Morpholino Treatment Improves Muscle Function and Pathology of Pitx1 Transgenic Mice

Oligonucleotide Therapeutics © The American Society of Gene & Cell Therapy original article original article

Morpholino Treatment in Pitx1 Transgenic Mice

Sachchida Nand Pandey1, Yi-Chien Lee1,2, Toshifumi Yokota3 and Yi-Wen Chen1,4

1Center for Genetic Medicine Research, Children’s National Medical Center, Washington, DC, USA; 2Department of Biochemistry and Molecular Biol- ogy, George Washington University, Washington, DC, USA; 3Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, Alberta, Canada; 4Department of Integrative Systems Biology, George Washington University, Washington DC, USA

Paired-like homeodomain transcription factor 1 (PITX1) of the D4Z4 region and allows DUX4 mRNA to be transcribed.10 was proposed to be part of the disease mechanisms of Second, an intact polyadenylation signal in the region distal to the facioscapulohumeral muscular dystrophy (FSHD). We gen- last repeat of D4Z4, which allows DUX4 transcripts from the last erated a tet-repressible muscle-specific Pitx1 transgenic D4Z4 repeat to be polyadenylated thus stable for protein transla- mouse model which develops phenotypes of muscular dys- tion. The combination leads to the aberrant expression of DUX4 trophy after the PITX1 expression is induced. In this study, and downstream molecular changes involved in FSHD.11,12 we attempted to block the translation of PITX1 protein Our previous study showed that paired-like homeodomain using morpholinos. Three groups of the transgenic mice transcription factor 1 (PITX1) was specifically upregulated in the received intravenous injections of phosphorodiamidate muscle of patients with FSHD and may mediate DUX4-induced morpholino oligomers (PMO) (100 mg/kg), octaguanidin- myopathy in FSHD via the regulatory pathway involving DUX4, ium dendrimer-conjugated morpholino (vivo-morpholino) PITX1, and p53.11,13,14 To study the effect of PITX1 overexpression (10 mg/kg), or phosphate-buffered saline (PBS) after the in skeletal muscles, we generated a tet-repressible muscle-specific PITX1 expression was induced. Immunoblotting data Pitx1 transgenic mouse model in which expression of PITX1 in showed that PITX1 expression in the triceps and quadri- skeletal muscles can be controlled by oral administration of doxy- ceps was significantly reduced 70% and 63% by the vivo- cycline.13 We showed that overexpression of PITX1 in skeletal morpholino treatment, respectively. Muscle pathology of muscles lead to phenotypes of muscular dystrophy, which share the mice treated with the vivo-morpholino was improved similarity to FSHD, including muscle atrophy, muscle weakness, 13 by showing 44% fewer angular-shaped atrophic myofi- and angular-shaped atrophic myofibers. In this study, we used bers. Muscle function determined by grip strength was the Pitx1 transgenic model to investigate the feasibility of sup- significantly improved by the vivo-morpholino treatment. pressing a pathogenic protein in vivo using morpholinos. The study showed that systemic delivery of the vivo- Phosphorodiamidate morpholino oligomers (PMOs) are short morpholino reduced the PITX1 expression and improved chains of ~25 morpholino subunits. Each subunit is comprised of the muscle phenotypes. Aberrant expression of DUX4 a nucleic acid base, a morpholino ring, and a nonionic phospho- 15 from the last unit of the D4Z4 array has been proposed to rodiamidate intersubunit linkage. PMOs have been used to block ′ be the cause of FSHD. The findings of this study suggest translation initiation by targeting the 5 UTR of a gene; to modify that the same principle may be applied to suppress the pre-mRNA splicing in the nuclei by targeting splice junctions or aberrantly expressed DUX4 in FSHD. splice regulatory sites; and to inhibit miRNA processing and activ- 5June2013 ity by targeting mature miRNA or pri-miRNA.16 Several advan- Received 5 June 2013; accepted 4 November 2013; advance online tages of using morpholinos to suppress gene expression include: publication 17 December 2013. doi:10.1038/mt.2013.263 4November2013 INTRODUCTION 1. Morpholinos have higher binding affinity than equivalent 10.1038/mt.2013.263 Facioscapulohumeral muscular dystrophy (FSHD) is a dominant DNA-based antisense oligos, which allows them to invade muscular dystrophy with a prevalence of 1:20,000, which is the RNA secondary structure therefore increases the probabil- 1 Molecular Therapy third common muscular dystrophy. The majority of patients with ity of designing effective oligos. FSHD carry a deletion of the D4Z4 repeats in the 4q35 subtelo- 2. With their requirement for greater complementarity 22 meric region. Unaffected individuals have 11–150 copies of the with their target RNAs, morpholinos cause less off-target D4Z4 repeats while patients have only 1 to 10 copies.2–5 Each D4Z4 expression modulation. repeat contains a double homeobox protein 4 (DUX4), which is 3. The morpholinos are stable in cells and do not induce 2 cytotoxic when ectopically expressed in cells based on in vivo and immune responses.15,17,18 ex vivo studies.6–9 Recent studies showed that a combination of two 390 genomic features is required to cause FSHD. First, the contrac- In this study, we used two types of morpholinos, PMO tion of the D4Z4 repeats, which leads to DNA hypomethylation and octaguanidinium dendrimer-conjugated morpholino 396 The first two authors contributed equally to this work. 00feb2014 Correspondence: Yi-Wen Chen, Center for Genetic Medicine Research, Children’s National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010, USA. E-mail: [email protected] 17December2013 390 www.moleculartherapy.org vol. 22 no. 2, 390–396 feb. 2014 © The American Society of Gene & Cell Therapy Morpholino Treatment in Pitx1 Transgenic Mice

(vivo-morpholino). The octaguanidium dendrimer conjugation a 5 * improves delivery of the morpholino by increasing its ability of * penetrating the cell membrane.19 The findings will help us to 4 Control (PBS) evaluate the strategy as a potential therapeutic mean for FSHD. ** Pitx1 (PBS) Pitx1 (PMO) Pitx1 (VM) RESULTS 3

In this study, we evaluated the PMO and vivo-morpholino targeting xpression the same 25 bases DNA sequence located at the translation start site e 2 of the Pitx1 mRNA transcript. To improve the delivery efficiency, the vivo-morpholino was conjugated with a triazine core scaffold Relativ featuring eight guanidinium head groups to facilitate cell penetra- 1 tion.19 Considering the higher delivery efficiency and potential toxicity of the vivo-morpholino, a lower dosage (10 mg/kg) was used, in 0 comparison to the dosage of the PMO (100 mg/kg). Both morpho- PBS PBS PMO VM linos were delivered by intravenous injections weekly for 6 weeks. CC DT DT DT DT DT DT

In this study, we would like to determine whether the morpholino PITX1 treatments can suppress PITX1 protein expression and prevent or slow down the disease progression. Therefore, we chose to start the Vinculin morpholinos injections at the same time when the Pitx1 overexpression was induced in the Pitx1 transgenic mice. Expression of b 5 Control (PBS) the PITX1 protein, muscle function of the Pitx1 transgenic mice * Pitx1 (PBS) and muscle pathology were evaluated after the treatments. ** * Pitx1 (PMO) 4 Pitx1 (VM) The vivo-morpholino against Pitx1 suppressed PITX1 protein expression in the Pitx1 transgenic mice 3

To determine whether the overexpressed PITX1 protein was xpression e knocked down by the morpholinos, immunoblotting was con- 2 ducted using proteins from the triceps and quadriceps of the mice treated with either the PMO or vivo-morpholino. ThePitx1 Relativ transgenic mice treated with phosphate-buffered saline (PBS) 1 were used to determine the PITX1 expression level without treatment, while the baseline expression of PITX1 in normal muscles 0 was determined using muscles from littermates that carry only PBS PBS PMO VM one of the two transgenes. Our results showed that 70% of the CC DT DT DT DT DT DT PITX1 expression was knocked down by the vivo-morpholino PITX1 treatment (P < 0.05) in the triceps compared to the PBS treatment (Figure 1a), while the PMO did not significantly affect Vinculin the PITX1 expression. Similarly, the vivo-morpholino treatment reduced 63% of the PITX1 expression in the quadriceps (P < 0.05) Figure 1 Suppression of paired-like homeodomain transcription factor 1 (PITX1) protein expression in skeletal muscle by vivo-morpho- (Figure 1b), while the PMO treatment had no effect on PITX1 lino treatment. Expression levels of PITX1 protein in (a) triceps and expression. Immunohistochemistry was performed to visualize (b) quadriceps of the Pitx1 transgenic mice and control mice. Data are localization of the PITX1 proteins in myonuclei (Figure 2). The presented as mean ± SEM, control mice received PBS (n = 5); Pitx1 trans- results showed no visible PITX1 staining in control muscles while genic mice received PBS (n = 4); Pitx1 transgenic mice received PMO (n = 3); Pitx1 transgenic mice received VM (n = 5). “*” indicates P < a large number of PITX1-positive nuclei were observed in muscle 0.05 and “**” indicates P < 0.01. C, control mice; DT, Pitx1 transgenic; sections of the Pitx1 transgenic mice treated with PBS. The vivo- PBS, phosphate-buffered saline; PMO, phosphorodiamidate morpholino morpholino treatment reduced the number of PITX1-positive oligomers; VM, vivo-morpholino. nuclei, while PMO treatment did not show obvious effect. The results of immunoblotting and immunohistochemistry showed The vivo-morpholino treatment reduced atrophic that the vivo-morpholino treatment, but not the PMO treatment, myofibers in thePitx1 transgenic mice reduced the expression of PITX1 protein significantly. To examine the pathological improvement of the Pitx1 trans- The PMO and vivo-morpholino used in this study were designed genic (TRE-Pitx1/mCK-tTA) mice treated with the PMO and to block protein translation instead of inducing RNA degradation. vivo-morpholino, histology of the triceps and quadriceps of the To determine whether the vivo-morpholino treatment also affect the mice was examined and percentages of angular-shaped myofi- expression of Pitx1 at the mRNA level, we performed real-time quan- bers were determined (Figure 2). The control samples showed no titative reverse transcription polymerase chain reaction (qRT-PCR) angular-shaped atrophic fibers, while a large number of angular- and showed that the mRNA level of the Pitx1 in mice treated with shaped atrophic fibers (16%) and some fibers with central nuclei vivo-morpholino was not affected by the treatment (Figure 3). were observed in muscle sections of the Pitx1 transgenic mice

Molecular Therapy vol. 22 no. 2 feb. 2014 391 © The American Society of Gene & Cell Therapy Morpholino Treatment in Pitx1 Transgenic Mice

ae 0.06 Pitx1 (PBS) Pitx1 (VM)

0.05

0.04

bf xpressio n

e mRNA 0.03

* Relativ * cg 0.02 * * 0.01

Figure 3 The vivo-morpholino treatments did not affect Pitx1 mRNA level in the skeletal muscles of the Pitx1 transgenic mice. Data are presented as mean ± SEM, Pitx1 transgenic mice received PBS (n = 4); dh Pitx1 transgenic mice received VM (n = 5). PBS, phosphate-buffered saline; VM, vivo-morpholino.

* 25 * ** **

20 ** Figure 2 The paired-like homeodomain transcription factor 1 (PITX1) protein is reduced by vivo-morpholino treatment in the quadricep muscle. (a) PITX1 expression is not detected in control mice. Nuclear localization of the PITX1 in muscles of the Pitx1 15 transgenic mice (TRE-Pitx1/mCK-tTA) treated with (b) PBS or (c) PMO. (d) Vivo-morpholino treatment reduced the number of Pitx1 positive nuclei. H&E staining of control mice showed (e) healthy muscle fibers. Pitx1 transgenic (TRE-Pitx1/mCK-tTA) mice treated with (f) PBS and (g) 10 PMO showed large number of angular shape fibers. (h) Pitx1 transgenic (TRE-Pitx1/mCK-tTA) mouse treated with the vivo-morpholino % of angular fibers/total fibers showed fewer angular fibers in the muscles. Arrows indicate myofibers 5 with central nuclei and asterisks indicate angular atrophic myofibers. Scale bar: 100 µm.

0 treated with PBS. Mice treated with PMO did not show improve- Mice: Control Pitx1 Pitx1 Pitx1 ment of pathology (21%). However, the mice treated with the Treatment: PBS PBS PMO V M vivo-morpholino showed a 44% (P < 0.05) reduction of angular- shaped atrophic fibers (9%) (Figure 4). In addition, the PITX1 Figure 4 The Pitx1 transgenic mice (TRE-Pitx1/mCK-tTA) treated 2 with the vivo-morpholino showed significantly lower percentage of expression was highly correlated (r = 0.84) with the percentage angular fibers than the control. Data are presented as mean ± SEM, of angular fibers in each sample. The results showed that vivo- control mice received PBS (n = 5); Pitx1 transgenic mice received PBS morpholino treatment reduced PITX1 expression and the num- (n = 4); Pitx1 transgenic mice received PMO (n = 3); Pitx1 transgenic ber of atrophic myofibers. mice received VM (n = 5). “*” indicates P < 0.05 and “**” indicates P < 0.01. PBS, phosphate-buffered saline; PMO, phosphorodiamidate morpholino oligomers; VM, vivo-morpholino. The vivo-morpholino treatment improved grip strength of the Pitx1 transgenic mice without overt improved by the vivo-morpholino treatment (P < 0.05). We did toxicity not observe significant improvement of grip strength in the mice To determine whether the muscle strength was improved by the treated with PMO (Figure 5a,b). PMO and vivo-morpholino treatments, grip strength of both the To determine whether the vivo-morpholino treatment caused forelimb and hindlimb of the Pitx1 transgenic mice were per- hepatic or renal toxicity in the mice, blood serum enzymes formed. The Pitx1 transgenic mice treated with PBS were signifi- were examined (Supplementary Figure S1). Alanine transami- cantly weaker than the control mice as previously reported.13 The nase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase muscle strength of the Pitx1 transgenic mice was significantly (GGT), and bilirubin were tested for liver toxicity. Blood urea

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ab substitution of the five-membered-ring sugar of DNA with six- 6 16 membered-morpholino rings. In addition, negatively charged * * phosphate linkages are replaced by nonionic phosphorodiami- * 14 25 5 * date linkages. The structural changes make PMO more stable. * In addition, unlike other antisense approaches, morpholinos have 12 high binding affinity with less off-target effects.15,17,18 Using mor- 4 10 pholino to block the translation of specific mRNA is widely used to study functions of genes.26–29 For example, vivo-morpholinos 3 8 were used to suppress dopamine receptor 1 (DRD1), vesicular Kgf/Kg Kgf/Kg monoamine transporter 2 (VMAT2), and glucose transporter 4 6 2 (GLUT4) in skeletal muscle to study their functions in a mouse model of voluntary physical activity.30 However, less work has 4 been done by using this approach for treating diseases. Few cases 1 2 were reported, such as using modified morpholinos to suppress expression of viral proteins in mouse models.31–33 Our approach 0 0 showed that vivo-morpholinos can be delivered intravenously and Mice: Control Pitx1 Pitx1 Pitx1 Control Pitx1 Pitx1 Pitx1 Treatment: PBS PBS PMO V M PBS PBS PMO V M block protein translation in vivo, which provides an additional strategy for treating diseases caused by aberrant expression of Figure 5 Grip strength of the Pitx1 transgenic mice (TRE-Pitx1/ pathogenic proteins. mCK-tTA) treated with the vivo-morpholino was significantly improved. Grip strength test showed increase of (a) forelimb and In addition to blocking translation, the steric blocking mecha- (b) hindlimb grip strength in the Pitx1 transgenic mice treated with nism of morpholinos has also been used to alter splicing of pre- the vivo-morpholino. Data are presented as mean ± SEM, control mice mRNAs. The application of morpholinos in blocking splicing has received PBS (n = 5); Pitx1 transgenic mice received PBS (n = 4); Pitx1 been extensively studied in animal models of Duchenne muscular transgenic mice received PMO (n = 3); Pitx1 transgenic mice received vivo-morpholino (n = 5). “*” indicates P < 0.05. PBS, phosphate-buff- dystrophy (DMD). PMO against exon 23 donor site was reported ered saline; PMO, phosphorodiamidate morpholino oligomers; VM, to restore dystrophin expression in muscles of the mdx mice.34–36 vivo-morpholino. PMO has also been used to skip exon 6 and 8 in a dog model of DMD (CXMD) and exon 51 in human DMD to restore the dystro- nitrogen and creatine were tested for kidney toxicity. Creatine phin reading frame.37,38 The leakiness of the dystrophin-deficient kinase was tested for skeletal and cardiac muscle damage. These myofibers was believed to contribute to the success of the treat- enzymes did not show significant changes in response to the PMO ment. Since PMO is not charged and has been shown difficult nor the vivo-morpholino treatments. We concluded that there was to be delivered into intact skeletal muscles, the leakiness of the no obvious toxicity detected in mice treated with either type of myofibers allows the PMO to be delivered into the cells.39 In this morpholinos. study, the failure of delivering sufficient amount of PMO into the muscles of the Pitx1 mice supports the view that effective deliv- DISCUSSION ery of PMO relies on leaky membrane of the myofibers. In this Several approaches have been developed to suppress patho- study, we believed that the PMO could not be delivered into the genic proteins by sequestering or degrading the mRNA, includ- muscle fibers efficiently to suppress PITX1 expression. Normal ing shRNA, miRNA, siRNA, and antisense oligonucleotides.20–23 level of creatine kinase, an enzyme leaking out of muscles when Before DUX4 was considered as the causative gene of FSHD, damaged, in serum of all mice tested, further supported that the several candidate genes in the D4Z4 region were investigated for sarcolemmal membrane of the Pitx1 transgenic mice was intact. their involvement in FSHD, including FSHD region gene 1, FSHD On the contrary, vivo-morpholino treatment showed efficacy in region gene 2, and adenine nucleotide translocator 1.24 shRNA suppressing the PITX1 expression and rescued disease pheno- and miRNA against FSHD region gene 1 were designed and deliv- types in the Pitx1 transgenic mice although the vivo-morpholino ered using adeno-associated viral vector and were shown to suc- was delivered at a lower dose. The result demonstrated that vivo- cessfully reduce the FSHD region gene 1 expression in vivo.21,22 morpholino against a pathogenic protein effectively entered intact Later, miRNA against DUX4 was delivered by adeno-associated myofibers and suppressed the target gene. viral vector into mice ectopically expressing DUX4 and was able The dosages of PMO and vivo-morpholino used in this study to rescue the pathology induced by DUX4 expression.23 In this were determined based on previous studies in animal mod- study, we demonstrated that a modified antisense oligonucleotide, els.35,38,40,41 Previously, PMO at 100 mg/kg administered to mdx morpholino, can be used to successfully suppress the pathogenic mice showed efficacy without toxicity.40 The modification of the PITX1 protein and rescue the disease phenotypes, which offers vivo-morpholino significantly increases cellular uptake therefore an additional strategy for treating FSHD. Using this approach, much lower concentration is needed for delivery. The modifica- we can take advantages of using morpholino molecules, includ- tion also increases toxicity of the vivo-morpholino. The toxicity of ing higher affinity, more specificity, less off-target effects, and low vivo-morpholino and PMO has been extensively studied in mdx immunogenicity. mice.35,36,41,42 Vivo-morpholino and PMO showed no toxicity in Morpholinos are a short chain of morpholino rings. PMO mdx after treatment for as long as 5 and 12 weeks, respectively, have longer half-life comparing to oligonucleotides due to a determined by histological examination and serum enzyme levels

Molecular Therapy vol. 22 no. 2 feb. 2014 393 © The American Society of Gene & Cell Therapy Morpholino Treatment in Pitx1 Transgenic Mice

of liver and kidney.35,42 mdx mice treated with up to 1.5 g/kg PMO the pups in utero. Under these conditions, the TRE-Pitx1/mCK-tTA double for 6 months did not show signs of liver and kidney toxicity.36 transgenic mice were born at the expected Mendelian ratio. The pups con- Systemic delivery of vivo-morpholino in mdx mice (6 mg/kg) tinued to receive doxycycline through the mother’s milk. After weaning, all biweekly for 5 weeks did not show liver or kidney toxicity.35 A mice were maintained on water treated with doxycycline until they were entered into an experimental regiment. For all experiments, littermates car- recent study showed that 5 weekly injections of vivo-morpholino rying a single transgene (Pitx1 or tTA) were used as controls. Male patients 41 (12mg/kg) did not cause liver or kidney toxicity. While studies with FSHD generally are more affected than the female patients, including suggest that vivo-morpholino is generally well tolerated, PPMO, a age of onset and muscle weakness.47,48 Therefore, we studied only male mice PMO conjugated with arginine-riched peptides, targeting human to reduce experimental variations in this study. exon 50 (AVI-5038) was reported to cause mild tubular degenera- To identify mice carrying one or two of the transgenes (TRE-Pitx1 tion in the kidney of cynomolgus monkeys which received weekly and mCK-tTA), we collected a piece of tail tip and isolated DNA for injection (9 mg/kg) for 4 weeks.39 Although the cause of toxicity genotyping. The following primers were used for PCR amplification. was not known, one of the hypotheses was that the cationic prop- For Pitx1, forward 5′-TGGAGGCCACGTTCC AAAG-3′ and reverse ′ ′ erty of PPMO caused off-target effects which lead to renal toxicity. 5 -GTTCTTGAACCAGACCCGCAC-3 were used. For tTA, forward 5′-acagcgcattagagctgctt-3′ and reverse 5′-Ccccttctaaagggcaaaag-3′ were We delivered the vivo-morpholino at a dose of 10 mg/kg weekly used. for 6 weeks and showed no detectable liver and kidney toxicity by blood serum enzyme readings, which agreed with the findings of Real-time qRT-PCR. Real-time qRT-PCR was performed as previously previous studies. described.49 Briefly, total RNA samples were isolated from triceps of mice Efficacious downregulation of a physiologically expressed tar- treated with the vivo-morpholino and PBS. Total RNA (1 µg) was reverse get gene to lower than its normal expression level can potentially transcribed using oligo (dT) primer (0.5 µg/µl). Primer sequences used for ′ ′ be toxic to the cells, therefore careful evaluation of the dosages and mouse Pitx1 gene were (forward) 5 -TGGAGGCCACGTTCCAAAG-3 and (reverse) 5′-GTTCTTGAACCAGACCCGCAC-3′; for ribosomal RNA knock-down efficacy is necessary when the approach is used. In 18S: 5′-TAGCCTTCGCCATCACTGCCATTA-3′ and (reverse) 5′-AACC addition, genes that are not normally expressed in vital organs may TGGCTGTACTTCCCATCCTT-3′. ABI 7900HT (Applied Biosystems, be more suitable targets for this approach. DUX4 which expresses Foster City, CA) was used for mRNA quantification. Briefly, cDNA was in germline cells but not other healthy tissues seems to be such a added to SYBR Green PCR Master Mix (Applied Biosystems). Samples target.43 Generating a DUX4 mouse model has been challenging were amplified in triplicate by using the following thermal cycling condi- due to its cytotoxic nature when ectopically expressed. Previously, tions: 95 °C for 10 minutes, followed by 40 cycles of amplification at 95 °C we reported that PITX1 was a direct transcriptional target of DUX4 for 15 seconds, followed by 60 °C for 1 minute to allow for denaturing and specifically upregulated in muscles of patients with FSHD. In and annealing extension. All primers were tested for nonspecific ampli- cons and primer dimers by visualizing PCR products on 2% agarose gels our conditional muscle-specific Pitx1 transgenic model, overex- before performing qRT-PCR. The ΔΔCT method was used to determine pressing PITX1 lead to muscular dystrophy phenotypes including expression values of Pitx1 relative to ribosomal RNA 18S as described pre- muscle atrophy, muscle weakness, and angular shaped atrophic viously.49 T-test was used to determine statistical significance (P < 0.05). myofibers, which was also reported in FSHD.13,44–46 In this study, we showed that protein expression level of PITX1 was positively Morpholino injections. Morpholinos have been shown to effectively block correlated with the percentage of angular-shaped atrophic fibers in translation when sequences near the translation start site are targeted (−80 to +20 where +1 is the A of the AUG translation start site).50 The morpho- the muscle. The finding suggested that the upregulation of PITX1 lino oligos (5′- TCATGCCTCCCTTGAAGGCGTCCAT-3′) used in this in FSHD may contribute to this specific pathology. In addition, study were designed and synthesize by Gene Tools, LLC (Philomath, OR), we demonstrated that the pathogenic protein, PITX1, can be sup- which targets the first 25 bases from the start codon (+1 to +25 where +1 pressed by the vivo-morpholino without affecting the mRNA level. is the A of the AUG) of the Pitx1 (NM_011097.2). In general, Gene Tools The findings provide a proof of principle of using vivo-morpholino designs morpholino molecules by analyzing the first 25 bases of coding to suppress a pathogenic protein in vivo. Considering DUX4 tran- sequence then slide the 25-base window upstream until a 25-base target scripts from the last D4Z4 repeat in the patients were stabilized by is found that satisfies the general requirements for a optimal morpholino polyadenylation for translation11,12 and the aberrant expression of oligo (i.e., 40–60% GC content without significant self-complementarity or stretches of 4 or more Gs) (http://www.gene-tools.com/node/18). In PMO, the DUX4 protein is believed to be the cause of FSHD, morpholi- negatively charged phosphate linkages are replaced by noncharged back- nos that can block protein translation of the DUX4 may potentially bone, which causes difficulty for delivering the molecule into the cells. To be used to treat FSHD. overcome the problem, vivo-morpholino was conjugated with a triazine core scaffold featuring eight guanidinium head groups, which increases MATERIALS AND METHODS delivery efficiency.19 In this study, both the PMO and vivo-morpholino The tet-repressible muscle-specific Pitx1 transgenic mouse. All animal pro- were dissolved in PBS (100 µl) and delivered directly by intravenous injec- cedures were approved by the institutional animal care and use committee tions. The doses of the PMO and vivo-morpholino used were 100 and (IACUC) at the Children’s National Medical Center in Washington, DC. 10 mg per kg of body mass, respectively. They were delivered by intrave- Two transgenic mouse lines (TRE-Pitx1 and mCK-tTA) were crossbred in nous injections weekly for 6 weeks. order to generate the tet-repressible muscle-specific Pitx1 transgenic mice The oral doxycycline was discontinued to induce Pitx1 transgene (TRE-Pitx1/mCK-tTA) as previously reported.13 The TRE-Pitx1 mouse expression when the mice were 8 weeks old. At the same time, total eight line carries a construct containing the Pitx1 gene, driven by a tetracycline male TRE-Pitx1/mCK-tTA mice received either PMO (n = 3) or vivo- response element (TRE). The mCK-tTA mouse line carries a construct con- morpholino (n = 5) treatment weekly for 6 weeks. A group of TRE-Pitx1/ taining the tTA gene, driven by a muscle specific creatine kinase promoter mCK-tTA male littermates (n = 4) received weekly PBS injections as the (mCK). The female mice received drinking water with doxycycline (200 μg/ control of the experiment. Male littermates carrying only one transgene ml with 5.0% sucrose) in order to suppress the Pitx1 transgene expression in (TRE-Pitx1 or mCK-tTA) received PBS injections (n = 5) for 6 weeks to

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provide a baseline value (normal expression level) of the PITX1. One ACKNOWLEDGMENTS week after the last injection, the mice were euthanized, and skeletal This study is supported by NIH/NIAMS1R01AR052027 and muscles including quadriceps and triceps were collected for protein and R01AR052027-03S1. Y-W.C. is partially supported by NIH/ RNA assays. NICHD1R24HD050846 and DOD W81XWH-10-1-0659.

Muscle functional test. The grip strength test (Grip Strength Meter; SUPPLEMENTARY MATERIAL Columbus Instruments, Columbus, OH) was performed at week 6 after the Figure S1. The morpholino treatments did not affect the levels of Pitx1 transgene induction to determine the effects of treatment on muscle blood serum enzymes of the Pitx1 transgenic mice (TRE-Pitx1/ function. The test was performed according to previously published pro- mCK-tTA) cedure.13 Briefly, the grip strength is tested by holding the mouse over the grid of the instrument to allow the mouse grip the steel bars. Then the References 1. Padberg, G. (1982). Facioscapulohumeral disease. In: Thesis. University of Leiden: mouse was pulled away from the force meter until it released the grid. Leiden, The Netherlands. The meter recorded the maximum force that was applied. The mouse was 2. Hewitt, JE, Lyle, R, Clark, LN, Valleley, EM, Wright, TJ, Wijmenga, C et al. (1994). Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral then tested once a day for five consecutive days; five measurements were muscular dystrophy. Hum Mol Genet 3: 1287–1295. recorded for each test. The largest measurements from each of the five tests 3. van Deutekom, JC, Wijmenga, C, van Tienhoven, EA, Gruter, AM, Hewitt, JE, Padberg, were averaged and normalized to body weight (kg). GW et al. (1993). FSHD associated DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit. Hum Mol Genet 2: 2037–2042. 4. Wijmenga, C, Hewitt, JE, Sandkuijl, LA, Clark, LN, Wright, TJ, Dauwerse, HG et al. Histological and immunohistochemical assays. Immediately after mus- (1992). Chromosome 4q DNA rearrangements associated with facioscapulohumeral cle dissection, the whole muscle was snap-frozen in isopentane cooled muscular dystrophy. Nat Genet 2: 26–30. 5. Winokur, ST, Bengtsson, U, Feddersen, J, Mathews, KD, Weiffenbach, B, Bailey, H et with liquid nitrogen, then stored at −80 °C until sectioning. A Leica CM al. (1994). The DNA rearrangement associated with facioscapulohumeral muscular 1900 cryostat (Walldorf, Baden-Wurttenberg, Germany) was used to pre- dystrophy involves a heterochromatin-associated repetitive element: implications for a role of chromatin structure in the pathogenesis of the disease. Chromosome Res 2: pare cryosections for all the following histological analyses. Hematoxylin 225–234. and eosin staining was conducted using 8 µm sections. Five random non- 6. Bosnakovski, D, Xu, Z, Gang, EJ, Galindo, CL, Liu, M, Simsek, T et al. (2008). An overlapping fields of the tissue sections were imaged using Nikon Eclipse isogenetic myoblast expression screen identifies DUX4-mediated FSHD-associated molecular pathologies. EMBO J 27: 2766–2779. E800 microscope (Nikon, Tokyo, Japan), RT slider camera (Diagnostic 7. 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