Vol. 18 No. 3, March 2008
`
`Neuromuscular disorders: NMO rl:,1)
`v. 18, no. 3 (Mm 2G08)
`General Ccllection
`V\/1 ND37GB
`2003-05-08 0~1f:ri
`
`18 (3) 193-276
`ISSi~ 0960--8966
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`Associate Editors
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`

`

`Neuromuscular Disorders
`Volume 18 Number 3
`Cited in: Current Contents/Life Sciences, Elsevier BIOBASE/Current Awaren
`Index Medicus, MEDLINE, Neuroscience Citation Index, Reference Up
`Science Citation Index, SciSearch
`CONTENTS
`Research papers
`Hand weakness in Duchenne muscular
`dystrophy and
`its relation to physical
`disability
`F.l. Mattar, C. Sobreira
`Natural history of CMT1A including Qol: A
`2-year prospective study
`L. Padua, D. Pareyson, I. Aprile, T. Cavallaro,
`A. Quattrone, N. Rizzuto, G. Vita, P. Tonali,
`A. Schenone
`Novel mitochondrial tRNALeu(CUN) transition
`and D424 partial deletion in a patient with a
`facioscapulohumeral phenotype
`M. Filosto, P. Tonin, M. Scarpelli, C. Savio,
`F. Greco, M. Mancuso, G. Vattemi, V. Govoni,
`N. Rizzuto, R. Tupler, G. Tomelleri
`Differential
`diagnosis
`of muscular
`hypotonia in infants: The kyphoscoliotic
`type of Ehlers-Danlos syndrome (EDS VI)
`U. Yi§, E. Dirik, C. Chambaz, B. Steinmann,
`C. Giunta
`Expression of caveolar components in
`primary desminopathy
`A. Shinde, S. Nakano, M. Sugawara,
`I. Toyoshima, H. Ito, K. Tanaka, H. Kusaka
`GABAergic miniature spontaneous activity
`is increased in the CA1 hippocampal region
`of dystrophic mdx mice
`L. Graciotti, A. Minelli, D. Minciacchi,
`A. Procopio, G. Fulgenzi
`Reduced muscle necrosis and long-term
`benefits in dystrophic mdx mice after cV1q
`(blockade of TNF) treatment
`H.G. Radley, M.J. Davies, M.D. Grounds
`
`Microarray analysis of mdx mice expressi~_g
`high
`levels of utrophin: _T_herapeu ,c
`implications for dystrophin deficiency
`D. Baban, K.E. Davies
`Adenovirus and adeno-assocIa e
`· t d virus(cid:173)
`mediated delivery of human myo
`phos(cid:173)
`le
`phorylase cDNA and Lacz cDNA to muse .
`in the ovine model of McArdle's disease.
`Expression and re-expression of glycogen
`phosphorylase
`.
`J.McC. Howell, K.R. Walker, L. Davies,
`ati 248
`E. Dunton, A. Everaardt, N. Laing, G. Karp
`Workshop reports
`155th ENMC workshop: Polymeras_e ga~~;
`and disorders of mitochondrial d
`synthesis, 21-23 September 2007, Naar en,
`The Netherlands
`P.F. Chinnery, M. Zeviani
`149th ENMC International Workshop ~~~
`1st TREAT-NMD Workshop on: "Pla~nl II
`Phase 1/11 Clinical trials using Syst~mica i~
`Delivered Antisense Oligonucleot1des
`Duchenne Muscular Dystrophy"
`F. Muntoni, K.D. Bushby, G. van Ommen
`Letter to the Editor
`Genotype-phenotype analysis in patients
`with giant axonal neuropathy
`G. Kuhlenbaumer
`Update in Neuromuscular Disorders
`WMS online application form
`WMS - 13th International Congress
`Forthcoming meetings
`
`239
`
`259
`
`268
`
`z76
`
`II
`ill
`1V
`
`193
`
`199
`
`204
`
`210
`
`215
`
`220
`
`227
`
`NMD Gene Table is published:
`- online at http://www.musclegenetable.org
`- last pri_nted is~ue NMD 18/1_. This print versi~n is also freely available
`at http./lwww.1ournals.elsev1erhealth.comfpenodicalsfnmd
`Ill\\ 111\111 \\I\\ \\1\111\ II\ \Ill \11\\ 11111
`0960·8966(200803)18:3;1-M
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`ELSEVIER
`
`Neuromuscular Disorders 18 (2008) ~68-275
`
`www.elscvicr.com/locatc/nmd
`
`Workshop report
`149th ENMC International Workshop and 1st TREAT-NMD
`Workshop on: "Planning Phase 1/11 Clinical trials using
`Systemically Delivered Antisense Oligonucleotides 111
`Duchenne Muscular Dystrophy"
`
`Francesco Muntoni a,*, Kate D. Bushby h, Gcrtjan van Ommen c
`
`" /Juhowir: Ne11m11111.mt!ar Crntre. lmrirurc of Child I lcolrh ,( (irear Om11111d Street / lo.,11iral. 311 (,"11i//iml Srn-,·t , l.11111/1111 II '('/ N I J:/ /, / //.;
`h /11stit11te of //1111 11111 Cic11ctics, /11tcmari111111/ Cl'lltri· j i,r l.i/i-, /\',•11 ·rnstli· 11111111 "f\'111', //A"
`< l.cid('// U11il'crsiry M,·dirnl ( ',•11tcr, /.l'idc11, '/'lw N,·rl11·rl,1111/s
`
`Recdwd 15 November ~007
`
`Key1rnrds: Antiscnsc oligonuclcotidcs: Ducl1e1111e muscular dystrophy: Therapeutic trials: Exo11 skippi11g
`
`I. lntrocluction
`
`Thirty-one participants from 7 countries (Australia;
`Italy; The Netherlands;
`England; France; Germany;
`USA) attended the second ENMC workshop on antisense
`in Duchcnnc muscular Dystrophy
`oligonuclcotides
`(DMD). The topic of this workshop was on "Planning
`Phase 1/11 Clinical Trials Using systemically delivered
`in Duchenne Muscular
`Antiscnsc Oligonuclcotides
`Dystrophy (DMD)" and followed a similar workshop held
`intramuscular administration or
`in 2004 focused on
`antiscnsc oligonuclcotidcs or AONs. The workshop was
`organized with the support of the TREAT-NMD EU
`Network of Excellence (www.treat-nmd.eu) and Parent
`Project Muscular Dystrophy (PPMD), and was allended
`by representative of the two companies involved in the
`current intramuscular injection trials, Proscnsa ror the
`2-O-methyl phosphorothioate RNA modified and A VI
`the phosphorodiamidatc morpholino
`for
`Biopharma
`oligomers (PMOs or more commonly "morpholinos").
`Although, the PMO backbone is based on synthetic
`subunits, not regular nucleotides, for the sake or simplicity
`we will refer to all antiscnsc oligomcrs as AONs.
`
`• Corresponding author. Tel .: ·I 44 20X :18.~ :l:!'J5: fax : f 4.J 208 74(,21 87.
`/;'-111ai/ addrc.1s: f.mu11to11i l11'id1. ucl.ac.uk ( I-". l\luntoni).
`
`0%0-8%6/S - sec front matter r, 'J 2007 Elsevier 11.V. All rights reserved .
`doi: I 0.1 O I r,/j .nmd.2007. 11 .0 IO
`
`Specific aims of the meeting were for 2 consortia, one
`from the Netherlands and one from England, currently
`in volved in intramuscularly administered AON trials, to
`present the progress of their respective studies; and for
`members or these consortia and representatives from other
`international groups at different planning stages or the use
`or AONs in DMD, lo discuss various aspects related to the
`best strategics lo plan future systemic AON trials. Issues
`the
`issues on
`included methodological
`discussed also
`(2-O-methyl
`the AONs used
`different backbone or
`phosphorothioalc modified [2OMcl'S] and morpholino)
`oligomers; safely, regulatory, and ethical aspects.
`DMD is a severe muscle wasting condition with onset
`in early childhood, progressive muscle weakness and
`is
`It
`disability and ultimately reduced life expectancy.
`caused by mutations in the DMD gene that lead to the
`failure to produce the corresponding muscle protein called
`dyslrophin. Most of these mutations arc out-of-frame
`deletions. Laboratory studies over the last decade han:
`the addition of small molecules named
`that
`shown
`antisense oligonucleotides or oligomers (AONs) to cultun:d
`patient muscle cells, and their injection into muscles or the
`1111/x mouse model for DMD can restore the production or
`the protein dystrophin [1,2]. Although this correction is
`only temporary, it induces improved function of the paticnt
`the repeated
`cells and mouse muscle. More recently,
`systemic (intravenous) administration or /\ONs was shown
`
`Th is materia.l w as copied.
`
`

`

`F ,\/1111/()lli ct al. I Nc11r"11111srn/11r Dis"rdas 18 (2////8) 268-275
`
`269
`
`to be capable of restoring a sustained dystrophin
`expression in the mouse model of DMD, and this was
`followed by a significant functional improvement of the
`mouse muscle function [3-6]. If safe and equally effective
`in people, the repeated systemic administration of AONs
`could therefore be an effective tool to slow down the
`disease progression in DMD boys.
`
`2. Phase I/Ila trials on intramuscular AON administration
`
`Two representatives of the Dutch Consortium, Jan
`Vcrschuurcn and Judith van Dcutckom, presented the data
`of the recently completed IM injection trial of a 2OMcPS
`AON to induce skipping of cxon 51, funded by the Dutch
`Duchcnnc Parent Project, ScntcrNovcm (funded by the
`of Economic Alfairs)
`and ZonMw
`Department
`( Dutch M RC, funded by the Department of llcalth) and
`Association Frarn;aisc contrc lcs Myopathics (AFM) and
`sponsored by Proscnsa. This study was aimed at defining
`safety and local dystrophin restoration or IM administered
`2OMcPS AON in a single muscle in DMD boys [7]. As
`part of the pre-screening program, boys had a muscle
`MRI to document adequate preservation of the target
`muscle, the tibialis anterior (which was invariably the least
`affected muscle in the lower leg of affected boys), and a
`skin biopsy
`for MyoD
`transrcction and myogcnic
`conversion of fibroblasts and in vitro testing of response
`to the administration of AON. Four boys were included
`between the age of IO and 13, carrying deletions of cxons
`50, 52, 48 50 and 49 50. They received a single dose of
`0.8 mg or the 2OMcPS cxon 51 AON in one tibialis
`anterior muscle using an EMG guided needle. A muscle
`biopsy or the same tibialis anterior was performed 4 weeks
`after the injection of the AON and dystrophin protein and
`transcript analysed in detail. The results in all 4 boys were
`extremely encouraging with robust levels of dystrophin
`skipped transcript visible I month after the AON injection,
`and the percentage of dystrophin positive fibres in the
`tibialis anterior biopsy comprised between (i4'1/,, and 97%
`in the 4 children studied. In view or the unequivocal
`positive results in these 4 patients, the decision was made
`not to recruit additional patients into this study
`the
`original plans were to recruit a total or 4 (1 DMD boys.
`i11tra111uscular
`administration
`or
`Reassuringly
`the
`the 2OMcPS was well
`tolerated with no apparent
`i111la111111atory response to the administration of the AON.
`Two representatives of the rvtDEX consortium, France(cid:173)
`sco M untoni and Maria Kina Ii, illustrated the status or the
`study in UK. This study is runded by the Department or
`I lcalth, sponsored by I111perial ( 'ollcge and run in collabo(cid:173)
`ration with A VI Biopharnia. This study is similar to the
`Dutch trial. the main differences being that (i) the AON
`injected will he a 30mn n1orpholino. (ii) the study is a dose
`escalation study: (iii) one e:-;tl'nsor digitorun, hrcvis (EDB)
`of older children ( 12 17 yrs) will ll'l·l'ive the PMO AON
`administration while till' l·ontr:ilatl·ral EDB will rl·ceivc a
`sha1\l injection. At the end of the study an open biopsy will
`
`be performed on both muscles to allow quantitation and
`differentiation of dystrophin production following the
`administration of the AONs from the background of
`dystrophin that the patient might produce, including
`revcrtant fibres. Nine DMD boys will be studied, three
`receiving the lowest dose, 3 an intermediate dose and 3
`the highest dose, with the recruitment of this latter group
`only considered in case the results from the previous
`patient groups are equivocal. A muscle MRI protocol has
`been devised to pre-screen patients in order to confirm
`the preservation of the EDB, and preparatory studies have
`indicated that most DMD boys up to the age of 16 have
`sufliciently well preserved EDB muscle to be eligible for
`the study. Also the MDEX consortium protocol requires
`for each patient to be studied by MyoD translcction of skin
`fibroblasts and subsequent AON treatment to confirm
`lcasibility of AON-induced dystrophin
`restoration
`in vitro; in addition a detailed neuropsychiatric questionnaire
`was devised in order to be able to monitor expectations and
`impact of the trial on individuals. At the time of the
`workshop the study was in the process of completing
`regulatory authorization.
`As part or the preparatory studies, the M DEX consor(cid:173)
`tium has studied whether rcvcrtant fibres increase with
`age in DMD boys. Previous studies performed in the
`111dx mouse have suggested that this could be the case.
`Twelve boys who have had a muscle biopsy at diagnosis
`and which was available for further evaluation were
`recruited into this study; these boys had muscle biopsies
`during planned surgical procedures of either the EDB mus(cid:173)
`cles (9 cases) or paraspinal muscles (3 cases) on average 7
`years following the original diagnostic quadriceps muscle
`biopsy. In none of these 12 patients was there an increase
`of the frequency of revertant fibres with age, at least as
`far as the studied muscles were concerned. This informa(cid:173)
`tion is helpful as it suggests that any dystrophin produced
`following the AON administration is the likely result or the
`AON-induced cxon skipping and not naturally occurring
`rcvcrtants. provided that the number or rcvcrtants in the
`original muscle biopsy is negligible (a threshold of 5%
`was arbitrarily agreed).
`
`3. Preclinical studies focused on systemic AON
`:ulministration
`
`Judith van Dcutckom presented the recent results or the
`systemic administration or a 2OMcPS AON against
`cxon 23 in the 1111I.\' mouse. These studies focused on
`different mode or administration (IV, subcutaneous [SC];
`intrapcritoneal [IP]) and results were validated not only
`using semiquantitative assays, but also by an AON-spccilic
`hyhridi1.atio11 assay to measure tissue levels or 2OMcPS
`AON. This method developed by Proscnsa allowed assess(cid:173)
`ment or the hiodistribution or the 2OMcPS in a number or
`nrgans
`(including
`liver
`and
`kidney)
`scrum
`and
`muscle. While there were signilicant dillcrcnccs in the
`pharmacokinctics or the AON in several organs rollowing
`
`Th is m a t e ria I was co pcied
`
`

`

`270
`
`F M1111/011i cl al. I Ne11ro11111.1·rn!ar Di.wmlas 18 (20/JS) 268 275
`
`different routes of administration, the differences were less
`obvious in skeletal muscle. Interestingly the 2OMePS AON
`uptake in the dystrophic muscle environment was signifi(cid:173)
`cantly better compared to wild-type mice. This suggests
`that lower AON dosage might be sufficient in muscular
`dystrophy compared to controls. Treatment schedule up
`to 100 mg/kg/week IV for 6 weeks using 2OMe AONs
`resulted in progressive increase in dystrophin production,
`with levels approaching 25'1/ri of normal individual muscles.
`Repeated administration of higher dosage (up to 250 mg/
`kg/IV) within a shorter time frame resulted in more robust
`dystrophin production (up to 40'1/r, in individual muscles)
`and also the appearance of the expression of some dystro(cid:173)
`phin in cardiac muscle. Restoration of dystrophin expres(cid:173)
`sion in these mdx mice was associated with a decrease of
`serum CK, and significant improvement of mobility in
`the mdx mice, as suggested by Rotarod studies.
`Annemieke Aartsma-Rus presented the results of com(cid:173)
`parative studies aimed at determining the effect of different
`backbone chemistries (2OMePS and PMO) and different
`AON length on the elliciency of cxon skipping in the
`humanized mouse model (hDM D) which carries the entire
`human dystrophin locus as a transgcnc [8]. As there is no
`muscle degeneration in this model, muscle damage is chem(cid:173)
`ically induced before the administration of the AONs. The
`results of these studies demonstrated a significant effect of
`AON length, which was dependent on the cxon targeted,
`and which affected not always in a convergent way the dif(cid:173)
`ferent backbone studied.
`Dominic Wells discussed his results on the administra(cid:173)
`tion of PM Os. He first presented evidence on the longevity
`of PM Os: after a single IM injection, stable levels of skip(cid:173)
`ping were recorded for up to 14 weeks, while protein levels
`were stable up to 10 weeks and then gradually declined.
`Local administration was less influenced by regeneration
`and the dystrophic environment compared to the 2OMePS.
`He also highlighted remarkable differences in the nu111ber
`of dystrophin positive fibres in different muscles following
`intravenous administration, and stressed the relevance or
`the choice of which muscle to study following syste111ic
`AON administration both in preclinical but potentially
`also in clinical trials. Current work is focused on dose esca(cid:173)
`lation PMO studies, on effect of fibrosis, and physiological
`rescue of muscle with particular attention to resistance to
`eccentric exercise.
`Ian Graham presented studies on the administration of
`single escalating IV doses of PMO (111dx) ranging from I
`to I 00 mg/kg, followed by analysis at 4 and 8 weeks to
`establish the duration and possible accumulation of effect.
`A clear dose relationship regarding dyslrophin expression
`was established, the lowest dosage not producing any
`expression, and rare dyslrophin positive fibres appearing
`after a dose of 2.5 mg/kg, and robust dystrophin expres(cid:173)
`sion at the higher dosage, with up to 60% of positive fibres
`in individual muscle al the highest dosage used.
`Qi Lu presented the data from his group on recent
`in vitro and in vivo studies on AONs. Regarding the
`
`in vitro studies, he is generating a series of stably transfcc(cid:173)
`ted C2C 12 cell lines carrying an EG F Tagged splicing
`transgene with the relevant dystrophin cxons and intron
`boundaries for rapid screening of different AONs. These
`transgencs could also be used for generating transgenic ani(cid:173)
`mal models in the future. Regarding the in vivo studies, he
`conducted dose escalation studies in 111dx to determine a
`dose response using PMOs He reminded that in
`the
`recently published work on bodywide cllicacy of intrave(cid:173)
`nous PMOs, the dose used was approximately 80 mg/kg
`[3]. A range of dosages were acutely administered to 111dx
`mice, from I 5 to 300 mg/kg. Robust skipping was repro(cid:173)
`ducibly detected already with dosages between 50 and
`100 mg/kg, but not at lower PMO dosages. In addition
`comparative analysis of IV compared lo IP showed that
`IV was superior in targeting muscle and inducing exon
`skipping.
`Terry Partridge discussed collaborative work performed
`with Dr. Takeda on the beagle dyslrophic dog, which
`presents several advantages compared
`lo
`the golden
`retriever, both in term of its s111allcr size and the 111orc
`abundant litter. This animal carries a splice site mutation
`which results in skipping cxon 7, and in order to restore
`the reading fra111c using AONs, exon (i and 8 require
`targeting. This can be achieved with a cocktail of different
`AONs, although the skipping of exon 8 invariably also
`leads lo the removal of cxon 9 (but without affecting the
`reading frame). Importantly there arc differences between
`the AONs co111binalion which have an optimal effect in
`cultured cells from the dog (2 AONs) co111parcd to the
`in vivo situation, where 3 AONs arc required. One beagle
`dyslrophic dog received IV PMOs between the age of 5
`and 7 months. The treatment with the AONs combination
`resulted
`in apparent
`functional
`i111provc111cnt of the
`running speed compared to lillermales and immunohisto(cid:173)
`che111ical analysis showed that dystrophin expression had
`been restored in most muscles, with the exception of the
`heart. Because of the need lo give a mixture of morpholinos
`the dose used was high ( 1.2 g or the cocktail per weekly
`infusion for 5 weeks, i.e., a total of (i g. Considering the
`weight of the dog a mean of ~IO kg across that time
`period, the dose used was 120 mg/kg). l lowevcr, there were
`no obvious side effects of the chronic treatment. Partridge
`also discussed facilities
`in Washington for performing
`controlled experiments in dystrophie mice using activity
`cages where treadmills considerably increase the level or
`exercise or mice.
`
`3.1. Other A ON hack hones or co-ad111i11istratio11 1rith
`co111po111ul.1· .f,1r i11creasi111; 11111.w·le uptake <!/' A ONs
`
`A number of speakers presented experimental work on
`different AON backbone to increase muscle targeting.
`Annemieke Aarlsma-Rus showed that locked nucleic acids
`(LNA) have very high aflinity but also high self" annealing
`properties and reduced spccilicily compared to ~O1\lcPS.
`Both she and Mallhew Wood co111111cnted that the peptide
`
`Th is material was rnpcied
`
`

`

`F ,\/11111011i ct al. I Nc1m111111.1T11lar Di.wmlcrs /8 (:!008) :!68 -275
`
`271
`
`nucleic acid (PNA) AONs uptake in cell cultures
`is
`significantly affected by their uncharged backbone, while
`their in vivo behavior is much better. Matthew Wood also
`presented data on PNAs conjugated with different
`compounds (arginine rich peptide for example), aimed at
`increasing the muscle uptake, although no significant
`increase in their efficacy was demonstrated in vivo.
`Steve Wilton compared modified PMOs (peptide conju(cid:173)
`gate) to improve muscle targeting e01cicncy in mdx mice.
`Different acute and chronic regimens were evaluated, with
`AONs being administered
`intrapcritoncally (IP). The
`results or these studies suggested that peptide conjugated
`PM Os targeted muscle much more efficiently than standard
`PMOs (between 5 and 10 times more clliciently); however
`increased toxicity or the peptide modified PMO was
`observed. in contrast to the good tolerability or the unmod(cid:173)
`ified PMO. Recently. he also started to work on positively
`charged PM Os. I le linally discussed that the different back(cid:173)
`bone modilication while affecting the cllicicncy or uptake,
`do not appear to change the cllicacy or splicing, i.e .. the
`hierarchies or cllicacy or individual AONs obtained with
`2OMcPS backbone is recapitulated by other chemistry
`such as PMOs [9].
`Qi Lu indicated that modilicd PMOs (Vivoportcr) were
`at least 3 times 111ore effective compared to ordinary PM Os.
`I le also showed ongoing work using PM Os and polymer to
`increase 111usclc uptake following IV delivery.
`Dominic Wells showed the effect of delivering PM Os to
`the heart in combination with diagnostic ultrasound and
`microbubbles. Microbubbles arc indeed routinely use in
`radiology and cardiology. and the use or diagnostic ultra(cid:173)
`sound focused to the heart can induce microbubblc burst(cid:173)
`ing and
`this appears to facilitate PMO targeting to
`cardio111yocytcs. Indeed. this delivery method resulted in
`significant improvement in exon skipping in the heart of
`mdx mice. although not to levels seen in skeletal muscle.
`These results might point towards differences in trallicking
`and/or endothelial barriers between skeletal and cardiac
`muscles. Judith van Deutckom also showed that the
`administration of repeated high dosage 2OMePS over a
`short period or low dosagl! over a long period was indeed
`capable or inducing cardiac cxon skipping in 11uf.,· mice.
`
`3.2. 'f'oxicologicaf and dcli1 ·1'l'_l' rnnsidcrations .fi1r systc111ic
`dcli1·c•1y trials
`
`Sjef de Kimpe su111marised the toxicology work per(cid:173)
`formed to take the 2OMePS AON into clinical trials.
`hut also more in general the issue or toxicology and issues
`related to AON administration. J\ON toxicity in general
`can be divided into hybridisation dependent and hybrid(cid:173)
`isation independent toxicity . The lirst indicates the cl1'cct
`that an AON can ha\'l' 011 thl· targl't (tllll much or a golld
`thing. which is not a co11l·crn for D11cl1L'1111L' application).
`hut also off-targl'l. Such sL·querll'L' off-larget RNA dlles
`1101 ncl'd to ha\'c a I00''. 11 homology as for Sllllle backbone
`chemistries still hybridisL' significantly despite mismatches.
`
`The hybridisation
`independent
`toxicology
`relates
`to
`unknown motifs present
`in
`the AON
`sequence
`which might trigger unspecific reactions (such as toll
`receptor-mediated
`immune
`response or complement
`activation). The hybridisation
`independent
`toxicology
`relate generally to the backbone structure or (un)known
`sequence motifs. An example is the CpG motif that can
`cause a prominent
`immunostimulatory
`response via
`activation of toll-like receptor, TLR9. Such motifs can
`be avoided in the sequence selection for AONs. Much
`of the hybridisation independent effects arc influenced
`by
`the backbone AON structure. For example first
`generation AONs, phosphorothiatcd DNA oligonucleotidcs,
`interacted with proteins or the coagulation cascade causing
`a transient increase in APTT, but without clinical evidence
`of enhanced bleeding. In toxicity studies rodents arc
`especially sensitive to immunostimulatory effects of first
`generation oligonucleotidcs. These do not appear
`in
`monkeys. Many of such effects noticed with first generation
`AONs arc ameliorated or even absent in second generation
`AONs.
`like
`the 2OMe modifications
`[10]. Another
`advantage of 2OMe is the extended terminal half life ( ~4
`wks), making once a week or less frequent administration
`feasible. Moreover, 2OMe modified AONs can be
`administered
`subcutaneously
`(as an
`alternative
`to
`intravenous infusions) and this has been employed clini(cid:173)
`cally w