Stephen B. Shrewsbury

Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study

Summary Background We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. Method We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5–15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. Findings 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1–10·6) to 16·4% (10·8–22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. Interpretation The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. Funding UK Medical Research Council; AVI BioPharma.

Safety Pharmacology and Genotoxicity Evaluation of AVI-4658

Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations. Restoration of dystrophin by exon skipping was demonstrated with the phosphorodiamidate morpholino oligomers (PMO) class of splice-switching oligomers, in both mouse and dog disease models. The authors report the results of Good Laboratory Practice–compliant safety pharmacology and genotoxicity evaluations of AVI-4658, a PMO under clinical evaluation for DMD. In cynomolgus monkeys, no test article–related effects were seen on cardiovascular, respiratory, global neurological, renal, or liver parameters at the maximum feasible dose (320u2009mg/kg). Genotoxicity battery showed that AVI-4658 has no genotoxic potential at up to 5000 μg/mL in an in vitro mammalian chromosome aberration test and a bacterial reverse mutation assay. In the mouse bone marrow erythrocyte micronucleus test, a single intravenous injection up to 2000u2009mg/kg was generally well tolerated and resulted in no mutagenic potential. These results allowed initiation of systemic clinical trials in DMD patients in the United Kingdom.

Repeat-Dose Toxicology Evaluation in Cynomolgus Monkeys of AVI-4658, a Phosphorodiamidate Morpholino Oligomer (PMO) Drug for the Treatment of Duchenne Muscular Dystrophy:

AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) drug designed to restore dystrophin expression in a subset of patients with Duchenne muscular dystrophy (DMD). Previous reports demonstrated this clinical proof-of-principle in patients with DMD following intramuscular injection of AVI-4658. This preclinical study evaluated the toxicity and toxicokinetic profile of AVI-4658 when administered either intravenously (IV) or subcutaneously (SC) to cynomolgus monkeys once weekly over 12 weeks, at doses up to the maximum feasible dose of 320 mg/kg per injection. No drug-related effects were noted on survival, clinical observations, body weight, food consumption, opthalmoscopic or electrocardiographic evaluations, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic evaluations. Drug-related microscopic renal effects were dose-dependent, apparently reversible, and included basophilic granules (minimal), basophilic tubules (minimal to moderate), and tubular vacuolation (minimal to mild). These data establish the tolerability of AVI-4658 at doses up to and including the maximum feasible dose of 320 mg/kg by IV bolus or SC injection.

Clinical Expert Panel on Monitoring Potential Lung Toxicity of Inhaled Oligonucleotides: Consensus Points and Recommendations

Oligonucleotides (ONs) are an emerging class of drugs being developed for the treatment of a wide variety of diseases including the treatment of respiratory diseases by the inhalation route. As a class, their toxicity on human lungs has not been fully characterized, and predictive toxicity biomarkers have not been identified. To that end, identification of sensitive methods and biomarkers that can detect toxicity in humans before any long term and/or irreversible side effects occur would be helpful. In light of the publics greater interests, the Inhalation Subcommittee of the Oligonucleotide Safety Working Group (OSWG) held expert panel discussions focusing on the potential toxicity of inhaled ONs and assessing the strengths and weaknesses of different monitoring techniques for use during the clinical evaluation of inhaled ON candidates. This white paper summarizes the key discussions and captures the panelists perspectives and recommendations which, we propose, could be used as a framework to guide both industry and regulatory scientists in future clinical research to characterize and monitor the short and long term lung response to inhaled ONs.

The Oligonucleotide Safety Working Group (OSWG).

The past decade saw a shift from the development of oligonucleotides as a mainly academic interest to an active industry. Today many small and medium sized companies are developing oligonucleotides for therapeutic use, and even large companies are keeping a close watch on progress. Recognizing that this new class of pharmaceutical compound faces regulatory challenges that perhaps might not be best served by the current rules for small molecules (under which they are currently regulated) or biological products (Table 1) , the Food and Drug Administration (FDA) and Drug Information Association (DIA) convened the Oligonucleotide-Based Therapeutics Conference in 2007. This conference was attended by academics, regulators, and industrial scientists all seeking clarification of the path forward for early, preclinical, and clinical development for a multitude of oligonucleotide-based compounds for a range of diseases, which in many cases have hitherto not been the targets of conventional drug treatment using small molecules. At this inaugural meeting, it was agreed to establish an Oligonucleotide Safety Working Group (OSWG) for ongoing discussion such that emerging scientific data from academia and industry could be shared amongst participants and with regulatory authority members. This forum was felt to be the best way to collectively tackle the formidable hurdles involved with developing a completely new class of therapeutic. Since then, the DIA and FDA have organized and run several successful follow-up Oligonucleotide meetings that complement the activities of other societies and organizations. The members of OSWG have been very involved with the planning and execution of these meetings. In addition, because safety is a very broad term, the OSWG has established a number of subcommittees run by, and composed of, members representing the following aspects of safety assessment, specifically as related to oligonucleotides:

O.16 Current progress and preliminary results with the systemic administration trial of AVI-4658, a novel phosphorodiamidate morpholino oligomer (PMO) skipping dystrophin exon 51 in Duchenne muscular dystrophy (DMD)

the dystrophin gene. We are developing methods to deliver therapeutic genes to muscles throughout the body to either replace the missing dystrophin gene or to help compensate for the lack of dystrophin. We show that shuttle vectors derived from adeno-associated virus type 6 (rAAV6) are able to deliver genes to muscles throughout the body of adult mice when injected directly into the bloodstream. rAAV6 delivery results in highly efficient gene expression in skeletal and cardiac muscle that persists for the lifespan of the mouse. To accommodate the limited cloning capacity of rAAV vectors we have designed a variety of different micro-dystrophin vectors, and a recently modified micro-dystrophin with alterations in the hinge domains increases functionality. We have begun testing these AAV vectors in wild type and dystrophic dogs, and in wild type non-human primates. These studies revealed a cellular immune response directed against the AAV capsid proteins, but which could be blocked by short-term immune suppression, leading to long-term dystrophin expression. We have observed that delivery of AAV6 vectors into various veins and arteries of the dog results in efficient gene transfer to downstream muscles, but does not lead to whole body gene transfer. Instead, it appears that vector will need to be delivered into multiple vascular sites to target muscles body wide. These results suggest that a combination of intravascular AAV delivery coupled with transient immune suppression could lead to an effective therapy for DMD.

P03 Exon skipping and dystrophin restoration in Duchenne muscular dystrophy patients after systemic phosphorodiamidate morpholino oligomer treatment

1FC2.1 Exon skipping and dystrophin restoration in Duchenne Muscular Dystrophy patients after systemic phosphorodiamidate morpholino oligomer treatment S. Cirak1 *, V. Arechavala-Gomeza1, M. Guglieri2, L. Feng1, S. Torelli1, K. Anthony1, M.E. Garralda3, D.J. Wells4, G. Dickson5, M.J.A. Wood6, S. Wilton7, V. Straub2, S.B. Shrewsbury8, C. Sewry9, J.E. Morgan1, K. Bushby2, F. Muntoni1. 1UCL Institute of Child Health The Dubowitz Neuromuscular Centre, London, United Kingdom, 2Institute of Human Genetics, Newcastle University, Newcastle, United Kingdom, 3Academic Unit of Child and Adolescent Psychiatry, Imperial College St Mary’s Campus, London, United Kingdom, 4Royal Veterinary College, London, United Kingdom, 5Royal Holloway University of London, United Kingdom, 6Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom, 7Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, Australia, 8AVI BioPharma, Bothell, WA, United States, 9Centre for Inherited Neuromuscular Disorders, Robert Jones and Agnes Hunt NHS Trust, Oswestry, United Kingdom

O04 Results of a systemic antisense study in Duchenne muscular dystrophy

The UK MDEX Consortium (http://www.mdex.org.uk/) is involved in close collaboration with AVI BioPharma, in clinical trials using antisense oligonucleotides (AOs) to induce exon skipping in boys with DMD. In 2008 we completed a dose-escalation IM study of a morpholino (PMO) AO, AVI-4658, which induces skipping exon 51 in dystrophin mRNA. In February 2009 we initiated a dose escalation study in ambulant DMD boys aged 5–15 years with deletions benefitting from skipping exon 51. This study consists of 12 weekly administrations of AVI-4658 followed by a muscle biopsy to assess dystrophin expression at baseline and 14 weeks. Clinical parameters are followed for 26 weeks, consisting of safety (adverse events, physical examinations, laboratory tests), muscle, pulmonary and cardiac function, and pharmacokinetics at 1, 6 and 12 doses. A Data Safety Monitoring Board guides dose escalation decisions. Cohorts 1–5 completed 12 weeks of dosing (January 2010), while cohort 6 will complete dosing in March 2010 and clinical observations in June. No drug related SAEs or severe drug related AEs have been reported so far. To date, single doses of 900mg and cumulative exposure exceeding 10,000mg have been well tolerated. Exon skipping and dystrophin protein expression in the cohorts analysed so far indicates a dose response in exon skipping and protein expression in the cohorts up to 4.0mg/kg, with data in the 10.0 and 20.0mg/kg cohorts available soon. These results suggest that AVI-4658 has the potential to lead to the development of a drug that could play a role in the treatment of DMD.