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July 2019 Progress in the Treatment of Muscular Dystrophies

Progress in the Treatment of Muscular Dystrophies July 20191 ______

With Vertex’s announcements last month that it is

• expanding its collaboration with CRISPR Therapeutics on gene editing for Duchenne and myotonic dystrophy including an upfront payment of $175 million, and • acquiring all outstanding shares of Exonics Therapeutics, a gene editing company focused on Duchenne and other neuromuscular diseases for $245 million these rare diseases are getting the focused funding and attention from big pharma required to bring novel, effective treatments to the market (Genetic Engineering & Biotechnology News 2019). With a small patient population and a challenging breadth of disease types and underlying biological and genetic causes, the dystrophies have historically lacked attention from the biopharma industry as a value-driver for new drug development. New technical capabilities in gene therapy and gene editing, and seed funds from philanthropic organizations focused on de- risking early-stage drug development have changed the playing field. In this report, we provide an overview of the current state of funding, therapeutics in development and novel technical approaches towards treating this group of challenging diseases.2

Overview Muscular dystrophies are genetic disorders characterized by progressive muscle weakness and loss of muscle function. Duchenne and Becker muscular dystrophy (DMD & BMD) are the most prevalent forms with 300-600 affected male babies born in the US every year. Generally diagnosed when patients are between two to five years old, it is caused by absence or reduction of the muscle protein dystrophin. DMD is one of the most rapidly progressing dystrophies. Patients are typically wheelchair bound by 12 years old and die in the second to third decade of life. There is no cure for any of the dystrophies. Current standard of care is palliative and includes the use of steroids and ventilators. The cost to families and patients, and to the healthcare system for rare diseases like muscular dystrophy is significant. In 2016 the average annual cost per patient for treating orphan diseases was five times greater than other diseases, $140,000 compared to $27,750. DMD itself has a large economic impact.

1 Report by NelsenBiomedical.com; Primary Author: D’anna Nelson, PhD Candidate at the University of Minnesota. Contact: [email protected] 2 Companies included in this report are those with later stage clinical programs and are only a subset of the over 85 companies with a stated focus in muscular dystrophy therapeutics.

In the US alone in 2012, DMD cost $1.21 billion including hospital stays, caregiver’s lost work, and lost productivity (Luxner 2018). While there are over 30 different types of muscular dystrophy, most can be classified into eight categories based on the specific muscle groups affected. Across categories, muscular dystrophies are very heterogeneous with differing age of onset, rates of disease progression, and incidence rates that vary by country and race (populations of European descent often have higher prevalence). In order of incidence these groups include: 1. Duchenne and Becker muscular dystrophy (DMD & BMD) is the most prevalent category. Global incidence rates of 4.78 and 1.53 per 100,000 males, respectively (Mah et al. 2014). The United States DMD/BMD diagnosis rate is roughly 15 out of every 100,000 males between the ages of 5-24 years (Emery 2002). 2. Myotonic muscular dystrophy is the second most prevalent affecting 11 out of 100,000 people [Northern England, (Norwood et al. 2009)]. 3. The remaining 6 categories—facioscapulohumeral (FSHD), limb-girdle (LGMD), congenital, distal, oculopharyngeal, and Emery-Dreifuss—cumulatively account for approximately another 11 out of 100,000 people [Northern England, (Norwood et al. 2009)]. Both young biotechnology companies and large players such as Roche and Pfizer are using a variety of approaches to develop therapeutics for these diseases. Progress is being made. However, finding a cure, even within a subset of dystrophies such as DMD, may be years into the future. Current activity is focused on targeting the most prevalent mutation types and therefore the larger patient populations.

Clinical Trials

There are limited numbers of new treatments in development for muscular dystrophies. There are 76 interventional studies currently listed as active or enrolling. Two-thirds are specific to Duchenne and Becker. Other dystrophies do have a few targeted trials but the numbers are drastically lower (Figure 1A)3. FSHD, with the most clinical trials after DMD/BMD, has only five active trials specific to its pathology. Dr. Mittal, former CEO and president of the Jain Foundation suggested that the focus on DMD is caused by two factors: the earlier discovery/study of the causative gene and the intense advocacy by parents of children afflicted with a lethal disease (personal communication, 2019).

When assessed by trial phase, there are roughly an equivalent number of trials in all phases of development (Figure 1B). Sponsors are equally dispersed with 1/3 industry, 1/3 institutional and the remaining 1/3 a mix of industry, academic, government, and non-profit/advocacy groups (Figure 1C).

3 There are over 30 types of muscular dystrophy.

Limited drug development is the norm for rare neuromuscular diseases. For example, amyotrophic lateral sclerosis (ALS/Lou Gehrig’s) has 86 active or recruiting interventional trials and Charcot-Marie-Tooth has only 51 (ClinicalTrials.gov). Other rare genetic disorders not affecting muscle have marginally more drug development activity. Cystic fibrosis has 128 currently active clinical trials and sickle cell disease has 156. When one considers that the clinical trials for muscular dystrophy cover multiple distinctive dystrophies caused by different genes, the number of trials per genetically distinct disease is significantly lower for the muscular dystrophies.

Figure 1: Muscular dystrophy trials by dystrophy type, phase and funding source

A. Clinical Trials: Type of B. Clinical Trials: Phase C. Clinical Trials: Muscular dystrophy Sponsor Phase I Phase I/II Academic/Hospital DMD/BMD FSHD Phase II Phase II/III Industry LGMD Other/Multiple Phase III N/A Mixed/Other

17 13 18 23 23 2 13 5

52 16

2 14 30

Source: clinicaltrials.gov

Scientific Funding

NIH funding is also disparate between muscular dystrophy and other rare genetic disorders and diseases.

When one compares NIH funding of all 30+ muscular dystrophies to other rare diseases, muscular dystrophy comes up short. Cumulatively there was $145 million for muscular dystrophy research in FY20184. Sickle cell disease and Cystic fibrosis, both singular diseases, received $104 and $83 million in FY2018, respectively. Muscular dystrophy with $145 million may seem like a generous amount but when divided by 30 types of dystrophy, it is only $4.8 million per type, a figure much lower than Sickle cell and Cystic fibrosis. In Figure 2A, the

4 NIH categories of muscular dystrophy, DMD/BMD, FSHD, myotonic, and congenital.

FY2018 NIH funding is divided by patient number within the USA. It is clear to see that funding for muscular dystrophy is incredibly low per patient while cystic fibrosis funding per patient is almost five times higher.

Figure 2: NIH funding comparisons on three rare diseases

Source: NIH RePORT report.nih.gov/categorical_spending.aspx

In total NIH funding for 2018 • DMD/BMD received $32 million • Myotonic received $13 million • FSHD received $11 million • Congenital muscular dystrophy received $9 million. There was also additional $81 million given to muscular dystrophy research that was not classified by muscular dystrophy type (NIH, 2018). The Department of Defense (DoD) has also awarded $26.4 million to DMD research between FY2011-FY2018 as part of the Congressional Special Interest Medical Research Programs (CDMRP, 2019). DMD is the only muscular dystrophy type funded by this DoD program.

While NIH funding for muscular dystrophy is increasing, it has a long way to go before it is similar to other rare diseases on a per disease or per patient basis. More funding is needed, especially to make progress on rare types of the disease that may be overshadowed by DMD/BMD. Fortunately many new therapeutic approaches being developed for DMD may have the potential to be modified to address other types of muscular dystrophies.

DMD & BMD Therapeutic Approaches

DMD/BMD represent a monogenic disease of differing severity. DMD is the result of a mutation within the dystrophin gene that leads to complete absence of the dystrophin protein. A

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 4 shortened but still partially functional dystrophin protein resulting from a non-expressed exon(s) generally results in the milder BMD (Aartsma-Rus et al. 2006).

The current standard of care for DMD boys is corticosteroid treatment. Steroids have been found to improve muscle strength, improve respiratory function, and prolong ambulation in multiple clinical trials (Goto et al. 2016; Biggar et al. 2006). Steroid therapy however will only slow disease progression and has multiple associated side effects such as weight gain, weakened bones, cataracts, and metabolic disorders among others (Falzarano et al. 2015). There are currently two steroid options for DMD patients, prednisone and deflazacort. Deflazacort’s primary advantage is a decrease in side effects (Shieh 2018).

Another steroid, Vamorolone, is currently in Phase II clinical trials. Pre-clinical studies suggest this steroid may be as effective as current options but with fewer side effects (Heier et al. 2013). While improved steroid treatments may benefit patients, at their most efficacious, steroids will still only delay disease progression. A number of disease-modifying therapeutic strategies are being explored for the treatment of DMD. These include both dystrophin compensatory strategies as well as treatments to ameliorate DMD without directly modulating dystrophin.

1. Dystrophin Compensatory Strategies

The most direct way to address DMD is to restore dystrophin protein. If dystrophin is restored to high enough levels, this tactic could cure the disease rather than just treating downstream secondary effects of the disease. Below is a discussion of three primary strategies that are being explored to restore dystrophin expression.

A) Exon Skipping

The Concept: Antisense oligonucleotides (single stranded nucleic acid analogs; AOs) are utilized to sequence- specifically bind splice sites of precursor mRNA and exclude the targeted exon from the final mRNA product. This mechanism can be used to skip a mutated dystrophin exon that has a premature stop codon or a mutation that causes a frame shift. The resulting product is a slightly shortened dystrophin mRNA that will create a slightly internally truncated, but functional, dystrophin protein.

The Impact: Exon skipping has shown limited success to date. It is still unclear if the current therapeutics in development will be successful at slowing disease progression, let alone address the larger goal of curing the disease.

One Example: Eteplirsen (Exondys51) is the only approved disease-modulating therapy for DMD in the United States. Developed by Sarepta Therapeutics and approved by the FDA in 2016, this antisense oligonucleotide (AO) drug’s mode of action is to ‘skip’ over dystrophin exon

51 and allow a shortened, functional dystrophin protein to be produced. It is estimated that Eterplirsen could be efficacious for the 14% of DMD patients with mutations in exon 51 (Lim, Maruyama, and Yokota 2017)..

Eteplirsen itself has been controversial on many fronts. Approved based on a small number of patients tested without a placebo control group, initial results showed a 51% mean increase in the number of dystrophin positive fibers at 48 weeks post-treatment. However, additional testing found that the mean increase in dystrophin positive fibers to be 17.4% at 48 weeks and the actual level of dystrophin protein after 180 weeks of treatment to be <1% that of healthy individuals (Lim, Maruyama, and Yokota 2017). It is estimated that patients need at least 10- 20% dystrophin to become only mildly affected or asymptomatic.

The Controversy on Eteplirsen

• FDA advisory panel voted 7 to 6 that there was not enough evidence that Eteplirsen would provide clinical benefit. • Dr. Janet Woodcock, Director of the CDER, pushed for approval arguing that Sarepta wouldn’t be able to continue working on better DMD drugs if its value crashed due to disapproval of Eteplirsen (Biospace 2016). • FDA Commissioner Dr. Robert Califf sided with Woodcock but he called for correction or retraction of a 2013 study supporting Eteplirsen, saying the study is “now known to be misleading, [and] should probably be retracted by its authors” (APhA 2016). • Two years after FDA approved Eteplirsen, the European Medicines Agency rejected Eteplirsen in September 2018 due to similar concerns about small study size, lack of placebo control, and questionable efficacy (Biospace 2018). • FDA requires an additional trial to demonstrate stronger clinical benefit.

Eteplirsen is on the market in the U.S. with growing revenues of ~$154 million in 2017, ~$301 million in 2018, and an expected $418 million in 2019 (House 2019). Sarepta also has clinical data for two additional exon skipping therapies; Golodirsen which has been submitted to the FDA and awaiting an FDA decision in August 2019, and Casimersen which Sarepta expects to submit to the FDA for approval sometime in 2019. Neither of these therapies seem more efficacious than Eteplirsen and struggle with similar incredibly low dystrophin protein levels (Pagliarulo 2019). While therapeutic improvement may be limited, proof of the safety and utility of the technical approach has been valuable to set the stage for improvements.

Improvements and alternatives:

In addition to Sarepta, three additional companies have exon skipping drugs in clinical trials (Table 1). While the current exon skipping drugs in clinical trials have shown limited dystrophin expression (<1%), the next generation of exon skipping compounds are focused on higher expression and efficacy. Many exon skipping oligos, such as Sarepta’s Eteplirsen, are formed from a phosphorodiamidate morpholino oligomer (PMO). Preclinical data has shown that by conjugating a peptide to the PMO and creating a peptide PMO (PPMO), the oligo becomes capable of better penetration into muscle, including notable increases in the heart and

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 6 diaphragm (Shieh 2018). Sarepta has a new exon 51 PPMO and is currently recruiting for a phase I clinical trial which if more efficacious, could replace eteplirsen which is only a PMO (NCT03375255).Wave Life Sciences is taking it one step further by producing stereopure oligonucleotide preparations rather than the sterorandom preparations of its competitors, a factor that could improve their efficacy. An additional alternative being pursued by Audentes Therapeutics is use of adeno-associated virus vectors to deliver exon skipping reagents into muscle cells. While Audentes’ work is still preclinical, they hope to start a Duchenne phase I/II trial in the fourth quarter of 2019 (Audentes Therapeutics 2019).

Table 1: Exon Skipping for DMD

FDA Dystrophin Current Clinical Trial Exon Approval Company Product Production Status targeted Status Sarepta Therapeutics Phase III (NCT02255552). Phase II for young patients Eteplirsen < 1% (6-48 months). 51 Approved Casimersen/S Phase III recruiting RP-4045 ~1.7% (NCT02500381) 45 N/A Golodirsen/SR Phase III recruiting P-4053 ~1% in Phase I/II (NCT02500381) 53 Submitted Phase I recruiting SRP-5051 N/A 51 N/A (NCT03375255)

BioMarin No consistent Drisapersen increase N/A 51 Rejected Wave Life Sciences

Phase I (NCT03508947) WVE-210201/ had positive safety profile. Suvodirsen N/A Phase II/III in July 2019. 51 N/A 44, 45, 53, Pre-clinical N/A Pre-clinical development 54, 55 N/A Nippon Shinyaku Phase I/II showed NS- dystrophin increases 065/Vitolarsen in 14/16 patients Phase II (NCT02740972) 53 N/A Daiichi Sankyo Co.

DS-5141b Not consistent Phase I/II 45 N/A

Source: (Wave Life Sciences 2018), (Nippon Shinyaku Co., Ltd. 2018), (Wave Life Sciences n.d.).

Beyond single mutations: Many DMD mutations occur within hotspots of the dystrophin gene. An antisense oligonucleotide cocktail that skips the exon 45-55 cassette, for example, could be

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 7 efficacious to over 60% of DMD patients (Echigoya et al. 2018). In preclinical models skipping a cassette of exons has worked and was able to produce dystrophin protein post exon skipping (Echigoya et al. 2018). Deletion of exon 45-55 is associated with mild BMD in multiple studies and suggests that therapeutic skipping of an exon 45-55 cassette may be able to convert DMD patients into mild BMD patients, a drastic improvement in quality of life (Aslesh, Maruyama, and Yokota 2018).

Technical challenges of antisense oligo approaches for exon skipping therapeutics include:

• Continual dosing: targeting the dystrophin transcript instead of the gene requires continual dosing throughout the patient’s life. • Achieving sufficient expression levels: current therapies are only achieving ~ 1% dystrophin levels but patients will need a minimum of 10-20% dystrophin. • Regulatory hurdles: particularly if a cocktail of multiple AONs will be used (Aslesh, Maruyama, and Yokota 2018).

B) Stop Codon Read-through

The Concept: Stop codon read-through promotes erroneous stop codons in dystrophin mRNA to be bypassed, allowing for production of full-length dystrophin protein.

The Impact: Around 15% of DMD patients have nonsense mutations that create premature stop codons leading to lack of dystrophin protein. Drugs that avoid the stop codon and allow production of dystrophin could provide therapeutic benefit if enough dystrophin is produced. Early results using this approach are intriguing.

One Example: PTC Therapeutics has developed Ataluren (Translarna) for stop codon readthrough and gained conditional approval in the EU in 2014 (Lim, Maruyama, and Yokota 2017). However after initially showing mild increases in dystrophin expression in early clinical trials, a later Phase 2b trial did not replicate these results and no significant dystrophin increase was seen in the enrolled DMD patients (Barthélémy and Wein 2018). Nevertheless, Ataluren did show modest improvement in patients’ six minute walk test in Phase III trials within a pre- specified patient subset (McDonald et al. 2017).

More on Ataluren

• FDA is requiring an additional Phase III trial to confirm improvements in the six minute walk test. • PTC Therapeutics is also doing a Phase II trial to evaluate increases in dystrophin protein levels after treatment. • An international drug registry of patients on Ataluren compared to natural history data showed Ataluren treated patients lost ambulation up to five years later than non-treated

DMD patients (BioPharm Insight 2018). The current data at best shows a selective slowing of disease progression for some patients.

Improvements and alternatives:

Gentamicin, an antibiotic, has also been repurposed and used as a stop codon readthrough drug for nonsense mutation DMD patients. Following 6 months of dosing, 3/12 patients showed dystrophin level over 10%, a level which could be therapeutically beneficial (Malik et al. 2010).

Current challenges to using stop codon approaches include:

• Dosing. Stop codon readthrough drugs would need to be taken for the lifetime of the patient. • Efficacy. Possible routes to improvement could include higher dosage, better entry of the drug into muscle, and higher affinity of the drug at binding to dystrophin RNA and facilitating readthrough. • Heterogeneity. The effectiveness of stop codon readthrough varies between patients of different ages and disease progression. • Insertional fidelity. A random amino acid inserted into the protein in place of the stop codon may produce less than fully functional protein if the swapped amino acid had specific functions that can no longer be fulfilled.

C) Gene Therapy:

The Concept: Gene therapy seeks to deliver a new, non-mutated version of the gene rather than altering the endogenous gene or RNA. For muscular dystrophy the delivery mechanism of choice has been utilization of adeno-associated virus (AAV), which easily transduces muscle cells. Gene therapy is arguably the DMD therapy with the greatest potential to impact disease at the current time. However, the dystrophin gene is much too large to fit within AAV and therefore truncated, micro- dystrophin genes containing only the genetic regions with critical assigned functions have been created.

Early human DMD clinical trial data has shown astounding levels of micro-dystrophin expression post gene therapy in skeletal muscle, suggesting that current delivery and dosing regimens are feasible (Sarepta Therapeutics 2018). However, it is too early to determine how well the miniaturized dystrophins actually work to restore muscle function long term. The resultant micro- dystrophin protein is hypothesized to be able to drastically slow disease progression but likely will not be capable of curing patients. It is unknown if micro-dystrophin gene therapy will turn DMD patients into much milder BMD patients (as BMD patient mutations endogenously create somewhat similar internally truncated dystrophin proteins) or if micro-dystrophin has the potential to be even more impactful than that.

Examples: Three companies are in a heated race for DMD gene therapy results. Sarepta Therapeutics, Solid Biosciences, and Pfizer are all using AAV capsids and muscle specific promoters although they vary in choice of viral capsid, promoter, and which dystrophin domains have been included in the micro-dystrophin.

In October 2018, Sarepta released updated data on 4 patients dosed with micro-dystrophin in their Phase I/II trial (NTC03375164). All patients showed:

• Robust micro-dystrophin expression with a mean greater than 80% of fibers being dystrophin positive and mean of almost 96% normal dystrophin protein levels when muscle biopsies where adjusted for fat and fibrotic tissue (90 days post treatment). • Decreases in serum creatine kinase (which suggests muscle membranes are less damaged/leaky as compared to pre-treatment). • Improvements in functional endpoints as assessed by the North Star Ambulatory Assessment (Sarepta Therapeutics 2018).

A final update on these 4 patients at 270 days post treatment was released in March 2019. These data still showed micro-dystrophin expression and large improvements in serum creatine kinase and functional endpoints, but it does appear like the improvements may have reached their maximum:

• Serum creatine kinase is drastically lower than pre-treatment, however the creatine kinase levels at day 270 are consistently higher for all patients than they were at the minimal levels observed between days 60 and 90. • North Star Ambulatory Assessment is still showing functional improvements although improvement appears to have plateaued between days 180 and 270 post-treatment. • No evidence of immune reaction to the micro-dystrophin protein. • Sarepta is now enrolling patients in a larger phase II placebo-controlled study and have already dosed more than 10 patients (Sarepta Therapeutics 2019).

Solid Biosciences’ Phase I/II clinical trial (NCT03368742) is still recruiting patients due to early safety concerns. In January 2018, the chair of Solid’s scientific advisory board, James Wilson5, resigned citing “emerging concerns about the possible risks” (Fidler 2018). Solid dosed their first patient in February 2018 but within days the patient was hospitalized with reduced blood cell counts and complement activation. Solid’s trial was put on clinical hold by the FDA in mid-2018, but has now resumed (Solid Biosciences 2018). In February 2019, Solid released three month post-treatment data for three patients. Unlike the high micro-dystrophin levels seen in Sarepta’s trial, Solid reported that in one patient micro-dystrophin levels were below 5% of normal protein

5 Dr. Wilson, a pioneer in the gene therapy field, co-led the early gene therapy trial that killed Jesse Gelsinger via systemic inflammatory response to the vector.

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 10 levels and only~10% of fibers were expressing detectable micro-dystrophin. In the other two patients, Solid was only able to detect micro-dystophin expression at very low levels by one detection method (immunofluorescence) and could not detect any expression by a second method (western blot). Solid Biosciences was quick to point out that the vector dosage they used was 4 times lower than the dose used by Sarepta. Solid plans to continue with the dose escalation portion of their study (Solid Biosciences 2019).

Pfizer has also released initial data for the first six patients dosed for its Phase Ib trial (NCT03362502). Micro-dystrophin expression levels were 10-60% of normal levels at 2 months post treatment as measured by mass spectrometry (Pfizer 2019). The average micro-dystrophin expression was 23.6% and 29.5% for the two doses Pfizer utilized; mean expression levels that are considerably lower than those achieved by Sarepta. All 6 patients exhibited some level of immune response to the gene therapy with the most severe reaction requiring an 11 day hospitalization. Due to the immune response, Pfizer has halted dosing additional patients but plans to eventually include additional subjects in this trial. Genethon, partnered with Sarepta, is also expected to start a Phase I micro-dystrophin gene therapy trial this year (BioPharm Insight 2018).

Technical and clinical challenges:

• To what extent can micro-dystrophin gene therapy slow DMD disease progression? • Can patients be treated early enough, before muscle damage has built up? • How long will AAV gene therapy last given that AAV genomes don’t integrate? • Is re-dosing feasible and safe if micro-dystrophin expression declines and re-dosing is required? • Are there other vectors, viral and transposons, which would be improvements?

AAV gene therapy is also being pursued for other muscular dystrophies as well. Myonexus Therapeutics, funded by CincyTech LLC, Rev1 Ventures and The Jain Foundation (Business Wire 2017) has five gene therapies aimed at treating limb-girdle muscular dystrophy. These five gene therapy constructs (three of which are currently in clinical trial), were developed in the lab of Louise Rodino-Klapac, Ph.D., who is also co-inventor of Sarepta’s micro-dystrophin for DMD treatment. In February 2019, Sarepta exercised its exclusive rights to acquire Myonexus and its five LGMD gene therapies (Sarepta Therapeutics 2019). Results from three patients dosed with its MYO-101 gene therapy for LGMD2E showed a mean of 51% of fibers expressing the therapeutic protein (beta-sarcoglycan) with 36% of normal protein levels (Sarepta Therapeutics 2019). It is interesting to note the AAV viral dose used in this LGMD2E trial was equivalent to the dosage Solid Biosciences used with micro-dystrophin (and therefore 4x lower than the dose Sarepta used for micro-dystrophin). It is also important to note that gene therapy for many other muscular dystrophies, including LGMD2E, will be able to deliver the full gene via AAV versus a truncated version like micro-dystrophin.

Questions about the early gene therapy data

• Sarepta’s LGMD2E vector dosed at 5x1013 vg/kg showed expression of the therapeutic protein while Solid’s DMD micro-dystrophin at the same dose hardly had detectable expression. There are multiple differences between these two trials (the patient disease/background, the therapeutic construct, the promoter driving protein expression, the viral capsid used, etc). Which factors are influencing the drastic differences in therapeutic protein levels? • Sarepta’s LGMD2E patient data shows expression of the therapeutic protein in roughly half of the muscle fibers. If you ‘cure’ half the cells, and potentially allow for a more physically active patient, will you more rapidly damage and cause cell death in the remaining diseased muscle?

2. Non-dystrophin Compensatory Treatments

There are also multiple treatments under development to ameliorate DMD without directly modulating dystrophin. Two of the most frequently targeted mechanisms include utrophin modulation and myostatin antagonism. While many ‘surrogate’ methods such as myostatin antagonism could be widely applicable, it remains unclear whether the treatment of downstream effects or surrogate targets will improve quality of life. Some experts remain skeptical that these methodologies will make a difference if the actual disease-causing gene isn’t also being addressed.

A) Utrophin Modulation

When dystrophin is lost in DMD it leads to Figure 3: Dystrophin glycoprotein complex loss of a large membrane complex, the dystrophin glycoprotein complex (DGC; see figure 3. The DGC complex functions to connect the basal lamina to the intracellular actin network. This stabilizes the muscle membrane (sarcolemma) during contraction. Non-dystrophin therapeutic approaches are attempting to restore components of the DGC to the skeletal muscle membrane in hopes of partially or completely stabilizing the muscle membrane and restoring the mechanical and signaling functions of this complex. Source: (Campbell 2012) Utrophin is highly homologous to dystrophin and endogenously forms a complex similar to the DGC at the neuromuscular and myotendinous junctions. High levels of utrophin can restore DGC components along the whole muscle fiber by substituting utrophin for dystrophin. This mechanism has been shown to reduce dystrophic symptoms in mice (Barthélémy and Wein

2018). Increasing levels of other proteins, such as biglycan and laminin-1, can also increase utrophin and then lead to restoration of the glycoprotein complex (substituted with utrophin). Table 2 details current companies and institutions developing utrophin modulation strategies. Ezutromid, developed by Summit Therapeutics, showed increased utrophin protein and decreased muscle damage at 24 weeks but failed to meet primary or secondary endpoints by 48 weeks leading to Ezutromid’s termination (FierceBiotech 2018) and Summit Therapeutics’ exit from muscular dystrophy therapies. Trial enrollment for utrophin modulation, an indirect strategy, may be limited now that micro-dystrophin gene therapy trials, which address dystrophin loss directly, are showing promising clinical data.

B) Myostatin Antagonism

Another approach for improving muscle mass is by antagonizing myostatin. All muscular dystrophies lead to muscle loss and atrophy. Myostatin is the body’s endogenous negative regulator of muscle mass, i.e. the negative checkpoint that prevents muscle from growing too much. As a negative regulator of muscle growth, reduction of myostatin levels would allow more muscle growth which could potentially combat atrophy in many muscle diseases including muscular dystrophy. To block myostatin, therapeutics can either interfere with myostatin itself or block the myostatin receptor. Both approaches would potentially prevent myostatin signaling and relieve myostatin’s suppression of muscle growth. Current myostatin targeting strategies in development for muscular dystrophy are listed in Table 2. Myostatin inhibition is also being pursued for other neuromuscular indications which if successful could be extended to muscular dystrophy.

Table 2: Non-dystrophin Compensatory Treatments

Company Product Mechanism Current Clinical Trial Status Summit Therapeutics

Phase II discontinued after no Ezutromid Small molecule utrophin upregulation improvement by 48 weeks Pre-clinical in March 2017, may SMT022357 Small molecule utrophin upregulation now be discontinued Nationwide Children's Hospital/Sarepta Therapeutics Gene therapy delivery of A glycosyltransferase that leads to Galgt2 utrophin upregulation Phase I/II recruiting Tivorsan Pharmaceuticals Recombinant Extracellular matrix protein that human connects to the DGC, proposed to biglycan increase utrophin Phase I (not yet initiated) Prothelia

Recombinant laminin-111 Increases utrophin and integrin levels Expected in clinical trials in 2020 Pfizer

Domagrosum Terminated (Aug 2018), 1 year of ab Myostatin nuetralizing antibody treatment showed no benefit. Roche Talditercept Antibody/adnectin fusion. Binds & alfa/RG206 inhibits myostatin Phase II/III recruiting Acceleron Pharma Antibody/activin receptor fusion protein. ACE-031 Binds and inhibits myostatin. Discontinued, safety concerns Sources: (Biopharm Insight n.d.), (Martin et al. 2008), (Barthélémy and Wein 2018), (Pfizer 2018), (Wahl 2013).

Treatments for Other Muscular Dystrophies 1. Myotonic dystrophy The most common type of adult muscular dystrophy, myotonic dystrophy encompasses two types of dystrophy with different causative genes but a similar disease mechanism. This dystrophy is caused by expansion of nucleotide repeat sequences within the DMPK gene for type 1 (DM1) or within the CNBP/ZNF9 gene for type 2 (DM2) (LoRusso, Weiner, and Arnold 2018). Expanded repeats in DM1 and DM2 RNA gain new, toxic functions. These expanded repeats erroneously bind and sequester RNA binding proteins, preventing them from performing their normal tasks in RNA processing. The scope of RNA processing that is affected and the mechanisms by which this creates the disease pathology are not well understood. In DM1 (but

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 14 not DM2) larger repeat expansions lead to earlier onset and/or more severe the disease pathology. Current treatments are limited to palliative care for specific pathologies such as sodium channel blockers to treat myotonia [prolonged muscle contractions seen in a majority of patients (LoRusso, Weiner, and Arnold 2018)]. In recent clinical trials targeting different mechanisms only the Tideglusib trial showed clinical benefit in cognition, fatigue, and autism symptoms although not in muscle function. (Table 3).

Table 3: Myotonic Dystrophy Treatments

Current Clinical Trial Company Product Mechanism Status Ionis Pharmaceuticals Phase I/IIa trial Antisense AON binds DM1 expansion to suspended due to Oligonueclotide prevent RNA binding protein inadequate delivery to (AON) sequestration. muscle University of Versailles Oral Restore circulating DHEA levels. Phase II/III failed to show dehydroepiandrost DHEA is associated with muscle improved muscle erone (DHEA) strength. strength. Insmed/University of Rochester Insulin-like growth factor-1/insulin- like growth factor binding protein 3 Phase I/II failed to show SomatoKine fusion. Proposed to increase protein increase in muscle synthesis and muscle strength. strength or function. AMO Pharma Phase II improved cognition and reduced fatigue and autism Glycogen synthase kinase 3 (GSK3) symptoms. Phase II/III is inhibitor. GSK3 is hyperactive in recruiting soon Tideglusib DM1. (NCT03692312). Sources: (LoRusso, Weiner, and Arnold 2018), (Penisson-Besnier et al. 2008), (Heatwole et al. 2011), (Henriques, Muscular Dystrophy New Today 2017), (Lopes, Muscular Dystrophy News Today 2018).

2. Facioscapulohumeral muscular dystrophy (FSHD) FSHD is the third most common muscular dystrophy. Several clinical trials to modify pathology have shown interesting interim results for FSHD patients (Table 4). Unlike many other dystrophies caused by the lack of a protein, FSHD is caused by expression of the protein DUX4. The relationship between DUX4 expression and the loss of muscle in patients is only beginning to be understood. Strategies to treat FSHD include interfering with DUX4 RNA or protein using antisense oligos and small molecules as well as additional mechanisms to stop DUX4 from being expressed. Fulcrum Therapeutics is expected to begin a Phase 2 clinical trial for a treatment targeted at

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 15 reducing DUX4 levels in mid-2019. In addition, Fulcrum has preclinical programs focused on increasing utrophin (for DMD) and decreasing DMPK (for myotonic dystrophy). Fulcrum Therapeutics is poised to make an impact on this disease and is a company to watch going forward.

Table 4: Facioscapulohumeral Muscular Dystrophy Treatments

Company Product Mechanism Current Clinical Trial Status Generic

Reduce inflammatory changes Discontinued, no improvements in Prednisone seen in FSHD. muscle mass or strength. Beta2-adrenergic agonist, proposed to anabolically increase Conflicting results, may only be Albuterol muscle mass. effective in some muscle groups. aTyr Pharma Mimetic of endogenous protein that controls T-cell activation, Phase I/II and Ib/II for FSHD, early- Resolaris/ATYR1940 proposed to reduce immune cell onset FSHD, and LGMD2B. Has infiltration into muscle. shown improved muscle strength. Wyeth/Pfizer

Discontinued. Failed to show MYO-029 Myostatin neutralizing antibody improved muscle strength. Acceleron Pharma

Mimetic of follistatin (natural Phase II results expected May 2020. ACE-083 myostatin antagonist) Has shown increased muscle mass. Fulcrum Therapeutics Phase II trial expected to begin Losmapimod Inhibition of DUX4 expression enrollment in mid-2019. Sources: (Hamel and Tawil 2018), (Muscular Dystrophy New Today n.d.), (Wagner et al. 2008), (Henriques, Muscular Dystrophy News Today 2018), (BioSpace, 2019).

Novel Approaches For The Future 1. Gene-editing: CRISPR and similar gene editing approaches may have the potential to be transformative for DMD patients providing a single solution for a multitude of different mutation types. CRISPR has been utilized effectively to remove DMD mutation hotspots in both DMD patient derived induced pluripotent cells and in a humanized DMD mouse model (Echigoya et al. 2018). Additional CRISPR strategies have corrected DMD mutations in dog models of the disease (Amoasii et al. 2018). Exonics Therapeutics, a CRISPR based DMD therapy company launched in 2017, believes their technology could be adapted to cure 80% of DMD patients (FierceBiotech 2017). Biopharm Insight lists 58 therapeutic companies with CRISPR strategies for human diseases. Intellia Therapeutics, CRISPR Therapeutics, and Editas Medicine are considered the major human CRISPR therapeutics companies (Frost & Sullivan 2017). Two of these major players, CRISPR Therapeutics and Editas Medicine, are pursing CRISPR for DMD as part of their pipeline and will provide direct competition to Exonics Therapeutics.

• CRISPR Therapeutics has partnered with Anagenesis Biotechnologies, a cell therapy company that has designed a platform for differentiation of pluripotent cells into skeletal muscle stem cells. CRISPR Therapeutics plans to use this technology paired with ex vivo CRISPR editing to restore dystrophin expression and deliver healthy stem cells back to DMD patients (CRISPR Therapeutics 2016). • Editas Medicine is in preclinical development to target DMD mutations. • Sarepta Therapeutics in collaboration with Duke University is also pursuing CRISPR for DMD (Sarepta Therapeutics 2017). • Vertex Pharmaceuticals acquired Exonics Therapuetics and expanded their collaboration with CRISPR Therapeutics in June 2019, positioning themselves to pursue CRISPR strategies for both myotonic and Duchenne muscular dystrophy (Vertex Pharmaceuticals 2019). DMD isn’t the only dystrophy CRISPR is being utilized for. Preclinical work in DM1 has successfully utilized CRISPR to cleave out the expanded repeats present in myotonic dystrophy type 1 from both mouse and patient cell culture models (LoRusso, Weiner, and Arnold 2018). CRISPR could be a one-time treatment that could be fully curative depending on the editing strategy. But CRISPR relies on AAV delivery to human patients and delivery still needs to be optimized. 2. Cell Therapy: Cell therapy is not a new idea for muscular dystrophy research, but has struggled to get beyond preclinical research. For cell therapy to be effective long term, a cell type has to be able to be delivered intravenously, migrate to all muscles, fuse and repair current muscle, as well as populate the stem cell pool with corrected self-renewable cells. This is a long list of requirements that has not yet been achieved. While cell therapy for muscular dystrophy has not yet been shown clinically viable, it is an area that should be pursued. As patients lose muscle mass, the combination of a therapeutic to correct existing muscle with cell therapy to create new muscle may prove a powerful approach. There are many factors to consider when choosing a cell therapy strategy:

• Cell Type: stem cells (mesenchymal or induced pluripotent), myoblast cells, and mesoangioblasts are being explored. Healthy stem cells in animal models were successful at restoring dystrophin expression but have failed in human clinical trial (Barthélémy and Wein 2018). Mesenchymal stem cells have been shown in one study to be capable of either delaying muscle strength loss or increasing muscle strength in DMD patients (Rajput et al. 2015).

• Cell Source: healthy allogeneic cells can be used but may elicit an immune response. Autologous cells will not cause an immune reaction but would require ex vivo correction of the patient’s mutation. Ex vivo correction and transplantation of pluripotent cells in animal models has been successful multiple times (Barthélémy and Wein 2018).

• Delivery: Intramuscular delivery requires many injections, relies on delivered cells to migrate throughout the muscle (yet to be seen at significant levels), and cannot deliver to interior muscles such as the diaphragm and heart (Rai 2018). Intravenous delivery

requires the delivered cells to cross the endothelial border, a feat that only mesoangioblasts have been shown capable. Systemic delivery of allogeneic mesoangioblasts has improved muscle function in dystrophic animal models (Sampaolesi et al. 2006) but did not improve DMD patients’ muscle (Cossu et al. 2015).

• Viability: Every cell type being explored shows reduced viability upon delivery to the muscle. Additionally, ex vivo expansion of cells to allow delivery of a higher cell number has been shown to limit myogenic potency in vivo. A phase I/II clinical trial with allogeneic myoblast cells, a cell stage slightly more mature than stem cells, hopes to have a longer cell viability once delivered to patients (NCT02196467).

• Stem Cell Population: Myoblasts cannot create a corrected, self-renewing stem cell population while the other cell types discussed potentially can. Even with the current struggles in the skeletal muscle cell therapy field, the global market for skeletal muscle repair via cell therapy is expected to grow from $228 million in 2016 to $1.1 billion by 2022 (Rai 2018). One intriguing cell therapy product for DMD is CAP-1002 from Capricor Therapeutics. Currently in Phase I/II clinical trial, CAP-1002 cell therapy consists of dystrophin positive allogeneic cardiosphere-derived cells (CDCs). CDCs are known to release exosomes capable of modulating inflammation, fibrosis, and apoptosis. A Phase I/II trial using CAP-1002 showed improvements in upper limb function and cardiac function. Capricor has begun recruiting for a phase II trial with this therapeutic. 3. Small molecule interacting with RNA (SMiRNA): Expansion Therapeutics is in preclinical development with small molecules that would bind the expanded RNA repeats in Myotonic Dystrophy, thereby freeing the erroneously sequestered RNA binding proteins (Frost & Sullivan 2018). Expansion Therapeutics launched in January 2018 with $55.3 million from multiple venture capital firms (5AM Ventures, Kleiner Perkins, Novartis Venture Fund, Sanofi Ventures, RA Capital Management and Alexandria Venture Investments (Business Wire 2018).

Market Outlook Muscular dystrophy therapeutics are being pursued by a wide range of companies including some of the top pharmaceutical companies such as Roche and Pfizer, and there are a variety of approaches being explored to treat muscular dystrophy. However finding a cure, even within a subset of dystrophies such as DMD, appears to still be years in the future. Drug exploration is rationally targeting the most prevalent mutation types and therefore the larger patient populations. Knowledge gained in these early trials will hopefully allow any successfully therapies to be more cost-effectively transitioned to treat populations with even more rare mutations. Additionally, once there is a first wave of successful therapeutics, it may also be possible to combine successful therapies, a strategy that is already being explored in preclinical work. While rare/genetic disease therapies were valued at $102.6 billion in 2017 and expected to grow to $168.3 billion in 2023 with a CAGR of 8.6% (Frost & Sullivan, 2018), the portion that is

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 18 genetic disease therapies for DMD is tiny: 0.15% in 2017 and 0.34% in 2023 (Figure 4). The global market size for genetic modification therapies in DMD is small, estimated at $155 million in 2017, $256 million in 2018, reaching a market value of $572.5 million by 2023 (Bergin 2018). Given the limited market attractiveness, funding for translation and commercialization for these diseases has historically come from philanthropic organizations.

Figure 4: 2023 Market Size

$572.5 million

$168.3 billion

Rare/Genetic Disease Therapies

DMD Genetic Modiication Therapies

Sources: Frost & Sullivan. 2018. Novel Therapies for Rare and Genetic Diseases. June 28. Accessed December 29, 2018. https://cds.frost.com. Bergin, John. 2018. Genetic Modification Therapies Clinical Applications: Gene Therapies, Genetically Modified Cell Therapies, RNA Therapies and Gene Editing. BCC Research.

Venture Funding Both CureDuchenne and the Muscular Dystrophy Association (MDA) are utilizing philanthropic donations to accelerate drug development by providing grants to academic research groups as well as financing biotech companies. MDA is focusing on funding promising scientists mostly at the academic level and is currently supporting more than 175 research programs (Muscular Dystrophy Association 2018). CureDuchenne Ventures, formed in only 2014, has a portfolio of 16 projects with the majority going to companies pursuing therapeutics (CureDuchenne 2018). Both MDA and CureDuchenne also utilize their knowledge to suggest partnerships to push therapy candidates to the next level of discovery or testing. While MDA and CureDuchenne are some of the big associations, there are many more organizations that are donating funds for muscular dystrophy research such as Charley’s Fund (over $40 million) and Parent Project Muscular Dystrophy (over $45 million). DMD Therapeutics, a start-up in Seattle, Washington was launched solely on $400,000 raised from 3 DMD foundations (Ryan’s Quest, Michael’s Cause, and Pietro’s Fight (BioSpace 2018)). Venture capital funding is critical in launching biotechnology start-ups. Rare disease is becoming a very hot topic for venture capital as big pharma is becoming more willing to buy

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 19 small orphan drug companies (Nishka Mittal of OrbiMed Advisors). Ms. Mittal believes this is fueled by both patient advocacy highlighting the unmet need and the many FDA designations that reward or speed rare disease drugs to market, such as Fast Track, Priority Review, Breakthrough, and Accelerated Approval designations as well as the Orphan Drug Act’s 7 year market exclusivity (Mittal, N., personal communication, 2019). In 2012 and 2013, over $500 million/year venture capital funding went toward orphan disease drug development (Thomas and Wessel 2015), and more than 10% of total Series A funds went to rare disease startups in 2015 (Luxner 2018).

Figure 5: Venture Capital Investments 2010-2018

Figure 5A: Venture Capital Figure 5B: Venture Capital Investment in Rare and Orphan Investment in Muscular Dystrophy Disease 140 3000 120 2500 100 2000 80 1500 60

1000 Millions$ 40 Millions$ 500 20 0 0 2010 2011 2012 2013 2014 2015 2016 2017 2018 2010 2011 2012 2013 2014 2015 2016 2017 2018 Year Year

Source: Pitchbook.com

Between the years of 2010 and 2018 there were 845 venture capital investments in 286 companies working in the rare and/or orphan disease area (Pitchbook.com). During this same time period there were 110 venture capital deals for 33 companies involved in the muscular dystrophy field. While venture capital investment in rare/orphan diseases has shown an overall increased growth trajectory since 2010, there is not an obvious trend for muscular dystrophy (Figure 5). With the exception of 2017, venture capital investment in muscular dystrophy has been significantly lower since its peak in 2013.

Summary The muscular dystrophy field is making progress towards having treatments that drastically slow disease progression if not cure the disease entirely. Over the course of the next few years, patients dosed with micro-dystrophin gene therapy are expected to show reductions in disease progression never seen before. Exon skipping and stop codon read-through have the ability to provide improved therapeutic benefit over micro-dystrophin by allowing production of a more complete dystrophin protein, if these therapies can be modified to produce dystrophin at higher,

Progress in the Treatment of Muscular Dystrophies ©2019 Nelsen Biomedical, LLC 20 therapeutically relevant levels. For the multitude of dystrophies caused by mutations in genes small enough to fit in AAV, gene therapy has the potential to be the gold standard. Further in the future, CRISPR technologies may provide a curative treatment that doesn’t require constant surveillance and re-dosing. Even with the progress that has been made towards muscular dystrophy therapies many hurdles still exist including:

• Skeletal muscle is the largest organ in the human body accounting for 30-40% of body mass and is not an easily accessible organ (Janssen et al. 2000). • Genetic diagnosis is still lacking for many patients.6 Diagnosis at the genetic level is the only way for a definitive diagnosis. • Patients are often not diagnosed until after they have suffered significant muscle loss.7 • Limited patients for clinical trials.8 • Many dystrophies don’t have thorough natural history/disease progression studies, something that is valuable when assessing treatment efficacy. • Dystrophies progress slowly. We may need longer trials to see clinical benefit. While there is still much progress to be made, this is an exciting time for muscular dystrophy therapeutics. Despite most clinical trials targeting DMD, progress made in DMD has a high likelihood of being able to be retrofitted to treat other muscular dystrophies. A therapeutic ‘win’ for any muscular dystrophy is likely to be a win for all muscular dystrophies.

6 Dr. Mittal stresses the fact that multiple dystrophies can appear similar as well as the same dystrophy appearing drastically different between patients and disease stages. 7 Once muscle has been replaced by fat and fibrosis, it is unknown how well patients will be able to get muscle back even if the underlying cause of their dystrophy is fixed. 8 Many muscular dystrophies don’t have large registries of diagnosed patients.

Appendix

CRISPR/Cas9 Finances: CRISPR as a gene editing tool was pioneered in 2012 and since then NIH funding of projects mentioning CRISPR rose from USD 7.4 million to USD 603.3 million in 2016. The revenue generated by CRISPR in 2017 was estimated at USD 2 billion with an estimated increase to 25-30 billion by 2030 (Frost & Sullivan 2017).

FSHD Disease Mechanism: The genetic cause of FSHD type 1 is a reduction in the number of D4Z4 repeats on chromosome 4. FSHD patients will have only 1-10 D4Z4 repeats which allows for expression of DUX4 mRNA and protein. FSHD type 2, which accounts for 5% of patients, results when DUX4 is aberrantly allowed to be expressed due to mutations in other genes (SMCHD1 or DNMT3B). DUX4 is normally only expressed early in development and then suppressed. When DUX4 is expressed after development within FSHD skeletal muscle it is toxic to the muscle and leads to muscle cell death. How DUX4 affects muscle is not well understood but it has been implicated in changing RNA processing, inhibiting the regeneration of new muscle and inducing death of any muscle cell in which it is expressed (Hamel and Tawil 2018). Gene Therapy Affordability: If gene therapy for DMD does come to fruition it is unclear how financially accessible these treatments will be to patients. The first FDA approved in vivo gene therapy (Spark Therapeutics’ Luxturna) for a form of vision loss is priced at USD 850,000 for both eyes (BioPharm Insight 2018). Specialty treatments are generally purchased by the treating institution and then billed to insurance; however, this creates significant risk for the treating institution. Insurers and drug companies are looking at novel payment mechanisms to ensure patient access; in the case of Luxturna this has been through an outcomes-based rebate system. Spark Therapeutics will pay rebates at both short term (30-90 days) and long term (30 month) time points if patients’ outcomes do not meet a pre-specified threshold. Spark is also working on an installment payment system that would limit the large upfront cost to a patient’s current insurer (Spark Therapeutics 2018).

In May 2019, the FDA approved Zolgensma (Novartis/AveXis) an in vivo gene therapy for the neuromuscular disease spinal muscular atrophy (SMA). SMA results from lack of SMN1 protein and Zolgensma is AAV vector delivery of a functional SMN1 gene. Dosing of 15 babies with the most severe SMA type 1 resulted in all babies alive with motor function benefits at 24 months old; natural history studies at 20 months only see 8% of babies surviving event free. With such inarguable benefit in the clinic, Novartis bought Avexis in May 2018. The only other treatment available for SMA is Nusinersen/Spinraza from Biogen which requires continual dosing and is priced at $750,000 the first year and $375,000 each year after that. Novartis initially forecast the list price for Zolgensma to be as high as $4-5 million, although upon approval the pricing was listed as $2.125 million, a sum that still makes it the most expensive FDA approved drug (The Scientist 2019). The Institute for Clinical and Economic Review (ICER), a drug pricing watchdog,

suggests that to be cost effective, Spinraza would need to be priced below $65,000 (except for the first year) and that Zolgensma needs to be a maximum of $2 million (Lash 2019).

Newborn & Prenatal Screening: Most neuromuscular diseases are only diagnosed once a patient begins showing symptoms and currently Pompe disease and spinal muscular atrophy are the only neuromuscular diseases recommended during newborn screening in the U.S although not all states actually screen for them. There are multiple countries with optional DMD newborn screens which look for elevated creatine kinase (CK) levels in the blood, a general marker of muscle damage. Elevated CK will often lead to a DMD diagnosis but can also lead to diagnosis of a different muscle disease following muscle biopsy and mutational testing. This screening mechanism however will not identify all muscular dystrophies, was shown to miss roughly 6% of DMD cases in one study (Gatheridge et al. 2016) and can have many false positives potentially from injury during the birthing process. In October 2018, CureDuchenne partnered with Baebies, a newborn screening company to add DMD to Baebies screening platform (Baebies 2018). Prenatal testing for muscular dystrophy can also be carried out by looking for genetic mutations via chorionic villus sampling or amniocentesis. Similar genetic testing can be paired with IVF prior to embryo implantation. Prenatal testing is often only chosen in families that have a history of muscular dystrophy. Muscular dystrophy mutations can arise de novo, especially in the case of DMD where the immense size of the dystrophin gene leads to 1/3 of diagnosed patients having a de novo mutation (Leiden Muscular Dystrophy pages 2008). Dr. Mittal is a strong advocate for early diagnosis of patients so that once we have effective therapies, they can be treated before the patients lose significant muscle. Dr. Mittal would like to see everyone sequenced at birth to catch diseases like muscular dystrophy early on but admits that the policy and infrastructure to enable this is not currently in existence.

References

Aartsma-Rus, Annemieke, Judith C. T. Van Deutekom, Ivo F. Fokkema, Gert-Jan B. Van Ommen, and Johan T. Den Dunnen. 2006. “Entries in the Leiden Duchenne Muscular Dystrophy Mutation Database: An Overview of Mutation Types and Paradoxical Cases That Confirm the Reading-Frame Rule.” Muscle & Nerve 34 (2). John Wiley & Sons, Ltd: 135–44. doi:10.1002/mus.20586. Amoasii, Leonela, John C W Hildyard, Hui Li, Efrain Sanchez-Ortiz, Alex Mireault, Daniel Caballero, Rachel Harron, et al. 2018. “Gene Editing Restores Dystrophin Expression in a Canine Model of Duchenne Muscular Dystrophy.” Science (New York, N.Y.) 362 (6410). American Association for the Advancement of Science: 86–91. doi:10.1126/science.aau1549. APhA. 2016. FDA commissioner calls for retraction of eteplirsen study. September 23. Accessed January 13, 2019. https://pharmacist.com/article/fda-commissioner-calls- retraction-eteplirsen-study. Aslesh, Tejal, Rika Maruyama, and Toshifumi Yokota. 2018. “Skipping Multiple Exons to Treat DMD-Promises and Challenges.” Biomedicines 6 (1). Multidisciplinary Digital Publishing Institute (MDPI). doi:10.3390/biomedicines6010001. Audentes Therapeutics. 2019. Audentes Therapeutics Annonces Expansion of AAV Technology Platform and Pipeline with New Development Programs for Duchenne Muscular Dystrophy and Myotonic Dystrophy. April 8. Accessed July 9, 2019. https://audentestx.gcs- web.com/news-releases/news-release-details/audentes-therapeutics-announces- expansion-aav-technology Baebies. 2018. CureDuchenne Partners with Baebies to Accelerate Newborn Screening for Duchenne Muscular Dystrophy. October 3. Accessed January 27, 2019. https://www.baebies.com/cureduchenne-partners-with-baebies-to-accelerate-newborn- screening-for-duchenne-muscular-dystrophy/. Barthélémy, Florian, and Nicolas Wein. 2018. “Personalized Gene and Cell Therapy for Duchenne Muscular Dystrophy.” Neuromuscular Disorders 28: 803–24. doi:10.1016/j.nmd.2018.06.009. Bergin, John. 2018. Genetic Modification Therapies Clinical Applications: Gene Therapes, Genetically Modified Cell Therapies, RNA Therapies and Gene Editing. Bcc Research. Biggar, W.D., V.A. Harris, L. Eliasoph, and B. Alman. 2006. “Long-Term Benefits of Deflazacort Treatment for Boys with Duchenne Muscular Dystrophy in Their Second Decade.” Neuromuscular Disorders 16 (4): 249–55. doi:10.1016/j.nmd.2006.01.010. BioPharm Insight. 2018. Gene Therapy Report - April 2018. April 4. Accessed December 29, 2018. https://www.biopharm-insight.com.

BioPharm Insight. 2018. Orphan Drug Report - September 2018. September 27. Accessed December 29, 2018. https://www.biopharm-insight.com. BioPharm Insight. 2018. PTC Therapeutics Announces Initial Data from Patient Registry Demonstrating Translarna™ (ataluren) Slows Disease Progression in Children with Duchenne Caused by a Nonsense Mutation. October 6. Accessed December 29, 2018. https://www.biopharm-insight.com/biopharm/AccessPoint.aspx?action= DisplayAjaxFrame&transaction=42.0.3411433414. Biopharm Insight. n.d. Summit Therapeutics PLC. Accessed December 28, 2018. https://www.biopharm-insight.com. Biospace. 2019. Fulcrum Therapeutics Acquires Global Rights to Losmapimod, a Potential Disease-Modifying Therapy for Facioscapulohumeral Muscular Dystrophy. April 23. Accessed July 6, 2019. https://www.biospace.com/article/releases/fulcrum-therapeutics- acquires-global-rights-to-losmapimod-a-potential-disease-modifying-therapy-for- facioscapulohumeral-muscular-dystrophy/ Biospace. 2018. Going Its Own Way, European Regulators Reject Sarepta's Exondys 51 for DMD. September 21. Accessed January 13, 2019. https://www.biospace.com/article/going-its-own-way-european-regulators-reject-sarepta- s-exondys-51-for-dmd-fd1a-/?keywords=sarepta+FDA+approval. Biospace. 2016. This is Why Woodcock Oked Sarepta's DMD Drug. November 4. Accessed January 13, 2018. https://www.biospace.com/article/this-is-why-b-woodcock-b-oked- sarepta-s-dmd-drug-/?keywords=sarepta+FDA+approval. Business Wire. 2018. Expansion Therapeutics Raises $55.3 Million in Series A Financing to Advance Portfolio of Novel RNA Targeted Small Molecule Medicines to Treat Rare Diseases. January 3. Accessed January 12, 2018. https://www.businesswire.com/news/home/20180103005068/en/Expansion- Therapeutics-Raises-55.3-Million-Series-Financing. Business Wire. 2017. Myonexus Therapeutics Secures $2.5 Million Seed Financing to Clinically Advance Limb-Girdle Muscular Dystrophy (LGMD) Gene Therapies. December 13. Accessed January 14, 2019. https://www.businesswire.com/news/home/20171213005559/en/Myonexus- Therapeutics-Secures-2.5-Million-Seed-Financing. BusinessWire. 2018. CureDuchenne Partners with Baebies to Accelerate Newborn Screening for Duchenne Muscular Dystrophy. October 4. Accessed December 29, 2018. https://www.businesswire.com/news/home/20181004005420/en/. Campbell, Yvonne M. Kobayashi & Kevin P. 2012. "Chapter 66 - Skeletal Muscle Dystrophin- Glycoprotein Complex and Muscular Dystrophy." In Muscle, by Eric N. Olson Joseph A. Hill, 935-942. Academic Press. CDMRP. 2019. Duchenne Muscular Dystrophy. February 22. Accessed March 23, 2019. https://cdmrp.army.mil/dmdrp/default.

Cossu, G., S. C. Previtali, S. Napolitano, M. P. Cicalese, F. S. Tedesco, F. Nicastro, M. Noviello, et al. 2015. “Intra-Arterial Transplantation of HLA-Matched Donor Mesoangioblasts in Duchenne Muscular Dystrophy.” EMBO Molecular Medicine 7 (12): 1513–28. doi:10.15252/emmm.201505636. CRISPR Therapeutics. 2016. CRISPR Therapeutics and Anagenesis Biotechnologies Announce Strategic In-Licensing and Collaboration Agreement to Develop CRISPR/Cas9-based Cell Therapies for Muscle Diseases. June 8. Accessed December 29, 2018. http://ir.crisprtx.com/news-releases/news-release-details/crispr-therapeutics- and-anagenesis-biotechnologies-announce. CureDuchenne. 2018. CureDuchenne Ventures. Accessed March 23, 2019. https://www.cureduchenne.org/ventures/. Deyle, David R, and David W Russell. 2009. “Adeno-Associated Virus Vector Integration.” Current Opinion in Molecular Therapeutics 11 (4). NIH Public Access: 442–47. http://www.ncbi.nlm.nih.gov/pubmed/19649989. Echigoya, Yusuke, Kenji Rowel Q. Lim, Akinori Nakamura, and Toshifumi Yokota. 2018. “Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots : Prospects and Challenges.” Journal of Personalized Medicine 8 (Dmd). Multidisciplinary Digital Publishing Institute: 1–29. doi:10.3390/jpm8040041. Emery, Alan E. H. 2002. “The Muscular Dystrophies.” Lancet 359 (9307): 687–95. doi:10.1016/S0140-6736(03)14959-8. EvaluatePharma. 2018. Orphan Drug Report 2018. May. Accessed March 20, 2019. http://info.evaluategroup.com/rs/607-YGS-364/images/OD18.pdf. Falzarano, Maria, Chiara Scotton, Chiara Passarelli, Alessandra Ferlini, Maria Sofia Falzarano, Chiara Scotton, Chiara Passarelli, and Alessandra Ferlini. 2015. “Duchenne Muscular Dystrophy: From Diagnosis to Therapy.” Molecules 20 (10). Multidisciplinary Digital Publishing Institute: 18168–84. doi:10.3390/molecules201018168. Fidler, Ben. 2018. Exome. January 25. Accessed December 29, 2018. https://xconomy.com/boston/2018/01/25/solid-delays-ipo-discloses-duchenne-problems- raises-rivals-hackles/?single_page=true. FierceBiotech. 2017. Exonics to use CRISPR in an effort to treat majority of DMD boys. February 27. Accessed December 28, 2018. https://www.fiercebiotech.com/biotech/biotech-exonics-to-use-crispr-effort-to-treat- majority-dmd-boys. FierceBiotech. 2018. Summit dumps ezutromid after phase 2 Duchenne fail. June 27. Accessed December 28, 2018. https://www.fiercebiotech.com/summit-dumps-ezutromid-after- phase-2-duchenne-fail. Frost & Sullivan. 2017. Growth Insights on the Global CRISPR/Cas9 Tools Market, 2017. October 30. Accessed December 29, 2018. https://cds.frost.com.

Frost & Sullivan. 2018. Novel Therapies for Rare and Genetic Diseases. June 28. Accessed December 29, 2018. https://cds.frost.com. Frost & Sullivan. 2018. RNA Technologies Empowering Small Molecule Therapeutics. August 9. Accessed December 29, 2018. https://cds.frost.com.v Gatheridge, Michele A., Jennifer M. Kwon, Jerry M. Mendell, Günter Scheuerbrandt, Stuart J. Moat, François Eyskens, Cheryl Rockman-Greenberg, Anthi Drousiotou, and Robert C. Griggs. 2016. “Identifying Non–Duchenne Muscular Dystrophy–Positive and False Negative Results in Prior Duchenne Muscular Dystrophy Newborn Screening Programs.” JAMA Neurology 73 (1). American Medical Association: 111. doi:10.1001/jamaneurol.2015.3537. Genetic Engineering & Biotechnology News. 2019. Vertex Grows Gene Editing Presence, Acquiring Exonics and Expanding CRISPR Therapeutics Collaboration. June 10. Accessed July 9, 2019. https://www.genengnews.com/news/vertex-grows-gene-editing-presence- acquiring-exonics-and-expanding-crispr-therapeutics-collaboration/ Goto, Masahide, Hirofumi Komaki, Eri Takeshita, Yoshiki Abe, Akihiko Ishiyama, Kenji Sugai, Masayuki Sasaki, Yu-ichi Goto, and Ikuya Nonaka. 2016. “Long-Term Outcomes of Steroid Therapy for Duchenne Muscular Dystrophy in Japan.” Brain and Development 38 (9): 785– 91. doi:10.1016/j.braindev.2016.04.001. Hamel, Johanna, and Rabi Tawil. 2018. “Facioscapulohumeral Muscular Dystrophy: Update on Pathogenesis and Future Treatments.” Neurotherapeutics 15 (4). Springer International Publishing: 863–71. doi:10.1007/s13311-018-00675-3. Heatwole, Chad R, Katy J Eichinger, Deborah I Friedman, James E Hilbert, Carlayne E Jackson, Eric L Logigian, William B Martens, et al. 2011. “Open-Label Trial of Recombinant Human Insulin-like Growth Factor 1/Recombinant Human Insulin-like Growth Factor Binding Protein 3 in Myotonic Dystrophy Type 1.” Archives of Neurology 68 (1). NIH Public Access: 37–44. doi:10.1001/archneurol.2010.227. Heier, Christopher R, Jesse M Damsker, Qing Yu, Blythe C Dillingham, Tony Huynh, Jack H Van der Meulen, Arpana Sali, et al. 2013. “VBP15, a Novel Anti-Inflammatory and Membrane-Stabilizer, Improves Muscular Dystrophy without Side Effects.” EMBO Molecular Medicine 5 (10): 1569–85. doi:10.1002/emmm.201302621. Henriques, Carolina. 2017. Muscular Dystrophy New Today. June 1. Accessed December 22, 2018. https://musculardystrophynews.com/2017/06/01/amo-02-congenital-myotonic- dystrophy-treatment-gains-fda-fast-track/. Henriques, Carolina. 2018. Muscular Dystrophy News Today. May 2. Accessed December 23, 2018. https://musculardystrophynews.com/2018/05/02/ace-083-treat-fshd-earns-fda-fast- track-designation/. House, Douglas W. 2019. Sarepta down 5% on preliminary Exondys 51 sales. January 7. Accessed January 17, 2019. https://seekingalpha.com/news/3421096-sarepta-5- percent-preliminary-exondys-51-sales.

House, Douglas W. 2019. Sarepta down 5% on preliminary Exondys 51 sales. January 7. Accessed March 17, 2019. https://seekingalpha.com/news/3421096-sarepta-5-percent- preliminary-exondys-51-sales. House, Douglas W. 2018. Sarepta sees as much as $305M in Exondys 51 sales this year; shares up 1%. January 8. Accessed March 17, 2019. https://seekingalpha.com/news/3321700-sarepta-sees-much-305m-exondys-51-sales- year-shares-1-percent. Janssen, Ian, Steven B. Heymsfield, ZiMian Wang, and Robert Ross. 2000. “Skeletal Muscle Mass and Distribution in 468 Men and Women Aged 18–88 Yr.” Journal of Applied Physiology 89 (1): 81–88. doi:10.1152/jappl.2000.89.1.81. Lim, Kenji Rowel, Rika Maruyama, and Toshifumi Yokota. 2017. “Eteplirsen in the Treatment of Duchenne Muscular Dystrophy.” Drug Design, Development and Therapy Volume11 (February). Dove Press: 533–45. doi:10.2147/DDDT.S97635. Lash, Alex. 2019. Report: Biogen, Novartis Should Drop Prices of Billon-Dollar SMA Drugs. April 4. Accessed April 7, 2019. https://xconomy.com/boston/2019/04/04/report-biogen- novartis-should-drop-prices-of-billon-dollar-sma- drugs/?mc_cid=327d054d60&mc_eid=a19fdc7673. Leiden Muscular Dystrophy pages. 2008. Dystrophin (DMD) sequence variations. September. Accessed January 27, 2019. http://www.dmd.nl/database.html. Lopes, Jose Marques. 2018. Muscular Dystrophy News Today. March 21. Accessed December 22, 2018. https://musculardystrophynews.com/2018/03/21/amo-pharma-myotonic- dystrophy-therapy-improves-cognition-and-function/. LoRusso, Samantha, Benjamin Weiner, and W. David Arnold. 2018. “Myotonic Dystrophies: Targeting Therapies for Multisystem Disease.” Neurotherapeutics, October 18. doi:10.1007/s13311-018-00679-z. Luxner, Larry. 2018. Duchenne Needs ‘Thinking Outside the Box’ to Advance Treatments But Investors Taking Note, Advocate Says. June 1. Accessed December 29, 2018. https://musculardystrophynews.com/2018/06/01/ecrd2018-duchenne-needs-new-ways- to-advance-treatments-but-investors-interested-advocate-says/. Mah, Jean K., Lawrence Korngut, Jonathan Dykeman, Lundy Day, Tamara Pringsheim, and Nathalie Jette. 2014. “A Systematic Review and Meta-Analysis on the Epidemiology of Duchenne and Becker Muscular Dystrophy.” Neuromuscular Disorders 24 (6). Elsevier: 482–91. doi:10.1016/J.NMD.2014.03.008. Malik, Vinod, Louise R Rodino-Klapac, Laurence Viollet, Cheryl Wall, Wendy King, Roula Al- Dahhak, Sarah Lewis, et al. 2010. “Gentamicin-Induced Readthrough of Stop Codons in Duchenne Muscular Dystrophy.” ANN NEUROL 67: 771–80. doi:10.1002/ana.22024. Martin, Paul T., Rui Xu, Louise R. Rodino-Klapac, Elaine Oglesbay, Marybeth Camboni, Chrystal L. Montgomery, Kim Shontz, et al. 2008. “Overexpression of Galgt2 in Skeletal Muscle Prevents Injury Resulting from Eccentric Contractions in Both Mdx and Wild-Type

Mice.” AJP: Cell Physiology 296 (3): C476–88. doi:10.1152/ajpcell.00456.2008. McDonald, Craig M., Craig Campbell, Ricardo Erazo Torricelli, Richard S. Finkel, Kevin M. Flanigan, Nathalie Goemans, Peter Heydemann, et al. 2017. “Ataluren in Patients with Nonsense Mutation Duchenne Muscular Dystrophy (ACT DMD): A Multicentre, Randomised, Double-Blind, Placebo-Controlled, Phase 3 Trial.” The Lancet 390 (10101): 1489–98. doi:10.1016/S0140-6736(17)31611-2. Mittal, Nishka. 2019. "Personal Communication." March 12. Mittal, Plavi. 2019. "Personal Communication." February 20. Muscular Dystrophy Association. 2018. Grants at a Glance. Accessed March 23, 2019. https://www.mda.org/science/grants-at-a-glance. Muscular Dystrophy New Today. n.d. Resolaris (ATYR1940). Accessed December 23, 2018. https://musculardystrophynews.com/resolaris-atyr1940/. NIH. 2018. Estimates of Funding for Various Research, Condition, and Disease Categories (RCDC). May 18. Accessed March 23, 2018. https://report.nih.gov/categorical_spending.aspx. Nippon Shinyaku Co., Ltd. 2018. "NS-065/NCNP-01 (Viltolarsen) of Nippon Shinyaku’ in-house product." News Release, Kyoto, Japan. Norwood, Fiona L M, Chris Harling, Patrick F Chinnery, Michelle Eagle, Kate Bushby, and Volker Straub. 2009. “Prevalence of Genetic Muscle Disease in Northern England: In- Depth Analysis of a Muscle Clinic Population.” Brain : A Journal of Neurology 132 (Pt 11). Europe PMC Funders: 3175–86. doi:10.1093/brain/awp236. Pagliarulo, Ned. 2019. Sarepta to seek speedy approval of third DMD drug. March 28. Accessed April 6, 2019. https://www.biopharmadive.com/news/sarepta-to-seek-speedy- approval-of-third-dmd-drug/551522/. Penisson-Besnier, I., M. Devillers, R. Porcher, D. Orlikowski, V. Doppler, C. Desnuelle, X. Ferrer, et al. 2008. “Dehydroepiandrosterone for Myotonic Dystrophy Type 1.” Neurology 71 (6): 407–12. doi:10.1212/01.wnl.0000324257.35759.40. Pfizer. 2019. Pfizer Presents Initial Clinical Data on Phase 1B Gene Therapy Study for Duchenne Muscular Dystrophy (DMD). June 28. Accessed June 30, 2019. https://www.pfizer.com/news/press-release/press-release- detail/pfizer_presents_initial_clinical_data_on_phase_1b_gene_therapy_study_for_duch enne_muscular_dystrophy_dmd Pfizer. 2018. Pfizer Terminates Domagrozumab (PF-06252616) Clinical Studies for the Treatment of Duchenne Muscular Dystrophy . August 30. Accessed December 24, 2018. https://www.pfizer.com/news/press-release/press-release- detail/pfizer_terminates_domagrozumab_pf_06252616_clinical_studies_for_the_treatme nt_of_duchenne_muscular_dystrophy. Rajput, B S, Swarup K Chakrabarti, Vaishali S Dongare, Christina M Ramirez, and Kaushik D

Deb. 2015. “Human Umbilical Cord Mesenchymal Stem Cells in the Treatment of Duchenne Muscular Dystrophy: Safety and Feasibility Study in India.” Journal of Stem Cells 10 (2): 141–56. http://www.ncbi.nlm.nih.gov/pubmed/27125141. Rai, Sangeeta. 2018. Cell Therapy Processing: Global Markets and Technologies. Bcc Reseach.

Sampaolesi, Maurilio, Stephane Blot, Giuseppe D’Antona, Nicolas Granger, Rossana Tonlorenzi, Anna Innocenzi, Paolo Mognol, et al. 2006. “Mesoangioblast Stem Cells Ameliorate Muscle Function in Dystrophic Dogs.” Nature 444 (7119): 574–79. doi:10.1038/nature05282. Sarepta Therapeutics. 2018. For Investors. October 3. Accessed December 29, 2018. http://investorrelations.sarepta.com/news-releases/news-release-details/sarepta- therapeutics-announces-23rd-international-congress-world. Sarepta Therapeutics. 2018. Micro-dystrophin. Accessed December 29, 2018. https://www.duchennegenetherapy.com/. Sarepta Therapeutics. 2019. Micro-dystrophin Gene Therapy Update Conference Call. March 25. Accessed April 6, 2019. http://investorrelations.sarepta.com/events/event- details/sarepta-therapeutics-update-call. Sarepta Therapeutics. 2019. Sarepta Exercises Option to Acquire Myonexus Therapeutics. February 27. Accessed March 17, 2019. http://investorrelations.sarepta.com/news- releases/news-release-details/sarepta-exercises-option-acquire-myonexus-therapeutics. Sarepta Therapeutics. 2019. Sarepta Therapeutics Announces Positive and Robust Expression and Biomarker Data from the First Three-Patient Cohort Dosed in the MYO-101 Gene Therapy Trial to Treat Limb-Girdle Muscular Dystrophy Type 2E, or Beta- Sarcoglycanopathy. February 27. Accessed March 17, 2019. http://investorrelations.sarepta.com/news-releases/news-release-details/sarepta- therapeutics-announces-positive-and-robust-expression. Sarepta Therapeutics. 2017. Sarepta Therapeutics Signs Exclusive Global Collaboration with Duke University for Gene Editing CRISPR/Cas9 Technology to Develop New Treatments for Duchenne Muscular Dystrophy (DMD). October 31. Accessed December 29, 2018. https://globenewswire.com/news-release/2017/10/31/1169596/0/en/Sarepta- Therapeutics-Signs-Exclusive-Global-Collaboration-with-Duke-University-for-Gene- Editing-CRISPR-Cas9-Technology-to-Develop-New-Treatments-for-Duchenne- Muscular-Dystrophy-DMD.html. Sarepta Therapuetics. 2018. Sarepta Therapeutics Announces Partnership with Myonexus Therapeutics for the Advancement of Multiple Gene Therapy Programs Aimed at Treating Distinct Forms of Limb-Girdle Muscular Dystrophies. May 3. Accessed January 27, 2019. http://investorrelations.sarepta.com/news-releases/news-release- details/sarepta-therapeutics-announces-partnership-myonexus-therapeutics. Shieh, Perry B. 2018. “Emerging Strategies in the Treatment of Duchenne Muscular Dystrophy.”

Neurotherapeutics, October 9. doi:10.1007/s13311-018-00687-z. Solid Biosciences. 2018. Solid Biosciences Announces FDA Removes Clinical Hold on SGT- 001. June 18. Accessed December 29, 2018. https://investors.solidbio.com/news- releases/news-release-details/solid-biosciences-announces-fda-removes-clinical-hold- sgt-001. Solid Biosciences. 2019. Solid Biosciences Announces Preliminary SGT-001 Data and Intention to Dose Escalate in IGNITE DMD Clinical Trial for Duchenne Muscular Dystrophy. February 7. Accessed March 17, 2019. https://www.solidbio.com/about/media/press- releases/solid-biosciences-announces-preliminary-sgt-001-data-and-intention-to-dose- escalate-in-ignite-dmd-clinical-trial-for-duchenne-muscular-dystrophy-1. Spark Therapeutics. 2018. Spark Therapeutics Announces First-of-their-kind Programs to Improve Patient Access to LUXTURNA™ (voretigene neparvovec-rzyl), a One-time Gene Therapy Treatment. January 3. Accessed December 29, 2018. http://ir.sparktx.com/news-releases/news-release-details/spark-therapeutics-announces- first-their-kind-programs-improve. Taylor, Phil. 2018. Biogen pays $27.5M upfront for 2 AliveGen muscle drugs. July 24. Accessed December 24, 2018. https://www.fiercebiotech.com/biotech/biogen-pays-27-5m-upfront- for-two-alivegen-muscle-drugs. The Scientist. 2019. FDA Approves Gene Therapy for Spinal Muscular Atrophy. May 27. Accessed June 30, 2019. https://www.the-scientist.com/news-opinion/fda-approves- gene-therapy-for-spinal-muscular-atrophy-65935. Thomas, David, and Chad Wessel. 2015. “Venture Funding of Therapeutic Innovation: A Comprehensive Look at a Decade of Venture Funding of Drug R&D.” https://www.bio.org/sites/default/files/files/BIO-Whitepaper-FINAL.PDF. Vertex Pharmaceuticals. 2019. Vertex Expands into New Disease Areas and Enhances Gene Editing Capabilities Through Expanded Collarboration with CRISPR Therapeutics and Acquisition of Exonics Therapeutics. June 6. Accessed June 30, 2019. https://investors.vrtx.com/news-releases/news-release-details/vertex-expands-new- disease-areas-and-enhances-gene-editing Wagner, Kathryn R., James L. Fleckenstein, Anthony A. Amato, Richard J. Barohn, Katharine Bushby, Diana M. Escolar, Kevin M. Flanigan, et al. 2008. “A Phase I/IItrial of MYO-029 in Adult Subjects with Muscular Dystrophy.” Annals of Neurology 63 (5): 561–71. doi:10.1002/ana.21338. Wahl, Margaret. 2013. Update: ACE-031 Clinical Trials in Duchenne MD. May 1. Accessed April 6, 2019. https://www.mda.org/quest/article/update-ace-031-clinical-trials-duchenne- md. Wave Life Sciences. n.d. Pipeline. Accessed December 28, 2018. https://www.wavelifesciences.com/pipeline/.

Wave Life Sciences. 2018. Wave Life Sciences Announces Positive Phase 1 Results for WVE- 210201 in Duchenne Muscular Dystrophy (DMD). December 6. Accessed December 28, 2018. http://ir.wavelifesciences.com/news-releases/news-release-details/wave-life- sciences-announces-positive-phase-1-results-wve-210201.