Download Smn - Nationwide Children`s Hospital

Proximal Spinal Muscular Atrophy
•
•
•
•
•
•
autosomal recessive motor neuron disease
incidence: 1:10,000 live births
carrier frequency: 1:40-60
loss of spinal a motor neurons in anterior horn of spinal cord
atrophy of limb and trunk muscles
clinical grades
– Type I (Werdnig-Hoffmann): cannot sit or walk; death>2 yr
– Type II: able to sit but cannot walk unaided; death<4 yr
– Type III (Kugelberg-Welander): can walk unaided initially;
progressive proximal muscle weakness; normal lifespan
– Type IV (adult onset): very mild phenotype; normal lifespan
SMN in a healthy individual




90+% of SMN
transcript
10% SMN
transcript
Healthy
individual
SMN encoded by
two genes:
SMN1 and
SMN2
SMN1 produces
mostly full length
SMN protein
SMN2 produces
mostly truncated
SMN protein
SMN in an SMA patient




90+% of SMN
transcript
10% SMN
transcript
90% of SMN from
SMN2 lacks exon
7 and is rapidly
degraded
Results in low
SMN levels
*BUT*
The remaining
full-length SMN
partially
compensates
SMN2 copy
number is main
determinant of
phenotype
Modeling SMA in mice and points on SMA
1) SMA is due to reduced SMN levels not loss of SMN
2) SMN is found in all tissues
3) Removal of functional SMN is embryonic lethal
4) Removal of SMN from specific tissue using Cre/Lox results
in destruction of that tissue. Liver, Muscle, Nerve
?
Where and When are high levels of SMN required.
Does SMN2 produce sufficient SMN for normal function of most
tissues? How do Mild Missense mutations work?
What function of SMN is altered in SMA?
SMN2 Transgenic Mice
A
1 2
B
3
Br-Ca dherin
hom olog
ESTs
B
4
B
BBB
----->
H4F5
B
Ag1CA
PAC 215P15 (SMN-2)
----->
B
B B B
<------
SMN2
NAIPD5
35.5kb
NH 89 566 NH
2.4kb
Founders transmitting SMN-2
Mouse models of SMA
Loss of mSmn -/- or ( Schrank et al. PNAS 1997)
1 copy of SMN2 on Smn-/- background is embryonic lethal
SMN2+/+;Smn-/- mice have a severe SMA phenotype; die at 5.16 days
8 or 16 copies of SMN2 but Smn-/- complete normal but tail a little
shorter
(Hsieh-Li et al. NG 24 66-70 2000, Monani et al. HMG9 333-339 2000)
SMN2;D7 SMN2;Smn-/- mice survive 14-15 days on average
Le et al. HMG ( 2005)
SMN A2G;SMN2;Smn-/- mice have a mild phenotype; survive
to 1 year or more
( Monani et al. JCB 2003)
Measures of motor circuit function in SMA mice
Nerve Conduction studies in the severe SMA mouse
Recording
electrodes
Ground
electrode
Dr. Dave Arnold CMAP different at all time points, MUNE from day 7
Stimulating
electrodes
When are high SMN levels above those that occur in
SMA needed ?
Experiment :Correction of relatively severe SMA mice
Delta 7 SMA mice usually live 14days. ( 2 copies
SMN2 present in all mice )
1) Cre induction of activity with tamoxifen cause
mouse SMN to be produced at high levels
2) TRE promoter binds rtTA in presence of doxycycline
activates SMN and luciferase expression.
Conclusions from SMN inducible mice
Induction of SMN in SMA mice which have symptoms
does increase survival.
Best efficacy when induction occurs early. Newborn
screening and early introduction of therapy needs to be
considered as well as design of clinical trials in neonates.
Late SMN induction no effect on SMA mice
Removal of SMN induction in SMA mice after 28 days does not
result in a neuromuscular defect. Mice still die but do not show
abnormal NMJ Electrical physiology. End plate current amplitude
so quantal content of NMJ normal and facilitation reponse normal.
Critical postnatal window for high SMN levels
Where is SMN required?
over-expressed SMN in muscle or nerve
• High expression in nerve
prion92-SMN rescue
• High expression in mature muscle
HSA69-SMN no effect
• High expression in muscle, low in nerve
HSA63-SMN rescue
Question: What happens when one reduces (not eliminate) SMN in just neurons or
just muscle?
Use cre drivers to either delete or replace Smn exon 7 in a specific tissue
(with SMN2 giving low levels of SMN everywhere)
Cre expression pattern:
tdTomato reporter
JAX no.
003771
006410
012687
003966
007893
010802
muscle
Lumbar spinal cord
Nestin-Cre
line
Rat nestin-Cre
Mouse ChAT-IRES-Cre
Hu Synapsin-iCre
Rat synapsin-Cre
Mouse myf5-Cre
Mouse SOX2-Cre
ChAT-Cre
expression
all neurons and glia
cholinergic neurons
all neurons, vasculature, low in muscle
all neurons, variable in motor neurons
muscle precursors, some MN
ubiquitous (control)
tdTomatoRFP HB9:GFP
huSyn-Cre
ratSyn-Cre
Myf5-Cre
No difference in muscle physiology upon deletion
of Smn from muscle (Myf5-Cre)
Measurement of normalized specific force, strength
Tests muscle damage by measuring force drop
after repeated contractions
The expression of the Nestin-Cre driver in the spinal cord.
Nestin-Cre is a poor Motor neuron driver so not a surprise it does not correct SMA mice
completely
SMN is reduced or restored by Nestin and ChAT-Cre and survival increased or decreased
Some mice normal survival and motor neuron function
Conclusions
•Expression of SMN from two copies of
SMN2 is sufficient for normal function
and survival in muscle but not neurons.
•Deletion of Smn from muscle results in interneurons
proprioceptive neurons
normal muscle physiology and fiber size.
•High SMN expression in neurons is
sufficient for rescue of SMA mice.
•High SMN expression is required in
motor neurons and other neurons for
normal function.
motor neurons
•Replacement of SMN in Motor neurons
Restores EPC and quantal content.
Removal of high SMN from neurons
results in MUNE and CMAP typical of
SMA mice. Nestin/Chat Cre most severe.
High SMN expression http://universe-review.ca/I10-85-proprioceptors.jpg
is required in the motor
circuit. Maybe in autonomic neurons?
SMN is in a complex that assembles Sm protein onto snRNA
SMN and domains and missense mutations
Self association
36
Gemin2
binding
Sm binding
51
88
1
1
2a
A2G
D44V
Tudor
2b
52
28
252
156
K-rich
N-
Self association
P-rich
3
92
4
159
YG
5
210
280
6
242
-C
7
279
294
S262I
Y272C
Severe mutation
H273R
Mild mutation
T274I
G279V Q282A
A111G
I116F
E134K
•None of the missense mutations can rescue
•Smn-/- mice in the absence of SMN2 on their own
•Cannot function as homomers
•All mild missense mutations can rescue Smn-/- mice
•In the presence of SMN2
•Functional at heteromers
Adapted from: Sarachan et al. Biochem J. 2012
Martin R, et al Cell 2012
survival
(Smn ) alleles in mice and allelic complementation
snRNP assembly
SMN
mutant
-/-
1 SMN2
2 SMN2
embryonic lethal
5 days
Severe mutant alleles complement SMN2 poorly
SMN I116F
14 days
SMN Delta 7
14 days
Mild mutant alleles complement SMN2 well
N-terminal
SMNA111G
>250 days (Workman 2009)
SMNA2G
>250 days (Monani 2003)
SMND44V
45 days to date n=3
C-terminal
SMNQ282A
>250 days (n=25)
SMNT274I
>250 days
low
low
high
high
?
high
?
Mild SMN
mutation
2 copy SMN2
mSmn -/-
RESCUE
Mild SMN
mutation
1 copy SMN2
mSmn -/-
RESCUE
Mild SMN
mutation
0 copy SMN2
mSmn -/-
LETHAL
•Mild SMN alleles oligomerize with the small amount of SMN from 1 or 2 copies of SMN2,
to create a heteromeric complex that is functional in snRNP assembly and can rescue the
SMA mice but does not rescue Smn-/- mice. Do SMN missence alleles complement?
Complementation of SMN complex by the mutations
SMNA111G and SMNT274I
SMNA111G and SMNQ282A
A111G
T274I
No rescue
No rescue
Rescue of Smn-/- mice.
Fully capable of snRNP
assembly
Homomeric SMN complex
•N-terminal and C-terminal missense mutations can complement each other
•Completely consistent with Martin et al, 2012 (Van Duyne lab)
Thus no genetic evidence against snRNP assembly as key function
in SMA
Analysis of changes ( splicing and SMN) that occur in motor neurons with
Laser Capture Microdissection
Reduced Full length SMN in motor neurons of
SMA mice
Summary
1) SMA motor neurons pattern normally in SMA mice. There is no correlation
with SMN mutants that do or do not rescue SMA mice and the rescue of
axonal defects in fish.
2) SnRNP assembly is altered as are levels of certain snRNPs and there is
correlation of activity and correction.
3) Complementation indicates the functional SMN is oligomeric and requires
the C and N terminus of SMN to function correctly. Appears that when
lethality is rescued so is SMA thus likely that critical function is snRNP
assembly..
4) SMN2 splicing even less exon7 incorporation in motor neurons.
5) There are 20 (limited) splicing alterations in SMA motor neurons which
are not detectable in total spinal cord only in purified motor neurons. Which
is critical? ? Can scAAV9 expression determine which gene(s) suppress
SMA
Postnatal therapies for SMA
1) Drug compounds: That induce sufficient levels of SMN
from SMN2. PTC/Roche have new compounds that are
effective in mice. Science
2) Anti sense Oligonucleotide that alter splicing of SMN2
by blocking splice inhibitors and inducing SMN. Either MOE
( methoxyethyl) or MO ( morpholinos)
3) Gene therapy AAV9 –SMN does cross the blood barrier
Kevin Foust and Brian Kaspar Nationwide Children’s.
What happens with postnatal injections of scAAV9-SMN?.
Foust et al. (Nat Biotechnol 27, 59-65 (2009) and
Duque et al. Mol Ther 17, 1187-1196 (2009)
Oral Administration of D156844-04 to SMND7
SMA Mice
• SMND7 SMA mice receiving D156844-04 (3 mg/kg/day) at PND04
had a significant (~21%) increase in mean lifespan (17.0±0.5 days
vs. 14.0±0.4 days; C2=16.7)
• prenatal treatment (starting at E11) increased lifespan by ~38%
(18.0±0.5 days vs. 13.0±0.7 days; C2=15.0) Butchbach et al
HMG2010
Need drugs that give better induction of SMN from SMN2
PTC-SSN significantly improves body weight and motor
function in D7 SMA mice
 IP dosing 10 mg/kg once a day P3 – P23, oral dosing 30 mg/kg twice a day
P24 – P144
Body weight
Survival
45
100
Body weight  SEM (g)
Percent survival
40
75
50
MST=15d
MST=130d
25
SMA + vehicle (n=15)
SMA + PTC-SSN (n=16)
Het + vehicle (n=6)
25
50
75
100
125
30
25
20
15
SMA + vehicle (n=15)
SMA + PTC-SSN (n=16)
Het + vehicle (n=6)
10
5
0
0
35
0
150
0
Postnatal day
25
50
75
100
125
150
Postnatal day
 Mice do not have necrosis, exhibit normalized phenotype
Slide 25
© 2011 PTC Therapeutics, Inc.
SMN protein increase
(RT-qPCR in SMA fibroblasts)
(HTRF in SMA fibroblasts)
SMN protein
Fold relative to DMSO  SD
SMN2 alternative splicing correction
2.0
1.5
SMN2 FL
1.0
SMN2 D7
0.5
0.0
0.001
0.01
0.1
1
10
1.8
1.6
1.4
1.2
1.0
0.8
0.001
0.01
0.1
1
10
Concentration (M)
Concentration (M)
Gems count increase
(SMA fibroblasts)
Gems per 100 nuclei  SD
SMN2 alternative splicing correction
(end-point RT-PCR in SMA fibroblasts)
DMSO
SMN2 mRNA
Fold relative to DMSO  SD
PTC-SSN corrects SMN2 alternative splicing and increases
SMN protein and gems count in vitro in SMA patient cells
0.003 µM
3µM
FL
Δ7
50
Het (est.)
40
30
20
10 DMSO
0
0.0001 0.001
0.01
0.1
1
Concentration (M)
Slide 26
© 2011 PTC Therapeutics, Inc.
(ISS-N1) Antisense Oligo
Nucleotide Therapy for SMA
Methods
• Breeders: Smn+/-, SMN2+/+, ∆7+/+
Smn +/+
Smn +/Smn -/-
2µL Morpholino Injection
Scramble (control)
ISS-N1 (27ug, 40.5ug, 81ug)
ISS-N1 (27ug, 40.5ug, 81ug)
Average newborn mass: 1.5g
•Outcomes:
–Survival, mass
–Protein
–RNA quantification
Average Smn -/- survival: 14.6 days
RT-PCR after ASO injection
Spinal cord
Day
27 µg
14
7
21
28
55
45
65
Scramble
HiC WT
Full Length SMN2
SMN2∆7
Spinal cord
Day
81µg
14
21 28
45
55
65
Scramble
HiC
WT
Full Length SMN2
SMN2∆7
Brain
81µg
Day
7
14
21
28
45
55 65 Scramble HiC WT
Full Length SMN2
SMN2∆7
-primer specific to SMN2 (does not amplify ∆7 or Smn)
-HiC= 16 copies SMN2, Smn -/-
SMN increased after ASO delivery
3
2.5
2
Intensity
SMN/Actin
1.5
1
0.5
0
7 da y
28 da y
45 da y
45 da y
65 da y
65 da y
Scra m ble
HiC
Mouse/Day
Day
SMN
Actin
7
28
27 ug ISS-N1
45
65
WT Scramble HiCopy
Survival: ISS-N1 MO P0 ICV Injection
140
83
104
2mM
4mM
112
Median Survival (days)
120
100
80
60
40
20
0
6mM
Intraventricular Dose
Porensky et al Hum Mol Genet. 2012 Apr 1;21(7):1625-38.
Antisense chemistries used that work in SMA mice to date
MOE backbone
40ug CSF delivery survival 100 days
20ug CSF survival 20 days
Hua et al. 2010 Genes and Development 24:1634-44
Porensky et al 2012 Hum Mol Genet. Passini et al. Sci Transl Med. 2011 Mar 2;3(72):
21:1625-38. (Delat 7 Mouse)
Zhou et al 2013 Hum Gene Ther.
24:331-4 (Taiwanese mouse)
Multiple doses at 150ug/g systemically
and ICV Approx 200 days (Taiwanese mouse)
Hua et al 2011 Nature 478: 123-6
In Clinical trials via CSF delivery
Not in clinical trial
2-0-methyl ASOs have not been effective
Early SMN Restoration: ASO at P0
CMAP 12 days
40
*
30
20
*
MUNE 12 days
400
Estimated # of MU
Amplitude mV
A
*
300
200
100
10
0
0
SMA
B
MUNE 30 days
Amplitude mV
40
30
20
10
0
Estimated # of MU
CMAP 30 days
400
300
200
100
0
No fibrillations
With correction
Symptomatic ASO delivery: Survival, weight, and righting reflex
EIM non invasive measure
Measured parameters of EIM:
• Reactance (ohm)
• Resistance (ohm)
• Phase angle is calculated (degrees)
Muscle modeled as resistor-capacitor
circuits
Resistor: extra/intracellular fluids
Capacitor: cell membranes
Symptomatic ASO: EIM at P12
GENE DELIVERY TO TREAT SMA
PRE-CLINICAL STUDIES WITH THE SMN∆7 SMA
MOUSE MODEL

Mt
AAV2
ITR
scAAV9-SMN construct
CMV
enhancer

Chicken b-Actin
Promoter
SV40
Intron
Human SMN mRNA
BGH
Poly A
AAV
2 ITR
SMN∆7 SMA Mouse Model


Molecularly mimics disease
Severe model
Severe atrophy
 End-stage ~16 days of age

Le, et al. Human Mol Gen 14(6) 845-857
ONE-TIME GENE DELIVERY OF SCAAV9-SMN
RESCUES SEVERE MOUSE MODEL OF SMA
Evaluating scAAV9-GFP in Non-Human Primates
In Situ Hybridization Reveals Robust
Transduction in Ventral Spinal Cord
Intrathecal injection of scAAV9-shRNA in 5 day old piglets
to create large animal model of SMA what does it predict
Maps of the scAVV9 vectors
scAAV9-shRNA
mut ITR
scAAV9-SMN
mut ITR
shRNA1
H1 promoter
CBA promoter
CBA promoter
Intron
Intron
SMN
 Intrathecal injection
Head
Needle
Toward
tail
+ 1min
+ 2min
GFP
ITR
ITR
Transduction profile in scAAV9-SMN treated piglets
Group
Control
shRNA
SMN early
SMN late
number of
animal
6
5
5
5
Age at
sacrifice
79 ± 3
61 ± 7
71 ± 2
73 ± 3
Immunosuppresive
treatment
Prograf
Prograf + Cellcept
Prograf + Cellcept
Age at injection
Dose vector (vg/kg)
scAAV9-shRNA scAAV9-SMN scAAV9-shRNA scAAV9-SMN
PND5
6.5x1012
12
PND5
PND6
6.5x10
8x1012
12
PND5
PND33-36
6.5x10
8x1012
(Prograf = tacrolymus, FK506. Cellcept = Mycophenolate mofetil. Block T-cell response)
SMN
GFP
Merge
% of motor neurons
Spinal cord section from lumbar 6 segment stained for GFP and
human SMN (human specific antibody)
90
SMN
80
GFP
70
60
50
40
30
20
10
0
SMN
GFP
Merge
early
late
Transduction profile in scAAV9-SMN treated piglets
Group
Control
shRNA
SMN early
SMN late
number of
animal
6
5
5
5
Age at
sacrifice
79 ± 3
61 ± 7
71 ± 2
73 ± 3
Immunosuppressive
treatment
Prograf
Prograf + Cellcept
Prograf + Cellcept
Age at injection
Dose vector (vg/kg)
scAAV9-shRNA scAAV9-SMN scAAV9-shRNA scAAV9-SMN
PND5
6.5x1012
12
PND5
PND6
6.5x10
8x1012
12
PND5
PND33-36
6.5x10
8x1012
(Prograf = tacrolymus, FK506. Cellcept = Mycophenolate mofetil. Block T-cell response)
SMN
GFP
Merge
% of motor neurons
Spinal cord section from lumbar 6 segment stained for GFP and
human SMN (human specific antibody)
90
SMN
80
GFP
70
60
50
40
30
20
10
0
SMN
GFP
Merge
early
late
Intrathecal injection of scAAV9-shRNA leads to development of
symptoms resembling SMA
Smn mRNA levels from laser captured
motor neurons
Time course of symptoms progression
PND5
PND24-34
PND33-47
PND38-55
injection1st sign of weakness Mainly sitting Mainly crawling
Splayed gait
and sitting
position
Axons labelled
PND46-69
sacrifice
Control
Early rescue
Late rescue
No rescue
Correlation of MUNE, CMAP and Motor Neuron loss with SMN
restoration in the pig.
Motor neuron path also improved
CMAP
25
Early = day after shRNA
Late = First symptoms
20
MUNE
450
400
350
300
15
250
200
10
150
100
5
50
0
0
Motor neuron cresyl violet
120
60
control
50
40
30
Normal
100
shRNA
80
Pig SMN levels in motor neurons
Dorsa
l
shRNA
early
late
Early
rescue
60
Late
rescue
20
40
20
10
0
0
control
shRNA
early
late
Summary
scAAV9-SMN results in over a year survival of SMA mice survival. scAAV9
is even more effective in larger animals ( primates). Intravenous and
intrathecal delivery can be considered both target motor neurons. IND
given clinical phase 1 trial started. Early results positive no adverse
affects and still alive. Early indroduction
Made pig with SMA and corrected at different time points. Symptomatic
Rescue does work and CMAP an MUNE dose improve.
Antisense oligonucleotide (ASOs) that block ISS-N1 increase SMN and
and extends survival of SMA animals to over 100 days with a single ICV.
MOE ISSN1 in clinical trials for both type 1 and II/III with repeat
dosing
Drugs that induce SMN well do have a major impact on SMA mouse
survival. Roche/PTC and Norvartis in clinical trials
Clinical trials are in progress for all Dr Mendell reviewed gene therapy
trial. All looks good early introduction is likely to be key. Next steps can
you add a agent to get more from the remaining MNs in patients that have
had a large loss of MNs.
Acknowledgement
Arthur Burghes
Sandra Duque
Paul Porensky
Narasimhan Madabusi
Vicki McGovern
Xiaohui Li
Kathrin Marenhke
Anton Blatnik
Thanh Le
Corey Ruhno
W. Dave Arnold
Stephen Kolb
Daniel Schumperli
Philipp Odermatt
Brian Kaspar
Kevin Foust
Adam Bevan
Leah Schmelzer
Lyndsey Braun
Kathrin Meyer
Steve wilton
Mitrpant
Price
Sue Fletcher
Livio Pellizzoni
Luciano Saieva
Gabanella
Matteo Ruggi
Large animal suite in Wiseman Hall
Dondrae Coble
Lori Mattox
Crystal Sims
Michelle Creamer
. Mark Rich
Xueyong Wang
Re-introduction of SMN improves
electrophysiology of SMA piglets
Sciatic CMAP
Sciatic MUNE
(PND53-55)
450
20
15
10
*
# of motoneurons
Amplitude (mV)
25
(PND53-55)
control
400
shRNA
350
Early
300
Late
250
200
150
*
100
5
50
0
0
* P=0.00016
* P=0.00003
Group
shRNA
early SMN
late SMN
control
Number of pig
5
5
5
6
Fibrillations Percentage
5
100%
1
20%
3
60%
0
0%
vascular delivery of scAAV9-SMN: IND
September 20, 2013: FDA Approval to Nationwide Children’s Hospital for Phase I Clinical Trial of
Systemic (vascular) AAV9-Delivered SMN Gene For Spinal Muscular Atrophy.
The clinical trial is expected to begin in early 2014 and will be limited to Type I SMA patients, ages
birth to 9 months.
Principal Investigator Brian Kaspar, PhD,
Jerry Mendell, MD, director, Center for Gene Therapy at Nationwide Children’s
October 17, 2013: Nationwide Children’s Hospital received Fast Track designation from the U.S.
Food and Drug Administration for its scAAV9.CB.SMN gene therapy product for the treatment of
spinal muscular atrophy (SMA).
ICV delivery of scAAV9-SMN: pre-IND
Currently addressing:
dose response in the spinal cord
number of motor neurons transduced
Clinical Trials with ISIS-SMNRX
A) In type 2 and 3 SMA Multiple at 9mg (days 1, 29 and 85) results at 3
months in 3.7 point increase in HFMSE score. No change in CMAP in
single dose? Multiple dose is similar to single dose ? Why? All SMA open
trials have been positive.
B) does SMN induction need to occur early ?
C) Increase in SMN detected in CSF in multiple dose trial good!
C) 12 mg dose started.
D) Infant multiple dose trial started very interesting as early introduction will
occur.
Can morpholinos be used? Patent?
Nationwide children’s/OSU
Gene therapy with scAAV9-SMN in infants will start soon so interesting times.
IND approved
Acknowledgments
Burghes lab
Dr. Sandra Duque (Pig)
Dr. Vicki McGovern
Dr. Thanh Le
Dr. Paul Porensky
Dr. Dave Arnold (EMG)
r Steve Wilton
Dr. Chalermchai Mitrpant
Loren Price
Sue Fletcher
Price L,
r. Brian Kaspar Lab
Dr. Kevin Foust
Lyndsey Braun
Kathrin Meyer
• Funding
– National Institute of
Health (NINDS,NICHD)
– Families of SMA
– Miracles for Madison
– Matthew & Preston
Foundations
– SMA Angels
ISIS phase 1 single dose trial
No significant change in CMAP? How has the motor neuron improved?
ISIS-SMNRX Clinical trial
1) 2’-o-methoxyethyl modified (MOE) ASO drug
2) Corrects SMN2 splicing
3) Extends survival in SMA mouse model 20 days
vs 14 days on CNS delivery (Passani et al) .
Some tox at higher levels.
4) MOE distributes broadly in adult mice and
monkeys and adult mice. How important is charge in
distrubution
5) Long half life in CNS
6) First trial single bolus dose patient aged 2-14
7) All SMA open label trials have been positive ????