Intralingual and Intrapleural AAV Gene Therapy Prolongs Survival in a SOD1 ALS Mouse Model

Journal Pre-proof Intralingual and intrapleural AAV gene therapy prolongs survival in a SOD1 ALS mouse model Allison M. Keeler, Marina Zieger, Carson Semple, Logan Pucci, Alessandra Veinbachs, Robert H. Brown, Jr., Christian Mueller, Mai K. ElMallah PII:

S2329-0501(19)30156-1

DOI:

https://doi.org/10.1016/j.omtm.2019.12.007

Reference:

OMTM 365

To appear in:

Molecular Therapy: Methods & Clinical Development

Received Date: 25 July 2019 Accepted Date: 13 December 2019

Please cite this article as: Keeler AM, Zieger M, Semple C, Pucci L, Veinbachs A, Brown Jr RH, Mueller C, ElMallah MK, Intralingual and intrapleural AAV gene therapy prolongs survival in a SOD1 ALS mouse model, Molecular Therapy: Methods & Clinical Development (2020), doi: https://doi.org/10.1016/ j.omtm.2019.12.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s).

1

Intralingual and intrapleural AAV gene therapy prolongs survival in a SOD1 ALS mouse model

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Allison M. Keeler1,2,3, Marina Zieger1,2,3, Carson Semple3, Logan Pucci6, Alessandra Veinbachs3,

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Robert H. Brown, Jr.3,5, Christian Mueller2,3, Mai K. ElMallah1,2,3

4 5

1

Division of Pulmonary Medicine, 2Department of Pediatrics,3Horae Gene Therapy Center,

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4

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Massachusetts Medical School, Worcester MA 01655;

8

University, Durham, NC

Microbiology

and

Physiological

Systems

5

Department

of

Neurology,

University

of

6

Department of Pediatrics, Duke

9 10

Correspondence should be addressed to C.M. e-mail: [email protected] and

11

M.K.E., e-mail: [email protected]

12 13

All work was done in Worcester, MA, USA

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Mai K. ElMallah, 203 Research Drive, Durham, NC 27710, Ph: 9196843577; e-mail:

15

[email protected]

16 17 18 19

Short title: Intralingual and intrapleural injected gene therapy for ALS

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Abstract

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results in death

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from respiratory failure. No cure exists for this devastating disease, but therapy that directly

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targets the respiratory system has the potential to prolong survival and improve quality of life in

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some cases of ALS. The objective of this study is to enhance breathing and prolong survival by

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suppressing SOD1 expression in respiratory motor neurons using adeno-associated virus

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expressing an artificial microRNA targeting the SOD1 gene. AAV-miRSOD1 was injected in the

27

tongue and intrapleural space of SOD1G93A mice and repetitive respiratory and behavioral

28

measurements were performed until end stage. Robust silencing of SOD1 was observed in the

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diaphragm and tongue as well as systemically. Silencing of SOD1 prolonged survival by

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approximately 50 days, and delayed weight loss and limb weakness in treated animals

31

compared to untreated controls. Histologically, there was preservation of the neuromuscular

32

junctions in the diaphragm as well as number of axons in the phrenic and hypoglossal nerves.

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Although SOD1 suppression improved breathing and prolonged survival, it did not ameliorate

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the restrictive lung phenotype. Suppression of SOD1 expression in motor neurons that underlie

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respiratory function prolongs survival and enhances breathing until end stage in SOD1G93A ALS

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mice.

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1

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Introduction

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Amyotrophic lateral sclerosis (ALS) is a devastating, untreatable neurodegenerative

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disease. Patients with ALS die 3-5 years after diagnosis from respiratory failure, and death is

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accelerated if the bulbar muscles and motor neurons are affected early in the disease.1 Bulbar

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involvement leads to recurrent aspiration, choking and aggravation of respiratory disease.2,

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The most severely affected bulbar muscle is the tongue4 which atrophies as a result of loss of

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the hypoglossal motor neurons and neuromuscular junction (NMJ) disruption.5-7 In addition to

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tongue involvement, all individuals afflicted with ALS develop progressive diaphragm and

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intercostal weakness which results in inadequate ventilation and respiratory failure.8-10 As a

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result of the tongue and respiratory pathology, patients progressively develop decreased

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exercise tolerance, shortness of breath, early morning headaches and excessive daytime

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sleepiness. The rate of decline in respiratory function is directly related to mortality.11-13

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Furthermore, respiratory support with non-invasive positive pressure ventilation significantly

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prolongs survival.12, 14

3

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Approximately 5-10% of ALS is familial, and 20% of familial ALS is due to mutations in

53

the gene encoding Cu/Zn superoxide-dismutase 1 (SOD1).15 The exact mechanism of the

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SOD1 induced neurotoxicity is unclear but aggregations of mutant SOD1 result in a cascade of

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events that eventually leads to neuronal degeneration. The SOD1G93A mouse is the most

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commonly used ALS mouse model. It ubiquitously expresses the human SOD1 gene with the

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G93A mutation16 and recapitulates ALS pathophysiology, including motor neuron loss, axonal

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degeneration, muscle denervation, and limb paralysis.16-18 In addition, this mouse model has

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significant respiratory insufficiency, restrictive lung disease and hypoventilation.18 Further, the

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SOD1G93A mouse has orolingual motor deficits which initially appear as tongue motility

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abnormalities and then progress to tongue force weakness.19-21 This pathology significantly

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impacts breathing because the tongue genioglossal muscle contracts during breathing and

2

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maintains upper airway patency in the face of the negative intrathoracic pressure that occurs

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with each breath.

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There is no cure for ALS. Novel therapies aimed at silencing SOD1 include inhibitory

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short hairpin RNA, artificial microRNA and anti-sense oligonucleotides. Our group recently

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reported successful silencing of SOD1 in mice and non-human primates using adeno-

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associated viral (AAV) gene therapy encoding microRNA (AAV-miRSOD1).22-24 When injected into

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neonatal mice, survival was prolonged by 69 days22 whereas a systemic injection in adult mice

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resulted in extension of life span by 22-27 days.23 Despite this systemic therapy, respiratory

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insufficiency persisted.22, 23 We hypothesized that these animals eventually died of respiratory

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failure. Therefore, the goal of this study was to evaluate the impact of gene silencing therapy

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targeted to respiratory motor units on breathing and survival, with the ultimate goal of using this

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as an adjunct therapy to systemic or intrathecal delivery. Since respiratory support in ALS

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patients prolongs survival, we hypothesized that suppression of SOD1 expression in motor

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neurons that underlie respiration will prolong survival. We used a combination of intralingual

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(specifically genioglossal) and intrapleural AAV-miRSOD1 injections exploiting intramuscular

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delivery and retrograde axonal transport to target the entire motor unit: muscle, NMJ, motor

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axon, and motor neurons. The tongue genioglossal delivery targets both the muscle and

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hypoglossal motor neurons25,

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intercostal muscles as well as the phrenic and thoracic motor neurons.27 Our ultimate goal was

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to reduce expression of the mutant SOD1 in the tongue and respiratory motor units and thereby

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enhance breathing and prolong survival.

26

while intrathoracic delivery targets the diaphragm and

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Results:

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To assess the benefit of respiratory targeted gene therapy for ALS, we injected the

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SOD1G93A mutant mouse model with 1x1011 vector genomes via an intralingual injection and 3

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1x1011 vector genomes via an intrapleural injection of AAVrh.10 encoding an artificial mircoRNA

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targeting SOD1 (AAV-miR SOD1). SOD1G93A animals were injected as adults at approximately 60

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days of age with either the therapeutic vector, AAV-miRSOD1, or saline, and non-transgenic

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littermate animals were used as additional controls. All animals were followed longitudinally until

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they were not able to right themselves within 30 seconds after being placed on either side.

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Respiratory directed therapy results in efficient muscle and CNS transduction as well as

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SOD1 mRNA levels silencing.

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To assess the level of transduction and SOD1 mRNA silencing, we evaluated vector

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genome expression in treated SOD1G93A mice. Efficient targeting of the AAV-miR SOD1 vector was

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observed in the tongue, intercostal muscles, lung and diaphragm (Fig 1A). To assess the extent

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of systemic distribution, we examined vector genome levels in the liver, heart and hindlimb.

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Injections targeting the tongue and thoracic cavity led to a considerable amount of vector

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genomes in the liver, heart and hindlimb (Fig 1B). Vector genomes were also detected in the

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medulla and throughout the entire spinal cord (Fig 1C).

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hypoglossal nucleus and phrenic motor nucleus by intralingual and intrathoracic injections, we

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injected SOD1G93A mice with an AAVrh10 vector containing GFP. At end stage in the SOD1G93A

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mice we observed GFP staining of motor neurons in the hypoglossal motor nucleus and phrenic

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motor nucleus, respectively (Supplementary Figure 1).

To assess retrograde transport to the

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Next, we evaluated mRNA expression levels in treated and untreated SOD1G93A mice.

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Transduction within the tongue and respiratory muscles led to robust silencing of SOD1 mRNA

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by 64% in the tongue and 80% in the diaphragm (Fig 1D).The lung had a more modest

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reduction of SOD1 mRNA (35%).

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evidenced by a decrease in expression by 86%, 87% and 89%, in the liver, heart and hindlimb

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respectively (Fig 1E). Suppression of SOD1 mRNA was also observed in the medulla and spinal

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cord ranging from 23%-45% (Fig 1F).

Efficient silencing was also observed systemically – as

4

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Delayed disease onset and prolonged survival in AAV-miR SOD1 treated SOD1 mice

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Animal strength and behavior were assessed every 30 days until disease onset and then

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every week thereafter. AAV-miRSOD1 therapy targeting the respiratory system significantly

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enhanced survival by an average of 50 days in treated compared to untreated SOD1G93A mice

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(Fig 2A). Weight loss, neurological symptoms and muscle strength were also assessed. A

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significant improvement in weight gain was observed in treated compared to untreated

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SOD1G93A mice with some treated mice maintaining weight until 190 days of life (Fig 2B).

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Neurological screening using a standard ALS neurological screening metric28 revealed that,

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compared to untreated SOD1G93A mice, AAV-miRSOD1 treated SOD1G93A mice had a slower

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neurological decline (Fig 2C). Four-limb grip strength test (Fig 2D) and inverted screen testing

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(Figure 2E) revealed less rapid deterioration of muscle strength in AAV-miRSOD1 treated mice

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compared to untreated SOD1G93A mice. Overall, AAV-miRSOD1 treated SOD1G93A mice showed

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enhanced survival, weight gain and neurological improvement.

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Enhancement of breathing in AAV-miR SOD1 treated animals SOD1G93A mice

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Whole body plethysmography (WBP) was used to assess the therapeutic benefits of

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tongue and respiratory directed AAV-miRSOD1 on respiratory function. WBP measures

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frequency, lung volumes and flow rates in awake spontaneously breathing animals. At 90 days

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and 130-149 days, no significant differences in breathing parameters were observed between

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the treated and untreated SOD1G93A mice or between the SOD1G93A and non-transgenic

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littermates (Fig 3A-E). At the final time point from 150-169 days, few untreated animals

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remained (n=2) and they had a decreased respiratory rate (frequency) compared to the

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remaining AAV-miRSOD1 treated SOD1G93A mice (n=8) (Fig 3A). In addition, the untreated

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SOD1G93A mice had decreased tidal volumes compared to non-transgenic littermates (Fig 3B;),

5

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and decreased minute ventilation (Fig 3C) compared to both the AAV-miRSOD1 treated SOD1G93A

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mice and the non-transgenic littermates ( for both). Untreated SOD1 mice had decreased peak

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expiratory flow (PEF) and decreased peak inspiratory flow (PIF) compared to non-transgenic

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littermates suggesting increased weakness in respiratory muscles (Fig 3E). Furthermore,

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compared to the AAV-miRSOD1 treated SOD1G93A mice, the untreated mice had decreased PIF

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and PEF ). PIF and PEF reflect inspiratory muscle strength and expiratory muscle strength

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respectively. Thus, the AAV-miRSOD1 treated SOD1G93A, behaved similarly to non-transgenic

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littermates, in that they survived to this time point and maintained respiratory muscle strength,

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frequency, tidal volume and minute ventilation. Thus, overall breathing decline was attenuated

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in the SOD1G93A mice treated with AAV-miRSOD1, whereas surviving untreated SOD1G93A mice

147

had declines in several in respiratory parameters.

148

To assess the pulmonary mechanics of AAV-miRSOD1 treated animals, forced

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oscillometry techniques were used at disease endpoint. The total respiratory resistance, central

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airway resistance and tissue resistance were all increased in both the AAV-miRSOD1 treated and

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untreated SOD1G93A animals (Supplementary Fig 2A-C). Similarly, the inspiratory capacity and

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respiratory compliance were decreased at endpoint in both untreated and AAV-miRSOD1 treated

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animals. Thus, no significant improvement occurs in pulmonary mechanics in either untreated

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or treated SOD1G93A mice.

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Preservation of motor neurons and corresponding peripheral synaptic terminals

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The impact of therapy on motor neuron survival was examined using histological

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quantification. Specifically, we quantified cresyl violet stained hypoglossal and cervical ventral

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motor neurons at end point (Fig 4). Non-transgenic littermates had significantly more motor

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neurons in both hypoglossal and cervical ventral regions than both the untreated or treated

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animals suggesting that at disease endpoint motor neuron death was similar between treated

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and untreated animals. 6

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To assess the effect of respiratory directed therapy on axonal integrity of hypoglossal

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and the phrenic nerve, these were collected from animals at endpoint, with non-transgenic

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littermates being age-matched to the treated group (Fig 5). In the AAV-miRSOD1 treated mice,

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there was a preservation of axonal numbers at end stage in the hypoglossal and phrenic

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nerves. However, despite the preservation of axon numbers, the size of the axons were

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significantly smaller (Fig 5D).

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To assess the impact of distal nerve degeneration, we assayed the neuromuscular

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junctions in the tongue and diaphragm at endpoint.

Within the diaphragm, the untreated

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animals revealed significant disruption of the neuromuscular junctions (Fig 6). By contrast, in

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the AAV-miRSOD1 treated diaphragm, there was some preservation of presynaptic and

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postsynaptic components of the neuromuscular junction (Fig 6). Within the tongue of the

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untreated animals there was significant denervation of neuromuscular junctions (Supplementary

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Fig 3). AAV-miRSOD1 treatment mitigated this denervation, but this improvement was not as

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robust as in the diaphragm (Fig 6 and Supplementary Figure 3).

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Discussion

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The most significant outcome of this study is that targeting respiratory motor neurons

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using AAVrh.10 expressing an artificial microRNA (miRNA) against the SOD1 (AAV-miRSOD1)

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significantly prolongs survival in adult SOD1G93A ALS mice. In addition, respiratory function was

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preserved until end stage disease and disease onset was delayed. This study highlights the

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importance of treating the respiratory system in ALS.

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AAV gene therapy for ALS

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It is difficult to directly compare AAV gene therapy animal studies because of variations

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in injection routes, capsid variants, vector constructs and genomes, titration methods of different 7

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labs, vector dosages, and age of animals when treated. However, similar studies using different

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routes of injection have been performed in SOD1 mouse models with varying results. Recent

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studies from our group documented that an SOD1-targeting microRNA delivered by AAV9

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prolongs survival by ~70 days in SOD1G93A mice when injected in the neonatal period.22

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However in a more direct comparison, data from our group using the same capsid and similar

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vector, AAVrh.10-mir SOD1, was given to adult mice intravenously, and survival was prolonged

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only by 22-27 days.23 Similarly, a recent study using AAVrh10 to express a small nuclear RNA

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that skips exon 2 out of frame, reported significant improvement in survival in mice treated at

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both adult and neonatal ages.29 In a subset of mice, limb function was completely preserved in

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their study, comparable to our earlier observation.22 In addition, other groups used AAV2 or

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AAV9 to deliver therapeutic genes, such as insulin growth factor 1 (IGF-1)30-33 into muscles,

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including the muscles of respiration. The goal of both these studies was to use retrograde

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transport of AAV to transduce the motor neurons of interest.30,

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enhancement in survival ~30-37 day when the muscles of respiration such as the intercostal

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muscles were targeted versus ~10-12 days without intercostal muscle injections.30, 32

201

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Both studies saw significant

AAVrh10 was an ideal choice for this present study because of its ability to cross the

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blood brain barrier and to transduce motor neurons by retrograde transport.34,

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AAVrh10 is safe and efficacious in both mice and non-human primate models.23, 24 The utility of

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AAVrh10 is underscored by proof-of-principle and safety data generated with this vector design

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in cynomolgus macaques.24 In this study, we injected 2x1011vg of AAV expressing the miRSOD1,

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which is 1/10th of the dose that was injected systemically (Borel et al.) or 1/39th of the dose co-

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injected intracerebroventricular and systemically in adult mice (Biferi et al.) and we report an

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improvement in survival by 50 days, whereas other reported 22-27 days and 63 days

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respectively.

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Respiratory involvement in ALS 8

35

In addition,

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All individuals afflicted with ALS ultimately succumb to inadequate ventilation and

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respiratory failure secondary to diaphragm and intercostal muscle weakness.8-10 These muscles

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are essential for breathing and are innervated by the phrenic nerve in the ventral horn of C3-C5

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and by the intercostal motor neurons in T1-T12 in the thoracic spinal cord. The largest increase

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in lifespan to date in an SOD1 mouse model was reported in a study that also targeted the

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respiratory muscles. Specifically, this study used a lentiviral-shRNA delivered by a series of

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intramuscular injections that included hindlimb, facial, tongue, diaphragm and intercostal

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muscles.36 Through an intralingual and intrapleural injection of AAV gene therapy, our goal was

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to target the entire motor unit of the tongue, diaphragm and intercostal muscles. Specifically, we

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sought to target the nerves and motor neurons innervating these muscles. Using the AAVrh10-

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GFP we found positive GFP staining in the phrenic and hypoglossal motor nuclei

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(supplementary figure 1). In addition, at end-stage in AAV-miRSOD1 treated animals, we report

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improvements in neuromuscular junction in the diaphragm, and preservation of axonal integrity

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of the phrenic nerve, but the survival of the hypoglossal and cervical ventral motor neurons was

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not significantly different. Treated animals showed evidence of greater respiratory muscle

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strength as seen with preserved peak inspiratory flow and peak expiratory flow. However, motor

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neuron loss and restrictive lung disease were still evident at end stage. We previously reported

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restrictive lung disease in SOD1G93A mice.18 Unfortunately, AAV-miRSOD1 therapy targeting the

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respiratory system did not prevent restrictive lung disease and at end stage mice succumbed to

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respiratory failure. A recent report utilizing muscle specific kinase (MuSK) antibody therapy

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preserved neuromuscular junctions in the diaphragm but did not improve diaphragm strength or

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breathing.37 This suggest the importance of not only preserving the NMJ but also the motor

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neurons. The eventual decline in respiratory function and restrictive lung disease may be

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caused by inefficient targeting of all motor neurons present in motor unit of diaphragm and

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intercostal muscles and combined targeting strategies such as injections directly into intercostal

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and diaphragm in combination with intravenous, intracerebral ventricle or intrathecal infusion. 9

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Since positive pressure ventilation enhances survival and quality of life, combination therapy

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has the ability to treat respiratory insufficiency and enhance survival and quality of life.

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Tongue involvement in ALS

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Patients with ALS who initially present with bulbar symptoms have a worse prognosis

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and rapidly progressive disease course.1 The most severely affected bulbar muscle is the

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tongue.4 These abnormalities of the tongue are a result of atrophy due to loss of the

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hypoglossal motor neurons7, which control the intrinsic and most extrinsic muscles of the

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tongue.6

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The hypoglossal motor neurons are also important in maintaining upper airway patency

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as these cells regulate the shape, stiffness and position of the tongue.6, 38-40 Contraction of the

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extrinsic tongue muscles dilate and stiffen the pharyngeal lumen and prevent collapse in the

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face of negative inspiratory pressures.41, 42 Thus, degeneration of hypoglossal motor neurons in

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ALS results in upper airway obstruction, oral phase dysphagia, and recurrent aspiration. With

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our dual injection strategy, we were able to significantly silence expression of SOD1 within the

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tongue. Interestingly this tongue correction correlated with maintenance of weight and increased

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survival in treated animals.

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Although our approach targeted the respiratory system, it is important to note that we did

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see systemic distribution of virus which correlated to significant reduction in SOD1 mRNA

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expression in the liver, heart and limb skeletal muscle. We believe that the systemic distribution

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of vectors in combination with the directed lingual treatment enhanced survival in these animals.

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Thus, we propose that future gene therapy approaches should consider a dual injection of

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systemic and intralingual or intrathecal and intralingual to ensure that the hypoglossal motor

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neurons are targeted. If successful, we believe this will allow patients better control of their

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tongue, maintenance of upper airway patency and decreased aspiration of secretion. 10

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In conclusion, this study illustrates the importance of targeting the respiratory system in

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the treatment of ALS. Ideally a therapy would target the entire CNS with focus on both motor

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units responsible for respiration and ambulation.

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demonstrates the degree to which respiratory rescue can enhance survival. In addition, a robust

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improvement in survival in adult animals was documented at considerably lower doses then

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previously published. Correction of hindlimb paralysis is an important functional benchmark in

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treated ALS, and indeed we saw modest but significant improvement in limb strength in our

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study. However, because respiratory failure is the ultimate cause of death in this disease, future

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therapeutics should also target the respiratory system.

However, our directed approach clearly

272 273 274

Materials and Methods

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Animals: All experimental procedures were approved by the Institutional Animal Care and Use

276

Committee at the University of Massachusetts Medical School.

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randomly assigned to AAVrh10-H1-miRSOD1 (N=8, 7F, 1M) vs PBS group (N=8, 6F, 1M).

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Littermate non-transgenic controls (N=8, 7F, 1M) were injected with PBS. Injections were

279

administered by an investigator blinded to the groups. Mice transgene copy numbers were

280

monitored by RT-qPCR according to Prize for Life and Jackson Laboratory guidelines such that

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a drop in 3/4Ct (1/2 cycle) value from the internal control. Any mice with less than 20 copies or

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more than 25 copies was excluded.

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AAV Vectors and injection methods: AAVrh10-H1-miRSOD1 vectors (hereafter AAV-miR SOD1),

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targeting the human SOD1 sequence, were generated as described.23, 24 The vector construct

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contains mutated ITR to create dsAAV vectors, a single miR sequence driven by H1 promoter

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and contains Rabbit poly-globin Poly-A tail (clinical H1 construct described in

11

SOD1G93A animals were

24

). All vector

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doses were per animal, at 1x1011 vector genomes (vg) or PBS were injected into each location

288

at a volume of 40µl in the tongue and 400µl in the intrapleural space described for a total vector

289

dosage of 2 x1011 vg per animal.25-27 Injections were performed by an investigator blinded to the

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groups and the study drug. The intralingual injections were performed as previously described25

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and the intrapleural injections were performed as previously described.27 SOD1G93A animals

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were injected at approximately 60 days of age with either AAV-miRSOD1 or phosphate buffered

293

solution (PBS), and non-transgenic littermates were used as additional controls. All animals

294

were followed longitudinally.

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Behavior Testing: Four limbs grip strength and inverted screen testing was determined by a an

296

investigator blinded to the experimental groups, as previously described.43

297

strength test was performed using mesh screen on Alemno digital grip strength meter

298

(Holzkirchen, Germany) to determine peak force. Each grip test was repeated twice with 15

299

minute rest period, and the maximum force recorded. Inverted screen was performed using

300

square wire mesh rotated 180 degrees. Animals were assessed for latency to fall up to 2

301

minutes, again this was repeated twice per session with 15 minute rest period and longest hang

302

time recorded. For the neurological screening score assessment, investigator blinded to groups

303

scored animals according to Prize for Life and Jackson Laboratory guidelines where 0= full

304

extension of hind legs from midline when suspended by tail, 1= collapse of leg extension

305

towards midline or trembling, 2= toes curl under at least 2x when walking or any foot dragging,

306

3=paralysis or minimal joint movement and 4= mouse can not right itself within 30 seconds after

307

being placed on either side.

308

Ventilation: Ventilation was quantified using whole-body plethysmography in unrestrained,

309

unanesthetized mice as previously described.18,

310

inside a 3.5” × 5.75” Plexiglas chamber (SCIREQ, Montreal, Ca) and data were collected in 10

311

second intervals. The Drorbaugh and Fenn equation44 was used to calculate respiratory 12

43

Four-limb grip

Awake, non-restrained mice were placed

312

volumes including tidal volume and minute ventilation. Mice immediately underwent 10 minute

313

hypercapnic challenge (FiO2: 0.21; FiCO2: 0.07; nitrogen balance) followed by a subsequent

314

60-90 minute eupnea period when they were exposed to normoxic air (FiO2: 0.21; nitrogen

315

balance).

316

Measures of Pulmonary mechanics: Pulmonary mechanics were performed at study end

317

point using forced oscillometry (FlexiVent system, SCIREQ, Montreal, Ca) at baseline and in

318

response to incremental doses of methacholine as previously described.45 In brief, animals

319

were anesthetized with an intraperitoneal injection (i.p.) injection of a mixture of ketamine

320

(90mg/kg, Animal Health International) and xylazine (4.5mg/kg, Propharma), and a tracheotomy

321

was performed followed by insertion of a pre-calibrated cannula into the trachea. Spontaneous

322

respiratory effort was prevented using a neuromuscular blocking agent (pancuronium bromide:

323

2.5 mg/kg, Hospira, Inc., Lake Forest, IL). Respiratory mechanics were obtained and calculated

324

using FlexiWare software (SCIREQ, Montreal, Ca.) as previously described.18, 43, 45, 46

325

Vector Genomes: gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia,

326

CA) from the tissues harvested from AAV and PBS injected animals at study endpoint.43 RT-

327

qPCR was carried out using custom Tagman Gene Expression Master Mix (Applied

328

Biosystems) and Tagman probes (Applied Biosystems) for custom probe against Rabbit

329

polyglobin polyA tail (Probe: /56-FAM/ATGAAGCCCCTTGAGCATCTGACTTCT/36-TAMSp/,

330

Primer 1: GCCAAAAATTATGGGGACAT and Primer 2: ATTCCAACACACTATTGCAATG) with

331

plasmid control standard curve.

332

Analysis of RNA knock down: RNA was isolated with TRIzol® (Invitrogen) and Direct-zol RNA

333

Mini Prep Kit (Zymogen), then transcribed into complementary DNA using the High Capacity

334

RNA to cDNA Kit (Applied Biosystems). RT-qPCR was carried out using Taqman Gene

335

Expression Master Mix (Applied Biosystems) and Taqman probes (Applied Biosystems) for

13

336

human SOD1 (Hs00533490_m1), and mouse hypoxanthine-guanine phosphoribosyltransferase

337

Hprt (Mm01545399_m1) as normalization gene.

338

described.22

339

Immunostaining for Neuromuscular Junctions: The tongue samples were embedded in

340

optimal cutting temperature (O.C.T.) compound and frozen. 20 µm thick sections were cut for

341

the tongue and stained by immunohistochemistry. Briefly, the tissue was incubated with alpha-

342

bungarotoxin, Alexa Fluor 594 conjugated, 1:1,000 (Invitrogen #B-13422) for 2 h at room

343

temperature, washed in PBS and then tissue was incubated at least 24 h in a mixer of the two

344

primary antibodies against neurofilament, 1:400 (chicken polyclonal to neurofilament NF-H, IgY,

345

EnCorBiotechnology Inc. #CPCA-NF-H) and synaptotagmin, 1:200 (ZNP1 synaptotagmin,

346

Zebrafish International research Centre, znp-1 #090811). The following day, the tissue was

347

washed in PBS, incubated in an anti-mouse Alexa 488, 1:200 and in an anti-chicken Alexa 647,

348

1:1,000 secondary antibodies for at least 24 h. The sections were washed in PBS, covered with

349

fluorescent mounting media and coversliped. Diaphragm was not sectioned and immunostained

350

using the same method, except of the incubation time was extended to at least 72 h at 4°C.

351

Quantification neuromuscular junction integrity: Neuromuscular junctions (NMJs) in tongue

352

and diaphragm were imaged using z-series maximized projection on a Leica DM5500B

353

microscope equipped with a 40x oil objective and fluorescent camera. Randomly selected equal

354

area images were used to evaluate integrity of presynaptic (immunolabeled with synaptotagmin

355

and neurofilament) and postsynaptic (labeled with bungarotoxin) structural components of each

356

NM junction found in the field using the ‘Count’ function of the Adobe Photoshop software. At

357

least 10 images were analyzed for each animal for Diaphragm n=4 for SOD1G93A, AAV-miRSOD1

358

n=5 for non-transgenic, for tongue n=4 for AAV-miRSOD1 and non-transgenic, n=5 for SOD1G93A .

359

A “Normal” NMJ unit was considered to have well-structured presynaptic and postsynaptic

360

components defined by colocalization of axon terminals with fluorescently labeled AChRs. In 14

Relative expression was calculated as

361

contrast, incomplete, or denervated, NMJs have Ach receptor clusters on muscle fibers that are

362

not contacted by an axon as observed as an absence of anti-neurofilament staining.47, 48

363

Cresyl Violet staining and quantification: The medulla, and cervical spinal cord tissues were

364

extracted and post fixed by immersion in 4% paraformaldehyde for at least 48 h and then

365

transferred to 30% sucrose for cryoprotection and until sinking and were embedded in optimal

366

cutting temperature (O.C.T.) compound and frozen. 40 µm thick medullar and cervical spinal

367

cord sections were mounted on glass slides and stained with cresyl violet stain pre-warmed to

368

60°C and coverslipped with Permount mounting medium (Thermo Fisher Scientific, Hampton

369

NH). Images were taken on a Leica DM5500B bright-field microscope. Every 6th section was

370

counted for a total of approximately 16 sections from each region in each mouse. Obtained

371

values were averaged across each experimental group of animals and the average of motor

372

neurons per each section was graphed.

373

Toluidine Blue staining and quantification of axonal numbers: The hypoglossal and phrenic

374

nerves were fixed in 2.5% glutaraldehyde in 0.1 M Sodium Cacodylate buffer pH 7.2 overnight

375

at 4°C, postfixed in 1% Osmium tetroxide in 0.1 M S odium Cacodylate buffer and then

376

embedded in epoxy resin. Blocks were trimmed to orient the nerves in order to obtain precise

377

1.5 µm thick cross sections on a Leica Ultracut E ultramicrotome. To quantify number of axons

378

sections were counterstained with toluidine blue, coverslipped with Permount mounting medium

379

(Thermo Fisher Scientific, Hampton NH) without dehydration in ethanol and the use of xylene as

380

described elsewhere (ref). Once dried completely, slides were coverslipped with Permount

381

mounting medium (Thermo Fisher Scientific, Hampton NH) without dehydration in ethanol and

382

the use of xylene. Images were taken on a Leica DM5500B bright-field microscope. The number

383

of axons was manually quantified using PhotoShop software. At least 3 mice were analyzed per

384

experimental group.

15

385

Statistics: All statistical analyses were performed using Prism Software (version 7, Graphpad,

386

La Jolla, CA).

387

between all four treatment groups. A 2-way ANOVA was performed for weights, strength testing

388

and measures of respiratory mechanics. A mixed-effects model was used to take into account

389

missing animals that did not survive to the later time points. Multiple comparisons between

390

groups and comparisons to baselines wereperformed using the Fisher’s LSD. For the vector

391

genomes analysis was performed using the Student’s t-test, and significance for the enzyme

392

assay and ventilation data was determined by one way ANOVA. Significance was considered

393

at a p value < 0.05.

Survival Long-rank (Mantel-Cox) test was performed for survival analysis

394 395

Acknowledgements: NIH NICHD K08 HD077040-01A1, NIH NINDS 1R21NS098131-01

396

(MKE), P01 HL131471, R01-NS08868 (CM) NIH/NINDS, ALS FindingACure, ALSOne, Angel

397

Fund, Celluci Fund, Project ALS, Rosenfeld Fund, Target ALS (RHB).

398 399

Author Contributions: Conceptualization: AMK and MKE; Methodology: AMK, MKE, RHB,

400

CM; Formal Analysis: AMK, LP, MKE Investigation: AMK, MZ, CS, AL; Resources: RHB, CM,

401

MKE; Writing-Original Draft: AMK, MKE, Writing- Review & Editing: AMK, MZ, CM, RHB, MKE;

402

Visualization: AMK; Project Administration: AMK, MKE; Funding Acquisition: MKE

403

Disclosure/Conflict of Interest: CM and RHB are co-founders of Apic Bio. Apic Bio has

404

licensed patents related to this work in which CM and RHB are both inventors.

405 406

Figure Legends:

407

Figure 1: Intralingual and Intrathoracic injections of AAV-miRSOD1 are systemically

408

distributed and lead to widespread suppression of SOD1 expression. 16

409

Quantitative PCR estimates of vector genomes are shown in (A) peripheral tissues (liver, heart,

410

hindlimb) (B) respiratory muscles; and (c) four regions of the CNS. RT-PCR estimates of levels

411

of SOD1 mRNA are illustrated for extracted (D)peripheral tissues (liver, heart, hindlimb) (E)

412

respiratory muscles and (F) four regions of the CNS. Error bars represent ± SEM (A-C) and ±

413

Positive and Negative Error (D-F). n=4, Student’s T-test to determine significance in panels D, E

414

and F comparing untreated to treated. * designates p<0.005

415 416

Figure 2: AAV-miRSOD1 treated animals show increased survival and improved

417

maintenance of weight and strength. (A) A Kaplan-Meier percent survival plot documents an

418

increase in 36% cumulative survival of 50 days in AAV-mirSOD1 mice.

419

(Mantel-Cox) test p <0.0001(B) Percent change in body weight plotted from day 60 until

420

endpoint. (C) Changes in the neurological screening from day 60 to endpoint. This ranges from

421

0 the mouse appears normal to 4 the mouse cannot right itself within 30 seconds of being

422

placed on either side. (D) Four-limb grip strength measured in centiNewtons (cN). (E) Inverted

423

screen hanging test time, plotted to a maximum of 120 seconds. With exception of (A), data are

424

represented as mean ± standard error of the mean (SEM) 1-way ANOVA was performed for

425

each time point p<0.05 . Multiple comparisons between groups were performed using the

426

Fisher’s LSD. N=8 at start of experiment, N/8 is listed for each time point for SOD1 and mir

427

treated animals. For non-transgenics n were the following at each timepoint (not from humane

428

endpoint but for age matched controls) 160d n=7, 175d n=6, 190d n=3.. * designates

429

significantly different from non-transgenic littermates and #designates significantly different from

430

untreated SOD1.

Survival Long-rank

431 432

Figure 3: AAV-miRSOD1 animals display improvement in breathing. Whole-body

433

plethysmography of awake spontaneously breathing animals. Data are reported as a percent of

17

434

response to 10-minute period of hypercapnia compared to baseline breathing. At untreated

435

SOD1G93A endpoint (151-169 days), significant differences are noted between AAV-miRSOD1 in

436

(A)frequency and Non-transgenic littermates in (B) tidal volume. (C) Minute ventilation findings

437

for both treated and non-transgenic littermates were significantly different from untreated

438

animals at untreated SOD1 endpoint. No differences were observed in (D) peak inspiratory flow

439

between any group, but significant differences were noted between non-transgenic littermates

440

and untreated animals in (E) peak expiratory flow. All data are represented as mean ± standard

441

error of the mean (SEM). Starting N’s were n=8 for AAV-miRSOD1, n=5 untreated SOD1G93A , and

442

n=8 for NTG. Only 2 untreated animals survived to day 150-169 timepoint. A mixed-effects

443

model was used to take into account missing animals that did not survive to the later time

444

points. Multiple comparisons between groups were performed using the Fisher’s LSD.

445

designates p<0.05.

446

Figure 4: Motor neuron death is observed in both treated and untreated SOD1 animals.

447

Representative images of cresyl violet staining for motor neurons within the hypoglossal nuclei

448

were stained from tissues harvested at their respective end-points (A) untreated SOD1G93A

449

animals untreated (137 days) (C) AAV-miRSOD1 treated SOD1G93A (190 days) and (E) non-

450

transgenic littermate controls (192 days). B, D and F are higher magnifications of the boxed

451

area in panels A, C and E, respectively. Scale bars for A,C,E are 100µm and B,D,F are 50µm.

452

Panels G and H: Motor neuron quantification. All data are represented as mean ± standard error

453

of the mean (SEM). n=4 for AAV-miRSOD1, and n=3 for untreated SOD1G93A , and n=6 for NTG

454

(n=5 for phrenic), 1-way ANOVA was performed with uncorrected Fisher’s LSD

455

comparisons test * designates p<0.05

456

Figure 5: AAVmiRSOD1 preserves axons within the hypoglossal and phrenic nerves.

457

Representative cross-sections of hypoglossal (A,C,E) and phrenic (B,D,F) nerves stained with

458

toluidine Blue at endpoint for treated and untreated SOD1G93A mice at their respective endpoints 18

*

multiple

459

and age-matched non-transgenic littermate controls. Non-transgenic littermate (172days) (A-B),

460

AAV-mirSOD1(190days) (C-D) and untreated (156 days) (E-F).

461

performed within the hypoglossal (G) and phrenic (H) nerves. All data are represented as mean

462

± standard error of the mean (SEM). n=3 for all groups, 1-way ANOVA was performed with

463

uncorrected Fishers LSD multiple comparisons test * designates p<0.05.

464

Figure 6: AAVmiRSOD1 preserves neuromuscular junctions within the diaphragm.

465

Neuromuscular junctions (NMJ) were analyzed in untreated SOD1G93A and AAV-miRSOD1 mice at

466

their respective endpoints and age-matched non-transgenic littermate controls.

467

Immunofluorescence staining with rhodamine conjugated α-bungarotoxin (red), and antibodies

468

against anti-synaptotagmin (ZNP-1) and neurofilament-200 (gray). Denervation of NMJ is

469

depicted by dashed circles. Representative images for non-transgenic littermate control (205

470

days), AAV-miRSOD1 (205 days) and untreated SOD1G93A (137 days). (B) Quantification of

471

percent innervated NMJs is depicted. All data are represented as mean ± standard error of the

472

mean (SEM). n=4 for AAV-miRSOD1 and untreated SOD1G93A , and n=5 for NTG. 1-way ANOVA

473

was performed with uncorrected Fishers LSD multiple comparisons test * designates p<0.05.

Quantification of axons was

(A)

474 475

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