Natural history of Canavan disease revealed by proton magnetic resonance spectroscopy (1H-MRS) and diffusion-weighted MRI.

Canavan disease is a childhood leukodystrophy caused by mutations in the gene for human aspartoacylase ( ASPA), which leads to an abnormal accumulation of the substrate molecule N-acetyl-aspartate (NAA) in the brain. This study was designed to model the natural history of Canavan disease using MRI and proton magnetic resonance spectroscopy ( (1)H-MRS). NAA and various indices of brain structure (morphology, quantitative T1, fractional anisotropy, apparent diffusion coefficient) were measured in white and gray matter regions during the progression of Canavan disease. A mixed-effects statistical model was used to fit all outcome measures. Longitudinal data from 28 Canavan patients were directly compared in each brain region with reference data obtained from normal, age-matched pediatric subjects. The resultant model can be used to non-invasively monitor the natural history of Canavan disease or related leukodystrophies in future studies involving drug, gene therapy, or stem cell treatments.

Immune responses to AAV in a phase I study for Canavan disease.

BACKGROUND: Canavan disease is a rare leukodystrophy with no current treatment. rAAV-ASPA has been developed for gene delivery to the central nervous system (CNS) for Canavan disease. This study represents the first use of a viral vector in an attempt to ameliorate a neurodegenerative disorder. METHODS: Subjects received intracranial infusions via six cranial burr holes. Adeno-associated virus, serotype 2 (AAV2), mediated intraparenchymal delivery of the human aspartoacylase cDNA at a maximum dose of 1 x 10(12) vector genomes per subject. The immune response and safety profiles were monitored in the follow-up of ten subjects. RESULTS: Following rAAV2 administration, we found no evidence of AAV2 neutralizing antibody titers in serum for the majority of subjects tested (7/10). In a subset (3/10) of subjects, low to moderately high levels of AAV2 neutralizing antibody with respect to baseline were detected. In all subjects, there were minimal systemic signs of inflammation or immune stimulation. In subjects with catheter access to the brain lateral ventricle, cerebrospinal fluid was examined and there was a complete absence of neutralizing antibody titers with no overt signs of brain inflammation. CONCLUSIONS: rAAV2 vector administration to the human CNS appears well tolerated. The low levels of immune response to AAV2 detected in 3/10 subjects in this study suggest at this dose and with intraparenchymal administration this approach is relatively safe. Long-term monitoring of subjects and expansion to phase II/III will be necessary in order to make definitive statements on safety and efficacy.

Effects of AAV-2-mediated aspartoacylase gene transfer in the tremor rat model of Canavan disease.

The tremor rat is a spontaneous epilepsy model with a seizure phenotype caused by a deletion in the aspartoacylase (ASPA) gene. The absence of ASPA expression in these animals results in undetectable levels of enzyme activity and the accumulation of the substrate N-acetyl-aspartate (NAA) in brain, leading to generalized myelin vacuolation and severe motor and cognitive impairment. In support of human gene therapy for CD, recombinant adeno-associated viral vector (AAV-2) expressing ASPA was stereotactically delivered to the tremor rat brain and effects on the mutant phenotype were measured. AAV-ASPA gene transfer resulted in elevated aspartoacylase bioactivity compared to untreated mutant animals and elicited a significant decrease in the pathologically elevated whole-brain NAA levels. Assessment of motor function via quantitative rotorod testing demonstrated that rats injected with AAV-ASPA significantly improved on tests of balance and coordinated locomotion compared to animals receiving control vectors. This study provides evidence that AAV-2-mediated aspartoacylase gene transfer to the brain improves biochemical and behavioral deficits in tremor rat mutants (tm/tm) and supports the rationale of human gene transfer for Canavan disease.

Functional regulatory regions of human transcription factor MEF2C.

Myocyte enhancer-binding factor 2C (MEF2C), a transcription factor expressed at high levels in muscle and brain, is implicated in the terminal differentiation and post-mitotic survival of neurons. In this study MEF2C deletion mutants and naturally-occurring isoforms were transfected into COS and P19 cells with two different reporter genes, to test the relative transcriptional activities of the MEF2C constructs. Deletion of parts of the carboxy terminus, in particular amino acids 387-473, enhanced transcriptional activation. A region rich in serine, threonine, proline, and tyrosine from amino acids 312-367 was sufficient to activate transcription at low levels when coupled to amino acids 1-86, which contain the DNA-binding (MADS/MEF) domain of MEF2C, but also depended on amino acids 87-311 for full effect. A construct with amino acids 312-350 missing showed significantly less transcriptional activation than proteins containing this sequence. MEF2C constructs were uniformly localized to the cell nucleus by immunostaining with an antibody to the constant N-terminal region of MEF2C. Western blot and gel shift studies of extracts from transfected cells and from in vitro transcription/translation suggest that variation in the amount of protein expressed or in DNA-binding properties does not account for observed differences in transcriptional activation. This structural information may be useful for elucidating the mechanisms of MEF2C in interacting with other factors to regulate target genes.

Viral vectors as part of an integrated functional genomics program.

Over the past decade, viral vectors have slowly gained mainstream acceptance in the neuroscience and genetics communities for the in vivo study of gene function [1]. Using stereotactic techniques, it is possible to characterize neuroanatomical relationships through the delivery of neurotropic viral vectors to specific brain regions. More sophisticated studies combine viral vectors with other methods of genetic manipulation such as germline transgenic mice. As more is learned about the properties of different viral vectors, it has become possible to use viral vectors to test hypotheses about the function of genes, through targeted in vivo delivery to the central nervous system (CNS). The effects of gene expression in the brain can be measured on the molecular, biochemical, electrophysiological, morphological, and behavioral levels. We propose that viral vectors should be considered as part of an integrated functional genomics platform in the CNS.

Viral-based gene transfer to the mammalian CNS for functional genomic studies.

A fundamental problem in neuroscience has been the creation of suitable in vivo model systems to study basic neurological phenomena and pathology of the central nervous system (CNS). Somatic cell genetic engineering with viral vectors provides a versatile tool to model normal brain physiology and a variety of neurological diseases.

Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.

This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette.

Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease.

With the ultimate goal of developing safe and effective in vivo gene therapy for the treatment of Canavan disease and other neurological disorders, we developed a non-viral lipid-entrapped, polycation-condensed delivery system (LPD) for central nervous system gene transfer, in conjunction with adeno-associated virus (AAV)-based plasmids containing recombinant aspartoacylase (ASPA). The gene delivery system was tested in healthy rodents and primates, before proceeding to preliminary studies in 2 children with Canavan disease. Toxicity and expression testing was first carried out in human 293 cells, which demonstrated effective transduction of cells and high levels of functional ASPA activity. We performed in vivo toxicity and expression testing of LPD/pAAVaspa and LPD/pAAVlac in rodents, which demonstrated widespread gene expression for more than 10 months after intraventricular delivery, and local expression in deep brain nuclei and white matter tracts for more than 6 months after intraparenchymal injections, with no significant adverse effects. We also performed intraventricular delivery of LPD/pAAVaspa to 2 cynomologous monkeys, with 2 additional monkeys receiving LPD and saline controls. None of the monkeys demonstrated significant adverse effects, and at 1 month the 2 LPD/pAAVaspa monkeys were positive for human ASPA transcript by reverse transcriptase polymerase chain reaction of brain tissue punches. Finally, we performed the first in vivo gene transfer study for a human neurodegenerative disease in 2 children with Canavan disease to assess the in vivo toxicity and efficacy of ASPA gene delivery. Our results suggest that LPD/pAAVaspa is well tolerated in human subjects and is associated with biochemical, radiological, and clinical changes.

Multi-site partitioned delivery of human tyrosine hydroxylase gene with phenotypic recovery in Parkinsonian rats.

Parkinson's disease (PD) is a leading candidate for neurological gene therapy, given our increasing knowledge of the functional anatomy of the striatonigral system and the localized nature of the affected cell populations. Here we report that stereotactic introduction of a human tyrosine hydroxylase (TH-2) gene using multi-site partitioned doses resulted in behavioral recovery in 6-OHDA-lesioned rats, with transient 100% recovery observed in some animals. We also show correlation between numbers of TH-immunoreactive cells and loss of apomorphine induced rotation, with a near-linear relationship between TH expression and phenotypic recovery. Furthermore, the data suggest that only a fraction of striatal cells need to be transduced in order to exert phenotypic effects, and therefore TH partitioned gene transfer may have clinical potential in PD.

Global CNS gene transfer for a childhood neurogenetic enzyme deficiency: Canavan disease.

The neurogenetic prototypic disease on which we chose to test our gene therapy strategy is Canavan disease (CD). CD is an autosomal recessive leukodystrophy associated with spongiform degeneration of the brain. At present the disease is uniformly fatal in affected probands. CD is characterized by mutations in the aspartoacylase (ASPA) gene, resulting in loss of enzyme activity. In this review, recent evidence is summarized on the etiology and possible treatments for CD. In particular, we discuss two gene delivery systems representing recent advances in both viral and liposome technology: a novel cationic liposome-polymer-DNA (LPD) complex, DCChol/DOPE-protamine, as well as recombinant adeno-associated virus (AAV) vectors.

Long-term development for girls and boys at age 16-18 as related to birth weight and gestational age.

The present study was based on data from a longitudinal research program which consisted of 12,032 children, born in the Stockholm area in 1953 of which there were 494 children born with low birth weight (LBW, 2500 g or less). For all children at age 16 it was apparent that adjustment and psychiatric disturbances as well as juvenile delinquency were not related to birth weight and gestational age. LBW girls born at term, had significantly lower school grades, at age 16, than NBW (normal birth weight) girls. NBW boys born pre-term had lower school grades than NBW boys born at term. It is suggested that childhood development is gender related; in girls the birth weight--and in boys the length of the pregnancy was related to school marks at age 16. For boys at 18 years of age at the military draft, it was shown that LBW boys had smaller body size and lower IQ-test scores as compared to NBW boys. Additionally the length of the pregnancy was related to some measures of body size but not to IQ-test scores.

School marks and IQ-test scores for low birth weight children at the age of 13.

The present study was based on data from a longitudinal research program. The cohort consisted of 12,079 children, born in the Stockholm area in 1953. There were 494 children born with low birth weight (LBW; 2500 g or less). The results of the present study showed, that the LBW children had significantly lower school marks and intelligence-test scores (numerical, verbal and logical abilities) at the age of 13 than the normal birth weight children (NBW). For girls reared in non-manual socio-economic status (SES), decreased school marks and IQ-test scores were related to birth weight, and this was especially pronounced for LBW girls born after pregnancy week 37. For boys, however, no decreased school marks and IQ-test scores were related to birth weight and gestational age, with the exception of verbal ability for LBW boys born after pregnancy week 37 reared in non-manual SES.