Effective knockdown-replace gene therapy in a novel mouse model of DNM1 developmental and epileptic encephalopathy.

Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wild-type replacement. We evaluated such a knockdown-replace strategy in a mouse model of DNM1 disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons-which resulted in growth delay and lethal seizures evident by postnatal week three-and delivered to newborn pups an AAV9-based vector encoding a ubiquitously expressed, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 cDNA. Pups receiving RNAi or cDNA alone fared no better than untreated pups, whereas the vast majority of mutants receiving modest doses survived with almost full growth recovery. Synaptic recordings of cortical neurons derived from treated pups revealed that significant alterations in transmission from inhibitory to excitatory neurons were rectified by bivalent vector application. To examine the mutant transcriptome and impact of treatment, we used RNA sequencing and functional annotation clustering. Mutants displayed abnormal expression of more than 1,000 genes in highly significant and relevant functional clusters, clusters that were abrogated by treatment. Together these results suggest knockdown-replace as a potentially effective strategy for treating DNM1 and related genetic neurodevelopmental disease.

A self-complementary AAV proviral plasmid that reduces cross-packaging and ITR promoter activity in AAV vector preparations.

Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

Putting 'X' into Context: The Diversity of 'Candidatus Phytoplasma pruni' Strains Associated with the Induction of X-Disease.

Recurrent epiphytotics of X-disease, caused by 'Candidatus Phytoplasma pruni,' have inflicted significant losses on commercial cherry and peach production across North America in the last century. During this period, there have been multiple studies reporting different disease phenotypes and, more recently, identifying different strains through sequencing core genes, but the symptoms have not, to date, been linked with genotype. Therefore, in this study we collected and assessed differing disease phenotypes from multiple U.S. states and conducted multilocus sequence analysis on these strains. We identified a total of five lineages associated with the induction of X-disease on commercial Prunus species and two lineages that were associated with wild P. virginiana. Despite a century of interstate plant movement, there were regional trends in terms of lineages present, and lineage-specific symptoms were observed on P. avium, P. cerasus, and P. virginiana, but not on P. persica. Cumulatively, these data have allowed us to define "true" X-disease-inducing strains of concern to the stone fruit industry across North America, as well as potential sources of infection that exist in the extraorchard environment.

First report of 'Candidatus Phytoplasma pruni' associated with X-disease on sweet cherry (Prunus avium L.) in Canada.

British Columbia (BC) is the lead producer of sweet cherries in Canada with more than 2,000 ha in production and a farm gate value of over CAD$100 million annually. Since 2010, an outbreak of little cherry disease caused by Little cherry virus 1 (LChV1) and Little cherry virus 2 (LChV2), as well as X-disease (XD) caused by 'Candidatus Phytoplasma pruni' has caused significant economic losses in neighboring Washington State (WA), USA. LChV1 and LChV2 have long been known to occur in BC (Theilmann et al. 2002); however, 'Ca. P. pruni' has not yet been reported in BC. Due to its geographical proximity to WA State, the BC cherry industry expressed significant concerns about the possible presence of the phytoplasma in cherry orchards. Accordingly, the main objective of this study was to survey cherry orchards to determine whether 'Ca. P. pruni' was present in symptomatic trees in BC. A total of 118 samples of leaves and fruit stems from individual symptomatic trees were collected prior to harvest from nine cherry orchards and one nectarine orchard in the Okanagan and Similkameen Valleys in BC. Characteristic symptoms included small and misshapen fruit with poor color development. Samples were submitted to AGNEMA, LLC (Pasco, WA) for testing using qPCR TaqMan assays for LChV1 (Katsiani et al. 2018), LChV2 (Shires et al. 2022) and 'Ca. P. pruni' (Kogej et al. 2020). Test results showed 21 samples (17.8%) from three cherry orchards positive for LChV2 and 2 samples (1.7%) from one cherry orchard positive for 'Ca. P. pruni'. In order to confirm the identification of 'Ca. P. pruni', part of the 16S ribosomal RNA gene was amplified by nested PCR using the P1/P7 followed by R16F2n/R2 primer sets (Gundersen and Lee 1996) and Sanger sequenced. BC-XD-Pa-1 (GenBank Acc. No. OR539920) and BC-XD-Pa-2 (OR537699) were identical to one another and showed 99.92% identity to the 'Ca. P. pruni' reference strain CX-95 (JQ044397). Analysis using iPhyClassifier (Zhou et al. 2009) indicated that they were 16SrIII-A strains. Interestingly, the two partial 16S sequences showed 100% nucleotide identity to strain 10324 (MH810016) and others from WA. For additional confirmation, partial secA (Hodgetts et al. 2008) and secY (Lee et al. 2010) translocases were amplified and sequenced. As with the 16S sequences, secY sequences (OR542980, OR542981) showed 99.92% nucleotide identity to strain CX-95 (JQ268249), and 100% to strain 10324 (MH810035). The secA sequences (OR542978, OR542979) had nucleotide identities of 99.77% to strain CX (MW547067), and 100% to the Green Valley strain from California (EU168733). Accordingly, 'Ca. P. Pruni' was confirmed to be present in sweet cherry samples from BC. 'Ca. P. Pruni'-related strains have been previously reported to occur in Canada in commercial poinsettias (Euphorbia pulcherrima) (Arocha-Rosete et al. 2021). To our knowledge, this is the first report of 'Ca. P. Pruni' in sweet cherry in Canada. Due to the important economic value of sweet cherries in BC, these findings are highly significant and represent the first steps towards the development of a surveillance system for early detection of XD, and consequent implementation of management strategies, including vector control. As required by federal and provincial regulations, cherry trees infected with LChV2 and 'Ca. P. Pruni' found in the survey were removed by the growers.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3+ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3+ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3 + macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3 + phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3 + macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.

Post-Translational Modifications of the DUX4 Protein Impact Toxic Function in FSHD Cell Models.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. Although the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 post-translational modifications (PTMs) and their impact on the toxic function of DUX4. METHODS: We immunoprecipitated DUX4 protein and performed mass spectrometry to identify PTMs. We then characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics, and biochemical assays in HEK293 and human myoblast cell lines. RESULTS: We identified 17 DUX4 amino acids with PTMs, and generated 55 DUX4 mutants designed to prevent or mimic PTMs. Five mutants protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate FSHD biomarkers. These mutagenesis results suggested that DUX4 toxicity could be counteracted by serine/threonine phosphorylation and/or inhibition of arginine methylation. We therefore sought to identify modifying enzymes that could play a role in regulating DUX4 PTMs. We found several enzymes capable of modifying DUX4 protein in vitro, and confirmed that protein kinase A (PKA) and protein arginine methyltransferase (PRMT1) interact with DUX4. INTERPRETATION: These results support that DUX4 is regulated by PTMs and set a foundation for developing FSHD drug screens based mechanistically on DUX4 PTMs and modifying enzymes. ANN NEUROL 2023;94:398-413.

Bacterial Endosymbionts Identified From Leafhopper (Hemiptera: Cicadellidae) Vectors of Phytoplasmas.

Insects often harbor bacterial endosymbionts that provide them with nutritional benefit or with protection against natural enemies, plant defenses, insecticides, and abiotic stresses. Certain endosymbionts may also alter acquisition and transmission of plant pathogens by insect vectors. We identified bacterial endosymbionts from four leafhopper vectors (Hemiptera: Cicadellidae) of 'Candidatus Phytoplasma' species by direct sequencing 16S rDNA and confirmed endosymbiont presence and identity by species-specific conventional PCR. We examined three vectors of Ca. Phytoplasma pruni, causal agent of cherry X-disease [Colladonus geminatus (Van Duzee), Colladonus montanus reductus (Van Duzee), Euscelidius variegatus (Kirschbaum)] - and a vector of Ca. Phytoplasma trifolii, the causal agent of potato purple top disease [Circulifer tenellus (Baker)]. Direct sequencing of 16S identified the two obligate endosymbionts of leafhoppers, 'Ca. Sulcia' and 'Ca. Nasuia', which are known to produce essential amino acids lacking in the leafhoppers' phloem sap diet. About 57% of C. geminatus also harbored endosymbiotic Rickettsia. We identified 'Ca. Yamatotoia cicadellidicola' in Euscelidius variegatus, providing just the second host record for this endosymbiont. Circulifer tenellus harbored the facultative endosymbiont Wolbachia, although the average infection rate was only 13% and all males were Wolbachia-uninfected. A significantly greater percentage of Wolbachia-infected Ci. tenellus adults than uninfected adults carried Ca. P. trifolii, suggesting that Wolbachia may increase this insect's ability to tolerate or acquire this pathogen. Results of our study provide a foundation for continued work on interactions between leafhoppers, bacterial endosymbionts, and phytoplasma.

Draft Genome Sequence of a Washington Isolate of "Candidatus Phytoplasma pruni".

Illumina sequencing of a Prunus avium tree with X-disease symptoms was performed to obtain a draft genome of "Candidatus Phytoplasma pruni." The genome consists of 14 contigs covering 588,767 bp. This is the first metagenome to be sequenced from the current X-disease epidemic in stone fruit in the Pacific Northwest.

The DUX4 protein is a co-repressor of the progesterone and glucocorticoid nuclear receptors.

DUX4 is a transcription factor required during early embryonic development in placental mammals. In this work, we provide evidence that DUX4 is a co-repressor of nuclear receptors (NRs) of progesterone (PR) and glucocorticoids (GR). The DUX4 C-ter and N-ter regions, including the nuclear localization signals and homeodomain motifs, contribute to the co-repressor activity of DUX4 on PR and GR. Immunoprecipitation studies, using total protein extracts of cells expressing tagged versions of DUX4 and GR, support that these proteins are physically associated. Our studies suggest that DUX4 could modulate gene expression by co-regulating the activity of hormone NRs. This is the first report highlighting a potential endocrine role for DUX4.

A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication. Overexpression of WT PMP22 in Schwann cells destabilizes the myelin sheath, leading to demyelination and ultimately to secondary axonal loss and disability. No treatments currently exist that modify the disease course. The most direct route to CMT1A therapy will involve reducing PMP22 to normal levels. To accomplish this, we developed a gene therapy strategy to reduce PMP22 using artificial miRNAs targeting human PMP22 and mouse Pmp22 mRNAs. Our lead therapeutic miRNA, miR871, was packaged into an adeno-associated virus 9 (AAV9) vector and delivered by lumbar intrathecal injection into C61-het mice, a model of CMT1A. AAV9-miR871 efficiently transduced Schwann cells in C61-het peripheral nerves and reduced human and mouse PMP22 mRNA and protein levels. Treatment at early and late stages of the disease significantly improved multiple functional outcome measures and nerve conduction velocities. Furthermore, myelin pathology in lumbar roots and femoral motor nerves was ameliorated. The treated mice also showed reductions in circulating biomarkers of CMT1A. Taken together, our data demonstrate that AAV9-miR871-driven silencing of PMP22 rescues a CMT1A model and provides proof of principle for treating CMT1A using a translatable gene therapy approach.

Meeting report: the 2021 FSHD International Research Congress.

Facioscapulohumeral muscular dystrophy (FSHD) is the second most common genetic myopathy, characterized by slowly progressing and highly heterogeneous muscle wasting with a typical onset in the late teens/early adulthood [1]. Although the etiology of the disease for both FSHD type 1 and type 2 has been attributed to gain-of-toxic function stemming from aberrant DUX4 expression, the exact pathogenic mechanisms involved in muscle wasting have yet to be elucidated [2-4]. The 2021 FSHD International Research Congress, held virtually on June 24-25, convened over 350 researchers and clinicians to share the most recent advances in the understanding of the disease mechanism, discuss the proliferation of interventional strategies and refinement of clinical outcome measures, including results from the ReDUX4 trial, a phase 2b clinical trial of losmapimod in FSHD [NCT04003974].

Improved Detection of Little Cherry Virus-2 Using a Hydrolysis Probe to Manage the Pacific Northwest Little Cherry Disease Epidemic.

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington State. This virus is insect vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions, we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington State. This information was used to develop an up-to-date reverse transcription real-time quantitative PCR assay, which was then optimized, validated, and compared with four previously published assays of a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting <10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

Human miRNA miR-675 inhibits DUX4 expression and may be exploited as a potential treatment for Facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating myopathy caused by de-repression of the DUX4 gene in skeletal muscles. Effective therapies will likely involve DUX4 inhibition. RNA interference (RNAi) is one powerful approach to inhibit DUX4, and we previously described a RNAi gene therapy to achieve DUX4 silencing in FSHD cells and mice using engineered microRNAs. Here we report a strategy to direct RNAi against DUX4 using the natural microRNA miR-675, which is derived from the lncRNA H19. Human miR-675 inhibits DUX4 expression and associated outcomes in FSHD cell models. In addition, miR-675 delivery using gene therapy protects muscles from DUX4-associated death in mice. Finally, we show that three known miR-675-upregulating small molecules inhibit DUX4 and DUX4-activated FSHD biomarkers in FSHD patient-derived myotubes. To our knowledge, this is the first study demonstrating the use of small molecules to suppress a dominant disease gene using an RNAi mechanism.

Effect of 'Candidatus Phytoplasma pruni' Infection on Sweet Cherry Fruit.

In sweet cherry (Prunus avium), infection by 'Candidatus Phytoplasma pruni' results in small fruit with poor color and taste, rendering the fruit unmarketable. Yet the disease pathology is poorly understood, particularly at the cultivar level. Therefore, in this study we examined the physiological effects of Ca. P. pruni infection across a range of cultivars and locations in eastern Washington. We found that infection could be separated into early and established stages based on pathogen titer, which correlated with disease severity, including fruit size, color, and sugar and metabolite content. Furthermore, we observed that the effects of early-stage infections were largely indistinguishable from healthy, uninfected plants. Cultivar- and location-specific disease outcomes were observed with regard to size, color, sugar content, and citric acid content. This study presents the first in-depth assessment of X-disease symptoms and biochemical content of fruit from commercially grown sweet cherry cultivars known to be infected with Ca. P. pruni.

A stromal progenitor and ILC2 niche promotes muscle eosinophilia and fibrosis-associated gene expression.

Despite the well-accepted view that chronic inflammation contributes to the pathogenesis of Duchenne muscular dystrophy (DMD), the function and regulation of eosinophils remain an unclear facet of type II innate immunity in dystrophic muscle. We report the observation that group 2 innate lymphoid cells (ILC2s) are present in skeletal muscle and are the principal regulators of muscle eosinophils during muscular dystrophy. Eosinophils were elevated in DMD patients and dystrophic mice along with interleukin (IL)-5, a major eosinophil survival factor that was predominantly expressed by muscle ILC2s. We also find that IL-33 was upregulated in dystrophic muscle and was predominantly produced by fibrogenic/adipogenic progenitors (FAPs). Exogenous IL-33 and IL-2 complex (IL-2c) expanded muscle ILC2s and eosinophils, decreased the cross-sectional area (CSA) of regenerating myofibers, and increased the expression of genes associated with muscle fibrosis. The deletion of ILC2s in dystrophic mice mitigated muscle eosinophilia and impaired the induction of IL-5 and fibrosis-associated genes. Our findings highlight a FAP/ILC2/eosinophil axis that promotes type II innate immunity, which influences the balance between regenerative and fibrotic responses during muscular dystrophy.

Is Upregulation of Sarcolipin Beneficial or Detrimental to Muscle Function?

Sarcolipin (SLN) is a regulator of sarco/endo plasmic reticulum Ca2+-ATPase (SERCA) pump and has been shown to be involved in muscle nonshivering thermogenesis (NST) and energy metabolism. Interestingly, SLN expression is significantly upregulated both during muscle development and in several disease states. However, the significance of altered SLN expression in muscle patho-physiology is not completely understood. We have previously shown that transgenic over-expression of SLN in skeletal muscle is not detrimental, and can promote oxidative metabolism and exercise capacity. In contrast, some studies have suggested that SLN upregulation in disease states is deleterious for muscle function and ablation of SLN can be beneficial. In this perspective article, we critically examine both published and some new data to determine the relevance of SLN expression to disease pathology. The new data presented in this paper show that SLN levels are induced in muscle during systemic bacterial (Salmonella) infection or lipopolysaccharides (LPS) treatment. We also present data showing that SLN expression is significantly upregulated in different types of muscular dystrophies including myotubular myopathy. These data taken together reveal that upregulation of SLN expression in muscle disease is progressive and increases with severity. Therefore, we suggest that increased SLN expression should not be viewed as the cause of the disease; rather, it is a compensatory response to meet the higher energy demand of the muscle. We interpret that higher SLN/SERCA ratio positively modulate cytosolic Ca2+ signaling pathways to promote mitochondrial biogenesis and oxidative metabolism to meet higher energy demand in muscle.

Titer and distribution of little cherry virus 2 in Prunus avium.

Little cherry virus 2 (LChV-2) is a causal agent of little cherry disease, which produces small, misshapen fruit with poor color and taste. As LChV-2 symptoms are only present near harvest, molecular detection is essential for effective control. Therefore, we determined the titer and distribution of this virus in infected trees over time. While initial infections were found to be basipetal, in field trees, early-stage infection was characterized by uneven distribution and low titer, concentrated in woody stems. In contrast, established infections were systemic, and detection was consistent across tissues. These data provide improved sampling recommendations for the detection of LChV-2.

Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes.

Facioscapulohumeral muscular dystrophy (FSHD) arises from epigenetic changes that de-repress the DUX4 gene in muscle. The full-length DUX4 protein causes cell death and muscle toxicity, and therefore we hypothesize that FSHD therapies should center on inhibiting full-length DUX4 expression. In this study, we developed a strategy to accomplish DUX4 inhibition using U7-small nuclear RNA (snRNA) antisense expression cassettes (called U7-asDUX4). These non-coding RNAs were designed to inhibit production or maturation of the full-length DUX4 pre-mRNA by masking the DUX4 start codon, splice sites, or polyadenylation signal. In so doing, U7-asDUX4 snRNAs operate similarly to antisense oligonucleotides. However, in contrast to oligonucleotides, which are limited by poor uptake in muscle and a requirement for lifelong repeated dosing, U7-asDUX4 snRNAs can be packaged within myotropic gene therapy vectors and may require only a single administration when delivered to post-mitotic cells in vivo. We tested several U7-asDUX4s that reduced DUX4 expression in vitro and improved DUX4-associated outcomes. Inhibition of DUX4 expression via U7-snRNAs could be a new prospective gene therapy approach for FSHD or be used in combination with other strategies, like RNAi therapy, to maximize DUX4 silencing in individuals with FSHD.