Kimberlee Michals Matalon

Knock‐out mouse for Canavan disease: a model for gene transfer to the central nervous system

Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N‐acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock‐out mouse for Canavan disease that can be used for gene transfer.

Phenylketonuria Scientific Review Conference: State of the science and future research needs

New developments in the treatment and management of phenylketonuria (PKU) as well as advances in molecular testing have emerged since the National Institutes of Health 2000 PKU Consensus Statement was released. An NIH State-of-the-Science Conference was convened in 2012 to address new findings, particularly the use of the medication sapropterin to treat some individuals with PKU, and to develop a research agenda. Prior to the 2012 conference, five working groups of experts and public members met over a 1-year period. The working groups addressed the following: long-term outcomes and management across the lifespan; PKU and pregnancy; diet control and management; pharmacologic interventions; and molecular testing, new technologies, and epidemiologic considerations. In a parallel and independent activity, an Evidence-based Practice Center supported by the Agency for Healthcare Research and Quality conducted a systematic review of adjuvant treatments for PKU; its conclusions were presented at the conference. The conference included the findings of the working groups, panel discussions from industry and international perspectives, and presentations on topics such as emerging treatments for PKU, transitioning to adult care, and the U.S. Food and Drug Administration regulatory perspective. Over 85 experts participated in the conference through information gathering and/or as presenters during the conference, and they reached several important conclusions. The most serious neurological impairments in PKU are preventable with current dietary treatment approaches. However, a variety of more subtle physical, cognitive, and behavioral consequences of even well-controlled PKU are now recognized. The best outcomes in maternal PKU occur when blood phenylalanine (Phe) concentrations are maintained between 120 and 360 μmol/L before and during pregnancy. The dietary management treatment goal for individuals with PKU is a blood Phe concentration between 120 and 360 μmol/L. The use of genotype information in the newborn period may yield valuable insights about the severity of the condition for infants diagnosed before maximal Phe levels are achieved. While emerging and established genotype-phenotype correlations may transform our understanding of PKU, establishing correlations with intellectual outcomes is more challenging. Regarding the use of sapropterin in PKU, there are significant gaps in predicting response to treatment; at least half of those with PKU will have either minimal or no response. A coordinated approach to PKU treatment improves long-term outcomes for those with PKU and facilitates the conduct of research to improve diagnosis and treatment. New drugs that are safe, efficacious, and impact a larger proportion of individuals with PKU are needed. However, it is imperative that treatment guidelines and the decision processes for determining access to treatments be tied to a solid evidence base with rigorous standards for robust and consistent data collection. The process that preceded the PKU State-of-the-Science Conference, the conference itself, and the identification of a research agenda have facilitated the development of clinical practice guidelines by professional organizations and serve as a model for other inborn errors of metabolism.

Adeno-associated virus-mediated aspartoacylase gene transfer to the brain of knockout mouse for canavan disease.

Canavan disease (CD) is an autosomal recessive leukodystrophy caused by deficiency of aspartoacylase (ASPA). Deficiency of ASPA leads to elevation of N-acetyl-L-aspartic acid (NAA) in the brain and urine. To explore the feasibility of gene transfer to replace ASPA in CD, we generated a knockout mouse and constructed an AAV vector that encodes human ASPA cDNA (hASPA) followed by green fluorescent protein (GFP) after an intraribosomal entry site. We injected CD mice with rAAV-hASPA-GFP in the striatum and thalamus or injected rAAV-GFP identically into control animals. Three to five months after the injection, we determined the presence of ASPA in the CD mouse brain by ASPA activity assay, GFP expression, and Western blot analysis. While rAAV-GFP-injected animals displayed undetectable levels of ASPA, all detection methods revealed significant ASPA levels in rAAV-hASPA-GFP-injected CD mice. We evaluated the functional effects of rAAV-hASPA-GFP-mediated ASPA expression by standard histological methods, magnetic resonance spectroscopy (MRS) for in vivo NAA levels, and magnetic resonance imaging of CD mice. rAAV-hASPA-injected animals displayed a remarkable lack of spongiform degeneration in the thalamus. However, pathology in sites unrelated to the injected areas showed no improvement in histopathology. The improvement in thalamic neuropathology was also detectable via in vivo MRI. MRS revealed that in vivo NAA levels were also reduced. These data indicate that rAAV-mediated ASPA delivery may be an interesting avenue for the treatment of CD.

Molecular Basis of Canavan's Disease From Human to Mouse

Canavans disease is an autosomal recessive disorder caused by aspartoacylase deficiency. The deficiency of aspartoacylase leads to increased concentration of N-acetylaspartic acid in brain and body fluids. The failure to hydrolyze N-acetylaspartic acid causes disruption of myelin, resulting in spongy degeneration of the white matter of the brain. The clinical features of the disease are hypotonia in early life, which changes to spasticity, macrocephaly, head lag, and progressive severe mental retardation. Although Canavans disease is panethnic, it is most prevalent in the Ashkenazi Jewish population. Research at the molecular level led to the cloning of the gene for aspartoacylase and development of a knockout mouse for Canavans disease. These developments have afforded new tools for research in the attempts to understand the pathophysiology of Canavans disease, design new therapies, and explore methods for gene transfer to the central nervous system. (J Child Neurol 2003;18:604—610).

Canavan disease: Prenatal diagnosis and genetic counseling☆

Canavan disease is a severe leukodystrophy more common among Ashkenazi Jews. The enzyme defect, apartoacylase, has been identified, and the gene cloned. Only two mutations account for over 98% of all Jewish alleles with Canavan disease. The carrier frequency among healthy Jews is 1:37-58. Carrier detection and prenatal diagnosis can be accurately carried out using molecular analysis. When mutations are unknown, analysis of amniotic fluid for NAA using stable isotope dilution technique can be used for prenatal diagnosis.

Metabolic changes in the knockout mouse for Canavan's disease: implications for patients with Canavan's disease.

Canavans disease is an autosomal recessive disorder caused by aspartoacylase deficiency, which leads to accumulation of N-acetylaspartic acid in the brain and blood and an elevated level of N-acetylaspartic acid in the urine. The brain of patients with Canavans disease shows spongy degeneration. How the enzyme deficiency and elevated N-acetylaspartic acid cause the pathophysiology observed in Canavans disease is not obvious. The creation of a knockout mouse for Canavans disease is being used as a tool to investigate metabolic pathways in the mouse and correlate them with the patients with Canavans disease. The level of glutamate is lower in the knockout mouse brain than in the wild-type mouse brain, similar to what we have found in children with Canavans disease, and so are the levels of γ-aminobutyric acid (GABA). The level of aspartate is higher in the Canavans disease mouse brain. The activity of aspartate aminotransferase, an enzyme involved in the malate-aspartate shuttle, is lower in the Canavans disease mouse brain. The lower weight of the Canavans disease mouse was in direct proportion to low total-body fat and bone mineral density. These changes might be similar to what is seen in patients with Canavans disease and could have therapeutic implications. (J Child Neurol 2003;18:611—615).

High levels of orexin A in the brain of the mouse model for phenylketonuria: possible role of orexin A in hyperactivity seen in children with PKU

Phenylketonuria (PKU) is a metabolic disorder caused by phenylalanine hydroxylase deficiency leading to increased levels of phenylalanine in the brain. Hyperactivity is reportedly induced by a high level of orexin A, and therefore orexin A content was studied in the PKU mice. Hypothalamus and brain stem had higher levels of orexin A compared to cerebrum and cerebellum both in wild type and PKU mice brains as observed by radioimmunoassay method. Interestingly, all these regions of the brain in PKU mouse showed a higher level of orexin A compared to the wild type. Heart and plasma also had higher levels of orexin A in PKU compared to the wild type. Immunohistochemical analysis revealed an increased number of orexin A–stained cells in the brain and heart of PKU mouse compared to the wild type. This is the first report of increased level of orexin in the PKU mouse brain. Hyperactivity is commonly observed in children with PKU; thus these findings suggest that orexin A is a contributing factor for the hyperactivity.

Chapter 31 – The Mucopolysaccharidoses

The mucopolysaccharidoses are a group of inherited disorders caused by specific enzyme deficiencies in the degradation of the glycosaminoglycans (mucopolysaccharides). Enzyme deficiencies result in the accumulation of glycosaminoglycans in lysosomes of various tissues and in the excessive excretion of partially degraded glycosaminoglycans in urine. Clinical manifestations of the mucopolysaccharidoses depend on the specific enzyme deficiency, the end organ affected, and the accumulation of glycosaminoglycans in the affected organs. In diseases in which the brain is not involved, there is no mental retardation. On the other hand, if the brain is affected and other somatic manifestations are minimal, the coarse features that are characteristic of the mucopolysaccharidoses are not as prominent. Specific degradative lysosomal enzyme deficiencies have been identified for all the mucopolysaccharidoses. The glycosaminoglycans that are stored and excreted in the urine of the various mucopolysaccharidoses are dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin 4/6 sulfates.

Chapter 32 – The Mucolipidoses

The mucolipidoses emerged as a unique group of inherited disorders that originally were considered to be part of the mucopolysaccharidoses. The storage material within tissues or cultured skin fibroblasts includes lipids and glycosaminoglycans. These disorders are not characterized by mucopolysacchariduria.