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Patrick Aebischer

Publications and source records attributed to Patrick Aebischer.

35 records · Page 2Linked to original sources
PubMed2004

Lentiviral-mediated gene transfer to model triplet repeat disorders.

This chapter describes the potential use of viral-mediated gene transfer in the central nervous system as a new strategy in developing animal models of neurodegenerative diseases. To illustrate the approach, procedures for the production of lentiviral vectors encoding polyQ proteins are provided, as well as methods for the determination of viral titers, in vitro infection, and basic protocols for in vivo studies in rodents.

Animals↗
PubMed2003

Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum.

The ability to overexpress full-length huntingtin or large fragments represents an important challenge to mimic Huntington's pathology and reproduce all stages of the disease in a time frame compatible with rodent life span. In the present study, tetracycline-regulated lentiviral vectors leading to high expression levels were used to accelerate the pathological process. Rats were simultaneously injected with vectors coding for the transactivator and wild type (WT) or mutated huntingtin (TRE-853-19Q/82Q) in the left and right striatum, respectively, and analyzed in the 'on' and 'off' conditions. Overexpression of TRE-853-19Q protein or residual expression of TRE-853-82Q in 'off' condition did not cause any significant neuronal pathology. Overexpressed TRE-853-82Q protein led to proteolytic release of N-terminal htt fragments, nuclear aggregation, and a striatal dysfunction as revealed by decrease of DARPP-32 staining but absence of NeuN down-regulation. The differential effect on the DARPP-32/NeuN neuronal staining was observed as early as 1 month after injection and maintained at 3 months. In contrast, expression of a shorter htt form (htt171-82Q) did not require processing prior formation of nuclear aggregates and caused decrease of both DARPP-32 and NeuN neuronal markers at one month post-injection suggesting that polyQ pathology may be dependent on protein context. Finally, the reversibility of the pathology was assessed. Huntingtin expression was turn 'on' for 1 month and then shut 'off' for 2 months. Recovery of DARPP-32 immunoreactivity and clearance of huntingtin aggregates were observed in animals treated with doxycycline. These results suggest that a tetracycline-regulated system may be particularly attractive to model Huntington's disease and induce early and reversible striatal neuropathology in vivo.

Animals↗
PubMed2003

Comparative study of GDNF delivery systems for the CNS: polymer rods, encapsulated cells, and lentiviral vectors.

Glial cell line-derived neurotrophic factor (GDNF) holds great promise for the treatment of Parkinson's disease. In humans, its intracerebroventricular administration leads to limiting side effects. Direct parenchymal delivery using mechanical means, or cell and gene therapy represent potential alternatives. In the present study, a representative of each of these three approaches, i.e. polymer rods, genetically modified encapsulated cells and lentiviral vectors was analyzed for its ability to release GDNF in the striatum of rats. One week post-surgery, GDNF was detected over a distance of 4 mm with all three methods. At 4 weeks GDNF staining diminished with rods and to a lesser extent with encapsulated cells, whereas it increased with lentiviral vectors. Nanogram range of GDNF was measured with all methods at 1 week. At 4 weeks, GDNF levels decreased significantly with rods, whereas they remained stable with encapsulated cells and lentiviral vectors. We conclude that all three methods investigated allow striatal delivery of GDNF, but the time during which it needs to be released will determine the approach chosen for clinical application.

Animals↗
PubMed2003

Prevention of the initial host immuno-inflammatory response determines the long-term survival of encapsulated myoblasts genetically engineered for erythropoietin delivery.

The present study investigates the respective roles of both the host immune response and the metabolic requirements in determining the long-term survival of erythropoietin-secreting myoblasts within encapsulating polymer membranes. Hollow-fiber capsules loaded with a high density of erythropoietin-secreting C(2)C(12) myoblasts survived poorly in the subcutaneous tissue of syngeneic mice, inducing variable hematocrit responses. To determine the role and the nature of the host response, recipients were treated with anti-inflammatory (diclofenac) and immunosuppressive (dexamethasone, FK506) agents. Only immunosuppressive drugs led to improved graft survival after 5 weeks of implantation, indicating an immune process as the cause of cell death. Furthermore, transient blocking of this process allowed long-term preservation of the implanted cells (> 100 days). The formation of necrotic cell cores inside densely packed devices elicited a local chronic inflammatory reaction. Hence, implants were designed to limit early cell death by inserting a supporting matrix and decreasing the number of loaded cells. The most efficient erythropoietin delivery was obtained with matrix-containing capsules that had received the lowest myoblast density. These results highlight the critical role of initial engraftment in the long-term acceptance of encapsulated myoblasts and the need to limit early cell death in the device to prevent subsequent host immuno-inflammatory responses.

Animals↗
PubMed2003

Presence of Gal-alpha1,3Gal epitope on xenogeneic lines: implications for cellular gene therapy based on the encapsulation technology.

Exposure to human serum induces the lysis of xenogeneic cells through natural antibodies and complement activation. The carbohydrate Galactose-alpha1,3-Galactose (Gal-alpha1,3-Gal) epitope, has been shown to be the principal antigenic determinant on target cells. This reaction is, therefore, particularly important for xenogeneic cell-based therapy. As a first step toward the evaluation of the impact of this phenomenon for encapsulated xenogeneic cells, we have evaluated the presence of the Gal-alpha1,3Gal epitope on two cell lines currently being used for the systemic delivery of protein in the periphery or the treatment of neurodegenerative diseases. In the second part of the study, we have tested and compared human serum and cerebrospinal fluid (CSF) for the presence of xenoreactive natural antibodies (XNAs) and their potential impact on the survival of xenogeneic cells. Fluorescence-activated cell sorting analysis indicated that baby hamster kidney (BHK) cells expressed low levels of the alpha-Gal epitope, whereas mouse myoblast C2C12 cells were extensively stained with the specific IB4-lectin. There was a direct correlation between serum killing and the level of Gal-alpha1,3-Gal epitope expression on these cells. Importantly, we showed that CSF did not lyse BHK and C2C12 cells as determined by cytotoxic crossmatch assays. The reaction was specific as the addition of soluble Gal-alpha1,3-Gal sugar to human serum effectively reduced cell killing, and the overproduction of alpha-1,3-galactosyltransferase in BHK cells significantly increased inactivation by human serum. To interfere with this antibody-antigen reaction and develop cell lines particularly suitable for cell-based therapy, we either selected C2C12 clones expressing low levels of Gal-alpha1,3-Gal or high levels of alpha-1,2-fucosyltransferase. These cells were found to be resistant to complement-mediated cytolysis. These strategies may, therefore, protect encapsulated xenogeneic cells transplanted in the periphery or the central nervous system even in an unlikely event of a blood-brain barrier breakage and the post-transplantation development of an antibody response.

Animals↗
PubMed2002

Glial cell line-derived neurotrophic factor released by synthetic guidance channels promotes facial nerve regeneration in the rat.

Regeneration of the human facial nerve after lesion is often limited, leading to severe functional impairments, in particular when repair is delayed for several months, when cross-facial nerve grafts have to be performed, or in elderly patients. To improve the outcome, the potential accelerating and maturating effects of the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) on nerve regeneration were assessed using an axotomy model of the rat facial nerve. One-centimeter-long synthetic guidance channels releasing the neurotrophic factors over several weeks were used to bridge an 8 mm nerve gap, a distance that does not allow regeneration in the absence of growth factors. Nerve cables regenerated in the presence of GDNF showed a large number of myelinated axons 6 weeks after grafting (871 +/- 373, n = 5), whereas only 106 +/- 86 (n = 5) myelinated axons were counted in the presence of NT-3. Retrograde labeling with fluorogold revealed 981 +/- 450 (n = 5) and 53 +/- 38 (n = 5) retrogradely labeled motoneurons in the facial nucleus in the presence of GDNF and NT-3, respectively. No regenerated axons or retrogradely labeled cells were observed in the absence of growth factors (n = 6). These results demonstrate that GDNF, as previously described for the sciatic nerve, a mixed sensory and motor nerve, is also very efficient in promoting regeneration of the facial nerve, an essentially pure motor nerve. GDNF may therefore be useful in improving facial nerve regeneration in the clinic.

Animals↗
PubMed2002

Lentiviral-mediated RNA interference.

RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNAs are capable of binding to a specific mRNA sequence and causing its degradation. The recent demonstration of a plasmid vector that directs siRNA synthesis in mammalian cells prompted us to examine the ability of lentiviral vectors to encode siRNA as a means of providing long-term gene silencing in mammalian cells. The RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (EGFP). This construct successfully silenced EGFP expression in two stable cell lines expressing this protein, as analyzed by fluorescence microscopy, flow cytometry, and Western blotting. The silencing, which is dose dependent, occurs as early as 72 hr postinfection and persists for at least 25 days postinfection. The ability of lentiviruses encoding siRNA to silence genes specifically makes it possible to take full advantage of the possibilities offered by the lentiviral vector and provides a powerful tool for gene therapy and gene function studies.

Cell Line↗
PubMed2002

Neurospheres modified to produce glial cell line-derived neurotrophic factor increase the survival of transplanted dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to increase the survival of dopamine neurons in a variety of in vitro and in vivo model systems. Therefore, it constitutes an important therapeutic protein with the potential to ameliorate dopamine neuronal degeneration in Parkinson's disease or to support dopamine neuronal replacement strategies. However, biophysical and practical considerations present obstacles for the direct delivery of the GDNF protein to CNS neurons. Here we show that rodent neural precursor cells isolated and expanded in culture as neurospheres (NS) can be genetically modified to express green fluorescent protein (GFP) or to release GDNF using lentiviral constructs. GDNF-NS increased the fibre outgrowth of primary embryonic dopamine neurons in cocultures, showing that the protein was released in biologically significant quantities. Furthermore, after transplantation into the 6-hydroxydopamine-lesioned rat striatum, GDNF-NS significantly increased the survival of cografted primary dopamine neurons. However, this was not reflected in behavioural recovery in these animals. We found that, by 6 weeks, few cells expressed GDNF or GFP, suggesting either that transgene expression was down-regulated over time or that the cells died. This may explain the initial effects on dopamine neuronal survival within the graft but the lack of long-term effect on subsequent fibre outgrowth and behaviour. Providing sustained levels of neural precursor-mediated transgene expression can be achieved following transplantation in the future; this approach may prove beneficial as an alternative therapeutic strategy in the cell-based management of Parkinson's disease.

Animals↗
PubMed2002

Simultaneous GDNF and BDNF application leads to increased motoneuron survival and improved functional outcome in an experimental model for obstetric brachial plexus lesions.

Motoneurons of the neonate rat respond to proximal axonal injury with morphologic and functional changes and ultimately with neuronal death. Recent studies showed that both glial cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) reduce induced degeneration of motoneurons after axotomy and avulsion. Whether rescued motoneurons are functionally intact has been argued. In the present investigation, the authors have used a proximal crush lesion of the brachial plexus in neonatal rats as the experimental model of neuronal injury. This allowed the authors to study the effects of trophic factor administration on injured motoneurons and the relationship between motoneuron survival and extremity function. Trophic factors were locally released by small polymer implants in a low-dose slow-release mode. Six groups of 10 animals were prepared: BDNF, GDNF, GDNF/BDNF, control, sham, and normals. The number of surviving motoneurons was determined by retrograde tracer techniques using Fluorogold and Fastblue. Extremity function was quantitatively evaluated with functional muscle testing at day 56. The results of this study demonstrate that trophic factors applied separately had no effect, whereas combined trophic factor application (GDNF/BDNF group) had a dramatic rescue effect on motoneuron survival as compared with the control groups, which also effected significantly greater strength. The authors conclude that a combination of trophic factors leads to enhanced motoneuron survival, with improved voluntary function as the animal enters adulthood so that exogenous trophic support of motoneurons might have a role in the treatment of all types of severe neonatal plexopathies, maintaining the viability of motoneurons until reconstructive surgery provides them with a pathway for regeneration and endogenous trophic support.

Animals↗
PubMed2002

Lentivirally delivered glial cell line-derived neurotrophic factor increases the number of striatal dopaminergic neurons in primate models of nigrostriatal degeneration.

The primate striatum contains tyrosine hydroxylase (TH)-immunoreactive (ir) neurons, the numbers of which are augmented after dopamine depletion. Glial cell line-derived neurotrophic factor (GDNF) strongly modulates the viability and phenotypic expression of dopamine ventral mesencephalic neurons. The effect of GDNF on TH-ir neurons intrinsic to the striatum has yet to be investigated. In the present study, stereological counts of TH-ir striatal neurons in aged and parkinsonian nonhuman primates revealed that GDNF delivered via a lentiviral vector (lenti-) further increased the number of these cells. Aged monkeys treated with lenti-GDNF displayed an eightfold increase in TH-ir neurons relative to lenti-beta-galactosidase-treated monkeys. Unilateral 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine treatment alone in young monkeys resulted in a bilateral eightfold increase in TH-ir striatal cells. This effect was further magnified sevenfold on the side of lenti-GDNF treatment. These cells colocalized with the neuronal marker neuronal-specific nuclear protein. Some of these cells colocalized with GDNF-ir, indicating that an alteration in phenotype may occur by the direct actions of this trophic factor. Thus, GDNF may mediate plasticity in the dopamine-depleted primate brain, which may serve to compensate for cell loss by converting striatal neurons to a dopaminergic phenotype.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2002

Allogeneic beta-islet cells correct diabetes and resist immune rejection.

Allogeneic MHC-incompatible organ or cell grafts are usually promptly rejected by immunocompetent hosts. Here we tested allogeneic beta-islet cell graft acceptance by immune or naive C57BL/6 mice rendered diabetic with streptozotocin (STZ). Fully MHC-mismatched insulin-producing growth-regulated beta-islet cells were transplanted under the kidney capsule or s.c. Although previously or simultaneously primed mice rejected grafts, STZ-treated diabetic mice accepted islet cell grafts, and hyperglycemia was corrected within 2-4 weeks in absence of conventional immunosuppression. Allogeneic grafts that controlled hyperglycemia expressed MHC antigens, were not rejected for >100 days, and resisted a challenge by allogeneic skin grafts or multiple injections of allogeneic cells. Importantly, the skin grafts were rejected in a primary fashion by the grafted and corrected host, indicating neither tolerization nor priming. Such strictly extralymphatic cell grafts that are immunologically largely ignored should be applicable clinically.

Animals↗
PubMed2002

Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length.

A new strategy based on lentiviral-mediated delivery of mutant huntingtin (htt) was used to create a genetic model of Huntington's disease (HD) in rats and to assess the relative contribution of polyglutamine (CAG) repeat size, htt expression levels, and protein length on the onset and specificity of the pathology. Lentiviral vectors coding for the first 171, 853, and 1520 amino acids of wild-type (19 CAG) or mutant htt (44, 66, and 82 CAG) driven by either the phosphoglycerate kinase 1 (PGK) or the cytomegalovirus (CMV) promoters were injected in rat striatum. A progressive pathology characterized by sequential appearance of ubiquitinated htt aggregates, loss of dopamine- and cAMP-regulated phosphoprotein of 32 kDa staining, and cell death was observed over 6 months with mutant htt. Earlier onset and more severe pathology occurred with shorter fragments, longer CAG repeats, and higher expression levels. Interestingly, the aggregates were predominantly located in the nucleus of PGK-htt171-injected rats, whereas they were present in both the nucleus and processes of CMV-htt171-injected animals expressing lower transgene levels. Finally, a selective sparing of interneurons was observed in animals injected with vectors expressing mutant htt. These data demonstrate that lentiviral-mediated expression of mutant htt provides a robust in vivo genetic model for selective neural degeneration that will facilitate future studies on the pathogenesis of cell death and experimental therapeutics for HD.

Animals↗
PubMed2002

Long-term doxycycline-regulated secretion of erythropoietin by encapsulated myoblasts.

We developed an ex vivo gene therapy approach for the regulated delivery of therapeutic proteins based on the implantation of encapsulated, genetically engineered C(2)C(12) myoblasts. We investigated doxycycline-based regulation of gene expression to modulate the secretion of erythropoietin (EPO) from encapsulated myoblasts in a mouse model. An autoregulatory tet-off system provided high induction levels with low basal expression in the noninduced state. Stable C(2)C(12) clones constitutively secreted between 25 and 50 IU mouse EPO/10(6)cells/24 hours in the on-state. The clone C15, selected for its in vivo survival characteristics, displayed a desirable secretion profile when encapsulated. Devices released 5 IU EPO per capsule in the on-state, with EPO levels being undetectable upon the addition of doxycycline (dox). Capsules subcutaneously implanted in DBA/2J mice demonstrated a tightly regulated secretion of EPO through up to four on-off cycles during a period lasting 40 weeks. Hematocrits could be modulated between basal levels (40-50%) and elevated levels (70-90%) through the presence or absence of dox in the drinking water. Hematocrit returned to normal levels, paralleling the kinetics observed following capsule explantation, 6 to 8 weeks following dox administration to polycythemic mice. The results of this study suggest that encapsulation and implantation of a tet-off regulated C(2)C(12) cell clone represents a safe method for the controlled long-term delivery of proteins in vivo.

Animals↗
PubMed2002

GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap.

The present work was performed to determine the ability of neurotrophic factors to allow axonal regeneration across a 15-mm-long gap in the rat sciatic nerve. Synthetic nerve guidance channels slowly releasing NGF and GDNF were fabricated and sutured to the cut ends of the nerve to bridge the gap. After 7 weeks, nerve cables had formed in nine out of ten channels in both the NGF and GDNF groups, while no neuronal cables were present in the control group. The average number of myelinated axons at the midpoint of the regenerated nerves was significantly greater in the presence of GDNF than NGF (4942 +/-1627 vs. 1199 +/-431, P < or = 0.04). A significantly greater number of neuronal cells in the GDNF group, when compared to the NGF group, retrogradely transported FluoroGold injected distal to the injury site before explantation. The total number of labelled motoneurons observed in the ventral horn of the spinal cord was 98.1 +/-23.4 vs. 20.0 +/-8.5 (P < or = 0.001) in the presence of GDNF and NGF, respectively. In the dorsal root ganglia, 22.7% +/- 4.9% vs. 3.2% +/-1.9% (P +/-0.005) of sensory neurons were labelled retrogradely in the GDNF and NGF treatment groups, respectively. The present study demonstrates that, sustained delivery of GDNF and NGF to the injury site, by synthetic nerve guidance channels, allows regeneration of both sensory and motor axons over long gaps; GDNF leads to better overall regeneration in the sciatic nerve.

Animals↗
PubMed2002

Seizure suppression by adenosine-releasing cells is independent of seizure frequency.

PURPOSE: Intraventricular cellular delivery of adenosine was recently shown to be transiently efficient in the suppression of seizure activity in the rat kindling model of epilepsy. We tested whether the suppression of seizures by adenosine-releasing grafts was independent of seizure frequency. METHODS: Adenosine-releasing cells were encapsulated and grafted into the lateral brain ventricle of rats kindled in the hippocampus. During 4 weeks after grafting, electric test stimulations were delivered at a frequency of either once a week or 3 times per week. Seizure activity was evaluated by visual scoring of seizure severity and by the recording of EEGs. RESULTS: Adenosine released from encapsulated cells exerted potent antiepileptic activity for >/=2 weeks. One week after grafting, treated rats displayed a complete protection from clonic seizures, and a protection from focal seizures was observed in the majority of animals. Seizure suppression was accompanied by a reduction of afterdischarges in EEG recordings. The protective efficacy of the grafted cells was the same irrespective of whether electrical test stimulations were delivered 1 or 3 times per week. Rats receiving control grafts continued to display full clonic convulsions. CONCLUSIONS: This study demonstrated that the frequency of test stimulations did not influence the seizure-suppressive potential of adenosine-releasing grafts. Thus the local delivery of adenosine is likely to be effective in seizure control over a threefold range of seizure-discharge frequency.

Adenosine↗
PubMed2002

Delivery of erythropoietin by encapsulated myoblasts in a genetic model of severe anemia.

BACKGROUND: Existing animal models of anemia inadequately reflect the hematocrit usually present in chronic renal failure (CRF) patients and do not permit long-term treatment studies. The transgenic mouse strain 134.3LC (Epo-TAg(H)) displays a severe chronic anemia resembling that observed clinically during CRF, while displaying an active, normal life span. This phenotype makes it a particularly interesting mouse model for testing erythropoietin (Epo)-based gene transfer strategies. METHODS: Ex vivo gene therapy was employed to administer mouse Epo to homozygous anemic Epo-TAg(H) mice. Encapsulated C(2)C(12) myoblasts genetically engineered to secrete 163 IU mouse Epo/10(6) cells/day were subcutaneously transplanted on the dorsal flank of the mice. Efficacy of delivered Epo was monitored by weekly measurements of animal hematocrit. RESULTS: Most treated homozygous Epo-TAg(H) mice displayed only a transient rise in hematocrit before eventually decreasing to levels as low as 3%. Administering the immunosuppressor anti-CD4+ monoclonal antibody (mAb) to homozygous Epo-TAg(H) mice, beginning at the time of implantation, permitted a rise in hematocrit that remained stable at elevated levels in cases of continued immunosuppression. CONCLUSIONS: Mice having the T antigen insertion in both Epo alleles appeared to develop an immune response to the natural mouse Epo delivered by encapsulated cells. By preventing this reaction using immunosuppression, we demonstrate that encapsulated myoblasts can deliver therapeutic doses of mouse Epo systemically and restore hemopoiesis in a genetic model of severe anemia.

Anemia↗