Systemic PPMO-mediated dystrophin expression in the Dup2 mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease that arises due to the loss of dystrophin expression, leading to progressive loss of motor and cardiorespiratory function. Four exon-skipping approaches using antisense phosphorodiamidate morpholino oligomers (PMOs) have been approved by the FDA to restore a open reading frame, resulting in expression of a functional but internally deleted dystrophin protein, but in patients with single-exon duplications, exon skipping has the potential to restore full-length dystrophin expression. Cell-penetrating peptide-conjugated PMOs (PPMOs) have demonstrated enhanced cellular uptake and more efficient dystrophin restoration than unconjugated PMOs.

Splice modulating antisense oligonucleotides restore some acid-alpha-glucosidase activity in cells derived from patients with late-onset Pompe disease.

Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-α-glucosidase activity in patients, and a progressive decline in mobility and respiratory function. Enzyme replacement therapy is one therapeutic option, but since not all patients respond to this treatment, alternative interventions should be considered. One GAA mutation, c.

Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy.

Objective: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping.

Methods: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28.

Functional muscle recovery following dystrophin and myostatin exon splice modulation in aged mdx mice.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 3500-5000 newborn boys. It is due to mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibres and their replacement with fibro-adipogenic tissue. Out-of-frame mutations in the DMD gene can be modified by using antisense oligonucleotides (AONs) to promote skipping of specific exons such that the reading frame is restored and the resulting protein produced, though truncated, is functional.

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value.

Eteplirsen treatment for Duchenne muscular dystrophy: Exon skipping and dystrophin production.

Objective: To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen.

Methods: Clinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg.

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule.

Alternative splice forms of CTLA-4 induced by antisense mediated splice-switching influences autoimmune diabetes susceptibility in NOD mice.

Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology.

Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo.

Background: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogues that silence expression of specific genes. We studied whether PPMOs targeted to essential genes in Acinetobacter lwoffii and Acinetobacter baumannii are active in vitro and in vivo.

Methods: PPMOs were evaluated in vitro using minimum inhibitory concentration (MIC) and viability assays, and in vivo using murine pulmonary infection models with intranasal PPMO treatment.

Development of novel bioanalytical methods to determine the effective concentrations of phosphorodiamidate morpholino oligomers in tissues and cells.

Phosphorodiamidate morpholino oligomers (PMO) are neutrally charged, sequence-specific antisense agents that interfere with targeted gene expression. PMO have been shown to be highly specific and potent therapies after cellular uptake, yet methods to detect PMO in tissue and inside the cell are limited. We offer in this report novel methods for the detection of cellular resident PMO using flow cytometry-fluorescence in situ hybridization (flow FISH) and a sandwich hybridization technique to quickly and sensitively quantify tissue resident PMO.

Lymphocytic choriomeningitis virus infection in FVB mouse produces hemorrhagic disease.

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever in humans. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. Preclinical testing for development of antiviral or therapeutics is in part hampered due to a lack of an immunologically well-defined rodent model that exhibits similar acute hemorrhagic illness or sequelae compared to the human disease.

CD8+ T cell responses to a viral escape mutant epitope: active suppression via altered SHP-1 activity.

One mechanism viruses use to subvert immune surveillance is through mutation of MHC contact residues of antigenic epitopes that weaken T cell recognition to the point that the immune system is ignorant of the infection. However, in contrast to ignorance, results presented herein demonstrate that intracellular signaling does occur upon stimulation with a lymphocytic choriomeningitis virus-derived escape mutant as demonstrated by the sustained activation of Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1). In addition to the increased SHP-1 activity, we found that the mutated epitope failed to induce oxidation of SHP-1, further enhancing enzymatic activity.

Murine pregnancy leads to reduced proliferation of maternal thymocytes and decreased thymic emigration.

During mammalian pregnancy the maternal thymus undergoes significant involution, and then recovers in size after birth. The mechanism behind this involution is not known, but it has been suggested that elevated levels of hormones during pregnancy induce the involution. We have recently shown that injection of 17beta-oestradiol into mice causes loss of early thymocyte precursors and inhibits proliferation of developing thymocytes.

Early growth response gene 1 provides negative feedback to inhibit entry of progenitor cells into the thymus.

The size of the thymus can be greatly influenced by changes in the small number of early progenitors in the thymus. However, it is not known whether thymic cellularity feeds back to regulate the recruitment, survival, and expansion of progenitors. The transcription factor early growth response gene 1 (Egr1) has been implicated in controlling proliferation and survival in many cell types.

An IL-7-dependent rebound in thymic T cell output contributes to the bone loss induced by estrogen deficiency.

The bone wasting induced by estrogen deficiency is, in part, a consequence of increased T cell production of the osteoclastogenic cytokine TNF-alpha. This phenomenon is due to an expansion of T cells, but the responsible mechanism is unknown. We now show that ovariectomy (ovx) disregulates T lymphopoiesis and induces bone loss by stimulating, through a rise in IL-7 levels, both thymic-dependent differentiation of bone marrow-derived progenitors and thymic-independent, peripheral expansion of mature T cells.

Control of recent thymic emigrant survival by positive selection signals and early growth response gene 1.

Early growth response gene 1 (Egr1) is a transcriptional regulator whose expression can be induced by multiple signals including the TCR. Egr1 has been shown to promote positive selection, but an investigation of its role in T cell homeostasis has not been reported. The possibility that similar signals control both positive selection and peripheral T cell homeostasis led us to investigate the role of Egr1 in the maintenance of peripheral T cells.

Prostate specific antigen bounce after simultaneous irradiation for prostate cancer: the relationship to patient age.

Purpose: We report on the relationship of patient age to prostate specific antigen (PSA) bounce after brachytherapy for prostate cancer.

Materials And Methods: From 1992 to 1997, 1,011 consecutive men with stage T1-T2NxM0 prostate cancer were treated with simultaneous irradiation, transperineal prostate I-125 implant followed by external beam irradiation. No patients received neoadjuvant hormones.

Signal regulatory protein (SIRPalpha), a cellular ligand for CD47, regulates neutrophil transmigration.

Recent studies have demonstrated that CD47 plays an important role in regulating human neutrophil (PMN) chemotaxis. Two ligands for CD47, thrombospondin and SIRPalpha, have been described. However, it is not known if SIRP-CD47 interactions play a role in regulating PMN migration.