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Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease, caused by mutations in DMD gene leading to absence of dystrophin. 5-6% of DMD patients have mutations in exons 6-8 encoding for the second calponin homology domain of dystrophin. To test the efficiency of a novel vectorized exon skipping (VES) for patients with these mutations, we developed a multiple VES constructs that was screened in vitro in HEK293 and patient fibroblasts differentiated into myotubes to select our best VES candidates that mediate exon 6-8 skipping. In addition, we tested our lead candidates in a new humanized hDMD mdx mouse called hDMDm7 that was generated using CRISPR/Cas9. This mouse model does not express murine dystrophin and additionally contains a nonsense mutation in exon 7 (hDMDm7) resulting in no human dystrophin expression. Intramuscular (I.M.) injections using selected VES constructs were performed for hDMDm7 mice at 6 and 7 weeks of age. Exon skipping was evaluated by RT-PCR, dystrophin expression by WB and IF, and muscle force using electrophysiology. This mouse model presents a reduction of muscle integrity with centronucleation (>90% at 3 and 6 months of age) using H&E staining. Little to no dystrophin expression was determined by western blot, and the average percent drop over 10 consecutive eccentric contractions was 59% in hDMDm7 vs 9% in hDMD. Four lead VES constructs were selected and evaluated in the hDMDm7. One VES construct was tested effective in muscle force measurement at 3 months of post I.M. injection, which brought the percent drop of eccentric contraction back to 40%. This exon skipping strategy was able to restore muscle functions following I.M. treatment for hDMDm7 mouse model. This VES for exons 6-8 could be beneficial for 5-6% of DMD patients. [ABSTRACT FROM AUTHOR]

Exon skipping, a promising modality to treat Duchenne muscular dystrophy (DMD) is based on restoring the reading frame of the DMD pre-mRNA and to make truncated but functional dystrophin. Conventional exon skipping using antisense oligonucleotides has some limitations – a need of repeated injections due to less stability and limited tissue penetration in major affected tissues (e.g., heart). To overcome these, we and others used AAV.U7 antisense delivery. U7 small nuclear RNA carrying antisense sequence improves the stability and adeno-associated virus (AAV) increases tropism. This approach is currently being tested in a clinical trial for DMD exon 2 duplication. In this study, we applied this vectorized exon skipping strategy (VES) approach to a mutational hot spot in the DMD gene: the exon 44. We evaluated our lead candidate using intramuscular (IM) and systemic (IV) pre-clinical dose escalation study. Humanized DMD mice with exon 45 deletion were used. We evaluated exon skipping by RT-PCR, proteins restoration using immunostaining, muscle function using force transducer, muscle co-ordination and strength using rotarod and hang wire, and behavior test with open field apparatus. 3-months post-IM, around 85% of exon skipping was achieved, resulting in around 90% of truncated dystrophin expression and more than 50% improvement in eccentric contraction. 3-months post-IV dose escalation, around 80% of exon skipping was observed in heart and 30–60% in diaphragm, TA and gastrocnemius muscles. Dystrophin expression and improvement in force generation in dose-dependent manner was noted. Macrophage infiltration, muscle fibrosis, rotarod, hang wire and open field test revealed improvement after treatment with the highest IV dose. To conclude, our lead candidate induces efficient DMD exon 44 skipping, resulting in dystrophin production and muscle strength improvement in DMD. This AAV.U7-exon 44 skipping vector represents a promising candidate for ∼6-12% of DMD patients. [ABSTRACT FROM AUTHOR]

In biological systems, spatial organization and function are interconnected. Here we present photoselective sequencing, a new method for genomic and epigenomic profiling within morphologically distinct regions. Starting with an intact biological specimen, photoselective sequencing uses targeted illumination to selectively unblock a photocaged fragment library, restricting the sequencing-based readout to microscopically identified spatial regions. We validate photoselective sequencing by measuring the chromatin accessibility profiles of fluorescently labeled cell types within the mouse brain and comparing with published data. Furthermore, by combining photoselective sequencing with a computational strategy for decomposing bulk accessibility profiles, we find that the oligodendrocyte-lineage-cell population is relatively enriched for oligodendrocyte-progenitor cells in the cortex versus the corpus callosum. Finally, we leverage photoselective sequencing at the subcellular scale to identify features of chromatin that are correlated with positioning at the nuclear periphery. These results collectively demonstrate that photoselective sequencing is a flexible and generalizable platform for exploring the interplay of spatial structures with genomic and epigenomic properties., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Duchenne muscular dystrophy (DMD) is typically caused by mutations that disrupt the DMD reading frame, but nonsense mutations in the 5' part of the gene induce utilization of an internal ribosomal entry site (IRES) in exon 5, driving expression of a highly functional N-truncated dystrophin. We have developed an AAV9 vector expressing U7 small nuclear RNAs targeting DMD exon 2 and have tested it in a mouse containing a duplication of exon 2, in which skipping of both exon 2 copies induces IRES-driven expression, and skipping of one copy leads to wild-type dystrophin expression. One-time intravascular injection either at postnatal days 0-1 or at 2 months results in efficient exon skipping and dystrophin expression, and significant protection from functional and pathologic deficits. Immunofluorescence quantification showed 33%-53% average dystrophin intensity and 55%-79% average dystrophin-positive fibers in mice treated in adulthood, with partial amelioration of DMD pathology and correction of DMD-associated alterations in gene expression. In mice treated neonatally, dystrophin immunofluorescence reached 49%-85% of normal intensity and 76%-99% dystrophin-positive fibers, with near-complete correction of dystrophic pathology, and these beneficial effects persisted for at least 6 months. Our results demonstrate the robustness, durability, and safety of exon 2 skipping using scAAV9.U7snRNA.ACCA, supporting its clinical use., Competing Interests: Since the performance of this work, Nationwide Children’s Hospital licensed the vector described herein to Audentes/Astellas Therapeutics. N.W., A.V., and K.M.F. have received royalty payments as a result of this license., (© 2022 The Authors.)

Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules, the spatially confined functional units of spermatogenesis. Here, we use Slide-seq, a spatial transcriptomics technology, to generate an atlas that captures the spatial gene expression patterns at near-single-cell resolution in the mouse and human testis. Using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased analysis systematically identifies spatially patterned genes and gene programs. Combining Slide-seq with targeted in situ RNA sequencing, we demonstrate significant differences in the cellular compositions of spermatogonial microenvironment between mouse and human testes. Finally, a comparison of the spatial atlas generated from the wild-type and diabetic mouse testis reveals a disruption in the spatial cellular organization of seminiferous tubules as a potential mechanism of diabetes-induced male infertility., Competing Interests: Declaration of interests F.C. and E.Z.M. are listed as inventors on a pending patent application related to the development of Slide-seq. F.C. and E.Z.M. are paid consultants of Atlas Bio. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The uridine-rich 7 (U7) small nuclear RNA (snRNA) is a component of a small nuclear ribonucleoprotein (snRNP) complex. U7 snRNA naturally contains an antisense sequence that identifies histone premessenger RNAs (pre-mRNAs) and is involved in their 3' end processing. By altering this antisense sequence, researchers have turned U7 snRNA into a versatile tool for targeting pre-mRNAs and modifying splicing. Encapsulating a modified U7 snRNA into a viral vector such as adeno-associated virus (also referred as vectorized exon skipping/inclusion, or VES/VEI) enables the delivery of this highly efficacious splicing modulator into a range of cell lines, primary cells, and tissues. In addition, and in contrast to antisense oligonucleotides, viral delivery of U7 snRNA enables long-term expression of antisense sequences in the nucleus as part of a stable snRNP complex. As a result, VES/VEI has emerged as a promising therapeutic platform for treating a large variety of human diseases caused by errors in pre-mRNA splicing or its regulation. Here we provide an overview of U7 snRNA's natural function and its applications in gene therapy.

Here we describe TRACE (T7 polymerase-driven continuous editing), a method that enables continuous, targeted mutagenesis in human cells using a cytidine deaminase fused to T7 RNA polymerase. TRACE induces high rates of mutagenesis over multiple cell generations in genes under the control of a T7 promoter integrated in the genome. We used TRACE in a MEK1 inhibitor-resistance screen, and identified functionally correlated mutations.