Chronic lung injury and impaired pulmonary function in a mouse model of acid ceramidase deficiency.

Farber disease (FD) is a debilitating lysosomal storage disorder (LSD) caused by a deficiency of acid ceramidase (ACDase) activity due to mutations in the gene ASAH1. Patients with ACDase deficiency may develop a spectrum of clinical phenotypes. Severe cases of FD are frequently associated with neurological involvement, failure to thrive, and respiratory complications. Mice homozygous ( Asah1P361R/P361R) for an orthologous patient mutation in Asah1 recapitulate human FD. In this study, we show significant impairment in lung function, including low compliance and increased airway resistance in a mouse model of ACDase deficiency. Impaired lung mechanics in Farber mice resulted in decreased blood oxygenation and increased red blood cell production. Inflammatory cells were recruited to both perivascular and peribronchial areas of the lung. We observed large vacuolated foamy histiocytes that were full of storage material. An increase in vascular permeability led to protein leakage, edema, and impacted surfactant homeostasis in the lungs of Asah1P361R/P361R mice. Bronchial alveolar lavage fluid (BALF) extraction and analysis revealed accumulation of a highly turbid lipoprotein-like substance that was composed in part of surfactants, phospholipids, and ceramides. The phospholipid composition of BALF from Asah1P361R/P361R mice was severely altered, with an increase in both phosphatidylethanolamine (PE) and sphingomyelin (SM). Ceramides were also found at significantly higher levels in both BALF and lung tissue from Asah1P361R/P361R mice when compared with levels from wild-type animals. We demonstrate that a deficiency in ACDase leads to sphingolipid and phospholipid imbalance, chronic lung injury caused by significant inflammation, and increased vascular permeability, leading to impaired lung function.

Detection of F8 mutations in carriers and patients with severe hemophilia A. Identification of a novel mutation.

The molecular diagnosis of haemophilia A (HA) patients has many benefits including diagnosis confirmation and inhibitor risk development prediction. In female carries of a mutation, the molecular diagnosis allows for genetic counseling and prenatal diagnosis, which have become part of the comprehensive care for HA in many countries. Therefore, the aim of this study was to determine the F8 mutations in severe HA (sHA) patients and female carriers. In 12 patients with sHA, the presence of the intron 22 and intron 1 inversions was investigated using an inverse and a conventional PCR method, respectively. In patients negative for the inversions, the F8 gene was screened through conformation sensitive gel electrophoresis (CSGE) and further sequencing. The causative mutation was successfully identified in 6/12 patients, including the novel mutation p.G190C. The mothers of these six patients and those of seven other sHA patients molecularly diagnosed in a previous work were investigated for the presence of the genetic alterations found in their sons. All mothers were found to be carriers. This is the first study conducted in Venezuela which directly analyzes the F8 gene in potential carrier mothers to specifically identify the presence of the mutation that was detected in their sons, and complements a previous study on sHA patients. Our findings will facilitate the implementation of regular screening of HA carriers in our country and will allow a better care of bleeding symptoms and genetic counseling.

Development and Optimization of a Target Engagement Model of Brain IDO Inhibition for Alzheimer's Disease.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO1) inhibition is a promising target as an Alzheimer's disease (AD) Disease-modifying therapy capable of downregulating immunopathic neuroinflammatory processes. METHODS: To aid in the development of IDO inhibitors as potential AD therapeutics, we optimized a lipopolysaccharide (LPS) based mouse model of brain IDO1 inhibition by examining the dosedependent and time-course of the brain kynurenine:tryptophan (K:T) ratio to LPS via intraperitoneal dosing. RESULTS: We determined the optimal LPS dose to increase IDO1 activity in the brain, and the ideal time point to quantify the brain K:T ratio after LPS administration. We then used a brain penetrant tool compound, EOS200271, to validate the model, determine the optimal dosing profile and found that a complete rescue of the K:T ratio was possible with the tool compound. CONCLUSION: This LPS-based model of IDO1 target engagement is a useful tool that can be used in the development of brain penetrant IDO1 inhibitors for AD. A limitation of the present study is the lack of quantification of potential clinically relevant biomarkers in this model, which could be addressed in future studies.

Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing: Targeting Mobilized CD34+ Cells for Correction of Fabry Disease.

Fabry disease is a rare lysosomal storage disorder (LSD). We designed multiple recombinant lentivirus vectors (LVs) and tested their ability to engineer expression of human α-galactosidase A (α-gal A) in transduced Fabry patient CD34+ hematopoietic cells. We further investigated the safety and efficacy of a clinically directed vector, LV/AGA, in both ex vivo cell culture studies and animal models. Fabry mice transplanted with LV/AGA-transduced hematopoietic cells demonstrated α-gal A activity increases and lipid reductions in multiple tissues at 6 months after transplantation. Next we found that LV/AGA-transduced Fabry patient CD34+ hematopoietic cells produced even higher levels of α-gal A activity than normal CD34+ hematopoietic cells. We successfully transduced Fabry patient CD34+ hematopoietic cells with "near-clinical grade" LV/AGA in small-scale cultures and then validated a clinically directed scale-up transduction process in a GMP-compliant cell processing facility. LV-transduced Fabry patient CD34+ hematopoietic cells were subsequently infused into NOD/SCID/Fabry (NSF) mice; α-gal A activity corrections and lipid reductions were observed in several tissues 12 weeks after the xenotransplantation. Additional toxicology studies employing NSF mice xenotransplanted with the therapeutic cell product demonstrated minimal untoward effects. These data supported our successful clinical trial application (CTA) to Health Canada and opening of a "first-in-the-world" gene therapy trial for Fabry disease.

Towards in vivo amplification: Overcoming hurdles in the use of hematopoietic stem cells in transplantation and gene therapy.

With the advent of safer and more efficient gene transfer methods, gene therapy has become a viable solution for many inherited and acquired disorders. Hematopoietic stem cells (HSCs) are a prime cell compartment for gene therapy aimed at correcting blood-based disorders, as well as those amenable to metabolic outcomes that can effect cross-correction. While some resounding clinical successes have recently been demonstrated, ample room remains to increase the therapeutic output from HSC-directed gene therapy. In vivo amplification of therapeutic cells is one avenue to achieve enhanced gene product delivery. To date, attempts have been made to provide HSCs with resistance to cytotoxic drugs, to include drug-inducible growth modules specific to HSCs, and to increase the engraftment potential of transduced HSCs. This review aims to summarize amplification strategies that have been developed and tested and to discuss their advantages along with barriers faced towards their clinical adaptation. In addition, next-generation strategies to circumvent current limitations of specific amplification schemas are discussed.

Detection of F8 mutations in carriers and patients with severe hemophilia A: Identification of a novel mutation / Detección de mutaciones en el gen F8 en mujeres portadoras y en pacientes con hemofilia A: Identificación de una mutación nueva

The molecular diagnosis of haemophilia A (HA) patients has many benefits including diagnosis confirmation and inhibitor risk development prediction. In female carries of a mutation, the molecular diagnosis allows for genetic counseling and prenatal diagnosis, which have become part of the comprehensive care for HA in many countries. Therefore, the aim of this study was to determine the F8 mutations in severe HA (sHA) patients and female carriers. In 12 patients with sHA, the presence of the intron 22 and intron 1 inversions was investigated using an inverse and a conventional PCR method, respectively. In patients negative for the inversions, the F8 gene was screened through conformation sensitive gel electrophoresis (CSGE) and further sequencing. The causative mutation was successfully identified in 6/12 patients, including the novel mutation p.G190C. The mothers of these six patients and those of seven other sHA patients molecularly diagnosed in a previous work were investigated for the presence of the genetic alterations found in their sons. All mothers were found to be carriers. This is the first study conducted in Venezuela which directly analyzes the F8 gene in potential carrier mothers to specifically identify the presence of the mutation that was detected in their sons, and complements a previous study on sHA patients. Our findings will facilitate the implementation of regular screening of HA carriers in our country and will allow a better care of bleeding symptoms and genetic counseling.

El diagnóstico molecular de pacientes con hemofilia A (HA) tiene múltiples beneficios, incluyendo la confirmación del diagnóstico y la predicción del riesgo de desarrollar inhibidores. En mujeres portadoras de una mutación, el diagnóstico molecular permite el consejo genético y el diagnóstico prenatal, los cuales son parte de la atención integral de HA en muchos países. Así, el objetivo de este estudio fue determinar mutaciones en el gen F8 en pacientes con HA severa (HAs) y en mujeres portadoras. En 12 pacientes con HAs, la presencia de la inversión del intrón 22 y el intrón 1 fue investigada utilizando una PCR inversa y una convencional, respectivamente. En pacientes negativos para cualquiera de las inversiones, el gen del F8 fue analizado a través de la técnica de electroforesis en gel sensible a conformación (CSGE) y posterior secuenciación. La mutación causante de la enfermedad fue identificada en 6/12 pacientes, incluyendo la mutación nueva p.G190C. Las madres de estos seis pacientes y las de otros siete pacientes de HAs diagnosticados en un estudio previo y fueron investigadas para la presencia de alteraciones genéticas encontradas en sus hijos. Todas las madres resultaron ser portadoras. Éste es el primer estudio realizado en Venezuela donde se analiza directamente el gen F8 en portadoras potenciales para identificar específicamente la presencia de una mutación que fue detectada en sus hijos, y complementa un estudio previo con pacientes HAs. Nuestros hallazgos facilitarán la implementación del análisis regular de portadoras de HA en nuestro país y permitirán un mejor cuidado de los síntomas de sangrado y consejo genético.