FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3136361381> ?p ?o ?g. }

• W3136361381 endingPage "340" @default.
• W3136361381 startingPage "325" @default.
• W3136361381 abstract "Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial. Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial. The X-linked DMD gene, which encodes the dystrophin protein, consists of 79 exons and at least 7 promoters. Mutations in DMD result in either the severe Duchenne or milder Becker muscular dystrophy (DMD or BMD)—collectively, dystrophinopathies—with the predominant determinant of phenotype being whether or not the mutation maintains an open reading frame (ORF) that allows expression of a partially functional protein.1Monaco A.P. Bertelson C.J. Liechti-Gallati S. Moser H. Kunkel L.M. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus.Genomics. 1988; 2: 90-95Crossref PubMed Scopus (907) Google Scholar Deletions of one or more exons constitute the most common mutation class in the dystrophinopathies, accounting for approximately 65% of all mutations.2Dent K.M. Dunn D.M. von Niederhausern A.C. Aoyagi A.T. Kerr L. Bromberg M.B. Hart K.J. Tuohy T. White S. den Dunnen J.T. et al.Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort.Am. J. Med. Genet. A. 2005; 134: 295-298Crossref PubMed Scopus (110) Google Scholar,3Flanigan K.M. Dunn D.M. von Niederhausern A. Soltanzadeh P. Gappmaier E. Howard M.T. Sampson J.B. Mendell J.R. Wall C. King W.M. et al.United Dystrophinopathy Project ConsortiumMutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort.Hum. Mutat. 2009; 30: 1657-1666Crossref PubMed Scopus (210) Google Scholar One therapeutic approach for these mutations has been exon skipping, in which pre-mRNA splicing is altered by the use of antisense oligomers directed to exons adjacent to those containing mutations; the resultant mRNA contains a larger deletion, but one in which the reading frame is restored.4Aartsma-Rus A. Antisense-mediated modulation of splicing: therapeutic implications for Duchenne muscular dystrophy.RNA Biol. 2010; 7: 453-461Crossref PubMed Scopus (61) Google Scholar, 5Aartsma-Rus A. Fokkema I. Verschuuren J. Ginjaar I. van Deutekom J. van Ommen G.J. den Dunnen J.T. Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations.Hum. Mutat. 2009; 30: 293-299Crossref PubMed Scopus (394) Google Scholar, 6Mendell J.R. Rodino-Klapac L.R. Sahenk Z. Roush K. Bird L. Lowes L.P. Alfano L. Gomez A.M. Lewis S. Kota J. et al.Eteplirsen Study GroupEteplirsen for the treatment of Duchenne muscular dystrophy.Ann. Neurol. 2013; 74: 637-647Crossref PubMed Scopus (511) Google Scholar Exon duplications are less frequent, accounting for 6%–11% of all mutations in large dystrophinopathy cohort studies.2Dent K.M. Dunn D.M. von Niederhausern A.C. Aoyagi A.T. Kerr L. Bromberg M.B. Hart K.J. Tuohy T. White S. den Dunnen J.T. et al.Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort.Am. J. Med. Genet. A. 2005; 134: 295-298Crossref PubMed Scopus (110) Google Scholar,7Aartsma-Rus A. Van Deutekom J.C. Fokkema I.F. Van Ommen G.J. Den Dunnen J.T. 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. 2006; 34: 135-144Crossref PubMed Scopus (456) Google Scholar The most common single exon duplicated is exon 2 (Dup2), accounting for around 10% of all DMD duplication mutations and thus around 0.6%–1.1% of all dystrophinopathy mutations. Duplications of exon 2 result in an altered reading frame, with a premature stop codon in the new reading frame within the exon 3 sequence. Consistent with this, the majority (80%) of Dup2 patients present with a typical DMD phenotype, with loss of ambulation before the age of 15 years. The remainder meet a common classification for BMD, with later loss of ambulation, although nearly all of these outliers have lost ambulation by the early 20s (unpublished data). In contrast, only one person has been described with a deletion of exon 2 (Δ2).8Wein N. Vulin A. Falzarano M.S. Szigyarto C.A. Maiti B. Findlay A. Heller K.N. Uhlén M. Bakthavachalu B. Messina S. et al.Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.Nat. Med. 2014; 20: 992-1000Crossref PubMed Scopus (77) Google Scholar Although Δ2 also results in a frameshift and premature stop codon in a new reading frame in the exon 3 sequence, this person was essentially asymptomatic, except for myalgias and elevated serum creatine kinase (CK). The difference between the Dup2 and the Δ2-associated phenotypes was explained by the identification of an internal ribosome entry site (IRES) within the DMD exon 5, which allows for cap-independent translational initiation in the case of an exon 2 deletion but is nonfunctional in the presence of an exon 2 duplication.8Wein N. Vulin A. Falzarano M.S. Szigyarto C.A. Maiti B. Findlay A. Heller K.N. Uhlén M. Bakthavachalu B. Messina S. et al.Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.Nat. Med. 2014; 20: 992-1000Crossref PubMed Scopus (77) Google Scholar Translation from this IRES results in the expression of a dystrophin isoform missing the calponin-homology 1 (CH1) domain within the N-terminal actin binding domain 1 (ABD1), but which is nevertheless highly functional; expression of the same isoform is found in patients across North America carrying the DMD p.Trp3X founder allele, resulting in mildly symptomatic BMD with ambulation into the seventh or eighth decade.9Flanigan K.M. Dunn D.M. von Niederhausern A. Howard M.T. Mendell J. Connolly A. Saunders C. Modrcin A. Dasouki M. Comi G.P. et al.DMD Trp3X nonsense mutation associated with a founder effect in North American families with mild Becker muscular dystrophy.Neuromuscul. Disord. 2009; 19: 743-748Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar As a result of these clinical observations, we may assume that skipping of exon 2 is a therapeutic approach that offers a very broad therapeutic window. Differing from the case with skipping for deletion mutations, which results in creation of a BMD-like internally truncated transcript, skipping a single copy of exon 2 would restore an entirely normal mRNA transcript. However, skipping of both copies of exon 2 results in a stable transcript subject to translation, via the exon 5 IRES, of a dystrophin isoform that is highly stable, as demonstrated by multiple patients with an exon 1 nonsense mutation who express the same isoform with symptoms of only myalgias and hyperCKemia.9Flanigan K.M. Dunn D.M. von Niederhausern A. Howard M.T. Mendell J. Connolly A. Saunders C. Modrcin A. Dasouki M. Comi G.P. et al.DMD Trp3X nonsense mutation associated with a founder effect in North American families with mild Becker muscular dystrophy.Neuromuscul. Disord. 2009; 19: 743-748Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar To test this as a potential therapeutic approach, we developed a mouse model with a duplication of exon 2 (Dup2).10Vulin A. Wein N. Simmons T.R. Rutherford A.M. Findlay A.R. Yurkoski J.A. Kaminoh Y. Flanigan K.M. The first exon duplication mouse model of Duchenne muscular dystrophy: A tool for therapeutic development.Neuromuscul. Disord. 2015; 25: 827-834Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar In this mouse dystrophin expression is essentially absent, and pathologic features and physiologic defects are essentially identical to the common mdx mouse model, which carries a nonsense mutation in exon 23 (rendering it unsuitable for testing exon 2 skipping therapy). In contrast to exon skipping using a phosphorodiamidate morpholino oligomer (PMO),6Mendell J.R. Rodino-Klapac L.R. Sahenk Z. Roush K. Bird L. Lowes L.P. Alfano L. Gomez A.M. Lewis S. Kota J. et al.Eteplirsen Study GroupEteplirsen for the treatment of Duchenne muscular dystrophy.Ann. Neurol. 2013; 74: 637-647Crossref PubMed Scopus (511) Google Scholar,11Mendell J.R. Goemans N. Lowes L.P. Alfano L.N. Berry K. Shao J. Kaye E.M. Mercuri E. Eteplirsen Study Group and Telethon Foundation DMD Italian NetworkLongitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy.Ann. Neurol. 2016; 79: 257-271Crossref PubMed Scopus (339) Google Scholar we designed modified U7snRNAs with terminal antisense oligonucleotide sequences that target exon 2 definition elements. The U7snRNAs, delivered in an adeno-associated virus (AAV) genome, are transcribed but never translated and are stable for long-term inducement of exon skipping.12Schümperli D. Pillai R.S. The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein.Cell. Mol. Life Sci. 2004; 61: 2560-2570Crossref PubMed Scopus (104) Google Scholar, 13Goyenvalle A. Babbs A. Wright J. Wilkins V. Powell D. Garcia L. Davies K.E. Rescue of severely affected dystrophin/utrophin-deficient mice through scAAV-U7snRNA-mediated exon skipping.Hum. Mol. Genet. 2012; 21: 2559-2571Crossref PubMed Scopus (69) Google Scholar, 14Goyenvalle A. Vulin A. Fougerousse F. Leturcq F. Kaplan J.C. Garcia L. Danos O. Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping.Science. 2004; 306: 1796-1799Crossref PubMed Scopus (391) Google Scholar, 15Vulin A. Barthélémy I. Goyenvalle A. Thibaud J.L. Beley C. Griffith G. Benchaouir R. le Hir M. Unterfinger Y. Lorain S. et al.Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.Mol. Ther. 2012; 20: 2120-2133Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar We designed two such U7snRNAs, targeting either the exon 2 splice acceptor (sequence A) or splice donor (sequence C) sites, and cloned four copies (two of each sequence) into an AAV genome that we packaged within a self-complementary AAV9 vector (scAAV9.U7.ACCA) to demonstrate efficient skipping in human-derived cell culture and preliminary mouse studies.8Wein N. Vulin A. Falzarano M.S. Szigyarto C.A. Maiti B. Findlay A. Heller K.N. Uhlén M. Bakthavachalu B. Messina S. et al.Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.Nat. Med. 2014; 20: 992-1000Crossref PubMed Scopus (77) Google Scholar Here we present dose-escalation studies of both intramuscular (i.m.) and systemic delivery of scAAV9.U7.ACCA that show efficient skipping of exon 2, along with increased expression of properly localized dystrophin that restores muscle function. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window due to utilization of the IRES with complete exon 2 exclusion, and suggest that patients harboring other mutations 5′ of the exon 5 IRES may potentially benefit from expression of this highly functional dystrophin isoform via the same mechanism. To determine if we could titrate skipping of exon 2 to a wild-type (WT) transcript, we performed an i.m. dose escalation in the tibialis anterior (TA) muscle by delivering 5 single doses at half-log increments. RT-PCR was used to evaluate the amount of exon 2 skipping seen at the mRNA level, where potential transcripts include a duplication of exon 2 (Dup2), a single copy of exon 2 (WT), or zero copies of exon 2 (Δ2) (Figure 1). The results show an expected dose response, with the lowest dose of 3.2 × 109 vg resulting in 11% WT transcript, and the incremental dose of 9.1 × 109 vg showing both the WT and Δ2 transcripts, each comprising at least 10% of the total transcript. At the highest dose of 3.2 × 1011 vg, only 20% of the transcript is Dup2, while the Δ2 transcript represents almost 75% of the total transcript (Figure 1). Dystrophin immunofluorescence (IF) analysis on frozen TA sections shows a similar progressive dose response for both dystrophin-positive fibers (Figure 2A) and dystrophin signal intensity (Figure 2B), confirmed by a significant linear trend from left to right. The mean proportion of dystrophin-positive fibers ranged from 27% up to 71% across all i.m. dose groups in contrast to <20% in all untreated Dup2 sections, and dystrophin signal intensity peaked around 52% of median WT intensity at the sarcolemma. Representative images confirm proper sarcolemmal localization at all 5 doses tested (Figure 2C) and demonstrate sparse sarcolemmal dystrophin staining at low doses that increases to include the vast majority of fibers at higher doses, consistent with the RT-PCR analysis. Dystrophin protein was further quantified by immunoblot (IB), confirming the progressive dose response and showing 62% WT dystrophin levels at the highest dose of 3.2 × 1011 vg (Figure 3).Figure 3Immunoblot analysis following i.m. injection of scAAV9.U7.ACCA in the TA in Dup2 miceShow full caption(A) Assessment of dystrophin expression by immunoblotting shows a dose-dependent increase in dystrophin protein that is confirmed by quantification of the dystrophin band doublet (red) normalized to actinin (green). (B) Quantification results reported as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed using one-way ANOVA with a post hoc test for linear trend from left to right; ∗∗∗∗p < 0.0001 for trend.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Assessment of dystrophin expression by immunoblotting shows a dose-dependent increase in dystrophin protein that is confirmed by quantification of the dystrophin band doublet (red) normalized to actinin (green). (B) Quantification results reported as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed using one-way ANOVA with a post hoc test for linear trend from left to right; ∗∗∗∗p < 0.0001 for trend. To determine the degree of functional rescue, in vivo muscle function studies were performed on additional TA muscles 4 weeks after a single i.m. injection of 3.2 × 1011 vg, measuring both force (absolute and specific) and force drop following repeated eccentric contractions. Treated Dup2 muscle showed 45% higher mean absolute force (Figure 4A) and 64% higher specific force (Figure 4B) relative to untreated Dup2, although the dataset lacked sufficient statistical power for the difference in specific force to reach statistical significance. Despite the near-complete correction of absolute force, specific force in treated Dup2 muscles remained significantly different from Bl6. The Dup2 mouse is generally larger than control Bl6 mice,10Vulin A. Wein N. Simmons T.R. Rutherford A.M. Findlay A.R. Yurkoski J.A. Kaminoh Y. Flanigan K.M. The first exon duplication mouse model of Duchenne muscular dystrophy: A tool for therapeutic development.Neuromuscul. Disord. 2015; 25: 827-834Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and this difference in size may account for the discrepancy between recovery of the absolute and specific force, as specific force takes into account muscle cross-sectional area. Similarly, treated Dup2 muscles show a significant partial rescue of force drop following repeated eccentric contractions but remain more sensitive to this injury than Bl6 muscles (Figure 4C). With an ultimate goal of systemic delivery to reach all skeletal muscles, heart, and diaphragm, we next undertook systemic intravenous (i.v.) dose-escalation studies in order to establish a minimal efficacious dose (MED) necessary for designing IND-enabling toxicology studies, as well as for eventual clinical translation. We initially performed single tail vein injections of 6 ascending doses and analyzed outcomes 4 weeks later. The results of RT-PCR analysis (Figure 5) show that the Dup2 transcript was the major species at the lowest doses (2.9 × 1011 vector genomes per kilogram (vg/kg) to 2.9 × 1012 vg/kg), accounting for at least 60% of transcripts. Higher doses, ranging from 9.4 × 1012 vg/kg to 7.6 × 1013 vg/kg, were associated with increased skipping of either one or two copies of exon 2. At the highest dose of 7.6 × 1013 vg/kg, skipped transcripts represented 42%–96% of total transcripts in different muscle types. Notably, both the WT and the Δ2 transcripts are therapeutic, given the presence of the dystrophin exon 5 IRES. IF images and analysis (Figure 6) are consistent with RT-PCR results, showing modest increases in dystrophin-positive fibers and dystrophin signal intensity at lower doses and robust restoration of properly localized dystrophin at higher doses across all skeletal muscles. At the highest dose of 7.6 × 1013 vg/kg, dystrophin is present in 69%–92% of fibers on average across different skeletal muscles, with a global mean of 81% for pooled data from all skeletal muscles (Figure 6A). Dystrophin signal intensity restoration at the sarcolemma was less robust, with a global mean of 56% of Bl6 intensity for pooled data from all skeletal muscles at highest dose, which is 4.1-fold higher than untreated Dup2 intensity (Figure 6B). Quantification of dystrophin expression by immunoblot confirms a dose-related increase in dystrophin protein, reaching a global mean of 34% of Bl6 expression, 4.4-fold higher than untreated Dup2, at the highest dose of 7.6 × 1013 vg/kg (Figure 7).Figure 7Immunoblot analysis following delivery of scAAV9.U7.ACCA confirms dystrophin expression 4 weeks post-injectionShow full caption(A and B) Representative images of immunoblots (A) show increase in dystrophin protein (red) normalized to actinin (green), and quantification (B) confirms a significant increase in dystrophin protein expression with increasing dose. Sample lanes marked with a red X were omitted due to technical issues. Quantification reported as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed on pooled data from all skeletal muscles using one-way ANOVA with a post hoc test for linear trend from left to right; ∗p < 0.05 for trend. Lower panel displays quantification results broken down by individual muscles.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Representative images of immunoblots (A) show increase in dystrophin protein (red) normalized to actinin (green), and quantification (B) confirms a significant increase in dystrophin protein expression with increasing dose. Sample lanes marked with a red X were omitted due to technical issues. Quantification reported as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed on pooled data from all skeletal muscles using one-way ANOVA with a post hoc test for linear trend from left to right; ∗p < 0.05 for trend. Lower panel displays quantification results broken down by individual muscles. In order to further define a clinically relevant minimal efficacious dose, and to assess the longevity of dystrophin expression, we injected another cohort of Dup2 mice with the same set of 6 i.v. doses and analyzed dystrophin expression and muscle function 12 weeks post-injection. With the exception of IF, only results for the 3 highest doses are shown due to a negligible effect at the 3 lower doses. RT-PCR analysis (Figure 8) shows increased exon 2 skipping in all doses, with single or double skipping of exon 2 representing at least 75% of the total transcript at the highest dose of 7.6 × 1013 vg/kg. IF shows a marked increase in dystrophin expression at the 3 highest doses, with near complete average restoration of dystrophin-positive fibers (85%) and complete restoration of mean sarcolemmal dystrophin intensity (104% of Bl6 intensity) at the highest dose in all muscles (Figure 9). Immunoblot analysis corroborates progressively increasing dystrophin levels at the 3 upper doses, with mean dystrophin content of 52% of Bl6 at the highest dose of 7.6 × 1013 vg/kg (Figure 10).Figure 9I.v. delivery of scAAV9.U7.ACCA results in dose-dependent expression of dystrophin protein at 12 weeks post-injectionShow full caption(A and B) Dystrophin immunofluorescence confirms sustained dystrophin expression 12 weeks after systemic injection of scAAV9.U7.ACCA in four representative skeletal muscles, as shown by dystrophin-positive fibers (A) and dystrophin signal intensity normalized to Bl6 signal (B). Diaphragm Bl6 tissue images collected with identical exposure settings were not available for analysis, so diaphragm intensities are normalized to the mean of dystrophin intensities in the other three Bl6 skeletal muscles. Data presented as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed on pooled data from all skeletal muscles using one-way ANOVA with a post hoc test for linear trend from left to right; ∗∗∗∗p < 0.0001 for trend. Right panel of (A) and (B) displays the quantification results broken down by each muscle. (C) Representative images of diaphragm (Dia) and triceps (Tri) sections from the 5 highest dose groups confirm correct dystrophin localization (red) and reflect fiber dystrophin positivity. Images were processed uniformly with automatic shading correction, rolling-ball background subtraction, and denoising as a part of preparation for quantitative analysis (see Figure S2B for unprocessed images). The color-coded heatmaps of each image reflect the percent of the fiber perimeter with dystrophin-positive pixels. Fibers that have dystrophin around ≥30% of the perimeter are considered dystrophin positive. The color scale indicates the conversion between color and % dystrophin-positive perimeter.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Immunoblot analysis demonstrates continued dystrophin expression 12 weeks after i.v. injection of scAAV9.U7.ACCAShow full caption(A) Immunoblots for dystrophin show a sustained increase in dystrophin protein expression (red) normalized to actinin (green) across four different muscles 23 weeks after treatment. Sample lanes marked with a red X were omitted due to technical issues. (B) Immunoblot quantification confirms a significant increase in dystrophin protein expression with increasing dose. Quantification reported as mean ± 95% CI (shaded region), with individual data points representing individual samples. Quantification reported as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed on pooled data from all skeletal muscles using one-way ANOVA with a post hoc test for linear trend from left to right; ∗∗∗∗p < 0.0001 for trend. Bottom panel displays quantification results broken down by individual muscles.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Dystrophin immunofluorescence confirms sustained dystrophin expression 12 weeks after systemic injection of scAAV9.U7.ACCA in four representative skeletal muscles, as shown by dystrophin-positive fibers (A) and dystrophin signal intensity normalized to Bl6 signal (B). Diaphragm Bl6 tissue images collected with identical exposure settings were not available for analysis, so diaphragm intensities are normalized to the mean of dystrophin intensities in the other three Bl6 skeletal muscles. Data presented as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed on pooled data from all skeletal muscles using one-way ANOVA with a post hoc test for linear trend from left to right; ∗∗∗∗p < 0.0001 for trend. Right panel of (A) and (B) displays the quantification results broken down by each muscle. (C) Representative images of diaphragm (Dia) and triceps (Tri) sections from the 5 highest dose groups confirm correct dystrophin localization (red) and reflect fiber dystrophin positivity. Images were processed uniformly with automatic shading correction, rolling-ball background subtraction, and denoising as a part of preparation for quantitative analysis (see Figure S2B for unprocessed images). The color-coded heatmaps of each image reflect the percent of the fiber perimeter with dystrophin-positive pixels. Fibers that have dystrophin around ≥30% of the perimeter are considered dystrophin positive. The color scale indicates the conversion between color and % dystrophin-positive perimeter. (A) Immunoblots for dystrophin show a sustained increase in dystrophin protein expression (red) normalized to actinin (green) across four different muscles 23 weeks after treatment. Sample lanes marked with a red X were omitted due to technical issues. (B) Immunoblot quantification confirms a significant increase in dystrophin protein expression with increasing dose. Quantification reported as mean ± 95% CI (shaded region), with individual data points representing individual samples. Quantification reported as mean ± SEM, with individual data points representing individual samples. Statistical analysis was performed on pooled data from all skeletal muscles using one-way ANOVA with a post hoc test for linear trend from left to right; ∗∗∗∗p < 0.0001 for trend. Bottom panel displays quantification results broken down by individual muscles. Functional studies performed on the TA muscle of treated Dup2 mice 12 weeks after injection also showed progressively increasing absolute and specific force at the 3 highest doses, culminating in complete rescue of absolute force (Figure 11A) and partial rescue of specific force (Figure 11B). Muscle force after multiple eccentric contractions again showed a partial rescue, with intermediate force drop observed in treated Dup2 that was significantly different from both untreated Dup2 and Bl6 muscles (Figure 11C). Due to the prevalence of cardiomyopathy among patients with DMD, successful dystrophin restoration in the heart is an important aspect of the potential clinical benefits of gene therapies. Immunoblot and IF results reflect effective dystrophin expression in treated Dup2 hearts at time points of 4 weeks and 12 weeks post-injection (Figure 12). Immunoblots showed dystrophin levels reaching just over 40% at the higher doses at both time points (Figure 12B), and average dystrophin signal intensity at the sarcolemma reached 73% of Bl6 levels (5.2-fold higher than untreated Dup2) at the highest dose of 7.6 × 1013 vg/kg (Figure 12C). Systemic delivery of scAAV9.U7.ACCA induces robust skipping of exon 2 within DMD mRNA in skeletal muscle, heart, and diaphragm of Dup2 mice. This skipping results in two tran" @default.
• W3136361381 created "2021-03-29" @default.
• W3136361381 creator A5033024298 @default.
• W3136361381 creator A5039313795 @default.
• W3136361381 creator A5041177612 @default.
• W3136361381 creator A5050280688 @default.
• W3136361381 creator A5059015837 @default.
• W3136361381 creator A5079070501 @default.
• W3136361381 date "2021-06-01" @default.
• W3136361381 modified "2023-10-16" @default.
• W3136361381 title "Pre-clinical dose-escalation studies establish a therapeutic range for U7snRNA-mediated DMD exon 2 skipping" @default.
• W3136361381 cites W1533447420 @default.
• W3136361381 cites W1947548159 @default.
• W3136361381 cites W1963623233 @default.
• W3136361381 cites W1969922725 @default.
• W3136361381 cites W1987768824 @default.
• W3136361381 cites W2011383034 @default.
• W3136361381 cites W2018862827 @default.
• W3136361381 cites W2024998665 @default.
• W3136361381 cites W2028353886 @default.
• W3136361381 cites W2044337709 @default.
• W3136361381 cites W2051628498 @default.
• W3136361381 cites W2062056760 @default.
• W3136361381 cites W2072832813 @default.
• W3136361381 cites W2077301034 @default.
• W3136361381 cites W2090849645 @default.
• W3136361381 cites W2091953158 @default.
• W3136361381 cites W2099540110 @default.
• W3136361381 cites W2136106574 @default.
• W3136361381 cites W2137703332 @default.
• W3136361381 cites W2142169677 @default.
• W3136361381 cites W2234725241 @default.
• W3136361381 doi "https://doi.org/10.1016/j.omtm.2021.03.014" @default.
• W3136361381 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8047432" @default.
• W3136361381 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33898631" @default.
• W3136361381 hasPublicationYear "2021" @default.
• W3136361381 type Work @default.
• W3136361381 sameAs 3136361381 @default.
• W3136361381 citedByCount "17" @default.
• W3136361381 countsByYear W31363613812021 @default.
• W3136361381 countsByYear W31363613812022 @default.
• W3136361381 countsByYear W31363613812023 @default.
• W3136361381 crossrefType "journal-article" @default.
• W3136361381 hasAuthorship W3136361381A5033024298 @default.
• W3136361381 hasAuthorship W3136361381A5039313795 @default.
• W3136361381 hasAuthorship W3136361381A5041177612 @default.
• W3136361381 hasAuthorship W3136361381A5050280688 @default.
• W3136361381 hasAuthorship W3136361381A5059015837 @default.
• W3136361381 hasAuthorship W3136361381A5079070501 @default.
• W3136361381 hasBestOaLocation W31363613811 @default.
• W3136361381 hasConcept C126322002 @default.
• W3136361381 hasConcept C2776056953 @default.
• W3136361381 hasConcept C71924100 @default.
• W3136361381 hasConceptScore W3136361381C126322002 @default.
• W3136361381 hasConceptScore W3136361381C2776056953 @default.
• W3136361381 hasConceptScore W3136361381C71924100 @default.
• W3136361381 hasLocation W31363613811 @default.
• W3136361381 hasLocation W31363613812 @default.
• W3136361381 hasOpenAccess W3136361381 @default.
• W3136361381 hasPrimaryLocation W31363613811 @default.
• W3136361381 hasRelatedWork W1506200166 @default.
• W3136361381 hasRelatedWork W1995515455 @default.
• W3136361381 hasRelatedWork W2039318446 @default.
• W3136361381 hasRelatedWork W2048182022 @default.
• W3136361381 hasRelatedWork W2080531066 @default.
• W3136361381 hasRelatedWork W2604872355 @default.
• W3136361381 hasRelatedWork W2748952813 @default.
• W3136361381 hasRelatedWork W2899084033 @default.
• W3136361381 hasRelatedWork W3032375762 @default.
• W3136361381 hasRelatedWork W3108674512 @default.
• W3136361381 hasVolume "21" @default.
• W3136361381 isParatext "false" @default.
• W3136361381 isRetracted "false" @default.
• W3136361381 magId "3136361381" @default.
• W3136361381 workType "article" @default.