Persistent Expression of Dopamine-Synthesizing Enzymes 15 Years After Gene Transfer in a Primate Model of Parkinson's Disease.

Restoring dopamine production in the putamen through gene therapy is a straightforward strategy for ameliorating motor symptoms for Parkinson's disease (PD). In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity-based primate model of PD, we previously showed the safety and efficacy of adeno-associated viral (AAV) vector-mediated gene delivery to the putamen of three dopamine-synthesizing enzymes (tyrosine hydroxylase [TH], aromatic l-amino acid decarboxylase [AADC], and guanosine triphosphate cyclohydrolase I [GCH]) up to 10 months postprocedure. Although three of four monkeys in this study have previously undergone postmortem analysis, one monkey was kept alive for 15 years after gene therapy to evaluate long-term effects. Here, we report that this monkey showed behavioral recovery in the right-side limb that remained unchanged for 15 years, at which time euthanasia was carried out owing to onset of senility. Immunohistochemistry of the postmortem brain from this monkey revealed persistent expression of TH, AADC, and GCH genes in the lesioned putamen. Transduced neurons were broadly distributed, with the estimated transduction region occupying 91% of the left postcommissural putamen. No signs of cytotoxicity or Lewy body pathology were observed in the AAV vector-injected putamen. This study provides evidence of long-term safety and efficacy of the triple-transduction method as a gene therapy for PD.

Prevention of cerebral embolism progression by emergency surgery of the left atrial myxoma.

A 21-year-old woman developed left hemiparesis during work and was hospitalized. Her National Institutes of Health Stroke Scale score was 4. Hyperintense areas in the left basal ganglia, corona radiata, and cortex of the temporal lobe were found by brain diffusion-weighted magnetic resonance imaging, indicating acute cerebral infarction. Echocardiography showed a giant mass of diameter 7 × 4 cm in the left atrium. Therefore, she was diagnosed with cerebral embolism due to a left atrial myxoma. Currently, thrombolytic therapy may continue to be effective because the embolic source may be composed of tumor tissue itself. In case of atrial myxoma, we considered that the use of tPA as emergency treatment in all patients with infarction by atrial myxoma may be questioned. Thus, cardiac tumor extraction was performed the next day after hospitalization without thrombolytic therapy. The excised myxoma measured 7 × 6 × 4 cm. The patient recovered and her neurological symptoms also improved. Furthermore, her National Institutes of Health Stroke Scale score improved to 0. Thirteen days after admission, the patient was discharged from our hospital. Cardiac myxoma is often associated with a high risk of embolic episodes, which emphasizes the need for prompt surgical excision as soon as the diagnosis is confirmed.

Preoperative high-dose steroid has long-term beneficial effects for myasthenia gravis.

Previous studies addressing preoperative steroid treatment have revealed that control of myasthenia gravis (MG) with steroids prior to surgery appeared to stabilize postoperative status. The purpose of our study was to clarify the clinical benefits of the preoperative programmed high-dose steroid treatment on the long-term outcomes of MG patients. We retrospectively reviewed the records of 171 MG patients who were followed up after undergoing thymectomy in our hospital between 1988 and 2006. One hundred and thirteen patients in the programmed treatment group had received preoperative steroid treatment, while 58 patients received no steroid treatment during the preoperative period. Clinical remission, which was defined as the achievement of the modified pharmacologic remission (PR) for at least 1 year, and clinical benefits were compared between the two groups. With regard to the remission after thymectomy, Kaplan-Meier life-table curves for patients in the preoperative steroid treatment group versus those for patients in the no steroid preoperative treatment group revealed a significantly higher probability of the PR in the preoperative steroid treatment group (log-rank test, P < 0.01). This study might be the first, as per our knowledge, to indicate that preoperative programmed high-dose steroid treatment has long-term beneficial effects for MG patients.

[Food-borne botulism].

Botulism is a neuroparalytic disease caused by neurotoxins produced by Clostridium botulinum, and classically presents as palsies of cranial nerves and acute descending flaccid paralysis. Food-borne botulism is the most common form of botulism, and caused by preformed neurotoxins produced by Clostridium botulinum. Electrophysiological studies play an important role in the early diagnosis. Confirmation of the diagnosis is based on the detection of botulinum toxins in the patient's serum or stool. In Japan, decades ago, botulism type E occurred, though only sporadically, almost every year, but in recent years, has dramatically decreased in frequency. Botulism is a curable disease when treated early and adequately. Differential diagnosis of cranial nerves and limb muscle palsies with rapid exacerbation should include food-borne botulism.

[Food-borne botulism].

Botulism is a neuroparalytic disease caused by neurotoxins produced by Clostridium botulinum. Food-borne botulism is a kind of exotoxin-caused food intoxication. Although this disease is rarely reported in Japan now, it is a cause of great concern because of its high mortality rate, and botulism cases should be treated as a public health emergency. Botulism classically presents as acute symmetrical descending flaccid paralysis. Its diagnosis is based on the detection of botulinum toxins in the patient's serum or stool specimens. Electrophysiologic tests of such patients show reduced compound muscle action potentials (CMAPs), low amplitudes and short durations of motor unit potentials (MUPs), and mild waning in repetitive low-frequency stimulations. Single fiber electromyography (EMG) is particularly useful for the diagnosis of botulism. We report a case of food-borne botulism that we had encountered. An 83-year-old man with rapidly progressive diplopia, dysphagia, and tetraplegia was hospitalized; he required intensive care, including artificial ventilatory support. Electrophysiologic tests yielded findings compatible with botulism. We made a clinical diagnosis of food-borne botulism and administered antitoxin on the seventh disease day. The patient's motor symptoms started ameliorating several days after the antitoxin injection. Subsequently, botulinum toxin type A was detected in the patient's serum specimen by using a bioassay, and the type A gene and silent B gene were detected in his serum specimen by using polymerase chain reaction (PCR). C. botulinum was also obtained from stool culture on the 17th and 50th disease days. Botulism is a curable disease if treated early. Although it is a rare condition, it should always be considered in the differential diagnosis of patients with rapid onset of cranial nerve and limb muscle palsies.

[Practice of botulism].

Botulism is a rare but potentially life-threatening motor and autonomic paralytic syndrome caused by the action of neurotoxins released by Clostridium botulinum. The modern syndromes of botulism have 6 forms: 4 naturally occurring syndromes and 2 human-induced syndromes. Food-borne botulism is caused by ingestion of foods contaminated with botulinum toxin. Wound botulism is caused by C. botulinum infection of a wound and in situ toxin production. Infant botulism and adult intestinal botulism are caused by intestinal colonization and in situ toxin production. Air-borne botulism results from aerosolization of botulinum toxin, whereas iatrogenic botulism occurs due to injection of a large dose of the toxin. Another human-induced form may occur when the aerosolized toxin is released as an act of bioterrorism. All forms of botulism produce a similar syndrome: symmetrical cranial motor nerve palsies followed by descending, symmetrical motor weakness, which may progress to respiratory failure requiring intubation and respiratory support. The clinical diagnosis can be confirmed by electrophysiological studies. Laboratory diagnosis includes serum analysis of toxin by bioassay in mice. Analysis of stool, vomitus, and suspected food items may reveal the toxin. In wound botulism, isolation of C. botulinum from the wound site is diagnostic. The mainstays of therapy are meticulous intensive care including respiratory support, when necessary, and timely administration of the antitoxin.

A phase I study of aromatic L-amino acid decarboxylase gene therapy for Parkinson's disease.

Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinson's disease (PD). We evaluated the safety, tolerability, and potential efficacy of adeno-associated virus (AAV) vector-mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinson's Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[(18)F]fluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.

Viral-mediated temporally controlled dopamine production in a rat model of Parkinson disease.

Regulation of gene expression is necessary to avoid possible adverse effects of gene therapy due to excess synthesis of transgene products. To reduce transgene expression, we developed a viral vector-mediated somatic regulation system using inducible Cre recombinase. A recombinant adeno-associated virus (AAV) vector expressing Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor alpha (CreER(T2)) was delivered along with AAV vectors expressing dopamine-synthesizing enzymes to rats of a Parkinson disease model. Treatment with 4-hydroxytamoxifen, a synthetic estrogen receptor modulator, activated Cre recombinase within the transduced neurons and induced selective excision of the tyrosine hydroxylase (TH) coding sequence flanked by loxP sites, leading to a reduction in transgene-mediated dopamine synthesis. Using this strategy, aromatic L-amino acid decarboxylase (AADC) activity was retained so that l-3,4-dihydroxyphenylalanine (L-dopa), a substrate for AADC, could be converted to dopamine in the striatum and the therapeutic effects of L-dopa preserved, even after reduction of TH expression in the case of dopamine overproduction. Our data demonstrate that viral vector-mediated inducible Cre recombinase can serve as an in vivo molecular switch, allowing spatial and temporal control of transgene expression, thereby potentially increasing the safety of gene therapy.

Intramuscular injection of AAV-GDNF results in sustained expression of transgenic GDNF, and its delivery to spinal motoneurons by retrograde transport.

Adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for amyotrophic lateral sclerosis (ALS) and other motor neuron diseases. The purpose of this report was to investigate transgenic GDNF expression at different time points post AAV mediated GDNF intramuscular delivery. An AAV vector was constructed to encode a recombinant fusion of GDNF tagged with a FLAG sequence at the C-terminal (AAV-GDNF) to distinguish it from its endogenous counterpart. A single intramuscular injection of AAV-GDNF led to substantial expression of transgenic GDNF which remained for at least 10 months in transduced gastrocnemius muscle. This transgenic GDNF was distributed in a large number of myofibers, mainly in the vicinity of the sarcolemma and predominantly concentrated at the sites of neuromuscular junctions (NMJs). Furthermore, transgenic GDNF, but not beta-galactosidase expressed as a control, was detected in the motoneurons that projected axons to the injected muscles, thus, indicating retrograde axonal transportation of the transgenic GDNF. This study provides a basis for a strategy of intramuscular AAV-GDNF delivery to protect motoneurons as a possible means of ALS treatment.

Recombinant adeno-associated viral vectors bring gene therapy for Parkinson's disease closer to reality.

The recombinant adeno-associated viral (rAAV) vector is a powerful tool for delivering therapeutic genes into mammalian brains. In rodents and non-human primates, a substantial number of striatal neurons can be transduced with high titer rAAV vectors by simple stereotaxic injection. Efficient and long-term expression of genes for dopamine (DA)-synthesizing enzymes in the striatum restored local DA production and achieved behavioral recovery in animal models of Parkinson's disease (PD). Moreover, sustained expression of a glial cell line-derived neurotrophic factor gene in the striatum rescued nigral neurons and led to functional recovery in a rat model of PD, even when treatment was delayed until after the onset of progressive degeneration. These results suggest that gene therapy using rAAV vectors may become a novel and feasible treatment for PD.

Neuroprotective effects of glial cell line-derived neurotrophic factor mediated by an adeno-associated virus vector in a transgenic animal model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for ALS and other motor neuron diseases. Because adeno-associated virus (AAV) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy of intramuscular delivery of the GDNF gene mediated by an AAV vector (AAV-GDNF) in the G93A mouse model of ALS. We show here that AAV-GDNF leads to substantial and long-lasting expression of transgenic GDNF in a large number of myofibers with its accumulation at the sites of neuromuscular junctions. Detection of GDNF labeled with FLAG in the anterior horn neurons, but not beta-galactosidase expressed as a control, indicates that most of the transgenic GDNF observed there is retrogradely transported GDNF protein from the transduced muscles. This transgenic GDNF prevents motoneurons from their degeneration, preserves their axons innervating the muscle, and inhibits the treated-muscle atrophy. Furthermore, four-limb injection of AAV-GDNF postpones the disease onset, delays the progression of the motor dysfunction, and prolongs the life span in the treated ALS mice. Our finding thus indicates that AAV-mediated GDNF delivery to the muscle is a promising means of gene therapy for ALS.

[Determination of medullasin levels for the diagnosis of multiple sclerosis].

Medullasin levels in granulocytes of patients with neurological diseases and healthy volunteers were determined by the enzyme immunoassay using mouse monoclonal antibodies against human medullasin and o-phenylenediamine-H2O2 as the detection system of the enzyme activity. One hundred twenty-one out of 159 patients with multiple sclerosis (76.1%) showed positive results (above means of normals + 2SD) in this test, while only 16.9% (24/142) of patients with non-inflammatory neurological diseases had positive results. This enzyme immunoassay method for medullasin is considered to be an useful paraclinical test for the diagnosis of multiple sclerosis.

Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes.

One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.