Journal: Molecular Therapy Advances

Article Title: Preclinical evaluation of INS1201 AAV9-micro-dystrophin via CSF administration as a potential therapy for Duchenne muscular dystrophy

doi: 10.1016/j.omta.2026.201707

Figure Lengend Snippet: INS1201 construct design and gastrocnemius histopathological correction (A) Full-length dystrophin protein is represented (top) as a reference for the micro-dystrophin gene contained within INS1201 (bottom). The design of the INS1201 vector utilizes the following functional elements from 5ʹ to 3ʹ, which are flanked on either side by an ITR: a muscle-derived MHCK7 promoter, SV40 intron for transcript stability, the micro-dystrophin gene, and SV40 poly(A) signal. Together, these elements have been combined to create an AAV9-encapsidated genome, INS1201, a single-stranded AAV vector designed to express high levels of functional micro-dystrophin in skeletal and cardiac muscle. (B) Histopathological evaluation of gastrocnemius muscle: treatment groups include (from left to right): (1) mdx (−) control (vehicle), (2) 8.0E+09 vg of INS1201, (3) 2.0E+11 vg of INS1201, (4) 4.0E+11 vg of INS1201, (5) 8.0E+11 vg of INS1201, and (6) WT mice. Panel rows are (from top to bottom) as follows: (A) H&E staining, (B) picrosirius red staining for collagen and fast green staining for non-collagenous tissue, (C) laminin (red) staining, and (D) dystrophin (green) staining of EDL muscle tissue. Scale bars: 100 μm. AAV, adeno-associated virus; ABD, actin binding domain; DbBD, dystrobrevin binding domain; EDL, extensor digitorum longus; H&E, hematoxylin and eosin; nNOS BD, neuronal nitric oxide synthase binding domain; SBD, syntrophin binding domain; WT, wild-type.

Article Snippet: Note, the monoclonal dystrophin antibody used in these studies, MANEX1A (4C7), produced by the Developmental Studies Hybridoma Bank, IA, USA, recognizes the first three amino acids of the n-terminus of dystrophin and therefore detects both micro-dystrophin and full-length dystrophin.

Techniques: Construct, Plasmid Preparation, Functional Assay, Derivative Assay, Control, Staining, Virus, Binding Assay

Journal: Molecular Therapy Advances

Article Title: Preclinical evaluation of INS1201 AAV9-micro-dystrophin via CSF administration as a potential therapy for Duchenne muscular dystrophy

doi: 10.1016/j.omta.2026.201707

Figure Lengend Snippet: Quantitative improvements in skeletal muscle histopathology Following INS1201 treatment via ICV delivery at ∼ p28, gastrocnemius, tibialis anterior, EDL, diaphragm, and soleus muscles were evaluated for (A) fibrosis, by quantifying the percent collagen in picrosirius red-stained tissue, (B) average fiber size diameter, (C) membrane dystrophin expression, and (D) percent dystrophin positive fibers. Analyzed groups include WT ( n = 5) and mdx mice ( n = 8–11/group for all muscles except soleus that had n = 4–8/group) administered vehicle or INS1201 at 8.0E+9, 2.0E+11, 4.0E+11, and 8.0E+11 vg/animal. Values presented are mean ± SD. From ordinary 1-way ANOVA with Bonferroni correction comparing individual groups with mdx (−) vehicle control mice: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ∗∗∗∗ p < 0.0001. ANOVA, analysis of variance; au, arbitrary unit; Avg., average; EDL, extensor digitorum longus; H&E, hematoxylin and eosin; ICV, intracerebroventricular; SD, standard deviation; vg, vector genomes; WT, wild-type.

Article Snippet: Note, the monoclonal dystrophin antibody used in these studies, MANEX1A (4C7), produced by the Developmental Studies Hybridoma Bank, IA, USA, recognizes the first three amino acids of the n-terminus of dystrophin and therefore detects both micro-dystrophin and full-length dystrophin.

Techniques: Histopathology, Muscles, Staining, Membrane, Expressing, Control, Standard Deviation, Plasmid Preparation

Journal: Molecular Therapy Advances

Article Title: Preclinical evaluation of INS1201 AAV9-micro-dystrophin via CSF administration as a potential therapy for Duchenne muscular dystrophy

doi: 10.1016/j.omta.2026.201707

Figure Lengend Snippet: Long-term durability of efficacy, biodistribution, and micro-dystrophin protein expression in mdx mice treated with INS1201 (A) Plantar torque frequency curves as a function of body weight in mdx mice at ∼ p400 following ICV administration of INS1201 at ∼ p28. mdx (−) control mice (vehicle/untreated; n = 13) exhibited significantly reduced plantar flexor torque at stimulation frequencies of 60–160 Hz relative to WT control mice ( n = 6). This reduction was partially mitigated following ICV administration of INS1201. mdx mice treated with INS1201 showed significant improvements in plantar torque at the doses of 4.0E+11 vg ( n = 8), 8.0E+11 vg ( n = 14), and 1.6E+12 vg ( n = 10) when stimulated at 80 to 160 Hz relative to mdx (−) control mice. Significant increases in maximal torque were also observed in the INS1201 2.0E+11 vg dose group ( n = 8) at the lower frequencies of 60 and 80 Hz. Values presented are mean ± SD. Statistical testing was conducted using an ordinary two-way ANOVA with Bonferroni’s correction comparing individual groups with mdx (−) control mice. (B) Improvement in gastrocnemius dystrophic histopathology at approximately 1 year after ICV administration of INS1201. Treatment groups include age-matched animals at ∼ p400 (from left to right): (1) mdx (−) control, (2) INS1201 4.0E+11 vg, (3) INS1201 8.0E+11 vg, (4) INS1201 1.6E+12 vg, and (5) WT mice. Scale bars noted on WT images (100 μm) apply to all images in the panel. (A) H&E staining, (B) picrosirius red staining for collagen and fast green staining for non-collagenous tissue, (C) laminin (red) staining, and (D) dystrophin (green) staining of gastrocnemius muscle tissue. (C) Quantification of percent collagen in muscle tissue by picrosirius red staining to assess fibrosis in gastrocnemius muscle tissue collected from mdx mice at approximately 1 year after INS1201 administration. Values presented are mean ± SD. Groups are WT ( n = 4); mdx (−) control mice (vehicle/untreated, n = 8); and INS1201 doses of 4.0E+11 vg, 8.0E+11 vg, and 1.6E+12 vg (n = 8–9 mice per group). (D) INS1201 vg biodistribution in gastrocnemius and heart muscle tissue collected from mdx mice at approximately 1 year after INS1201 administration. Data shown on a log scale. Values presented are mean ± SD ( n = 6–10 mice per group). (E) Clear membrane dystrophin expression was observed in the heart of mdx mice at approximately 1 year after treatment for all three INS1201 doses assessed (4.0E+11 vg, 8.0E+11 vg, and 1.6E+12 vg). Laminin (red) staining and dystrophin (green) staining of heart tissue. Western blot analysis of micro-dystrophin protein in the (F) gastrocnemius and (G) heart muscles showed clear dose-response, as animals in each INS1201 dose group expressed micro-dystrophin protein at this time point. Chemiluminescence intensity was measured, and dose-response was confirmed in graphs (H) and (I) for gastrocnemius and heart, respectively. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. ANOVA, analysis of variance; H&E, hematoxylin and eosin; ICV, intracerebroventricular; SD, standard deviation; vg, vector genomes; WT, wild-type.

Article Snippet: Note, the monoclonal dystrophin antibody used in these studies, MANEX1A (4C7), produced by the Developmental Studies Hybridoma Bank, IA, USA, recognizes the first three amino acids of the n-terminus of dystrophin and therefore detects both micro-dystrophin and full-length dystrophin.

Techniques: Expressing, Control, Histopathology, Staining, Membrane, Western Blot, Muscles, Standard Deviation, Plasmid Preparation

Journal: Molecular Therapy Advances

Article Title: Preclinical evaluation of INS1201 AAV9-micro-dystrophin via CSF administration as a potential therapy for Duchenne muscular dystrophy

doi: 10.1016/j.omta.2026.201707

Figure Lengend Snippet: INS1201 biodistribution in WT mouse and NHP muscle tissue (A–C) The vg of INS1201 per diploid genome in the diaphragm, rib with muscle#6/#7, triceps, quadriceps, tibialis anterior, EDL, tongue, and heart collected from WT C57BL/6J mice at (A) 3, (B) 6, and (C) 12 weeks after INS1201 administration. Mice were dosed via ICV injection at p28, with a single dose per animal of 2.0E+11 vg, 4.0E+11 vg, or 8.0E+11 vg; n = 5 mice per dosing group. (D) Western blot analysis was performed on mdx mouse tissue to confirm the presence of micro-dystrophin protein expression in select skeletal and cardiac muscles and to confirm the absence of micro-dystrophin protein expression in brain, spinal cord, DRG, and liver. (E) The vg of INS1201 per diploid genome in the diaphragm, biceps, triceps, quadriceps, gastrocnemius, tibialis anterior, deltoid, and heart collected from NHPs (1–3 years of age at dosing) 3 weeks after administration in a non-GLP study. NHPs were dosed via IT injection, with a single dose per animal of 2.5E+13 vg, 5.0E+13 vg, 1.0E+14 vg, or 2.0E+14 vg; n = 2 NHPs per group. (F) The vg of INS1201 per diploid genome in the diaphragm, rib with muscle #6/#7, triceps brachii, quadriceps, gastrocnemius, tibialis anterior, soleus, biceps brachii, deltoid, and heart collected from NHP (7–12 months of age at dosing) 3 months after administration in a GLP-compliant toxicology study. NHP were dosed via IT injection with a single dose per animal of 5.28E+13 vg, 1.09E+14 vg, or 3.05E+14 vg; n = 3 NHPs per group. Data shown on a log scale. Error bars are mean ± SD. DRG, dorsal root ganglia; EDL, extensor digitorum longus; FFB, final formulation buffer (vehicle used in study); GLP, Good Laboratory Practice; ICV, intracerebroventricular; IT, intrathecal; NHP, nonhuman primate; SD, standard deviation; vg, vector genomes; WT, wild-type.

Article Snippet: Note, the monoclonal dystrophin antibody used in these studies, MANEX1A (4C7), produced by the Developmental Studies Hybridoma Bank, IA, USA, recognizes the first three amino acids of the n-terminus of dystrophin and therefore detects both micro-dystrophin and full-length dystrophin.

Techniques: Injection, Western Blot, Expressing, Muscles, Formulation, Standard Deviation, Plasmid Preparation

Journal: Molecular Therapy Advances

Article Title: Preclinical evaluation of INS1201 AAV9-micro-dystrophin via CSF administration as a potential therapy for Duchenne muscular dystrophy

doi: 10.1016/j.omta.2026.201707

Figure Lengend Snippet: INS1201 RNA expression Qualitative RT-PCR analysis of INS1201 micro-dystrophin mRNA expression from muscle tissues and organs collected from NHPs in the 3-month GLP-compliant toxicology study. RNA transcript expression was evaluated in samples from four animals within the study: one vehicle control animal (NHP #3) and the three animals dosed via IT injection with a single dose per animal of 3.05E+14 vg (NHPs #10, #11, and #12). (A) INS1201 mRNA expression was observed in each skeletal and cardiac muscle examined from the NHPs dosed with INS1201 but not in the vehicle control NHPs. Muscles examined included gastrocnemius, tibialis anterior, biceps, triceps, deltoid, quadriceps, soleus, diaphragm, rib with intercostal muscle #6/7, and heart. (B) Limited INS1201 mRNA expression was detected within the midbrain of two of the three NHPs dosed with INS1201 but was not observed in other brain regions analyzed including the cerebrum, cerebellum, striatum, diencephalon, and pons. mRNA expression was not observed in the vehicle control samples. (C) INS1201 mRNA expression was not detected within any region of the spinal cord or DRG or injection site in NHPs dosed with INS1201 or vehicle. (D) INS1201 micro-dystrophin mRNA expression was not detected in NHPs dosed with INS1201 or vehicle within any peripheral organs and non-muscle tissues analyzed, including the adrenal gland, epididymis, kidney, liver, lungs, testes, thymus, skin, small intestine, spleen, and sciatic nerve. (E) Evaluation of mouse DRG following INS1201 delivery via the CSF revealed healthy cells and normal histopathology in all dose groups (image is from animal administered highest dose, 8.0E+11 vg). CSF, cerebrospinal fluid; DRG, dorsal root ganglion; GLP, Good Laboratory Practice; IT, intrathecal; mRNA, messenger RNA; NHP, nonhuman primate; NTC, no template control; positive, positive control; −RT, no reverse transcriptase; RT, reverse transcriptase; PCR, polymerase chain reaction; SC, spinal cord; SI, spike in; vg, vector genomes.

Article Snippet: Note, the monoclonal dystrophin antibody used in these studies, MANEX1A (4C7), produced by the Developmental Studies Hybridoma Bank, IA, USA, recognizes the first three amino acids of the n-terminus of dystrophin and therefore detects both micro-dystrophin and full-length dystrophin.

Techniques: RNA Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Injection, Muscles, Histopathology, Positive Control, Reverse Transcription, Polymerase Chain Reaction, Plasmid Preparation

Journal: Skeletal Muscle

Article Title: Functional and structural pathologies in skeletal muscle of a rat model of Duchenne muscular dystrophy

doi: 10.1186/s13395-026-00419-4

Figure Lengend Snippet: CRISPR/Cas9-mediated deletion of full-length rat dystrophin. A Organization of the rat Dmd that spans ~ 2.3 mbp of the X chromosome. The 5 exons targeted for removal – 22–26 (in gray) and the two flanking exons (21 & 27 – in green) are shown in more detail (WT). The two guide RNAs (5’ and 3’ gRNAs) aided in the Cas9-mediated deletion of 18,307 bps that include exons 22–26 (MDR). B The 5’ and 3’ untranslated regions (unfilled magenta boxes) flank the 79 exons (filled magenta boxes) that contribute coding sequence of full-length dystrophin protein. Indicated with a closer view of exons 21–27 is the number of nucleotides each exon contributes (in parentheses). The 5 deleted exons (in gray) together contribute 800 bp of protein coding sequence, whose removal leads to a frame shift and a premature termination codon within the region of the transcript coded by exon 27 (*, MDR). C Immunoblotting confirms loss of full-length dystrophin (427 kDa; black arrow) and presence of predicted 106 kDa N-terminal fragment (gray double arrow) in 4 mo MDR gastrocnemius and heart lysates, and D immunofluorescence confirms loss of dystrophin staining at the sarcolemma of muscle fibers and cardiomyocytes (scale bars = 25 μm). E-F Immunoblotting was also performed for dystrophin glycoprotein complex (DGC) members β-dystroglycan (β-DG), α-dystrobrevin (α-DB), and syntrophins ( n = 3). Data are displayed as mean ± SEM with individual values and were analyzed using Student’s T-tests (α = 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The following primary antibodies were used for immunofluorescence in the present study: dystrophin (1:100; MANEX1011B, Clone 1C7; DSHB), β-dystroglycan (1:100; MANDAG2, Clone 7A11; DSHB), utrophin (1:50; VP-U579; Vector Laboratories), α-sarcoglycan (1:50; VP-A105; Vector Laboratories); syntrophins (1:2000; #11425; Abcam), perilipin 1 (1:500; #9349; Cell Signaling Technology), anti-Laminin β2 antibody (1:10,000; [ ]), embryonic MHC (1:100; F1.652; DSHB), type I myofibers (1:50; BA-D5; DSHB), Type IIa myofibers (1:50; SC-71; DSHB), type IIx myofibers (1:20; 6H1; DSHB), pan-muscle MHC(1:100; MF 20; DSHB), COMP (1:800; #NBP2-92658; Noveus Biologicals), α-SMA (1:1000; #7817; Abcam), SMOC2 (1:500; #AF5140; R&D Systems), and PDGFRα (1:500; #AF1062; R&D Systems).

Techniques: CRISPR, Sequencing, Western Blot, Immunofluorescence, Staining