CRISPR-Cas9 correction in the DMD mouse model is accompanied by upregulation of Dp71f protein.

Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a deficiency in the dystrophin protein. The most frequent types of disease-causing mutations in the DMD gene are frameshift deletions of one or more exons. Precision genome editing systems such as CRISPR-Cas9 have shown potential to restore open reading frames in numerous animal studies. Here, we applied an AAV-CRISPR double-cut strategy to correct a mutation in the DMD mouse model with exon 8-34 deletion, encompassing the N-terminal actin-binding domain. We report successful excision of the 100-kb genomic sequence, which includes exons 6 and 7, and partial improvement in cardiorespiratory function. While corrected mRNA was abundant in muscle tissues, only a low level of truncated dystrophin was produced, possibly because of protein instability. Furthermore, CRISPR-Cas9-mediated genome editing upregulated the Dp71f dystrophin isoform on the sarcolemma. Given the previously reported Dp71-associated muscle pathology, our results question the applicability of genome editing strategies for some DMD patients with N-terminal mutations. The safety and efficacy of CRISPR-Cas9 constructs require rigorous investigation in patient-specific animal models.

In-Frame Deletion of Dystrophin Exons 8-50 Results in DMD Phenotype.

Mutations that prevent the production of proteins in the DMD gene cause Duchenne muscular dystrophy. Most frequently, these are deletions leading to reading-frame shift. The "reading-frame rule" states that deletions that preserve ORF result in a milder Becker muscular dystrophy. By removing several exons, new genome editing tools enable reading-frame restoration in DMD with the production of BMD-like dystrophins. However, not every truncated dystrophin with a significant internal loss functions properly. To determine the effectiveness of potential genome editing, each variant should be carefully studied in vitro or in vivo. In this study, we focused on the deletion of exons 8-50 as a potential reading-frame restoration option. Using the CRISPR-Cas9 tool, we created the novel mouse model DMDdel8-50, which has an in-frame deletion in the DMD gene. We compared DMDdel8-50 mice to C57Bl6/CBA background control mice and previously generated DMDdel8-34 KO mice. We discovered that the shortened protein was expressed and correctly localized on the sarcolemma. The truncated protein, on the other hand, was unable to function like a full-length dystrophin and prevent disease progression. On the basis of protein expression, histological examination, and physical assessment of the mice, we concluded that the deletion of exons 8-50 is an exception to the reading-frame rule.

Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression.

High expectations have been set on gene therapy with an AAV-delivered shortened version of dystrophin (µDys) for Duchenne muscular dystrophy (DMD), with several drug candidates currently undergoing clinical trials. Safety concerns with this therapeutic approach include the immune response to introduced dystrophin antigens observed in some DMD patients. Recent reports highlighted microutrophin (µUtrn) as a less immunogenic functional dystrophin substitute for gene therapy. In the current study, we created a human codon-optimized µUtrn which was subjected to side-by-side characterization with previously reported mouse and human µUtrn sequences after rAAV9 intramuscular injections in mdx mice. Long-term studies with systemic delivery of rAAV9-µUtrn demonstrated robust transgene expression in muscles, with localization to the sarcolemma, functional improvement of muscle performance, decreased creatine kinase levels, and lower immunogenicity as compared to µDys. An extensive toxicity study in wild-type rats did not reveal adverse changes associated with high-dose rAAV9 administration and human codon-optimized µUtrn overexpression. Furthermore, we verified that muscle-specific promoters MHCK7 and SPc5-12 drive a sufficient level of rAAV9-µUtrn expression to ameliorate the dystrophic phenotype in mdx mice. Our results provide ground for taking human codon-optimized µUtrn combined with muscle-specific promoters into clinical development as safe and efficient gene therapy for DMD.

New species of Ameletus Eaton, 1885 from the Russian Far East with notes on Ameletus camtschaticus Ulmer 1927 (Ephemeroptera: Ameletidae).

The male imagoes, larvae, and eggs of Ameletus allengaensis sp. nov. and Ameletus sirotskii sp. nov. from the Russian Far East are described. Based on the structure of the male genitalia, the imago and larvae of A. allengaensis sp. nov. and A. sirotskii sp. nov. are similar to those of A. camtschaticus, but the discovery of these new species and separation from A. camtschaticus were confirmed by studies of the morphology of the larvae and male imago, as well as molecular analysis. Identity of various developmental stages of the new species were confirmed by analysis of the mitochondrial gene cytochrome oxidase 1 (COI) DNA barcode, which was also used to reconstruct the phylogenetic relationships within the genus Ameletus. The intraspecific sequence divergence based on the Kimura-2-parameter (K2P) distance ranged from 0.0-2.5%, whereas the interspecific sequence divergence based on the K2P distance ranged from 6.2-7.9% within A. sirotskii sp. nov., A. allengaensis sp. nov. and A. camtschaticus. Male imagoes of A. allengaensis sp. nov., A. sirotskii sp. nov., and A. camtschaticus can be distinguished by the size and location of small denticles on the ventral plate of the penis. The larvae of A. allengaensis sp. nov. differ from those of A. sirotskii sp. nov. by the size of gills I and II. In A. allengaensis sp. nov., gill I is almost twice as small as gill II; in A. sirotskii sp. nov., gill I is only slightly smaller than gill II. Both new species differ from A. camtschaticus by gill II, which does not have an anal rib on the anal margin.