Mapping and targeted viral activation of pancreatic nerves in mice reveal their roles in the regulation of glucose metabolism.

A lack of comprehensive mapping of ganglionic inputs into the pancreas and of technology for the modulation of the activity of specific pancreatic nerves has hindered the study of how they regulate metabolic processes. Here we show that the pancreas-innervating neurons in sympathetic, parasympathetic and sensory ganglia can be mapped in detail by using tissue clearing and retrograde tracing (the tracing of neural connections from the synapse to the cell body), and that genetic payloads can be delivered via intrapancreatic injection to target sites in efferent pancreatic nerves in live mice through optimized adeno-associated viruses and neural-tissue-specific promoters. We also show that, in male mice, the targeted activation of parasympathetic cholinergic intrapancreatic ganglia and neurons doubled plasma-insulin levels and improved glucose tolerance, and that tolerance was impaired by stimulating pancreas-projecting sympathetic neurons. The ability to map the peripheral ganglia innervating the pancreas and to deliver transgenes to specific pancreas-projecting neurons will facilitate the examination of ganglionic inputs and the study of the roles of pancreatic efferent innervation in glucose metabolism.

Assessment of disease severity in late infantile neuronal ceroid lipofuscinosis using multiparametric MR imaging.

BACKGROUND AND PURPOSE: LINCL is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene that encodes for tripeptidyl peptidase 1, a lysosomal enzyme necessary for the degradation of products of cellular metabolism. With the goal of developing quantitative noninvasive imaging biomarkers sensitive to disease progression, we evaluated a 5-component MR imaging metric and tested its correlation with a clinically derived disease-severity score. MATERIALS AND METHODS: MR imaging parameters were measured across the brain, including quantitative measures of the ADC, FA, nuclear spin-spin relaxation times (T2), volume percentage of CSF (%CSF), and NAA/Cr ratios. Thirty MR imaging datasets were prospectively acquired from 23 subjects with LINCL (2.5-8.4 years of age; 8 male/15 female). Whole-brain histograms were created, and the mode and mean values of the histograms were used to characterize disease severity. RESULTS: Correlation of single MR imaging parameters against the clinical disease-severity scale yielded linear regressions with R2 ranging from 0.25 to 0.70. Combinations of the 5 biomarkers were evaluated by using PCA. The best combination included ADC, %CSF, and NAA/Cr (R2=0.76, P<.001). CONCLUSIONS: The multiparametric disease-severity score obtained from the combination of ADC, %CSF, and NAA/Cr whole-brain MR imaging techniques provided a robust measure of disease severity, which may be useful in clinical therapeutic trials of LINCL in which an objective assessment of therapeutic response is desired.

Bidirectional regulation of emotional memory by 5-HT1B receptors involves hippocampal p11.

Cognitive impairments are common in depression and involve dysfunctional serotonin neurotransmission. The 5-HT1B receptor (5-HT(1B)R) regulates serotonin transmission, via presynaptic receptors, but can also affect transmitter release at heterosynaptic sites. This study aimed at investigating the roles of the 5-HT(1B)R, and its adapter protein p11, in emotional memory and object recognition memory processes by the use of p11 knockout (p11KO) mice, a genetic model for aspects of depression-related states. 5-HT(1B)R agonist treatment induced an impairing effect on emotional memory in wild type (WT) mice. In comparison, p11KO mice displayed reduced long-term emotional memory performance. Unexpectedly, 5-HT(1B)R agonist stimulation enhanced memory in p11KO mice, and this atypical switch was reversed after hippocampal adeno-associated virus mediated gene transfer of p11. Notably, 5-HT(1B)R stimulation increased glutamatergic neurotransmission in the hippocampus in p11KO mice, but not in WT mice, as measured by both pre- and postsynaptic criteria. Magnetic resonance spectroscopy demonstrated global hippocampal reductions of inhibitory GABA, which may contribute to the memory enhancement and potentiation of pre- and post-synaptic measures of glutamate transmission by a 5-HT(1B)R agonist in p11KO mice. It is concluded that the level of hippocampal p11 determines the directionality of 5-HT(1B)R action on emotional memory processing and modulates hippocampal functionality. These results emphasize the importance of using relevant disease models when evaluating the role of serotonin neurotransmission in cognitive deficits related to psychiatric disorders.

Progress and prospects: gene therapy clinical trials (part 1).

Over the last two decades gene therapy has moved from preclinical to clinical studies for many diseases ranging from single gene disorders such as cystic fibrosis and Duchenne muscular dystrophy, to more complex diseases such as cancer and cardiovascular disorders. Gene therapy for severe combined immunodeficiency (SCID) is the most significant success story to date, but progress in many other areas has been significant. We asked 20 leaders in the field succinctly to summarize and comment on clinical gene therapy research in their respective areas of expertise and these are published in two parts in the Progress and Prospect series.

Neurological deterioration in late infantile neuronal ceroid lipofuscinosis.

BACKGROUND: Late infantile neuronal ceroid lipofuscinosis (LINCL) is associated with progressive degeneration of the brain and retina starting in early childhood. METHODS: Thirty-two individual neurologic, ophthalmologic, and CNS imaging (MRI and MRS) assessments of 18 children with LINCL were analyzed. Disease severity was followed by two rating scales, one previously established but modified to solely assess the brain and exclude the retinal disease (modified Hamburg LINCL scale), and a newly developed scale, with expanded evaluation of the CNS impairment (Weill Cornell LINCL scale). RESULTS: For the 18 children, the Weill Cornell scale yielded a closer correlation with both age and time since initial clinical manifestation of the disease than did the modified Hamburg scale. There were no significant differences as a function of age or time since initial manifestation of the disease in the rating scales among the most frequent CLN2 mutations (G3556C, 56% of all alleles or C3670T, 22% of all alleles). Measurements of cortical MRS N-acetyl-aspartate content, MRI ventricular, gray matter and white matter volume, and cortical apparent diffusion coefficient correlated to a variable degree with the age of the children and the time since initial clinical manifestation of the disease. All imaging measurements correlated better with the Weill Cornell CNS scale compared to the modified Hamburg LINCL scale. CONCLUSION: The data suggest that the Weill Cornell late infantile neuronal ceroid lipofuscinosis (LINCL) scale, together with several of the MRI measurements, may be useful in the assessment of severity and progression of LINCL and for the evaluation of novel therapeutic strategies.

Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation.

This gene transfer experiment is the first Parkinson's Disease (PD) protocol to be submitted to the Recombinant DNA Advisory Committee. The principal investigators have uniquely focused their careers on both pre-clinical work on gene transfer in the brain and clinical expertise in management and surgical treatment of patients with PD. They have extensively used rodent models of PD for proof-of-principle experiments on the utility of different vector systems. PD is an excellent target for gene therapy, because it is a complex acquired disease of unknown etiology (apart from some rare familial cases) yet it is characterized by a specific neuroanatomical pathology, the degeneration of dopamine neurons of the substantia nigra (SN) with loss of dopamine input to the striatum. This pathology results in focal changes in the function of several deep brain nuclei, which have been well-characterized in humans and animal models and which account for many of the motor symptoms of PD. Our original approaches, largely to validate in vivo gene transfer in the brain, were designed to facilitate dopamine transmission in the striatum using an AAV vector expressing dopamine-synthetic enzymes. Although these confirmed the safety and potential efficacy of AAV, complex patient responses to dopamine augmenting medication as well as poor results and complications of human transplant studies suggested that this would be a difficult and potentially dangerous clinical strategy using current approaches. Subsequently, we and others investigated the use of growth factors, including GDNF. These showed some encouraging effects on dopamine neuron survival and regeneration in both rodent and primate models; however, uncertain consequences of long-term growth factor expression and question regarding timing of therapy in the disease course must be resolved before any clinical study can be contemplated. We now propose to infuse into the subthalamic nucleus (STN) recombinant AAV vectors expressing the two isoforms of the enzyme glutamic acid decarboxylase (GAD-65 and GAD-67), which synthesizes the major inhibitory neurotransmitter in the brain, GABA. The STN is a very small nucleus (140 cubic mm or 0.02% of the total brain volume, consisting of approximately 300,000 neurons) which is disinhibited in PD, leading to pathological excitation of its targets, the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNpr). Increased GPi/SNpr outflow is believed responsible for many of the cardinal symptoms of PD, i.e., tremor, rigidity, bradykinesia, and gait disturbance. A large amount of data based on lesioning, electrical stimulation, and local drug infusion studies with GABA-agonists in human PD patients have reinforced this circuit model of PD and the central role of the STN. Moreover, the closest conventional surgical intervention to our proposal, deep brain stimulation (DBS) of the STN, has shown remarkable efficacy in even late stage PD, unlike the early failures associated with recombinant GDNF infusion or cell transplantation approaches in PD. We believe that our gene transfer strategy will not only palliate symptoms by inhibiting STN activity, as with DBS, but we also have evidence that the vector converts excitatory STN projections to inhibitory projections. This additional dampening of outflow GPi/SNpr outflow may provide an additional advantage over DBS. Moreover, of perhaps the greatest interest, our preclinical data suggests that this strategy may also be neuroprotective, so this therapy may slow the degeneration of dopaminergic neurons. We will use both GAD isoforms since both are typically expressed in inhibitory neurons in the brain, and our data suggest that the combination of both isoforms is likely to be most beneficial. Our preclinical data includes three model systems: (1) old, chronically lesioned parkinsonian rats in which intraSTN GAD gene transfer results not only in improvement in both drug-induced asymmetrical behavior (apomorphine symmetrical rotations), but also in spontaneous behaviors. In our second model, GAD gene transfer precedes the generation of a dopamine lesion. Here GAD gene transfer showed remarkable neuroprotection. Finally, we carried out a study where GAD-65 and GAD-67 were used separately in monkeys that were resistant to MPTP lesioning and hence showed minimal symptomatology. Nevertheless GAD gene transfer showed no adverse effects and small improvements in both Parkinson rating scales and activity measures were obtained. In the proposed clinical trial, all patients will have met criteria for and will have given consent for STN DBS elective surgery. Twenty patients will all receive DBS electrodes, but in addition they will be randomized into two groups, to receive either a solution containing rAAV-GAD, or a solution which consists just of the vector vehicle, physiological saline. Patients, care providers, and physicians will be blind as to which solution any one patient receives. All patients, regardless of group, will agree to not have the DBS activated until the completion and unblinding of the study. Patients will be assessed with a core clinical assessment program modeled on the CAPSIT, and in addition will also undergo a preop and several postop PET scans. At the conclusion of the study, if any patient with sufficient symptomatic improvement will be offered DBS removal if they so desire. Any patients with no benefit will simply have their stimulators activated, which would normally be appropriate therapy for them and which requires no additional operations. If any unforeseen symptoms occur from STN production of GABA, this might be controlled by blocking STN GABA release with DBS, or STN lesioning could be performed using the DBS electrode. Again, this treatment would not subject the patient to additional invasive brain surgery. The trial described here reflects an evolution in our thinking about the best strategy to make a positive impact in Parkinson Disease by minimizing risk and maximizing potential benefit. To our knowledge, this proposal represents the first truly blinded, completely controlled gene or cell therapy study in the brain, which still provides the patient with the same surgical procedure which they would normally receive and should not subject the patient to additional surgical procedures regardless of the success or failure of the study. This study first and foremost aims to maximally serve the safety interests of the individual patient while simultaneously serving the public interest in rigorously determining in a scientific fashion if gene therapy can be effective to any degree in treating Parkinson's disease.

Tension pneumocephalus resulting from iatrogenic subarachnoid-pleural fistulae: report of three cases.

BACKGROUND: Symptomatic pneumocephalus may result from a cerebrospinal fluid leak communicating with extradural air. However, it is a rare event after thoracic surgical procedures, and its management and physiology are not widely recognized. METHODS: During the past 2 years, we have identified 3 patients who developed pneumocephalus after thoracotomy for tumor resection. Only 1 patient had a discernible spinal fluid leak identified intraoperatively. Two patients experienced delayed spinal fluid drainage from their chest tubes and subsequently developed profound lethargy, confusion, and focal neurologic signs. The third patient was readmitted to the hospital with a delayed pneumothorax and altered mental status. Radiographic imaging in all patients showed significant pneumocephalus of the basilar cisterns and ventricles. RESULTS: The first 2 patients were managed by discontinuation of the chest tube suction and bedrest. The third patient underwent surgical reexploration and nerve root ligation. All 3 patients had resolution of their symptoms within 72 hours. CONCLUSIONS: Pneumocephalus is a rare, but serious, complication of thoracotomy. Previous patients reported in the literature have been managed with reoperation to ligate the nerve roots. However, the condition resolved nonoperatively in 2 of our patients. Discontinuation of chest tube suction may be definitive treatment and is always the important initial management to decrease cerebrospinal fluid extravasation into the pleural space and allow normalization of neurologic symptoms.

Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-gamma.

We investigated the feasibility of local treatment or tumor vaccination with a herpes simplex virus (HSV) type 1-defective vector. The vector was engineered to express murine interferon-gamma (IFN-gamma) for experimental gene therapy against mouse glioma Rous sarcoma virus (RSV). The murine IFN-gamma gene was driven by the cytomegalovirus promoter. The helper virus (tsk) was thermosensitive; consequently, this vector could only proliferate at 31 degrees C. A high level of murine IFN-gamma expression was confirmed in vitro and in vivo by immunohistochemistry using anti-mouse IFN-gamma monoclonal antibody. This engineered vector (dvHSV/MulFN-gamma) inhibited the proliferation of mouse glioma RSV cells in vitro, and an intratumoral (i.t.) local injection of the vector caused i.t. necrosis in vivo. The immunological effect of dvHSV/MulFN-gamma was also examined in a mouse glioma RSV cell implantation model. A subcutaneous (s.c.) implant of 1 x 10(6) mouse glioma RSV cells after treatment with dvHSV/MulFN-gamma was rejected. However, the implant after treatment with an engineered HSV-defective vector containing an antisense nucleotide sequence of the murine IFN-gamma gene was not rejected. In addition, in another group of mice in which RSV cells treated with dvHSV/MulFN-gamma were implanted into a femoral (s.c.) region and nontreated RSV cells were implanted into a contralateral femoral (s.c.) region, the implanted RSV cells were rejected. The rejection of the implanted mouse glioma RSV was blocked by anti-asialo GM1, which was known to inhibit natural killer cell activity. These results revealed that the HSV-defective vector could realize a high efficiency of transfection to glioma cells through short-time treatment, and that the IFN-gamma gene transferred to the cells had the effect of tumor vaccination, which was suggested be related to natural killer cells. In conclusion, dvHSV/MulFN-gamma may be useful for the gene therapy of malignant glioma through either i.t. local injection or a practical tumor vaccination with ex vivo gene transfer.

Peroral gene therapy of lactose intolerance using an adeno-associated virus vector.

Gene therapy is usually reserved for severe and medically refractory disorders because of the toxicity, potential long-term risks and invasiveness of most gene transfer protocols. Here we show that an orally administered adeno-associated viral vector leads to persistent expression of a beta-galactosidase transgene in both gut epithelial and lamina propria cells, and that this approach results in long-term phenotypic recovery in an animal model of lactose intolerance. A gene 'pill' associated with highly efficient and stable gene expression might be a practical and cost-effective strategy for even relatively mild disorders, such as lactase deficiency.

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.

Prospects for gene therapy in pediatric neurosurgery.

Gene therapy represents a powerful tool for both the study and potential treatment of pediatric neurological diseases. The majority of strategies for brain gene therapy have focused upon the use of modified viruses as vehicles for efficient delivery of genes into cells of the central nervous system. Retroviruses were originally the most popular vehicles for gene transfer outside the brain; however, these only function in actively dividing cells and have thus been limited to developmental neurobiology and treatment of brain tumors. Viruses with DNA-based genomes can transfer genes to both dividing and nondividing cells such as neurons, and these include adenovirus, adeno-associated virus and herpes simplex virus. Each system has special features, and the choice of vehicle may be based upon a variety of factors including toxicity or immunogenicity of the vector in vivo, size of the gene which can be inserted, titer of virus which can be obtained and technical difficulty in generating reagent grade viruses. Pediatric patients present unique opportunities for gene therapy, and inherited genetic defects and brain tumors are among the pediatric disorders which would most benefit from this new field. Preclinical studies using each of these systems in a variety of models of pediatric CNS disease have proven promising. Several ongoing studies have been initiated for treatment of pediatric brain tumors, and a protocol for treatment of an inherited neurological defect has recently achieved approval for initial clinical trials. Continued advances in gene therapy technology and delivery systems combined with the explosion of available genetic information should make gene therapy an increasingly important tool for the future of pediatric neurosurgery.

In situ PCR for in vivo detection of foreign genes transferred into rat brain.

Here we describe the use of in situ PCR to detect a viral transgene in rat brain. Previously, we have reported in vivo gene transfer by using a defective herpes simplex viral vector in mammalian brain (Kaplitt, M.G., Pfaus, J.G., Kleopoulos, S.P., Hanlon, B.A., Rabkin, S.D., Pfaff, D.W., Mol. Cell. Neurosci. 2 (1991) 320-330). For detection of the LacZ transgene, we have used histochemical staining for the protein product, beta-galactosidase, and in situ hybridization for its mRNA, but the DNA itself cannot be reliably detected with conventional methods. Therefore we have adapted the technique of in situ PCR, so that we may detect minute quantities of transgenic vector DNA following in vivo gene. The brain sections, prefixed, were treated with PBS-detergent before PCR amplification to increase permeability for peptides and oligonucleotides across cellular barriers in brain tissue. Pretreatment with detergent retained better brain morphology than the more widely used proteinase treatment. The PCR mixture containing dNTPs, primers, digoxigenin-dUTP (Dig-dUTP) and buffer was loaded onto each brain section. Slides containing brain sections were placed in an aluminum boat and then on the block of the thermal cycler. Temperature was brought to 82 degrees C before adding Taq polymerase ('hot start' method). Dig-labeled PCR amplified fragments were then detected by alkaline-phosphatase-linked anti-digoxigenin-antibody. Positive signals were seen within the nucleus of transduced neurons, indicating presence of viral DNA. Enhanced specificity was observed with the use of Dig-labeled primers which eliminates the possibility of non-specific viral DNA detection through primer-independent reactions. Overall, this technique can serve not only as an internal control for transgene presence during comparisons of experimental groups of animals, but may also have clinical applications including the detection of viral infection in human brain such as HIV in pathology specimens.

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.

Adeno-associated viral vector gene transfer into leptomeningeal xenografts.

Leptomeningeal carcinomatosis is a painful and debilitating complication of cancer. Indwelling reservoirs provide continuous assess to the subarachnoid space, making leptomeningeal cancer potentially amenable to gene therapy. Adeno-associated virus (AAV) is a defective virus not associated with any human disease. We used an AAV vector to transduce medulloblastoma (DAOY) cells in a nude rat model of leptomeningeal disease. After intraventricular injection of vector carrying the bacterial lacZ gene, beta-galactosidase positive cells were found in the implanted tumor and in ependymal and subependymal cells but not in underlying normal brain parenchyma. No evidence of virally-mediated toxicity was noted in the animals. The results of this pilot study demonstrate that AAV vectors may be used to transfer and express foreign genes in established leptomeningeal tumors.

Adenoviral vector-mediated expression of B-50/GAP-43 induces alterations in the membrane organization of olfactory axon terminals in vivo.

B-50/GAP-43 is an intraneuronal membrane-associated growth cone protein with an important role in axonal growth and regeneration. By using adenoviral vector-directed expression of B-50/GAP-43 we studied the morphogenic action of B-50/GAP-43 in mature primary olfactory neurons that have established functional synaptic connections. B-50/GAP-43 induced gradual alterations in the morphology of olfactory synapses. In the first days after overexpression, small protrusions originating from the preterminal axon shaft and from the actual synaptic bouton were formed. With time the progressive formation of multiple ultraterminal branches resulted in axonal labyrinths composed of tightly packed sheaths of neuronal membrane. Thus, B-50/GAP-43 is a protein that can promote neuronal membrane expansion at synaptic boutons. This function of B-50/GAP-43 suggests that this protein may subserve an important role in ongoing structural synaptic plasticity in adult neurons and in neuronal membrane repair after injury to synaptic fields.

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.

Defective viral vectors as agents for gene transfer in the nervous system.

Viral vectors have attracted great interest as vehicles for gene therapy. Due to concerns regarding continued viral gene expression in several systems, new approaches have been sought for gene transfer in the nervous system. This article reviews the general concepts and basic biology of defective viral vectors. These are vectors which can package into a viral coat but contain no viral genes, thereby allowing efficient gene transfer in the absence of viral gene expression in target cells. The defective herpes simplex virus (HSV) vector has been applied to numerous interesting questions in neurobiology. The inability to completely eliminate helper viruses has raised concern regarding the application of this vector to human disease. The adeno-associated virus (AAV) vector has recently been introduced into the nervous system. This vector harbors no viral genes, however helper viruses can also be completely eliminated from the system. Although the smaller size may limit the range of applications for this vector, it has received great interest as a potential agent for gene therapy in the nervous system. Potential future directions are discussed as well.

Long-term gene transfer in porcine myocardium after coronary infusion of an adeno-associated virus vector.

BACKGROUND: Viral vector-mediated gene transfer into the heart represents a potentially powerful tool for studying both cardiac physiology as well as gene therapy of cardiac disease. We report here the use of a defective viral vector, which expresses no viral gene products, for gene transfer into the mammalian heart. Previous studies have used recombinant viral vectors, which retained viral genes and yielded mostly short-term expression, often with significant inflammation. METHODS: An adeno-associated virus vector was used that contains no viral genes and is completely free of contaminating helper viruses. The adeno-associated virus vector was applied to rat hearts by direct intramuscular injection; adeno-associated virus was also infused into pig hearts in vivo via percutaneous intraarterial infusion into the coronary vasculature using routine catheterization techniques. RESULTS: Gene transfer into rat heart yielded no apparent inflammation, and expression was observed for at least 2 months after injection. Infusion into pig circumflex coronary arteries resulted in successful transfer and expression of the reporter gene in cardiac myocytes without apparent toxicity or inflammation; gene expression was observed for at least 6 months after infusion. CONCLUSIONS: We report the use of adeno-associated virus vectors in the cardiovascular system as well as successful myocardial gene transfer after percutaneous coronary artery infusion of viral vectors in a large, clinically relevant mammalian model. These results suggest that safe and stable gene transfer can be achieved in the heart using standard outpatient cardiac catheterization techniques.

Continuous renewal of the axonal pathway sensor apparatus by insertion of new sensor molecules into the growth cone membrane.

BACKGROUND: Growth cones at the tips of growing axons move along predetermined pathways to establish synaptic connections between neurons and their distant targets. To establish their orientation, growth cones continuously sample for, and respond to, guidance information provided by cell surfaces and the extracellular matrix. To identify specific guidance cues, growth cones have sensor molecules on their surface, which are expressed differentially during the temporospatial progress of axon outgrowth, at levels that depend on the pattern of neural activity. However, it has not been elucidated whether a change in gene expression can indeed change the molecular composition and, hence, the function of the sensor apparatus of growth cones. RESULTS: We have constructed adenoviral gene transfer vectors of the chicken growth cone sensor molecules axonin-1 and Ng-CAM. Using these vectors, we initiated the expression of axonin-1 and Ng-CAM in rat dorsal root ganglia explants during ongoing neurite outgrowth. Using specific surface immunodetection at varying time points after infection, we found that axonin-1 and Ng-CAM are transported directly to the growth cone and inserted exclusively in the growth cone membrane and not in the axolemma of the axon shaft. Furthermore, we found that axonin-1 and Ng-CAM do not diffuse retrogradely, suggesting that the sensor molecules are integrated into multimolecular complexes in the growth cone. CONCLUSIONS: During axon outgrowth, the pathway sensor apparatus of the growth cone is continuously updated by newly synthesized sensor molecules that originate directly from the transcription/translation machinery. Changes in the expression of sensor molecules may have a direct impact, therefore, on the exploratory function of the growth cone.