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.

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.

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.

Oral feeding of an immunodominant MHC donor-derived synthetic class I peptide prolongs graft survival of heterotopic cardiac allografts in a high-responder rat strain combination.

The efficacy of two synthetic major histocompatibility complex (MHC)-derived DA (RT1.Aa) 25-mer peptides (residues 56-80 and 96-120) to modulate alloreactivity was tested in Lewis (RT1.A1) responder animals. The DA peptide 56-80, but not peptide 96-120, induced delayed-type hypersensitivity (DTH). DTH was significantly reduced by oral feeding of peptide 56-80, P = 0.004. In addition, oral feeding of this peptide in combination with a short course of cyclosporin A (CsA) prolonged graft survival of 60% of heterotope transplanted DA cardiac allografts in Lewis recipient rats. Long-term survivors developed low levels of allo-antibodies against donor tissue as compared to rejecting animals and increased levels of interleukin-4 (IL-4) within the allograft. Similarly, IL-4-secreting splenocytes were identified by flow cytometry in these animals, indicating a Th2-type cytokine pattern. However, graft survival was particularly limited to cardiac allografts because donor-type skin grafts were acutely rejected in tolerant animals. It is interesting that residue alignment of peptide 56-80 to the motif of the RT1.A1 molecule showed a preferred class I motif within this sequence, suggesting indirect presentation of this peptide to recipient T cells. Thus, peptide 56-80 appears to represent a dominant epitope that can be exploited for establishing tolerance in this transplantation strain combination.

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.

Restorative gene therapy approaches to Parkinson's disease.

Perhaps one of the most exciting developments in brain research of the past decade is the advent of genetic intervention in human neurologic disease. Although there are a variety of gene transfer approaches, none of which has been perfected, gene therapy is now science fact and no longer science fiction. As technology progresses, some vectors will prove more effective for certain disease categories than others; it is too early to predict definitively which vector would be most effective for therapy in Parkinson's disease and other movement disorders. Nonetheless, it is likely that within the next year or two a gene therapy trial will be instituted in human patients with Parkinson's disease. The potential for an impact on the symptoms and progression of this disease is significant. Clinicians may be on the threshold of a new era of intervention for Parkinson's disease and other neurologic diseases, based on bypassing traditional but less selective drug-extracellular receptor interactions and instead focusing on genetic modulation of specific intracellular processes. The continuing development of small incremental changes of new dopamine agonists and pharmacologic agents will likely pale in comparison to the specificity of intracellular genetic manipulation.

In vivo expression of therapeutic human genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector.

An adeno-associated virus (AAV) vector, expressing genes for human tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC), demonstrated significantly increased production of dopamine in 293 (human embryonic kidney) cells. This bicistronic vector was used to transduce striatal cells of six asymptomatic but dopamine-depleted monkeys which had been treated with the neurotoxin MPTP. Striatal cells were immunoreactive for the vector-encoded TH after stereotactic injection for periods up to 134 days, with biochemical effects consistent with dopamine biosynthetic enzyme expression. A subsequent experiment was carried out in six more severely depleted and parkinsonian monkeys. Several TH/aadc-treated monkeys showed elevated levels of dopamine near injection tracts after 2.5 months. Two monkeys that received a beta-galactosidase expressing vector showed no change in striatal dopamine. Behavioral changes could not be statistically related to the vector treatment groups. Toxicity was limited to transient fever in several animals and severe hyperactivity in one animal in the first days after injection with no associated histological evidence of inflammation. This study shows the successful transfection of primate neurons over a period up to 2.5 months with suggestive evidence of biochemical phenotypic effects and without significant toxicity. While supporting the idea of an in vivo gene therapy for Parkinson's disease, more consistent and longer lasting biochemical and behavioral effects will be necessary to establish the feasibility of this appraoch in a primate model of parkinsonism.

Adenovirus vector-mediated gene transfer into human epileptogenic brain slices: prospects for gene therapy in epilepsy.

As a first step in the development of a gene therapy approach to epilepsy, we evaluated the ability of adenovirus vectors to direct the transfer into and expression of a marker gene in human brain slices obtained from patients undergoing surgery for medically intractable epilepsy. Following injection of adenovirus vectors containing the Escherichia coli lacZ gene into hippocampal and cortical brain slices, lacZ mRNA, beta-galactosidase protein, and enzymatic activity were detected, confirming successful gene transfer, transcription, and translation into a functional protein. Transfected cells were predominantly glial, with some neurons expressing beta-galactosidase as well. These results support the potential of adenovirus vectors to transfer genetic information into human epileptogenic brain, resulting in expression of the gene into a functional protein. These findings also have implications for the development of gene therapy approaches to certain seizure disorders. A number of potential therapeutic approaches are discussed, including the elevation of inhibitory neurotransmitter or neuropeptide levels, expression or modulation of postsynaptic receptors, and manipulation of signal transduction systems.

Analysis of harvest morbidity and radiographic outcome using autograft for anterior cervical fusion.

STUDY DESIGN: Retrospective study of 184 autologous iliac crest bone grafts used for anterior cervical fusion in 144 procedures. OBJECTIVES: To evaluate the effect of autologous iliac crest bone graft harvest site on operation and recovery and to identify patients at risk for harvest morbidity. SUMMARY OF BACKGROUND DATA: Although autologous iliac crest bone graft is considered the most successful grafting material, concerns about harvest morbidity provide a rationale for considering allograft. Data about the use of autograft therefore would assist spinal surgeons in selecting the appropriate substrates for fusion after anterior cervical decompression. METHODS: Statistical analysis based on patient gender, smoking history, obesity, and medical or pharmacologic risk factors for wound healing was used to evaluate morbidity after patient interviews and examinations. Limited assessment of radiographic outcome also was performed. RESULTS: A second operation because of donor site morbidity was performed in four patients (2.8%), but only one (0.7%) with meralgia paresthetica had permanent sequelae. Superficial wound infection or dehiscence occurred in 5.6% of patients, with a disproportionate number of women, obese patients, and those with medical risk represented. Protracted wound symptoms of pain and poor cosmesis were reported in 2.8% and 3.5% of patients, respectively, and also were found in a significant number of female and obese patients. Evidence of fusion was present in 97% of cases. CONCLUSION: Autologous iliac crest bone graft harvest results in minimal major morbidity when regional anatomy is respected and careful technique is observed. The identification of patients at risk for minor complications suggests that allograft may be appropriate in these patients; however, prospective comparison is required to identify whether graft material or technical factors determine fusion success and relative benefit.

Direct gene transfer into human epileptogenic hippocampal tissue with an adeno-associated virus vector: implications for a gene therapy approach to epilepsy.

PURPOSE: Virus vectors capable of transferring genetic information into human cells provide hope for improved therapy in several neurological diseases, including epilepsy. We evaluated the ability of an adeno-associated virus (AAV) vector to transfer and cause expression of a lacZ marker gene in brain slices obtained from patients undergoing temporal lobectomy for control of medically intractable seizures. METHODS: Human brain slices were injected with an AAV vector (AAVlacZ) encoding Escherichia coli beta-galactosidase and incubated for as long as 24 h. The presence of lacZ mRNA. beta-galactosidase protein and enzymatic activity were assayed by reverse transcriptase polymerase chain reaction (rtPCR), immunocytochemistry, and the X-Gal technique, respectively. RESULTS: AAVlacZ directed the expression in human epileptogenic brain of E. coli beta-galactosidase that had functional activity. Expression was observed in < or =5 h and was sustained for as long as the slices were viable. Morphological analysis indicated that neurons were preferentially transfected, and there was no evidence of cytotoxicity. CONCLUSIONS: Our results confirm the feasibility of using AAV vectors to transfer genes into the human CNS and in particular, into neurons. Replacement of the lacZ gene with a functional gene modulating hippocampal neuronal physiology, might allow a localized genetic intervention for focal seizures based on the stereotaxic or endovascular delivery of such a vector system into the appropriate brain region.

Parkinson's disease: the case for novel treatment strategies.

Over the past 30 years, significant strides have been made toward improving symptomatic therapy of patients with Parkinson's disease. However, limitations persist, and the incremental improvement each new drug offers a patient yields only a minimal advantage therapeutically. Therefore, to alter meaningfully the natural course of this debilitating disorder, which becomes more prevalent each year in our society, significant novel therapeutic approaches must be tested. Genetic intervention in Parkinson's disease may offer an entirely different method for treating this disease in the future, based not only on symptomatic therapy, but also on neuroprotective and/or neuroregenerative therapy which would alter the natural history of relentless progression.

MR of thoracic cord compression caused by epidural extramedullary hematopoiesis in myelodysplastic syndrome.

Spinal cord compression caused by extramedullary hematopoiesis is a rare complication of chronic anemic states, most frequently occurring in patients with beta-thalassemia. We report the MR appearance of extramedullary hematopoiesis resulting in cord compression in a patient with a myelodysplastic syndrome, which was isointense with the spinal cord on T1-weighted images and markedly hypointense on fast spin-echo T2-weighted images, and that demonstrated enhancement.

Further characterization and in situ localization of chain-like aggregates of the gliding bacteria Myxococcus fulvus and Myxococcus xanthus.

For the first time, chain-like aggregates, called "strands," have been enriched from crude cell wall preparations of liquid-grown vegetative cells of two strains of Myxococcus xanthus. These strands are highly isomorphic to macromolecular structures, previously described for Myxococcus fulvus (Lünsdorf and Reichenbach, J. Gen. Microbiol. 135:1633-1641, 1989). The strands are morphologically composed of ring elements, consisting of six or more peripheral protein masses and possibly three small central masses. The ring elements are linked by two parallel strings of filamentous proteins, called elongated elements, which keep the ring elements at a constant distance. The overall dimensions of the ring elements are 16.6 +/- 1.0 nm (n = 55) for M. xanthus Mx x48 and 16.4 +/- 1.5 nm (n = 37) for M. xanthus DK 1622. The distance between the ring elements, as a measure of the length of the elongated elements, is 16.6 +/- 1.1 nm (n = 59) for strain Mx x48 and 15.5 +/- 0.6 nm (n = 41) for strain DK 1622. Characteristically, the strands and oligomeric forms thereof show a strict association with the outer membrane. In situ studies of freeze-fractured cells of M. fulvus showed ring elements, isomorphic to those described for M. xanthus, within the periplasm; they appeared in parallel rows just below the outer membrane but not in direct contact with the cytoplasmic membrane. A three-dimensional model summarizes the morphological data. It is hypothesized that the chain-like strands, as building blocks of a more complex belt-like continuum, represent the peripheral part of the gliding machinery, which transforms membrane potential energy into mechanical work.

Functional and biosynthetic aspects of sialic acid diversity.

Sialic acids comprise a large family of N- and O-substituted neuraminic acid derivatives as components of glycoconjugates. N-Glycolylneuraminic acid is formed from N-acetylneuraminic acid by the action of the CMP-N-acetylneuraminic acid hydroxylase studied in various animals. O-Methylated sialic acids originate from the action of S-adenosylmethionine-8-O-methyltransferase studied in starfish. Sialic acids are O-acetylated at diverse positions by the action of acetyl-CoA-4-O- and -7-O-acetyltransferases found in various animals and, leading to the O-acetylation of sialic acid glycerol side chain, also in man. Some properties of these enzymes are described and biological implications discussed.

Outcome analysis for adults with spondylolisthesis treated with posterolateral fusion and transpedicular screw fixation.

The outcomes of 52 adult patients with symptomatic low-grade spondylolisthesis treated with autologous posterolateral arthrodesis and pedicle screw fixation were retrospectively reviewed. Although a 90% rate of successful fusion was obtained using this technique, only 60% of patients were considered to have good outcomes. Treatment failures consisted mostly of back pain and were not predicted by preoperative symptoms. Compensation claims and smoking had very significant adverse impacts on both employment and pain results despite high fusion rates, particularly in patients under the age of 55 years. Overall, patients who required more than one operation demonstrated poor outcomes compared to those who only needed one. However, patients with at least two prior operations or preoperative pseudoarthrosis fared particularly poorly, whereas those who had undergone only one prior surgery and had no attendant compensation issue reported good results. A trend toward poor outcome was observed in patients with postlaminectomy spondylolisthesis, versus those with isthmic or degenerative etiologies. Gender did not exert an impact on outcome. The authors conclude that autologous posterolateral arthrodesis combined with pedicle screw fixation resulted in a high fusion rate, and contributed to successful outcomes in the treatment of certain subgroups of adults with spondylolisthesis. In the absence of other risk factors, patients may obtain significant benefit from surgery despite older age and a single failed operation. Careful patient selection appears critical in predicting the maximum benefit from this technique.

Prospects for gene therapy in Parkinson's disease.

Numerous advances in in vivo and ex vivo gene-therapy approaches to Parkinson's disease offer promise for direct clinical trials in patients in the next several years. These systems are predicated on introducing gene that encode enzymes responsible for dopamine biosynthesis or neurotrophic factors that may delay nigrostriatal degeneration or facilitate regeneration. We review the current status of experimental approaches to gene therapy for Parkinson's disease. Comparative advantages and disadvantages of each system are enumerated, and preclinical trials of some of the systems are evaluated. Although the specific in vivo or ex vivo methods used for gene transfer into the brain are likely to be supplanted by newer technology over the next decade, the principles and approaches developed in current studies likely will remain the same.

Subvocal speech, reading rate, and comprehension.

The relationship of subvocal speech and reading rate to comprehension of 25 children, ranging from 8 to 15 years of age, was investigated by means of electromyographic (EMG) recordings taken while the subjects silently read two meaningful passages. The first was orthographically regular, and the second was composed of approximately sixty percent homophones, Labial muscle action recordings, latencies, and comprehension measures were obtained. Variables derived from these measures were used to predict reading age. Profiles derived from the EMGs provided information about how each reader processed the information from the reading passages. The empirical results of the study provide strong support for the valuable role of subvocal speech in the extraction of information and the importance of readers demonstrating the ability to use flexibility of the reading process when reading for meaning.

Transfection of human lactotroph adenoma cells with an adenovirus vector expressing tyrosine hydroxylase decreases prolactin release.

Pituitary adenomas are common intracranial neoplasms, for which surgery and radiation are usually not curative. In attempting to develop gene therapy as a better approach to treating pituitary adenomas, we chose lactotroph adenomas as a model. The rationale for the use of this model is based on the observation that dopamine agonists decrease prolactin secretion by lactotroph adenomas, and also decrease their size. We transfected primary cultures of human lactotroph adenoma cells with an adenovirus vector containing a cDNA which encodes a human tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. Transfection induced expression of tyrosine hydroxylase and increased production of dopamine, resulting in the predicted biologic effect of decreased prolactin secretion. These results demonstrate the potential for gene therapy of lactotroph adenomas and perhaps other pituitary adenomas, which are less amenable to pharmacologic treatment than lactotroph adenomas.