Therapy of Genetic Disorders Novel Therapies for

Authors: Jane T Seto Niclas E Bengtsson Jeffrey S Chamberlain

Publish Date: 2014/03/11

Volume: 2, Issue: 2, Pages: 102-112

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Abstract

Duchenne muscular dystrophy is an inherited progressive musclewasting disorder caused by mutations in the dystrophin gene An increasing variety of approaches are moving towards clinical testing that all aim to restore dystrophin production and to enhance or preserve muscle mass Gene therapy methods are being developed to replace the defective dystrophin gene or induce dystrophin production from mutant genes Stem cell approaches are being developed to replace lost muscle cells while also bringing in new dystrophin genes This review summarizes recent progress in the field with an emphasis on clinical applicationsDuchenne muscular dystrophy DMD is the most common and severe form of childhood muscular dystrophy affecting approximately 1 in 5000 boys 1 The disease is characterized by progressive muscle weakness with delayed motor and speech milestones and elevated serum creatine kinase levels Patients demonstrate reduced motor skills by age 3–5 and typically lose ambulation by 12 years of age Glucocorticoids prednisone or deflazacort are prescribed to slow disease progression but to date there is no cure for this disease Despite intensive clinical attention to respiratory support and management of cardiac complications 2 most patients do not live beyond age 30DMD is caused by loss of function mutations in the Xlinked DMD gene commonly called ‘dystrophin’ resulting in near complete deficiency of dystrophin in cardiac skeletal and smooth muscles The types of mutations that lead to DMD include point mutations that can encode premature termination codons nonsense mutations or “stop codons” deletions or exon–intron splice site mutations that disrupt the normal protein or open reading frame ORF of the dystrophin messenger RNA mRNA partial gene duplications that disrupt the normal ORF and rarely point mutations or deletions that disrupt critical functional domains of dystrophin Mutations that enable expression of partially functional dystrophin lead to the milder Becker muscular dystrophy BMD Clinical observations of a BMD patient who remained ambulatory past age 70 despite absence of 46  of the dystrophin gene 3 lent proof of principle that restoring some functional dystrophin expression can ameliorate the disease severity of DMD Expression of 20–30  of normal dystrophin levels is sufficient to avoid muscular dystrophy in mice and humans while lower levels can lead to various severities of BMD 4 5Functional dystrophin can be restored through rAAVmediated gene replacement exon skipping mutation suppression not shown or cell therapies not shown For gene replacement minimally essential regions of dystrophin are removed to fit the limited carrying capacity of rAAV vectors and generate short dystrophin constructs microdystrophins Despite the truncation microdystrophins restored dystrophin protein production and ~90  of strength in dystrophic mice Larger dystrophin constructs minidystrophins that incorporate additional regions necessary for recruiting important dystrophin binding partners eg nNOS result in even greater physiological improvements Minidystrophins are delivered in pieces using multiple rAAV vectors and reassembled in muscle cells by various methods such as homologous recombination In exonskipping synthetic antisense oligonucleotides AON are specifically designed to anneal to precursor mRNA premRNA and alter RNA splicing to restore normal openreading frames resulting in the production of a smaller but functional dystrophin These techniques can be used in combination with cell therapies to correct genetic mutations prior to transplantation back into the patient in ex vivo therapiesApproximately 10  of DMD mutations are singlebase “point” mutations that introduce premature stop codons into the dystrophin mRNA One strategy to restore dystrophin expression in patients with such nonsense mutations is mutation suppression specifically of premature termination codon PTC mutations Nonsense mutations signal an end to protein translation and result in production of a shortened nonfunctional protein that is usually unstable and degraded in the cell These ‘PTC’ mutations account for ~10  of DMD cases 6 see also http//wwwumdbe/DMD/W DMD/indexhtml Certain antibiotics such as gentamicin can induce translational readthrough of PTC mutations At high concentrations the binding of gentamycin to the protein production machinery in cells induces readthrough of PTCs in mRNA inserting a new amino acid to continue translation of the complete protein 7 However serious doselimiting toxicities preclude the use of gentamicin in patients for this purpose Drug discovery programs led to the identification of PTC124 also known as ataluren a compound shown to be more potent in PTC readthrough than gentamicin 8 although recent in vitro studies have raised questions regarding the genuine readthrough ability of ataluren 9 10 11 Regardless of the mechanism ataluren was somewhat effective in restoring functional dystrophin expression in mdx mice resulting in improved strength and decreased injury in response to exerciseinduced damage 8 A phase I study in healthy volunteers established safety and tolerability at doses exceeding preclinical efficacy 12 Phase 2b studies over 48 weeks involving DMD and BMD patients aged 5–20 years showed that patients receiving a lowdose treatment 20 mg/kg experienced a significantly slower disease progression demonstrating a 29m improvement in a 6min walk test 6MWT compared to patients who received placebo http//clinicaltrialsgov/show/NCT01182324 http//wwwmusculardystrophyorg/assets/0003/5427/201207 ataluren updatepdf Intriguingly patients who received a higher dose 40 mg/kg showed a similar decline as the placebo group suggesting that ataluren may have an inverted bellshaped dose–response curve As of March 2013 a phase 3 study of ataluren was initiated with recruitment restricted to DMD patients with PTC stop codon mutations This will be a randomized doubleblind placebocontrolled study with higher power to determine the potential efficacy and safety of lowdose ataluren http//clinicaltrialsgov/show/NCT01826487The majority of DMD cases arise from partial dystrophin gene deletions or duplications or from mutations that affect the normal splicing of the dystrophin RNA transcript into mRNA Each of these types of gene mutations is a problem when it disrupts the normal dystrophin mRNA open reading frame such that a functional dystrophin protein can not be produced in muscle cells An approach to restore the normal mRNA reading frame involves exon skipping This can be induced by short synthetic fragments of nucleic acids known as “antisense oligonucleotides” AONs which are designed to bind anneal with RNA sequences that regulate how a gene’s initial RNA transcript is spliced into a functional mRNA By altering RNA splicing near the mutation on the precursor mRNA premRNA the cell can be tricked into making a slightly shorter than normal mRNA that lacks a mutation and that carries an otherwise normal open reading frame Fig 1 This strategy is theoretically applicable to ~80  of all DMD patients 13 Recent drug development for exon skipping has targeted exon 51 of the DMD gene as this could be applied to ~13  of boys with DMD Two types of AONs have been taken into clinical trials each made using different nucleic acid chemistries Drisapersen previously known as PRO051 GSK2402968 was developed by Prosensa and GSK The other was eteplirsen AVI4658 developed by Sarepta Therapeutics Therapeutic efficacy of these AONs has been demonstrated in several mouse models for DMD mdx and the dystrophic cxmd dog 14 15 16 17 Both compounds demonstrated safety and efficacy by direct intramuscular injections in patients with restoration of dystrophin expression in the majority of muscle fibers near the injection site without severe treatmentrelated adverse effects 18 19Initial phase 1/2A studies for both drisapersen and eteplirsen appeared promising but disappointingly neither compound succeeded in demonstrating significant improvements in the primary outcome measure the 6MWT in larger cohorts In early phase 1/2A drisapersen trials involving 12 patients a subset of patients who received the higher doses 40 and 60 mg/kg/week showed some variable de novo dystrophin expression and demonstrated a mean improvement of 35 m in the 6MWT after 12 weeks of treatment 20•• However phase 3 trials where 125 patients aged 5–16 years were administered 6 mg/kg/week over 48 weeks did not result in statistically significant improvements in the 6MWD or in secondary measures of motor function when all patients were analyzed However Prosensa recently announced that when the analysis was confined to patients younger than 7 a 49m difference was observed in the 6MWT in patients on a 96week extension trial Some of these studies also revealed kidney toxicity and low platelet counts in a few patients at high drug doses To date these latest results have only been discussed at conferences and in press releases http//wwwgskcom/media/pressreleases/2013/gskandprosensaannounceprimaryendpointnotmetinphaseiiihtml http//wwwthestreetcom/story/11854354/1/glaxodmddrugtiedtoserioussideeffectshospitalizationshtml http//cureduchennecom/blog/prosensareportsinitialfindingsfromthefurtherclinicaldataanalysesofdrisapersenforthetreatmentofduchennemusculardystrophy/ GSK recently announced that it would not continue studies of drisapersen and is returning rights to the drug to Prosensa which will continue its development http//usgskcom/html/medianews/pressreleases/2014/prosensaregainsrightstodrisapersenfromgskandretainsrighhtmlSimilarly early phase 2 studies for eteplirsen showed low and variable increases in dystrophin expression in 7 out of 19 patients all but one who received the highest dose of 10 or 20 mg/kg over 12 weeks There was a posttreatment increase in dystrophin protein expression from 9 to 16  of normal controls three patients showed between 15 and 55  dystrophinpositive myofibers Notably restored dystrophin was shown to be functional and restored βdystroglycan αsarcoglycan and nNOS expression in patients with exon 49–50 deletions in keeping with the nNOS binding domain being encoded in exons 42–45 Inflammatory cell infiltrate was also reduced in the 10–20 mg/kg cohorts suggesting that restored dystrophin is tolerated by the immune system 21 22•• In a second more recent phase 2 study increased dosage of eteplirsen was further assessed in a doubleblind placebocontrolled test in 12 DMD boys aged 7–13 years 23 Patients were administered with placebo 30 mg/kg/week or 50 mg/kg/week of eteplirsen for 24 weeks followed by an openlabel extension study at the two doses through week 48 Results showed that at least 12 weeks of treatment was required to produce significant increases in dystrophin At 48 weeks the mean increase in dystrophinpositive fibers was 47  and the four patients in the placebo/delayed group after 12 weeks of treatment showed a significant increase of 38  Similarly functional assessment by the 6MWT shows a divergence between the placebo and the eteplirsen groups at week 12 after which the treated group no longer showed a significant decline and stabilized The placebo/delayed cohort likewise appeared to stabilize by 36 weeks 12 weeks after treatment Comparison of the 48weektreated group to the delayed group showed a significant difference of 67 m by week 48 in the 6MWT Recent natural history data have shown that patients who could walk 350 m at baseline tend to stabilize over 1 year 24 and mean baseline 6MWT for all patients in the eteplirsen study was 350 m 23 In a very recent press release Sarepta announced continued stabilization of the 6MWT test in the phase 2b study where patients were continued on highdose drug 30–60 mg/kg for 120 weeks http//investorrelationssareptatherapeuticscom/phoenixzhtmlc=64231p=irolnewsArticleID=1891149highlight=

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