Samedan Ltd Pharmaceutical Publishers

Advanced search

Publications

European Pharmaceutical
Contractor

European Biopharmaceutical
Review

International Clinical Trials

Pharmaceutical Manufacturing
and Packing Sourcer

Innovations in Pharmaceutical
Technology

Subscribe Online

Pharmaceutical Directory

Industry Events Calendar

News and Press Releases

Employment Opportunities

Advertising with us

Air Transport Publications

home > ebr > summer 2010 > therapy in motion

PUBLICATIONS

European Biopharmaceutical Review Therapy in Motion
Juliet A Ellis and Francesco Muntoni at University College, London, provide an overview of how RNA therapy has progressed for Duchenne muscular dystrophy The concept of RNA therapeutics was introduced in 1996, but it is only in the last few years that significant progress has been made in the research and clinical environment. The term ‘antisense oligonucleotide’ (AO) refers to a wide range of oligonucleotide designs that bind to and modulate RNA function through indirectly affecting downstream activities. Early clinical trials with AO confirmed that their use as a therapeutic approach was safe in humans at a commercially competitive price; however their efficacy was often insufficient. The aim of most of the early AO studies was to knock down a target RNA in order to induce less of the deleterious protein product (such as tumour growth factors and viral mRNAs). This necessitated rapid suppression of genes which often had a fast turnover. However, more recent applications targeting mRNA with slow turnover and stabilising it from nonsense-mediated decay by inducing exon skipping have brought a new lease of life to AO, and allowed the development of novel formulations and delivery mechanisms. Targeting rare genetically determined diseases, particularly rare orphan diseases (ROD; defined as not affecting more than five in 10,000 persons within the EU and fewer than a total of 200,000 in the US) where effective treatments are lacking, is an area of rapid expansion. Significant clinical trial progress has been made with the ROD Duchenne muscular dystrophy (DMD), and there are others such as myotonic dystrophy type I (DM-1), dysferlinopathies, spinal muscular atrophy 1 (SMA 1), amyotrophic lateral sclerosis, ataxia telangectasia and cerebellar ataxis in preclinical development. The financial implications behind developing AOs to treat ROD, which in many instances arise from a different specific mutation, are breathtaking, requiring substantial long-term funding commitments from pharmaceutical companies. Moreover, with RNA as the target molecule, a personalised medicine approach can be implemented in order to maximise patient benefit. RNA OLIGONUCLEOTIDES AS A THERAPEUTIC PLATFORM The central dogma of molecular genetics states that DNA as the encoder of genetic information is transcribed into a messenger RNA molecule (mRNA), which in turn is translated into protein. A vital intermediate is the processing of a pre-messenger RNA (pre-mRNA), which involves the splicing out of introns (nonprotein coding), generating a mature mRNA. Splicing also allows for the inclusion or exclusion of specific exons (protein coding), thereby yielding more than one mRNA molecule per pre-mRNA transcript and maximising proteomic diversity. However, by manipulating splicing – so called splice-modulation therapy – we can indirectly correct a variety of genetic mutations (1). To do this, short (20 to 30 nucleotides) AOs homologous to the exon-intron boundary surrounding the mutated exon in the pre-mRNA are used to steer pre-mRNA splicing to ‘skip’ the mutated exon or exons (since multi-exon skipping is also possible), while restoring the open reading frame. This generates a protein of at least partial functionality (see Figure 1). DMD affects young boys who develop progressive severe muscle weakness and wasting, becoming wheelchair bound in their teens. The condition is life-limiting, from a decline in respiratory and cardiac function, and even with supportive ventilation and corticosteroid therapy, death occurs in the mid-20s (2). It is the most common fatal genetic disorder to affect children on a global basis (incidence 1:3,500 boys) with an estimated global health cost of €257 million per annum (3). Mutations leading to DMD disrupt the open reading frame so as to abolish production of the protein dystrophin. Functionally, dystrophin cross-links the muscle cell membrane to the internal actin cytoskeleton, acting as a molecular shock absorber to cushion muscle cells from the forces imposed on them during movement. Importantly, inframe mutations also occur, resulting in the significantly milder Becker muscular dystrophy (BMD). Here, only central parts of the protein are missing; the ends of the dystrophin protein are retained, allowing for partial functionality. DMD is an excellent model for exon-skipping therapy for three reasons. Theoretically it could restore the open reading frame in 83 per cent of cases, and thus ‘conversion’ to the milder BMD with potentially stabilising disease progression. Secondly, the existence of a few dystrophin-positive (‘revertant’) fibres in many patients implies that an intrinsic alternative splicing mechanism exists to correct mutations. Finally, there is no other treatment available to address this disease pathogenesis. A similar corrective splicing technique is currently in preclinical stages for SMA 1, a neurodegenerative disease where there is the loss of the SMN1 (survival of motor neuron 1) gene, leading to selective motor neuron degeneration. SMN2 is nearly identical to SMN1 but has a nucleotide replacement that causes exon 7 skipping, resulting in a truncated, unstable version of the SMA protein. SMN2 is present in all SMA patients, and corrective SMN2 splicing is in preclinical trials. However, this requires suppressing the endogenous signals to splice exon 7, to induce retention rather than skipping and this is proving more challenging. To date, the most effective method involves the AO into viral expression vectors incorporating U7 snRNA sequences (4). AO can also be administered to block inappropriate DNA-protein interactions, as occurs in DM-1. Here, expansion of a trinucleotide repeat in the 3’ untranslated region leads to the formation of hairpin-like structures. These interact with RNA binding proteins triggering the aberrant RNA processing and splicing associated with muscle development. The expanded region is thus a therapeutic target, since blocking the interaction with proteins triggering incorrect splicing is likely to alleviate the clinical symptoms. AO treatment on both transgenic models for DM-1 and patient cells suggests that this approach is clinically viable (5, 6). CHEMISTRY & TISSUE DELIVERY All therapeutic oligonucleotides contain a fraction of non-natural nucleotides. Modifications to the base, sugar and phosphate, as well as 5’ and 3’ ‘caps’, can increase stability against endogenous nuclease attack, in addition to increasing target affinity, biological potency and cellular distribution (7). Of note are three very different chemistries currently in preclinical and/or clinical trials for DMD. These are phosphorodiamidate morpholino oligonucleotides (PMOs), 2’-Omethyl phosphorothioate oligonucleotides (PS oligomers) and peptide nucleic acid (PNA) (see Figure 2). Both the PNA and PMO-type have a stable, uncharged backbone, whereas the PS oligomers are negatively charged due to the addition of O-methyl residues on the 2’ ribose position (8). This difference affects their respective delivery and skipping efficiencies. Both a PS oligomer and a PMO have been used successfully in clinical trials by injecting a single muscle in DMD patients to skip exon 51 (9,10). This affirms the validity of the technique. Nevertheless, expression is non-uniform across the treated muscles, individual patient variation is observed and in vitro experiments suggest neither type of AO easily delivers to the heart. Therefore, by far the greatest challenge for the clinical and marketing success of RNA therapeutics lies with specific, uniform tissue delivery and patient tolerability. PS oligomers are of a large molecular size and are charged, so spontaneous cell membrane uptake is restricted. AO treatment on DMD patients has progressed well because the muscle degenerative pathology allows efficient AO cellular uptake through the damaged cell membrane. However, DMD is a systemic disease, ideally requiring systemic delivery and preferably through an oral or intravenous route to reduce patient discomfort. Animal experiments show that this is possible, but high AO doses are required to give even a moderate dystrophin-positive fibre response (11). To maintain lower AO doses, which are comparable with acceptable safety and toxicology data, improved targeting systems are now being investigated. Examples of delivery methodologies being investigated include: Encapsulating AO in nanoparticles which biodegrade, releasing the AO in the target tissue Attaching altered, naturally occurring, cell-penetrating peptides (CPP) to the 5’ end of the AO, which direct cell uptake of their attached baggage Nucleic acid-based aptamers that target specific cell surface proteins (12) The most success has been seen with PMO and PNA-type AOs conjugated to a combination of CPPs, which can be administered intravenously, yielding a far superior uptake into multiple tissues, including the heart, at low AO doses (13). REGULATORY AFFAIRS RNA therapeutics have been in clinical trials for over 15 years, with the first approved for clinical use for age-related macular degeneration in 2004. In this time, over 3,000 patients and healthy volunteers have been in clinical trials for AO therapy for up to, and beyond, one year. Currently, about 60 AOs are in clinical trials, but despite this, AOs remain relatively unknown in the market place. There are several reasons for this. RNA medicines present with a mode of action very different to all other currently licensed medicinal products. AOs exist as several different chemistry classes and each individual AO is sequence-specific. So is safety data required on every AO or just for each class? Moreover, certain AO sequences present atypical toxicity profiles and these need to be screened out. Regulators require a comprehensive portfolio on the pharmacodynamic, genotoxic, pharmacokinetic and toxicology profiles of a medicinal product from several confirmatory sources, before they are able to make a valid judgement for licensing. This is currently lacking for AO. The situation is compounded by the very few licensing requests received to date and with an insufficient pool of safety data for the regulators to draw upon, sufficient guidelines are lacking. To move forward, concerted efforts by both pharma and clinicians to produce the necessary safety data and clinical protocol design must occur, while simultaneously liaising with the regulators at each step to ensure swift orphan drug status approval. Recently a landmark meeting was held at the European Medicines Agency, to address how the current regulatory guidelines for orphan drugs are applicable to AO therapy on DMD patients (14). The regulators were asked, since it is both financially non-viable and temporally demanding to obtain safety data on the 30 or so AOs needed to treat the DMD populace, if the necessary safety data could be extrapolated from one AO to another. In reply, the conditions on safety data-gathering mentioned above were stipulated as needing to be met first, before any precept could be issued on the minimum number of AOs required for full toxicology analysis. In addition, they ruled that both biochemical and clinical outcome measures confirming clear, functional benefit to the patient were required. Indeed a more proactive interaction with patients and advocacy groups to help in clinical trial design was encouraged to aid this process, since patients are in the best position to know what improves their life quality. The meeting stimulated a positive relationship with the regulators, and future meetings are planned to ensure a fast, but safe way forward. So why has RNA therapy progressed so well for DMD over other RODs? A strong contributory factor is the pre-existence of globally acquired patient registries on ambulant DMD boys as part of an EU funded Clinical Network Translational Research in Europe for the Assessment and Treatment of Neuromuscular Diseases (TREAT-NMD). As well as providing natural histories, they include standardised clinical care procedures – which facilitate both the planning and interpretation of multicentre trials – making it an attractive industrial tool (15). If RNA therapy is to progress as a general form of treatment, the way forward is to generate disease-specific patient registries. Indeed, global registries for Limb-Girdle muscular dystrophy (LGMD) are currently being collected, and at least one type of LGMD is amenable to the exon skipping protocols used for DMD (16). Targeted splice modulation is thus likely to be a treatment of the future where there are clear divisions between types of mutations causing mild or severe forms of the disorder in question. The experience gathered by studying these rare genetic diseases will most certainly help the development of therapeutic intervention for more common genetic and acquired disorders affecting metabolism, or the heart and brain. References Wood MJ, Gait MJ and Yin H, RNA-targeted splice-correction therapy for neuromuscular disease, Brain 133: pp957-972, 2010 Bushby K, Finkel R, Birnkrant DJ, Case LE, Clemens PR, Cripe L, Kaul A, Kinnett K, McDonald C, Pandya S, Poysky J, Shapiro F, Tomezsko J and Constantin C, Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management, Lancet Neurol 9: pp77-93, 2010 Australian Access Economics; 2007 Meyer K, Marquis J, Trub J, Nlend Nlend R, Verp S, Ruepp MD et al, Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation, Hum Mol Gens 18: pp546-555, 2009 Wheeler TM, Sobczak K, Lueck JD, Osborne RJ, Lin X, Dirksen RT et al, Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA, Science 325: pp336-339, 2009 Mulders SA, van den Broek WJ, Wheeler TM, Croes HJ, van Kuik- Romeijn P, de Kimpe SJ et al, Triplet-repeat oligonucleotidemediated reversal of RNA toxicity in myotonic dystrophy, Proc Natl Acad Sci USA 106: pp13,915- 13,920, 2009 Wilson C and Keefe AD, Building oligonucleotide therapeutics using non-natural chemistries, Curr Opin Chem Biol 10: pp607-614, 2006 Yin H, Lu Q and Wood M, Effective exon skipping and restoration of dystrophin expression in peptide nucleic acid antisense oligonucleotides in mdx mice, Mol Ther 16: pp38-45, 2008 Van Deutekom JC, Janson AA, Ginjaar IB et al, Local dystrophin restoration with antisense oligonucleotie PRO051, New Engl J Med 357: pp2,677-2,686, 2007 Kinali M, Arechavala-Gomeza V, Feng L et al, Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in DMD: a single-blind, placebocontrolled, dose-escalation, proofof- concept study, Lancet Neurol 8: pp918-928, 2009 Lu QL, Rabinowitz A, Chen YC, Yokota, T, Yin H, Alter J et al, Systemic delivery of antisense oligonucleotide restores dystrophin expression in body-wide skeletal muscles, Proc Natl Acad Sci USA 102: pp198-203, 2005 Zhou J and Rossi JJ, The therapeutic potential of cellinternalizing aptamers, Curr Top Med Chem 9: pp1,144-1,157, 2009 Yin H, Moulton HM, Betts C, Seow Y, Boutilier J, Iverson PL et al, A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophindeficient mdx mice, Hum Mol Genet 18: pp4,405-4,414, 2009 Muntoni F, The development of antisense oligonucleotide therapies for Duchenne muscular dystrophy: Report of a TREAT-NMD workshop hosted by the European Medicines Agency (EMA) on September 25th 2009, Neuromuscl Dis 20: pp355-362, 2010 Bushby K et al, Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care, Lancet Neurol 9: pp177-189, 2010 Aartsma-Rus A, Singh KH, Fokkema IF, Ginjaar IB, van Ommen GJ, Dunnen JT and van der Maarel SM, Therapeutic exon-skipping for dysferlinopathies?, Eur J Hum Genet, in press, PMID 20145676, 2010
Read full article from PDF >>



Rate this article	You must be a member of the site to make a vote.	Average rating:	0

There are no comments in regards to this article.

Therapy in Motion