Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, ...while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.
The mammalian urea cycle (UC) is responsible for siphoning catabolic waste nitrogen into urea for excretion. Disruptions of the functions of any of the enzymes or transporters lead to elevated ...ammonia and neurological injury. Carbamoyl phosphate synthetase 1 (CPS1) is the first and rate-limiting UC enzyme responsible for the direct incorporation of ammonia into UC intermediates. Symptoms in CPS1 deficiency are typically the most severe of all UC disorders, and current clinical management is insufficient to prevent the associated morbidities and high mortality. With recent advances in basic and translational studies of CPS1, appreciation for this enzyme's essential role in the UC has been broadened to include systemic metabolic regulation during homeostasis and disease. Here, we review recent advances in CPS1 biology and contextualize them around the role of CPS1 in health and disease.
Impairment of excretion and enzymatic processing of nitrogen, for example, because of liver or kidney failure, or with urea cycle and creatine synthesis enzyme defects, surprisingly leads to ...primarily neurologic symptoms, yet the exact mechanisms remain largely mysterious. In guanidinoacetate N-methyltransferase (GAMT) deficiency, the guanidino compound guanidinoacetate (GAA) increases dramatically, including in the cerebrospinal fluid (CSF), and has been implicated in mediating the neurological symptoms in GAMT-deficient patients. GAA is synthesized by arginine-glycine amidinotransferase (AGAT), a promiscuous enzyme that not only transfers the amidino group from arginine to glycine, but also to primary amines in, for example, GABA and taurine to generate γ-guanidinobutyric acid (γ-GBA) and guanidinoethanesulfonic acid (GES), respectively. We show that GAA, γ-GBA, and GES share structural similarities with GABA, evoke GABA
receptor (GABA
R) mediated currents (whereas creatine methylated GAA and arginine failed to evoke discernible currents) in cerebellar granule cells in mouse brain slices and displace the high-affinity GABA-site radioligand
Hmuscimol in total brain homogenate GABA
Rs. While γ-GBA and GES are GABA agonists and displace
Hmuscimol (EC
/IC
between 10 and 40 μM), GAA stands out as particularly potent in both activating GABA
Rs (EC
~6 μM) and also displacing the GABA
R ligand
Hmuscimol (IC
~3 μM) at pathophysiologically relevant concentrations. These findings stress the role of substantially elevated GAA as a primary neurotoxic agent in GAMT deficiency and we discuss the potential role of GAA in arginase (and creatine transporter) deficiency which show a much more modest increase in GAA concentrations yet share the unique hyperexcitability neuropathology with GAMT deficiency. We conclude that orthosteric activation of GABA
Rs by GAA, and potentially other GABA
R mimetic guanidino compounds (GCs) like γ-GBA and GES, interferes with normal inhibitory GABAergic neurotransmission which could mediate, and contribute to, neurotoxicity.
Adeno-associated viruses (AAVs) are being increasingly used as gene therapy vectors in clinical studies especially targeting central nervous system (CNS) disorders. Correspondingly, host immune ...responses to the AAV capsid or the transgene-encoded protein have been observed in various clinical and preclinical studies. Such immune responses may adversely impact patients' health, prevent viral transduction, prevent repeated dosing strategies, eliminate transduced cells, and pose a significant barrier to the potential effectiveness of AAV gene therapy. Consequently, multiple immunomodulatory strategies have been used in attempts to limit immune-mediated responses to the vector, enable readministration of AAV gene therapy, prevent end-organ toxicity, and increase the duration of transgene-encoded protein expression. Herein we review the innate and adaptive immune responses that may occur during CNS-targeted AAV gene therapy as well as host- and treatment-specific factors that could impact the immune response. We also summarize the available preclinical and clinical data on immune responses specifically to CNS-targeted AAV gene therapy and discuss potential strategies for incorporating prophylactic immunosuppression regimens to circumvent adverse immune responses.
Dementia and autoimmune diseases are prevalent conditions with limited treatment options. Taurine and homotaurine (HT) are naturally occurring sulfonate amino acids, with taurine being highly ...abundant in animal tissues, but declining with age in the blood. HT is a blood-brain barrier permeable drug under investigation for Alzheimer’s disease. HT also has beneficial effects in a mouse model of multiple sclerosis likely through an anti-inflammatory mechanism mediated by GABA
A
receptor (GABA
A
R) agonism in immune cells. While both taurine and HT are structural GABA analogs and thought to be GABA mimetics at GABA
A
Rs, there is uncertainty concerning their potency as GABA mimetics on native GABA
A
Rs. We show that HT is a very potent GABA mimetic, as it evokes GABA
A
R-mediated currents with an EC
50
of 0.4 μM (vs. 3.7 μM for GABA and 116 µM for taurine) in murine cerebellar granule cells in brain slices, with both taurine and HT having similar efficacy in activating native GABA
A
Rs. Furthermore, HT displaces the high affinity GABA
A
R ligand
3
Hmuscimol at similarly low concentrations (HT IC
50
of 0.16 μM vs. 125 μM for taurine) in mouse brain homogenates. The potency of taurine and HT as GABA
A
R agonists aligns with endogenous concentrations of taurine in the blood and with HT concentrations achieved in the brain following oral administration of HT or the HT pro-drug ALZ-801. Consequently, we discuss that GABA
A
Rs subtypes, similar to the ones we studied here in neurons, are plausible targets for mediating the potential beneficial effects of taurine in health and life-span extension and the beneficial HT effects in dementia and autoimmune conditions.
Propionic acidaemia is a rare disorder caused by defects in the propionyl-coenzyme A carboxylase α or β (PCCA or PCCB) subunits that leads to an accumulation of toxic metabolites and to recurrent, ...life-threatening metabolic decompensation events. Here we report interim analyses of a first-in-human, phase 1/2, open-label, dose-optimization study and an extension study evaluating the safety and efficacy of mRNA-3927, a dual mRNA therapy encoding PCCA and PCCB. As of 31 May 2023, 16 participants were enrolled across 5 dose cohorts. Twelve of the 16 participants completed the dose-optimization study and enrolled in the extension study. A total of 346 intravenous doses of mRNA-3927 were administered over a total of 15.69 person-years of treatment. No dose-limiting toxicities occurred. Treatment-emergent adverse events were reported in 15 out of the 16 (93.8%) participants. Preliminary analysis suggests an increase in the exposure to mRNA-3927 with dose escalation, and a 70% reduction in the risk of metabolic decompensation events among 8 participants who reported them in the 12-month pretreatment period.
Impairment of excretion and enzymatic processing of nitrogen, for example, because of liver or kidney failure, or with urea cycle and creatine synthesis enzyme defects, surprisingly leads to ...primarily neurologic symptoms, yet the exact mechanisms remain largely mysterious. In guanidinoacetate N‐methyltransferase (GAMT) deficiency, the guanidino compound guanidinoacetate (GAA) increases dramatically, including in the cerebrospinal fluid (CSF), and has been implicated in mediating the neurological symptoms in GAMT‐deficient patients. GAA is synthesized by arginine–glycine amidinotransferase (AGAT), a promiscuous enzyme that not only transfers the amidino group from arginine to glycine, but also to primary amines in, for example, GABA and taurine to generate γ‐guanidinobutyric acid (γ‐GBA) and guanidinoethanesulfonic acid (GES), respectively. We show that GAA, γ‐GBA, and GES share structural similarities with GABA, evoke GABAA receptor (GABAAR) mediated currents (whereas creatine methylated GAA and arginine failed to evoke discernible currents) in cerebellar granule cells in mouse brain slices and displace the high‐affinity GABA‐site radioligand 3Hmuscimol in total brain homogenate GABAARs. While γ‐GBA and GES are GABA agonists and displace 3Hmuscimol (EC50/IC50 between 10 and 40 μM), GAA stands out as particularly potent in both activating GABAARs (EC50 ~6 μM) and also displacing the GABAAR ligand 3Hmuscimol (IC50 ~3 μM) at pathophysiologically relevant concentrations. These findings stress the role of substantially elevated GAA as a primary neurotoxic agent in GAMT deficiency and we discuss the potential role of GAA in arginase (and creatine transporter) deficiency which show a much more modest increase in GAA concentrations yet share the unique hyperexcitability neuropathology with GAMT deficiency. We conclude that orthosteric activation of GABAARs by GAA, and potentially other GABAAR mimetic guanidino compounds (GCs) like γ‐GBA and GES, interferes with normal inhibitory GABAergic neurotransmission which could mediate, and contribute to, neurotoxicity.
Neurological impairment is the primary manifestation of enzyme defects in urea cycle and creatine biosynthesis pathways, as well as in hepatic and uremic encephalopathy (HE and UE). In HE and UE, generally because of liver and kidney failure, respectively, toxins like ammonium, glutamine, arginine, urea, and guanidino compounds (GCs) accumulate and lead to a poorly understood neuropathology. We show that guanidinoacetate (GAA) and two other guanidino compounds (γ‐GBA and GES) are structural GABA analogs and high‐affinity GABA mimetics. Guanidinoacetate acts at pathophysiologically relevant concentrations and interference with GABAergic neurotransmission is a likely molecular mechanism for causing neuropathology.
Chronic inflammation drives synaptic loss in multiple sclerosis (MS) and is also commonly observed in other neurodegenerative diseases. Clinically approved treatments for MS provide symptomatic ...relief but fail to halt neurodegeneration and neurological decline. Studies in animal disease models have demonstrated that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) exhibits anti-inflammatory, neuroprotective and regenerative properties. Anti-inflammatory actions appear to be mediated primarily by two receptors, VPAC1 and VPAC2, which also bind vasoactive intestinal peptide (VIP). Pharmacological experiments indicate that another receptor, PAC1 (ADCYAP1R1), which is highly selective for PACAP, provides protection to neurons, although genetic evidence and other mechanistic information is lacking. To determine if PAC1 receptors protect neurons in a cell-autonomous manner, we used adeno-associated virus (AAV2) to deliver Cre recombinase to the retina of mice harboring floxed PAC1 alleles. Mice were then subjected to chronic experimental autoimmune encephalomyelitis (EAE), a disease model that recapitulates major clinical and pathological features of MS and associated optic neuritis. Unexpectedly, deletion of PAC1 in naïve mice resulted in a deficit of retinal ganglionic neurons (RGNs) and their dendrites, suggesting a homeostatic role of PAC1. Moreover, deletion of PAC1 resulted in increased EAE-induced loss of a subpopulation of RGNs purported to be vulnerable in animal models of glaucoma. Increased axonal pathology and increased secondary presence of microglia/macrophages was also prominently seen in the optic nerve. These findings demonstrate that neuronal PAC1 receptors play a homeostatic role in protecting RGNs and directly protects neurons and their axons against neuroinflammatory challenge.
Chronic inflammation is a major component of neurodegenerative diseases and plays a central role in multiple sclerosis (MS). Current treatments for MS do not prevent neurodegeneration and/or neurological decline. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to have anti-inflammatory, neuroprotective and regenerative properties but the cell type- and receptor-specific mechanisms are not clear. To test whether the protective effects of PACAP are direct on the PAC1 receptor subtype on neurons, we delete PAC1 receptors from neurons and investigate neuropathologigical changes in an animal model of MS. The findings demonstrate that PAC1 receptors on neurons play a homeostatic role in maintaining neuron health and can directly protect neurons and their axons during neuroinflammatory disease.
Display omitted
•PAC1 plays a homeostatic role in maintaining a subset of neurons in the eye.•PAC1 directly protects neurons and their axons in the experimental autoimmune encephalomyelitis model of multiple sclerosis.•Under an inflammatory condition, deletion of PAC1 leads to more microglia in the optic nerve.
The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy ...progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.
Display omitted
Present-day therapy for the urea cycle disorder carbamoyl phosphate synthetase deficiency is incomplete, resulting in CNS injury or neonatal death. Here, Lipshutz and colleagues demonstrate a dual AAV approach to overcome the cDNA size restriction, resulting in enzyme expression with improved ammonia control and ureagenesis restoration, both supporting long-term survival.
PDF
