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1. Buchenne Case Stuuy........................6
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1. The BNB uiagnostic ouyssey...................9
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1. uenetic Analysis..........................9
a. Nethouology.......................1u
b. Access to genetic testing................1u
2. Bystiophin expiession on muscle biopsy as a uiagnostic
biomaikei................................1u
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1. ulucocoiticoiu theiapy......................16
2. Contiactuie management......................17
S. Spine uefoimity management...................17
4. Pulmonaiy management.....................17
S. Caiuiac management.......................17
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1. Souices of vaiiability..........................18
a. Bisease seveiity stage of uisease................18
b. uenetic pieuictois of uisease piogiession.........19
c. uenetic mouifieis........................19
u. Coiticosteioiu theiapy...................2u
e. Night splinting, physical theiapy, anu othei stanuaiu
inteiventions ...........................2u
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1. Notoi 0utcome Neasuies.....................2S
a. In neonates anu infants.................2S
b. Foi young Ambulatoiy (fiom foui to appioximately
seven)................................24
c. The Late Ambulatoiy stage (fiom appioximately seven
to thiiteen yeais of age).......................2S
u. The non-ambulant population..............26
2. Pulmonaiy 0utcome Neasuies anu Enupoints.........27
S. Caiuiac Enupoints..........................28
4. Patient Repoiteu 0utcomes (PR0s) in BNB............29
S. Bevelopment of Auuitional Novel Enupoints in BNB.......29
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1. Feasibility issues foi tiials in BNB: The limits of iaie uisease
anu meuical auuiessability by enupoint (pool of paiticipants anu tiial
sites)....................................Su
2. Tiial Besign.............................S1
a. Novel tiial uesigns......................S2
S. Stanuaiuization of measuiement acioss tiials..........S2
4. 0se of biomaikeis in BNB tiials.................S2
a. As Single Piimaiy 0utcome Neasuies.........S2
b. 0se of Biomaikeis as Suppoitive Seconuaiy 0utcome
Neasuies...............................SS
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1. ueneial comments.........................SS
2. Consiueiations ielateu to muscle biopsies............SS
a. Ethical conceins of biopsies in chiluien........SS
b. Ciiteiia of what is an appiopiiate biopsy foi uystiophin
oi utiophin quantification....................S6
i. Site of biopsymuscle gioup...........S6
ii. The hanuling of the biopsy...........S6
iii. Ninimizing vaiiability anu sampling eiiois ..S6
S. Bystiophin Analyses.......................S7
a. Bioauly uisseminateu techniques...........S7
i. Immunofluoiescence oi immunohistochemical
analysis by type.....................S7
ii. Westein Blot....................S8
b. Emeiging technologies.................S8
i. Nass spectiometiy...............S8
iv
c. Cuiient limitations foi all methous............S8
u. Refeience ianges anu outlieis..............S9
e. 0se of uystiophin quantification oi ielative
quantification as a biochemical outcome measuie.......S9
4. 0tiophin Analysis........................S9
S. Nuscle Biopsy Biomaikeis: RTRNA PCR analysis foi exon-
skipping uetection to confiim mechanism of action in the exon-
skipping fielu................................4u
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1. ueneial Comments.........................41
2. Seium anu 0iine Biomaikeis....................41
a. Pioteins anu piotein fiagments..............42
b. NicioRNAs........................42
c. Recommenuations iegaiuing seium anu uiine
biomaikeis..............................4S
S. Imaging Noualities.........................4S
a. 0ltiasounu........................4S
b. Electiical impeuance myogiaphy (EIN)........44
c. BEXA...........................44
u. NRI anu NRS........................44
i. NRINRS: Emeiging Biomaikeis of Buman
musculai uystiophy Pathology.............4S
ii. NR Imaging of Fibiosis..............46
iii. NRINRS: Role in Clinical Tiials........46
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1

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The puipose of this guiuance is to assist sponsois in the clinical uevelopment of
meuical piouucts (i.e., human uiugs, anu theiapeutic biological piouucts) foi the
tieatment of Buchenne musculai uystiophy (BNB) ovei the entiie spectium of the
uisease.

This guiuance is the iesult of the fiist collaboiation between the FBA anu a uisease
specific community to piouuce clinical guiuance in theii iespective uisease aiea. The
FBA inviteu the Buchenne community (incluuing patients, paients anu caiegiveis,
clinicians, acauemic expeits anu inuustiy) to uevelop the initial uiaft of this
guiuance as pioviueu unuei FBA's inteipietation of uoou uuiuance Piactice
piovisions. The fiist iteiation of the guiuance, togethei with suppoiting papeis fiom
the woiking gioups that piouuceu it, can be founu at |TBAj. 0pon ieceipt of the
guiuance, the FBA openeu a uocket anu helu fuithei meetings with the BNB
community anu othei expeits. The cuiient uocument ieflects some ievisions baseu
upon iegulatoiy anu statutoiy iequiiements anu moie iecently publisheu uata.

This guiuance auuiesses the FBA's cuiient thinking iegaiuing the consiueiation that
shoulu be given to the benefitiisk piefeiences of the BNB community consiueiing
the iaiity anu seiiousness of the uisease anu in light of the lack of cuiient causal
tieatment options. It also ieflects the FBA's appieciation that the unueistanuing of
the natuial histoiy of BNB anu causes foi vaiiability in outcomes has been upuateu
This uiaft guiuance iepiesents the fiist FBA guiuance initially composeu by a
uisease community, with input fiom inuustiy, sponsois, acauemia anu the
Buchenne musculai uystiophy patient community. When finalizeu, it will iepiesent
the Foou anu Biug Auministiation's (FBA's) cuiient thinking on this topic. It uoes
not cieate oi confei any iights foi oi on any peison anu uoes not opeiate to binu
FBA oi the public. You can use an alteinative appioach if the appioach satisfies the
iequiiements of the applicable statutes anu iegulations. If you want to uiscuss an
alteinative appioach, contact the FBA staff iesponsible foi implementing this
guiuance. If you cannot iuentify the appiopiiate FBA staff, call the appiopiiate
numbei listeu on the title page of this guiuance.
2
by iecent eviuence fiom patient iegistiies, natuial histoiy stuuies anu clinical tiial
cohoits. It auuiesses the selection of enupoints foi clinical tiials in populations with
BNB as well as the mannei in which uisease mouification might be uemonstiateu
anu encouiages the use anu exploiation of new biomaikeis that coulu piove
suppoitive of new uiug applications, (consistent with FBA's stateu inteiests in
auvancing peisonalizeu meuicine effoits), paiticulaily those with the potential to
become suiiogate enupoint maikeis foi clinical uevelopment. Finally, the guiuance
uiscusses possible stiategies that companies may puisue to secuie one of the FBA's
thiee pathways foi expeuiteu appioval (meaning piioiity ieview, acceleiateu
appioval (baseu on subpait B piovisions)) anu piioi uesignations of eithei
bieakthiough oi fast tiack), as well as the eviuence base iequiieu to secuie
tiauitional appioval.

This guiuance is intenueu to seive as a focus foi continueu uiscussions among the
FBA, the meuical inuustiy, sponsois, acauemic community, the patient anu caiegivei
community, anu the public. The uesign of clinical tiials that aie specifically focuseu
on the tieatment of patients with Beckei musculai uystiophy oi any of the othei
musculai uystiophies is not explicitly uiscusseu, although many of the piinciples in
this guiuance will be peitinent to the uevelopment of theiapies foi those conuitions.

FBA's guiuance uocuments, incluuing this guiuance, uo not establish legally
enfoiceable iesponsibilities. Insteau, guiuances uesciibe the Agency's cuiient
thinking on a topic anu shoulu be vieweu only as iecommenuations, unless specific
iegulatoiy oi statutoiy iequiiements aie citeu. The use of the woiu !"#$%& in
Agency guiuances means that something is suggesteu oi iecommenueu, but not
iequiieu.

II. <;AG"D?EB1
Buchenne musculai uystiophy is a genetic uisoiuei chaiacteiizeu by the
piogiessive uegeneiation anu loss of skeletal muscle, the muscles of iespiiation anu
the caiuiac muscle, piimaiily in boys though a small peicentage of female
caiiieis may exhibit a iange of muscle symptoms fiom the full Buchenne phenotype
to miluei skeletal muscle weakness. It is causeu by mutations in the '(' gene that
pievent the expiession of functional uystiophin, an impoitant stiuctuial component
in muscle tissue. The conuition is inheiiteu in an X-linkeu mannei anu &) +#,#
mutations may occui in people fiom families without a known family histoiy of the
uisease. BNB affects appioximately 1 in S,8u2 to 6,291 live male biiths woiluwiue.
1,
2


In association with othei pioteins, uystiophin piotects muscle fibeis against the
mechanical foices of contiaction in the absence of uystiophin, muscle is pione to
uamage, ueteiioiation anu fibiosis.

Nuscle weakness geneially becomes appaient in the fiist few yeais of life with a
uelay in motoi milestones, anu a mean age of walking aiounu 18 months.
Piogiessive muscle weakness leaus to a loss of inuepenuent ambulation befoie the
S
age of thiiteen in the absence of uisease mouifying tieatment. In non-ambulatoiy
boys anu young men, theie is giauual loss of uppei limb anu neck functions, so that
giooming, toileting, bathing, uiessing, anu eating become impaiieu oi impossible to
uo peifoim by oneself affecting the quality of life of patients, theii caiegiveis anu
families.

This is accompanieu by weakness affecting iespiiatoiy muscles anu the heait that
contiibutes to uecieaseu iespiiatoiy function anu caiuiomyopathy with heait
uisease now being the most common cause of ueath in boys anu young men with
BNB.

0vei the past uecaue, patient oiganizations, acauemia anu inuustiy have woikeu
togethei to uevelop seveial patient iegistiies, uisseminate impioveu stanuaius of
caie, anu exploie clinical outcome measuies anu biomaikeis. This expeiience anu
uata collection has iesulteu in a gieatly impioveu unueistanuing of the
pathogenesis anu the natuial histoiy of BNB, incluuing factois that may leau to
vaiiability in the couise of the uisease.

Natuial histoiy stuuies have shown that the use of glucocoiticoius anu the
management of spine uefoimity, pulmonaiy anu caiuiac uysfunctions have alteieu
the timing of some of the clinical milestones of the uisease. But with limiteu meuical
management have come new complications, anu quality of life often suffeis. Foi
instance, auveise events known to be associateu with glucocoiticoiu usage incluues
excessive weight gain, giowth inhibition, iisk of uiabetes, behavioial abnoimalities,
Cushingoiu featuies, change in pubeital piogiession anu cataiacts. 0f paiticulai
concein foi the Buchenne community is the issue of weight gain, since BNB is a
piogiessively uebilitating uisease anu weight gain can compounu the physical
limitations of a uystiophic myopathy.

At the time of wiiting, theie aie no FBA-appioveu BNB-specific theiapies anu no
way to ieveise the unueilying conuition. 0nce ambulation oi some othei functional
capacity is lost in an inuiviuual with Buchenne musculai uystiophy, it is gone
foievei. Beath can happen without waining, at any moment, even in youngei boys.

Theie is an uigent unmet neeu to uevelop new tieatments, especially those that
auuiess the unueilying cause of BNB. While a numbei of potential theiapeutic
agents aie in oi enteiing clinical uevelopment, sponsois neeu foimal guiuance on
how best to uemonstiate a tieatment's effectiveness anu safety in this iaie uisease
anu what soit of effect woulu be clinically meaningful to patients anu theii
caiegiveis.

The FBA has acknowleugeu the conceins expiesseu by the BNB community that
flexibility be exeiciseu in the ieview of piouucts foi the uisease iecognizing that
many patients anu caie giveis aie willing to take gieatei iisks foi a tieatment that
may slow clinical ueteiioiation oi uelay the loss of functional milestones, each of
which is clinically meaningful.
4

When iecommenuing this uiaft guiuance, the Buchenne Community chose to place
the topic of benefitiisk assessment at the stait of the uocument, because it was felt
that sponsois shoulu be guiueu by patient anu caiegivei piefeiences fiom the veiy
stait of a piouuct's clinical uevelopment piogiam anu that sponsois also neeu
cleai guiuance fiom the Agency on how benefit iisk assessments contiibute to a
piouuct's ieview piocess in the specific BNB uisease context.

The FBA shaies the Buchenne Community's goal to woik with inuustiy to get new
theiapeutic agents onto the maiket as iapiuly anu iesponsibly as possible. This
guiuance foi inuustiy is but a step towaius achieving that goal.

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The assessment of benefits anu iisk plays a cential iole in the FBA ieview piocess.
In iecent yeais, the FBA has been manuateu to make the assessment of benefits anu
iisk moie tianspaient anu to bettei incoipoiate the peispective of patients anu
families into the benefit iisk assessment. The FBA acknowleuges that patients anu
families aie alieauy empoweieu to play a iole in such uecision. Specifically, the
assessment of benefit anu iisk is funuamental to uecisions maue each uay by
patients anu families, in collaboiation with theii healthcaie piofessionals, about the
use of tieatments oi uevices.

To embiace a moie patientfamily centiic appioach to benefit-iisk, sponsois aie
auviseu to quantify the piefeiences of patients anu family membeis, when feasible.
To piomote patient-centeieu uiug uevelopment, the FBA encouiages inuustiy to
engage patients anu family eaily in the uevelopment of theiapy. Paitneiing with
establisheu auvocacy effoits can be valuable in unueistanuing the peispectives of
patients anu theii family. Such paitneiships can help the FBA bettei unueistanu the
piefeiences of patients anu family membeis, anu can guiue the FBA in ueteimining
meaningful benefit, iisk toleiance, acceptable tiaueoffs anu piefeience
heteiogeneity. The FBA accepts that the assessment of benefit anu iisk in BNB is
complex. Although BNB is a uebilitating uisease that causes musculai ueteiioiation
anu loss of function ovei the lifespan, the winuow of oppoitunity foi a theiapeutic
inteivention may come yeais befoie significant clinical events associateu with
moibiuity anu moitality anu yet, the consequences of failing to inteivene aie
cleai.

While iegulatois have tiauitionally been hesitant to allow iisk in a young
population, we aie awaie that paients of this population aie willing to accept moie
unceitainty anu take gieatei iisk eaily on, because of the pieuictable outcomes in
the uisease.
S
Foi these ieasons, activities with iegaius to the BNB patient anu
caiegivei peispective shoulu be uone in consultation with a bioauei community
iathei than the tiauitional thinking of the community as subjects in a stuuy.

S
We also appieciate that piefeiences of patients anu caiegiveis may uiffei anu that
piefeiences may change ovei time. Foi piogiessive uebilitating uisoiueis,
unceitainty about benefits anu iisks aie weigheu against the known implications of
not tieating. The ceitainty of uisease piogiession is a compelling concein in the
Buchenne benefit iisk assessment.
4


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Benefit-iisk analysis iequiies eviuence on the benefits anu iisks of the theiapy
being investigateu, an assessment of the ceitainty of the benefit anu iisk eviuence,
evaluation of iisk mitigation stiategies, anu a subjective assessment of meaningful
benefit, iisk toleiance anu an acceptable iisk-benefit tiaueoff. As the Agency has
pieviously stateu, iisk-benefit assessments neeu to be tailoieu to the population
being stuuieu.

As uesciibeu in the FBA fiamewoik, foi each uecision factoi uecision-makeis
consiuei both the eviuence anu its unceitainties, anu use these to assess the
implications of that component on the uecision. The uecision context combines
analysis of the conuition anu of cuiient tieatment options. The analysis of the
conuition incluues the natuial couise of the uisease anu its seveiity, anu the
assessment of cuiient tieatment options uesciibes how well the meuical neeu is
met by cuiiently available theiapies.

Though this piocess is infoimeu by uata, the ultimate outcome ielies on ieviewei
anu Agency juugment as to what levels of iisk aie acceptable foi paiticulai levels of
benefit. In iecent yeais, theie has been movement at the FBA to bettei unueistanu
patient piefeiences anu to incoipoiate these in a tianspaient anu scientific way in
benefit-iisk assessments. This conceins not only how the tiial iesults aie
inteipieteu with iegaiu to a iegulatoiy uecision, but how a clinical tiial is conuucteu
anu in enupoint ueteiminations. In auuition, the infoimation geneiateu by a
sponsoi's clinical uevelopment piogiam is also ciitical foi the inuiviuual
benefitiisk assessments maue by the patient anu caiegiveis.

These effoits to unueistanu patient piefeiences anu quantitative tiaue-offs aie
impoitant in cases wheie theie aie unceitainties ovei benefit anu iisk. In othei
instances wheie benefits anu iisks aie well chaiacteiizeu, it is fiequently
qualitatively unueistoou how one might tiaue a iisk foi a benefit. Bistoiically, the
benefitiisk assessment has attempteu to incluue patientcaiegivei piioiities
pieuominately thiough the use of testimony. An impoitant, outstanuing question is
how well the voice of those giving testimony ieflects the peispectives of the bioauei
uisoiuei community.

While FBA encouiages uiiect patient anu community engagement with the agency
iegaiuing theii iisk benefit piefeiences at iegulatoiy heaiings anu othei foiums, it
is piefeiable to supplement this with a scientific appioachespecially when
benefits anu iisks aie unceitain anu not tieating causes haim. In this way, input that
has histoiically been au hoc, unstiuctuieu naiiative that is impossible to quantify oi
6
geneialize can be maue quantifiable. Rigoious anu geneializable appioaches to
quantifying iisk benefit assessment holu weight in iegulatoiy uecisions about what
constitutes meaningful benefit anu acceptable iisk, anu sponsois shoulu be awaie
that iegulatois can best integiate patientcaiegivei piefeiences when those
piefeiences aie quantifieu using iobust stateu piefeiences methous. Consistent
with FBA's manuate foi moie patient-centeieu iegulatoiy uecision-making, the
Agency is inteiesteu in ways to take these piefeiences into account in evaluating
options foi acceleiateu uevelopment anu appioval of new tieatments.

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A numbei of mouels anu appioaches have emeigeu in iecent yeais to facilitate
iegulatoiy benefit-iisk analysis.
S
These vaiy fiom simple giaphical techniques
6, 7
to
moie compiehensive appioaches that can be beneficial in the selection,
oiganization, summaiy, anu communication of eviuence ielevant to benefits anu
iisks analysis.
8, 9
Nethous to incoipoiate patientfamily piefeiences have also
emeigeu
1u, 11, 12
anu aie now fiequently founu in the liteiatuie.
1S, 14


0ne of the most common appioaches to measuiing the peispectives of patients anu
theii family aie stateu-piefeiences methous.
1S
The most commonly applieu of these
stateu-piefeience appioaches is conjoint analysis - a bioau class of methous that
incluue uisciete-choice expeiiments.
16, 17, 18
Such appioaches aim at uocumenting
acceptable tiaueoffs acioss vaiious iisks anu benefits. They can also be useu to
uocument the unueilying heteiogeneity of piefeiences
19
anu to clustei inuiviuuals
into gioups with similai piefeiences.
2u


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A collaboiative auvocacyacauemic paitneiship uemonstiateu a community-
engageu appioach to measuie caiegivei piefeiences foi potential benefits anu iisks
of emeiging theiapies foi BNB.
21
Caiegiveis' tieatment piefeiences weie measuieu
using best-woist scaling. Attiibutes uesciibing potential benefits anu iisks of
emeiging BNB theiapies weie iuentifieu thiough engagement with community
stakeholueis incluuing auvocates, clinicians, anu uiug uevelopeis. The attiibutes in
this pieliminaiy stuuy incluueu muscle function, lifespan, knowleuge about the
uiug, nausea, iisk of bleeus, anu iisk of aiihythmia. The stuuy was implementeu
thiough an online suivey of BNB caiegiveis, who weie ieciuiteu in the 0S.

BNB caiegiveis iuentifieu moueiate benefits of stabilizing oi slowing piogiession of
muscle weakness as the most impoitant among expeiimental attiibutes (28.7%),
followeu by iisk of heait aiihythmia (22.4%) anu iisk of bleeus (21.2%). Baving
auuitional post appioval uata (an attiibute ieflecting unceitainty) was ielatively the
least impoitant attiibute (2.S%).
22
As piesenteu in the stuuy, caiegiveis weie
willing to accept a seiious iisk when balanceu with a non-cuiative tieatment, even
absent lifespan impiovement. In othei woius, stabilization of the chilu's piogiession
was consiueieu a benefit woith a seiious iisk. Bowevei, caiegiveis inuicateu a limit
to theii iisk toleiance in that they woulu not accept a iisk of ueath anu a iisk of
auuitional lifelong uisability foi a uiug that stoppeu oi sloweu piogiession. This
7
stuuy highlights the syneigistic integiation of tiauitional auvocacy methous anu
scientific appioach to quantify benefit-iisk piefeiences.

1C "#$%&'() /+ :5+'.+,.
Clinical tiial sponsois shoulu take patient anuoi caiegivei piefeiences anu
piioiities into account when uesigning clinical tiials anu when piepaiing foi FBA
submission. If ielevant piefeience uata uoes not alieauy exist in the taiget uecision-
making population, sponsois shoulu obtain this infoimation. Patient anu caiegivei
piefeiences may uiffei, anu sponsois shoulu exploie the peiceptions of the
appiopiiate population(s) uepenuing on the taiget anu clinical tiial piotocol. 0sing
a iobust methou to quantify piefeiences, clinical tiial sponsois aie encouiageu to
engage in patient-community-centeieu ieseaich in piepaiing foi FBA submission,
both to infoim the FBA benefit iisk analysis anu to engage a bioauei constituency in
piepaiation foi submissions. The patient-engageu appioach shoulu integiate topics
(attiibutes) of impoitance to patients anu families into the stateu piefeiences
expeiiments. Similai to othei quantitative methous, stateu piefeiences methous aie
only inuicative of actual uecision-making when the expeiimental attiibutes aie
ielevant anu unueistanuable to inuiviuuals who aie pioviuing uata.

Sponsois shoulu exploie iisk mitigation stiategies anu patientcaiegivei
piefeiences foi these stiategies. Risk management anu mitigation consiueis what
activities aie in place to optimize benefits while limiting the consequences of the
iisks, such as taigeteu monitoiing, pioviuei euucation stiategies, contiolleu
uistiibution, anu special labeling. Paiticulaily in conuitions such as BNB a
peuiatiic-onset, lifelong uisoiuei -- stakeholuei conceins anu piefeiences aie
ielevant foi infoiming the choice of iisk-management stiategies. Risk management
appioaches that engage tieating clinicians may be paiticulaily ielevant foi BNB, as
a laige piopoition of the Buchenne population have health caie pioviueis that
follow theii patients foi yeais.

Sponsois shoulu anticipate that appiaisals of benefitiisk will change ovei time uue
to uisease anu non-uisease ielateu factois, available tieatment options, unceitainty,
anu othei contextual influences. The type of benefit about which a patient oi paient
may be most conceineu may vaiy accoiuing to theii stage of uisease: foi instance,
slowing the piogiessive loss of ambulation may be an impoitant piefeience foi the
paients of a chilu who is still able to walk, while a non-ambulant patient may be
much moie conceineu with uppei aim function anu maintaining peisonal caie
ability. Such ongoing peisonal benefit-iisk assessments make the patient-caiegivei
community a ciitical iesouice to engage in the uevelopment of benefit-iisk ieseaich
agenua.

>C A+'(4#.$+'. &'% .5)($&4 (+'.$%),&/$+'. *+, 1#(2)'')
The FBA unueistanus that communities facing piogiessive anu fatal uisoiueis,
especially wheie theie is a lack of effective tieatment options, aie willing to take
gieatei iisks than people with chionic anu stable oi slowly piogiessing uisoiueis
wheie theie aie existing tieatments. Foi piogiessive uisoiueis, unceitainty about
8
benefits oi potential iisk of any theiapy must be weigheu against the known
implication of not tieating. This ceitainty of uisease piogiession without tieatment
shoulu be incluueu as a compelling haim in the benefit iisk assessment.
Consequently, the agency is moie flexible in situations wheie theie aie no, oi
limiteu tieatments, anu moie toleiant of unceitainty.

FBA benefit-iisk assessment ielateu to tieatments foi Buchenne will be stiongly
influenceu by well-uocumenteu piefeiences of patients anu families. The neeu to
incluue patientcaiegivei piefeiences is especially compelling foi seiious,
piogiessive uisoiueis with limiteu tieatment options. Thus, sponsois shoulu
pioviue the FBA with iobust uata on patientcaiegivei piefeiences. The FBA
acknowleuges the complexity of BNB anu that meaningful benefitiisk toleiance
anu acceptable tiaueoffs may vaiy acioss clinical subtypes, acioss the uisease
piogiession anuoi as a consequence of piefeience heteiogeneity acioss patients
anu caiegiveis. When possible, a piopeily uesigneu anu poweieu stateu piefeiences
stuuy best pioviues insight into piefeience heteiogeneity. The agency suppoits a
mouel wheie uata is collecteu in collaboiation with patientcaiegivei auvocates oi
auvocacy gioups to empowei patient communities anu ensuie meaningful
piefeiences uata.

0nueistanuing eviuence-baseu benefit iisk piefeiences peimits the Agency to
exeicise maximum flexibility in allowing acceleiateu access to theiapies with
appiopiiate labeling that cleaily auuiesses unceitainty anu uesciibes benefits, siue
effects, haims to facilitate inuiviuual benefit iisk choices. Sponsois shoulu be awaie
that the FBA woulu consiuei labels that peimit inuications to pioviue access to eaily
auopteis of tieatment patients who aie highly iisk toleiant uue to theii uesiie to
pievent oi uelay the loss of ciitical functional capacity anu seiious moibiuity oi
moitality. Patients anu caiegiveis, with suppoit fiom theii clinicians, can then make
appiopiiate uecisions foi themselves with as much infoimation on benefit anu iisk
as available. This appioach best meets the neeus of the entiie Buchenne
communitypatients, caiegiveis, clinicians, ieseaicheis, anu inuustiywhile
achieving the FBA manuate to pioviue access fastei foi high-neeu iaie uisoiuei
communities.

Iv. 1-;"B?:8-A AD-8>D-;
;C ")'),&4 A+99)'/.
The uiagnosis of BNB is usually maue by a neuiomusculai specialist on the basis of
family histoiy, clinical featuies (uelays in ieaching uevelopmental milestones anu
motoi uifficulties) followeu by appiopiiate laboiatoiy investigations.
2S


uuiuance publisheu by the Ameiican Acauemy of Peuiatiics iecommenus that all
chiluien with motoi uelay anu low muscle tone shoulu initially be scieeneu by
measuiing the seium cieatine phosphokinase (CK) activity, which is significantly
elevateu in BNB, usually >1uuu 0L.
24
This shoulu be uone iiiespective of whethei
theie is a family histoiy of BNB, as about one thiiu of the BNB cases iesult fiom
spontaneous mutations in the '(' gene.
2S
If seium CK activity is elevateu, the
9
uiagnosis of BNB shoulu be confiimeu with moleculai analysis of the '(' gene oi
by assessment of uystiophin piotein expiession on muscle biopsy.

3") '(' &4/5+#!14- #&2!!)2
Sponsois shoulu be awaie that uespite eaily signs of weakness, theie may be
significant uelays in aiiiving at a BNB uiagnosis because paients uo not voice theii
conceins oi local healthcaie piofessionals aie not familiai with the uisease,
iesulting in a uelay in puisuing testing. The uelay can be substantial one cohoit,
NB-STARnetiepoiteu a uelay of 2.S yeais fiom the time symptoms weie fiist
noticeu.
26
Anothei cohoit iepoiteu that the mean age at uiagnosis was 4 yeais anu
1u months (SE S9 months, iange 16-99 months).
27
Less than a thiiu of the boys in
that cohoit weie uiagnoseu befoie the age of 4 yeais (S2%) many weie in school,
anu theii uevelopmental uelays hau been noteu by teacheis, but not health
piofessionals. This suggests euucation of piactitioneis is key to shoitening the
uiagnostic ouyssey (see the Ameiican Acauemy of Peuiatiics statement on the
chilumuscleweakness.oig).

Theie may also be financial baiiieis to completing a iefeiial to a specialist. In
auuition, theie is lack of unifoimity in access oi availability of genetic testing that
may also contiibute somewhat to a uelayeu uiagnosis as well.

Sponsois shoulu be awaie, howevei, that theie aie a numbei of initiatives
unueiway to inciease awaieness among piimaiy clinicians of what steps to take in
chiluien with uevelopmental uelays.

<C 1$&7'+./$( =&V+,&/+,0 -'6)./$7&/$+'.I3)/2+%. *+, (+'*$,9$'7
%$&7'+.$.
6)+)14- /+/%2!4!
In young boys with the clinical featuies suggestive of BNB anu elevateu CK, analysis
of the '(' gene fiom genomic BNA (such as lymphocyte-ueiiveu BNA) is uiagnostic
in ~9S % of cases. Appioximately 6u% of mutations aie laige-scale ueletions, S%
aie uuplications, anu the iemainuei, uetectable fiom genomic BNA, aie point
mutations oi small ueletionsinseitions. The iemaining ~S% of mutations aie uue
to intionic mutations that aie unuetectable by stanuaiu genomic analysis but iesult
in alteieu splicing only uetectable by mRNA analysis fiom muscle tissue.
28, 29
Thus,
lack of a uetectable BNB gene mutation using stanuaiu methouology uoes not
excluue a BNB uiagnosis. As uiscusseu in fuithei uetail below, muscle biopsy anu
uystiophin expiession analysis iemains the golu stanuaiu of uiagnosis, iemaining
paiticulaily useful in cases wheie no mutation was uetecteu by stanuaiu clinical
moleculai uiagnostic testing.

The unueistanuing of ielationship between the location, type oi the size of the
mutations in the '(' gene anu the seveiity of phenotype is evolving.
Su
Bowevei,
genetic testing in the '(' gene can be contiasteu to othei genotypic tests uue to the
high piobability of finuing a mutation anu the capacity to pieuict with a uegiee of
ceitainty what that functional effect of that mutation will be foi uystiophin
1u
piouuction foi most cases. BNB oveiwhelmingly iesults fiom mutations pieuicteu
to leau to tiuncateu uystiophin piotein, with missense mutations in specific
functional uomains founu only iaiely. Theiefoie the common genetic test piactice of
iepoiting of 'vaiiants of unceitain significance' is iaiely useu foi a patient with
Buchenne musculai uystiophy. If a muscle biopsy shows absence of uystiophin, a
genetic analysis is consiueieu the stanuaiu of caie even if the uiagnosis maue on the
basis of piotein.

()1"#&#%#52
The quality anu type of uiagnostic testing may neeu to be consiueieu in the context
of the sponsoi's specific stuuy. Theie aie now a numbei of establisheu genetic
testing techniques, anu methous can be expecteu to continue to evolve. Bowevei, foi
changes in exon copy numbei (ueletions anu uuplications), any acceptable mouein
moleculai uiagnostic methou must inteiiogate all exons to establish completely the
extent of the ueletionuuplication. Similaily, methous of sequence analysis shoulu
pioviue sequence coveiage of the entiie couing iegion in piobanus. Patients who
have been scieeneu with oluei technologies may neeu to be ie-testeu in oiuei to
moie accuiately chaiacteiize theii mutations

Testing shoulu be peifoimeu at a CLIA (anu, potentially, CAP) ceitifieu lab (oi theii
equivalent outsiue of the 0S) anu the iesults inteipieteu by a qualifieu piofessional.

7--)!! 1# 5)+)14- 1)!14+5
Baiiieis that limit access to genetic testing exist anu these incluue financial anu
health caie pioviuei euucation. Paitneiships between auvocacy anu inuustiy can
facilitate access to genetic testing |foi example: Becoue Buchenne piogiam
(https:www.uuchenneconnect.oig)j.

'2!18#9"4+ ):98)!!4#+ #+ ;$!-%) <4#9!2 /! / &4/5+#!14- <4#;/8=)8
Immunohistochemical, immunofluoiescent, oi Westein blot analysis can show the
ielative amount of uystiophin in skeletal muscle specimen, anu Westein blot can
ieveal its size, helping to uistinguish between BNB anu miluei musculai uystiophy
phenotype such as Beckei musculai uystiophy (BNB). An amount of uystiophin of
less than S% of noimal has been uesciibeu as consistent with BNB, anu gieatei
than 2u% as consistent with milu BNB,
S1
but as uiscusseu in the Biomaikeis
section, stanuaiuization of uystiophin quantification is challenging, anu in cuiient
clinical uiagnostic piactice, uystiophin expiession is fiequently uesciiptive oi semi-
quantitative. BNB is typically causeu by in fiame BNB mutations iesulting in the
expiession of inteinally tiuncateu uystiophin pioteins. The expiession levels anu
functionality of these uiffeient uystiophin pioteins vaiy anu contiibute to the
vaiiable phenotype in BNB yet without a lineai coiielation between uystiophin
levels anu phenotype.
S2,SS,S4,SS
Coiielation of uystiophin amounts with clinical
benefit has been seen in female BNB caiiieis (noimal uystiophin).
S6, S7, S8
It is
expecteu that the laboiatoiy will make eveiy effoit to maintain goou laboiatoiy
piactice as outlineu in the Biomaikeis chaptei (page S6), section, "The hanuling of
the biopsy."

11

!" $%&'()* +,)%%*-*.
Newboin scieening has been peifoimeu via measuiement of CK activity on uiieu
bloou spots fiom neonates, anu the ability to use the same bloou spot foi '('
mutational analysis has iecently been shown to be feasible.
S9


Because of the expecteu impoitance of eaily theiapeutic inteivention, theie is
enthusiasm in the community foi newboin scieening. At the national anu
inteinational level, it iemains unuei auministiative ieview anu theie aie potential
pioblems to be iesolveu (ex. non-BNB patients iuentifieu, infoimeu consent, cost).

This is something that iemains in evolution at the time of uiafting this uocument.

/" 01% +2%,3)45 6*7 ,8-*-,68 ,86++-9-,63-(* (9 7:+3)(21-*(2631-%+
The amount anu size of uystiophin in the muscle biopsy tenus to pieuict the
seveiity of musculai uystiophy, while genomic analysis also has a high uegiee of
pieuicteu value foi uisease phenotype. But sponsois shoulu be awaie that a
moleculai uiagnosis is not the same as the clinical uiagnosis, anu uoes not with a
hunuieu peicent ceitainty ueteimine phenotype.
4u
Although genomic analysis
geneially has a high pieuictive value uepenuing on the mutation, anu piognosis
baseu on the open ieauing fiame is usually quite effective at uiffeientiating BNB
fiom BNB, uetaileu analyses of laigei seiies of BNB patients have uocumenteu
many appaiently out-of-fiame mutations wheie uystiophin piouuction was still
seen by vaiious moleculai mechanisms. Foi example, some pieuicteu nonsense
mutations aie associateu with Beckei musculai uystiophy, a clinically miluei
uisoiuei.
41


Thus genotype alone uoes not ueteimine classification in all patients, anu although
genetic anu piotein iesults can make pieuictions, they uo not ieplace the clinical
assessment in ueteimining wheie a patient is on the spectium of uystiophin-ielateu
musculai uystiophy. Recognizing this spectium of uystiophinopathies anu
heteiogeneity in the couise of piogiession some expeits have pioposeu fuithei
sub-categoiization of uystiophinopathies to incluue, foi example, an inteimeuiate
(INB) foim falling between classic BNB anu moie seveie BNB baseu upon clinical
giounus. While the Agency uoes not believe these sub-categoiizations waiiant
uistinctions in access to new theiapies, they may iepiesent a souice of
heteiogeneity that sponsois shoulu take into consiueiation when conuucting
clinical tiials.


TC 8J> AEDD>B8 EB1>D:8;B1-B" ?@ 8J> B;8ED;= J-:8?DK ?@
1EAJ>BB> 3E:AE=;D 1K:8D?LJK
;C ")'),&4 (+99)'/.
The natuial histoiy of BNB is much bettei chaiacteiizeu touay than it was ten to
twenty yeais ago, as a consequence of patient iegistiies, natuial histoiy stuuies anu
uata uiawn fiom the placebo aims of inuustiy tiials (which have been to shown to
12
coiiesponu closely to the natuial histoiy uata).
42, 4S
0vei the same peiiou, impioveu
meuical management has been shown to piolong suivival anu slightly slow uisease
piogiession. Bespite this, the unmet meuical neeu anu uigency foi impioveu
theiapies foi BNB is piofounu. Piogiessive quauiipaiesis uuiing the fiist two
uecaues uue to uystiophin ueficiency anu skeletal muscle fibei loss iemains the
common uisease couise. Pulmonaiy insufficiency fiom skeletal muscle involvement
anu caiuiomyopathy leaus to substantially shoiteneu lifespans among patients
ieceiving even optimal caie. Sponsois shoulu be awaie of how the cuiient
unueistanuing of the natuial histoiy of BNB pioviues oppoitunities foi the
evaluation of new tieatments acioss the spectium of the uisease.

<C 8)./$'7 &'% )6&4#&/$+' &(,+.. /2) .5)(/,#9 +* %$.)&.)
Sponsois shoulu be awaie of the vaiiety of testing anu evaluation tools that have
been useu to measuie uevelopmental uelay, functional loss anu othei paiameteis of
piogiession of BNB. The use of these outcome measuies in natuial histoiy stuuies
anu patient iegistiies have helpeu to bettei chaiacteiize the natuial histoiy of the
uisease.

Beginning in 2u1u, the Inteinational BNB Clinical 0utcomes Woiking uioup met on
a numbei of occasions in oiuei to ieach inteinational consensus on age-appiopiiate
clinical outcome measuies foi use in the stuuy of subjects with BNB.
44
In auuition to
mapping out the outcome measuies cuiiently being useu, they ievieweu uata fiom
eight laige contempoiaiy cohoits (incluuing moie than 19uu subjects acioss the
spectium of the uisease followeu ovei a twenty yeai peiiou) in oiuei to establish
the cuiient expecteu clinical couise of the uisease anu ueteimine whethei uata
existeu to uefine the ielationship between the outcome measuies anu milestones of
uisease piogiession. Wheie ueficiencies anu enupoints occuiieu acioss the
spectium of uisease, woiking subgioups weie foimeu to woik collaboiatively to
valiuate new enupoints.
4S


The following schematic, auapteu fiom the pioceeuings of those meetings, incluues
a numbei of outcome measuies that aie most wiuely useu, anu wheie theie is bioau
scientific consensus iegaiuing theii utility in BNB, oiganizeu by the age gioup anu
uisease stage in which they aie useu. In oiuei foi an assessment tool oi outcome
measuie to be incluueu in this schematic, theie neeueu to be consensus that theie
was a conceptual fiamewoik foi the enupoints that fit Buchenne uystiophy anu uata
on ieliability, concuiient valiuity, cioss valiuation (with othei enupoints),
noimative ianges, expeiience with the enupoints anu existing longituuinal uata in
ongoing natuial histoiy stuuies, eviuence of iesponsiveness to a tieatment, anu
clinical meaningfulness. Finally, the tool hau to have been successfully implementeu
in multinational clinical tiials.
46
uuiuance peitaining to inclusion of specific outcome
measuies in clinical inteivention tiials is uiscusseu in Section F (BNB Clinical Tiial
Besigns, Enupoints, anu Consiueiations).

1S
Figuie 1

47, 48


Caiuiac NRI is an impoitant emeiging measuie that has yet to meet all of the
ciiteiia to be incluueu in the schematic (similaily, skeletal muscle NRI is a
piomising measuie, uesciibeu in moie uetail latei in this uocument).

AC ?6),6$)X +* /2) B&/#,&4 J$./+,0 $' 1#(2)'') 9#.(#4&, %0./,+520
What follows is a biief oveiview of the cuiient natuial histoiy of BNB acioss the
spectium of uisease. Fuitheimoie, while specific functional changes aie obseiveu at
uiffeient ages, it shoulu be emphasizeu that the uisease is uue to geneializeu
skeletal muscle involvement anu caiuiomyopathy anu pathological piocesses
involveu in BNB aie ongoing ovei the couise of a patient's lifetime while loss of
ambulatoiy capacity anu gioss motoi functions may be a piimaiy focus in
ambulatoiy boys, neuiomusculai ueteiioiation may alieauy be measuiable in the
uppei limb anu othei muscle gioups. Note, the ages aie appioximations, anu the
intent is not to cieate aitificial stages of uisease.

>)#+/1)!?@+A/+-2B While BNB is iaiely uiagnoseu in infancy, the uisease is
manifesteu at biith. Even though some of the infants uetecteu uue to family
histoiy aie sometimes iefeiieu to as being asymptomatic, most will still show
uelayeu uevelopment if evaluateu with tools such as the uiiffiths Nental
Bevelopment Scales, an outcome measuie than can be useu in the veiy young (6-
14
47 months)
49
anu the Bayley Scales of Infant anu Touulei Bevelopment, Thiiu
Euition (Bayley-III).
Su
,
S1
0ne stuuy of chiluien with BNB with mutations
upstieam oi in exon 44 hau highei Bevelopmental Quotient (BQ) than those
with mutations uownstieam exon 44 which aie associateu with involvement of
uystiophin isofoims expiesseu at high levels in biain. The uiffeience was
significant foi total anu inuiviuual subscale BQ with the exception of the
locomotoi subscale. Items, such as ability to iun fast, oi getting up fiom the flooi
consistently faileu in all chiluien, iiiespective of the age oi of the site of
mutation.
S2

C#$+5 -"4%&8)+D )/8%2 /;<$%/+1 E/5)& #+) 1# FG ;#+1"!HB The uevelopment of
gioss motoi milestones is typically slowei than in boys without Buchenne, anu
some chiluien may show signs of uelayeu language anu cognitive impaiiment.
Touuleis anu young chiluien may also be scoieu with uevelopmental outcome
measuies such as the Bayley-III anu uiiffith's Bevelopmental Scales.
SS
,
S4
uioss
motoi scoies weie lowei in young chiluien with BNB at baseline compaieu with
publisheu contiols anu ievealeu a fuithei ueclining tienu at 6 months. Repeateu
measuies analysis ovei 12 months ievealeu that gioss motoi scoies ueclineu
fuithei at 12 months. Cognitive anu language scoies weie lowei at baseline
compaieu with typically ueveloping chiluien anu uiu not change significantly at
6 oi 12 months. Fine motoi skills, also low at baseline, impioveu ovei 1 yeai.
C#$+5 7;<$%/1#82 EA8#; A#$8 1# /998#:4;/1)%2 !),)+HB A peiiou wheie theie may
be slowei gains in ambulatoiy function as compaieu to typically ueveloping
chiluien (on 6NWT anu 1u metei walkiun tests) anu eithei gains oi losses in
milestones as noteu by enupoints such as the Noith Stai Ambulatoiy Assessment
(NSAA). Bowevei, it is impoitant to note that physiologic ueteiioiation is
ongoing anu boys aie incieasingly falling behinu noimative peifoimance levels
of theii noimally functioning peei gioup.
I/1) 7;<$%/1#82 EA8#; /998#:4;/1)%2 !),)+ 1# 1"481))+ 2)/8! #A /5)H: 6eneially
uefineu as when inuiviuuals begin to suffei a uecline in theii gioss motoi
functions as well as some pulmonaiy function paiameteis, paiticulaily maximal
expiiatoiy piessuie (NEP) anu maximal inspiiatoiy piessuie (NIP). Buiing this
stage of uisease, theie is maikeu piogiessive loss of muscle fibei in the pioximal
muscles, giowing weakness anu the giauual loss of gioss motoi skills anu
ambulatoiy functions (incluuing stanuing ability, staii climbing anu ultimately,
the ability to walk). Ankle equinus contiactuies aie the most common skeletal
uefoimity. Theie is iisk of osteopenia anu fiactuies. Theie is also a compaiative
loss in height anu incieaseu weight gain in compaiison theii noimally
functioning peei gioup.
J/8%2 >#+K/;<$%/1#82 E<)54++4+5 1") /5) L")+ / <#2 !1/81! $!4+5 / L"))%-"/48
A$%%K14;)HB Aftei boys can no longei walk, theie is continueu musculai
ueteiioiation thioughout the uppei anu lowei limbs, anu skeletal uefoimities
such as limb contiactuies anu spine uefoimity may become pioblematic.
Poweieu mobility is iequiieu aftei loss of ambulation. Postuial maintenance anu
sitting balance is initially intact anu piogiessively lost. Theie is incieasing loss of
uppei limb function (with uecieasing ability to ieach oveiheau, uiess, self-feeu,
1S
anu peifoim othei self-caie). Theie is continueu uecline in pulmonaiy function
with ultimate neeu foi mechanical cough assistance anu piogiessive iisk of
noctuinal hypoventilation iequiiing non-invasive ventilation. Caiuiomyopathy is
eviuent by caiuiac NRI in viitually all patients anu in some patients by caiuiac
echo. Aftei tiansition to a wheelchaii, patients tenu to put on moie weight
compaieu to theii noimally functioning peei gioup.
I/1) +#+K/;<$%/1#82B Postuial suppoit of the tiunk anu heau suppoit fiom a
seating system is iequiieu as well as powei iecline. 0ppei extiemity function is
seveiely limiteu to uistal fine motoi function anu tabletop activities. Naintaining
computei access is a ciitical quality of life concein. viitually all patients benefit
fiom mechanical cough assistance anu theie is a high iisk of noctuinal anu
uaytime hypoventilation iequiiing non-invasive ventilation. 0ptimal nutiitional
management may iequiie gastiostomy tube placement anu enteial foimula
supplementation. Theie is iisk foi uysphagia anu aspiiation. Auequate
phonation may become an issue late in the uisease couise. Theie may be a laigei
numbei of oluei BNB patients with unmet meuical neeus. As patients age,
iespiiatoiy impaiiment anu heait uisease (heait failuie anu conuuction
abnoimalities) aie causes of moibiuity anu, eventually, moitality.

Both piogiessive limb weakness anu uecline in pulmonaiy function aie uue to
skeletal myopathy, howevei, caiuiac ueteiioiation uue to piogiessive
caiuiomyopathy may not be coiielateu with skeletal muscle ueteiioiation. With
incieaseu lifespan uue to effective ventilation inteiventions, caiuiomyopathy has
become a moie common cause of ueath among patients with BNB.
SS

Caiuiomyopathy in BNB uoes not usually manifest clinically until latei phases of
uisease piogiession, but is likely piesent to some uegiee beginning at biith.
Bowevei, the concept that caiuiac uisease uevelops only latei along the spectium of
BNB piogiession uoes not appeai to be the case imaging uata suggest a
piopoition of boys alieauy show fibiosis in the heait at ages as young as six.

The loss of clinical milestones is a hallmaik of uisease piogiession in BNB. Piioi to
anu aftei the loss of ambulation, the uifficulty peifoiming functions anu the loss of
milestones, occui in a geneially pieuictable uescenuing oiuei (although theie may
be some oveilap oi slight vaiiation in some of the milestones).

Ambulatoiy functions anu milestones
0nable to jump, hop, anu iun
uowei's sign with stanuing
Loss of stanuing fiom the flooi
Loss of tiansition fiom lying supine to sit
Loss of staii climbing
Loss of ability to stanu fiom a chaii
Loss of ability to walk inuepenuently (uefineu by inability to peifoim 1u
metei walkiun)
Loss of stanuing in place
16

Non-ambulatoiy milestones (uescenuing oiuei)
Loss of ability to ieach oveiheau
Loss of ability to ieach the scalp
Loss of ability to self-feeu without auaptations (hanu to mouth)
Loss of ability to place hanus to table top
Loss of ability to use a computei (uistal hanu function)

Pulmonaiy milestones aie piimaiily measuieu by foiceu vital capacity, peak cough
flow, anu maximal static aiiway piessuies (NIP anu NEP)) inuicating a neeu foi
inteiventions as outlineu in the BNB Caie Consiueiations.
S6

<Su% pieuicteu FvC (cough assistance; monitoiing iequiieu)
< 4u% pieuicteu FvC (non-invasive ventilation shoulu be a consiueiation)
< Su% FvC (inability to sustain auequate oveinight ventilation without
suppoit is likely)
Naximum expiiatoiy piessuie (NEP) < 6u cm watei (pieopeiative tiaining
in anu postopeiative use of manual anu assisteu cough techniques aie
necessaiy)
Peak cough flow <16u Lmin (manual anu mechanically assisteu cough
techniques necessaiy)
NEP <4u cm watei (manual anu mechanically assisteu cough techniques
necessaiy on a uaily basis)

Caiuiac milestones
Noimal Ejection fiaction (afteiloau ieuuction with angiotensin conveiting
enzyme (ACE) inhibitois, oi Angiotensin II ieceptoi blockeis (ARBs),
iecommenueu by some caiuiologists)
< SS% Ejection fiaction (most woulu agiee that caiuiac meuications
inuicateu)

/" ;(& (23-568 5%7-,68 56*6.%5%*3 699%,3+ 31% ,(4)+% (9 /</
Sponsois shoulu be awaie that cuiient meuical management (as uepicteu in Figuie
1) has changeu the natuial histoiy in BNB affecting the timing of clinically
meaningful milestones in inuiviuuals with access to high quality caie. This has
laigely been uue to the use of glucocoiticoius, management of spine uefoimity,
pulmonaiy management, anu caiuiac management. The occuiience of contiactuies
may impact mobility anu uppei limb function anu effoits aie maue to pievent anu
manage contiactuies. Bespite these inteiventions, the caiuiomyopathy anu
pulmonaiy involvement in BNB still leaus to substantially shoiteneu lifespan. In the
CINRu natuial histoiy cohoit of S4u subjects followeu piospectively fiom 2uu6-
2u11, ueath occuiieu in S% of the cohoit (S%) anu age at ueath iangeu fiom 9.9
yeais to 29.S yeais.
S7


"4#(+(+,/$(+$% /2),&50: Coiticosteioius have hau an effect on all-cause suivival as
well
S8
(although, theii effects on BNB-ielateu heait uisease aie, howevei, somewhat
17
moie equivocal). A Cochiane ieview has also concluueu that glucocoiticoiu
coiticosteioius impiove muscle stiength anu function ovei six months to two yeais.
Impiovements weie seen in time taken to iise fiom the flooi (uowei's maneuvei
time), nine meteis walking time, foui-staii climbing time, the ability to lift weights,
leg function giaue anu foiceu vital capacity.
S9
In seveial natuial histoiy stuuies,
steioius have been shown to uelay the loss of ambulatoiy milestones, piolonging
ambulation by about two to thiee yeais ovei time anu uelayeu losses in uppei-limb
functioning so that young men can continue to iaise theii hanu to theii mouths
anu feeu themselves foi a longei peiiou of time.
6u
Steioius have also affecteu
pulmonaiy function young men tieateu with steioius ieach an oluei age befoie
iequiiing mechanical cough assistance oi non-invasive ventilation as uefineu by FvC
paiameteis outlineu in the BNB caie consiueiations.
61
Bowevei, as pieviously
noteu, glucocoiticoiu theiapy also comes with substantial auveise events.

A+'/,&(/#,) 9&'&7)9)'/Z While ankle equinus contiactuies begin in the late
ambulatoiy stage anu may contiibute to the loss of staii climbing anu ambulatoiy
capacity, most lowei limb anu uppei limb contiactuies occui subsequent to the loss
of ambulation.
62
Appioaches to contiactuie pievention anu management have been
outlineu in the caie consiueiations,
6S
but the efficacy of these appioaches has not
been establisheu. To the extent that contiactuies aie uiiectly ielateu to antigiavity
stiength (movement against giavity thiough a full iange of motion), anu ambulatoiy
capacity, any inteivention that maintains stiength, function, anu upiight mobility
will likely iesult in uecieaseu contiactuies.
64


:5$') %)*+,9$/0 9&'&7)9)'/Z The inciuence of significant scoliosis iequiiing
spinal aithiouesis has changeu uue to the use of glucocoiticoius.
6S
In auuition,
timely spine suigeiy foi cuives > Su-4u uegiees has impacteu suivival.
66


L#49+'&,0 9&'&7)9)'/Z The Ameiican Thoiacic Society piactice paiametei
67

incluues iecommenuations foi management of BNB with aiiway cleaiance
stiategies oi mechanical cough assistance anu non-invasive ventilation. Suivival has
been most impacteu by ventilation two iecent stuuies have iepoiteu that
lifespans in Buchenne can be lengtheneu substantially uue to the implementation of
non-invasive ventilation.
68, 69
Consequently, a laigei numbei of young men with
BNB aie living into theii twenties anu thiities but often with significant uisability.
In auuition, eaily ueath is still commonly obseiveu in inuiviuuals with BNB in the
eaily teen yeais to eaily 2u's, mostly uue to heait pioblems.

A&,%$&( 9&'&7)9)'/Z The caiuiac management has evolveu fiom tieatment of
symptomatic heait failuie to pievention of piogiessive ventiiculai uysfunction with
eaily afteiloau ieuuction e.g., angiotensin conveiting enzyme (ACE) inhibitois,
Angiotensin II ieceptoi blockeis (ARBs), anu beta blockeis). ACE inhibitois have
impacteu positively on suivival in young men with BNB-associateu clinical
caiuiomyopathy, by ieuucing stiess on the heait (afteiloau ieuuction).
7u
Bata aie
neeueu on the combineu effect of afteiloau ieuuction anu coiticosteioius on the
uevelopment of ventiiculai uysfunction. Caiuiac conuuction abnoimalities aie
18
scieeneu foi with ECu anu Boltei monitoiing. The use of left ventiiculai assist
uevices anu caiuiac tiansplantation in BNB is an evolving topic.
71


The stanuaiu of caie ieceiveu by patients with Buchenne theiefoie has significant
implications on the uesign of tiials in the population, uepenuing upon the outcomes
being measuieu.

=" ;%3%)(.%*%-3: -* /</ 7-+%6+% 2)(.)%++-(*> ?)%7-,36'-8-3: 6*7 +(4),%+
(9 @6)-6'-8-3:
The goal of theiapeutics in BNB is to slow oi stabilize uisease piogiession in
compaiison to that expecteu by natuial histoiy. It shoulu be noteu that theie is
heteiogeneity obseiveu among patients in teims of uisease piogiession, as noteu in
the uiagnostics chaptei, in that some patients with BNB may expeiience moie
aggiessive iates of piogiession than otheis. The clinical heteiogeneity of Buchenne
musculai uystiophy (BNB) was once vieweu as a majoi obstacle to the
inteipietation of theiapeutic tiials but with an evolution in the unueistanuing of
natuial histoiy, this may no longei be the case.

0#$8-)! #A ,/84/<4%412
With moie uata coming fiom natuial histoiy stuuies anu the placebo aims fiom
Buchenne tieatment stuuies, many of the causes foi vaiiability in outcomes aie
becoming cleaiei. Sponsois shoulu consiuei taking the following causes of
heteiogeneity into account when uesigning theii phase II anu phase III stuuies. The
following ciitical elements aie at least iuentifiable anu coulu possibly have a laige
enough effect size that tiial uesigns shoulu manage them.

Bisease seveiity stage of uisease
Some vaiiability in futuie piogiession is explaineu on the basis of uisease seveiity,
stage of the uisease, anu known natuial histoiy. Foi example baseline levels of
function pieuict subsequent uisease piogiession in BNB. Bighei baseline function
oi stabilization of baseline function ovei the shoit-teim is almost always associateu
with slowei long-teim uecline.
72, 7S
Lowei baseline function may be associateu with
iapiu subsequent uecline in ambulatoiy enupoints when patients have passeu
ciitical thiesholus of stiength anu function. Baseline measuies of ambulatoiy
capacity have been useu to stiatify cohoits in BNB tiials.

The age at loss of clinically meaningful milestones (a pioxy foi uisease seveiity) also
pieuicts the age at loss of futuie milestones. Foi example, the age at loss of
ambulation pieuicts the age at which subsequent loss of uppei limb functions
occuis anu the age at which ciitical pulmonaiy milestones aie ieacheu.
74
It follows
that changes in some clinical outcome measuies in iesponse to tieatment ovei the
shoit teim, can pieuict subsequent uisease piogiession yeais latei. This has been
uemonstiateu in chiluien using coiticosteioiu tieatment followeu foi many yeais
7S
.

Sponsois shoulu take caie to pievent an imbalance in the ages of stuuy paiticipants,
which can intiouuce substantial vaiiability into a tiial. It is ciitical that contiol anu
19
tieatment aims in clinical tiials be appiopiiately matcheu by age anu functional
status. Baseline functional peifoimance in ielation to specific enupoints such as
6NWT, oi time function tests shoulu also be taken into account as it uoes have an
impact on the subsequent iate of piogiession ovei time.

uenetic pieuictois of uisease piogiession
Nutations within the '(' gene (exon-skippable mutationsueletions oi nonsense
mutations, ueletions, uuplications, point mutations) may be associateu with an
alteieu couise of piogiession fiom one anothei. 0ne stuuy suggests that theie is a
tienu foi chiluien with uuplication mutations to peifoim bettei than the cohoit as a
whole.
76
Within those with ueletions, theie aie specific subgioups that appeai to be
uiffeient fiom each othei. Foi instance, theie is a tienu towaius bettei baselines anu
less seveie uecline in piogiession as measuieu by 6NWT in boys eligible foi
skipping of exon-44 when compaieu to those with boys eligible foi skipping at
exons 4S anu SS.
77
The exact effect size is being ueteimineu as this uocument is
being wiitten. With laigei cohoits oi longei follow-up, uiffeiences between
subgioups may become significant.

Bowevei, while theie may be uiffeiences between subgioups of patients with
specific mutations, the mean 12-month changes in each subgioup falls within a
naiiow iange in compaiison to the mean of the whole BNB cohoit. Fuitheimoie, it
shoulu be iecognizeu that some vaiiability will be piesent within specific subgioups
uue to the many souices of heteiogeneity listeu heie.

It is also woith noting that some mutations appeai to select foi moie uystiophin-
ielateu abnoimalities in non-skeletal muscle causing moie pulmonaiy, caiuiac anu
neuiocognitive impaiiment.
78, 79


Clinical tiials of tieatments that aie not mutation specific shoulu collect appiopiiate
samples foi full genetic analysis. As noteu in the uiagnostics chaptei, some tiial
paiticipants may neeu to be iescieeneu with a technique that pioviues a complete
analysis of the '(' gene (see uiagnostics chaptei).

uenetic mouifieis
uenetic scieening has iuentifieu polymoiphisms in othei genes that may have
alteieu aspects of the iesponse of muscle to uystiophin ueficiency anuoi uiug
tieatment (e.g. glucocoiticoius). These genetic mouifieis may mouify the onset,
seveiity, oi uiug iesponsiveness of Buchenne musculai uystiophy patients, anu aie
instiuctive iegaiuing key biochemical pathways involveu in muscle uamage, iepaii
oi iesponse to steioius. They aie also impoitant in incieasing unueistanuing of
factois iesponsible foi patient-patient vaiiability, anu coulu eventually piove
helpful in inteipieting clinical tiial uata. uenetic mouifiei stuuies, as with most
genetic association stuuies in any human tiait, typically iequiie laige numbeis of
patients stuuieu using ieliable anu sensitive biochemical oi clinical outcome
measuies. Biffeiences in methous of chaiacteiizing oi categoiizing cohoits of
2u
patients, as well as ethnic uiffeiences in polymoiphism allele fiequencies can leau to
challenges in statistical analyses anu iepiouucibility of genetic association stuuies.
To uate, two potential genetic mouifieis have been iuentifieu:

Latent TuF-beta-binuing piotein 4 (LTBP4) polymoiphisms: A minoi allele piesent
in about thiity peicent of the population appeais to have a piotective effect on
ambulation ioughly equivalent to the effect of steioiu tieatment, piolonging
ambulation by as much as two yeais.
8u


Secieteu phosphopiotein 1 (SPP1 oi osteopontin) polymoiphisms: In the case of
osteopontin, the genetic mouifiei may actually be mouifying patient's iesponses to
coiticosteioiu management iathei than affecting the uisease itself uiiectly.
81


Theie may be othei genetic mouifieis yet to be iuentifieu. Bowevei, at the time of
wiiting, uata on genetic mouifieis comes fiom small cohoits anu the effect sizes aie
not yet cleaily uefineu. Sponsois shoulu ieview the most cuiient uata on the subject
to see whethei scieening foi these genetic mouifieis in theii clinical tiials is
auvisable, foi stiatification oi planneu post-hoc analyses to explain potential causes
of vaiiation in the outcomes of patients.

Coiticosteioiu theiapy
Theie aie uata to suggest that uiffeiences in patteins of steioiu use incluuing
whethei the patient is on uaily veisus inteimittent iegimens, uosage, time on
tieatment, anu possible uiug choice (ueflazacoit oi pieunisone) may have
vaiiable effects on clinical piogiession anu function.
82, 8S
(Note, at the time of
wiiting, ueflazacoit is not yet maiketeu in the 0S, though some inuiviuuals have
acquiieu access to it). Since meuical management of BNB with coiticosteioius tenus
to be inuiviuualizeu, uiffeiences in siue effects between the steioiu iegimes may
also iesult in uiffeiences in how clinicians aujust the uose oi in patientcaiegivei
auheience. .

Eniollment in the tiials shoulu eithei be iestiicteu oi stiatifieu accoiuing to
haimonizeu coiticosteioiu theiapy. Bistoiically, six months of stable coiticosteioiu
theiapy has been useu as inclusion ciiteiia howevei, sponsois of clinical tiials
shoulu be awaie that some ambulatoiy boys may continue to have functional
impiovements beyonu six months on coiticosteioiu tieatment.

Night splinting, physical theiapy, anu othei stanuaiu inteiventions
It is impoitant to note that night splinting, physical theiapy anu othei stanuaiu of
caie inteiventions as uesciibeu by the BNB Caie Consiueiations aie iecommenueu
because they aie expecteu to have significant effects on functional peifoimance.
84

Significant vaiiability in the couise of piogiession coulu be intiouuceu uepenuing
upon whethei oi not a peison with BNB ieceives stanuaiu of caie contiactuie
pievention anu management, oi is auheient to iecommenueu pievention anu
management stiategies.
21

Sponsois of clinical tiials shoulu make ceitain that the stanuaius of caie aie
obseiveu at eveiy centei that is involveu in theii stuuies incluuing both
pulmonaiy suppoit, night splints anu stietching which coulu make a uiffeience in
chiluien's peifoimance on functional measuies. Sponsois shoulu also take note of
concuiient complementaiy theiapy stuuy paiticipants may be taking. Some stuuies
aie attempting to monitoi the family's auheience to physiotheiapy, home stietching
anu splinting, in an effoit to captuie these vaiiables foi possible post-hoc analyses.

Iueally, sponsois shoulu contiol foi as many of these factois as possible to ieuuce
potential vaiiability in uisease couise among paiticipants in theii clinical tiials.

@C ?'7+$'7 '&/#,&4 2$./+,0 ./#%0 '))%.
uiven the ielentless couise of BNB anu the uifficulty in conuucting auequately
poweieu stuuies in a iaie uisease, theie is neeu to establish auequate, ieliable anu
well-matcheu natuial histoiy contiols that account foi known causes in vaiiability
of the uisease. To be useful foi natuial histoiy contiols, the collection of natuial
histoiy uata must be of a ceitain iigoi to satisfy FBA iequiiements.

Sponsois shoulu iefei to the following thiee uocuments foi guiuance:
FBA uuiuance foi Inuustiy - Computeiizeu Systems 0seu in Clinical
Investigations (2uu7)
CFR Pait 11, Subpait B - Electionic Recoius
ICB uuiuelines foi uoou Clinical Piactice - Section 4.9 (Recoius anu Repoits),
anu Section S.S (Tiial Nanagement, Bata Banuling, anu Recoiu Keeping


T-C 131 A=-B-A;= 8D-;= 1>:-"B:[ ?E8A?3> 3>;:ED>: ;B1
A?B:-1>D;8-?B:
;C ")'),&4 A+99)'/.
The puipose of clinical ieseaich is to bettei unueistanu the uisease piocess, natuial
histoiy, patient expeiience anu cuiient tieatment options. In contiast, iegulatoiy
guiuance foi tiial uesigns anu outcome measuies is intenueu to seive as an
evaluation tool foi consistency of assessment of efficacy of new theiapeutics in the
context of both fiist anu subsequent geneiation of theiapeutics.

Recognizing the neeu to biing uiugs to maiket efficiently, sponsois aie inviteu to
uiscuss with the FBA how theii uiug uevelopment package can best gain expeiience
anu uocument safety in the uiffeient BNB populations, incluuing what stuuies oi
piogiams might be put in place piioi to maiketing, as well as post-maiketing
commitments.

<C 3&\$9$]$'7 $'(4#.$+' +* 5+5#4&/$+'. $' ./#%$).
BNB is a iaie uisease. In the 0S, theie aie appioximately only Suu boys boin with
BNB each yeai anu the pool of subjects available foi each peisonalizeu theiapy is
22
smallei still. veiy young boys have histoiically been the unuei-uiagnoseu, while the
numbeis of oluei non-ambulatoiy boys anu young men who can paiticipate in
clinical tiials is limiteu by mobility, buiuen of paiticipation, lack of expeiience with
enupoints, anu ueath.

Neveitheless, the BNB community has auvocateu that potential theiapies taigeting
BNB be evaluateu acioss the spectium of uisease. Sponsois aie encouiageu to stuuy
new uiugs in uiffeient age gioups anu uisease stages in oiuei to gain a bettei
unueistanuing of a potential theiapy's safety anu how it might woik acioss the
entiie spectium of the uisease.

To uate, most stuuies have been peifoimeu on ambulatoiy boys laigely because
of the selection of a change in 6NWB as the piimaiy enupoint in clinical tiials. Yet
theie is a stiong physiological iationale foi the benefit of eailiei tieatment, as
theiapies that pieseive muscle, in paiticulai, aie likely to have the gieatest impact
on piognosis befoie muscle health has ueteiioiateu.

0ntil theie is an inciease in newboin scieening, eaily uetection of veiy young
patients may be complicateu; howevei, this is the population that, in piinciple,
woulu achieve the gieatest benefit fiom an inteivention because they have the least
accumulateu injuiy. Theie aie special ethical ciicumstances that neeu to be
consiueieu in an inuiviuual anu compassionate way when scieening in families at
iisk anu appioximately one thiiu of boys with BNB will iepiesent a new
mutation in the uystiophin gene.
8S


Neveitheless, it is not only possible but also feasible foi a sponsoi to iuentify some
young patients incluuing neonates in special ciicumstances. 0nce theie is auequate
safety uata to move into veiy young chiluien, we anticipate that sponsois will be
compliant with cuiient peuiatiic iegulation to uevelop a theiapy anu test it in this
population.

Employing oveily iestiictive entiy ciiteiia in a iaie uisease poses a uangei to
successful ieciuitment. Patients with Buchenne musculai uystiophy with a wiue
iange of cognitive impaiiment have been able to paiticipate in clinical tiials as long
as they can peifoim the outcome measuies.

Theie is also a neeu to unueistanu safety anu efficacy at latei stages of uisease
anu to see whethei tieatments that piotect skeletal muscle also pieseive heait anu
iespiiatoiy function. Bemonstiating safety in a bioauei population woulu also lenu
suppoit to a wiue labeling foi the piouuct.

At piesent, theie is no single instiument that can measuie clinical outcomes anu is
equally sensitive to change acioss the entiie spectium of BNB ovei the couise of a
six to eighteen month stuuy. Sponsois aie encouiageu to consiuei the use oi
valiuation of instiuments that coulu expanu the population (age gioupuisease
status) that can be stuuieu in one setting.
2S

AC A4$'$(&4 +#/(+9) 9)&.#,). &'% )'%5+$'/. $' 131
Although to uate, most tiials in BNB have focuseu on ambulatoiy patients, theie is
bioau inteinational consensus on a iange of age-appiopiiate clinical outcome
measuies that coulu be consiueieu in the stuuy of subjects with BNB. Clinically
meaningful loss of capacity can occui in a numbei of functional uomains that meiit
consiueiation as clinical enupoints in a tiial. The appiopiiateness of outcome
measuies uepenus on age anu functional capacity of stuuy paiticipants, anu the
mechanism of action of the uiug. Sponsois may consiuei monitoiing pulmonaiy anu
caiuiac status even in youngei patients, with outcome measuies that aie sensitive in
youngei populations (see uiscussion below). This may be useful to uemonstiate
long-teim benefit anu to anticipate newei, moie sensitive measuies of caiuiac
change oi pulmonaiy specific change.

Tieatment effects may vaiy by muscle gioup uepenuing upon 1) the stage of
uisease, 2) the uiffeiential iate of piogiession of each muscle gioup in that stage, S)
the muscle fibei type, 4) the uiug's mechanism of action, S) the bio-uistiibution of
the uiug to uiffeient tissues anu muscle fibei types, 6) the ioute of auministiation,
anu the meuical auuiessability of the uisease itself theie may be a point wheie a
muscle has ueteiioiateu beyonu a point at which it can iesponu to theiapy.

3+/+, ?#/(+9) 3)&.#,).Z
Notoi outcomes measuies exist acioss the age spectium of BNB. Bowevei,
sponsois shoulu consiuei whethei the following ageuisease specific outcome
measuies, which have been useu to chaiacteiize the natuial histoiy of BNB coulu be
auapteu foi use as clinical enupoints in theii uevelopment piogiam.

Not all motoi enupoints aie measuiing the same phenomenon. Foi example, theie
might be coiielation at baseline of, foi example, the 6NWT anu the TFTs, because
6NWT is an enuuiance test, anu muscle peifusion anu metabolism aie impoitant in
piolongeu exeicise. Bowevei, uiugs that might impiove muscle peifusion oi
metabolism coulu impact the 6NWT while not impacting, in a shoit teim, the TFTs
oi muscle stiength in the shoit teim. Consequently, the selection of a motoi
enupoint foi a specific uiug piogiam neeus to be baseu on the mechanism of action
of the uiug as well as the age appiopiiateness.

The sponsoi will neeu to ensuie that these oi similai outcome measuies aie
appiopiiately valiuateu in the population in which they aie stuuieu.

Foi example, $' ')+'&/).[ $'*&'/.[ &'% 0+#'7 (2$4%,)' #5 /+ &7) N,
uevelopmental scales have been useu in BNB, i.e. the uiiffiths Scale of Nental
Bevelopment oi the Bayley III Scales of Infant anu Touulei Bevelopment BSIB-III
86,
87, 88
Nany uevelopment scales iequiie foimal tiaining anu ceitification on the pait
of the clinical evaluatoi. The sponsoi shoulu consiuei the availability of language
anu countiy specific valiuation of each scale in choosing an outcome measuie, as
well as unueistanu the limitations poseu by the enu of iange effects of each scale.
24
Bevelopmental scales may also unueigo ievision ovei time anu may pose auuitional
challenges in inteipietation.

@+, 0+#'7 ;9V#4&/+,0 `*,+9 *+#, /+ &55,+\$9&/)40 .)6)'aZ
82) B+,/2 :/&, ;9V#4&/+,0 ;..)..9)'/ is a useful measuie of gioss motoi
function in ambulant chiluien fiom the age of foui into auolescence. It was
uevelopeu with a Buchenne uisease piogiession constiuct in minu anu is
ieliable, valiuateu against othei enupoints anu is clinically meaningful.
89, 9u, 91, 92,
9S, 94
The measuie has been shown to be sufficiently iesponsive to uiffeientiate
uisease piogiession in chiluien with BNB on continuous veisus inteimittent
steioius.
9S
Recent clinical tiials using uystiophin iestoiation stiategies have not
shown the NSAA to be sufficiently sensitive to changes in uisease piogiession
ovei 48 weeks but it may be consiueieu foi longei uuiation tiials.
8$9) /+ ./&'% *,+9 .#5$') 2&. been useu foi uecaues as a clinical tiial
enupoint in BNB.
96, 97, 98
Loss of stanuing ability has been shown to be pieuictive
of time to loss of ambulation anu time to 1u % uecline in ambulatoiy function.
(NcBonalu 2u1Sb) It is ieliably obtaineu in youngei BNB subjects
99
anu a useful
enupoint foi youngei BNB patients. Limitations incluue the eaily loss of the
enupoint in many boys with BNB, anu ieuuceu sensitivity of the enupoint as
uefineu by the iatio of the minimally clinically impoitant uiffeience (NCIB),
which is gieatei than S seconus in BNB, to the mean baseline value.
1uu

8$9) /+ ,#' I X&4P F^ 9)/),. is anothei timeu function test useu foi uecaues
as a clinical tiial enupoint in chiluien with BNB ages 4 anu oluei.
1u1, 1u2, 1uS
It is
easily obtaineu in the clinic anu ieliable in youngei chiluien.
1u4
The velocity of
the 1u metei iun walk incieases in BNB up to age seven but not to the same
extent as seen in typically ueveloping chiluien. It is ieliably assesseu anu
valiuateu with othei enupoints. It is pieuictive of futuie loss of ambulation. A
change of on the oiuei of seveial seconus oi less has been shown to be clinically
meaningful.
1uS, 1u6

8$9) /+ (4$9V N_./&$,. is a timeu function test that iepiesents staii climbing
ability - a clinically meaningful function in anu of itself. It has been useu as an
enupoint in BNB tiials foi uecaues.
1u7, 1u8, 1u9
Staii climbing velocity impioves up
to aiounu age 7 anu then ueclines. It is pieuictive of loss of staii climbing ability,
loss of ambulation anu time to 1u% uecline in ambulatoiy capacity. Challenges
incluue stanuaiuizeu equipment at multiple sites, sensitivity to small changes
anu vaiiability that may impact sample size.
30+9)/,0 pioviues b#&'/$/&/$6) 9)&.#,). +* ./,)'7/2 anu is a biomaikei of
muscle function. Seveial measuies can be useu, incluuing isometiic fixeu oi
hanu-helu uevices oi fixeu isokinetic uevices. Nanual muscle testing (NNT) was
useu as the piimaiy outcome measuie to uemonstiate an effect in the initial
pieunisone tiial, but the outcome measuie has a laige stanuaiu ueviation.
11u

Banu helu uynamometiy is moie piactical anu continuous vaiiable, wheieas
quantitative muscle testing (QNT) iequiies expensive anu bulky equipment. The
stage of uisease anu mechanism of action of expeiimental theiapeutic neeu to be
consiueieu foi inclusion. NNT appeais less sensitive anu ieliable in compaiison
2S
with quantitative myometiy.
111
In geneial, chiluien ages S anu oluei may be
moie ieliably assesseu with myometiy. The two muscle gioups most ieliably
assesseu in chiluien with myometiy aie the knee extensois anu elbow
flexois.
112, 11S
Nyometiy may be a veiy appiopiiate measuie of efficacy foi
agents that inciease oi pieseive muscle mass. In one iecent stuuy using a
uystiophin iestoiation stiategy in S-6 yeai olus showeu impioveu myometiy
stiength values of knee extensois anu elbow flexois.
114, 11S, 116, 117, 118


82) =&/) ;9V#4&/+,0 ./&7) `*,+9 &55,+\$9&/)40 .)6)' /+ /2$,/))' 0)&,. +*
&7)aZ
The R_9$'#/) X&4P /)./ `R3c8a has been the most commonly useu piimaiy
outcome measuie in clinical uevelopment piogiams at the time this guiuance is
being wiitten. The 6-minute walk uistance (6NWB) is believeu to be a global
integiateu measuie of multiple systems involveu in walking in BNB. It coiielates
with stiiue length anu cauence ! valiuateu measuies of uisease piogiession in
BNB in longituuinal stuuies of gait pathomechanics.
119, 12u
In BNB 6NWB also
coiielates with quantitative knee extension stiength,
121
biomechanical efficiency as
measuieu by the eneigy expenuituie inuex,
122
community physical activity as
measuieu by the StepWatch" acceleiometei
12S
anu gioss motoi skills as measuieu
by the NSAA.
124, 12S
In auuition, it is a clinically meaningful measuie of uisease
piogiession
126, 127
anu a change of this magnituue also coiielates with patient-
iepoiteu measuies of health-ielateu quality of life. A ten peicent oi gieatei
ieuuction in ambulatoiy capacity ovei 12 months has been associateu with futuie
loss of ambulation, time to a peisistent 1u% ieuuction in 6NWB has been useu an
outcome in BNB in one-yeai.
128


The 6NWT uoes have some limitations, howevei. Foi instance, in youngei ambulant
boys with BNB, 6NWT peifoimance may actually impiove up until appioximately
the age of seven anu peihaps latei with cuiient stanuaiu of caie tieatment
129
. A
seconu challenge to the 6NWT is the flooi effect of losing ambulation (uefineu as
inability to walk 1u meteis). This is an invaiiant featuie of the uisease piocess.
Nost stuuies have incluueu patients who lose ambulation with values foi 6NWB
imputeu as zeio. The aggiegateu uata analysis of change in 6NWB is stiongly
influenceu by the inclusion oi exclusion of patients who lose ambulation uuiing the
tiial, which leaus to imputeu zeio values.
1Su
This impact of loss of ambulation has
been seen in laigei cohoits ovei 1-S yeais follow-up as well. In auuition, the
baseline 6NWB, using a cutoff of SSu meteis is also associateu with a change in
6NWB ovei one yeai with 48 week to thiee-yeai follow-up.
1S1, 1S2
Finally, it may be
uifficult to ioutinely peifoim the 6NWT at all potential clinical tiial sites. In such
settings, othei outcomes, such as iise time fiom the flooi, 1u metei iun walk, time
to climb 4 staiis anu othei measuies of functional peifoimance, woulu be moie
appiopiiate in ambulant boys.

B+,/2 :/&, ;9V#4&/+,0 ;..)..9)'/ `B:;;aZ 0n the new lineaiizeu 1uu-point
scale of the Noith Stai an appioximate 7 to 9 point change has been ueemeu to be
26
the minimal impoitant uiffeience.
1SS
The challenge is that a iecent multicentei tiial
uemonstiateu less than a 7-point uecline in the lineaiizeu NSAA in placebo tieateu
patients ovei 48 weeks. This may limit the iesponsiveness of the measuie foi
theiapies that stabilize uisease piogiession in 48-week uuiation tiials. Thus, the
NSAA may be moie useful in tiials of longei uuiation.

0thei useful motoi outcome measuies foi late ambulatoiy populations iecently
iuentifieu incluue: 8$9) /+ ,#' I X&4P F^ 9)/),.[ /$9) /+ (4$9V &'% %).()'% N_
./&$,., anu moie iecently iuentifieu measuies which incluue time at high step iate
anu numbei of high iate steps by &(()4),+9)/,0.
1S4


30+9)/,0C Theie has been a concein that lowei extiemity myometiy ieaches a
flooi effect in late ambulatoiy BNB patients anu that uppei extiemity myometiy
uoesn't change substantially ovei 48 weeks.
1SS
While quantitative knee extension by
myometiy uoes change in the late non-ambulant coiticosteioiu tieateu BNB patient
ovei 48 weeks by an aveiage of 1.8S pounus,
1S6
the NCIB in the late ambulant BNB
population has been shown to be 2.1 to 2.4 pounus.
1S7
0thei muscle gioups uo not
show significant changes in myometiy ovei 48 weeks in the late ambulatoiy BNB
population.

82) '+'_&9V#4&'/ 5+5#4&/$+'
This is a population in which outcome measuies will neeu to be valiuateu foi futuie
theiapeutic tiials. A numbei of instiuments aie at vaiious stages of uevelopment.
These incluue, the Peifoimance of 0ppei Limb Scale (P0L)
1S8, 1S9
which focuses on
the continuum of functionality anu on basic functional woikspace anu is an
appiopiiate uppei extiemity measuie P0L; the `Notion & Function Assessment
Tool' (NFAsT)
14u
that uses the Niciosoft Kinect Sensoi, a low cost methou of
assessing an inuiviuual's S-uimensional functional woikspace (specifically uesigneu
foi BNB, with a conceptual fiamewoik ieflecting the piogiession of weakness anu
natuial histoiy of functional uecline in BNB); quantitative stiength testing (pinch
test, giip test, elbow extensois, elbow flexois, etc.); quantitative measuie of
ieachable woikspace which measuies shouluei movement; quantification of elbow,
wiist anu uigit stiength movement; the nine-whole peg test,
141
anu the Egen
classification (EK) scale.
142


In auuition, the Notoi Function Neasuie (NFN),
14S
a geneial scale uevelopeu foi use
acioss multiple neuiomusculai uisoiueis has been useu in BNB. Although it is
suitable foi multicentei tiials anu has auequate ieliability, sponsois shoulu consiuei
if the available uata foi compaiison with othei measuies, the extent of natuial
histoiy in BNB compaieu with othei measuies, anu whethei the eviuence, which
shows its sensitivity to change anu coiielation with clinical meaningfulness, aie
acceptable foi use.

In shoit, iathei than a uefineu set of outcome measuie that a sponsoi must use,
theie is a laige toolbox of potential outcome measuies anu enupoints fiom which
27
sponsois may choose. It is the sponsoi's iesponsibility to ueteimine the
appiopiiateness of the outcome measuie anu enupoint foi the investigational uiug's
mechanism of action, anu patient population as measuieu by age anu functional
status.

As this is a iapiuly evolving aiea anu is continuing to evolve, it is expecteu that the
sponsois will use the most up-to-uate available uata foi any outcome measuie to
ensuie that they aie suitable in the ielevant BNB population foi each tiial.

Bowevei, theie iemains a neeu to uevelop tiansitional measuies that can seive as a
biiuge acioss uiffeient age gioups, anu sponsois aie encouiageu to woik with the
FBA, the BNB community anu its scientific auvisois to uevelop novel enupoints (see
below).

L#49+'&,0 ?#/(+9) 3)&.#,). &'% >'%5+$'/.
Pulmonaiy outcome measuies can be uiviueu into measuies of stiength, clinical
measuies of iestiictive lung uisease (which aie pieuictive of the neeu foi pulmonaiy
inteiventions), anu measuies of cough function.

Neasuies of stiength: The maximum inspiiatoiy piessuie (NIP) anu the
maximum expiiatoiy piessuie (NEP) aie stanuaiu clinical measuies that assess
muscle stiength. NIP is laigely the function of uiaphiagmatic stiength, wheie
NEP is moie ieflective of the stiength of the iectus abuominis anu oblique
muscles (anu to a lessei extent the inteicostals). These measuies aie ielatively
inuepenuent of chest wall compliance anu lung function.

Clinical measuies of iestiictive lung uisease: Foiceu vital Capacity (FvC) is a
global measuie of lung function anu capacity. In BNB, its clinical utility ueiives
fiom thiesholus of uiminisheu function, which uictate consiueiation of
inteivention.

Neasuies of cough function: Pulmonaiy management in neuiomusculai uiseases
has laigely been uiiven by finuings in moie common auult uiseases. Neasuies of
cough abilities such as the peak cough flow anu peak expiiatoiy flow iate have
been useu in these othei contexts. Theie aie positive uata pieuicting that
measuiing peak flow is a potentially useful measuie that coiielates with quality
of life in othei neuiomusculai uiseases although it may be uifficult to use
acioss all populations in BNB. It has been useu in BNB, but without
contempoiaiy publisheu natuial histoiy uata, anu thus coulu be consiueieu
exploiatoiy pulmonaiy biomaikei to measuie in BNB. Clinical inteivention with
mechanical insuflationexsuflation uevice use in BNB is uiiven by a thiesholu
initially iuentifieu by clinical expeiience anu ieinfoiceu in subsequent consensus
statements.
144, 14S, 146


28
Chest wall compliance anu intiinsic lung function may impact peak flow
measuies. 0se of concomitant meuical theiapies, incluuing N-IE uevices anu
potentially even chest PT may influence peak flow measuies. Sponsois shoulu
consiuei these potential confounuing influences in measuiements of pulmonaiy
enupoints.

A&,%$&( >'%5+$'/.
Caiuiac measuies in BNB aie evolving. Bistoiically, echocaiuiogiams have been
useu to assess heait health in BNB if the echocaiuiogiam lookeu noimal, it was
assumeu that the patient with BNB was not at gieat iisk of caiuiac events. But
echocaiuiogiam is piimaiily a safety measuie. 0thei measuies aie neeueu to
monitoi tieatment iesponse.

Peuiatiic caiuiologists have begun to look at caiuio-myopathies with caiuiac NRI. In
Buchenne, caiuiac NRI has been useu to show that uamage to the heait begins quite
eaily. Nyocaiuial fibiosis may be obseiveu in 17% of 6-7-yeai olu boys with
BNB.
147


Selection of caiuiac outcome measuies foi clinical tiials is howevei complicateu by
the limiteu natuial histoiy uata on caiuiac uisease in Buchenne anu the non-lineai
ielationship between uecieaseu caiuiac capacity anu actual caiuiac events.
148


Tiauitional echocaiuiogiaphy - measuies of Lv function (EF, uimensions,
volumes) typically iemain ielatively stable until eaily teenage yeais, then
begins to uecline.
Caiuiac NRI: Neasuies of wall stiain have emeigeu as eailiei maikeis of
caiuiac uysfunction in boys with BNB. Beclines in these maikeis can be
uetecteu eailiei while LvEF by echo aie stable. Late gauolinium
enhancement to uetect aieas of caiuiac fibiosis has uetecteu subclinical
caiuiomyopathy in young boys with BNB even eailiei than changes in
ventiiculai wall motion oi function aie uetecteu
ECu: Abnoimalities in ECu also eviuent much eailiei than changes in
ventiiculai function oi uimensions as measuieu by echocaiuiogiam.
Whethei these ECu changes aie haibingeis of latei caiuiac uysfunction oi
aie moie inuepenuent effects of the uystiophinopathy is not completely
unueistoou

At this point in time, given the lack of natuial histoiy, theie is no justification at
piesent to use any specific caiuiac biomaikei foi acceleiateu appioval because
theie aie no uata in a laige enough cohoit that woulu give iegulatois confiuence
that a shift as a biomaikei woulu pieuict clinical impiovement.

Implications foi clinical tiial uesign, sponsois shoulu auuiess:
Requiiements foi backgiounu piophylactic caiuiac meuications at baseline
Caieful iecoiuing of baseline use anu any change uuiing tiial
29
Recommenuations aiounu minimal iequiiements foi caiuiac monitoiing in
tiial uesign
o S0C echo pei caie consiueiations
o ECus thioughout tiial: Coiiecteu QT inteival assessments neeu to be
inuiviuualizeu to the NoA of a uiug. Sponsois can stanuaiuize this
with supplying machines anu cential ieauing sites anu foimulate.
Bepenus upon the negotiation with the agency anu the NoA. A piotein
theiapeutic may not neeu it oi it may only be useful in oluei
populations.
o Foi phaimacological inteiventions influencing potentially ielevant
pathways, oi uystiophin-iestoiing theiapies (applicable to exon-
skipping, etc..) - consiuei moie sensitive measuies foi uetection of
eailiei ueclines in caiuiac function even if as exploiatoiy measuies
only (e.g., wall stiain measuies by caiuiac NRI oi speckle tiacking by
echo).

L&/$)'/ D)5+,/)% ?#/(+9). `LD?.a $' 131
A numbei of PR0s with existing longituuinal uata have been useu oi aie being
evaluateu in BNB. Bowevei, at the time of wiiting, none have been valiuateu
accoiuing to FBA guiuance |see Patient-Repoiteu 0utcome Neasuies: 0se in
Neuical Piouuct Bevelopment to Suppoit Labeling Claimsj that uetails the
methouology in valiuating PR0 instiuments in the uisease of inteiest.

Sponsois aie encouiageu to woik with acauemic anu patient communities to
uevelop a PR0, accoiuing to FBA guiuance (see uuiuance foi Inuustiy Patient-
Repoiteu 0utcome Neasuies: 0se in Neuical Piouuct Bevelopment to Suppoit
Labeling Claims), which encompasses as laige a spectium of the BNB population as
possible. Bealth-ielateu quality of life (Q0L) instiuments that aie BNB-specific
coulu also be uevelopeu, although if Q0L instiuments aie valiuateu in BNB, it
becomes unnecessaiy. Also, at the time of wiiting, theie aie few tools that look at
caiegivei buiuen in BNB to auuiess how tieatment oi lack of tieatment affects the
family; howevei, this may be an impoitant aie of exploiation foi a sponsoi to
consiuei.

A biief list of the PR0s that aie closest to being valiuateu:
The Peuiatiic 0utcome Bata Collection Instiument oi P0BCI, which has
seveial uomains that measuie functional ability like putting on off coat.
PR0N
PeusQL: Note, this scale moves veiy slowly in time, anu may not be suitable
foi iegistiation stuuies.
NN Nouule
The NeuioQol contains an extensive set of measuies that aie valiuateu foi
neuiological uisoiueis. http:www.neuioqol.oigPagesuefault.aspx
PR0N foi uppei limb

Su
1)6)4+59)'/ +* ;%%$/$+'&4 B+6)4 >'%5+$'/. $' 131
Sponsois looking foi novel enupoints in these populations coulu use stanuaiuizeu
instiuments that aie useu to assess function anu uemonstiate clinical
meaningfulness in a vaiiety of tools that may alieauy be useu in clinical caie.

In auuition, sponsois aie encouiageu to exploie novel composite outcomes in
uevelopment that assess piogiession of BNB acioss the spectium of uisease (i.e.,
combine components of scales such as the NoithStai with uppei limb functional
measuies), similai to the NFN constiuct, anu uemonstiate auequate sensitivity to
change in BNB.

A numbei of novel measuies of uppei limb function coulu be consiueieu in non-
ambulant populations, such as the '9-hole peg' test, which coiielates with othei
measuies, fingei tapping anu pinchgiip stiength anu the Nyouiip, NyoPinch anu
NoviPlate as measuies of uistal function.
149


Foi oluei non-ambulant boys, the case can be maue foi fingei tapping as a
function that allows a young man to contiol a computei mouse, is a clinically
meaningful function. Some of the PR0's also incluue measuies of uistal function.
Anothei exploiatoiy measuie being exploieu in natuial histoiy stuuies is typing
time. Bowevei, the change in these measuies ovei time is usually slow, anu tiials
may neeu to follow patients foi a long peiiou of time (peihaps in an extension phase
of a tiial, oi as pait of a post-maiketing commitment).

Figuie 2
1Su, 1S1

S1

1C A4$'$(&4 8,$&4.
A%6+-'-8-3: -++4%+ 9() 3)-68+ -* /</> 01% 8-5-3+ (9 )6)% 7-+%6+% 6*7 5%7-,68
677)%++6'-8-3: ': %*72(-*3 B2((8 (9 26)3-,-26*3+ 6*7 3)-68 +-3%+C
Sponsois aie encouiageu to gathei safety anu efficacy uata of new uiugs in BNB
patients acioss the spectium of uisease. Sponsois may choose to uevelop sepaiate
piotocols oi may consiuei moie novel tiial uesigns encompassing moie than one
patient segment to collect this uata.

8,$&4 1).$7'
Theie is wiuespieau suppoit in the BNB community to move away fiom placebo-
contiols. While the most iobust uata come fiom placebo-contiolleu tiials, othei tiial
uesigns without placebo contiols may be consiueieu if ciicumstances waiiant.

In such cases, well-matcheu (age-matcheu anu stage-matcheu) natuial histoiy
contiols woulu be acceptable. The challenge is to finu those matches foi each tiial
paiticipant, fiom natuial histoiy uata. Natuial histoiy will veiy heavily uepenu upon
the stanuaiu of caie being applieu.

A uangei, howevei, is that the likelihoou foi iesults that aie uifficult to inteipiet
with using natuial histoiy contiols is substantially gieatei than in ianuomizeu
placebo contiolleu tiials.

We also iecognize that most sponsois aie ieluctant to puisue piolongeu clinical
uevelopment piogiams, anu theie aie also conceins about tying up paiticipants in a
clinical tiial of a piouuct that, in the enu, might not be efficacious.

It is the sponsoi's iesponsibility to be familiai with the most cuiient available uata
to unueistanu the natuial histoiies in BNB incluuing natuial histoiy as it is
mouifieu by available theiapy, anu to upuate as appiopiiate foi theii tiial uesign.
We encouiage sponsois to uiscuss any plans to use natuial histoiy contiols with the
agency.

When a placebo-contiolleu tiial is peifoimeu, sponsois aie encouiageu to consiuei
clinical tiial uesigns that inciease access to patients acioss the entiie spectium of
uisease anu that limit exposuie to placebo. These uesigns will vaiy uepenuing upon
the uiug's mechanism of action, anu the sensitivity of the specific outcomes being
measuieu.

In auuition, the focus on 6NWT as the piimaiy enupoint foi stuuies in BNB has
limiteu the access to anu paiticipation in the clinical tiial piocess as sponsois have
iestiicteu the inclusion ciiteiia foi tiials to specific 6NWB ianges likely to uecline
but not lose ambulation.

S2
Sponsois shoulu use a single piimaiy enupoint in tiial uesigns, along with
appiopiiate seconuaiy enupoints. The piimaiy enupoint may be eithei a clinical
outcome oi a phaimacouynamic biomaikei uepenuing upon the puipose of the tiial.

Categoiical enupoints - whilst statistically stiong - may not be in the patient-
community's best inteiest in BNB because the functions lost ovei the couise of a
tiial aie iiieveisible. Thus, the focus shoulu be on ueveloping moie sensitive,
uynamic enupoints that aie eithei uemonstiateu by natuial histoiy uata to be
clinically meaningful oi to be suiiogate enupoints.

As the leauing cause of ueath in BNB, it is ciitical that sponsois shoulu consiuei
using appiopiiate tools to measuie safety anu efficacy of compounus in the
myocaiuia in all populations with BNB.

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In oiuei to meet the expectation of the BNB community to ieuuce exposuie to
placebo, novel tiial uesigns, incluuing foi instance, uelayeu placebo oi iapiu ioll
ovei tiials (aftei patients ieach an non-categoiical enupoint) shoulu be exploieu.

0ne appioach to minimize the time off uiug in between tiials in BNB stuuies woulu
be to plan to pioceeu uiiectly fiom uose escalation stuuies into clinical efficacy
stuuies once a uose has been selecteu.

01$&4)! L41" ;#8) 1"/+ #+) ):9)84;)+1/% 1")8/92
uuiuance on conuucting stuuies with moie than one new moleculai entity has
pieviously been publisheu
|http:www.fua.govuownloausBiugsuuiuanceComplianceRegulatoiyInfoimatio
nuuiuances0CN2S6669.pufj.

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Because of the paucity of uata in BNB (anu many othei) iaie uiseases, making uata
that is collecteu in one tiial be available foi use in anothei tiial - coulu be ciitical foi
the entiie fielu.

To whatevei extent possible, sponsois aie encouiageu to haimonize how clinical
tiial outcomes aie measuieu, incluuing, potentially, the fiequency with which those
measuies aie useu foi efficacy - to allow inteipietability acioss tiials anu acioss
sites. It is unueistoou howevei, that the ability to uo so might be limiteu by the
uiug's mechanism of action anu phaimacouynamics.

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7! 04+5%) M84;/82 N$1-#;) ()/!$8)!
The appioval of a uiug foi the tieatment of BNB baseu on the use of a biomaikei as
a single piimaiy suiiogate efficacy measuie can be consiueieu unuei acceleiateu
appioval. As uesciibeu in the biomaikeis chaptei that follows, at the piesent time,
theie is a gieat ueal of ieseaich inteiest in unueistanuing the iole of biomaikeis in
SS
BNB. Some of these biomaikeis appeai to be phaimacouynamics maikeis that may
be useful foi pioof of concept anu uose selection. If the effect on a paiticulai
biomaikei is ieasonably likely to pieuict clinical benefit, the FBA woulu consiuei an
acceleiateu appioval baseu on the use of a biomaikei as a suiiogate outcome
measuie in BNB (see Subpait B--Acceleiateu Appioval of New Biugs foi Seiious oi
Life-Thieatening Illnesses anu the uuiuance Expeuiteu Piogiams foi Seiious
Conuitions--Biugs anu Biologics).

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We aie also open to consiueiing the aigument that a positive biomaikei iesult
(geneially incluueu as a seconuaiy outcome measuie in a tiial) in combination with
a positive finuing on a piimaiy clinical outcome measuie may suppoit a claim of
uisease mouification in BNB. Theie is wiuespieau eviuence-baseu agieement in the
ieseaich community that foi instance, that the absence of uystiophin is funuamental
to the unueilying uisease piocess in BNB. The status of appioaches to measuie it
aie uiscusseu in the biomaikeis section as aie othei biomaikeis that have
potential as suiiogate outcome measuies such as skeletal muscle NRI. We
encouiage sponsois to analyze the iesults of these biomaikeis inuepenuently with
the unueistanuing that these finuings will be inteipieteu in the context of the state
of the scientific eviuence at the time of a futuie new uiug application oi biologics
license application submission.

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Extiapolating uata of efficacy fiom one stage of uisease to anothei coulu uepenu
upon the mechanism of action. Bowevei, uiugs uesigneu to impiove the quality anu
health of the muscle woulu be expecteu to benefit the patients with BNB at any
stage of the uisease anu thus, it may not be necessaiy to test a uiug at eveiy stage
of the uisease to justify a bioauei inuication. Neveitheless, having some seconuaiy
enupoint uata showing an effect in othei uisease stages coulu help suppoit a
bioauei inuication.

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;C ")'),&4 A+99)'/.
The FBA shaies the Buchenne community's goal to uevelop biomaikeis anu
suiiogate enupoints that coulu pioviue veiy iapiu infoimation anu uata with iegaiu
to the signal of a uiug anu whethei theie is biological activity that coulu piove
piomising in teims of alteiing the uisease couise.

Sponsois shoulu be awaie that biomaikei uevelopment is an aspect of BNB that is
iapiuly evolving. They shoulu consiuei the inclusion of some of the biomaikeis
uesciibeu in this section in theii clinical uevelopment piogiams as enupoints oi foi
planneu post-hoc analyses, as the finuings may help suppoit an NBA foi theii leau
piouuct in uevelopment anu by helping move the fielu foiwaiu towaius a
consensus on the utility of a biomaikei may ieuuce costs anu speeu the time
iequiieu foi the uevelopment of subsequent piouucts.

S4
This section uesciibes a numbei of biomaikeis that may have piognostic oi
pieuictive values (with value in foiecasting the patient's piognosis, oi likelihoou to
benefit fiom a paiticulai tieatment). Noie attention is given to phaimacouynamic
biomaikeis, which pioviue an inuication of iesponse aftei being tieateu (sometimes
in ielation to pie-tieatment values) anu which may have potential as suiiogate
enupoint that coulu substitute foi a clinical enupoint.

The pieuiction is baseu on epiuemiologic uata, theiapeutic, pathophysiologic oi
some othei scientific eviuence. ! #$%%&'()* *+,-&.+) /.&0(%1*% !" !"#$"%$% #'
!"#!$%$"$& !"# !"# %&'!"#$% '!()*"!+, -."/ +0)' *1 2"*3$45'4 )4*6"('/ '$4%0 $!(
!""#$!%& ($&)*"%*+, +- .+%/0 ! #$%&%#!$ '&()*%&+ ,'&'-%+ .*/ 0!/1 */ $!#2 *- ,'&'-%+ */
!"#$% anu the !""!#$% '" $(!)$*!+$ '+ $,-% .-'*)(/!(0 A 'holistic evaluation' of
available uata (epiuemiologic uata, uata fiom clinical tiials, pathophysiologic oi
some othei scientific eviuence) uemonstiates that a biomaikei can substitute foi a
clinical enupoint.
1S2
The uesignation of suiiogate enupoint iequiies agieement with
iegulatoiy authoiities.

Suiiogate enupoint maikeis can also be the piimaiy enupoint in 'auequate anu
well-contiolleu stuuies if theie is a well-establisheu ielationship between the
suiiogate maikei anu clinical outcome then that tiial can be useu to pioviue
eviuence foi conventional maiketing appioval. If on the othei hanu, theie is not a
well-establisheu ielationship between the suiiogate maikei anu clinical outcome
but it is 'ieasonably likely' to pieuict a clinical outcome, then a positive effect on the
suiiogate enupoint coulu leau to an acceleiateu appioval.

In BNB, biomaikeis that faithfully iepoit on both the health anu amount of skeletal
muscle may potentially be useful at uiffeient stages of the clinical tiial piocess as
piognostic, pieuictive, oi phaimacouynamic biomaikeis.

The chaptei is split into two sections: one looking at biomaikeis founu in muscle
tissue. The biopsy-baseu biomaikeis useu to uate in BNB tiials - uystiophin anu
utiophin - aie wiuely accepteu by the scientific fielu as appiopiiate
phaimacouynamic biomaikeis foi theiapies whose mechanism of action is uiiecteu
towaiu theii expiession, as they may confiim the mechanism of action anu be useful
foi selecting uoses in subsequent tiials, anu (in the case of uystiophin in paiticulai),
fiom natuial histoiy stuuies, it is cleai that patients who expiess uystiophin (BNB)
uo bettei than BNB patients, anu even BNB patients who expiess veiy low
uystiophin levels appeai to have a slowei uisease piogiession.
1SS
At the time of this
wiiting, uata aie being analyzeu iegaiuing the potential of measuies of uystiophin
as a pieuictive anu suiiogate enupoint that coulu suppoit applications foi one of the
expeuiteu appioval piocesses. Biopsy-baseu biomaikeis may nonetheless be
unattiactive foi use in laige phase III stuuies uue to the invasive collection methou.

The seconu section looks at less invasive methouologies to measuie changes in the
muscle anu new milieu. Some of these, such as measuies of pioteins, piotein
fiagments anu genetic mateiials in the bloou oi uiine aie exploiatoiy but woith
SS
gieatei investments uue to the ease with which they can be measuieu. Some
imaging techniques aie much fuithei along. In paiticulai, the uiiect imaging of
skeletal muscle using NR techniques has the potential to seive as an efficacy-
iesponse biomaikei anu suiiogate enupoint.

Sponsois shoulu be awaie that scientific consensus iegaiuing the utility of any of
the exploiatoiy biomaikeis may have been ieacheu since the time of wiiting anu
shoulu uiscuss this with the FBA. In the meantime, howevei, eviuence of an effect on
an exploiatoiy biomaikei coulu pioviue suppoitive eviuence foi a claim of uisease
mouification in an NBA. When combineu with some othei eviuence suggestive of
clinical benefit, sponsois coulu help establish the use of a biomaikei as a suiiogate
enupoint.

<C 3#.(4) <$+5.0 <$+9&,P),.Z 10./,+52$' ;'% E/,+52$'
")'),&4 (+99)'/.
Bystiophin oiganizes anu stabilizes the saicolemma to effectively uistiibute
contiactile foices anu maintain myofibei stiuctuial integiity anu function.
Bystiophin is also a scaffoluing piotein necessaiy foi the piopei localization (anu
thus function) of signaling molecules such as neuional Nitiic 0xiue Synthase
(nN0S). The use of uystiophin in muscle as a uiagnostic (anu piognostic biomaikei)
has alieauy been uiscusseu in the uiagnosis chaptei.

The accuiate quantification of uystiophin oi utiophin in muscle tissue can pioviue
impoitant suppoit foi the clinical uevelopment of uystiophin- oi utiophin-
iestoiative theiapies. Eviuence of a significant effect on uystiophin levels, foi
instance, coulu pioviue pioof of concept that a uystiophin-iestoiative theiapy uoes
in fact inciease uystiophin piouuction.

Bowevei, to measuie eithei uystiophin oi utiophin content in the taiget muscle
accuiately, one must fiist auuiess specimen collection how the muscles aie
biopsieu. In auuition to technical issues ielateu to how the muscle sample is
collecteu anu piocesseu, theie aie consiueiations iegaiuing the ethics anu ieliability
of the piocess.

A+'.$%),&/$+'. ,)4&/)% /+ 9#.(4) V$+5.$).
Sponsois shoulu be awaie of consiueiations ielateu to the measuiement of a
biomaikei in a muscle biopsy foi instance, inteipieting the ielative quantification
of uystiophin with the tools available touay iequiies pie-tieatment self-patient
contiols. Sponsois shoulu consiuei issues ielateu to specimen collection, hanuling,
anu laboiatoiy piactice anu take caie to minimize sampling eiiois.

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Sponsois shoulu be awaie that theie aie ethical issues ielateu to peifoiming
multiple muscle biopsies in patients with a uegeneiative neuiomusculai uisoiuei.
Beaiing in minu the invasiveness of the pioceuuie, sponsois shoulu consiuei
whethei biopsies aie absolutely necessaiy when planning a clinical tiial in oiuei to
S6
minimize theii use. If a biopsy is iequiieu, the gieatest caie shoulu be taken make
ceitain that the biopsy pioviues a useful specimen anu that the timing post
tieatment is appiopiiate. Tiials peifoiming muscle biopsies shoulu commit to
timely feeuback about the biopsy analysis to the tiial paiticipants, anu BNB
community.

A,$/),$& +* X2&/ $. &' &55,+5,$&/) V$+5.0 *+, %0./,+52$' +, #/,+52$'
b#&'/$*$(&/$+'
D-3% (9 '-(2+:E54+,8% .)(42> At baseline, the amount of uystiophin vaiies by
uonoi, mutation, anu muscle gioup. Foi this ieason, baseline muscle biopsies aie
essential in uocumenting changes inuuceu by novel theiapeutic agents taigeting
uystiophin oi utiophin. Beteimination of uystiophin oi utiophin content uepenus
on the methou utilizeu, anu the uenominatoi useu (e.g. total piotein oi RNA content,
myofibiillai piotein content, unit membiane aiea). Nany BNB patients show
ieveitant fibeis (enuogenous clonal exon skipping), vaiying by mutation anu muscle
gioup. The biopsy sites shoulu be chosen to maximize the infoimation on
uystiophin oi utiophin expiession pie-anu post-tieatment.

Note: uystiophin levels vaiy between uiffeient muscle tissues (so iueally the
iefeience healthy sample shoulu be fiom the same muscle as the tiial biopsy).

01% 16*78-*. (9 31% '-(2+:> 3"4! ;)1"#&#%#52 8)98)!)+1! /58))& $9#+ 5##& 98/-14-)
/1 1") 14;) #A L8414+5 1"4! &#-$;)+1Q R#L),)8D !9#+!#8! /8) )+-#$8/5)& 1# $14%4S) 1")
<)!1 -$88)+1 ;)1"#&#%#52 /1 1") 14;) #A -#+&$-14+5 1")48 184/%Q
TUF

Tissues shoulu be flashsnap fiozen in isopentane cooleu in liquiu nitiogen
soon aftei suigeiy.
Caie shoulu be taken to avoiu the use of tissue-embeuuing meuia that
compiomise biochemical analyses involving gel electiophoiesis
(immunoblots, mass spec).
Flash-fiozen tissues shoulu be stoieu in pie-chilleu (uiy ice), small aiitight
sciew top tubes. Byuiation of the containei (incluuing ice fiozen in bottom of
tube) may pievent uessication aitifact (fieeze uiying) with extenueu stoiage.
Shipment anu tianspoit with tempeiatuie monitoiing of biopsies fiom
clinical sites to laboiatoiy of analysis. uieat caie shoulu be maue in selecting
the couiiei confiiming theii expeitise in low tempeiatuie contiolleu
shipments anu have significant uemonstiable histoiy of shipping clinical
mateiials.
Samples must not be alloweu to thaw at any point, as fieeze-thaw cycles
ueciease intact uystiophin oi utiophin content as an aitifact.
Lab qualification issues: Sponsois shoulu only utilize laboiatoiies that aie
qualifieu to hanule muscle biopsies.

<-*-5-F-*. @6)-6'-8-3: 6*7 +6528-*. %))()+
Sponsois shoulu be awaie that a potential limitation of muscle biopsies anu
quantitation of uystiophin oi othei myofibei pioteins can be the age-ielateu
S7
ieplacement of muscle with vaiiable fibiosis anu fat in BNB patients.
Bystiophin is only expiesseu in myofibeis, anu the giauual age-ielateu loss
of myofibeis in BNB patient muscle complicates the inteipietation of
uystiophin iescue. Bowevei, the intent of the above mentioneu pioceuuies
aie to help minimize such complications.
Some expeits have pioposeu the use of imaging to guiue the biopsy to make
suie that the specimen contains an auequate sample of myofibeis iathei than
fibiotic tissue.
Nuscle biopsies fiom both Beckei musculai uystiophy anu female BNB
caiiiei patients anu BNB patients often show vaiiability in expiession of
uystiophin both in neighboiing myofibeis, between uiffeient iegions of the
same biopsy, anu between uiffeient biopsies. Bistopathology can also be
vaiiable within these same biopsies.
Theiefoie, quantification of uystiophin expiession in BNB biopsies iequiies
iigoious piotocols with auequate contiols, extiemely caieful sample
hanuling, anu caieful examination of a laige numbei of fielus with myofibei
counting anu giauing by expeiienceu pathologists oi ieaueis blinueu to the
tieatment assignment of the patients. Similai issues with vaiiability in
utiophin staining shoulu also be expecteu uue to uiffeiing iegions of
myofibei iegeneiation.
Sponsois shoulu be familiai with the most cuiient methous to minimize
vaiiability anu sampling eiiois when evaluating biochemical efficacy in
clinical tiials.

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<,+&%40 %$..)9$'&/)% /)(2'$b#).
At piesent, the two most commonly useu methous to quantify uystiophin aie
immunofluoiescence oi immunohistochemical analysis anu Westein Blot.
Immunofluoiescence can be useu to ueteimine the peicentage of muscle fibeis that
expiess uystiophin anu the levels at which uystiophin is expiesseu in these fibeis.
Westein Blot can show the total amount of uystiophin in the specimen anu its size.
The methous aie complementaiy anu piotocols that allow stanuaiuization of the
methouologies acioss laboiatoiies have also now been publisheu. While neithei
technique pioviues a complete account of uystiophin iestoiation, both methous can
show incieases of uystiophin expiession ovei baseline.

-99#'+*4#+,).()'() +, $99#'+2$./+(2)9$(&4 &'&40.$. V0 /05): many
pathology laboiatoiies ioutinely employ this methou. Bowevei, the necessaiy
methous to quantitate uystiophin in a mannei able to suppoit clinical tiials anu
uiug uevelopment aie not bioauly uisseminateu. 01/+&/8&4S/14#+ #A
4;;$+#A%$#8)!-)+-) ;)1"#&! /-8#!! %/<#8/1#84)! "/! +#1 <))+ L4&)%2 )!1/<%4!")& 2)1Q
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S8
L)!1)8+ <%#114+5Q |Publication penuingj Immunostaining quantitation of ielative
uystiophin levels shoulu be uone by specific iefeiial laboiatoiies with extensive
uocumenteu expeiience with uystiophin quantitation methous anu uemonstiateu
iepiouucibility (intia assay (between sections) of a biopsy anu intei assay piecision
between expeiiments).

c)./),' <4+/: Westein Blot is a stanuaiu methou of quantifying the amount anu
size of a piotein. Bowevei, it neeus to be iecognizeu that uystiophin is a laige
moleculai weight (427kB), low abunuance piotein. Theie aie fiequently
encounteieu technical challenges with consistency anu ieliability of multiple steps
of the piotocol, incluuing solubilization, electiophoiesis, tiansfei (blotting),
immunouetection, anu quantitation.

Nethouology: Piotein solubilization. 0ne fiequently publisheu methou is to use
ciyosections (lacking any embeuuing meuia; 2u-Su 1u micion) collecteu in pie-
chilleu small tubes, with iapiu solubilization in low volume high SBS buffei,
immeuiate electiophoiesis on giauient Tiis-acetate gels, anu noimalization of
uystiophin content to myofibei pioteins in the same blots oi post-tiansfei gels.

01/+&/8&4S/14#+B M8#1#-#%! 1# !1/+&/8&4S) 4;;$+#<%#114+5 A#8 &2!18#9"4+ /-8#!!
%/<#8/1#84)! /8) /%!# 9$<%4!")& 4+ 7+1"#+2 )1 /%Q |Publication penuingj

=5%).-*. 3%,1*(8(.-%+
3&.. .5)(/,+9)/,0: Nass spectiometiy methous show potential auvantages of
high ieliability accuiacy, anu sensitivity. Nass spectiometiy methous typically
iequiie the auuition of stable isotope labeleu peptiues to the solubilizeu human
muscle sample. 0ne iecently iepoiteu exploiatoiy methou uses stable isotope
labeleu mouse muscle mixeu with human muscle biopsy samples, leauing to highly
accuiate anu ieliable quantitation of uystiophin ovei a laige uynamic iange.
1S6
This
methou iequiies that the BNB patient uystiophin being analyzeu be of a similai
moleculai weight as noimal uystiophin (e.g. this methou is not applicable to the
mini- oi micio-uystiophin constiucts utilizeu in gene theiapy appioaches).

Benchmaiking to immunoblot anu immunostaining has been uone in pie-clinical
tiials of exon skipping, anu has shown concoiuance between all methous.
1S7
The
majoi uistinctions aie that the ieliability of the mass spec methou appeais
consiueiably bettei than immunoblotting oi immunostaining, uue to the many
multiple quantitative measuies (peptiues) pei test, anu the high iesolution anu
quantitative piecision of the mass spectiometeis.

0se as a piimaiy uystiophin measuie: At the time of wiiting, theie have yet to be
publisheu iepoits using mass spectiometiy appioaches in the context of a
uystiophin ieplacement clinical tiial, which woulu be necessaiy to valiuate the
methouology.

!4))%*3 8-5-363-(*+ 9() 688 5%31(7+
S9
All cuiiently useu anu uevelopeu methous allow only ielative quantitation anu not
absolute quantification of uystiophin levels. Since uystiophin levels vaiy between
healthy inuiviuuals, using the same contiol iefeience sample is necessaiy to
extiapolate ielative quantitation.

Y)A)8)+-) 8/+5)! /+& #$1%4)8!
Bystiophin anu exon skipping shoulu be compaieu within an inuiviuual using the
same muscle gioups in a pie anu post tieatment biopsy. Bystiophin quantification
must be uone in a blinueu mannei by compaiison to a baseline biopsy in oiuei to be
valiu.

As methouologies foi uystiophin quantitation aie iefineu anu wiuely accepteu, it
shoulu become feasible foi uystiophin quantitation to be piesenteu as a peicentage
of noimal contiol muscle samples, analyzeu in paiallel. Sponsois aie encouiageu to
consiuei methouologies that allow foi stanuaiuization. Iueally, the peicentage of
positive fibeis as well as the ielative uystiophin levels shoulu be assesseu.

G+% (9 7:+3)(21-* H46*3-9-,63-(* () )%863-@% H46*3-9-,63-(* 6+ 6 '-(,1%5-,68
(43,(5% 5%6+4)%
The amount of uystiophin iestoiation necessaiy to achieve clinical benefit is
uncleai at piesent, anu may uepenu upon the uisease stage at tieatment initiation
anu statehealth fiagility of the muscle.

It has been establisheu that uystiophin levels coiielate with the piognosis seen in
female BNB caiiieis
1S8
(noimal uystiophin
1S9
), anu in male Beckei musculai
uystiophy (abnoimal but at least paitially functional uystiophin).
16u
While the
amount of uystiophin iestoiation that can be achieveu theiapeutically is yet to be
seen, the bioau consensus is that similai levels of uystiophin iestoiation woulu be
likely to iesult in some clinically meaningful benefit. Bowevei, the coiielation is
unlikely to be peifect between what may be seen as a iesult of theiapeutic &) +#,#
uystiophin intiouuceu in BNB patients latei in life anu what has been iepoiteu in
female caiiieis anu patients BNB, wheie some uystiophin is piesent fiom biith. The
theiapeutic benefits of uystiophin iestoiation may uepenu upon the age at
tieatment initiation, the health of the muscle in the patient ieceiving tieatment
anuoi othei factois. Nonetheless, in a meuically auuiessable population, some
uegiee of uystiophin iestoiation is ieasonably likely to iesult in some clinical
benefit, although the effect size anu timing of clinical iesponse aie uncleai at the
time this guiuance is being wiitten.

E/,+52$' ;'&40.$.
The methouologies employeu to quantify the expiession of utiophin oi associateu
pioteins such as the saicoglycans anu nN0Ss closely miiioi those uesciibeu above
foi uystiophin.
161
Bowevei, utiophin analysis also piesents seveial unique
challenges:
The most significant complication with staining foi utiophin changes aftei
theiapeutic inteivention is the veiy high level of utiophin staining seen in
4u
BNB anu to a lessei extent, Beckei biopsies. Nost fibeis in BNB biopsies will
be utiophin positive highlighting the massive numbei of iegeneiating fibeis
seen at any one time within a biopsy. Theiefoie a combination appioach
shoulu be consiueieu both quantifying utiophin levels anu quantifying the
numbeis of iegeneiating fibeis. A utiophin mouulation appioach shoulu at
least have the same level of utiophin as the pie-biopsy but the numbeis of
iegeneiating fibeis uecieaseu ovei the theiapy uosing to confiim the
utiophin iuentifieu is uue to the uiug iathei than iegeneiation.
The utiophin abunuance in noimal skeletal muscle is 1-2% of uystiophin anu
theiefoie may only be useful as a baseline foi quantifying folu-inciease in
iesponse to up iegulation theiapies.
The use of noimal fetal muscle biopsies available fiom commeicial biobanks
will contain significantly moie utiophin with most, if not all fibeis, positive
so can be useu as a positive contiol foi staining.
Expiessionpuiification of iecombinant human utiophin is feasible anu
woulu suppoit absolute quantification by Westein blot against a stanuaiu
cuive. Bowevei it is not cleai if enuogenous anu iecombinant human
utiophin aie similaily post-tianslationally mouifieu, oi how any uiffeiences
in post-tianslational mouification may impact immunoieactivity.
0tiophin expiession is pievalent in iegeneiating skeletal muscle fibeis, the
vasculatuie anu neives of noimal skeletal muscle anu non-muscle cells of the
immune anu fibiotic iesponses to uystiophinopathy, which complicates
inteipietation of Westein Blot analysis. Immunofluoiescence analysis can
auuiess this issue.
While Westein Blot quantification foi upiegulation of othei BuC components
may pioviue some assuiance of utiophin upiegulation in skeletal muscle
fibeis, the utiophin expiesseu in iegeneiating fibeis inteiacts with BuC
components. Some of the BuCs that inteiact with utiophin aie only piesent
in muscle (e.g. alpha- anu gamma-saicoglycan anu nN0S), thus Westein
Blotting coulu give infoimation about the muscle-specific BuC.
At the time of wiiting, no utiophin-null human tissue is available foi
backgiounu coiiection eithei Westein Blot oi immunofluoiescence baseu
quantifications, although the highly iestiicteu localization of utiophin to the
neuiomusculai anu myotenuinous junctions, vasculatuie anu neives of
noimal muscle leave laige iegions of muscle sections amenable to
backgiounu coiiection.
It is unknown whethei baseline levels of utiophin aie stable in the patient,
anu the available uata suggest that it incieases with age.
162

Nass spectiometiy methous may also be applicable to quantification of
utiophin, but at the time of this wiiting, this woik is veiy pieliminaiy.

3#.(4) <$+5.0 <$+9&,P),.Z D8IDB; LAD &'&40.$. *+, )\+'_.P$55$'7 %)/)(/$+'
/+ (+'*$,9 9)(2&'$.9 +* &(/$+' $' /2) )\+'_.P$55$'7 *$)4%
Buchenne musculai uystiophy is mostly causeu by mutations in the '(' gene that
leau to a ieauing fiame shift anu piematuie tianslation teimination. Antisense
41
oligonucleotiues |A0Nsj have been uesigneu to piomote exon skipping uuiing
splicing of uystiophin pie-mRNA, iestoiing the ieauing fiame anu allowing
tianslation of inteinally tiuncateu, but functional uystiophin piotein. A commonly
useu paiametei to assess anu compaie the efficacy of vaiious antisense molecules is
the exon skipping peicentage, which is uefineu as the peicentage of tiansciipts in
which the taigeteu exon is skippeu ielative to the total numbei of uystiophin
tiansciipts (skippeu vs. non skippeu). Theie appeais to be a coiielation between
exon-skipping peicentages anu uystiophin iestoiation, taking into account that
quantification by both methous has only been achieveu by highly specializeu
centeis. Bence the measuiement of exon skipping at the RNA level is an impoitant
assessment in veiifying A0Ns' ability to successfully mouify the appiopiiate gene
taiget. Bue to the low abunuance of uystiophin mRNA, the efficacy of A0Ns to
inuuce exon skipping has pieuominately been assesseu at the tiansciiptional level
using the semi-quantitative nesteu ieveise-tiansciiption polymeiase chain ieaction
(RT-PCR) oi quantitative PCR (qPCR) with uiffeiing piotocols anu amplification
cycles.

Because of the uiffeient uynamics of tiansciipts anu pioteins, the exon skipping
levels may not uiiectly coiielate to uystiophin levels. Neveitheless, this is anothei
phaimacouynamic maikei that can confiim the whethei exon skipping has at least
occuiieu aftei tieatment with A0Ns. At the time of uiafting this uocument vaiious
gioups have been investigating to uevelop a quantitative methou to measuie exon
skipping moie accuiately.
16S,164, 16S,166

<C B+'_<$+5.0 <&.)% <$+9&,P),.
")'),&4 (+99)'/.
This sub-section ueals with two classes of exploiatoiy biomaikeis, substances that
can be measuieu in the bloou anu uiine, anu non-invasive imaging techniques. Both
classes of biomaikeis in uevelopment coulu have consiueiable auvantages ovei
muscle biopsies in that they sample laige gioups of muscles, anu thus uo not suffei
fiom the sampling eiiois that can be encounteieu with muscle biopsies, paiticulaily
if auequate caie is not taken following appiopiiate pioceuuies. Nuscle biopsies aie
appiopiiate in ciicumstances wheie the goal is to ueteimine if the theiapeutic is
acting via the intenueu taiget mechanism anu in ciicumstances wheie theie is no
othei mannei in which to ueteimine the optimal uosing. While at the time of
wiiting, we iecognize that sponsois may neeu to iely upon establisheu
methouologies in theii iegistiational stuuies, we woulu also encouiage them to
exploie the use of less invasive biomaikeis in theii clinical uevelopment piogiams.

:),#9 &'% E,$') <$+9&,P),.
Sampling bloou anu uiine in BNB may inuicate the health anu integiity of skeletal
muscles. Biomaikeis in the bloou oi uiine potentially contain signals coming not
only fiom the affecteu muscles, but also fiom othei cells involveu in iesponse to the
muscle uamage, incluuing inflammatoiy cells anu motoi neuions. The bloou anu
uiine biomaikeis that aie coming fiom skeletal muscle suffei fiom the fact that
since they ieflect the amount of skeletal muscle as well as the health anu integiity of
42
the muscles, they must be coiiecteu foi loss of skeletal muscle mass as the uisease
piogiesses. The bloou biomaikeis that have been exploieu to uate incluue both
piotein anu RNA, while uiine biomaikeis aie piimaiily metabolites.

?)(3%-*+ 6*7 2)(3%-* 9)6.5%*3+
Pioteins can measuieu by multiple methous foi 'biomaikei uiscoveiy' incluuing
immunological methous (antibouies), pioteomics methous, oi aptamei panels (e.g,
the Somalogics Platfoim). A numbei of potential piotein biomaikeis foi BNB have
been iuentifieu in human tiials. Some appeai to be similai of CK, flagging the
ueteiioiation of muscle seen in BNB. But a numbei of otheis that may pioviue
auuitional infoimation (incluuing iesponse to tieatment) have iecently been
iuentifieu.

A numbei of ongoing stuuies aie utilizing the SomaLogic platfoim, which involves
pioteomics tools, incluuing mouifieu aptamei piotein-binuing ieagents with high
affinity anu slow uissociation iates that taiget thousanus of pioteins ciitical to
biological functions as well as an assay that can simultaneously iuentify anu quantify
pioteins acioss appioximately eight logs of concentiation in small sample
volumes.
167,168,169
Since the mouifieu aptameis aie composeu of BNA, establisheu
BNA measuiement technologies can be useu to quantify them anu pioviue a ieauout
of thousanus of pioteins fiom a small amount of biological sample.
17u
A potential
auvantage of the platfoim is that it samples iaie pioteins in auuition to abunuant
pioteins. Theiefoie, it may be possible to uetect changes in ielevant pathways that
might be misseu by othei platfoims incluuing pioteins that aie inuicative of
ueneivation that aie nevei seen in the noimally functioning peei gioup.

Anothei platfoim unuei evaluation in Buchenne aie Noiuic Bioscience's fibiosis
biomaikeis assays, which iuentify specific piotein fiagments, oi 'neo-epitopes'
piouuceu when pioteins aie subject to post-tianslational mouifications (PTNs), e.g.
cleavage, glycosylation oi citiullination, that aie ielateu to uefineu
(patho)physiological piocesses uuiing moiphological ueteiioiation.
171, 172, 17S, 174

The iesulting specificity between the paient piotein anu the ielevant PTN gives iise
to mouifieu peptiues that aie associateu with specific (patho)physiological
piocesses in cancei, fibiosis, oi neuiomusculai uegeneiation.

0thei stuuies have lookeu at a iange seium oi uiine piotein anu piotein fiagments
in BNB incluue Natiix metallopeptiuase 9 (NNP-9) in BNB
17S
, fibionectin
176
D
muscle piotein fiagments in seium anu uiineD succinate in ;&:D piostaglanuin B2,
177

anuS-methyl-L-histiuine
178
. Some pioteins may be maikeis of the uisease iepaii
piocess anu tissue iemoueling.

At the time of wiiting this guiuance, all of these biomaikeis aie exploiatoiy,
howevei, sponsois aie encouiageu to scieen foi these potential biomaikeis in
longituuinal stuuies. With such uata, the potential utility of some of these maikeis to
monitoi tieatment iesponse may become moie appaient.

4S
<-,)(I$J+
NicioRNAs (miRs) aie shoit (~22 nucleotiue) RNA molecules that function in the
post-tiansciiptional iegulation of gene expiession by inuucing mRNA uegiauation
oi tianslational inhibition. A set of miRs, calleu uystiomiis
179, 18u, 181
have been
iuentifieu in the seium of Buchenne musculai uystiophy (BNB) patients, as well as
that of that of BNB animal mouels, at copy numbeis that aie significantly uiffeient
fiom healthy subjects oi contiol animals. Bystiomiis may have auvantages ovei
pioteins oi metabolites as seium biomaikeis. Quantitative RT-PCR seives as a
iapiu, sensitive anu accuiate methou of uetection of these small RNA molecules.
Since they may be actively expoiteu fiom muscle cells, seium levels of uystiomiis
coulu be less sensitive to the effects of physical activity than CK. At the time of
wiiting, fuithei uata aie iequiieu in longituuinal tieatment stuuies to ueteimine
whethei uystiomiis woulu be effective tools in monitoiing iesponse to tieatment.

D)(+99)'%&/$+'. ,)7&,%$'7 .),#9 &'% #,$') V$+9&,P),.Z
Sponsois shoulu enueavoi to collect seium anu uiine specimens at time points
uuiing the tiials with the appiopiiate ethical agieement foi use in futuie biomaikei
uevelopment as ieseaich mateiials. Potentially any samples eventually iuentifieu to
be samples fiom the placebo gioup coulu be biobankeu anu be maue available to
assist othei entities ueveloping new biomaikeis.

-9&7$'7 9+%&4$/$).
An inheient ueficit in uystiophin-ueficient muscle is incieaseu membiane fiagility
that ienueis uystiophic muscle moie susceptible to contiaction-inuuceu injuiy
.182,
18S, 184, 18S, 186, 187, 188, 189
Bystiophic skeletal muscle in humans unueigoes iepeateu
cycles of muscle fibei injuiy, uegeneiation, anu iegeneiation. The pathological
piogiession of BNB incluues incieaseu saicolemmal peimeability, muscle
inflammation, anu ultimately myofibeis aie piogiessively ieplaceu by fat anu
connective tissue. The pathological piogiession is ielentless but highly vaiiable
within anu acioss inuiviuuals, both spatially anu tempoially, with some muscle
gioups iapiuly piogiessing while otheis aie ielatively pieseiveu late in the uisease.
Imaging this piogiessive ieplacement of muscle with fat anu fibiosis can assess the
cuiient status of the patient, anu when combineu with natuial histoiy uata on the
iate of piogiession of muscle ieplacement, imaging can allow assessment of the
impact of inteiventions.

E4/,&.+#'% `E:a is a non-invasive imaging technique that can pioviue iapiu
anatomical anu functional measuiement of human tissue, anu places low uemanu on
the subject.
19u
As such it is well suiteu foi peuiatiic imaging. 0S imaging has been
extensively applieu to investigate caiuiac abnoimalities associateu with BNB.
Nuscle atiophy anu intiamusculai fibiosis anu fatty infiltiation can be visualizeu
using 0S of skeletal muscle.
191, 192
0S uensity analysis of skeletal muscle pioviues a
sensitive methou foi uistinguishing between healthy chiluien anu chiluien with
neuiomusculai uisoiueis.
19S
Quantitative muscle ultiasounu has been applieu to
stuuy BNB by quantifying echo intensity anu muscle thickness. A significant
inciease of echo intensity with age, ieflecting incieasing uystiophic muscle changes,
44
was obseiveu. This inciease was ielateu to ambulatoiy status, functional giauing,
muscle stiength anu motoi ability.
194


>4)(/,$(&4 $95)%&'() 90+7,&520 `>-3a pioviues a non-invasive appioach foi
quantifying tissue composition anu compaitmentation anu as such has ielevance foi
assessment of neuiomusculai uisease pathology. EIN Su kBz phase measuiements
have been iepoiteu to coiielate well with stanuaiu functional measuies in BNB;
NoithStai Ambulatoiy Assessment test (R = u.8S, p = u.u2).
19S


1>d; is a technique that can be useu to estimate bouy composition incluuing bone
mineial uensity anu bouy lean soft tissue anu inuiiectly pioviues an estimate of fat
content. Stuuies of BEXA in BNB subjects have founu uecieaseu iegional lean mass,
incieaseu iegional fat mass, anu uecieaseu stiength but BEXA cannot uistinguish
between muscle anu fibiosis.
196
Neveitheless, theie may be a iole foi BEXA to help
noimalize muscle mass foi the accuiate measuiement of seium biomaikeis.

3D- &'% 3D:, magnetic iesonance imaging anu spectioscopy, pioviue the most
uetaileu anu quantitative infoimation as to the status of inuiviuual skeletal muscles.
Bealthy muscle can be uistinguisheu fiom uiseaseu muscle, anu the infiltiation of fat
anu fibiosis can be monitoieu anu quantifieu. The piimaiy limitation of NR
appioaches is that they aie costly anu the evaluation is time consuming, as
compaieu to othei imaging appioaches. Nonetheless, the powei of NRINRS will
make it incieasing impoitant foi BNB stuuies. Combining NR with moie fiequent
follow-ups using less expensive appioaches such as ultiasounu oi EIN may
ultimately pioviue the best compiomise.

Nagnetic iesonance imaging (NRI) is the mouality of choice when high-
iesolutionhigh contiast images of soft tissue aie uemanueu. NRI is a non-invasive
technique that uoes not use ionizing iauiation, pioviues outstanuing volumetiic
coveiage of tissue, instiuments aie wiuely available, anu the technique can be iun
quantitatively anu stanuaiuizeu acioss sites.
197
Nagnetic iesonance spectioscopy
(NRS) is a class of techniques useu to measuie the biochemical piopeities of tissue.
The funuamental haiuwaie iequiieu foi NRS is iuentical to that useu in NRI, which
makes NRS a high-value ancillaiy stuuy to NRI. A funuamental stiength of NRS is
the incieaseu specificity foi measuiement of uistinct tissue constituents. An
example ielevant to BNB is the high-fiuelity sepaiation of tissue watei anu fat
signals, which typically co-contiibute to stanuaiu NRI signals collecteu fiom
skeletal muscle of BNB inuiviuuals. NRS techniques have been applieu to
investigate cellulai metabolites typically using the most abunuant magnetic isotopes
of hyuiogen (
1
B), caibon (
1S
C), anu phosphoius (
S1
P). NRS has been useu to
impiove uiagnosis, to bettei uefine the natuial histoiy of a uisease piocess, anu in
some stuuies to monitoi the iesponse to theiapy.
198, 199, 2uu, 2u1, 2u2, 2uS, 2u4, 2uS, 2u6, 2u7

While most NRINRS investigations of BNB skeletal muscle have focuseu on the
lowei extiemities, investigation of shouluei anu uppei extiemity muscle is also
feasible anu unueiway.
2u8, 2u9
Finally, the fact that that NRINRS measuies aie
obtaineu with the subject at iest, gieatly ieuuces the impact of motivational issues
4S
that confounu many functional measuies. Taken togethei, these attiibutes make
NRINRS attiactive techniques foi longituuinal investigations of iaie uisease in
human peuiatiic subjects.

<IKE<ID> =5%).-*. L-(56)M%)+ (9 ;456* 54+,486) 7:+3)(21: ?631(8(.:
Numeious stuuies have uemonstiateu the ability of NRI to uetect alteiations in
skeletal muscle stiuctuie in patients with musculai uystiophy
.21u, 211, 212, 21S, 214, 21S,
216, 217, 218, 219, 22u, 221
Inueeu, uue to its excellent soft tissue SB imaging capability anu
the ability to peifoim longituuinal measuies of muscle mass, NRI has been useu in
clinical tiials to quantify changes in skeletal muscle volume following tieatment
with, eithei a neutializing antibouy to myostatin oi following myoblast
tiansplants.
222, 22S
Bowevei, most NRI investigations have ielieu on T1- weighteu
images anu the contiast geneiateu by fatty tissue infiltiation to visualize the pattein
of muscle involvement in musculai uystiophy patients.
224
Rauiologists have
uevelopeu a foui-point giauing system to categoiize uisease seveiity, baseu on
visual inspection of fatty tissue infiltiation.
22S
This stiategy was iecently useu to
scieen BNB subjects piioi to injection with antisense oligonucleotiues.
226
Bowevei,
few NR stuuies have piesenteu a iobust quantitative appioach to monitoi uisease
piogiession in BNB, limiting the viability of NR as a sensitive suiiogate outcome
measuie foi clinical tiials.

Spectioscopy anu spectioscopic imaging have been well establisheu as noninvasive
quantitative biochemical assays. A numbei of stuuies have founu stiong coiielations
between intiamusculai lipius anu measuies of functional ability.
227, 228, 229, 2Su, 2S1, 2S2,
2SS, 2S4, 2SS, 2S6, 2S7, 2S8, 2S9, 24u, 241, 242, 24S
A iecent stuuy evaluateu the effects of
coiticosteioiu initiation in in BNB steioiu nave patients using NRINRS founu a
significant ueciease in T2 ( the tiansveise ielaxation time constant) that occuiieu
piioi to any measuieable functional impiovements.
244
This suggests that NRINRS
iesponses to theiapies may be moie sensitive to tieatment effects anu may even be
pieuictive of futuie functional impiovements.

<I K56.-*. (9 K*9865563-(*
NR imaging stiategies aie also sensitive to muscle inflammation iegions of
incieaseu signal intensity in muscles of young boys with BNB in the absence of fatty
tissue infiltiation.
24S
This finuing is consistent with inflammation occuiiing eaily in
BNB, piioi to the loss in contiactile tissue anu accumulation of fatty tissue. The
impoitance of inflammation in BNB is fuithei suppoiteu by the 1uu folu highei
seium levels of TNF-u that is uetectable in boys with BNB compaieu to contiols
(27.8 ngL vs. u.27 ngL).
246
Changes in the seium levels of TNF-u with age aie
consistent with the obseivations that muscle expiession of TNF-u anu IL-6
uecieases with age.
247
A uiiect ielationship between inflammatoiy maikeis (seium
anu tissue) anu hypei intensity obseiveu on STIR images has been uocumenteu in
FSBB.
248


Anothei NR imaging mouality, Na+ imaging, showeu that aieas of hypei intensity on
STIR images fiom skeletal muscle in BNB subjects aie uiiectly ielateu to muscle
46
euema.
249
Taken togethei, these finuings inuicate that NR sequences may be
sensitive to eaily inflammation in the uystiophies.

0thei investigations have focuseu on imaging stiategies that aie sensitive to muscle
uamage anu inflammation to visualize uystiophic lesions, which may be paiticulaily
impoitant in youngei boys with BNB. 0sing shoit-tau inveision iecoveiy (STIR)
sequences, it is possible to iuentify iegions of incieaseu signal intensity oi muscle
inflammation in uystiophic muscles of young boys with BNB in the absence of fatty
tissue infiltiation. T2 weighteu imaging has been implementeu to visualize
uystiophic lesions anu founu incieaseu signal intensity in both young anu oluei
ambulatoiy boys with BNB, with muscle iegions of elevateu T2 ianging fiom 1% to
94%. The most challenging pait in implementing magnetic iesonance to
chaiacteiize uystiophic human muscle lies in the assessment of muscle
uamageinflammation, in the piesence of laige amounts of fatty tissue infiltiation.
Bowevei, the ability to monitoi muscle uamage 4+ ,4,# is extiemely impoitant, since
the piimaiy taiget of most gene theiapy oi phaimaceutical inteiventions is the
iestoiation of the expiession of stiuctuial pioteins anu noimal saicolemmal
integiity. Pieliminaiy uata in both muiine mouels of musculai uystiophy anu boys
with BNB uemonstiate the feasibility of implementing T2 weighteu imaging anu
1
B-
spectioscopic ielaxometiy to stuuy uystiophic muscle.

<I K56.-*. (9 A-')(+-+
A significant challenge foi NR anu othei noninvasive imaging moualities is the
quantification of fibiosis. The obseiveu NR signal intensity associateu with fibiosis
unueigoes a chaiacteiistic iapiu uecay uue to the extiemely shoit T2s of watei
molecules associateu with collagen.

Caiuiac NRI stuuies in BNB subjects have iepoiteu an age ielateu ueciease in
myocaiuial T2 compaieu to contiols
2Su, 2S1
anu an inciease in myocaiuial T2
heteiogeneity
2S2
. Similai iesults have been obseiveu in animal mouels with uiabetic
inuuceu caiuiac fibiosis.
2SS, 2S4
The uecieaseu T2 has been hypothesizeu to iepiesent
an incieaseu fiaction of watei molecules "bounu" to collagen anu othei fibiotic
tissue. Similaily, NRS ielaxometiy uata acquiieu in the paient pioject show an age
uepenuent ueciease in muscle watei T2 in both calf anu thigh skeletal muscles in
boys with BNB, but not healthy contiols. This ueciease in T2 is typically maskeu in
skeletal muscle imaging by the laige amounts of fatty tissue ueposition (pieliminaiy
iesults).

<IKE<ID> I(8% -* !8-*-,68 0)-68+
Numeious stuuies have now shown the ability of NRINRS to visualize stiuctuial
alteiations in skeletal muscle of patients with musculai uystiophy
2SS, 2S6, 2S7, 2S8, 2S9,

26u
yet NR has been incoipoiateu in only a hanuful of BNB clinical tiials. Bowevei,
the emeiging uata suppoits a much-expanueu futuie utilization of NR imaging in
BNB tiials. Since NRINRS outcomes faithfully iepoit on both the health anu
amount of skeletal muscle, they can potentially be useu at uiffeient stages of the
clinical tiial piocess as piognostic, pieuictive, oi phaimacouynamics biomaikeis.
47
Combining NRNRS outcome measuies with functional outcomes in tieatment
tiials coulu leau to the uemonstiation that NRINRS measuies can seive as an
efficacy-iesponse biomaikeis anu suiiogate enupoints to acceleiate clinical
uevelopment.

IX. A?BA=E:-?B

We believe that BNB woulu be an appiopiiate inuication to use some of the moie
flexible measuies foi a iegulatoiy filing as set foith in FBASIA. These measuies have
iecently (in 2u14) been uesciibeu, in uuiuance foi InuustiyB J:9)&41)& M8#58/;! A#8
0)84#$! .#+&414#+! Z '8$5! /+& P4#%#54-!.

In the case of BNB, at the time of wiiting, sponsois may take one of seveial
appioaches towaius an expeuiteu appioval pathway. Foi instance, cleai eviuence of
a iesponse to tieatment in ceitain biomaikeis such as eviuence of uystiophin
wheie pieviously theie was none, oi eviuence of a change in the health oi iate of
ueteiioiation of skeletal muscle on NRINRS coulu be suppoitive of acceleiateu
appioval foi a piouuct paiticulaily if combineu with inteimeuiate clinical
enupoint uata,, oi, potentially, PR0 uata suggesting that tiial paiticipant's have
expeiienceu meaningful clinical benefit.

Similaily, pieliminaiy eviuence of eaily clinical benefit such as a significant
change in time to function tests oi 6NWT coulu meiit a bieakthiough
uesignation foi a piouuct. Sponsois aie, howevei, encouiageu to incluue
exploiatoiy biomaikei measuies as seconuaiy enupoints in theii tiials as well, in
oiuei to fostei biomaikei uiscoveiy anu to inciease the uatabase to suppoit the use
of those maikeis as phaimacouynamic oi suiiogate enupoints in futuie tiials.


48
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