Methods 99 (2016) 91–98
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Methods
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Gene therapy in monogenic congenital myopathies ⇑ Xuan Guan a,b,c,1, Melissa A. Goddard a,b,c,1, David L. Mack b,c, Martin K. Childers b,c, a Department of Physiology and Pharmacology, School of Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA b Department of Rehabilitation Medicine, University of Washington, Seattle, WA, USA c Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA article info abstract
Article history: Current treatment options for patients with monogenetic congenital myopathies (MCM) ameliorate the Received 22 April 2015 symptoms of the disorder without resolving the underlying cause. However, gene therapies are being Received in revised form 10 September developed where the mutated or deficient gene target is replaced. Preclinical findings in animal models 2015 appear promising, as illustrated by gene replacement for X-linked myotubular myopathy (XLMTM) in Accepted 7 October 2015 canine and murine models. Prospective applications and approaches to gene replacement therapy, using Available online 14 October 2015 these disorders as examples, are discussed in this review. Ó 2015 Elsevier Inc. All rights reserved. Keywords: Gene therapy Muscle disease Animal models Muscular dystrophy Myopathy
1. Introduction to monogenic congenital myopathies (MCM) stability during muscle contraction [3] (Table 1). The symptoms are visible as early as 2–3 years of age, a progressive decrease in Current treatment options for patients with monogenetic con- striated muscle function, starting from proximal muscle such as genital myopathies (MCM) ameliorate the symptoms of the disor- legs and pelvis and eventually involve the whole body. Most der without resolving the underlying cause. However, therapies are patients are wheelchair-dependent starting from early teen. The being developed where the mutated or deficient gene target is average life expectancy is around 25 years (
Table 1 A comparison of some of the monogenic congenital myopathies presently under study.
Duchenne muscular X-linked myotubular Facioscapulohumeral muscular Myotonic dystrophy (DM) Limb-girdle dystrophy (DMD) myopathy (XLMTM) dystrophy (FSHD) muscular dystrophy (LGMD) 2C and 2D Inheritance Single gene mutation on Single gene mutation at q28 on Autosomal dominant, Autosomal dominant. DM1 Autosomal the X chromosome the X chromosome [17] contraction of D4Z4 repeat on CTG triplet repeats expansion recessive. Single chromosome 4q35 and toxic of DMPK gene locates on gene mutation on gain of function of the DUX4 chromosome 19 [19]. DM2 chromosome 13 gene [18] CCTG tetranucleiotide repeat and 17 (2C and expansion of ZNF9 gene on 2D, respectively) chromosome 3 [20] Molecular Deficiency of the protein Deficiency of the phosphatase Toxic protein product of DUX4 Malfunctioned DMPK and ZNF9 Deficiency of biology dystrophin myotubularin [17] [18] proteins gamma and alpha sarcoglycan (2C and 2D, respectively) Clinical Weakness of the skeletal Weakness of the skeletal Initial weakness of facial, Muscle wasting and myotonic; Muscle wasting symptoms muscles; respiratory muscles [10], wheelchair scapula and humeral muscle, heart conduction block; primarily involve insufficiency in teens; dependence [10], respiratory progressively involving other cataract; infertility proximal muscle cardiac dysfunction; insufficiency at birth [21];no muscles; sparing respiratory such as hip and leading cause of death is cardiac phenotype; leading muscle shoulder cardiorespiratory failure cause of death is respiratory dysfunction [22] Demographics Presentation around 2– Affects 1 in 5000 live male Affects 12/100,000 [24] Affects 1/8000 people Up to 68% of 3 years of age; average life births [4]; presentation typi- worldwide. Type 1 most individuals with expectancy of 25 years cally at birth [23]; average life common in most countries [25] childhood onset expectancy of 29 months [9] and 10% with adult onset [26] Histology Increased fiber size Centrally located nucleus[27]; Non-specific fiber necrosis, Fiber atrophy, internal nuclei, Variation of fiber variability; cycling of fiber variably-sized myofibers with increased variation in fiber size, pyknotic nuclear clumps, diameter, fiber regeneration and an abnormally large number of internal nuclei, fiber type lipofuscin accumulation, degeneration and degeneration small fibers; organelle abnor- variability, connective tissue increased fiber size variation regeneration, mality and ‘‘necklace fibers” and fat proliferation. [30] split fibers, ring [28] Mononuclear cell filtration [29] fibers
consider when design gene therapies. High vector titers may be to treat diseases [31]. This occurs through modified expression of required to reach an effective dose [11], increasing the chance of genes of interest to trigger alterations of certain biological func- adverse effects in patients. In addition, the need to treat respiratory tions. Gene therapy targets living cells, primarily because cell’s muscles as well as the heart in DMD and XLMTM may complicate intrinsic gene expression machinery is indispensible to mediate delivery. Improvements in delivery methods [12] and in vector the production of therapeutic molecules, including protein, shRNA characterization to increase efficiency may address this problem and microRNA. [13]. Vector modification may also ensure more efficient delivery Since classical gene therapy acts on native tissues, the abun- to the muscle and improve safety by reducing off-target delivery dance of target cells largely determines the effect of gene therapy. to organs like the liver [11,14]. Tissue-specific promoters is another This is especially true in congenital myopathies. In the advanced strategy to secure tissue-specific transgene expression. Immune stage of diseases, such as DMD and XLMTM, surviving myocytes response is a major concern, particularly in genetically-null are so limited that even if the function of individual myofibers patients who may have antibodies against the gene product pro- were fully restored, there would be no appreciable functional duced by the treatment [15] and immunosuppression before and improvement on tissue level. The advent of stem cell technology, during treatment may have to be considered. There are also chal- especially the discovery of induced pluripotent stem cells (iPSCs) lenges specific to each disease. For example, the large size of the [32,33], has the potential to overcome this hurdle. Pluripotent stem dystrophin gene limits the choice of vector to be used in treatment. cells may be able to replenish tissue loss through their indefinite Overexpression of c-sarcoglycan in LGMD patients may exacerbate self-replicating potential and capacity to be converted into nearly the condition [16]. Significant wasting in XLMTM patients leaves all cell types within the body. The advantages of combining stem very little muscle to treat and, due to the young age of the patients, cell therapy with gene therapy have been demonstrated in several selecting an appropriate and reproducible outcome measure may animal studies, in which vectors were administered ex vivo and prove difficult. We will be discussing new developments that modified donor cells were later engrafted into native tissue [34– address these concerns, including modifications of the vector and 36]. the combination of gene therapy with other approaches. Various gene therapy strategies target gene expression and reg- ulatory network at different levels. For example, genetic sequence can be permanently inserted into genome for long-term expres- 2. Gene therapy sion, using retrovirus or lentivirus [31]. With the development of genome modification tools [37] such as clustered regularly inter- 2.1. What is gene therapy spersed short palindromic repeats (CRISPR) enzymes and Tran- scription activator-like effector nuclease (TALEN), the technical Gene therapy is defined as the introduction of nucleic acids, barrier of in situ editing eukaryotic genomic DNA has been sub- including DNA, RNA and their analogs into cells of living organism stantially lowered. These techniques hold the potential to seam- X. Guan et al. / Methods 99 (2016) 91–98 93 lessly restore the genome to a disease-free state without the intro- Table 2 duction of foreign genetic elements, eliminating the widely shared The different strategies for various monogenic myopathies. concern of increased tumorigenic risk. Alternatively, the therapeu- Gene therapy strategy Diseases tic genetic sequence can be designed to persist within cells as a Gene addition DMD [39], XLMTM [2], LGMD2D [43] stable episome, allowing maintenance of long-term expression Gene correction DMD [3] without genomic integration [31]. Gene subtraction FSHD [42], Myotonic dystrophy [44] Introduced nucleic acids can be further divided into protein- coding and non-coding sequence. While protein-coding sequences serve as templates for protein production, non-coding nucleic acids sired adverse effects. Gene therapy functions to modulate the function to modulate epigenetic processes controlling gene expres- production of native proteins and is therefore more specific sion. A good example is RNA interference, in which microRNA and effective. (miRNA) [38] or small interfering RNA (siRNA) [38] bind to mRNA Long-term duration of efficacy molecules to halt protein translation and mediate mRNA Certain vectors, such as lentivirus, retrovirus or AAV [31], degradation. demonstrate persistent effects leading to extended phenotypic correction/disease remission. This is in stark contrast to conven- 2.2. What will be administered tional drugs, requiring repetitive dosing to reach a steady-state drug concentration for stable effects. Based on the mechanism of action, gene therapy can be catego- rized into gene addition, gene correction or gene subtraction. In 2.3.1. Gene therapy clinical trails for congenital myopathies gene addition, exogenous genetic sequence is introduced into cells. Ongoing clinical trails are mainly targeting diseases with defec- Gene correction alters the diseased loci. Attenuating or silencing tive dystrophoglycan complex, including DMD, BMD and LGMD. the expression of single or a network of genes is known as gene Local delivery of full-length dystrophin plasmid to patients’ radi- subtraction. alis muscle results in detection of dystrophin mRNA and protein The root causes of diseases dictate the gene therapy strategy, in 6 out of 9 patients [45]. Encouraged by this study, various gene which differs dramatically among diseases (Table 2). Genomic therapy strategies have been employed in clinical trails aiming to mutations sometimes interrupt the reading frame of the protein- restore dystrophin expression, including read-through agents (Ata- coding genes, leading to the absence of proteins, as occurs with luren/PTC124, Arbekacin Sulfate/NPC14 and Gentamicin), exon dystrophin [39] and myotubularin [40] in DMD and XLMTM. Con- skipping oligonucleotides (AVI-4658, Drisapersen, sequently the focus of gene therapy has been devoted to supple- Pro044/045/053, SRP4045/4043 and NS-065/NCNP-01) and virus ment a protein-coding sequence to replace the defective gene. mediated delivery of protein encoding genes, such as minidys- Alternately, the mRNA splicing event can be modified to bypass trophin and Follistin. Mendell et al. reported delivery of truncated the mutated region, restoring the reading frame with a truncated minidystrophin gene (NCT00428935) elicited dystrophin specific T but functional protein product [41]. In other cases, where the dis- cells, without direct visualization of the protein in muscle, suggest- ease is caused by pathogenic overexpression, it is imperative to ing immune response to be a major hurdle for successful gene ther- silence the gene expression [42]. Once such example is facioscapu- apy [15]. The Phase 2a study of the read-through agent ataluren lohumeral muscular dystrophy (FSHD), which is caused by the reported 61% patients demonstrated increase of dystrophin expres- overexpression of the myopathic DUX4 gene [18]. sion after a course of 28 days treatment (NCT00264888) [46]. Intramuscular injection of the exon skipping agent AVI-4658 2.3. Advantages of gene therapy in treating congenital myopathies (NCT00159250) resulted in 17% increase of the mean dystrophin signal, reaching 22% to 32% of the healthy control [47]. For LGMD, The goal of gene therapy in treating congenital myopathies is AAV packaged alpha and gamma sarcoglycan are now being tested two-folds. For some monogenic diseases, the current technology in clinical trails. Local injection of AAV1.tMCK.hSGCA is adequate to completely restore the genetic defect in somatic (NCT00494195, NCT01976091) led to persistent a-sarcoglycan cells, representing a cure that is unachievable by any other meth- expression up to 6 months and augment muscle fiber size ods. Recent findings in canine and mouse models of XLMTM are [43,48]. A trial with AAV1-c-sarcoglycan vector in LGMD 2C good examples, where a single treatment recovered animals with patients has completed though result has not yet been disclosed a severe monogenic disease to normal function [2]. For myopathies (NCT01344798). The ongoing clinical trials are summarized in with complex genetic makeup, a more realistic goal is to delay dis- the Table 3. ease progression and preserve muscle function in order to main- tain life quality. There are several advantages associated with gene therapies in 3. Vector toolbox comparison to conventional pharmacotherapies. A critical element of gene therapy is the vector, the vehicle that Complete rectification of the abnormal genetic code facilitates the transfer of genetic material. Due to the size and neg- There are currently very limited therapeutic options that effec- ative charge of ribonucleic acid, shuttles are needed to carry the tively target congenital myopathies. Most available drugs are cargo across biological barriers to reach target cells. The vector largely symptom-alleviating agents with transient effect. In toolbox is composed of viral vectors and non-viral vectors. Viruses contrast, gene therapy is designed to target the root genetic have naturally evolved sophisticated machinery to target cells with cause and thus represent a potential cure for some monogenic high efficiency, making them the ideal tool for gene transfer. A diseases. summary of the advantages and disadvantages of commonly used High specificity viral vectors is outlined in Table 4. Conventional pharmacotherapy utilizes natural or synthetic AAV is the preferred viral vector for many ongoing clinical trials, small molecules aiming to alter certain biological functions. largely due to the fact that AAV efficiently targets post-mitotic However, it is difficult to identify high-specificity molecules parenchymal cells, such as neurons and skeletal myofibers, which that only interact with the molecule of interest. As a result, are usually inpermissive to other vectors [50]. Unlike integrating pleiotropic effects of these agents are the main source of unde- viral vectors, AAV exists as epichromosome, reducing the likeli- 94 X. Guan et al. / Methods 99 (2016) 91–98
Table 3 Viral vectors have been widely used in ongoing gene therapy Ongoing gene therapy clinical trails for congenital myopathy. clinical trials [1]. Despite their differences, however, the use of NCT number Conditions Interventions Phases any viral-based vector carries with it certain risks, including NCT02354781 DMD rAAV1.CMV.huFollistin344 Phase 1/Phase tumorigenicity, immunogenicity and limited cargo space. Most of 2 these risks can be minimized by non-viral vectors. On the other NCT01519349 BMD rAAV1.CMV.huFollistatin344 Phase 1 hand, the main drawbacks of non-viral vectors, including the NCT02255552 DMD eteplirsen injection Phase 3 capacity to cross various biological barriers and stability, have been NCT01918384 DMD NPC-14 Phase 2 largely addressed by late breakthroughs. For example, material NCT02376816 DMD rAAVrh74.MCK.micro- Phase 1 Dystrophin science has provided various lipid-based and polymer-based DNA NCT01247207 DMD/ Ataluren Phase 3 vectors, many of which have been tested in clinical trials [55]. BMD Nucleic acid chemistry evolution has resulted in the development NCT01557400 DMD/ Ataluren Phase 3 of nucleic acid analogs, such as 20-O-methyl-phosphorothioate BMD 0 NCT01826487 DMD Ataluren Phase 3 (2 OMe) and morpholino phosphorodiamidate oligonucleotide NCT02090959 DMD Ataluren Phase 3 (PMO). While retaining the same nucleo-base to enable Watson– NCT02329769 DMD PRO044 Phase 2 Crick base pairs with natural nucleotides, ribonuclease-resistant NCT01826474 DMD PRO045 Phase 1/Phase moieties have replaced natural ribose ring and backbone phospho- 2 diester linkage. These modifications lead to increased molecular NCT01910649 DMD/ Drisapersen Phase 2 BMD stability against enzyme degradation. Direct infusion of analog NCT01957059 DMD PRO053 Phase 1/Phase oligonucleotides is associated with efficient targeting [56–59]. 2 20OMe and PMO have been employed as antisense oligonucleotide NCT02310906 DMD SRP-4053 Phase 1/Phase (AON) to mediate exon skipping in DMD treatment (Table 5). Both 2 0 NCT02500381 DMD SRP-4045/SRP-4053 Phase 3 Drisapersen (2 OMePS AON to exon 51) and Eteplirsen (PMO mor- NCT02081625 DMD NS-065/NCNP-01 Phase 1 pholino to exon 51) have also demonstrated restored dystrophin NCT01976091 LGMD2D scAAVrh74.tMCK.hSGCA Phase 1/Phase expression and even mild clinical improvement as measured by 2 6 min walk [60,61]. hood of insertional mutagenesis while maintain long-term trans- 4. Routes of delivery gene expression. Another advantage of AAV is that the vector gen- ome can be pseudotyped with alternative capsids. The viral capsid The success of gene therapy is largely determined by the effi- largely determines tissue tropism, gene transfer efficacy and the cient delivery of vectors to target tissues directly determines the vector’s dose-dependent toxicity. For example AAV 1, 6, 9 trans- success. Effective delivery approaches include direct injection, duce muscle with high efficiency [51]. This array of available sero- locoregional perfusion and systemic delivery (see Tables 6–8). types allows the vector to be tailored to different applications. Though many natural AAV variants have been identified, efforts 4.1. Direct injection are being devoted to engineer synthetic viral capsids to address specific clinical challenges. Mutating tyrosine residues (Y445F Direct injection into the target tissue is commonly used to and Y731F) in the AAV6 capsid can improve the skeletal muscle determine the efficacy of a potential new therapy [2,62–64]. This gene transfer [52]. Moreover, retention of AAV vector in liver has method ensures that the desired organ receives the necessary ther- been a problem of systemic infusion. Vectors that ‘‘de-target” liver apeutic dose and restricts the treatment to that organ, reducing have been created through randomly mutating the surface- off-target effects. However, distribution may be limited, even exposing region of AAV9 capsid (AAV9.45 and AAV9.61) [53] or within the injected muscle [65]. For example, XLMTM dogs treated by engineering a chimeric capsid with AAV2 and AAV8 (AAV2i8) by AAV8-MTM1 injection into the cranial tibialis of the hindlimb [54]. Both strategies redirected the vector away from liver while show improvements in the strength, size and histopathology of maintaining high transduction efficiency to skeletal muscle. the treated limb [2] but show a continued progression of the
Table 4 Advantages and disadvantages of the various types of viral vector.
Viral vector Advantage Disadvantages Retrovirus Stable integration Only infect dividing cells Allowing to be pseudotyped Size limit of 8 kb Insertional mutagenesis associated tumor risk Low titers [49] Lentivirus Stable integration for persistent transgene expression Possibility of insertional mutagenesis Infect both dividing and non-dividing cells Allowing to be pseudotyped with the VSVG (vesicular stomatitis virus G), making concentration easier Adenovirus Large cargo space (36 kb adenoviral genome) Strong immune response associated toxicity High titer Transient expression due to immune response Permissiveness to non-dividing cells Persists as non-integrating episome [49] Adeno-associated virus Easy to concentrate Limited cargo space around 4 kb [31]; Availability of numerous serotypes makes it possible to choose a vector with a degree of selectivity for the desired target cell type Transducing non-dividing cells Mostly remain non-integrating episome Naturally non-pathogenic X. Guan et al. / Methods 99 (2016) 91–98 95
Table 5 Table 7 Some of the treatments currently under clinical trial for DMD (Abbr: SC, subcuta- Available dog models for monogenic congenital myopathies. neous; IV: intravenous). Mutation Phenotype Drug name Description Clinical trial Delivery DMD dog models number route Golden retriever X-linked Dystrophin deficient GSK2402968 (Pro051)/ 20OMePS AON to NCT01803412 SC muscular mutation Muscle hypertrophy and Drisapersen exon 51 dystrophy [101] weakness Pro044 20OMePS AON to NCT01037309 SC/IV Contracture exon 44 Esophageal dysfunction due to Pro045 20OMePS AON to NCT01826474 SC enlargement of the tongue exon 45 Cardiac phenotype AVI-4658 (Eteplirsen) PMO morpholino to NCT02255552 IV LGMD 2C dog models exon 51 SG-deficient Boston Mutation Reduced a-sarcoglycan; b-and terrier [102] unknown c-sarcoglycan absent Muscular dystrophy SG-deficient Mutation a-, b- and c-sarcoglycan absent Chihuahua [92] unknown Muscular dystrophy Table 6 SG-deficient cocker Mutation Reduced a- and b-sarcoglycan; Available mouse models for monogenic congenital myopathies. spaniel [92] unknown c-sarcoglycan absent Muscular dystrophy Mutation Phenotype XLMTM dog models DMD mouse models XLMTM dog Missense Myotubularin deficient Mdx mouse [95] Spontaneous point Similar but milder muscle [2,104,105] mutation Centronuclear myopathy mutation in exon phenotype compared to in exon Severely affected 23 human patients Skeletal muscle wasting, result- Mdx52 mouse Exon 52 deletion Absence of shorter isoforms ing in pronounced and progres- [96] Dp260 and Dp140 sive weakness Mdx/Utrphin Utrophin knockout Severe phenotype with Shortened lifespan double on the basis of Mdx early deterioration knockout mouse [97] LGMD 2C mouse models 4.3. Systemic delivery c-Sarcoglycan Exon 2 deletion Muscular dystropy null mouse Progressive but mild In systemic delivery, a potential gene therapy vector is intro- [98] Gait abnormalities duced to the entire body. This is of particular importance in con- Reduced activity genital myopathies where cardiorespiratory failure is the leading Increased serum CK Pseudohypertrophy of the cause of death but there are effects of the disease throughout the diaphragm body [72,73]. As with locoregional perfusion, the vascular endothe- XLMTM mouse models lium could hinder distribution to the skeletal muscle and car- XLMTM Deletion of exon 4 Centronuclear myopathy diorespiratory systems most affected by disease [71,74]. Systemic knockout causes an absence Severely affected dosing also increases the likelihood of off-target gene delivery, mouse [99] of myotubularin Skeletal muscle wasting, which may require the use of additional safety measures like resulting in pronounced and progressive weakness tissue-specific promoters. Finally, higher doses may be required Shortened lifespan for systemic delivery, due to circulating antibodies and filtration MTM1 p.R69C C to T point muta- Milder phenotype by the liver. However, modifications of the vector or immunosup- mutant tion in exon 4 pression can address these problems [75–77]. mouse [100]
5. Preclinical disease model systems disease including muscle weakness, impaired ambulation and Reliable model systems are indispensible for the critical transi- early death [2,66]. This limited distribution is therefore disadvan- tion from bench to bedside. It is required by the FDA the potency tageous when the disease affects multiple systems or the entire and toxicity of vectors be validated on several levels of preclinical body, as is often the case with congenital myopathies. Also, direct models, before entering clinical trials. The following section sum- treatment of organs like the heart or diaphragm may require inva- marizes model systems at different level from cell culture to ani- sive surgery or complicated techniques [67,68], which can be diffi- mal models. cult in chronically ill-patients.
5.1. Cell culture models 4.2. Locoregional perfusion Cell culture models can be used to test the potency and cellular Locoregional perfusion, where a limb is isolated before intravas- toxicity of a biological therapy. There are several advantages, such cular infusion under high pressure, is another approach to gene as intricate manipulation of experiment conditions and consider- therapy delivery. Despite the use of high pressure, it is a safe, rel- ably lower costs. Early phase vector validations are usually carried atively painless option for human patients [12] and has been used out in relevant cell cultures. Immortalized mammalian human successfully in animal models, including the XLMTM dogs, where embryonic kidney (HEK) 293 cells and C2 myoblasts were there was widespread clinical improvement [2,69,70]. However, employed to validate the vector expressing a truncated dystrophin vector titers may be reduced due to the low permeability of the protein [78]. Engineered HEK293 cells over-expressing pathogenic vascular endothelium [71] and, like direct injection, isolated DUX4 gene has been utilized to confirm the efficacy of RNAi vector locoregional perfusion does not address treatment of the cardiores- [42]. Cells derived from diseased tissues, such as myoblasts iso- piratory system. lated from animal models or human patients, have been utilized 96 X. Guan et al. / Methods 99 (2016) 91–98
Table 8 Comparison of cell culture, small animal and large animal models.
Cell culture Small animal Large animal Cost Cheap Relatively inexpensive Very expensive Mutations Harbors patient specific mutations Created through the knockdown or knock Often naturally-occurring mutations that Easy to engineer specific mutations out of related genes mirror human disease Phenotype Cellular phenotype May differ from human phenotype Often similar to human disease Size N/A Small size Larger size Organ size closer to that of humans Able to adapt test originally developed for use in the human clinic, increasing translational power Lifespan N/A Shorter lifespan may make assessing long Longer lifetime allows for extended study term effects difficult of a single organism Reproduction Primary cells have limited culture time Reproductive fecundity allows for Relatively slow reproduction with smaller iPS cells theoretically can be propa- multiple replicates for more fully powered litter size can limit study gated indefinitely experiments Expressivity Primary cells from patients with distinct Genetically very similar to wildtype Some variation in genetic background, genetic background outside of loci of interest even when using true littermate controls Immunological N/A Immune response may differ from that Immune response may differ from that seen in humans seen in humans
to test potency for vectors developed for DMD [79], Pompe disease been successfully adapted for use in dogs [22]. Many naturally- [80] and myotonic dystrophy [44]. occurring musculoskeletal diseases, similar to those seen in human Due to physiological differences between animal models and patients, have been identified and characterized [92] (Table 5) human patients, it is not rare therapies that are effective in dis- facilitating the use of that dog model for the pre-clinical assess- eased model animals failed to show benefits in clinical trials [81], ment of potential treatments [2,93,94]. including DMD [82]. Moreover, mutations may vary among AAV8-mediated delivery of myotubularin in the XLMTM dog patients, which requires vector personalization, such as oligonu- provides a successful example of gene therapy in preclinical prac- cleotides for exon skipping therapy [83]. Consequently a personal- tice. Due to the gene’s small size, a full-length canine MTM1 cDNA ized human cell culture system is highly desirable for potency was carried by the muscle-tropic AAV8. This was packaged with a testing. human desmin promoter to ensure muscle specific expression. The emergence of human iPSC offers a novel cell culture plat- Dogs were treated by direct injection and by locoregional hindlimb form to meet both these criteria. Unlimited disease relevant cells perfusion, where systemic effects were observed, likely due to with specific patient mutations could be generated. More impor- leakage. Treated dogs have near normal muscle strength, with tantly, these cells demonstrate disease-associated phenotypes the marked improvements in respiratory function and increased reflecting disease severity [84]. As a result, correction of these survival for dogs treated intravascularly. Indeed, these animals disease-associated phenotypes can be measured as efficacy. More- continue to survive more than two years after treatment and con- over, testing vectors on these personalized cells will not only tinue to thrive and breed. The size of the dog has allowed for the demonstrate the presence of the transgene product [85], but also inclusion of many at other relevant assessments at multiple time enables quantified measurement of biological activities, as points including neurological scoring, MRI, EMG and gait testing required by the FDA for gene therapy products. as we investigate potential outcome measures for translation of this therapy to human patients.
5.2. Animal models Acknowledgements While in vitro models allow for the testing of potential therapies in human cells, the ability to adequately recreate the complexities Funding: American Heart Association fellowship to X.G., Mus- of the human body is limited. As such, preclinical testing in an ani- cular Dystrophy Association to M.K.C, Association Française contre mal model remains the gold standard for investigating the efficacy les Myopathies to M.K.C.; NIH Grants R21 AR064503 and R01 and potential toxicity of a putative treatment. HL115001 to M.K.C.; Joshua Frase Foundation to M.K.C.; Where Non-mammalian models such as zebrafish have been invalu- There’s a Will There’s a Cure to DM and MKC; Peter Khuri Myopa- able to the understanding of disease mechanisms within a complex thy Research Foundation to M.K.C. organism, particularly in monogenetic neuromuscular disorders [86,87]. However physiological and phenotypic dissimilarities with humans limit their translational power [88,89] (Table 5). References Small mammalian models like rodents are often used in preclin- ical assessments of efficacy and toxicity. With extensive research [1] S.L. Ginn, I.E. Alexander, M.L. Edelstein, M.R. Abedi, J. Wixon, Gene therapy into study of the murine genome [90], mice in particular are a pow- clinical trials worldwide to 2012 – an update, J. Gene Med. 15 (2) (2013) 65– erful tool in the study of monogenetic disorders and knockdown or 77. [2] M.K. Childers, R. Joubert, K. Poulard, et al., Gene therapy prolongs survival and knockout mouse models have been developed to study muscu- restores function in murine and canine models of myotubular myopathy, Sci. loskeletal disease. Transl. Med. 6 (220) (2014). 220ra210. However the small size of the mouse as well as anatomical and [3] J.S. Chamberlain, Gene therapy of muscular dystrophy, Hum. Mol. Genet. 11 (20) (2002) 2355–2362. phenotypic differences make them less than ideal candidates for [4] V. Biancalana, A.H. Beggs, S. Das, et al., Clinical utility gene card for: the preclinical assessment of gene therapies [91]. Therefore, a lar- centronuclear and myotubular myopathies, Eur. J. Hum. Genet. 20 (10) ger animal model like a dog, where organ size more closely approx- (2012). [5] K. Amburgey, N. McNamara, L.R. Bennett, M.E. McCormick, G. Acsadi, J.J. imates that of humans, may be more suitable. Methods of Dowling, Prevalence of congenital myopathies in a representative pediatric functional assessment developed for use in the clinic have also united states population, Ann. Neurol. 70 (4) (2011) 662–665. X. Guan et al. / Methods 99 (2016) 91–98 97
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