But • Low efficiency transfer

Viral: Adeno-associated viruses

• No disease association • High efficiency transfer into skeletal muscle (AAV1, AAV6, AAV8, AAV9) • Reduced host immune response But • Can elicit immune responses • Limited transgene capacity (<5 kb)

Emerging viral gene therapy approaches • Gene replacement: lessons learned – Alpha-sarcoglycan – Microdystrophin

• Myostatin inhibition (follistatin) • Expression of other genes (Galgt2)

LGMD2D • Range of Phenotypes simulates dystrophinopathies – Duchenne-like (SCARMD) – Mild, later onset (Becker-like) – Aches / pains / cramps syndrome – Recurrent myoglobinuria • Calf Hypertrophy common • Serum CK 5000 – 15000 U/L • Cardiac disease is rare and cognitive function unimpaired

1,163bp

714 bp tMCK

DITR 145

In

132bp

hSCGA cDNA

211 bp pA

DITR 145

3.25 x 1011 vg

0.75ml

0.75 ml

• rAAV1.tMCK.hSGCA • 3.25x1011 vector genomes per EDB • Biopsy at 6 months

• Next step: planning underway for an vascular delivery trial (2015)

Emerging viral gene therapy approaches • Myostatin inhibition (follistatin) • Gene replacement – SGCA – lessons learned – Microdystrophin

• Expression of other genes (Galgt2)

DMD gene transfer with an AAV2.5.CMV.minidystrophin vector • Biopsied at 6 weeks (subjects #1, 3, 4, 6) or at 3 months (subects #2, 5) • No significant dystrophin expression • Development of T-cell immunity to the transgene (dystrophin) in 2/6 subjects

Next: an improved microdystrophin vector • Different transgene – a microdystrophin with different missing domains • Different promoter – a muscle specific “onswitch” for gene expression • Different viral “envelope” – AAVrh74 • First safety trials in human patients in the last quarter of 2015

Targeted Vascular Delivery to Gastrocnemius Muscle of Non-Human Primates

Femoral Artery

Sural Artery

Non-Human Primate 3 weeks post gene transfer AAV8.GFP by femoral artery

AAV8.MCK.Micro-dys.FLAG Expression at 8 weeks in Non-Human Primate

Rhesus macaques Micro-dys.FLAG 5 x 1012 vg Muscle Biopsy at 8wk Post delivery • Widespread gene expression without immune response • • • •

Anti-FLAG Ab Staining

Emerging viral gene therapy approaches • Myostatin inhibition (follistatin) • Gene replacement – SGCA – lessons learned – Microdystrophin

• Expression of other genes (Galgt2)

Muscle Fiber

Myostatin

Other negative regulators

Activin Receptor Type IIB

Myostatin and other negative regulators inhibit the growth of muscle tissue Source: Acceleron Pharma

McPherron et al, Nature 1997

Scheulke et al, NEJM 2004

Loss of myostatin attenuates severity of muscular dystrophy in mdx mice

Wagner et al, Ann Neurol. 2002 Dec;52(6):832-6.

• Wyeth sponsored 11 Center Trial (10 USA;1GB) Using MYO-029 antibody to myostatin – No Clinical Benefit – Muscle histology showed a trend toward increased muscle fiber size – Demonstrated safety of systemic delivery of a myostatin inhibitor in a clinical trial

ACE-031 is Designed to Increase Muscle Mass by Inhibiting Multiple Negative Regulators in TGF-β Superfamily Muscle Fiber ACE-031

Myostatin

Other negative regulators

Activin Receptor Type IIB

Myostatin and other negative regulators inhibit the growth of muscle tissue

ACE-031 inhibits the negative regulators, and rebuilds muscle Source: Acceleron Pharma

AAV1-FS

Control

Trial of Intramuscular injection of AAV1-FS (Jan 2012, NCH) • BMD (n=9) and sIBM (n=6) subjects with isolated or predominant quadriceps weakness • Safety and tolerability • Muscle strength and timed measure

E-06-009

Microdystrophin + follistatin Combination Treatment Restores Force & Resistance to Contraction Induced Damage

Emerging viral gene therapy approaches • Myostatin inhibition (follistatin) • Gene replacement – SGCA – lessons learned – Microdystrophin

• Expression of other genes (Galgt2)

Surrogate gene transfer: GALGT2 • Encodes an enzyme called GalNAc transferase

• Transfers a complex sugar molecule onto a few specific proteins, including dystroglycan • Complex sugar recognizable as a “CT antigen”

Dystrophin associated glycoprotein (DAG) complexes in skeletal muscle

Dystrophin-deficient mdx mice

Galgt2 transgenic mdx mice: 1. Upregulation of the Synaptic DAG Complex 2. No Development of Muscle Pathology

Nguyen, Jayasinha, Xia, Hoyte, and Martin PT (2002) Proc.

Natl. Acad. Sci. USA 99, 5616-21

Lack of muscle damage in Galgt2 transgenic (CT) mdx mice

Martin et al, Am J Physiol Cell Physiol 296: C476-488, 2009

Galgt2 shows broad therapeutic potential for treating multiple muscular dystrophies MDC1A dyW LGMD2D Sgca-/-

DMD mdx Nguyen et al (2002) Proc. Natl. Acad. Sci. USA 99, 5616-21 Hoyte et al. (2004) Am. J. Pathol. 164, 711-18 Xu et al. (2007) Neuromusc. Disord. 17, 209-20 Xu et al. (2007) Am. J. Pathol. 171, 181-99 Xu et al. (2009) Am. J. Pathol., 175, 235-47 Martin et al. (2009) Am. J. Physiol. 296,C476-88

1. Galgt2 is therapeutic in multiple forms of muscular dystrophy 2. Galgt2 is specific 3. Endogenous upregulation of Galgt2 ameliorates disease in mdx muscle

GALGT gene transfer • Based upon animal studies, it may be beneficial in multiple types of muscular dystrophy • The gene to be transferred is already expressed – immunity not likely to be a problem

GALGT clinical trial plans • FDA application (IND) to perform first-inhuman intramuscular injections to be submitted within two weeks • Safety trial of two escalating doses • Future trial of intravascular delivery • Seeking funds for the clinical production

Acknowledgments • Nationwide Children’s Hospital Center for Gene Therapy – Jerry Mendell, MD – Louise Rodino-Klapac, PhD – Brian Kaspar, PhD – Zarife Sahenk, MD – Paul Martin, PhD – Chris Shilling, MS

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