Christopher G. Janson

Gene Therapy of Canavan Disease: AAV-2 Vector for Neurosurgical Delivery of Aspartoacylase Gene (ASPA) to the Human Brain

This clinical protocol describes virus-based gene transfer for Canavan disease, a childhood leukodystrophy. Canavan disease, also known as Van Bogaert-Bertrand disease, is a monogeneic, autosomal recessive disease in which the gene coding for the enzyme aspartoacylase (ASPA) is defective. The lack of functional enzyme leads to an increase in the central nervous system of the substrate molecule, N-acetyl-aspartate (NAA), which impairs normal myelination and results in spongiform degeneration of the brain. No effective treatment currently exists; however, virus-based gene transfer has the potential to arrest or reverse the course of this otherwise fatal condition. This procedure involves neurosurgical administration of approximately 900 billion genomic particles (approximately 10 billion infectious particles) of recombinant adeno-associated virus (AAV) containing the aspartoacylase gene (ASPA) directly to affected regions of the brain in each of 21 patients with Canavan disease. Pre- and post-delivery assessments include a battery of noninvasive biochemical, radiological, and neurological tests. This gene transfer study represents the first clinical use of AAV in the human brain and the first instance of viral gene transfer for a neurodegenerative disease.

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

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.

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 β-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 × 105 neurons in vivo per microliter of vector, using the strongest expression cassette.

Long-Term Follow-Up After Gene Therapy for Canavan Disease

Gene therapy for Canavan disease results in a decrease in pathologically elevated N-acetyl-aspartate concentrations in the brain and long-term clinical stabilization. Gene Therapy for Canavan Disease Canavan disease is a fatal childhood neurodegenerative disorder for which there is no effective treatment. It is caused by a defect in a single gene (ASPA) that results in a deleterious buildup of N-acetyl-aspartate in the brain. This process starts at birth and is accompanied by a failure to form and maintain myelin, the protective sheath surrounding nerves. As a brain-specific disorder with simple Mendelian inheritance, Canavan disease represents an excellent target for enzyme replacement using gene therapy. Leone et al. now report the long-term results of gene therapy in 13 Canavan disease patients using adeno-associated viral vector delivery of the ASPA gene. The investigators found that gene therapy was safe and led to a decrease in N-acetyl-aspartate in the brain, together with decreased seizure frequency and clinical stabilization. Clinical stabilization was greatest in the youngest patients. Early detection and treatment with gene therapy–mediated enzyme replacement in the neonatal period may offer the best opportunity for a reduction in symptoms and long-term stabilization in patients with Canavan disease. Canavan disease is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and increased concentrations of the substrate N-acetyl-aspartate (NAA) in the brain. Accumulation of NAA results in spongiform degeneration of white matter and severe impairment of psychomotor development. The goal of this prospective cohort study was to assess long-term safety and preliminary efficacy measures after gene therapy with an adeno-associated viral vector carrying the ASPA gene (AAV2-ASPA). Using noninvasive magnetic resonance imaging and standardized clinical rating scales, we observed Canavan disease in 28 patients, with a subset of 13 patients being treated with AAV2-ASPA. Each patient received 9 × 1011 vector genomes via intraparenchymal delivery at six brain infusion sites. Safety data collected over a minimum 5-year follow-up period showed a lack of long-term adverse events related to the AAV2 vector. Posttreatment effects were analyzed using a generalized linear mixed model, which showed changes in predefined surrogate markers of disease progression and clinical assessment subscores. AAV2-ASPA gene therapy resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy, with some improvement in seizure frequency and with stabilization of overall clinical status.

Human Intrathecal Transplantation of Peripheral Blood Stem Cells in Amyotrophic Lateral Sclerosis

913 WE WRITE TO COMMENT BRIEFLY on the neurological literature on blood-derived neural stem cells, and to offer a case report regarding the safety of a simple procedure to introduce stem cells to the cerebrospinal fluid (CSF) compartment. For decades there has been evidence for cells of common neural and hematopoietic potential; this phenomenon was originally demonstrated by the presence of pluripotent cells resident in the adult brain that could form hematopoietic colonies in irradiated animals, as well as antigenic cross-reactivity between certain neural and hematopoietic cells (1–3). Recent studies have reproduced and confirmed the pleuripotency of neuro-hematopoietic cells, and showed that “blood can turn into brain” and vice-versa (4–6). Dr. W. Krivit at Harvard was the first to show that bone marrow transplantation (BMT) ameliorates some forms of central nervous system (CNS) genetic and metabolic disease (7), thought to be due to cellular margination and passage of the blood–brain barrier, which raised the intriguing idea of grafting blood cells directly to the human CNS. Indeed, administration of bone marrow-derived cells to the brain with neuronal engraftment has been demonstrated in animal models (8), and published reports now show blood stem cell differentiation along various neural lineages, including neurons and astrocytes. In an important clinical experiment conducted in Sweden, a group of patients received an intravenous (i.v.) label of bromodeoxyuridine (BrdU), which detects division of new cells. This work proved that neural progenitor cells exist in the brain of the adult human (9), thus generalizing an observation from primates and other animals that had generated much controversy, because it challenged a long-held tenet that brain cells could not be replenished or regenerate in situ. The precise source of neural progenitor cells is still a matter of debate and speculation, but the consensus is that they appear to be enriched around vascular tissues in the forebrain subventricular zone, the ependyma, and the dentate gyrus of the hippocampus. Given the close association between the vasculature and the sources of neural stem cells (10), one possibility is that neural stem cells arise in the adult human brain both from primordial brain stem cells and from migratory hematopoietic-derived cells. Considering the lack of effective drugs for amyotrophic lateral sclerosis (ALS) and accumulating evidence that primordial blood-derived cells can adopt a neuronal or glial fate, in 1999 we began exploring a blood stem cell selection protocol for application to ALS, in parallel with long-term plans for other experimental approaches. The objective was to bring to the clinic any promising drug or treatment protocol that had already been approved for human use. CD341 stem cell selection protocols are widely used in bone marrow transplants and are approved by the U.S. Food and Drug Administration (FDA), although clinical application for ALS is “off-label.” Immune ablation followed by BMT has already been performed in studies on multiple sclerosis and other primary CNS conditions; however, the mechanism we anticipated is one of cell grafting, CNS-specific differentiation, and local trophic effects, rather than the reconstitution of pharmacologically depleted immune cells. To demonstrate safety and generate preliminary data in support of dosing and method of deliver, we transplanted adult human marrow and fetal CD341 cells directly into the nonimmunosuppressed monkey spinal cord and intrathecal space. The monkeys underwent laminectomies, and xenogeneic human cells were introduced to the midline of the dorsal spinal cord at multiple sites using a Hamilton syringe and 30-gauge needle, without adverse behavioral or serological responses. The limited work available in the literature on cord blood transplants to superoxide dismutase (SOD1) mice also supported our ra-

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.

Mild-onset presentation of Canavan's disease associated with novel G212A point mutation in aspartoacylase gene.

We describe two sisters with a mild‐onset variant of Canavans disease who presented at age 50 and 19 months with developmental delay but without macrocephaly, hypotonia, spasticity, or seizures. Remarkably, both patients had age‐appropriate head control, gross motor development, and muscle tone. There were very mild deficits in fine motor skills, coordination, and gait. Both sisters had a history of strabismus, but otherwise vision was normal. The older child showed evidence of mild cognitive and social impairment, whereas language and behavior were normal for age in the infant. Both patients were found to be compound heterozygotes for C914A (A305E) and G212A (R71H) mutations in ASPA. Like all other known ASPA mutations, this previously unknown G212A mutation appears to have low absolute enzyme activity. Nevertheless, it is associated in these patients with an extremely benign phenotype that is highly atypical of Canavans disease. Biochemical and clinical data were evaluated using a generalized linear mixed model generated from 25 other subjects with Canavans disease. There were statistically significant differences in brain chemistry and clinical evaluations, supporting a distinct variant of Canavans disease. Future studies of ASPA enzyme structure and gene regulation in these subjects could lead to a better understanding of Canavans pathophysiology and improvements in ASPA gene therapy Ann Neurol 2006;59:428–431

Lithium citrate reduces excessive intra-cerebral N-acetyl aspartate in Canavan disease.

Our group has previously reported the first clinical application of lithium in a child affected by Canavan disease. In this study, we aimed to assess the effects of lithium on N-acetyl aspartate (NAA) as well as other end points in a larger cohort. Six patients with clinical, laboratory and genetic confirmation of Canavan disease were recruited and underwent treatment with lithium. The battery of safety and efficacy testing performed before and after sixty days of treatment included Gross Motor Function Testing (GMFM), Magnetic Resonance Imaging (MRI) Proton Magnetic Spectroscopy (H-MRS) as well as blood work. The medication was safe without any clinical or laboratory evidence for toxicity. Parental reports indicated improvement in alertness and social interactions. GMFM did not show statistically significant improvement in motor development. H-MRS documented an overall drop in NAA which was statistically significant in the basal ganglia. T1 measurements recorded on MRI studies suggested a mild improvement in myelination in the frontal white matter after treatment. Diffusion Tensor Imaging was available in two patients and suggested micro-structural improvement in the corpus callosum. The results suggest that lithium administration may be beneficial in patients with Canavan disease.

Treatment of spinocerebellar ataxia with buspirone

Preliminary data suggest potential benefit of 5-HT receptor agonists in the treatment of ataxias. We studied the effects of buspirone in a cohort of twenty patients with spinocerebellar ataxia (SCA). Twenty patients were treated in this double-blind, placebo controlled, cross-over trial with either buspirone HCl 30 mg twice daily or placebo for 3 months. Buspirone was not shown to be superior to placebo in the treatment of patients with SCA.

Improvement in clinical outcomes following optimal targeting of brain ventricular catheters with intraoperative imaging.

OBJECT The accurate placement of cerebral ventricular shunt catheters in hydrocephalus is an important clinical problem. Malfunction of shunts remains their most common complication and greatest liability, and the influence of catheter position on shunt function remains poorly defined. The objectives of this study were as follows: 1) determine the accuracy of intraventricular catheter placement with respect to a historically favored target, defined as a 1-cm radius sphere at the anterior lip of the ipsilateral foramen of Monro; 2) confirm that this target represents a satisfactory site for frontal and occipital catheter placement by examining whether inaccuracy is associated with more shunt failures; and 3) determine whether catheter trajectory, use of image confirmation, or other factors are associated with either the accuracy or the longevity of shunts. METHODS A retrospective cohort analysis was conducted on 236 patients with 426 ventricular shunts placed or revised at the University of Minnesota over a 10-year period. RESULTS Accuracy of shunt placement was optimal in 43.9% of patients and suboptimal or poor in 56.1% of patients. Time to failure was significantly affected by the accuracy of catheter placement with respect to the ipsilateral foramen of Monro, with a 57% higher risk of failure with suboptimal placement (hazard ratio [HR] 1.57, 95% CI 1.26-1.96; p < 0.001) and a 66% higher risk with poor placement (HR 1.66, 95% CI 1.45-1.89; p < 0.001) relative to optimal placement. The odds of highly suboptimal or unacceptable placement were significantly increased by lack of any intraoperative imaging (OR 5.89, 95% CI 2.36-14.65; p < 0.001). Use of a nonfrontal posterior trajectory also showed a trend toward poor placement (OR 1.64, p = 0.138). CONCLUSIONS The historical target for catheter tip placement within 1 cm of the foramen of Monro in the ipsilateral lateral ventricle was associated with significantly longer revision-free survival compared with other locations. This effect remained significant after adjusting for age and whether there was a prior history of shunting. The accuracy of catheter placement in both pediatric and adult patients was strongly associated with use of intraoperative fluoroscopic confirmation. In analyses comparing intraoperative fluoroscopy and no imaging, there was a non-statistically significant difference in the 3-year time to failure, but the worst-case scenario of catastrophic short-term failure was almost completely avoided with fluoroscopy. The authors conclude that accuracy of placement is critical for shunt survival, and that use of intraoperative imaging confirmation may optimize outcomes by avoiding the majority of unacceptable placements.