1111111111111111 IIIIII IIIII 111111111111111 111111111111111 111111111111111 1111111111 11111111
`US 20120190728Al
`
`c19) United States
`c12) Patent Application Publication
`Bennett et al.
`
`c10) Pub. No.: US 2012/0190728 Al
`Jul. 26, 2012
`(43) Pub. Date:
`
`(54) COMPOSITIONS AND METHODS FOR
`MODULATION OF SMN2 SPLICING IN A
`SUBJECT
`
`(76)
`
`Inventors:
`
`C. Frank Bennett, Carlsbad, CA
`(US); Gene Hung, San Diego, CA
`(US); Frank Rigo, Carlsbad, CA
`(US); Adrian R. Krainer,
`Huntington Square, NY (US);
`Yimin Hua, Jericho, NY (US);
`Marco A. Passini, Shrewsbury, MA
`(US); Lamya Shihabuddin,
`Brighton, MA (US); Seng H.
`Cheng, Natick, MA (US);
`Katherine W. Klinger, Sudbury,
`MA(US)
`
`(21) Appl. No.:
`
`13/380,021
`
`(22) PCT Filed:
`
`Jun.17,2010
`
`(86) PCT No.:
`
`PCT/USl0/39077
`
`§ 371 (c)(l),
`Apr. 12, 2012
`(2), ( 4) Date:
`Related U.S. Application Data
`
`(60) Provisional application No. 61/218,031, filed on Jun.
`17, 2009.
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 311713
`(2006.01)
`A61P 25128
`(2006.01)
`(52) U.S. Cl. ...................................................... 514/44 A
`ABSTRACT
`
`(57)
`
`Disclosed herein are compounds, compositions and methods
`for modulating splicing of SMN2 mRNA in a subject. Also
`provided are uses of disclosed compounds and compositions
`in the manufacture of a medicament for treatment of diseases
`and disorders, including spinal muscular atrophy.
`
`

`

`Patent Application Publication
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`Jul. 26, 2012 Sheet 1 of 13
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`US 2012/0190728 Al
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`con
`
`0
`
`1
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`2
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`4
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`6
`
`8
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`Figure 1
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`

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`Patent Application Publication
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`Jul. 26, 2012 Sheet 2 of 13
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`US 2012/0190728 Al
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`100
`
`80
`-g 60
`...
`ti. 40
`
`20
`
`0
`
`2
`Con O 05 1
`6
`4
`Monthsp~ 7-d Et/ ICVln'llston
`
`Figure 2
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`

`

`Patent Application Publication
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`Jul. 26, 2012 Sheet 3 of 13
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`US 2012/0190728 Al
`
`A
`
`B
`
`....,Na injec:tion (n"' 5)
`.._ASO 7.Sµg (n • 9)
`_._AC02!1119(n•7)
`
`1
`
`z
`
`~
`
`4
`
`5
`
`6
`
`7
`
`1
`
`e w n
`
`Week
`
`--+-Normal
`-ASO-20
`...,_ASO-10
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`
`8.0
`
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`
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`0
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`£ 4.0
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`~ 3.0
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`
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`
`0.0 -+----r-----r-----.--r----.-----,---,---,---,------,---,
`0 1 2 3 4 5 6 7 8 9 10 11 12
`
`Weeks
`
`Figure 3
`
`

`

`Patent Application Publication
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`Jul. 26, 2012 Sheet 4 of 13
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`US 2012/0190728 Al
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`SMN Western Blots of SMA
`
`Cervical
`
`Thoracic
`
`Lumbar
`
`100
`
`80
`
`100
`
`~ 80
`.!I! 60 ! 40
`
`z
`:i! 20
`Cl)
`
`4 µg dose, 16 days post-
`
`Figure 4
`
`

`

`Patent Application Publication
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`Jul. 26, 2012 Sheet 5 of 13
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`US 2012/0190728 Al
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`Body Weight
`8 Days
`
`Body Weight
`14 Days
`
`8
`
`6
`
`***
`
`I
`
`***
`
`16
`
`12
`'Ei
`._
`
`r:,,
`
`... .c: 8
`~
`
`***
`
`I
`1rlrlt
`
`I
`*** ***
`
`4
`
`Righting Reflex
`8 Days
`
`Righting Reflex
`14 Days
`
`60
`
`50
`E40
`>, g 30
`....
`j 20
`
`G)
`
`10
`
`60
`so
`E4o
`>, g 30
`.e
`j 20
`
`**
`
`**
`
`***
`
`Figure 5
`
`

`

`Patent Application Publication
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`Jul. 26, 2012 Sheet 6 of 13
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`US 2012/0190728 Al
`
`Grip Strength
`14 Days
`
`20
`
`16
`
`*
`
`I
`***
`
`4
`
`3
`
`e
`8 2
`
`II)
`
`1
`
`Hindlimb Splay
`14 Days
`
`*
`
`I
`***
`
`***
`
`**
`
`***
`
`i
`
`Figure 6
`
`

`

`Patent Application Publication
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`Jul. 26, 2012 Sheet 7 of 13
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`US 2012/0190728 Al
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`-.-ASO 15-mer (n=16, med=31.5d)
`-------ASO 18-mer (n=13, med=27.0d)
`-+-ASO 20-mer (n=19, med=28.0d)
`-111-ASO Scramble (n=18 med=16.0d)
`----Er- Untreated (n=13 med=16.0d)
`
`75
`
`50
`
`25
`
`-ca
`> -~
`
`:::s
`fl)
`
`0 +-r--r-r-T'"'T'"'1rr-f5111-r-r-r-~r-r,.,..,...,...,-,--rr,.,..,..,..,-,ll-m
`60
`70
`50
`20
`10
`0
`30
`40
`Age (d)
`
`Figure 7
`
`

`

`Patent Application Publication
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`Jul. 26, 2012 Sheet 8 of 13
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`US 2012/0190728 Al
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`ASO Treatment Increases Motor Neuron Cell Counts in the Spinal Cord
`
`Cervical
`
`Thoracic
`
`Lumbar
`
`4 µg dose, 16 days post-injection
`
`Figure 8
`
`

`

`Patent Application Publication
`
`Jul. 26, 2012 Sheet 9 of 13
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`US 2012/0190728 Al
`
`7
`
`~ 6
`(I) :e 5
`U) ti)
`CU 0
`(I) . . 4
`t;;;;."C u (I)
`3 -"C-
`.5 N
`ca o E
`-
`1 - 0
`LL •
`0
`C
`
`2
`
`Brain RT-PCR
`
`SMA
`
`SMA+20mer
`
`Figure 9
`
`

`

`Patent Application Publication
`
`Jul. 26, 2012 Sheet 10 of 13
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`US 2012/0190728 Al
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`Injections of 396443 at PO (8 ug) and P21 (20 ug) vs. PO alone (8 ug)
`
`100
`
`~
`
`- 75
`-iii 50
`> -~
`:::,
`U) 25
`
`PO vs. PO/P21
`p = 0.2363
`
`Q--h-.,....,.. .......... ~;;;....... .................. ,....,.. ................... ~ ...............
`0
`10
`20
`30
`40
`50
`60
`70
`Age (days)
`
`-ti- Untreated SMA(n = 33, med= 16d)
`_._ PO 396443 (n = 11, med = 26d)
`PO/P21 396443 (n = 26, med = 26d)
`
`Figure 10
`
`

`

`Patent Application Publication
`
`Jul. 26, 2012 Sheet 11 of 13
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`US 2012/0190728 Al
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`100---.
`
`PO vs. PO/P21
`p=0.0440
`
`_ 75
`
`'#, --i 50
`
`'!
`::,
`u, 25
`
`o"T""T"....,,-"T-r-T"""T"""llar--r~--r--,"""T"""1r---r-,.----r-,....,....,....,.................,.
`
`20
`
`30
`
`50
`40
`Age (days)
`
`60
`
`70
`
`PO 396443 (n = 6, med = 29.5d}
`PO/P21 396443 (n = 14, med= 35d)
`
`Figure 11
`
`

`

`Patent Application Publication
`
`Jul. 26, 2012 Sheet 12 of 13
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`US 2012/0190728 Al
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`Thoracic Cord
`
`40
`
`GI 30
`::J _;
`I- 20
`E
`"' 0)
`:, 10
`
`0
`
`Target
`}-
`concentration
`
`Cervical Cord
`
`cerebellum
`
`Lumbar Cord
`
`40
`
`~ 30
`
`.. .!/l
`
`I- 20
`E
`Cl
`g' 10
`
`0
`
`40
`
`a, 30
`::J
`j
`I- 20
`E
`i
`
`::J 10
`
`0
`
`2.0
`
`a, 1.5
`::J
`]
`~ 1.0
`I :, 0.5
`
`0.0
`
`25
`
`20
`Cl) ii: 15
`.!!l
`I- 10
`E
`Cl ci 5
`:::s
`
`0
`
`Left Caudate
`
`Pons
`
`1.5
`
`.. 1il 1.0
`
`en
`i=
`E
`Cl m 0.5
`
`::J
`
`0.0
`
`Frontal Cortex
`
`8
`
`Cl) 6
`:::s
`Ill
`.!!!
`I- 4
`E
`~
`Cl
`:I 2
`
`0
`
`Figure 12
`
`

`

`Patent Application Publication
`
`Jul. 26, 2012 Sheet 13 of 13
`
`US 2012/0190728 Al
`
`Q)
`-~
`
`~
`
`<( Go +--1------..le==*==~==~~~-=------ -sC4oo
`-sc200
`-sc100
`40 + - - - - - - - - - - - - - - - - - - - - - - - - - -HetControl
`
`...... Saline Control
`
`1
`
`11
`
`21
`
`31
`
`41
`
`61
`
`71
`
`81
`
`91
`
`51
`Days
`
`Figure 13
`
`

`

`US 2012/0190728 Al
`
`Jul. 26, 2012
`
`1
`
`COMPOSITIONS AND METHODS FOR
`MODULATION OF SMN2 SPLICING IN A
`SUBJECT
`
`SEQUENCE LISTING
`
`[0001] The present application is being filed along with a
`~eque~ce Listing in electronic format. The Sequence Listing
`1s provided as a file entitled 20100617 _CORE0086WOSEQ.
`bet, created Jun. 17, 2010, which is 5 Kb in size. The infor(cid:173)
`~ation in the ele_ctronic format of the sequence listing is
`mcorporated herem by reference in its entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Newly synthesized eukaryotic mRNA molecules,
`known as primary transcripts or pre-mRNA are processed
`before translation. Processing of the pre-mRNAs includes
`addition ofa 5' methylated cap and an approximately 200-250
`base poly(A) tail to the 3' end of the transcript. Processing of
`mRNA from pre-mRNA also frequently involves splicing of
`the pre-mRNA, which occurs in the maturation of90-95% of
`ma~alian mRNAs. Intrans ( or intervening sequences) are
`reg10ns of a pre-mRNA ( or the DNA encoding it) that are not
`included in the coding sequence of the mature mRNA. Exons
`are regions of a primary transcript that remain in the mature
`mRNA. The exons are spliced together to form the mature
`mRNA sequence. Splice junctions are also referred to as
`splice sites with the 5' side of the junction often called the "5'
`splice site," or "splice donor site" and the 3' side the "3' splice
`site" or "splice acceptor site." In splicing, the 3' end of an
`upstream exon is joined to the 5' end of the downstream exon.
`Thus the unspliced pre-mRNA has an exon/intron junction at
`the 5' end ofan intron and an intron/exon junction at the 3' end
`of an intron. After the intron is removed, the exons are con(cid:173)
`tiguous at what is sometimes referred to as the exon/exon
`junction or boundary in the mature mRNA. Cryptic splice
`sites are those which are less often used but may be used when
`the usual splice site is blocked or unavailable. Alternative
`splicing, defined as the splicing together of different combi(cid:173)
`nations of exons, often results in multiple mRNA transcripts
`from a single gene.
`[0003] Up to 50% of human genetic diseases resulting from
`a point mutation result in aberrant pre-mRNA processing.
`Such point mutations can either disrupt a current splice site or
`create a new splice site, resulting in mRNA transcripts com(cid:173)
`prised of a different combination of exons or with deletions in
`~xons._ Poi1;1t muta~ions also can result in activation of a cryp(cid:173)
`tic sphce site or disrupt regulatory cis elements (i.e. splicing
`enhancers or silencers) (Cartegni et al., Nat. Rev. Genet.,
`2002, 3, 285-298; Drawczak et al., Hum. Genet., 1992, 90,
`41-54!. Antisense oligonucleotides have been used to target
`~utat10n~ that lead to aberrant splicing in several genetic
`diseases m order to redirect splicing to give a desired splice
`product (Kole, Acta Biochimica Polonica, 1997, 44 231-
`n~.
`,
`[0004] Antisense compounds have also been used to alter
`the ratio of naturally occurring alternate splice variants such
`as the long and short forms ofBcl-x pre-mRNA (U.S. Pat. No.
`6,172,216; U.S. Pat. No. 6,214,986; Taylor et al., Nat. Bio(cid:173)
`technol. 1999, 17, 1097-1100) orto force skipping of specific
`exons containing premature termination codons (Wilton et
`al., Neuromuscul. Disord., 1999, 9, 330-338). U.S. Pat. No.
`5,627,274 and WO 94/26887 disclose compositions and
`methods for combating aberrant splicing in a pre-mRNA
`
`molecule containing a mutation using antisense oligonucle(cid:173)
`otides which do not activate RNAse H.
`[0005_] Proximal spinal muscular atrophy (SMA) is a
`genetic, neurodegenerative disorder characterized by the loss
`of spinal motor neurons. SMA is an autosomal recessive
`disease of early onset and is currently the leading cause of
`dea_th among infants. The severity of SMA varies among
`patlen!s and has thus been classified into three types. Type I
`SMA 1s the most severe form with onset at birth or within 6
`months and typically results in death within 2 years. Children
`with type I SMA are unable to sit or walk. Type II SMA is the
`intermediate form and patients are able to sit, but cannot stand
`or walk. Patients with type III SMA, a chronic form of the
`disease, typically develop SMA after 18 months of age (Lefe(cid:173)
`bvre et al., Hum. Mo!. Genet., 1998, 7, 1531-1536).
`[0006] The molecular basis ofSMA is caused by the loss of
`both copies of survival motor neuron gene 1 (SMNl ), which
`may also be known as SMN Telomeric, a protein that is part of
`a multi-protein complex thought to be involved in snRNP
`bio?enesis and recycling. A nearly identical gene, SMN2,
`which may also be known as SMN Centromeric, exists in a
`duplicated region on chromosome 5q13 and modulates dis(cid:173)
`ease severity. Expression of the normal SMNl gene results
`solely in expression of survival motor neuron (SMN) protein.
`Although SMNl and SMN2 have the potential to code for the
`same protein, SMN2 contains a translationally silent muta(cid:173)
`tion at position +6 of exon 7, which results in inefficient
`inclusion of exon 7 in SMN2 transcripts. Thus, the predomi(cid:173)
`n~t fo~ of SMN2 is a truncated version, lacking exon 7,
`which 1s unstable and inactive (Cartegni and Krainer, Nat.
`Genet., 2002, 30, 377-384). Expression of the SMN2 gene
`results in approximately 10-20% of the SMN protein and
`80-90% of the unstable/non-functional SMNdelta7 protein.
`S~ protein plays a well-established role in assembly of the
`sphceosome and may also mediate mRNA trafficking in the
`axon and nerve terminus of neurons.
`[0007] Antisense technology is an effective means for
`modulating the expression of one or more specific gene prod(cid:173)
`ucts, i~cluding alternative splice products, and is uniquely
`useful m a number of therapeutic, diagnostic, and research
`applications. The principle behind antisense technology is
`that an antisense compound, which hybridizes to a target
`nucleic acid, modulates gene expression activities such as
`transcription, splicing or translation through one of a number
`of antisense mechanisms. The sequence specificity of anti(cid:173)
`sense compounds makes them extremely attractive as tools
`for target validation and gene functionalization, as well as
`therapeutics to selectively modulate the expression of genes
`involved in disease.
`[0008] Certain antisense compounds complementary to
`SMN2 are known in the art. See for example, WO 2007 /
`002390; U.S. 61/168,885; Hua et al., American J. of Human
`Genetics (April 2008) 82, 1-15; Singh et al., RNA Bio. 6:3,
`1-10 (2009). Certain antisense compounds and methods dis(cid:173)
`closed herein posses desirable characteristics compared to
`such compounds and methods known in the art. Chimeric
`peptide nucleic acid molecules designed to modulate splicing
`of SMN2 have been described (WO 02/38738; Cartegni and
`Krainer, Nat. Struct. Biol., 2003, 10, 120-125).
`
`SUMMARY OF THE INVENTION
`
`[~009]
`In certain embodiments, the present invention pro(cid:173)
`vides methods comprising administering to a subject an anti(cid:173)
`sense compound comprising an antisense oligonucleotide
`
`

`

`US 2012/0190728 Al
`
`Jul. 26, 2012
`
`2
`
`complementary to intron 7 of a nucleic acid encoding human
`SMN2 pre-mRNA, wherein the antisense compound is
`administered into the cerebrospinal fluid. In certain embodi(cid:173)
`ments, the administration is into the intrathecal space. In
`certain embodiments, the administration is into the cere(cid:173)
`brospinal fluid in the brain. In certain embodiments, the
`administration comprises a bolus injection. In certain
`embodiments, the administration comprises infusion with a
`delivery pump.
`[0010]
`In certain embodiments, the antisense compound is
`administered at a dose from 0.01to10 milligrams ofantisense
`compound per kilogram of body weight of the subject. In
`certain embodiments, the dose is from 0.01 to 10 milligrams
`of antisense compound per kilogram of body weight of the
`subject. In certain embodiments, the dose is from 0.01 to 5
`milligrams of antisense compound per kilogram of body
`weight of the subject. In certain embodiments, the dose is
`from 0.05 to 1 milligrams of antisense compound per kilo(cid:173)
`gram of body weight of the subject. In certain embodiments,
`the dose is from 0.01 to 0.5 milligrams of anti sense compound
`per kilogram ofbody weight of the subject. In certain embodi(cid:173)
`ments, the dose is from 0.05 to 0.5 milligrams of antisense
`compound per kilogram of body weight of the subject.
`[0011]
`In certain embodiments, the dose is administered
`daily. In certain embodiments, the dose is administered
`weekly. In certain embodiments, the antisense compound is
`administered continuously and wherein the dose is the
`amount administered per day. In certain embodiments, the
`method comprises administering at least one induction dose
`during an induction phase and administering at least one
`maintenance dose during a maintenance phase. In certain
`embodiments, the induction dose is from 0.05 to 5.0 milli(cid:173)
`grams of anti sense compound per kilogram of body weight of
`the subject. In certain embodiments, the maintenance dose is
`from 0.01 to 1.0 milligrams of antisense compound per kilo(cid:173)
`gram of body weight of the subject. In certain embodiments,
`the duration of the induction phase is at least 1 week. In
`certain embodiments, the duration of the maintenance phase
`is at least 1 week. In certain embodiments, each induction
`dose and each maintenance dose comprises a single injection.
`In certain embodiments, each induction dose and each main(cid:173)
`tenance dose independently comprise two or more injections.
`In certain embodiments, anti sense compound is administered
`at least 2 times over a treatment period of at least 1 week. In
`certain embodiments, the treatment period is at least one
`month. In certain embodiments, the treatment period is at
`least 2 months. In certain embodiments, the treatment period
`is at least 4 months. In certain embodiments, the induction
`dose is administered by one or more bolus injections and the
`maintenance dose is administered by an infusion pump.
`[0012]
`In certain embodiments, the method comprises
`assessing the tolerability and/or effectiveness of the antisense
`compound. In certain embodiments, dose amount or fre(cid:173)
`quency of antisense compound is reduced following an indi(cid:173)
`cation that administration of the antisense compound is not
`tolerated. In certain embodiments, the dose amount or fre(cid:173)
`quency of antisense compound is maintained or reduced fol(cid:173)
`lowing an indication that administration of the anti sense com(cid:173)
`pound is effective. In certain embodiments, the dose of
`antisense compound is increased following an indication that
`administration of the antisense compound is not effective. In
`certain embodiments, frequency of administration of anti(cid:173)
`sense compound is reduced following an indication that
`administration of the antisense compound is effective. In
`
`certain embodiments, frequency of administration of anti(cid:173)
`sense compound is increased following an indication that
`administration of the antisense compound is not effective.
`[0013]
`In certain embodiments, the methods comprise co(cid:173)
`administration of the antisense compound and at least one
`other therapy. In certain embodiments, an antisense com(cid:173)
`pound and at least one other therapy are co-administered at
`the same time. In certain embodiments, an antisense com(cid:173)
`pound is administered prior to administration of the at least
`one other therapy. In certain embodiments, an antisense com(cid:173)
`pound is administered after administration of the at least one
`other therapy. In certain embodiments, the at least one other
`therapy comprises administration of one or more of valproic
`acid, riluzole, hydroxyurea, and a butyrate. In certain embodi(cid:173)
`ments, at least one other therapy comprises administration of
`trichostatin-A. In certain embodiments, the at least one other
`therapy comprises administration of stem cells. In certain
`embodiments, at least one other therapy is gene therapy. In
`certain embodiments, gene therapy is administered to the
`CSF and an antisense compound is administered systemi(cid:173)
`cally. In certain embodiments, gene therapy is administered
`to the CSF and an antisense compound is administered sys(cid:173)
`temically and to the CSF. In certain embodiments, the inven(cid:173)
`tion provides treatment regimens where initially, an antisense
`compound is administered to the CSF and systemically, fol(cid:173)
`lowed by gene therapy administration to the CSF and sys(cid:173)
`temic administration of antisense compound. In certain such
`embodiments, the subject is an infant at the time of initial
`treatment. In certain such embodiments, the subject is less
`that 2 years old. In certain embodiments, antisense compound
`is administered to the CNS of a subject until the subject is old
`enough for gene therapy. In certain such embodiments, anti(cid:173)
`sense compound is administered systemically throughout.
`[0014]
`In certain embodiments, the antisense compound is
`administered at a concentration of about 0.01 mg/ml, about
`0.05 mg/ml, about 0.1 mg/ml, about 0.5 mg/ml, about 1
`mg/ml, about 5 mg/ml, about 10 mg/ml, about 50 mg/ml, or
`about 100 mg/ml.
`[0015]
`In certain embodiments, inclusion of exon 7 of
`SMN2 mRNA in a motoneuron in the subject is increased. In
`certain embodiments, inclusion of exon 7 amino acids in
`SMN2 polypeptide in a motoneuron in the subject is
`increased.
`[0016]
`In certain embodiments, the invention provides
`methods of increasing inclusion of exon 7 ofSMN2 mRNA in
`a motoneuron in a subject comprising administering to the
`subject an antisense compound comprising an antisense oli(cid:173)
`gonucleotide complementary to intron 7 of a nucleic acid
`encoding human SMN2 and thereby increasing inclusion of
`exon 7 of SMN2 mRNA in the motoneuron in the subject.
`[0017]
`In certain embodiments, the invention provides
`methods of increasing inclusion of exon 7 amino acids in
`SMN2 polypeptide in a motoneuron in a subject comprising
`administering to the subject an antisense compound compris(cid:173)
`ing an antisense oligonucleotide complementary to intron 7
`of a nucleic acid encoding human SMN2 and thereby increas(cid:173)
`ing inclusion of exon 7 amino acids in SMN2 polypeptide in
`the motoneuron in the subject.
`[0018]
`In certain embodiments, the subject has SMA. In
`certain embodiments, the subject has type I SMA. In certain
`embodiments, the subject has type II SMA. In certain
`embodiments, the subject has type III SMA.
`[0019]
`In certain embodiments, a first dose is administered
`in utero. In certain embodiments, the first dose is adminis-
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`tered prior to complete formation of the blood-brain-barrier.
`In certain embodiments, a first dose is administered within 1
`week of birth of the subject. In certain embodiments, a first
`dose is administered within 1 month of birth of the subject. In
`certain embodiments, a first dose is administered within 3
`months of birth of the subject. In certain embodiments, a first
`dose is administered within 6 months of birth of the subject.
`In certain embodiments, a first dose is administered when the
`subject is from 1 to 2 years of age. In certain embodiments, a
`first dose is administered when the subject is from 1 to 15
`years of age. In certain embodiments, a first dose is adminis(cid:173)
`tered when the subject is older than 15 years of age.
`[0020]
`In certain embodiments, the subject is a mammal. In
`certain embodiments, the subject is a human.
`[0021]
`In certain embodiments, the methods comprise
`identifying a subject having SMA. In certain embodiments,
`the subject is identified by measuring electrical activity of one
`or more muscles of the subject. In certain embodiments, the
`subject is identified by a genetic test to determine whether the
`subject has a mutation in the subject's SMNl gene. In certain
`embodiments, the subject is identified by muscle biopsy.
`[0022]
`In certain embodiments, administering the antisense
`compound results in an increase in the amount of SMN2
`mRNA having exon 7 of at least 10%. In certain embodi(cid:173)
`ments, the increase in the amount of SMN2 mRNA having
`exon 7 is at least 20%. In certain embodiments, the increase in
`the amount ofSMN2 mRNAhaving exon 7 is at least 50%. In
`certain embodiments, the amount of SMN2 mRNA having
`exon 7 is at least 70%.
`[0023]
`In certain embodiments, administering of the anti(cid:173)
`sense compound results in an increase in the amount of SMN2
`polypeptide having exon 7 amino acids of at least 10%. In
`certain embodiments, wherein the increase in the amount of
`SMN2 polypeptide having exon 7 amino acids is at least 20%.
`In certain embodiments, the increase in the amount of SMN2
`polypeptide having exon 7 amino acids is at least 50%. In
`certain embodiments, the increase in the amount of SMN2
`polypeptide having exon 7 amino acids is at least 70%.
`[0024]
`In certain embodiments, the administering of the
`antisense compound ameliorates at least one symptom of
`SMA in the subject. In certain embodiments, the administer(cid:173)
`ing of the antisense compound results in improved motor
`function in the subject. In certain embodiments, the admin(cid:173)
`istering of the antisense compound results in delayed or
`reduced loss of motor function in the subject. In certain
`embodiments, administering of the antisense compound
`results in improved respiratory function. In certain embodi(cid:173)
`ments, the administering of the antisense compound results in
`improved survival.
`[0025]
`In certain embodiments, at least one nucleoside of
`the antisense oligonucleotide comprises a modified sugar
`moiety. In certain embodiments, at least one modified sugar
`moiety comprises a 2'-methoxyethyl sugar moiety. In certain
`embodiments, essentially each nucleoside of the antisense
`oligonucleotide comprises a modified sugar moiety. In certain
`embodiments, the nucleosides comprising a modified sugar
`moiety all comprise the same sugar modification. In certain
`embodiments, wherein each modified sugar moiety com(cid:173)
`prises a 2'-methoxyethyl sugar moiety. In certain embodi(cid:173)
`ments, each nucleoside of the antisense oligonucleotide com(cid:173)
`prises a modified sugar moiety. In certain embodiments, the
`nucleosides all comprise the same sugar modification. In
`certain embodiments, each modified sugar moiety comprises
`a 2'-methoxyethyl sugar moiety. In certain embodiments, at
`
`least one internucleoside linkage is a phosphorothioate inter(cid:173)
`nucleoside linkage. In certain embodiments, each inter(cid:173)
`nucleoside linkage is a phosphorothioate internucleoside
`linkage.
`[0026]
`In certain embodiments, the antisense oligonucle(cid:173)
`otide consists of 10 to 25 linked nucleosides. In certain
`embodiments, the antisense oligonucleotide consists of 12 to
`22 linked nucleosides. In certain embodiments, the antisense
`oligonucleotide consists of 15 to 20 linked nucleosides. In
`certain embodiments, the antisense oligonucleotide consists
`of 18 linked nucleosides.
`[0027]
`In certain embodiments, the antisense oligonucle(cid:173)
`otide is at least 90% complementary to the nucleic acid
`encoding human SMN2. In certain embodiments, the anti(cid:173)
`sense oligonucleotide is fully complementary to the nucleic
`acid encoding human SMN2. In certain embodiments, the
`oligonucleotide has a nucleobase sequence comprising at
`least 10 contiguous nucleobases of the nucleobase sequence
`SEQ ID NO: 1. In certain embodiments, the oligonucleotide
`has a nucleobase sequence comprising at least 15 contiguous
`nucleobases of the nucleobase sequence SEQ ID NO: 1. In
`certain embodiments, the oligonucleotide has a nucleobase
`sequence comprising the nucleobase sequence SEQ ID NO:
`1. In certain embodiments, the oligonucleotide has a nucleo(cid:173)
`base sequence consisting of the nucleobase sequence SEQ ID
`NO: 1.
`[0028]
`In certain embodiments, the antisense compound
`comprises a conjugate group or terminal group.
`[0029]
`In certain embodiments, the antisense compound
`consists of the antisense oligonucleotide.
`[0030]
`In certain embodiments, the antisense compound is
`also administered systemically. In certain embodiments, the
`systemic administration is by intravenous or intraperitoneal
`injection. In certain embodiments, systemic administration
`and the administration into the central nervous system are
`performed at the same time. In certain embodiments, sys(cid:173)
`temic administration and the administration into the central
`nervous system are performed at different times.
`[0031]
`In certain embodiments, the invention provides sys(cid:173)
`temic administration of anti sense compounds, either alone or
`in combination with delivery into the CSF. In certain embodi(cid:173)
`ments, pharmaceutical compositions are administered sys(cid:173)
`temically. In certain embodiments, pharmaceutical composi(cid:173)
`tions
`are
`administered
`subcutaneously.
`In
`certain
`embodiments, pharmaceutical compositions are adminis(cid:173)
`tered intravenously. In certain embodiments, pharmaceutical
`compositions are administered by intramuscular injection.
`[0032]
`In certain embodiments, pharmaceutical composi(cid:173)
`tions are administered both directly to the CSF ( e.g., IT and/or
`ICY injection and/or infusion) and systemically.
`[0033]
`In certain embodiments, the invention provides
`methods of administering to a subject having at least one
`symptom associated with SMA, at least one dose of an anti(cid:173)
`sense compound comprising an oligonucleotide consisting of
`15 to 20 linked nucleosides and having a nucleobase sequence
`which is 100% complementary to SEQ ID NO. 7 over its
`entire length, and wherein each nucleoside is a 2'-MOE modi(cid:173)
`fied nucleoside; and wherein at least one dose is between 0.1
`mg/kg and 5 mg/kg administered to the CSF. In certain such
`embodiments, the dose is between 0.5 mg/kg and 2 mg/kg. In
`certain embodiments, at least one dose is administered by
`bolus injection. In certain such embodiments, the dose is
`administered by bolus intrathecal injection. In certain
`embodiments, at least one second dose is administered. In
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`certain such embodiments, the second dose is administered at
`least 2 weeks after the first dose. In certain embodiments, the
`second dose is administered at least 4 weeks after the first
`dose. In certain embodiments, the second dose is adminis(cid:173)
`tered at least 8 weeks after the first dose. In certain embodi(cid:173)
`ments, the second dose is administered at least 12 weeks after
`the first dose. In certain embodiments, the second dose is
`administered at least 16 weeks after the first dose. In certain
`embodiments, the second dose is administered at least 20
`weeks after the first dose. In certain embodiments, the subject
`is under 2 years old at the time of the first dose. In certain
`embodiments, the subject is between 2 and 15 years old. In
`certain embodiments, the subject is between 15 and 30 years
`old. In certain embodiments, the subject is older than 30 years
`old. In certain embodiments, at least one symptom associated
`with SMA is reduced its progression has slowed. In certain
`embodiments, the oligonucleotide is ISIS396443.
`In certain embodiments, the invention provides
`[0034]
`methods of administering to a subject having at least one
`symptom associated with SMA, at least one dose of an anti(cid:173)
`sense compound comprising an oligonucleotide consisting of
`15 to 20 linkednucleosides and having anucleobase sequence
`comprising which is 100% complementary to SEQ ID NO. 7
`over its entire length, and wherein each nucleoside is a
`2'-MOE modifiednucleoside; and wherein at least one dose is
`administered systemically. In certain such embodiments, at
`least one dose is administered by bolus injection. In certain
`such embodiments, the dose is administered by bolus subcu(cid:173)
`taneous injection. In certain embodiments, the dose adminis(cid:173)
`tered is between 0.5 mg/kg and 50 mg/kg. In certain embodi(cid:173)
`ments, the dose is between 1 mg/kg and 10 mg/kg. In certain
`embodiments, the dose is between 1 mg/kg and 5 mg/kg. In
`certain embodiments, the dose is between 0.5 mg/kg and 1
`mg/kg. In certain embodiments, at least one second dose is
`administered. In certain such embodiments, the second dose
`is administered at least 2 weeks after the first dose. In certain
`embodiments, the second dose is administered at least 4
`weeks after the first dose. In certain embodiments, the second
`dose is administered at least 8 weeks after the first dose. In
`certain embodiments, the second dose is administered at least
`12 weeks after the first dose. In certain embodiments, the
`second dose is administered at least 16 weeks after the first
`dose. In certain embodiments, the second dose is adminis(cid:173)
`tered at least 20 weeks after the first dose. In certain embodi(cid:173)
`ments, the subject is under 2 years old at the time of the first
`dose. In certain embodiments, the subject is between 2 and 15
`years old. In certain embodiments, the subject is between 15
`and 30 years old. In certain embodiments, the subject is older
`than 30 years old. In certain embodiments, at least one symp(cid:173)
`tom associated with SMA is reduced its progression has
`slowed. In certain embodiments, the oligonucleotide is
`ISIS396443.
`In certain embodiments, the invention provides
`[0035]
`methods of administering to a subject having at least one
`symptom associated with SMA, at least one dose to the CSF
`and at least one systemic dose of an antisense compound
`comprising an oligonucleotide consisting of 15 to 20 linked
`nucleosides and having a nucleobase sequence which is
`100% complementary to SEQ ID NO. 7 over its entire length,
`and wherein each nucleoside is a 2'-MOE modified nucleo(cid:173)
`side. In certain such embodiments, the CSF dose is between
`0.1 mg/kg and 5 mg/kg. In certain embodiments, the systemic
`dose is between 0.5 mg/kg and 50 mg/kg. In certain embodi(cid:173)
`ments, at least one CSF dose is administered by bolus injec-
`
`tion. In certain such embodiments, at least one CSF dose is
`administered by bolus intrathecal injection. In certain
`embodiments, at least one systemic dose is administered by
`bolus injection. In certain such embodiments, at least one
`systemic dose is administered by subcutaneous injection. In
`certain embodiments, the CSF dose and the systemic dose are
`administered at the same time. In certain embodiments, the
`CSF dose and the systemic dose are administered at different
`times. In certain embodiments, the subject is