Deep postnatal phenotyping of a new mouse model of nonketotic hyperglycinemia.

Nonketotic hyperglycinemia due to deficient glycine cleavage enzyme activity causes a severe neonatal epileptic encephalopathy. Current therapies based on mitigating glycine excess have only limited impact. An animal model with postnatal phenotyping is needed to explore new therapeutic approaches. We developed a Gldc p.Ala394Val mutant model and bred it to congenic status in 2 colonies on C57Bl/6J (B6) and J129X1/SvJ (J129) backgrounds. Mutant mice had reduced P-protein and enzyme activity indicating a hypomorphic mutant. Glycine levels were increased in blood and brain regions, exacerbated by dietary glycine, with higher levels in female than male J129 mice. Birth defects were more prevalent in mutant B6 than J129 mice, and hydrocephalus was more frequent in B6 (40%) compared to J129 (none). The hydrocephalus rate was increased by postnatal glycine challenge in B6 mice, more so when delivered from the first neonatal week than from the fourth. Mutant mice had reduced weight gain following weaning until the eighth postnatal week, which was exacerbated by glycine loading. The electrographic spike rate was increased in mutant mice following glycine loading, but no seizures were observed. The alpha/delta band intensity ratio was decreased in the left cortex in female J129 mice, which were less active in an open field test and explored less in a Y-maze, suggesting an encephalopathic effect. Mutant mice showed no evidence of memory dysfunction. This partial recapitulation of human symptoms and biochemistry will facilitate the evaluation of new therapeutic approaches with an early postnatal time window likely most effective. Take home message: A mouse model of nonketotic hyperglycinemia is described that shows postnatal abnormalities in glycine levels, neural tube defects, body weight, electroencephalographic recordings, and in activity in young mice making it amenable for the evaluation of novel treatment interventions. Author contributions: Study concept and design: JVH, MHM, NB, KNMAnimal study data: MAS, HJ, NB, MHM, JC, CBBiochemical and genetic studies: MAS, RAVH, MWFStatistical analysis: NB, JVHFirst draft writing: JVH, NB, MHMCritical rewriting: MAS, NB, MHM, TAB, JC, MWF, KNM, JVHFinal responsibility, guarantor, and communicating author: JVH. Competing interest statement: The University of Colorado (JVH, MS, KNM, HJ) has the intention to file Intellectual property protection for certain biochemical treatments of NKH. Otherwise, the authors have stated that they had no interests that might be perceived as posing a conflict or bias to this subject matter. Funding support: Financial support is acknowledged form the NKH Crusaders, Brodyn's Friends, Nora Jane Almany Foundation, the Dickens Family Foundation, the Lucas John Foundation, Les Petits Bourdons, Joseph's Fund, the Barnett Family, Maud & Vic Foundation, Lucy's BEElievers fund, Hope for NKH, Madi's Mission NKH fund, and from Dr. and Ms. Shaw, and the University of Colorado Foundation NKH research fund. The study was supported by a grant (CNS-X-19-103) from the University of Colorado School of Medicine and the Colorado Clinical Translational Science Institute, which is supported by NIH/NCATS Colorado CTSA Grant Number UL1 TR002535. Contents are the authors' sole responsibility and do not necessarily represent official NIH views. All funding sources had no role in the design or execution of the study, the interpretation of data, or the writing of the study. Ethics approval on Laboratory Animal Studies: Mouse studies were carried out with approval from the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus (IACUC# 00413). Data sharing statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Clinical Pharmacology Perspective on Development of Adeno-Associated Virus Vector-Based Retina Gene Therapy.

Adeno-associated virus (AAV) vector-based gene therapy is an innovative modality being increasingly investigated to treat diseases by modifying or replacing defective genes or expressing therapeutic entities. With its unique anatomic and physiological characteristics, the eye constitutes a very attractive target for gene therapy. Specifically, the ocular space is easily accessible and is generally considered "immune-privileged" with a low risk of systemic side effects following local drug administration. As retina cells have limited cellular turnover, a one-time gene delivery has the potential to provide long-term transgene expression. Despite the initial success with voretigene neparvovec (Luxturna), the first approved retina gene therapy, there are still challenges to be overcome for successful clinical development of these products and scientific questions to be answered. The current review paper aims to integrate published experience learned thus far for AAV-based retina gene therapy related to preclinical to clinical translation; first-in-human dose selection; relevant bioanalytical assays and strategies; clinical development considerations including trial design, biodistribution and vector shedding, immunogenicity, transgene expression, and pediatric populations; opportunities for model-informed drug development; and regulatory perspectives. The information presented herein is intended to serve as a guide to inform the clinical development strategy for retina gene therapy with a focus on clinical pharmacology.

Quantification of clesrovimab, an investigational, half-life extended, anti-respiratory syncytial virus protein F human monoclonal antibody in the nasal epithelial lining fluid of healthy adults.

BACKGROUND: Clesrovimab (MK-1654) is an investigational, half-life extended human monoclonal antibody (mAb) against RSV F glycoprotein in clinical trials as a prophylactic agent against RSV infection for infants. METHODS: This adult study measured clesrovimab concentrations in the serum and nasal epithelial lining fluid (ELF) to establish the partitioning of the antibody after dosing. Clesrovimab concentrations in the nasal ELF were normalized for sampling dilution using urea concentrations from ELF and serum. Furthermore, in vitro RSV neutralization of human nasal ELF following dosing was also measured to examine the activity of clesrovimab in the nasal compartment. FINDINGS: mAbs with YTE mutations are reported in literature to partition ∼1-2 % of serum antibodies into nasal mucosa. Nasal: serum ratios of 1:69-1:30 were observed for clesrovimab in two separate adult human trials after urea normalization, translating to 1.4-3.3 % of serum concentrations. The nasal PK and estimates of peripheral volume of distribution correlated with higher extravascular distribution of clesrovimab. These higher concentration of the antibody in the nasal ELF corroborated with the nasal sample's ability to neutralize RSV ex vivo. An overall trend of decreased viral plaque AUC was also noted with increasing availability of clesrovimab in the nasal ELF from a human RSV challenge study. INTERPRETATION: Along with its extended half-life, the higher penetration of clesrovimab into the nasal epithelial lining fluid and the associated local increase in RSV neutralization activity could offer infants better protection against RSV infection.

Optimization of Peripheral Blood Mononuclear Cell Processing for Improved Clinical ELISpot Assay Performance.

Cell and gene therapies have demonstrated impressive therapeutic efficacy in various human diseases. Nevertheless, cellular immune response directed against these therapeutic agents is an obstacle for achieving long-lasting clinical efficacy. Therefore, it is crucial to develop robust assays to accurately monitor cellular immunogenicity towards these therapies. Enzyme-linked immunospot (ELISpot) assay is one of the primarily used methods for measuring cellular immune response in clinical programs, which requires isolation of the peripheral blood mononuclear cells (PBMCs). The quality of this clinical material is one of the most critical factors that impact the robust assessment of cellular immune responses. The optimal blood sample processing conditions, however, remain poorly understood. In this study, we examined the impact of blood sample processing time on the performance characteristics of ELISpot to measure antigen-specific cellular responses. Blood samples that were processed after overnight delay resulted in a loss of ELISpot signals. We subsequently optimized several parameters of sample processing, and successfully recovered ELISpot signals for the blood samples that are processed within 32 h. Furthermore, several mitigation strategies were employed that would potentially address the impact of granulocyte contamination on detection of antigen-specific cellular responses. Our investigation provides an extension of sample processing window for clinical studies and is significant for resolving the logistical challenge of whole blood sample shipment for timely PBMC preparation in cell/gene therapy clinical studies.

Phage-derived anti-idiotype and anti-YTE antibodies in development of MK-1654 pharmacokinetic and immune response assays.

Background: MK-1654 is a fully human monoclonal antibody with YTE mutations currently in phase III clinical trials for prophylactic use in protecting infants from human respiratory syncytial virus infection. Materials & methods: We generated anti-idiotype (anti-ID) and anti-YTE antibodies against MK-1654 by panning with MorphoSys HuCal phage libraries, and used the antibodies in the development of MK-1654 pharmacokinetic (PK) and immune response (IR) assays. Results: Detection of MK-1654 in nonhuman primate and human nasal wash samples showed combined use of anti-ID and anti-YTE antibodies can deliver desired sensitivity and accuracy in PK studies. IR studies showed anti-ID can serve as suitable positive control in neutralizing antibody assays. Conclusion: Phage-derived anti-IDs and anti-YTEs are suitable for PK and IR assays.

Survey Outcome on Immunogenicity Risk Assessment Tools for Biotherapeutics: an Insight into Consensus on Methods, Application, and Utility in Drug Development.

A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.

Pathogenic variants in GCSH encoding the moonlighting H-protein cause combined nonketotic hyperglycinemia and lipoate deficiency.

Maintaining protein lipoylation is vital for cell metabolism. The H-protein encoded by GCSH has a dual role in protein lipoylation required for bioenergetic enzymes including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase, and in the one-carbon metabolism through its involvement in glycine cleavage enzyme system, intersecting two vital roles for cell survival. Here, we report six patients with biallelic pathogenic variants in GCSH and a broad clinical spectrum ranging from neonatal fatal glycine encephalopathy to an attenuated phenotype of developmental delay, behavioral problems, limited epilepsy and variable movement problems. The mutational spectrum includes one insertion c.293-2_293-1insT, one deletion c.122_(228 + 1_229-1) del, one duplication of exons 4 and 5, one nonsense variant p.Gln76*and four missense p.His57Arg, p.Pro115Leu and p.Thr148Pro and the previously described p.Met1?. Via functional studies in patient's fibroblasts, molecular modeling, expression analysis in GCSH knockdown COS7 cells and yeast, and in vitro protein studies, we demonstrate for the first time that most variants identified in our cohort produced a hypomorphic effect on both mitochondrial activities, protein lipoylation and glycine metabolism, causing combined deficiency, whereas some missense variants affect primarily one function only. The clinical features of the patients reflect the impact of the GCSH changes on any of the two functions analyzed. Our analysis illustrates the complex interplay of functional and clinical impact when pathogenic variants affect a multifunctional protein involved in two metabolic pathways and emphasizes the value of the functional assays to select the treatment and investigate new personalized options.

Immunogenicity Risk Assessment of Spontaneously Occurring Therapeutic Monoclonal Antibody Aggregates.

Aggregates of therapeutic proteins have been associated with increased immunogenicity in pre-clinical models as well as in human patients. Recent studies to understand aggregates and their immunogenicity risks use artificial stress methods to induce high levels of aggregation. These methods may be less biologically relevant in terms of their quantity than those that occur spontaneously during processing and storage. Here we describe the immunogenicity risk due to spontaneously occurring therapeutic antibody aggregates using peripheral blood mononuclear cells (PBMC) and a cell line with a reporter gene for immune activation: THP-1 BLUE NFκB. The spontaneously occurring therapeutic protein aggregates were obtained from process intermediates and final formulated drug substance from stability retains. Spontaneously occurring aggregates elicited innate immune responses for several donors in a PBMC assay with cytokine and chemokine production as a readout for immune activation. Meanwhile, no significant adaptive phase responses to spontaneously occurring aggregate samples were detected. While the THP-1 BLUE NFκB cell line and PBMC assays both responded to high stress induced aggregates, only the PBMC from a limited subset of donors responded to processing-induced aggregates. In this case study, levels of antibody aggregation occurring at process relevant levels are lower than those induced by stirring and may pose lower risk in vivo. Our methodologies can further inform additional immunogenicity risk assessments using a pre-clinical in vitro risk assessment approach utilizing human derived immune cells.

Considerations in the Immunogenicity Assessment Strategy for Oligonucleotide Therapeutics (ONTs).

Oligonucleotide therapeutics (ONTs) are a diverse group of short synthetic nucleic acid-based molecules that exploit innovative intracellular molecular strategies to create novel treatments for a variety of medical conditions. ONT molecules (~7-15 kDa) reside between traditional large and small molecules, and there has been debate regarding their immunogenicity risk. To date, 13 ON drugs have been approved, and as the field is relatively new, there are currently no specific regulatory guidelines to indicate how to develop, validate, and interpret the immunogenicity assays of ONTs. Some investigators do not test for immune responses to ONs while others test for antibodies (Abs) to components within the formulation, which may or may not include aspects of characterization such as domain mapping of ONT conjugates. Similar to other biopharmaceuticals, the immunogenic properties of ONTs could be influenced by sequence, route, dosage, target population, co-medications, etc. The current anti-drug antibody (ADA) data for different approved ONTs suggest that their administration poses a low immunogenicity risk without any significant impact on pharmacokinetics (PK), pharmacodynamics (PD), and safety; nevertheless, until the field matures with data from many more ON drugs, it remains prudent to assess immunogenicity. The emphasis of this article is to highlight how current ADA methodologies might be applied to the development of ONTs, discuss factors that may pose immunogenicity risks, and provide the authors' current position on immunogenicity assessment strategies for ONTs. We also discuss assay parameters that may be appropriate for the detection and characterization of ADAs, including the evaluation of neutralizing ADAs, ADA isotyping, Abs to dsDNA, and pre-existing ADA. Immunogenicity risk assessments (IRAs) and early interactions with regulators will inform how to proceed in late stage/pivotal studies.

Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes protein agonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthase as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

Cerebrospinal fluid amino acids glycine, serine, and threonine in nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is caused by deficient glycine cleavage enzyme activity and characterized by elevated brain glycine. Metabolism of glycine is connected enzymatically to serine through serine hydroxymethyltransferase and shares transporters with serine and threonine. We aimed to evaluate changes in serine and threonine in NKH patients, and relate this to clinical outcome severity. Age-related reference values were developed for cerebrospinal fluid (CSF) serine and threonine from 274 controls, and in a cross-sectional study compared to 61 genetically proven NKH patients, categorized according to outcome. CSF d-serine and l-serine levels were stereoselectively determined in seven NKH patients and compared to 29 age-matched controls. In addition to elevated CSF glycine, NKH patients had significantly decreased levels of CSF serine and increased levels of CSF threonine, even after age-adjustment. The CSF serine/threonine ratio discriminated between NKH patients and controls. The CSF glycine/serine aided in discrimination between severe and attenuated neonates with NKH. Over all ages, the CSF glycine, serine and threonine had moderate to fair correlation with outcome classes. After age-adjustment, only the CSF glycine level provided good discrimination between outcome classes. In untreated patients, d-serine was more reduced than l-serine, with a decreased d/l-serine ratio, indicating a specific impact on d-serine metabolism. We conclude that in NKH the elevation of glycine is accompanied by changes in l-serine, d-serine and threonine, likely reflecting a perturbation of the serine shuttle and metabolism, and of one-carbon metabolism. This provides additional guidance on diagnosis and prognosis, and opens new therapeutic avenues to be explored.

Implementing a Clinical Immunogenicity Strategy using Preclinical Risk Assessment Outputs.

Immunogenicity to biologics is often observed following dosing in human subjects during clinical trials. Both product and host specific factors may be implicated in contributing to a potential immune response. However, even if such risk factors are identified and eliminated as part of the rational quality by design approaches, the outcome in clinic can be uncertain and challenging to predict. Several tools have been employed to identify these risk factors and consequent mitigation approaches implemented prior to dosing in humans. However, the complexity of the immune system with an interplay of network of immune cells involved in driving a long- term immune response as well as patient characteristics, can make it challenging to predict the outcome in clinic. This perspective will provide an insight into recent advances in the risk assessment approaches that are utilized during preclinical stage of development of a biologic. The outputs from such tools can help to rank order and select the most optimal candidate with the least likelihood of an immune response and can further drive the development of a clinical bioanalytical and immunogenicity monitoring strategy. Such a strategy can be proactively shared with the regulators along with the proposal to streamline clinical immunogenicity and personalizing the outcome based on pharmacogenomics and other patient-related factors. This paper provides a roadmap on performing risk assessments through a systematic identification of risks and their mitigations wherever possible. Recommendations on incorporating the key components of such risk assessments as part of the new regulatory submissions are also provided. Shorter abstract Immunogenicity to biologics is common during clinical trials. Both product and host specific factors have been implicated. Several risk assessment tools can be used to identify and mitigate the risk factors responsible for immunogenicity. An insight into recent advances in the risk assessment approaches will be presented. The outputs can define a risk score and guide the clinical bioanalytical and immunogenicity monitoring strategy. A roadmap on performing risk assessments through a systematic identification of risks and their mitigations wherever possible is provided. Best practices for a risk assessment strategy and recommendations on the content for IND and the Integrated summary of Immunogenicity are also provided.

Targeted disruption of pi-pi stacking in Malaysian banana lectin reduces mitogenicity while preserving antiviral activity.

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings show that pi-pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.

Identification and characterization of a residual host cell protein hexosaminidase B associated with N-glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product.

Host cell proteins (HCPs) are process-related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several monoclonal antibodies (mAbs) under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N-glycan heterogeneity profile. However, significant N-glycan degradation was observed for one mAb under accelerated and stressed stability conditions. The root cause for this instability was attributed to hexosaminidase B (HEXB), an enzyme known to remove terminal N-acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography-mass spectrometry (LC-MS)-based proteomics approach to be enriched in the impacted stability batches from mAb-1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb-1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry, and computational modeling provides a general strategy on how to study residual HCP for biotherapeutics development.

Ultra-long-acting tunable biodegradable and removable controlled release implants for drug delivery.

Here we report an ultra-long-acting tunable, biodegradable, and removable polymer-based delivery system that offers sustained drug delivery for up to one year for HIV treatment or prophylaxis. This robust formulation offers the ability to integrate multiple drugs in a single injection, which is particularly important to address the potential for drug resistance with monotherapy. Six antiretroviral drugs were selected based on their solubility in N-methyl-2-pyrrolidone and relevance as a combination therapy for HIV treatment or prevention. All drugs released with concentrations above their protein-adjusted inhibitory concentration and retained their physical and chemical properties within the formulation and upon release. The versatility of this formulation to integrate multiple drugs and provide sustained plasma concentrations from several weeks to up to one year, combined with its ability to be removed to terminate the treatment if necessary, makes it attractive as a drug delivery platform technology for a wide range of applications.

Identification of a novel biomarker for pyridoxine-dependent epilepsy: Implications for newborn screening.

Pyridoxine-dependent epilepsy (PDE) is often characterized as an early onset epileptic encephalopathy with dramatic clinical improvement following pyridoxine supplementation. Unfortunately, not all patients present with classic neonatal seizures or respond to an initial pyridoxine trial, which can result in the under diagnosis of this treatable disorder. Restriction of lysine intake and transport is associated with improved neurologic outcomes, although treatment should be started in the first year of life to be effective. Because of the documented diagnostic delay and benefit of early treatment, we aimed to develop a newborn screening method for PDE. Previous studies have demonstrated the accumulation of Δ1 -piperideine-6-carboxylate and α-aminoadipic semialdehyde in individuals with PDE, although these metabolites are unstable at room temperature (RT) limiting their utility for newborn screening. As a result, we sought to identify a biomarker that could be applied to current newborn screening paradigms. We identified a novel metabolite, 6-oxo-pipecolate (6-oxo-PIP), which accumulates in substantial amounts in blood, plasma, urine, and cerebral spinal fluid of individuals with PDE. Using a stable isotope-labeled internal standard, we developed a nonderivatized liquid chromatography tandem mass spectrometry-based method to quantify 6-oxo-PIP. This method replicates the analytical techniques used in many laboratories and could be used with few modifications in newborn screening programs. Furthermore, 6-oxo-PIP was measurable in urine for 4 months even when stored at RT. Herein, we report a novel biomarker for PDE that is stable at RT and can be quantified using current newborn screening techniques.

The genotypic spectrum of ALDH7A1 mutations resulting in pyridoxine dependent epilepsy: A common epileptic encephalopathy.

Pyridoxine dependent epilepsy (PDE) is a treatable epileptic encephalopathy characterized by a positive response to pharmacologic doses of pyridoxine. Despite seizure control, at least 75% of individuals have intellectual disability and developmental delay. Current treatment paradigms have resulted in improved cognitive outcomes emphasizing the importance of an early diagnosis. As genetic testing is increasingly accepted as first tier testing for epileptic encephalopathies, we aimed to provide a comprehensive overview of ALDH7A1 mutations that cause PDE. The genotypes, ethnic origin and reported gender was collected from 185 subjects with a diagnosis of PDE. The population frequency for the variants in this report and the existing literature were reviewed in the Genome Aggregation Database (gnomAD). Novel variants identified in population databases were also evaluated through in silico prediction software and select variants were over-expressed in an E.coli-based expression system to measure α-aminoadipic semialdehyde dehydrogenase activity and production of α-aminoadipic acid. This study adds 47 novel variants to the literature resulting in a total of 165 reported pathogenic variants. Based on this report, in silico predictions, and general population data, we estimate an incidence of approximately 1:64,352 live births. This report provides a comprehensive overview of known ALDH7A1 mutations that cause PDE, and suggests that PDE may be more common than initially estimated. Due to the relative high frequency of the disease, the likelihood of under-diagnosis given the wide clinical spectrum and limited awareness among clinicians as well as the cognitive improvement noted with early treatment, newborn screening for PDE may be warranted.

T cells establish and maintain CNS viral infection in HIV-infected humanized mice.

The human brain is an important site of HIV replication and persistence during antiretroviral therapy (ART). Direct evaluation of HIV infection in the brains of otherwise healthy individuals is not feasible; therefore, we performed a large-scale study of bone marrow/liver/thymus (BLT) humanized mice as an in vivo model to study HIV infection in the brain. Human immune cells, including CD4+ T cells and macrophages, were present throughout the BLT mouse brain. HIV DNA, HIV RNA, and/or p24+ cells were observed in the brains of HIV-infected animals, regardless of the HIV isolate used. HIV infection resulted in decreased numbers of CD4+ T cells, increased numbers of CD8+ T cells, and a decreased CD4+/CD8+ T cell ratio in the brain. Using humanized T cell-only mice (ToM), we demonstrated that T cells establish and maintain HIV infection of the brain in the complete absence of human myeloid cells. HIV infection of ToM resulted in CD4+ T cell depletion and a reduced CD4+/CD8+ T cell ratio. ART significantly reduced HIV levels in the BLT mouse brain, and the immune cell populations present were indistinguishable from those of uninfected controls, which demonstrated the effectiveness of ART in controlling HIV replication in the CNS and returning cellular homeostasis to a pre-HIV state.

Correction: The genetic basis of classic nonketotic hyperglycinemia due to mutations in GLDC and AMT.

The original supplementary information included with this article contained several minor errors. Corrected Supplementary Information accompanies this corrigendum.