RNA-seq in DMD urinary stem cells recognized muscle-related transcription signatures and addressed the identification of atypical mutations by whole-genome sequencing.

Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals' USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs' myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.

The Genetic Landscape of Dystrophin Mutations in Italy: A Nationwide Study.

Dystrophinopathies are inherited diseases caused by mutations in the dystrophin (DMD) gene for which testing is mandatory for genetic diagnosis, reproductive choices and eligibility for personalized trials. We genotyped the DMD gene in our Italian cohort of 1902 patients (BMD n = 740, 39%; DMD n =1162, 61%) within a nationwide study involving 11 diagnostic centers in a 10-year window (2008-2017). In DMD patients, we found deletions in 57%, duplications in 11% and small mutations in 32%. In BMD, we found deletions in 78%, duplications in 9% and small mutations in 13%. In BMD, there are a higher number of deletions, and small mutations are more frequent than duplications. Among small mutations that are generally frequent in both phenotypes, 44% of DMD and 36% of BMD are nonsense, thus, eligible for stop codon read-through therapy; 63% of all out-of-frame deletions are eligible for single exon skipping. Patients were also assigned to Italian regions and showed interesting regional differences in mutation distribution. The full genetic characterization in this large, nationwide cohort has allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, mutation locations along the gene, exon/intron architecture, and relevant protein domain, with effects on population genetic characteristics and new personalized therapies.

Increased excitability in tat-transgenic mice: role of tat in HIV-related neurological disorders.

HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND.

Biodistribution and molecular studies on orally administered nanoparticle-AON complexes encapsulated with alginate aiming at inducing dystrophin rescue in mdx mice.

We have previously demonstrated that intraperitoneal injections of 2'-O-methyl-phosphorothioate (2'OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles of mdx mice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were tracked in vivo as well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, in mdx mice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.

The DMD locus harbours multiple long non-coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms.

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.

Persistent dystrophin protein restoration 90 days after a course of intraperitoneally administered naked 2'OMePS AON and ZM2 NP-AON complexes in mdx mice.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2'-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.

Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches.

Identification of a deep intronic mutation in the COL6A2 gene by a novel custom oligonucleotide CGH array designed to explore allelic and genetic heterogeneity in collagen VI-related myopathies.

BACKGROUND: Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. METHODS: We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. RESULTS: A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. CONCLUSIONS: Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes.

Exon skipping-mediated dystrophin reading frame restoration for small mutations.

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2'OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations.

Identification and characterization of novel collagen VI non-canonical splicing mutations causing Ullrich congenital muscular dystrophy.

Splicing mutations occurring outside the invariant GT and AG dinucleotides are frequent in disease genes and the definition of their pathogenic potential is often challenging. We have identified four patients affected by Ullrich congenital muscular dystrophy and carrying unusual mutations of COL6 genes affecting RNA splicing. In three cases the mutations occurred in the COL6A2 gene and consisted of nucleotide substitutions within the degenerated sequences flanking the canonical dinucleotides. In the fourth case, a genomic deletion occurred which removed the exon8-intron8 junction of the COL6A1 gene. These mutations induced variable splicing phenotypes, consisting of exon skipping, intron retention and cryptic splice site activation/usage. A quantitative RNA assay revealed a reduced level of transcription of the mutated in-frame mRNA originating from a COL6A2 point mutation at intronic position +3. At variance, the transcription level of the mutated in-frame mRNA originating from a genomic deletion which removed the splicing sequences of COL6A1 exon 8 was normal. These findings suggest a different transcriptional efficiency of a regulatory splicing mutation compared to a genomic deletion causing a splicing defect.

Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies.

BACKGROUND: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene. RESULTS: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis. CONCLUSION: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.

HIV-1 tat protein modulates the generation of cytotoxic T cell epitopes by modifying proteasome composition and enzymatic activity.

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.

Inhibition of HIV-1 Tat activity correlates with down-regulation of bcl-2 and results in reduction of angiogenesis and oncogenicity.

The Tat protein of the human immunodeficiency virus type 1 promotes survival and growth and inhibits apoptosis of different cell types. These effects of Tat are attributed to the induction of bcl-2 gene expression. In this study we show that the blocking of both intracellular and extracellular Tat correlates with a decrease of bcl-2 transcripts, leading in vitro to a lower growth rate and attenuation of the transformed phenotype and in vivo to a reduced angiogenic and oncogenic activity of Tat-expressing cells. These results support the notion that bcl-2 is an effector of Tat-induced angiogenesis and oncogenesis and indicate that the blocking of Tat functions by immunoprophylactic, pharmacological, and gene therapy approaches may help to control oncogenesis during AIDS.