Reduced Efficacy of Anti-Aß Immunotherapy in a Mouse Model of Amyloid Deposition and Vascular Cognitive Impairment Comorbidity.

UNLABELLED: Vascular cognitive impairment and dementia (VCID) is the second most common form of dementia behind Alzheimer's disease (AD). It is estimated that 40% of AD patients also have some form of VCID. One promising therapeutic for AD is anti-Aß immunotherapy, which uses antibodies against Aß to clear it from the brain. While successful in clearing Aß and improving cognition in mice, anti-Aß immunotherapy failed to reach primary cognitive outcomes in several different clinical trials. We hypothesized that one potential reason the anti-Aß immunotherapy clinical trials were unsuccessful was due to this high percentage of VCID comorbidity in the AD population. We used our unique model of VCID-amyloid comorbidity to test this hypothesis. We placed 9-month-old wild-type and APP/PS1 mice on either a control diet or a diet that induces hyperhomocysteinemia (HHcy). After being placed on the diet for 3 months, the mice then received intraperotineal injections of either IgG2a control or 3D6 for another 3 months. While we found that treatment of our comorbidity model with 3D6 resulted in decreased total Aß levels, there was no cognitive benefit of the anti-Aß immunotherapy in our AD/VCID mice. Further, microhemorrhages were increased by 3D6 in the APP/PS1/control but further increased in an additive fashion when 3D6 was administered to the APP/PS1/HHcy mice. This suggests that the use of anti-Aß immunotherapy in patients with both AD and VCID would be ineffective on cognitive outcomes. SIGNIFICANCE STATEMENT: Despite significant mouse model data demonstrating both pathological and cognitive efficacy of anti-Aß immunotherapy for the treatment of Alzheimer's disease, clinical trial outcomes have been underwhelming, failing to meet any primary endpoints. We show here that vascular cognitive impairment and dementia (VCID) comorbidity eliminates cognitive efficacy of anti-Aß immunotherapy, despite amyloid clearance. Further, cerebrovascular adverse events of the anti-Aß immunotherapy are significantly exacerbated by the VCID comorbidity. These data suggest that VCID comorbidity with Alzheimer's disease may mute the response to anti-Aß immunotherapy.

Rescue of severely affected dystrophin/utrophin-deficient mice through scAAV-U7snRNA-mediated exon skipping.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.

Daily treatment with SMTC1100, a novel small molecule utrophin upregulator, dramatically reduces the dystrophic symptoms in the mdx mouse.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fibers leading to the gradual depletion of skeletal muscle. There is significant evidence demonstrating that increasing levels of the dystrophin-related protein, utrophin, in mouse models results in sarcolemmal bound utrophin and prevents the muscular dystrophy pathology. The aim of this work was to develop a small molecule which increases the levels of utrophin in muscle and thus has therapeutic potential. METHODOLOGY AND PRINCIPAL FINDINGS: We describe the in vivo activity of SMT C1100; the first orally bioavailable small molecule utrophin upregulator. Once-a-day daily-dosing with SMT C1100 reduces a number of the pathological effects of dystrophin deficiency. Treatment results in reduced pathology, better muscle physiology leading to an increase in overall strength, and an ability to resist fatigue after forced exercise; a surrogate for the six minute walk test currently recommended as the pivotal outcome measure in human trials for DMD. CONCLUSIONS AND SIGNIFICANCE: This study demonstrates proof-of-principle for the use of in vitro screening methods in allowing identification of pharmacological agents for utrophin transcriptional upregulation. The best compound identified, SMT C1100, demonstrated significant disease modifying effects in DMD models. Our data warrant the full evaluation of this compound in clinical trials in DMD patients.

Prevention of dystrophic pathology in severely affected dystrophin/utrophin-deficient mice by morpholino-oligomer-mediated exon-skipping.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.

Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract.

5-Hydroxytryptamine (serotonin) (5-HT) is a neurotransmitter with both central and peripheral functions, including the modulation of mood, appetite, hemodynamics, gastrointestinal (GI) sensation, secretion, and motility. Its synthesis is initiated by the enzyme tryptophan hydroxylase (TPH). Two isoforms of TPH have been discovered: TPH1, primarily expressed in the enterochromaffin cells of the gastrointestinal tract, and TPH2, expressed exclusively in neuronal cells. Mice lacking Tph1 contain little to no 5-HT in the blood and GI tract while maintaining normal levels in the brain. Because GI 5-HT is known to play important roles in normal and pathophysiology, we set out to discover and characterize novel compounds that selectively inhibit biosynthesis of GI 5-HT. Here, we describe two of a series of these inhibitors that are potent for TPH activity both in biochemical and cell-based assays. This class of compounds has unique properties with respect to pharmacokinetic and pharmacodynamic effects on GI serotonin production. Similar to the Tph1 knockout results, these TPH inhibitors have the ability to selectively reduce 5-HT levels in the murine GI tract without affecting brain 5-HT levels. In addition, administration of these compounds in a ferret model of chemotherapy-induced emesis caused modest reductions of intestinal serotonin levels and a decreased emetic response. These findings suggest that GI-specific TPH inhibitors may provide novel treatments for various gastrointestinal disorders associated with dysregulation of the GI serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.