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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 ????