Life-Limiting Peripheral Organ Dysfunction in Feline Sandhoff Disease Emerges after Effective CNS Gene Therapy.

OBJECTIVE: GM2 gangliosidosis is usually fatal by 5 years of age in its 2 major subtypes, Tay-Sachs and Sandhoff disease. First reported in 1881, GM2 gangliosidosis has no effective treatment today, and children succumb to the disease after a protracted neurodegenerative course and semi-vegetative state. This study seeks to further develop adeno-associated virus (AAV) gene therapy for human translation. METHODS: Cats with Sandhoff disease were treated by intracranial injection of vectors expressing feline ß-N-acetylhexosaminidase, the enzyme deficient in GM2 gangliosidosis. RESULTS: Hexosaminidase activity throughout the brain and spinal cord was above normal after treatment, with highest activities at the injection sites (thalamus and deep cerebellar nuclei). Ganglioside storage was reduced throughout the brain and spinal cord, with near complete clearance in many regions. While untreated cats with Sandhoff disease lived for 4.4 ± 0.6 months, AAV-treated cats lived to 19.1 ± 8.6 months, and 3 of 9 cats lived >21 months. Correction of the central nervous system was so effective that significant increases in lifespan led to the emergence of otherwise subclinical peripheral disease, including megacolon, enlarged stomach and urinary bladder, soft tissue spinal cord compression, and patellar luxation. Throughout the gastrointestinal tract, neurons of the myenteric and submucosal plexuses developed profound pathology, demonstrating that the enteric nervous system was inadequately treated. INTERPRETATION: The vector formulation in the current study effectively treats neuropathology in feline Sandhoff disease, but whole-body targeting will be an important consideration in next-generation approaches. ANN NEUROL 2023;94:969-986.

Intravenous delivery of adeno-associated viral gene therapy in feline GM1 gangliosidosis.

GM1 gangliosidosis is a fatal neurodegenerative disease caused by a deficiency of lysosomal ß-galactosidase. In its most severe form, GM1 gangliosidosis causes death by 4 years of age, and no effective treatments exist. Previous work has shown that injection of the brain parenchyma with an adeno-associated viral (AAV) vector provides pronounced therapeutic benefit in a feline GM1 model. To develop a less invasive treatment for the brain and increase systemic biodistribution, intravenous injection of AAV9 was evaluated. AAV9 expressing feline ß-galactosidase was intravenously administered at 1.5×1013 vector genomes/kg body weight to six GM1 cats at ∼1 month of age. The animals were divided into two cohorts: (i) a long-term group, which was followed to humane end point; and (ii) a short-term group, which was analysed 16 weeks post-treatment. Clinical assessments included neurological exams, CSF and urine biomarkers, and 7 T MRI and magentic resonance spectroscopy (MRS). Post-mortem analysis included ß-galactosidase and virus distribution, histological analysis and ganglioside content. Untreated GM1 animals survived 8.0 ± 0.6 months while intravenous treatment increased survival to an average of 3.5 years (n = 2) with substantial improvements in quality of life and neurological function. Neurological abnormalities, which in untreated animals progress to the inability to stand and debilitating neurological disease by 8 months of age, were mild in all treated animals. CSF biomarkers were normalized, indicating decreased CNS cell damage in the treated animals. Urinary glycosaminoglycans decreased to normal levels in the long-term cohort. MRI and MRS showed partial preservation of the brain in treated animals, which was supported by post-mortem histological evaluation. ß-Galactosidase activity was increased throughout the CNS, reaching carrier levels in much of the cerebrum and normal levels in the cerebellum, spinal cord and CSF. Ganglioside accumulation was significantly reduced by treatment. Peripheral tissues such as heart, skeletal muscle, and sciatic nerve also had normal ß-galactosidase activity in treated GM1 cats. GM1 histopathology was largely corrected with treatment. There was no evidence of tumorigenesis or toxicity. Restoration of ß-galactosidase activity in the CNS and peripheral organs by intravenous gene therapy led to profound increases in lifespan and quality of life in GM1 cats. These data support the promise of intravenous gene therapy as a safe, effective treatment for GM1 gangliosidosis.

Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: Modulation of glutamatergic synaptic transmission by N-methyl-D-aspartate receptors can produce rapid and sustained antidepressant effects. Rapastinel (GLYX-13), initially described as a N-methyl-D-aspartate receptor partial glycine site agonist, exhibits rapid antidepressant effect in rodents without the accompanying dissociative effects of N-methyl-D-aspartate receptor antagonists. METHODS: The relationship between rapastinel's in vitro N-methyl-D-aspartate receptor pharmacology and antidepressant efficacy was determined by brain microdialysis and subsequent pharmacological characterization of therapeutic rapastinel concentrations in N-methyl-D-aspartate receptor-specific radioligand displacement, calcium mobilization, and medial prefrontal cortex electrophysiology assays. RESULTS: Brain rapastinel concentrations of 30 to 100 nM were associated with its antidepressant-like efficacy and enhancement of N-methyl-D-aspartate receptor-dependent neuronal intracellular calcium mobilization. Modulation of N-methyl-D-aspartate receptors by rapastinel was independent of D-serine concentrations, and glycine site antagonists did not block rapastinel's effect. In rat medial prefrontal cortex slices, 100 nM rapastinel increased N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents and enhanced the magnitude of long-term potentiation without any effect on miniature EPSCs or paired-pulse facilitation responses, indicating postsynaptic action of rapastinel. A critical amino acid within the NR2 subunit was identified as necessary for rapastinel's modulatory effect. CONCLUSION: Rapastinel brain concentrations associated with antidepressant-like activity directly enhance medial prefrontal cortex N-methyl-D-aspartate receptor activity and N-methyl-D-aspartate receptor-mediated synaptic plasticity in vitro. At therapeutic concentrations, rapastinel directly enhances N-methyl-D-aspartate receptor activity through a novel site independent of the glycine coagonist site. While both rapastinel and ketamine physically target N-methyl-D-aspartate receptors, the 2 molecules have opposing actions on N-methyl-D-aspartate receptors. Modest positive modulation of N-methyl-D-aspartate receptors by rapastinel represents a novel pharmacological approach to promote well-tolerated, rapid, and sustained improvements in mood disorders.

NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-ß-Lactam That Modulates Synaptic Plasticity Processes Associated with Learning and Memory.

Background: N-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-ß-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil. Methods: The in vitro and in vivo pharmacological properties of NYX-2925 were examined. Results: NYX-2925 has a low potential for "off-target" activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100-500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01-1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour. Conclusions: NYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders.

IGFBP2 Produces Rapid-Acting and Long-Lasting Effects in Rat Models of Posttraumatic Stress Disorder via a Novel Mechanism Associated with Structural Plasticity.

Background: Posttraumatic stress disorder is an anxiety disorder characterized by deficits in the extinction of aversive memories. Insulin-like growth factor 1 (IGF1) is the only growth factor that has shown anxiolytic and antidepressant properties in human clinical trials. In animal studies, insulin-like growth factor binding protein 2 (IGFBP2) shows both IGF1-dependent and IGF1-independent pharmacological effects, and IGFBP2 expression is upregulated by rough-and-tumble play that induces resilience to stress. Methods: IGFBP2 was evaluated in Porsolt, contextual fear conditioning, and chronic unpredictable stress models of posttraumatic stress disorder. The dependence of IGFBP2 effects on IGF1- and AMPA-receptor activation was tested using selective receptor antagonists. Dendritic spine morphology was measured in the dentate gyrus and the medial prefrontal cortex 24 hours after in vivo dosing. Results: IGFBP2 was 100 times more potent than IGF1 in the Porsolt test. Unlike IGF1, effects of IGFBP2 were not blocked by the IGF1-receptor antagonist JB1, or by the AMPA-receptor antagonist 2,3-Dioxo-6-nitro-1,2,3,4 tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) in the Porsolt test. IGFBP2 (1 µg/kg) and IGF1 (100 µg/kg i.v.) each facilitated contextual fear extinction and consolidation. Using a chronic unpredictable stress paradigm, IGFBP2 reversed stress-induced effects in the Porsolt, novelty-induced hypophagia, sucrose preference, and ultrasonic vocalization assays. IGFBP2 also increased mature dendritic spine densities in the medial prefrontal cortex and hippocampus 24 hours postdosing. Conclusions: These data suggest that IGFBP2 has therapeutic-like effects in multiple rat models of posttraumatic stress disorder via a novel IGF1 receptor-independent mechanism. These data also suggest that the long-lasting effects of IGFBP2 may be due to facilitation of structural plasticity at the dendritic spine level. IGFBP2 and mimetics may have therapeutic potential for the treatment of posttraumatic stress disorder.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

Whole-Genome Shotgun Metagenomic Sequencing Reveals Distinct Gut Microbiome Signatures of Obese Cats.

Overweight and obesity are growing health problems in domestic cats, increasing the risks of insulin resistance, lipid dyscrasias, neoplasia, cardiovascular disease, and decreasing longevity. The signature of obesity in the feline gut microbiota has not been studied at the whole-genome metagenomic level. We performed whole-genome shotgun metagenomic sequencing in the fecal samples of eight overweight/obese and eight normal cats housed in the same research environment. We obtained 271 Gbp of sequences and generated a 961-Mbp de novo reference contig assembly, with 1.14 million annotated microbial genes. In the obese cat microbiome, we discovered a significant reduction in microbial diversity (P < 0.01) and Firmicutes abundance (P = 0.005), as well as decreased Firmicutes/Bacteroidetes ratios (P = 0.02), which is the inverse of obese human/mouse microbiota. Linear discriminant analysis and quantitative PCR (qPCR) validation revealed significant increases of Bifidobacterium sp., Olsenella provencensis, Dialister sp.CAG:486, and Campylobacter upsaliensis as the hallmark of obese microbiota among 400 enriched species, whereas 1,525 bacterial species have decreased abundance in the obese microbiome. Phascolarctobacterium succinatutens and an uncharacterized Erysipelotrichaceae bacterium are highly abundant (>0.05%) in the normal gut with over 400-fold depletion in the obese microbiome. Fatty acid synthesis-related pathways are significantly overrepresented in the obese compared with the normal cat microbiome. In conclusion, we discovered dramatically decreased microbial diversity in obese cat gut microbiota, suggesting potential dysbiosis. A panel of seven significantly altered, highly abundant species can serve as a microbiome indicator of obesity. Our findings in the obese cat microbiome composition, abundance, and functional capacities provide new insights into feline obesity. IMPORTANCE Obesity affects around 45% of domestic cats, and licensed drugs for treating feline obesity are lacking. Physical exercise and calorie restrictions are commonly used for weight loss but with limited efficacy. Through comprehensive analyses of normal and obese cat gut bacteria flora, we identified dramatic shifts in the obese gut microbiome, including four bacterial species significantly enriched and two species depleted in the obese cats. The key bacterial community and functional capacity alterations discovered from this study will inform new weight management strategies for obese cats, such as evaluations of specific diet formulas that alter the microbiome composition, and the development of prebiotics and probiotics that promote the increase of beneficial species and the depletion of obesity-associated species. Interestingly, these bacteria identified in our study were also reported to affect the weight loss success in human patients, suggesting translational potential in human obesity.

AAVrh10 vector corrects pathology in animal models of GM1 gangliosidosis and achieves widespread distribution in the CNS of nonhuman primates.

GM1 gangliosidosis is a rare, inherited neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes the lysosomal hydrolase acid ß-galactosidase (ß-gal). ß-gal deficiency leads to toxic accumulation of GM1 ganglioside, predominantly in the central nervous system (CNS), resulting in progressive neurodegeneration. LYS-GM101 is an AAVrh.10-based gene therapy vector carrying the human GLB1 cDNA. The efficacy of intra-cerebrospinal fluid injection of LYS-GM101 analogs was demonstrated in GM1 mouse and cat models with widespread diffusion of ß-gal and correction of GM1 ganglioside accumulation in the CNS without observable adverse effects. Clinical dose selection was performed, based on a good-laboratory-practice study, in nonhuman primates (NHPs) using the clinical LYS-GM101 vector. A broadly distributed increase of ß-gal activity was observed in NHP brain 3 months after intra-cisterna magna injection of LYS-GM101 at 1.0 × 1012 vg/mL CSF and 4.0 × 1012 vg/mL CSF, with 20% and 60% increases compared with vehicle-treated animals, respectively. Histopathologic examination revealed asymptomatic adverse changes in the sensory pathways of the spinal cord and dorsal root ganglia in both sexes and at both doses. Taken as a whole, these pre-clinical data support the initiation of a clinical study with LYS-GM101 for the treatment of GM1 gangliosidosis.

Rabies: who should care?

Rabies is the deadliest viral infection known, with no reliable treatment, and although it is entirely preventable, rabies continues to kill more than 60,000 people every year, mostly children in countries where dog rabies is endemic. America is only 1 generation away from the time when rabies killed more than 10,000 animals and 50 Americans every year, but 3 to 5 Americans continue to die annually from rabies. Distressingly, > 50,000 Americans undergo rabies prevention therapy every year after exposure to potentially rabid animals. While enormous progress has been made, more must be done to defeat this ancient but persistent, fatal zoonosis. In the US, lack of public awareness and ambivalence are the greatest dangers imposed by rabies, resulting in unnecessary exposures, anxiety, and risk. Veterinarians have a special role in informing and reassuring the public about prevention and protection from rabies. This summary of current facts and future advances about rabies will assist veterinarians in informing their clients about the disease.

7T MRI Predicts Amelioration of Neurodegeneration in the Brain after AAV Gene Therapy.

GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline ß-galactosidase (ß-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where ß-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and ß-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.

Modulation of CA3 afferent inputs by novelty and theta rhythm.

Models of hippocampal function suggest that the modulation of CA3 afferent input during theta rhythm allows for a rapid alternation between encoding and retrieval states, with each phase enhancing either extrinsic or intrinsic CA3 afferents, favoring either encoding or retrieval, respectively. Here, we show that during the initial exploration of a novel environment by rats, intrinsic CA3-CA3 synaptic inputs are attenuated on CA3 theta peaks, favoring extrinsic CA3 inputs, whereas extrinsic perforant path-CA3 synaptic inputs are attenuated on CA3 theta troughs, favoring intrinsic CA3 inputs. This modulation is absent when animals are re-exposed to the same environment 2 or 48 h later and thus habituates with familiarity, suggesting a process involved in learning. Modulation of CA3 synaptic inputs during novelty was blocked by atropine at a dose that blocks type 2 theta rhythm. Re-exposure to the same novel environment 48 h later in the absence of atropine did not result in habituation, but instead modulated CA3 synaptic responses as though the environment were novel and explored for the first time. The NMDA receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), administered in a dose that blocks long-term potentiation induction, did not alter CA3 synaptic modulation during initial exploration. However, like atropine, CPP blocked the habituation of synaptic modulation normally observed with re-exposure, as though the environment were novel and explored for the first time. Thus, as predicted theoretically, recurrent and cortical CA3 afferents are differentially modulated during phases of theta rhythm. This modulation is atropine sensitive and habituates in an NMDA receptor-dependent manner, suggesting an NMDA receptor-dependent process that, in conjunction with theta rhythm, contributes to encoding of novel information in the hippocampus.

Polyethylene glycol-b-poly(lactic acid) polymersomes as vehicles for enzyme replacement therapy.

AIM: Polymersomes are created to deliver an enzyme-based therapy to the brain in lysosomal storage disease patients. MATERIALS & METHODS: Polymersomes are formed via the injection method using poly(ethylene glycol)-b-poly(lactic acid) (PEGPLA) and bound to apolipoprotein E, to create a brain-targeted delivery vehicle. RESULTS: Polymersomes have a smallest average diameter of 145 ± 21 nm and encapsulate ß-galactosidase at 72.0 ± 12.2% efficiency. PEGPLA polymersomes demonstrate limited release at physiologic pH (7.4), with a burst release at the acidic pH (4.8) of the lysosome. PEGPLA polymersomes facilitate delivery of active ß-galactosidase to an in vitro model of GM1 gangliosidosis. CONCLUSION: The foundation has been laid for testing of PEGPLA polymersomes to deliver enzymatic treatments to the brain in lysosomal storage disorders for the first time.

AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease.

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme ß-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

Promiscuous tumor targeting phage proteins.

Cancer cell-specific targeting ligands against numerous cancer cell lines have been selected previously and used as ligands for cell-specific delivery of chemotherapies and various nanomedicines. However, tumor heterogeneity is one recognized problem hampering clinical translation of targeted anti-cancer medicines. Therefore, a novel class of targeting ligands is required that recognize receptors expressed between a variety of cancer phenotypes, identified here as 'promiscuous' ligands. In this work, promiscuous phage fusion proteins were first identified by a novel selection scheme to enrich for pan-cancer cell binding abilities, as indicated by conserved structural motifs identified previously in other cancer types. Additionally, peptide sequences containing a combination of motifs were identified to modulate binding. A panel of phage fusion proteins was studied for their specificity and selectivity for lung and pancreatic cancer cells. Phage displaying the fusion peptides GSLEEVSTL or GEFDELMTM, the two predominate clones with greatest binding ability, were used to modify preformed, doxorubicin-loaded, liposomes. These modified liposomes increased cytotoxicity up to 8.1-fold in several cancer cell lines when compared with unmodified liposomal doxorubicin. Taken together, these data indicate that promiscuous phage proteins, selected against different cancer cell lines, can be used as targeting ligands for treatment of heterogeneous tumor populations.

Combinatorial synthesis and screening of cancer cell-specific nanomedicines targeted via phage fusion proteins.

Active tumor targeting of nanomedicines has recently shown significant improvements in the therapeutic activity of currently existing drug delivery systems, such as liposomal doxorubicin (Doxil/Caelyx/Lipodox). Previously, we have shown that isolated pVIII major coat proteins of the fd-tet filamentous phage vector, containing cancer cell-specific peptide fusions at their N-terminus, can be used as active targeting ligands in a liposomal doxorubicin delivery system in vitro and in vivo. Here, we show a novel major coat protein isolation procedure in 2-propanol that allows spontaneous incorporation of the hydrophobic protein core into preformed liposomal doxorubicin with minimal damage or drug loss while still retaining the targeting ligand exposed for cell-specific targeting. Using a panel of 12 structurally unique ligands with specificity toward breast, lung, and/or pancreatic cancer, we showed the feasibility of pVIII major coat proteins to significantly increase the throughput of targeting ligand screening in a common nanomedicine core. Phage protein-modified Lipodox samples showed an average doxorubicin recovery of 82.8% across all samples with 100% of protein incorporation in the correct orientation (N-terminus exposed). Following cytotoxicity screening in a doxorubicin-sensitive breast cancer line (MCF-7), three major groups of ligands were identified. Ligands showing the most improved cytotoxicity included: DMPGTVLP, ANGRPSMT, VNGRAEAP, and ANDVYLD showing a 25-fold improvement (p < 0.05) in toxicity. Similarly DGQYLGSQ, ETYNQPYL, and GSSEQLYL ligands with specificity toward a doxorubicin-insensitive pancreatic cancer line (PANC-1) showed significant increases in toxicity (2-fold; p < 0.05). Thus, we demonstrated proof-of-concept that pVIII major coat proteins can be screened in significantly higher throughput to identify novel ligands displaying improved therapeutic activity in a desired cancer phenotype.

Rapastinel (GLYX-13) has therapeutic potential for the treatment of post-traumatic stress disorder: Characterization of a NMDA receptor-mediated metaplasticity process in the medial prefrontal cortex of rats.

Rapastinel (GLYX-13) is a NMDA receptor modulator with glycine-site partial agonist properties. It is a robust cognitive enhancer and shows rapid and long-lasting antidepressant properties in both animal models and in humans. Contextual fear extinction (CFE) in rodents has been well characterized and used extensively as a model to study the neurobiological mechanisms of post-traumatic stress disorder (PTSD). Since CFE is NMDA receptor modulated and neural circuitry in the medial prefrontal cortex (MPFC) regulates both depression and PTSD, studies were undertaken to examine the effects of rapastinel for its therapeutic potential in PTSD and to use rapastinel as a tool to study its underlying glutamatergic mechanisms. A 21-day chronic mild unpredictable stress (CUS) rat model was used to model depression and PTSD. The effects of CUS alone compared to No CUS controls, and the effects of rapastinel (3 mg/kg IV) on CUS-treated animals were examined. The effect of rapastinel was first assessed using CUS-treated rats in three depression models, Porsolt, sucrose preference, and novelty-induced hypophagia tests, and found to produce a complete reversal of the depressive-like state in each model. Rapastinel was then assessed in a MPFC-dependent positive emotional learning paradigm and in CFE and again a reversal of the impairments induced by CUS treatment was observed. Both synaptic plasticity and metaplasticity, as measured by the induction of long-term potentiation in rat MPFC slice preparations, was found to be markedly impaired in CUS-treated animals. This impairment was reversed when CUS-treated rats were administered rapastinel and tested 24 h later. Transcriptomic analysis of MPFC mRNA expression in CUS-treated rats corroborated the link between rapastinel's behavioral effects and synaptic plasticity. A marked enrichment in both the LTP and LTD connectomes in rapastinel-treated CUS rats was observed compared to CUS-treated controls. The effects of rapastinel on depression models, PEL, and most importantly on CFE demonstrate the therapeutic potential of rapastinel for the treatment of PTSD. Moreover, rapastinel appears to elicit its therapeutic effects through a NMDA receptor-mediated, LTP-like, metaplasticity process in the MPFC.

GLYX-13, a NMDA receptor glycine-site functional partial agonist, induces antidepressant-like effects without ketamine-like side effects.

Recent human clinical studies with the NMDA receptor (NMDAR) antagonist ketamine have revealed profound and long-lasting antidepressant effects with rapid onset in several clinical trials, but antidepressant effects were preceded by dissociative side effects. Here we show that GLYX-13, a novel NMDAR glycine-site functional partial agonist, produces an antidepressant-like effect in the Porsolt, novelty induced hypophagia, and learned helplessness tests in rats without exhibiting substance abuse-related, gating, and sedative side effects of ketamine in the drug discrimination, conditioned place preference, pre-pulse inhibition and open-field tests. Like ketamine, the GLYX-13-induced antidepressant-like effects required AMPA/kainate receptor activation, as evidenced by the ability of NBQX to abolish the antidepressant-like effect. Both GLYX-13 and ketamine persistently (24 h) enhanced the induction of long-term potentiation of synaptic transmission and the magnitude of NMDAR-NR2B conductance at rat Schaffer collateral-CA1 synapses in vitro. Cell surface biotinylation studies showed that both GLYX-13 and ketamine led to increases in both NR2B and GluR1 protein levels, as measured by Western analysis, whereas no changes were seen in mRNA expression (microarray and qRT-PCR). GLYX-13, unlike ketamine, produced its antidepressant-like effect when injected directly into the medial prefrontal cortex (MPFC). These results suggest that GLYX-13 produces an antidepressant-like effect without the side effects seen with ketamine at least in part by directly modulating NR2B-containing NMDARs in the MPFC. Furthermore, the enhancement of 'metaplasticity' by both GLYX-13 and ketamine may help explain the long-lasting antidepressant effects of these NMDAR modulators. GLYX-13 is currently in a Phase II clinical development program for treatment-resistant depression.