Mammalian Models of Spinal Muscular Atrophy

C H A P T E R

15 Mammalian Models of Spinal Muscular Atrophy A.H.M. Burghes1, C.J. DiDonato2,3, V.L. McGovern1, W.D. Arnold1 1The

Ohio State University, Columbus, OH, United States; 2Feinberg School of Medicine, Chicago, IL, United States; 3Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, United States

O U T L I N E Introduction241 Mouse Models of Spinal Muscular Atrophy

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Severe Spinal Muscular Atrophy Mouse Models

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The Use of Severe Spinal Muscular Atrophy Mouse Models to Determine Where High Survival Motor Neuron Expression Is Required247 The Use of Severe Spinal Muscular Atrophy Mouse Models to Determine When High Survival Motor Neuron Expression Is Required249 Mild Spinal Muscular Atrophy Mouse Models

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INTRODUCTION Spinal muscular atrophy (SMA) is an autosomal recessive disorder that results in motor neuron loss and muscle weakness. The main form of proximal SMA is caused by loss or mutation of the survival motor neuron 1 gene (SMN1) and retention of the SMN2 gene.1 This results in low levels of SMN proteins that are insufficient for motor neuron function. In humans, chromosomes that lack both SMN1 and SMN2 exist; however, a pair of these chromosomes is never found together in an individual.2,3 The simplest explanation is that complete loss of SMN1 and SMN2 is incompatible with survival. Indeed, in mice, the loss of the single Smn gene results in early embryonic lethality with death occurring very early in embryogenesis.4 The presence of the SMN1 and the SMN2 gene occurs only in humans.5 The only exception in SMN gene duplication occurs in the chimpanzee

Spinal Muscular Atrophy http://dx.doi.org/10.1016/B978-0-12-803685-3.00015-X

How Does Mouse Pathology Relate to Spinal Muscular Atrophy Patients?

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Electrophysiological Measures in Mouse Models

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Genetic Missense Mutations of Survival Motor Neuron253 Genetic Background Check

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Mouse Models Use in Therapeutic Testing and Large Mammalian Models of Spinal Muscular Atrophy254 Conclusions256 References256

that carries a duplication of the SMN1 gene.5 All other organisms contain only a single Smn gene equivalent to the SMN1 gene. Thus the deletion of the Smn gene in any mammalian organism is lethal. In contrast, nonmammalian species have maternal SMN (mRNA) contribution from the yolk and thus survive through various stages of embryonic development following deletion of Smn. In humans, the SMN1 and SMN2 genes, located on chromosome 5q13, are nearly identical. None of the nucleotide differences between the two genes result in a change in the encoded amino acid sequence. However, the one crucial difference is the C to T transition at the sixth nucleotide in exon 7 of SMN2. This alteration results in disruption of an exon splice modulator that causes the majority of SMN2 mRNA transcripts to lack exon 7.6–10 SMN mRNA transcripts, which lack exon 7, result in an SMN protein that does not oligomerize efficiently and is rapidly degraded.11–14 Some full-length SMN (∼10–20% of

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© 2017 Elsevier Inc. All rights reserved.

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15.  MAMMALIAN MODELS OF SPINAL MUSCULAR ATROPHY

transcripts) is produced from SMN2, and in humans, two copies of SMN2 produce sufficient SMN for the survival of most cell types. However, for unknown reasons, two copies of SMN2 are insufficient for normal motor neuron function.15 As complete loss of SMN is embryonic lethal, it appears all cells need some SMN to survive. The requirement of SMN is not surprising given that SMN functions in placing the Sm protein ring onto the small nuclear RNAs (snRNAs) which are then transported into the nucleus where they function in splicing (see Chapters 6 and 7). There are three important considerations when working with mouse models of SMA. First, SMA is not caused by complete loss of SMN protein. Instead SMA results from the reduced amount of SMN produced from the SMN2 gene15 (Table 15.1; also see Chapter 1). This situation has been recreated in animals using various methods. The first viable animal models were created by TABLE 15.1  SMN2 Modification of SMA Severity in Humans and Mouse SMN2 Modification of Homozygous Loss of SMN1

Copy# Human SMA

Mouse Models (Human SMN2 Transgene/Promoter)

0

No reports of an individual with zero copies of SMN2

Embryonic lethal Incompatible with cellular function4

1

Type 0: Weakness before or shortly after birth

Still born or die in utero17,26

2

Type 1 or 2: Nonambulatory (∼80% of SMA type 1 is associated with 2 copies)

6–8-day survival16,17 Addition of human SMN2 cDNA lacking exon 7 (SMNΔ7) extends survival to about 2 weeks28

3

Type 2 or 3: Ambulation more likely (∼80% of SMA type 2 is associated with 3 copies) In the case of type 3 individuals, about 50% have three copies and 45% have four copies35,137 When type 3 individuals are divided into 3a and 3b, then 62% of 3a have two or three copies, whereas 65% of 3b have four or five copies138,139 Elsheikh et al., showed that patient onset was stratified by three vs. four copies of SMN2140

Four copies of SMN2 results in features of necrosis starting in the tail at about 3 weeks but no overt weakness16

4

Type 4: Adult onset, ambulatory141 Most cases have 4–6 copies of SMN2138,139

>4

Individuals reported to be normal with increasing numbers of copies of SMN2142

transgenically introducing the SMN2 gene into mice containing a disruption of the mouse Smn gene.4,16,17 By intercrossing these mice, progeny contained two copies of SMN2, no functional mouse Smn (SMN2+/+; Smn−/−), and display hind limb weakness and death at 5 days of age. In contrast, the complete removal of SMN in any organism results in lethality. For example, selective deletion of Smn using Cre-loxP technology does not model human SMA, as this leads to complete removal of Smn in those cells that express Cre recombinase. Second, one must consider the particular SMN2 allele used and its location in the mouse genome. The SMN2 gene can be a randomly integrated transgene or it can be inserted into the mouse Smn locus. Different SMN levels and variable expression between tissues can result from different insertion sites of the transgene in the mouse genome. As such, different mouse models containing SMN2 transgene are not identical in their phenotypic presentation. For instance, the denervation state of various muscles is not the same in all mouse models of SMA (discussed later). One must know the model well to correctly interpret the experimental endpoints and outcome in a study. Finally, one must consider how well the model represents the major phenotypic features noted in patients with SMA. SMA mouse models must display reduced levels of SMN and ideally, muscle weakness. Given that the essential function of SMN is the assembly of snRNPs, which is disrupted in SMA one would expect alteration of splicing in the SMA mouse. Yet the possibility of differential intron sensitivity between species should be considered. If a particular intron is sensitive to SMN deficiency in humans due to its sequence and structure, this sensitivity might not replicate in the mouse. Furthermore, to show altered splicing of a particular intron in the mouse, SMN may need to be reduced to a level where other tissues/cell types become dysfunctional, resulting in changes that do not represent SMA in humans. This possibility is underscored by the fact that mouse models have a dramatic response to a very small SMN increase. For example, four SMN2 copies on a null Smn background results in a mouse with a relatively normal motor neuron physiology, whereas in humans, four copies of SMN2 results in type 3 SMA or type 4 SMA with pronounced motor neuron deficits (also see Chapter 1). The objective of this chapter is to provide an overview of the mammalian SMA models that have been developed and the insights that they have provided to our understanding of the disease.

MOUSE MODELS OF SPINAL MUSCULAR ATROPHY Normal phenotype with 8 copies of SMN217

Mouse models have been used most extensively to study SMA. The main factor contributing to the variability of SMA clinical severity is SMN2 copy number.18

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Severe Spinal Muscular Atrophy Mouse Models

Unlike humans, all other mammals have a single homolog of the SMN1 gene. The mouse Smn gene is 82% identical at the amino acid level, ubiquitously expressed, and similar to SMN1 as it is not alternatively spliced.19–21 Homozygous loss of the Smn gene results in embryonic lethality and massive cell death, but mice that are heterozygous for null alleles of Smn are phenotypically normal.1,4 Therefore, strategies have been utilized to incorporate a human SMN2 transgene onto a mutant Smn background or modify the endogenous Smn locus to produce reduced levels of Smn. A series of SMA mouse models with variable phenotypes and severities have been produced and the most commonly used are summarized in Table 15.2. The Jackson Laboratory (JAX) has an online reference guide for mouse models of SMA (https://www.jax.org/jax-mice-and-services/ customer-support/manuals-posters-and-guides/ jmcrs-manuals-guides/manuals-guides-models-spinalmuscular-atrophy). The success of advancing research and the rapid development of therapeutics in the SMA field has been aided by a centralized resource for various mouse lines namely the Jackson mouse repository directed by Dr. Lutz and in the case of SMA mice funded by the SMA foundation. Since the mice are under maximum barrier conditions, they can be rapidly obtained globally by investigators at all institutions.

SEVERE SPINAL MUSCULAR ATROPHY MOUSE MODELS The most commonly used mouse models of SMA contain a deletion or disruption of the mouse Smn gene and the addition of the human SMN2 gene.16,22 Mice containing the single copy integrant SMN2 89 and the two-copy integrant SMN2 Hung are the most commonly used SMN2 transgenic lines. In most cases, the SMN2 transgene gene is a random integrant except those by Osborne et al., which were specifically targeted to the endogenous Smn locus (Fig. 15.1).16,17,23–25 The mouse Smn null allele (Smn2A) was created by inserting the β-galactosidase gene in-frame into Smn exon 2A resulting in complete loss of Smn.4 One copy of SMN2 89 transgene on the Smn2A null background results in embryonic death, while two copies of SMN2 results in an SMA mouse (SMN2 89+/+; Smn2A/2A) in which embryonic motor axon development is normal, with no significant difference in motor neuron numbers at birth; however, by postnatal days 3–5, about 40% of the spinal and facial motor neurons are lost and mice rarely survive past 5 days of age.17,26 Furthermore, these mice show marked denervation in certain muscles such as the intercostals. The Jackson laboratory designates this strain as 5024. In addition to the low copy number line, a high copy number SMN2 line 566 was generated.

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When this is bred onto the Smn null background, mice hemi (8 copy)- or homozygous (16 copy) for this SMN2 transgenic line are normal, except for a slightly shorter tail (SMN2 566+/+; Smn2A/2A). This line tends to live longer than normal FVB/N mice and has completely normal motor unit electrophysiological characteristics,17 (unpublished observation, Arnold). A similar SMA mouse model was reported by others.16 In this particular line, the SMN2 transgene, SMN2 Hung, contains two tandem copies of the SMN2 gene. When placed onto a Smn exon 7 deletion background the mice survived about 10 days. This model is commonly referred to as the “Taiwanese SMA mouse.” It is notable in this case that the mouse Smn deletion allele used is not a null allele. As mentioned previously, Monani et al., used the Smn null allele (Smn2A) that was created by inserting the β-galactosidase gene in-frame into Smn exon 2A resulting in complete loss of Smn.4 Conversely, Hsieh-Li et al., used an Smn exon seven deletion (SmnH), generated by targeted replacement of Smn exon 7 with the hypoxanthine phosphoribosyltransferase (HPRT) selection cassette while maintaining the rest of the locus intact. This allele is capable of producing the transcript lacking exon 7 and as such is not a null allele. The JAX designation for this strain is 5058; SMN2 Hung+/−; SmnH/H. As the transgene has two copies of SMN2 per chromosome, a four copy number SMN2 mouse can be generated which is fertile and has a very mild phenotype, if any. Indeed, these mice do not show a strong denervation phenotype (unpublished observation, Arnold) and have necrosis of the tail that usually extends to the feet and ear pinnae within a few months of life. However, necrosis is not a common feature of SMA in humans. The most commonly used severe model of SMA is the Δ7SMA model, often referred to as “delta 7mice.” This model was created by adding the SMNΔ7 transgene along with the SMN2 transgene onto the Smn null background (SMNΔ7+/+; SMN2 89+/+; Smn2A/2A) (JAX 5025). Previously, studies in patient lymphoblasts suggested that SMN lacking exon 7 had toxic properties.27 However, the addition of a transgene expressing SMNΔ7 to SMN2 89+/+; Smn2A/2A increased survival from 5 to 13 days and demonstrated that SMNΔ7 RNA or protein is not toxic28. On a pure FVB/N background, the mice have a survival of 11 days and the phenotype is more severe on a C57/ Bl6.29 The genetic hybrid with a mixed genetic background is the longest-lived at 13–14 days. Overall, Δ7 SMA mice retain the characteristic pathology of severe SMA mice, including reduced SMN levels, inherent muscle weakness, motor coordination deficits, neuromuscular junction (NMJ) pathology, and motor neuron loss.28 This model has been extensively used for preclinical testing of therapeutics in many different laboratories; some of these therapeutics are currently in clinical development.

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TABLE 15.2  Mouse Models of SMA and Mice Used in SMA Research Description

References

SMN Delta 7 Mouse Model

Smn2A/2A; SMN2 89+/+; SMNΔ7+/+

SMN2 transgene on background of line89 with SMNΔ7 transgene; motor neuron loss by 9 days and survival till 14 days

28

Taiwanese Mouse Model

SmnH7/H7; SMN2 Hung+/–

Homozygous Smn knockout of Smn exon 7 replaced with HPRT gene, BAC SMN2 with two tandem copies of SMN2; mild SMA phenotype. If SMN2 Hung allele is homozygous (4 SMN2 copies) the mouse is normal

16

Low Copy SMN Mice

Smn2A/2A; SMN2 89+/+

Homozygous Smn knockout, two copies of SMN2 transgene. Survival 5 days

17

High Copy SMN Mice

Smn2A/2A; SMN2 566+/+

Eight copies of SMN2. Normal phenotype

17

Three-Copy N11/N46 Mice

Smn2A/2A; SMN2 11+/−; SMN2 46+/−

Three copies of SMN2 (line 11, single insertion; line 46, tandem insertion); average survival 15 or 22 days depending upon breeding

23

Burgheron Mouse

Smn2A/C; SMN2 89+/−

Mice are compound heterozygotes at the smn locus for the SmnC allele25 and Smn null allele4; average life span about 100 days; mice develop severe necrosis; parental line of transgene affects smn and survival

31

Smn+/–

Smn+/2A

Heterozygous SMA mice; reported to have 50% motor neuron loss by 1 year of age; compensation reported to be from CNTF expression; no survival alteration

4,98

2B-

Smn2B/–

Mutation of exonic enhancer (GGA-TTT) in central region of Smn exon 7; motor neuron loss, muscle weakness and atrophy at 21 days; survival about 28 days (C57; CD1 hybrid background)

102,105

SmnC−T/2A

SmnC–T/2A

Smn exon 7 nucleotide change from C to T to mimic SMN2 exon 7 sequence; induces smn exon 7 skipping but not to same extent as human SMN2; when mutation is combined with the null smn allele (smn C–T/−) mice still have a normal life span; if a phenotype is present, it is extremely mild

101

5058 Hemihybrid

SmnD7H7; SMN2 Hung+/–

F1 hybrid from intercross of homozygous Taiwanese mild (4 copy SMN2 on an SmnΔ7 background and heterozygous SmnΔ7- melki mice; low level of escapers; develops tail necrosis and hind limb pedal edema that becomes severe; effects rescued by increasing SMN expression; lifespan about 16 days

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Smn Alleles

Genotype

Description

References

Smn Null Allele

Smn2A/2A

Homozygous Smn knockout. LacZ gene placed in-frame with smn exon 2A; embryonic lethal

4

SmnD7 Allele

SmnD7/D7

Homozygous deletion of Smn exon 7 created by Cre deletion of floxed exon 7; embryonic lethal

64

SmnF7 Allele

SmnF7/F7

loxP-flanked (floxed) Smn exon 7; viable

64

Regeneron SmnA Allele

Smn1tm2Mrph

Smn exon 1–8 replaced with LacZ; null smn allele; homozygotes are embryonic lethal

25

Regeneron SmnB Allele

Smn1tm4(SMN2)Mrph

Hybrid smn allele that replaces a 2.2 kb segment of exon 7 and 8 with a 1.3 kb segment of human SMN2 exon 7 and 8; hybrid allele does not produce any full-length smn transcripts; homozygous state is embryonic lethal

25

Regeneron SmnC Allele

Smn1tm6(Smn1/SMN2)Mrph

Hybrid Smn allele and 1 genomic copy of SMN2 (42 kb in size) downstream of SMN2 exon 8; homozygotes are viable and have variable survival due to necrosis; have a very mild SMA phenotype

25

Regeneron SmnD Allele

Smn1tm5(Smn1/SMN2)Mrph

Hybrid Smn allele and contains three genomic copies of SMN2 (each 42 kb in size) downstream of SMN2 exon 8; homozygotes viable; survival may be impacted by necrosis; no phenotype

25

Tissue Specific SMN2 Transgenes

Genotype

Description

References

PRP-SMN Transgene

Smn2A/2A;

89+/+;

Wild-type SMN transgene under prion promoter expressed on severe SMA background; high level of expression in spinal cord and brain; survival >200 days. No overt phenotype, rescues severe phenotype

58

HSA69-SMN Transgene

Smn2A/2A; SMN2 89+/+; HSA69:SMN+/+

Wild-type SMN transgene under HSA promoter expressed on severe SMA background; high levels of expression in skeletal muscle; several different transgenic lines; no survival benefit over severe SMA mice when only expressed in muscle

58

SMN2 PrP:SMN+/+

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Genotype

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Mouse Models of SMA

HSA63-SMN Transgene

Smn2A/2A; SMN2 89+/+; HSA63:SMN+/+

Smn Missense Mutations Genotype

Wild-type SMN transgene under HSA promoter expressed on severe SMA background; high levels of expression in 58 skeletal muscle and very low in spinal cord; survival >160 days Description

References

Smn2A/2A; SMN2 89+/−; SMNA2G+/−

SMNA2G point mutation; lifespan >1 year; smaller mouse, muscle weakness, reduced number of motor neurons; 124 mice hemizygous for both SMN2 and point mutation; homozygous mice are indistinguishable from controls; mild SMA background; survival only occurs when SMN2 is present

SMNA111G

Smn2A/2A; SMN2 89+/−; SMNA111G+/+

SMN2 and point mutation can be in either a hemizygous or homozygous state. Rescued phenotype only when SMN2 is present. Restores snRNP assembly defects present in severe SMA mice. Life span >1 year

55

SMNVDQNQKE

Smn2A/2A; SMN2 89+/+; SMNVDQNKE+/-

Human SMN transgene that contains SMN exon 1–6 with additional motif; lifespan 2–6 days depending on transgenic line

55

SMN-RT

Smn2A/2A; SMN2 89+/+; SMN-RT

Modified Δ7 cDNA transgene that contains a point mutation at first stop codon to re-create a read-through protein; lifespan average is 34 days; if survival after 40 days, mild necrosis of ear, tail, and around eye but not feet; intermediate phenotype with NMJ defects; rescued with increased SMN expression strategies

93

Inducible SMN Lines

Genotype

Description

References

B-Geo Locus

Tg(Cag-B-geo-SMN2) E9Dscd

Conditionally expresses a human SMN2 cDNA targeted to the B-geo locus; useful for SMN induction or tissuespecific studies

81

2B-Neo

Smn1 tm1Cdid/tm1Cdid; CreEsr

32

CT-Neo

Smn1 tm2Cdid/tm2Cdid; CreEsr

Severe hypomorphs from combination of point mutations to induce smn exon 7 splicing and floxed Pgk-neo cassette; in conjunction with Cre-esr they can be used to increase smn expression through removal of floxed pgkneo cassette within intron 7; embryonic lethal when homozygote

Severe Inducible

Smn2B-Neo/2B-Neo; SMN2 89+/−

Embryonic lethality of 2B-neo homozygotes is rescued by SMN2; can be used to increase smn expression through removal of floxed pgk-neo cassette within intron 7; survival to about 5 days of age without Cre induction

30

Doxycycline Inducible

Rosa26-rtTA:SMN2 89+/+; Smn2A/2A; SMNΔ7+/+; tetOluciferase

Allows luciferase and SMN-inducible expression with doxycycline administration; mean survival is 13 days

82

Tamoxifen Inducible

SmnRes/Res; SMN2 89+/+; SmnΔ7+/+

Allows for smn induction from hybrid rescue allele (smnRes) on an SMNΔ7 background at varying time points by administration of tamoxifen; noninduced survival is about 13 days, variable survival depending upon other time points of induction

83

Chat-Cre, Myf5-Cre or MyoDi-Cre

SmnRes/Res; SMN2 89+/+; SmnΔ7+/+

Used for tissue-specific induction of smn on SMNΔ7 background; Chat-Cre survival increased from 15 to 23 days, less for myf5 or myoDi Cre + SMA mice

63

ChAT-Cre, Nestin Cre, Myf5-Cre, Sox2-Cre

Smn2A/Res; SMN2 89 +/+; SmnΔ7+/+ and Smn2A/F7; SMN2 89+/+; SmnΔ7+/+

Cre-loxP-mediated deletion or replacement of smn in tissues in which Cre is expressed; low levels of SMN present 47,62 in all tissues from SMN2 transgene; rescue upon expression of SMN in neurons and glia; no change in phenotype upon expression in muscle

Olig2-Cre

SmnF7/D7; SMN2 89+/+

Cre-loxP mediated deletion of Smn exon 7 directed to motor neuron progenitors; low levels of SMN present from SMN2 transgene; all tissue wild-type except motor neurons; lifespan of 70% of mice is > 1 year; early phenotype but mild

HB9-Cre

Smn2B-Neo/2B-Neo; SMN2 89+/−

Embryonic lethality of 2B-neo homozygotes is rescued by SMN2; specific increase of SMN in HB9 expression cells; 30 survival to about 5 days of age without Cre induction; HB9-Cre increases life span to 12 days; completely rescues motor neuron defects and unmasks other primary defects upon motor neuron correction

SEVERE SPINAL MUSCULAR ATROPHY MOUSE MODELS

III.  CELL AND ANIMAL SMA MODELS

SMNA2G

43

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FIGURE 15.1  SMN2 transgenic lines used in SMA research. Schematic representation of SMN2 transgenic lines indicating their location within the mouse genome, transgene size, and copy number. Genome walking was used to clone the integrations sites for Tg(SMN2)2Hung16 Tg(SMN2)89Ahmb58,126, and Tg(SMN2)CJD.24 Flourescence in situ hybridization was used to map Tg(SMN2)N11 and Tg(SMN2)N46 to chromosomes 18 and 3, respectively.23

There are two other less commonly used models of severe SMA: the three-copy N11/N46 SMN2 SMA mice23 and severe inducible SMA mice.30 Fig. 15.2 shows pictures of the severe Δ7 SMA mice at postnatal day 10 and the severe inducible mice at postnatal day 5. The threecopy N11/46 SMN2 SMA mice (SMN2N11gN46;Smn2A/2A) have, reduced compound muscle action potentials (CMAP) by postnatal day 15, NMJ pathology including diaphragm involvement, which resulted in early breathing impairments,23 and an average survival of 15 or 22 days depending upon how the cross was performed. While similar in survival to Δ7 SMA mice, there are some three-copy N11/N46 SMA mice that survive into adulthood, termed “escapers,” with severe hindlimb, ear, and tail necrosis. The percentage of “escapers” was dependent upon the parental origin of the SMN2 transgene. As

reported in 2015, Burgheron SMA mice have also differences in survival depending upon the paternal allele.31 Alternatively, it could simply be that the expression of one or both of these SMN2 transgenes is somehow gender influenced by the location where they integrated in the genome since they are random integrants (Fig. 15.1). To our knowledge, the Burgheron and the three-copy N11/N46 are the only SMA lines that have shown this epigenetic phenomenon. Severe inducible SMA mice were developed by breeding the SMN2 89 line onto the background of a mutant Smn2B-Neo allele. The 2B-neo allele contains a three nucleotide mutation in the core Smn exon 7 splice enhancer were ΗΤΡΑ2β1 and a LoxP flanked PGK-neomycin resistance cassette in intron 7 that is transcribed in the opposite direction to Smn, thus reducing Smn levels.32

III.  CELL AND ANIMAL SMA MODELS

DETERMINE WHERE HIGH SURVIVAL MOTOR NEURON EXPRESSION IS REQUIRED

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FIGURE 15.2  SMA mice models (A) Shows the SMNΔ7 SMA mouse a postnatal day 10. The black arrow indicates the affected mouse notice the marked size difference compared to a normal litter mate. (B) Shows the severe inducible mice the black arrow indicates affected individuals. (C) Shows the four copy SMN2 mice Taiwanese mild SMA mouse model (Jackson 5058) notice the marked tail necrosis.

Without the SMN2 transgene, Smn2B-Neo/2B-Neo embryos die by embryonic day 9 (E9) due to low Smn protein levels caused by first, an exon 7 knock-in mutation that alters Smn exon 7 splicing and second, hindrance from the floxed neomycin selection cassette within intron 7.32,33 One copy of the SMN2 89 transgene on this background rescued the embryonic lethality. These mice can be used as an inducible SMA model to address Smn temporal and spatial requirements. In all mouse models, a higher copy number of SMN2 ameliorates the SMA phenotype. This correlates with the motor neuron features in humans where higher copy number of SMN2 correlates with a milder phenotype34-37 (also see Chapters 1 and 2). This also indicated that the SMN2 gene could produce the required functional protein to correct SMA. These results confirmed SMN2 as a natural therapeutic target for the treatment of SMA.

THE USE OF SEVERE SPINAL MUSCULAR ATROPHY MOUSE MODELS TO DETERMINE WHERE HIGH SURVIVAL MOTOR NEURON EXPRESSION IS REQUIRED The pathological changes that occur in the severe SMA mouse models can be divided into neuronal and nonneuronal (also see Chapter 10). Neuronal dysfunction is the primary defining feature of SMA in both man and mouse models and multiple studies have detailed various aspects of motor unit dysfunction and the timing thereof.17,26,29,30,38–47 They are addressed in detail in Chapters 2, 8, 9, and 11. Additionally, when cultured primary motor neurons are obtained from severe mice with the genotype SMN2 89+/+; Smn2A/2A, they display shorter axons and a reduced growth cone size.48 In a similar manner, knockdown of SMN in the zebrafish leads to truncated and abnormally branched axons.49 There is also a reduced level of β-actin found at the growth cone of SMN2 89+/+; Smn2A/2A cultured motor neurons with the reduced actin believed to be causative of the truncated

axons.48 However, no truncated or branched axons are found in severe SMA mice SMN2 89+/+; Smn2A/2A. Thus in vivo there appear to be mechanisms that compensate and result in the normal growth of axon to their target.26,42 However, there are neurofilament accumulation lozenges (axonal swellings) in axon and deposits in the NMJ26,50 (also see Chapter 8). The findings of axonal defect along with detection of small amounts of SMN and reduced amounts of β-actin in the growth cone in cultured axons has led to the suggestion that SMN plays a role in localization of mRNA in the axon48,51,52 (see Chapters 7 and 8, SMN’s role in local mRNA processing). In the work reported in 2014, SMN has been localized in the NMJ terminals of mature motor neurons and it is currently not clear what other members of the SMN complex reside with SMN at this location.53 Currently, as discussed elsewhere, two major theories of how SMN deficiency gives rise to SMA exist. One indicates that the canonical function of SMN in snRNP assembly is disrupted leading to reduced snRNPs and an alteration in splicing.54–57 The second indicates a unique function to either assemble a complex onto mRNA or to be part of the complex assembled onto mRNA and control both transport and translation at the terminal region of the axon. These two theories have been discussed extensively in the literature15 and elsewhere in this book (see Chapter 7). Currently, whether one or all of these functions contribute to SMA has not been determined. In addition to motor neurons, it has been suggested that other tissues may play a role in SMA. Often this is based on observation of pathology in that tissue. However, if a particular tissue is critical, the phenotype should be reversible by SMN expression restricted to that tissue. The first attempt at tissue-specific expression of SMN used transgenic lines that express SMN under a tissue specific promoter.58 Expression of SMN under the skeletal actin promoter resulted in two lines that heavily expressed SMN in skeletal muscle, one line (HSA69) was completely restricted to muscle, the other line (HSA63) was leaky and did show low expression in nervous system (all tissues) and heavy expression in

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skeletal muscle and heart. These lines were crossed onto the severe SMA background SMN2 89+/+; Smn2A/2A. Line HSA69, with heavy SMN expression restricted to muscle, showed no impact on survival or any aspect of the phenotype, whereas the line HSA63 that was leaky did have a large impact. The Prion promoter was also used to drive SMN in neurons. This resulted in rescue of the severe SMA mice. However, there is a low level of RNA from the transgene in other tissues. This data indicated that expression of SMN in muscle did not impact SMA mice, but expression in neurons did have a major impact.58 Subsequent to this study the role of muscle has remained controversial with some studies reporting altered ability of myoblasts to form fibers.59–61 Additional studies in mouse models of SMA have reported defects in the myogenic program and reduced strength, but it has not been determined whether these findings are reversible with expression of SMN in muscle.61,62 Two further studies have been performed in SMNΔ7 mice. In the first study, Martinez et al.,63 used a line, where the mouse Smn gene is disrupted by the insertion of an inverted exon 7, that can be reverted to a functional mouse Smn. Upon reversion, the authors found a minimal improvement in the SMA phenotype with some increase in muscle fiber size (not seen by Iyer et al., see later), the use of either MyoD1-Cre or Myf5-Cre.63 The study of Iyer et al., used the same allele except it was paired with the classic null allele, and so only one of the mouse Smn alleles could revert to produce functional Smn. In addition, these authors used the Floxed (flanked by lox sites) exon 7 allele originally described by Fuiger et al.,64 to remove mouse Smn from the tissue where Cre was expressed in a mouse that has SMN2 and SMNΔ7. In the case of rescue in muscle using Myf5-Cre, no alteration in phenotype was observed including no alteration in the distribution of muscle fiber size. In the case of reduction of SMN levels to two copies of SMN2 specifically in muscle using Myf5-Cre, the muscle showed no defects in the ability to produce force at 2 months of age and the animals where in essence normal.62 Lastly, deletion of genes critical for the atrophy pathway in muscle in SMA either made the phenotype worse or had no effect.65 The results of these mouse studies indicate that deficiency of SMN in muscle does not have a major impact on the disease. However, while this does not advocate for the replacement of SMN in muscle per se, we do believe that the replacement of SMN in the CNS and enhancement of muscle function is likely to be important in treatment of patients that already present with SMA. In other words, the motor neurons that are remaining at a particular stage can be rescued and the muscle enhanced so as to perform more function with less nerve input. This is consistent with at least mild SMA cases showing minimal if any myopathic

changes unlike a muscle disorder such as Becker dystrophy.66 One feature of muscle that was not extensively investigated in the later studies was the role of satellite cells in regeneration of muscle. In mice, reduction in the ability of satellite cells to regenerate muscle requires challenging the muscle to undergo repair otherwise a phenotype is not observed. Thus it would be interesting to challenge muscle to repair in the situation where SMN had been reduced just in muscle and determine if repair is effective. Regardless of the direct importance of high levels of SMN in muscle, the use of therapies that enhance muscle function (independent of SMN) should be considered. This may be an important treatment strategy for patients, particularly those with longer duration of disease and more extensive motor neuron loss. In this situation, it may be possible to combine SMN restoring therapy with muscle-enhancing therapy to increase functional output from muscle with limited innervation. Alterations in neuronal cell types other than motor neurons have been suggested to play a role in SMA. Studies in severe SMA models or the SMNΔ7 mice have indicated potential defects in the autonomic nervous system that can alter blood vessels, heart rate (bradycardia), and the gut.30,39,43,47,67,68 Mentis et al., have suggested that the sensory input into the motor neuron from proprioceptive neurons is critical in the development of SMA,41 although Gogliotti et al., find that the postsynaptic densities are a motor neuron-dependent phenotype30 (see Chapter 9). In the SMNΔ7 mice, the reduction of SMN levels in motor neurons to that produced by two copies of SMN2 and SMNΔ7 using either an Oligo2-Cre driver or a ChAT-Cre driver results in mice that live a relatively normal life span but still have the electrophysiological defects in the motor neuron.43,47 Conversely, restoring high levels of SMN in SMA mice to just motor neuron using either the HB9-Cre in severe mice or ChatCre in SMNΔ7 mice results in minimal to no improvement in life span but rescue of the electrophysiological defects of the motor neuron.30,47,65,69 It is interesting to note that addition of the Nestin-Cre driver to the ChATCre driver in an SMA SMNΔ7 mouse results in high but chimeric expression in a large number of neurons as well as all astrocytes (glia) and a marked increase in survival as well as complete rescue of motor neuron function.47 Indeed, in some cases, animals have a normal survival indicating high expression in neurons and glia is sufficient to obtain complete rescue. Expression in glia has also been indicated to improve SMA mice in the studies of Rindt et al.,70 and tissue culture studies have also indicated the importance of astrocytes (see Chapter 10 for discussion of these studies). Other phenotypes, such as a marked cardiac bradycardia, clearly occur in severe SMA mice.71–73 However,

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DETERMINE WHEN HIGH SURVIVAL MOTOR NEURON EXPRESSION IS REQUIRED

these alterations have been suggested to occur due to defects in the autonomic nervous system.71,72 When the topic of the involvement of other tissues arises, there are a number of features that need to be kept in mind. First, the two commonly used SMA mouse models, the SMNΔ7 mice (SMNΔ7+/+; SMN2 89+/+; Smn2A/2A), and the severe Taiwanese mice (SMN2 Hung+/−; SmnH/H), are quite different regarding tissue specific phenotypes. Furthermore, the degree of denervation is not the same in these two models. Therefore, it is important to consider how the phenotypes of the SMNΔ7 and the severe Taiwanese mice (with two copies of SMN2) compare. While the survival of the SMNΔ7 and severe Taiwanese models are similar (2 weeks), the variability between the two models regarding the involvement of the motor system compared to other organ systems is significant.16,74 Indeed, Hua et al., reported that the longissimus capitis muscle in the severe Taiwanese model did not show any completely denervated NMJs75,75a. In contrast, the same muscle in the SMNΔ7 mouse shows partial-to-complete denervation of about 85% of synapses and complete innervation in about 15% of synapses.76 Given the weaker denervation phenotype in the severe Taiwanese mouse, a small increase in SMN in the CNS may be sufficient to overcome the CNS requirement. Furthermore, it is difficult to interpret the survival outcome of the Taiwanese mouse given the severity of dysfunction in several organ systems16,28,74,77,78 (DiDonato, personal observation). Iascone et al., have found using denervation as a quantitative readout for SMNΔ7 mice and comparing it to postmortem samples from type-1 patients on a muscle-by-muscle basis shows a remarkable overlap between the mouse and human neuromuscular phenotype.79 This indicates that the SMNΔ7 mouse model recapitulates the neuromuscular phenotype of patients. However, the SMNΔ7 mouse still has other organ system involvement such as the heart,73,77,80 which is not typical of most type-1 cases of SMA in the human. One must consider the mechanism by which deficiency of SMN gives rise to SMA in mouse and man. If disruption of splicing plays a major role, which appears quite likely, the sequence and structural variation of the intron could affect splicing of a particular gene and its sensitivity to SMN reduction. In mice, there is a very tight limit of SMN that results in SMA-like phenotypes. Conversely, in man, the range of SMN levels that give rise to SMA is broader. It is certainly possible that in mice in order to observe motor neuron phenotypes the SMN levels have to be pushed to lower levels than in man. Thus, when motor neuron phenotypes present the SMN level is low enough to cause additional changes that occur in the mouse but not in man.

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THE USE OF SEVERE SPINAL MUSCULAR ATROPHY MOUSE MODELS TO DETERMINE WHEN HIGH SURVIVAL MOTOR NEURON EXPRESSION IS REQUIRED An important question that has been addressed in mice is when restoration of SMN needs to occur to have a therapeutic effect. There are mouse lines with doxycycline-inducible SMN expression in an SMA background and inducible SMN alleles based on expression of Cre recombinase.32,81–83 In case of the Cre lines, Tamoxifeninducible Cre is used to induce high SMN expression levels, which results in permanently high SMN levels once induced. In contrast, the doxycyline-inducible SMN lines can be turned off and on by the removal or addition of doxycyline to the food. The tamoxifen-inducible lines have rapid induction of SMN expression, whereas the doxycyline lines take 3 days to reach full level of SMN expression. Both have been used in the SMNΔ7 mice to determine when high levels of SMN are required with consistent results. In summary, early introduction of SMN has the maximum impact with later induction having a reduced effect.82,83 However, it should be noted that a major impact did occur when the mice had symptoms. In other words, reintroduction of SMN works when the motor neuron is still there but cannot impact motor neurons once they have died. In addition, Le et al., reported that after rescue of SMA mice, removal of SMN induction at 28 days after induction at birth did not result in a marked neuromuscular phenotype.82 This was followed by Kariya et al., removing high levels of SMN at 21 days and the mouse being reliant on two copies of SMN2 in adulthood.50 Interestingly, this did not result in marked neuromuscular phenotypes but instead showed that peripheral nerve repair was dependent on high SMN in adulthood.50 The following studies indicate a critical window for SMN restoration and the importance of SMN in neuronal repair. In 2014, Feng et al., treated SMNΔ7 mice with a suboptimal dose (0.1 mg/kg as opposed to 3 mg/kg via IP for 23 days and then 0.3 mg/kg via oral gavage) of the drug compound SMN-C3 (PTC/Roche)84 that enhances incorporation of SMN exon 7 from SMN2 to produce a milder model of SMA.85 These mice had a median survival of 28 days and showed severe denervation and atrophy of severely affected muscles. The authors then tested full induction of SMN at P32 in mice that survived to this time point. The weight and survival of the animals showed no statistical improvement. In addition, the severely affected longissimus capitis muscle showed no statistical significant improvement in muscle cross-sectional area or fiber size. The EDL muscle showed a 20% increase in size as did mean muscle fiber size. However, a distribution

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of fiber sizes is not provided. It has been reported that reduction of SMN to SMA levels in muscle does not alter the fiber size distribution.62 In human SMA, the muscle in milder patients can range from showing clear group atrophy to the presence of triangulated fibers and very little change in fiber size.86 Even when there is atrophy present, there are often small muscle fibers, normal muscle fibers, and some hypertrophied fibers.86 The small fibers are most commonly thought of as denervated, whereas the larger fibers are innervated.86 Thus when modeling milder SMA, the muscle might be expected to follow a similar pattern. If SMN is increased later in the disease course, the remaining motor neurons might well be corrected and thus maintain more connections to muscle. This would result in the improvement of mean fiber size, but there would still be patches of denervated fibers as the number of motor neurons has not increased. Thus a small increase in muscle fiber size as reported for the EDL muscle could be due to improved maintenance of the remaining motor neurons a direct effect on muscle is possible, but consideration of whether this has relevance to SMA in humans is needed. Two other factors that showed a statistically significant improvement were tail length and necrosis; however, these do not directly relate to features seen in type-2 or type-3 SMA patients. The authors also showed a significant increase in vGLUT1 positive synapses on the cell body when switching to a high dose of the compound at 33 days, but this occurs on the remaining motor neurons. It is not yet clear how this observation translates to SMA in a human trial. The authors conclude that late symptomatic treatment had an impact on SMA.85 However, the question whether SMN upregulation affects denervation of muscle remains as severely affected muscle did not show an improvement of innervation and the EDL on morphological criteria of innervation remained fully innervated at PND60. We note that a milder mouse 2B/2B-Neo mouse on a pure C57/Bl6 background does show abnormalities of CMAP and motor unit number estimation (MUNE) and survives for longer than one year showing the denervation seen in type-3 SMA (DiDonato and Arnold, unpublished). We suggest that the major driver of the SMA phenotype in man is the dysfunction of the motor neuron and at least a portion of motor neurons are lost during the course of the disease. Motor neurons that are lost cannot be improved by increased SMN. Thus improvements that do occur are relative to the number of motor neurons that are retained. If these retained motor neurons have improved sprouting due to SMN restoration, then they could improve muscle innervation and fiber size. It seems likely that the more modest impact on NMJ denervation is the most relevant finding in the Feng et al.,85 study and this only improved slightly. The latter is consistent with studies in more severe mice where once the motor neuron is lost it is not possible to

obtain substantial recovery. In future, using this system, it would be interesting to perform treatment at earlier times and determine when motor neuron cell bodies are lost. It stills remains an open question how much affect treatment later in the disease can achieve. This will likely be answered by the current clinical trials. Given the slow progression of SMA, it is likely that loss of motor neurons can be stopped and a small response produced due to sprouting of existing motor neurons. However, without replacing the lost motor neurons, a large response to late therapeutic administration seems unlikely. One interesting observation in this regard is that myostatin inhibition or loss of atrogen have essentially no impact in severe SMA mice65,87,88 yet the treatment of drug-induced mild SMA mice do have a robust response to follistatin treatment.85 Indeed, we would suggest that the use of combined SMN restoration and follistatin treatment in symptomatic patients will likely give the best response. Since the remaining motor neurons will have high SMN, the increase in muscle performance for this small number of motor neurons may improve function.

MILD SPINAL MUSCULAR ATROPHY MOUSE MODELS Milder SMN2-containing SMA mice have also been generated; however, in all but two cases, these are on the very mild end of the expected phenotype. The Taiwanese 5058 homozygous mice (four copy number SMN2 Hung), often said to be a model of mild SMA, show tail necrosis but no overt weakness.16 Fig. 15.2 shows the 5058 mice at postnatal day 35 with tail necrosis. The measure of CMAP represents the electrophysiological output of motor neurons to a muscle or group of muscles. MUNE estimates the number of motor neurons that functionally innervate a muscle. CMAP and MUNE are typically both reduced in patients with SMA, but in milder forms of SMA, CMAP may normalize due to collateral sprouting. The 5058 Taiwanese mice with four copies of SMN2 show electrophysiological findings of preserved CMAP and minimally reduced MUNE and thus can be considered a model of extremely mild SMA (type 4 or even milder) (Personal Observation, Arnold). Recombineering was used to generate a series of SMN2 transgenic lines targeted to the endogenous mouse Smn locus. They are sometimes referred to as the secondgeneration SMN2 allelic series.25 In this approach, a hybrid Smn allele was created by replacing a 2.2 kb genomic fragment of murine Smn-containing exons 7 and 8 with a 1.3 kb genomic fragment of human SMN2-containing exons 7and 8 alone, or with one or three copies of the genomic region containing SMN2 (Fig. 15.1). However, it appears that the chimeric gene is at best poorly functional with very little full-length SMN from this allele

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Mild Spinal Muscular Atrophy Mouse Models

produced. Thus, in essence, the mice show phenotypes based on the copy number of intact human SMN2 genes. While different copy numbers can be generated with these lines only mice containing two or more copies of the entire human SMN2 genomic fragment survive postnatally. Thus this has resulted in a minimal number of new SMA lines. While the C/C line appears to have a minimal phenotype that is not in essence similar to type-3 SMA it has many issues, as do other lines, with necrosis of various organs (tail, feet, and ears). MUNE is not altered in these mice, and CMAP showed minimal change.89 These findings of reduced CMAP, but normal MUNE are surprising and reversed compared to that expected.25,90,91 In patients with SMA, there is generally a decrease in both CMAP and MUNE; however, CMAP can normalize in milder forms of SMA due to collateral sprouting.90–92 A major confounder in a series of mild SMA mice using human SMN2 transgenes is the necrosis that occurs in these models over time. For instance, more than 220 SmnC/C mice were bred for the EIM study with only 11 being able to be studied by the 1-year time point.89 This is also a major problem for the intermediate Burgheron mouse.31 In both cases, the mice have significant necrosis leading to difficulties obtaining reliable electrophysiological measures. These SMA mice can be generated from an intercross between SmnC/C and the severe SMN2 89 heterozygous mice.17 Perhaps the most interesting SMN2-containing line with an intermediate SMA phenotype is the “SMN-read-through” line, also known as the SMN-RT mouse.93 Genetically, this model is similar to the SMNΔ7 model; however, the Δ7 cDNA transgene has been replaced with a sequence modified SMNΔ7 read-through version in which the first stop codon is mutated. It should be noted that attaching tags to the C-terminus of full-length SMN appears to severely impact function of the protein, whereas additions to C-terminus of the delta 7 transcript enhance function.93 The addition of amino acids to delta 7 results in a slightly longer peptide that confers a greater degree of functionality by its increased activity in snRNP assembly assays, cytoplasmic localization, axonal extension assays, and SMN protein stability.12,94–97 Mice hemizygous for SMNRT on the severe SMA background (SMN2 89+/+; SMNRT+/−; Smn2A/2A) are not completely normal but have an intermediate phenotype. They have an average life span of 34 days, with about 25% living past 40 days of age and a few past 70 days. By P19, there was a significant increase in partially innervated muscle fibers with NMJs that had a perforated or fragmented morphology, which suggested a defect in synapse maturation. There was also a significant diminution of proprioceptive synapses onto L3–L5 lateral motor neurons but not motor neuron number when compared to control mice. Mice that lived past 40 days of age began to develop mild necrosis on the

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ears, tail, and/or eye but not feet. Mice homozygous for SMN-RT had similar lifespans and necrosis problems. Importantly, scAAV9-SMN administration to SMN-RT mice at P1 rescued all pathologies including the mild necrosis. Collectively, this study highlighted that SMNRT protein has functionality in vivo, does not hinder full-length SMN function, nor does it act as a dominant negative, and the SMN-RT model has utility for testing SMN-dependent therapeutics.93 Given the survival time of this model with the limited necrosis, it may also be useful for investigating SMN-independent therapeutic pathways. Lastly, as discussed earlier, Feng and colleagues generated a milder mouse by treating the SMNΔ7 mouse with the compound SMN-C3 at a suboptimal dose, this resulted in mice with clear atrophy and denervation (morphologically) and a median survival of 28 days.85 These mice also show necrosis and their response to late therapeutic induction of high SMN levels is discussed earlier. We note that while a number of authors use tail length as a read out of therapeutic impact, we would caution against using this measure as there is not a clear equivalent in the patients. Milder mouse models of SMA, which lack SMN2 but have reduced SMN levels, have also been generated. For example, Smn+/2A mice on a pure C57/Bl6 background have been reported with reduced motor neuron number.98 Yet, these mice demonstrate no overt phenotype and have normal muscle strength due to compensatory motor neuron sprouting and reinnervation.98 However, Smn+/2A mice on a predominantly FVB/N background showed no CMAP or MUNE abnormalities (unpublished observation, Arnold). The extensive ability of mouse motor neurons to sprout and compensate for lost motor neurons contributes to the difficulty in modeling mild SMA in mice.99 Several alleles that disrupt the splicing of mouse Smn exon 7 have been developed. The SmnC–T allele models the SMN2 C to T nucleotide change. Smn exon 7 alternative splicing occurs but not to the same extent as human SMN2 because the intronic sequences that are part of SMN2 exon 7 splicing regulation are not present in mice.100 When the allele is combined with the Smn2A null allele this mouse, SmnC–T/2A, essentially has no SMA phenotype or one that is extremely mild.101 In contrast, the Smn2B allele, which is a derivative of Smn2B-Neo allele, is a 3-nucleotide GGA-TTT alteration in the Tra2-B1 responsive, AG-rich enhancer within the central portion of Smn exon 7. This disruption mimics the level of alternative splicing seen in human SMN2 such that the predominant amount of Smn transcripts lack exon 7.32,33 When combined with the null Smn2A allele Smn2B/2A mice have SMN levels of about 15% compared to wild-type mice and a milder phenotype than SMNΔ7 mice. The Smn2B/2A mice have an average

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survival of 28 days in C57/BL6/J mixed background or 20 days in an FVB/N background, reduced CMAP amplitude, and by P18–P21 show abnormal neuromuscular junctions.45,61,102–104 The underlying cause of death in Smn2B/2A SMA mice remains unclear, but organ involvement outside of the motor unit appears to affect survival similar to the severe SMA models. This model has been used in testing therapeutics and has shown a very enhanced response to therapeutics103,105 in some case even without correction of CMAP size (an indirect measure of motor neuron function in vivo).103 This model currently appears to be the model most closely resembling a milder SMA phenotype. Its placement at a major repository such as the Jackson laboratory will facilitate its use. While this model does not have SMN2, the SMN2 inducers can be relatively easily tested in the current severe SMNΔ7 SMA mice or intermediate SMNRT mice and the Smn2B/− mice can be used to study the effect of SMN restoration at different time points, SMN-independent therapies as well as the pathobiology of milder SMA.

HOW DOES MOUSE PATHOLOGY RELATE TO SPINAL MUSCULAR ATROPHY PATIENTS? The clinical features of SMA in man are remarkably restricted to the degeneration of lower motor neurons and the associated features of chronic motor axonal loss. Phenotypically, there is a wide range of clinical severity ranging from onset prior to birth to mild weakness in adulthood. SMA is classically divided into three subtypes (types 1–3) where type-1 SMA is the most common with onset prior to 6 months of age and features of weakness preventing the ability to sit independently. Nonmotor features of the sensory, autonomic, gastrointestinal, endocrine, and cardiac defects have been noted in patients with SMA, but such cases are only sparsely reported in the literature.75,78,102,106–111 There are a number of important points to be considered in regards to these other organ and system phenotypes in humans and mice. First, these nonmotor pathological changes have mostly been reported in very severe cases, often with one copy of SMN2 and not in typical type-1 SMA with two copies of SMN2. Cases of SMA with one copy SMN2 have a very severe phenotype often with a fetal onset and are usually denoted as SMA type 0 or SMA type 1a.90 Furthermore, even if there are two copies of SMN2, the majority of genetic tests cannot determine if the SMN2 gene is intact with exon 1 and if the promoter is present. Thus it is important to consider the number of functional SMN2 alleles in a patient. It is likely that SMA cases with fetal onset have SMN levels that are

equivalent to one copy of SMN2. We favor designating these very severe forms of SMA as type 0 or type 1a. In type-0 cases, other organ involvement is possible, but in typical types 1–3, this does not appear to be the case. Secondly, different mice lines show various peripheral organ developmental defects that are most likely due to different levels of full-length SMN expression from the SMN2 transgene. Finally, one other tissue worth mentioning is the thalamus, which clearly shows pathology in human cases with type-I SMA (with two copies of SMN2), but this organ has not been studied in the mouse.15,109 As there is such tight regulation between generating a phenotype in an SMA mouse versus no phenotype, some of the observed mouse phenotypes may be specific to mice and not to classic type-1 SMA with two copies of SMN2. In the future, with treatment of motor neurons in the spinal cord resulting in extended survival, other pathologies or symptoms may be uncovered in humans.

ELECTROPHYSIOLOGICAL MEASURES IN MOUSE MODELS Prior to the availability of molecular diagnostic testing, electromyographic (EMG) features of motor neuron loss and reinnervation were an important tool for the diagnosis of SMA. The techniques of CMAP, MUNE, and EMG help identify and quantify motor axonal loss and show good correlation with severity of disease and functional level in SMA.92,112–115 In the early 2010s, electrical impedance myography (EIM), a bioimpedancebased procedure has been investigated for its utility in SMA.116,117 EIM allows a noninvasive assessment of localized muscle impedance that can be used to provide insight about muscle health and innervation status. EIM delivers a low intensity, alternating electrical current at varying frequencies to a localized area of muscle and the consequent voltages are measured.118 The voltages reflect changes in both muscle membrane health and size as well as local compositional changes to the muscle.119 Similar studies of CMAP, MUNE, EMG, and EIM can be applied in mouse models of SMA, but only occasionally these techniques have been utilized.90,103,119,120 Nevertheless, these studies demonstrate similar findings of motor neuron/axonal loss that are corrected with SMN-restoring therapies.90,120 Other electrophysiological techniques ex vivo have been frequently utilized to characterize disease phenotype in mouse models. Neuromuscular junction electrophysiological techniques demonstrate defects of reduced endplate current amplitude and quantal content at the NMJ.39,40,121,122 These findings are corrected with SMN restoration.123 The findings of ex vivo NMJ recordings provide important insight into

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Genetic Background Check

the pathogenesis of SMA, but it is more difficult to investigate how these findings relate to human SMA.

GENETIC MISSENSE MUTATIONS OF SURVIVAL MOTOR NEURON The availability of SMA mouse models allowed the study of missense mutation that occur in patients. SMN missense mutations have been placed into an SMA background.55,124 This work demonstrated that mild missense alleles are not functional on their own (do not rescue a Smn2A null mouse) but are capable of complementing SMN protein produced by SMN2 (Fig. 15.3). Of these, the SMNA2G mice with SMN2 are smaller and show weakness in grip strength but again have a very mild phenotype.124 In fact, the phenotype may have been lost upon importation to Jackson due to further backcrossing or only occurs when there is one copy of SMN2 and one copy of the transgene. There are no reports of backcrossing this transgene onto a C57/Bl6 background. The SMNA111G55 and SMNA2G represent mild SMN mutations. The severe SMN mutation SMNI116F was also examined as well as SMNE134K.125 These alleles either produce very little SMN protein or the protein does not function. A severe SMA mouse resulted when crossed onto an SMA mouse background. We have further investigated the SMN alleles SMND44V, SMNT274I, and SMNQ282A. All behave the same way with no rescue of a Smn2A null mouse but complete rescue of mice containing at least two copies of SMN2 89 (Burghes, McGovern, and Arnold, unpublished result). Rescue is dependent on the degree of expression of the allele. Thus low expression of SMNA111G on a SMN2 background results in minimal recovery of snRNP assembly and only a very minor change to phenotype.55 This indicates that all missense mutation by themselves are not functional, but instead these alleles act as complementors of the wildtype SMN from SMN2. Therefore, severe SMN alleles either do not oligomerize well with wild-type SMN or do not act as complementors, whereas mild alleles act as strong complementors. Lastly, we have found that amino terminal mutations SMNA2G and SMNA111G cannot complement each other, whereas N- and C-terminal mutations can. These pairs completely rescue SMA null mice (unpublished data). This allelic complementation indicates that the oligomer is the critical component of the SMN complex and that the amino and carboxyterminal domains of SMN have separate roles that are both required for function of the SMN complex (unpublished result). The occurrence of the complete lack of SMN2 genes occurs in about 5–10% of the population.37 Indeed, if a partial functionally SMN allele existed (i.e., has some function) in the absence of SMN2 then one might expect

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to rarely find patients that lacked SMN2 and possessed only the missense mutation. Since SMA patients with zero copies of SMN2 have not been identified to date, we feel the mouse experiments are indicative of what occurs in humans. Furthermore, complementation must be considered when assaying SMN missense alleles in cell culture as wild-type SMN is always present. In future, a complete analysis of all available missense mutations will be essential in defining missense allele function.

GENETIC BACKGROUND CHECK The original targeting of Smn alleles was performed in 129/SvJ embryonic stem cells and founders were backcrossed to FVB/N or C57/BL6 genetic backgrounds to breed with SMN2 transgenic founders. This resulted in SMA models with a mixed genetic background in which portions of the genome were still segregating. Many SMA models have been backcrossed to generate complete congenics on FVB/N or C57/BL6. Survival analysis studies have shown genetic background differences in all of the SMA models.17,28,103,105,126 Congenic Taiwanese severe FVB/N SMA mice live for 8 days versus variable phenotypes in a mixed background and death between 1 and 20 days.16,103,127–129 On a pure FVB/N background, the Δ7 SMA mice have a survival of 11 days and the phenotype is more severe on a C57/Bl6 background. The hybrid with the majority of the background in FVB/N appears to be the longest-lived (Cathleen Lutz, The JAX, personal communication).28 Interestingly, the opposite is observed in Smn2B/2A SMA mice. Mice in a predominantly C57Bl/6 background have a median survival of about 1 month of age with a broad min and max survival, whereas FVB/N Smn2B/2A mice have a median survival of 20 days and a short min–max range of about 17–25 days.103 Two interesting outcomes of the phenotypic strain differences are first, they can be used to our advantage to generate SMA models for specific purposes. For example, 5058 hemihybrid mice, which is an F1 hybrid between C57/BL6 and FVBN/N mice were used to double the lifespan of severe Taiwanese mice to address the therapeutic efficacy of the 8-mer 3UP8i.130 This spliceswitching oligo works through a different mechanism of altering SMN2 exon 7 splicing and showed an agedependent effect, such that Δ7 SMA and the standard Taiwanese SMA models were not useful for this particular study since the increased inclusion of exon 7 only became apparent at P8/P9, which is too late to produce rescue in these more severe mice.130 Second, the survival differences are indicative of modifier loci that alter disease progression albeit by relatively small degrees. Modifier screens have been performed in Caenorhabditis

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FIGURE 15.3  SMN functions as an oligomer. SMNA111G and SMNT274I are just two of the many missense mutations found in SMN. SMNA111G is located in the Tudor domain. SMNT274I is located in the oligomerization domain; however, this change at amino acid 274 does not affect oligomerization of SMN. We have indicated eight SMN molecules per oligomeric complex. In (A)–(E), some of the different SMN complexes that can form are represented. (A) Wild-type SMN complex. (B) A complex comprised of only a single SMN missense mutation is not functional. (C), (D). The mild SMN missense mutations can be complemented by wild-type SMN from SMN2 to form a functional SMN complex. E. The N-terminal and C-terminal SMN missense mutations can complement each other to make a functional SMN complex.

elegans and Drosophila,131,132 although no modifier has been identified in mice by either mapping modifier loci or examining the loci identified in lower organisms. One difficulty is whether the modifiers will be the same between these species in particular with the potential of different splicing sensitivities between species with SMN deficiency. While it is often argued that a pure background is an advantage in preclinical testing of treatments, we do not think this is particularly the case with Δ7SMA mice given its severe phenotype and a variance of about 3–5 days in terms of survival if one is employing standardized measures of severe debilitation. This may be different for the milder SMA models. In fact, the use of a mixed genetic background in severe mice, like Δ7SMA, is that not only is it representative of the human population, but it has the advantage of larger window of change when a treatment is given and effective (100-day survival increase). In the case of SMN2 induction, a primary target for SMA therapeutics, it is clear that relatively large effects of lifespan can be obtained if the treatment in question truly increases SMN to the appropriate level.78,84,123 The critical element in our opinion is the degree of change. A “big improvement” means that the variance of phenotype in the SMA mouse that is simply due to genetic background has clearly been overcome. Furthermore, given that humans are not a genetically uniform population, it seems that for a therapy to translate from mice to humans, it must be effective in mice with mixed genetic backgrounds. In the early testing of SMN inducing drugs, it was often stated that the SMNΔ7 mouse model was too severe and thus not suitable. However, the marked, reproducible increase in survival that can be obtained with an effective SMN inducing therapy (from 14 days to greater

than 100 days) is actually a strength of the model. Small subtle differences are often difficult to replicate. Furthermore, if a drug is effective at increasing SMN in the severe model, it will also likely increase SMN in a mild model of SMA. However, when testing non-SMN therapies, it would be advisable to include measures of recovery of motor neuron function. This leads to greater hope that the molecules can recapitulate the same effect in humans. The question would then become the timing of SMN restorative therapy for beneficial effect (see Chapter 11).

MOUSE MODELS USE IN THERAPEUTIC TESTING AND LARGE MAMMALIAN MODELS OF SPINAL MUSCULAR  ATROPHY Mouse models have been used to understand the pathophysiology of SMA in a wide range of studies, and this topic is discussed in detail in other chapters. Here, we wished to outline the models available and some of their key characteristics with regards to testing therapeutics. SMA mice, particularly those containing SMN2, have been used in the testing of therapeutics for SMA. It is now clear that SMN2 inducers can be effectively tested in these mice despite their severe phenotype. This can be seen as an advantage as significant extension of survival can be achieved when the therapeutics are effective.18 A number of SMN2-inducing therapies that increase SMN production from SMN2 are now in clinical trials including antisense oligonucleotides (IONIS), small molecules (Roche/PTC and Novartis), and gene therapy using scAAV9-SMN (AveXis) (see Chapters 16–19). All three of these strategies were tested in the available SMA mouse models. Molecules that do not act upon SMN2 must be

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FIGURE 15.4  SMA-like clinical symptoms in piglets and its progression. (A) Progression of the symptoms in the pig with reduced SMN levels. (B) Experimental set up for the recording of sciatic CMAP and MUNE responses. 1, recording electrodes E1 and E2; 2, anode and 3, cathode stimulating electrodes; 4, ground electrode. (C) Representative EMG recording from a control and an animal with reduced SMN levels (Injected with scAAV9-shSMN) showing fibrillations with positive wave morphology. (D) CMAP and MUNE at PND54 were significantly reduced in animals with reduced SMN levels (scAAV9-shSMN injected) (N = 4) compared to the noninjected control group (N = 6). The CMAP and MUNE values were preserved in the treated presymptomatic group that received scAAV9-shSMN to reduce SMN levels followed 24 h latter with a vector that restores SMN to the motor neurons (scAAV9-SMN) had values that were not significantly different from those of the unaffected controls. **p < .01. Figure from Duque SI, Arnold WD, Odermatt P, et al. A large animal model of spinal muscular atrophy and correction of phenotype. Ann Neurol. 2015;77(3):399–414.

tested in mammals by measuring electrophysiological endpoints to assess pertinent motor neuron function. In the case of muscle enhancers, physiological testing of muscle strength and force should be employed.18 To complement the mouse studies, it would be useful if a larger animal model of SMA existed. Hereditary canine SMA and feline SMA have been described, but these are not caused by reduced SMN levels.133,134 As reported in 2015, a pig model of SMA has been created by reducing levels of porcine SMN using scAAV9shRNA.135 While the knockdown of SMN does occur postnatally, whereas the reduced levels of SMN in SMA would be throughout development. The appearance of a clear denervation phenotype similar to SMA patients in our opinion indicates of high levels of SMN in the

postnatal period for the development of SMA. Previous studies have shown that motor neurons can be efficiently transduced when scAAV9 is delivered via intrathecal injection.77,135,136 The SMA pig was made by developing a shRNA that selectively knocks down pig SMN to levels observed in SMA spinal cord samples. The pigs develop a clear SMA-like phenotype including muscle weakness at approximately 30 days of age. The progression of the pig phenotype is shown in Fig. 15.4 along with the set up for electrophysiological recording and the CMAP and MUNE in the affected pigs. The weakness is particularly evident first in the hind limbs and then progresses to the front legs prior to euthanasia.135 The pigs display both reduced CMAP and MUNE mimicking the clinical presentation of SMA in humans. Rescue of the

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SMA-like weakness in pigs was also performed at different stages. Presymptomatic treatment was modeled by delivering scAAV9-SMN the day after injection of scAAV9-shRNA knockdown. The pigs in this paradigm were completely rescued with all weakness and electrophysiological parameters corrected. This demonstrated that the observed phenotypes are dependent on SMN. The development of the pig has allowed the question of treatment timing to be investigated in a large animal model. Substantial, although not complete, correction of the phenotype was also achieved with delivery of the scAAV9-SMN at postsymptomatic stages after hind limb weakness was evident. In the animals treated after symptomatic onset, CMAP was preserved but MUNE and motor neuron counts were reduced. This confirms that scAAV9-SMN halted progression of motor neuron loss and thus stabilized the animal.135 These studies suggest that early symptomatic treatment in humans may have a major impact on the SMA phenotype. When animals were treated symptomatically, motor neuron loss and MUNE were not completely rescued. As expected, once the motor neuron is lost, it cannot be replaced, but importantly it is likely that motor neuron loss can be stabilized and the remaining motor neurons can compensate through collateral sprouting if treated during early symptomatic stages. It is expected that with longer duration of symptoms and therefore more extensive motor neuron loss, the less robust the response is expected to be. In addition to questions about treatment timing, the comparison of the splicing changes that occur in pig and mouse can be used to look at intron sensitivity to SMN depletion and how similar these changes are in two mammalian models of SMA.

CONCLUSIONS The development of mammalian models of SMA has led to the identification and confirmation of many promising therapeutics including antisense oligonucleotides, gene therapy, and small drug molecules that are currently in clinical trials. Studies in animal models of SMA predict that these therapies will have a significant impact in humans. Yet it remains unclear how SMN protein reduction leads to selective loss of spinal motor neurons. The next step is to use mammalian models of SMA to identify the downstream targets of SMN deficiency. This will give a greater understanding of the disease pathology and open the field to new drug targets. Furthermore, the alteration of splicing of a particular gene due to SMN deficiency may not be the same in all mammals due to intron sensitivities as well as species sequence variance that causes differences in alternative splicing. Therefore, a careful study of SMA in rodents, pigs, and humans may reveal answers that cannot be

identified in just one species. Finally, alternative functions of SMN have been implicated in SMA such as a role in axonal outgrowth. Strong suppression of the SMA phenotype upon restoration of a particular pathway would give strong evidence of the importance of that pathway in SMA. If multiple functions of SMN are critical to SMA, then restoration of one pathway will suppress some of the phenotypes but not all. It will be important to perform this suppression to understand what functions contribute to SMA.

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III.  CELL AND ANIMAL SMA MODELS