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Biomedical subjects

F H Gage

Publications and source records attributed to F H Gage.

At least 109 records · Page 6Linked to original sources
PubMed1997

Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.

Successful gene therapy approaches will require efficient gene delivery and sustained expression of the transgene in recipients. A variety of methods, ranging from direct DNA delivery to infection with recombinant viruses containing foreign genes, have been developed, but they all have some major limitations that restrict their utility. We have described a human lentiviral (HIV)-based vector that can transduce non-dividing cells in vitro and deliver genes in vivo. With this vector, expression of transgenes in the brain has been detected for more than six months--the longest period tested so far. Because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein (VSVG; ref. 8), they can transduce a broad range of tissues and cell types. We now describe the ability of lentiviral vectors to introduce genes directly into liver and muscle. Sustained expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, can be observed for more than 22 weeks in the liver. Similar long-term expression (more than eight weeks) was observed in transduced muscle. In contrast, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukaemia virus (M-MLV), a prototypic retroviral based vector. At a minimum, 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10(7) infectious units (I.U.) of recombinant HIV vector. Furthermore, no inflammation of recruitment of lymphocytes could be detected at the site of injection. Animals previously transduced with a lentiviral vector can be efficiently re-infected with lentiviral vectors. Additionally, we show that the requirement for lentiviral accessory proteins to establish efficient transduction in vivo is tissue dependent.

Animals↗
PubMed1997

Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector.

The identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. A lentivirus-based vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral vector stock into the striatum and hippocampus of adult rats. Control brains were injected with a Moloney murine leukemia virus, adenovirus, or adeno-associated virus vector. The volumes of the areas containing transduced cells and the transduced-cell densities were stereologically determined to provide a basis for comparison among different viral vectors and variants of the viral vector stocks. The efficiency of infection by the lentivirus vector was improved by deoxynucleoside triphosphate pretreatment of the vector and was reduced following mutation of integrase and the Vpr-matrix protein complex involved in the nuclear translocation of the preintegration complex. The lentivirus vector system was able to efficiently and stably infect quiescent cells in the primary injection site with transgene expression for over 6 months. Triple labeling showed that 88.7% of striatal cells transduced by the lentivirus vector were terminally differentiated neurons.

Adult↗
PubMed1996

Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo.

Neurogenesis continues throughout adulthood in discrete regions. Proliferative zones include the subependymal zone, from where progenitors migrate along the rostral migratory pathway to differentiate into neurons in the olfactory bulb, and the hippocampal subgranular zone, where they migrate and differentiate into granule neurons. Progenitors isolated from adult subependymal zone exhibit in vitro neurogenesis when stimulated with epidermal or fibroblast growth factor. Cultured adult rat hippocampal progenitors (AHPs) grafted to adult rat hippocampus show site-specific neuronal differentiation. Here we investigate determinants of multipotentiality in the adult central nervous system, by grafting AHPs into homotypic (hippocampus) or heterotypic (the rostral migratory pathway) neurogenic sites or a heterotypic, non-neurogenic site (the cerebellum). We found that grafts into neurogenic, but not nonneurogenic sites, showed neuronal differentiation. Furthermore, AHPs grafted in the rostral migratory pathway migrated into the olfactory bulb, differentiating into tyrosine-hydroxylase-positive neurons, a non-hippocampus phenotype. These results reveal that AHP populations can respond to persistent neuronal differentiation cues in the adult central nervous system.

Adult↗
PubMed1996

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.

Animals↗
PubMed1996

The signal-dependent coactivator CBP is a nuclear target for pp90RSK.

We have examined the mechanism by which growth factor-mediated induction of the Ras pathway interferes with signaling via the second messenger cAMP. Activation of cellular Ras with insulin or NGF stimulated recruitment of the S6 kinase pp90RSK to the signal-dependent coactivator CBP. Formation of the pp90RSK-CBP complex occurred with high stoichiometry and persisted for 6-8 hr following growth factor addition. pp90RSK specifically recognized the E1A-binding domain of the coactivator CBP. In addition, like E1A, binding of pp90RSK to CBP was sufficient to repress transcription of cAMP-responsive genes via the cAMP-inducible factor CREB. By contrast with its effects on the cAMP pathway, formation of the pp90RSK-CBP complex was required for induction of Ras-responsive genes. These results provide a demonstration of cross-coupling between two signaling pathways that occurs at the level of a signal-dependent coactivator.

Adenovirus E1A Proteins↗
PubMed1996

Sprouting adult CNS cholinergic axons express NILE and associate with astrocytic surfaces expressing neural cell adhesion molecule.

To assess the cellular and molecular substrates for cholinergic axon growth in the adult central nervous system (CNS), we implanted grafts of control and nerve growth factor (NGF)-producing genetically modified fibroblasts within the striatum of rats. Sprouting cholinergic axonal processes that grew into grafts of NGF-producing fibroblasts were fasciculated and followed the surface of astrocytic processes for long distances within the grafts. The close and long distance anatomical relationship between the sprouted axons and the astrocytes supported previous ultrastructural evidence that astrocytes may serve as a cellular substrate for sprouting cholinergic axons in vivo. The sprouted axon processes were associated with the expression of nerve growth factor-inducible large external (NILE) glycoprotein on their surfaces. NILE expression was not seen in control grafts where there was an absence of cholinergic ingrowth. NILE has been demonstrated to play a role in axon fasciculation in a number of other neural systems. The astrocytic processes in both control and NGF-producing fibroblast grafts expressed neural cell adhesion molecule (NCAM), suggesting that NCAM-mediated adhesion may be responsible for the close relationship between the axons and astrocytes within the grafts. NGF-induced heterotypic interactions between neuronal NILE and astroglial NCAM may also be required for adult cholinergic axonal sprouting.

Acetylcholine↗
PubMed1996

In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

Amino Acid Sequence↗
PubMed1996

Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation.

The hippocampus is one of the few areas of the rodent brain that continues to produce neurons postnatally. Neurogenesis reportedly persists in rats up to 11 months of age. Using bromodeoxyuridine (BrdU) labeling, the present study confirms that in the adult rat brain, neuronal progenitor cells divide at the border between the hilus and the granule cell layer (GCL). In adult rats, the progeny of these cells migrate into the GCL and express the neuronal markers NeuN and calbindin-D28k. However, neurogenesis was drastically reduced in aged rats. Six-to 27-month-old Fischer rats were injected intraperitoneally with BrdU to detect newborn cells in vivo and to follow their fate in the dentate gyrus. When killed 4-6 weeks after BrdU labeling, 12- to 27-month-old rats exhibited a significant decline in the density of BrdU-positive cells in the granule cell layer compared with 6-month-old controls. Decreased neurogenesis in aging rats was accompanied by reduced immunoreactivity for poly-sialylated neural cell adhesion molecule, a molecule that is involved in migration and process elongation of developing neurons. When animals were killed immediately (12 hr) after BrdU injection, significantly fewer labeled cells were observed in the GCL and adjacent subgranular zone of aged rats, indicative of a decrease in mitotic activity of neuronal precursor cells. The reduced proliferation was not attributable to a general aged-related metabolic impairment, because the density of BrdU-positive cells was not altered in other brain regions with known mitotic activity (e.g., hilus and lateral ventricle wall). The decline in neurogenesis that occurs throughout the lifespan of an animal can thus be related to a decreasing proliferation of granule cell precursors.

Aging↗
PubMed1996

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

Animals↗
PubMed1996

Fibroblast growth factor-2 protects entorhinal layer II glutamatergic neurons from axotomy-induced death.

The entorhinal cortex is a major relay between the hippocampus and other cortical and subcortical regions. Glutamatergic axons from layer II neurons form the entorhinal cortical projection to the hippocampus via the perforant pathway. We have demonstrated previously that lesion of the perforant pathway causes the death of approximately 30% of entorhinal layer II (ECL2) neurons. To elucidate mechanisms contributing to neuronal death and to investigate strategies preventing it, we identified the phenotype of the vulnerable neuronal population. Sections were immunolabeled with antibodies to the neuronal markers NeuN, glutamate, and calbindin-D28k, and to receptors for fibroblast growth factor-2 (FGFR1) and NMDA (NMDAR1) and were examined using confocal microscopy. Calbindin immunoreactivity was strikingly lamina-specific to ECL2, where one-third of all ECL2 neurons were calbindin-positive. Localization of glutamate revealed that half of the glutamatergic ECL2 neurons coexpressed calbindin. Quantification using unbiased stereology at 9 weeks after lesion of the perforant pathway revealed that the only ECL2 neuronal population that experienced a significant (70%) loss (20% of the total) was the population of glutamatergic ECL2 neurons that did not coexpress calbindin. All ECL2 neurons expressed FGFR1; therefore, we tested the role of FGF-2 in the survival of glutamatergic ECL2 neurons. We grafted fibroblasts genetically engineered to express nerve growth factor or FGF-2 and found that only FGF-2 grafts prevented loss of the vulnerable glutamatergic/calbindin-negative neurons. We present a hypothesis for the selective vulnerability of these glutamatergic/calbindin-negative ECL2 neurons and address the role of FGF-2 in neuronal rescue.

Afferent Pathways↗
PubMed1996

Somatic gene therapy for nervous system disease.

Neurotrophic factors are target-derived molecules that prevent neuronal degeneration during development and, in some cases, during adulthood. They offer substantial promise as therapeutic agents in neurological disease by preventing cell loss and promoting axonal regeneration. However, the optimal means of delivering neurotrophic factors to the nervous system, and the CNS in particular, is an unresolved issue. Neurotrophic factors rarely influence only a single target neuronal population, hence broad delivery of neurotrophic factors to the nervous system may results in effects on multiple non-targeted neuronal populations. Ideally, neurotrophin delivery to the nervous system should be target-specific, regionally restricted, chronic, safe, well-tolerated and of sufficient concentration to elicit responses from target neurons. In this paper we discuss the use of somatic gene transfer methods to deliver neurotrophic factors to the CNS in a manner that seeks to meet the above criteria.

Animals↗
PubMed1996

Nerve growth factor delivery by gene transfer induces differential outgrowth of sensory, motor, and noradrenergic neurites after adult spinal cord injury.

Several neurotrophic factors have been identified that influence neuronal populations during central nervous system development, maturation, and senescence. To examine the responsiveness of the intact and the lesioned adult mammalian spinal cord to neurotrophic factors, primary rat fibroblasts were genetically modified to produce and secrete human nerve growth factor (NGF). These NGF-producing cells were then grafted to nonlesioned or lesioned adult rat spinal cords for periods of up to 1 year in vivo. Robust outgrowth of sensory and noradrenergic neurites was elicited by grafts in the previously nonlesioned spinal cord. Equally robust growth of sensory and noradrenergic neurites was observed in the lesioned spinal cord; in addition, partial sprouting of local motor neurites was elicited in the lesioned spinal cord. Thus, multiple neuritic populations of the adult spinal cord respond to neurotrophic factors by extending neurites, and this responsiveness is maintained and extended after major injury. Nerve growth factor delivered by somatic gene transfer may be a useful means of promoting axon regrowth in the injured spinal cord.

Adrenergic Fibers↗
PubMed1996

Long-term survival of transplanted basal forebrain cells following in vitro propagation with fibroblast growth factor-2.

The intracerebral transplantation of freshly dissected fetal tissue containing cholinergic neurons of the developing basal forebrain has been reported to reverse lesion-induced or age-related cognitive deficits in animal models of cholinergic neuronal degeneration. Grafts of cultured fetal neurons, however, have generally shown poor cellular survival and limited therapeutic benefit. We tested the hypothesis that recent advances in the identification of growth factors that promote the survival and propagation of fetal precursor cells in vitro would improve the long-term survival of cultured neurons following intracerebral implantation. Dissociated cells from gestational Day 14 rodent basal forebrain were grown in chemically defined media supplemented with 20 ng/ml basic fibroblast growth factor. Two weeks postplating, numerous cells were present in the cultures and showed immunoreactive labeling for a variety of markers, including glutamic acid decarboxylase, neuron-specific enolase, neurofilament proteins, glial fibrillary acidic protein and, occasionally, choline acetyltransferase. To determine if cultured basal forebrain cells would survive intracerebral implantation, the cells were implanted homotypically into the nucleus basalis magnocellularis. To enhance the potential for graft survival in vivo, cells were also implanted into the nucleus basalis magnocellularis following an ibotenic acid lesion and into the denervated frontal cortex. Animals sacrificed between 2 weeks and 7 months following transplantation showed good and comparable graft survival in all sites. Immunocytochemical analysis revealed that representative populations of cells observed in vitro survived for prolonged periods in vivo, even in sites distal from their normal cellular targets. Thus, neuronal populations expanded in vitro can successfully survive and maintain cellular phenotypes post-transplantation. These results suggest a potential for isolating and growing specific neuronal populations in vitro for intracerebral transplantation.

Animals↗
PubMed1996

TrkA activation is sufficient to rescue axotomized cholinergic neurons.

To test the molecular nature of the NGF receptor responsible for the ability of NGF to rescue septal cholinergic neurons following axotomy, we infused polyclonal antibodies that act as specific agonists of trkA (RTA) into the lateral ventricle of fimbria-fornix lesioned animals. Rats receiving chronic intraventricular infusions of RTA showed significantly more low affinity NGF receptor immunoreactive (p75NGFR-IR) neurons on the lesioned side than did control animals 2 weeks following unilateral fimbria-fornix lesion. RTA also initiated cholinergic sprouting. Infusions of RTA in combination with an antibody that blocks p75NGFR (REX) did not reduce the cell savings effect observed with RTA alone. However, animals infused with RTA plus REX demonstrated significantly less sprouting. These findings suggest that antibody-induced trkA activation is sufficient to mediate NGF-promoted survival of axotomized cholinergic neurons in vivo.

Animals↗
PubMed1996

Applications of gene therapy to the CNS.

Gene therapy is a new method with potential for treating a broad range of acquired and inherited neurologic diseases, where the causative gene defect or deletion has been identified. In addition to gene replacement the application of gene products that reduce cellular dysfunction or death represent new therapeutic options. Gene transfer techniques to express novel proteins using different viral vectors in vitro and in vivo, as well as animal models and human trials will be reviewed in this article. We will focus on a new lentiviral vector as a recent gene transfer method and degenerative disorders of the CNS, and their related model systems.

Adenoviridae↗
PubMed1996

Constitutive retinoid receptors expressed from adenovirus vectors that specifically activate chromosomal target genes required for differentiation of promyelocytic leukemia and teratocarcinoma cells.

Sufficient knowledge of transcription factor structure and function has accumulated to allow attempts at the rational design of novel transcription factors for the study of gene regulation and potential application in gene therapy. In the present studies, we have systematically evaluated the function of chimeric retinoid receptors generated by fusion with the transactivation domain of VP16 and expression in adenovirus vectors. By varying the location of fusion of the VP16 transactivation domain with the retinoic acid receptor (RAR) or retinoid X receptor (RXR), marked differences in the specificity of gene activation were obtained. Although several chimeric proteins activated both RAR and RXR target genes, fusion of the NT16 transactivation domain to the N terminus of RAR permitted specific activation of reporter genes containing retinoic acid response elements. In contrast, fusion of the VP16 transactivation domain to the C terminus of RXR permitted specific activation of reporter genes containing RXR response elements. When tested for their ability to activate chromosomal targets, the chimera consisting of VP16 linked to the N terminus of PAR was much more active in promoting the differentiation of HL-60 cells and NTera-2 cells than the chimera consisting of VP16 linked to the C terminus of RXR. These observations support the existence of two distinct retinoid signalling pathways predicted on the basis of biochemical and pharmacologic studies and provide direct evidence that the programs of differentiation elicited by retinoic acid in these cells are mediated by a specific subset of binding sites for RAR-RXR heterodimers. VP16-RAR and VP16-RXR fusion proteins should be of further use in dissecting the relative contributions of RARs and RXRs to specific programs of gene expression. Constitutive retinoid receptors may also be considered for use as novel tumor suppressor genes for genetically based treatment of retinoid-responsive cancers.

Adenoviridae↗
PubMed1996

Gene therapy in the adult primate brain: intraparenchymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration.

Gene therapy may be a useful means of delivering substances to the brain that are capable of preventing neuronal degeneration. In the present experiment, we determined whether intraparenchymal transplants of primary autologous cells genetically modified to produce nerve growth factor (NGF) would prevent injury-induced degeneration of cholinergic neurons. Cultured primary monkey fibroblasts were genetically modified to produce human NGF, and secreted 13.2 ng NGF/10(6) cells/h in vitro. Adult monkeys then underwent fornix transections to induce degeneration of basal forebrain cholinergic neurons, and received autologous grafts of either NGF-producing or control, beta-galactosidase-producing fibroblasts directly into the basal forebrain region. One month later, 61.7 +/- 8.9% of cholinergic neurons remained indentifiable in NGF-graft recipients compared to 26.2 +/- 5.0% in control graft recipients (P < 0.02). Neuronal protection correlated with the accuracy of graft placement: up to 92% protection from neuronal degeneration occurred when NGF-secreting grafts were accurately placed immediately adjacent to injured neurons. Thus, intraparenchymal NGF delivery to the adult primate brain by gene transfer can prevent the degeneration of basal forebrain cholinergic neurons. Gene therapy can target intraparenchymal brain sites for regionally specific neurotrophin delivery, thereby avoiding limitations imposed by diffusion of substances across the blood-brain barrier and through CNS parenchyma, while avoiding adverse effects of neurotrophic factors delivered in a non-directed manner to the central nervous system. The delivery of NGF by gene transfer to the brain merits further study as a means of preventing cholinergic neuronal degeneration in human disorders such as Alzheimer's disease.

Animals↗