Delivering adeno-associated virus (AAV) vectors into the central nervous system of nonhuman primates (NHPs) via the blood or cerebral spinal fluid is associated with dorsal root ganglion (DRG) toxicity. Conventional immune-suppression regimens do not prevent this toxicity, possibly because it may be caused by high transduction rates, which can, in turn, cause cellular stress due to an overabundance of the transgene product in target cells. To test this hypothesis and develop an approach to eliminate DRG toxicity, we exploited endogenous expression of microRNA (miR) 183 complex, which is largely restricted to DRG neurons, to specifically down-regulate transgene expression in these cells. We introduced sequence targets for miR183 into the vector genome within the 3′ untranslated region of the corresponding transgene messenger RNA and injected vectors into the cisterna magna of NHPs. Administration of unmodified AAV vectors resulted in robust transduction of target tissues and toxicity in DRG neurons. Consistent with the proposal that immune system activity does not mediate this neuronal toxicity, we found that steroid administration was ineffective in alleviating this pathology. However, including miR183 targets in the vectors reduced transgene expression in, and toxicity of, DRG neurons without affecting transduction elsewhere in the primate’s brain. This approach might be useful in reducing DRG toxicity and the associated morbidity and should facilitate the development of AAV-based gene therapies for many central nervous system diseases.

Peripheral coadministration of amylin and leptin produces enhanced suppression of food intake and body weight, but the central nuclei mediating these effects remain unclear. Because each of these peptides controls feeding via actions at the ventral tegmental area (VTA), we tested the hypothesis that the VTA is a site of action for the cooperative effects of leptin and amylin on energy balance control. First, we show that intra-VTA injection of amylin and leptin at doses of each peptide that are effective in reducing food intake and body weight when administered separately produces an enhanced suppression of feeding when administered in combination. We also demonstrate that subthreshold doses of both amylin and leptin cause significant hypophagia and body weight loss when coadministered into the VTA. Additionally, we provide evidence that VTA amylin receptor blockade significantly attenuates the ability of intra-VTA leptin to reduce feeding and body weight gain. Together, these data provide the first evidence that the VTA mediates the interaction of amylin and leptin to cooperatively promote negative energy balance. obesity; mesolimbic; reward; islet amyloid polypeptide OBESITY REPRESENTS A PREVALENT, COSTLY HEALTH PROBLEM in the US and worldwide (11,43,44), yet effective noninvasive treatment options for this disease are extremely limited. Many pharmacotherapeutic strategies to date have attempted to treat obesity by targeting a single feeding-related neuroendocrine system, but the vast majority of these monotherapy approaches have failed to produce meaningful and sustained reductions in body weight (41). Therefore, attention has been turned to the notion that combination pharmacotherapies targeting more than one neuroendocrine feeding-relevant system may be more effective for the treatment of obesity (8,49,51).The pancreatic hormone amylin has been identified as a leading candidate for the development of combination pharmaceutical strategies for the treatment of obesity (47, 51). Amylin acts centrally to promote negative energy balance (24,50), and several reports have demonstrated that amylin can enhance the anorectic effects of other feeding-related signals in an additive if not synergistic manner (6,10,22,35). Of particular interest is that amylin interacts cooperatively with the adipose-derived hormone leptin to produce enhanced suppression of food intake and body weight (13,15). This amylinleptin interaction has been observed in rodents (13,42,48,56,57,59) and humans (46,48). Indeed, several studies demonstrate that peripheral administration of amylin and leptin (or agonists for their receptors) produces greater weight loss than either compound alone (48,56,59). Of critical importance for the treatment of obesity is the fact that although humans and rodent models exhibit reduced sensitivity to the energy balance effects of leptin in the obese state (1, 39), amylin appears to restore leptin sensitivity (13, 48, 56), thereby improving weight loss when amylin and leptin are coadministered.Although the body we...

Krabbe disease is a lysosomal storage disease caused by mutations in the gene that encodes galactosylceramidase, in which galactosylsphingosine (psychosine) accumulation drives demyelination in the central and peripheral nervous systems, ultimately progressing to death in early childhood. Gene therapy, alone or in combination with transplant, has been developed for almost two decades in mouse models, with increasing therapeutic benefit paralleling the improvement of next-generation adeno-associated virus (AAV) vectors. This effort has recently shown remarkable efficacy in the canine model of the disease by two different groups that used either systemic or cerebrospinal fluid (CSF) administration of AAVrh10 or AAV9. Building on our experience developing CSF-delivered, AAV-based drug products for a variety of neurodegenerative disorders, we conducted efficacy, pharmacology, and safety studies of AAVhu68 delivered to the CSF in two relevant natural Krabbe animal models, and in nonhuman primates. In newborn Twitcher mice, the highest dose (1 × 10 11 genome copies [GC]) of AAVhu68.hGALC injected into the lateral ventricle led to a median survival of 130 days compared to 40.5 days in vehicle-treated mice. When this dose was administered intravenously, the median survival was 49 days. A single intracisterna magna injection of AAVhu68.cGALC at 3 × 10 13 GC into presymptomatic Krabbe dogs increased survival for up to 85 weeks compared to 12 weeks in controls. It prevented psychosine accumulation in the CSF, preserved peripheral nerve myelination, ambulation, and decreased brain neuroinflammation and demyelination, although some regions remained abnormal. In a Good Laboratory Practice-compliant toxicology study, we administered the clinical candidate into the cisterna magna of 18 juvenile rhesus macaques at 3 doses that displayed efficacy in mice. We observed no dose-limiting toxicity and sporadic minimal degeneration of dorsal root ganglia (DRG) neurons. Our studies demonstrate the efficacy, scalability, and safety of a single cisterna magna AAVhu68 administration to treat Krabbe disease. ID: NCT04771416.

It has been proposed that cholinergic neurons of the basal forebrain (BF) may play a role in vigilance state control. Since not all vigilance states have been studied, we evaluated cholinergic neuronal activation levels across spontaneously occurring states of vigilance, as well as during sleep deprivation and recovery sleep following sleep deprivation. Sleep deprivation was performed for two hours at the beginning of the light (inactive) period, by means of gentle sensory stimulation. In the rodent BF, we used immunohistochemical detection of the c-Fos protein as a marker for activation combined with labeling for choline acetyl-transferase (ChAT) as a marker for cholinergic neurons. We found c-Fos activation in BF cholinergic neurons was highest in the group undergoing sleep deprivation (12.9% of cholinergic neurons), while the spontaneous wakefulness group showed a significant increase (9.2%), compared to labeling in the spontaneous sleep group (1.8%) and sleep deprivation recovery group (0.8%). A subpopulation of cholinergic neurons expressed c-Fos during spontaneous wakefulness, when possible confounds of the sleep deprivation procedure were minimized (e.g., stress and sensory stimulation). Double-labeling in the sleep deprivation treatment group was significantly elevated in select subnuclei of the BF (medial septum/vertical limb of the diagonal band, horizontal limb of the diagonal band, and the magnocellular preoptic nucleus), when compared to spontaneous wakefulness. These findings support and provide additional confirming data of previous reports that cholinergic neurons of BF play a role in vigilance state regulation by promoting wakefulness.