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Biomedical subjects

Jean Bennett

Publications and source records attributed to Jean Bennett.

At least 19 recordsLinked to original sources
PubMed2006

In vivo delivery of recombinant viruses to the fetal murine cochlea: transduction characteristics and long-term effects on auditory function.

Congenital hearing deficits can be caused by a variety of genetic and acquired conditions. Complete reversal of deficits in the peripheral auditory system may require delivery of corrective genes to cochlear progenitor cells. We tested delivery of lentivirus and an array of recombinant adeno-associated viral (AAV) serotypes for efficiency and cellular specificity of transgene expression after in utero delivery to the developing mouse otocyst. Stability of expression and safety with respect to auditory function were then tested in those vectors that had the most favorable in utero cochlear transduction characteristics (AAV2/1, AAV2/8, and lentivirus). AAV2/1 was found to be the optimal vector for in utero cochlear gene transfer. It efficiently transduced progenitors giving rise to both inner and outer hair cells and supporting cells and had no adverse effect on cochlear cell differentiation. Further, it had no pathological effect on differentiated hair cells or the integrity of the auditory nerve or brain-stem nuclei as measured by auditory brain-stem response testing. AAV2/1 promises to be useful in further studies evaluating differentiation pathways of cochlear cells in health and disease and for developing gene-based therapies for congenital and acquired forms of peripheral hearing loss.

Animals↗
PubMed2006

Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection.

AAV2 delivery of the RPE65 gene to the retina of blind RPE65-deficient animals restores vision. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis (LCA), but toxicity and dose efficacy have not been defined. We studied ocular delivery of AAV-2/2.RPE65 in RPE65-mutant dogs. There was no systemic toxicity. Ocular examinations showed mild or moderate inflammation that resolved over 3 months. Retinal histopathology indicated that traumatic lesions from the injection were common, but thinning within the injection region occurred only at the two highest vector doses. Biodistribution studies at 3 months postinjection showed no vector in optic nerve or visual centers in the brain and only isolated non-dose-related detection in other organs. We also performed biodistribution studies in normal rats at about 2 weeks and 2 months postinjection and vector was not widespread outside the injected eye. Dose-response results in RPE65-mutant dogs indicated that the highest 1.5-log unit range of vector doses proved efficacious. The efficacy and toxicity limits defined in this study lead to suggestions for the design of a subretinal AAV-2/2.RPE65 human trial of RPE65-associated LCA.

Animals↗
PubMed2006

Eye movement recordings as an effectiveness indicator of gene therapy in RPE65-deficient canines: implications for the ocular motor system.

PURPOSE: To perform ocular motility recordings of infantile nystagmus (IN) in RPE65-deficient canines and determine whether they can be used as a motor indicator of restored retinal function to investigate the effects of gene therapy. METHODS: Treated and untreated canines were comfortably suspended in a custom-built sling and encouraged to fixate on distant targets at gaze angles varying between +/-15 degrees horizontally and +/-10 degrees vertically. Ocular motility recordings were made, using two distinct methods-infrared reflection and high-speed video. The resultant recordings from three untreated, four treated, and three pre- and post-treatment dogs were analyzed for using the eXpanded Nystagmus Acuity Function (NAFX), which yields an objective assessment of best potential visual acuity, based on the duration and repeatable accuracy of foveation and centralisation. RESULTS: During fixation, the untreated dogs exhibited large-amplitude, classic IN waveforms, including pendular and jerk in both the horizontal and vertical planes, which prevented them from keeping the targets within the area centralis (the region of highest receptor density, spanning +/-3 degrees horizontally by +/-1.5 degrees vertically, analogous to the fovea). Some untreated dogs also had small-amplitude (0.5-1 degrees), high-frequency (6-9 Hz) oscillations. Under the same conditions, successfully treated canines no longer exhibited clinically detectable IN. Their IN was converted to waveforms with very low amplitudes that yielded higher NAFX values and allowed target images to remain well within the area centralis. Of note, uniocular treatment appeared to damp the IN in both eyes. Behaviorally, the treated dogs were able to successfully navigate through obstacles more easily without inadvertent contact, a task beyond the untreated dogs' ability. CONCLUSIONS: Gene therapy that successfully restored retinal function also reduced the accompanying IN to such a great extent that it was not clinically detectable approximately 90% of the time in many of the dogs. IN improvement, as quantified by the NAFX, is an objective motor indicator of visual improvement due to gene therapy.

Animals↗
PubMed2006

Retinal ganglion cell loss induced by acute optic neuritis in a relapsing model of multiple sclerosis.

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are marked by inflammatory demyelinating lesions throughout the central nervous system, including optic nerve. Neuronal loss also occurs in MS and EAE lesions, but it is not known whether neuronal loss occurs secondary to inflammation, or as a primary process. In the current study, the relationship of inflammation to retinal ganglion cell (RGC) loss during acute optic neuritis is examined. RGCs were labelled with Flourogold, and EAE was induced in SJL/J mice by immunization with proteolipid protein peptide 139-151 (PLP). At various time points, RGCs were counted and optic nerves were examined for inflammatory cell infiltrates. No optic neuritis was detected prior to day 9 following immunization. Incidence of optic neuritis was 30% at day 9 and increased to over 70% by day 11, remaining high through day 18. In contrast, no RGC loss was detected in eyes with optic neuritis until day 14. A 43.1% reduction in RGC numbers at day 14 increased to 50.8% by day 18. No RGC loss occurred in eyes without optic neuritis. The fact that inflammation precedes RGC loss suggests that neuronal loss during optic neuritis occurs secondary to the inflammatory process.

Animals↗
PubMed2005

The distribution, concentration, and toxicity of enhanced green fluorescent protein in retinal cells after genomic or somatic (virus-mediated) gene transfer.

PURPOSE: The concentration of enhanced green fluorescent protein (EGFP) in individual photoreceptor cells of live mouse retina was quantified and correlated with physiological measurements of cell function. METHODS: EGFP protein levels in the retinas of mice injected subretinally by either one of two serotypes of adeno-associated virus (AAV; AAV2/5.CMV.EGFP; AAV2/2.CMV.EGFP) were quantified with a photon-counting confocal laser scanning microscope and compared with those of transgenic mice whose retinas expressed EGFP under the beta-actin (pbetaAct) or human L/M-cone opsin (pLMCOps) promoter. Single-cell suction pipette recordings of single rods and whole-field electroretinograms (ERGs) were performed to assess retinal cell function. RESULTS: The highest levels of EGFP (680 microM) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of pbetaAct.EGFP mice contained 270 microM EGFP, while their bipolars had 440 microM. The cones of pLMCOps.EGFP mice expressed 60 microM protein. The amplitudes of the major components of ERGs were within the normal range for all transgenic animals examined, and single cell recordings from living pbetaAct.EGFP rods were indistinguishable from those of controls. CONCLUSIONS: EGFP levels in individual cells of live mouse retinas can be quantified, so that the efficacy of gene transfer methods can be quantified. Concentrations of several hundred microM are not deleterious to normal function of photoreceptors and bipolar cells. This approach can also be used to quantify levels of biologically active EGFP fusion proteins.

Animals↗
PubMed2005

Deficiency of SPAG16L causes male infertility associated with impaired sperm motility.

The axonemes of cilia and flagella contain a "9+2" structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of M(r) approximately 71 x 10(3) (SPAG16L) and M(r) approximately 35 x 10(3) (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys-symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus.

Animals↗
PubMed2005

Spatial and temporal expression patterns of the choroideremia gene in the mouse retina.

PURPOSE: Choroideremia (CHM), an X-linked retinal disease, is caused by mutations affecting the CHM gene. This gene encodes REP-1, which functions in the covalent modifications of proteins involved in vesicle trafficking. The disease affects several cell types in the retina, but it is not known which cell types contribute directly or indirectly to disease progression. A study of the expression patterns of Chm and the related gene Chml in the mouse retina was undertaken in order to address this issue. METHODS: The expression patterns of Chm and Chml were determined by in situ hybridization. The localization of the Chm protein product, Rep-1, was determined spatially and temporally in the mouse retina by immunohistochemistry. RESULTS: Chm and Chml mRNA were found in every major layer of the retina in adult mice. During development, Rep-1 protein localization changes from a fairly diffuse pattern during embryogenesis to a more specific pattern at the time of retinal differentiation. In adulthood, Rep-1 localizes to distinct cellular compartments in multiple retinal cell types. CONCLUSIONS: Chm and Chml have the same broad expression profile in the mouse retina. In particular, the Chm transcript and corresponding protein are found in cell types other than those thought to be primarily affected in the human disease. These results have important implications for approaches with which to develop a relevant mouse model of choroideremia and for therapeutic strategies for this disease.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness.

The short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no detectable 11-cis-retinal, but markedly elevated retinyl esters. Subretinal AAV-RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Subretinal AAV-RPE65 vector is well tolerated and does not elicit high antibody levels to the vector or the protein in ocular fluids or serum. In long-term studies, wild-type cDNA is expressed only in target cells. Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations.

Animals↗
PubMed2005

Oxygen distribution and vascular injury in the mouse eye measured by phosphorescence-lifetime imaging.

Maps of the oxygen distribution in the retina of the mouse eye were obtained by phosphorescence-lifetime imaging. Phosphor dissolved in the blood was excited by modulated light and phosphorescence imaged through microscope optics with an intensified-CCD camera. Phosphorescence lifetimes and oxygen pressures were calculated for each pixel of the images. The resolution was sufficient to permit the detection of anomalies that result in reduced oxygen pressures in individual retinal capillaries. High-resolution maps of oxygen distribution in the retina can provide greater understanding of the role of oxygen and vascular function in diseases of the eye.

Animals↗
PubMed2005

Identifying photoreceptors in blind eyes caused by RPE65 mutations: Prerequisite for human gene therapy success.

Mutations in RPE65, a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA). Retinal gene therapy restores vision to blind canine and murine models of LCA. Gene therapy in blind humans with LCA from RPE65 mutations may also have potential for success but only if the retinal photoreceptor layer is intact, as in the early-disease stage-treated animals. Here, we use high-resolution in vivo microscopy to quantify photoreceptor layer thickness in the human disease to define the relationship of retinal structure to vision and determine the potential for gene therapy success. The normally cone photoreceptor-rich central retina and rod-rich regions were studied. Despite severely reduced cone vision, many RPE65-mutant retinas had near-normal central microstructure. Absent rod vision was associated with a detectable but thinned photoreceptor layer. We asked whether abnormally thinned RPE65-mutant retina with photoreceptor loss would respond to treatment. Gene therapy in Rpe65(-/-) mice at advanced-disease stages, a more faithful mimic of the humans we studied, showed success but only in animals with better-preserved photoreceptor structure. The results indicate that identifying and then targeting retinal locations with retained photoreceptors will be a prerequisite for successful gene therapy in humans with RPE65 mutations and in other retinal degenerative disorders now moving from proof-of-concept studies toward clinical trials.

Adolescent↗
PubMed2005

Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16.

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.

Algal Proteins↗
PubMed2005

Persistent fetal ocular vasculature in mice deficient in bax and bak.

BACKGROUND: The ocular fetal vasculature normally regresses by apoptosis but for unknown reasons fails to regress in the human disease persistent fetal vasculature. OBJECTIVE: To investigate whether proapoptotic Bcl-2 members, Bax and Bak, are involved in fetal vasculature regression. METHODS: Adult eyes from mice deficient in Bax and/or Bak were examined grossly and histologically for persistence of fetal vasculature. Vessels were identified by the presence of lumens and erythrocytes and by Factor VIII labeling. Eyes from postnatal day 7 mice were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) analysis to determine if deficiency of Bax and Bak results in defective developmental apoptosis. RESULTS: Only bax(-/-)bak(-/-) eyes retained fetal vasculature into adulthood. This vasculature consisted of a hyaloid artery emerging from the optic nerve head and intravitreal and perilental vessels but not a pupillary membrane. At postnatal day 7, wild-type but not bax(-/-)bak(-/-) eyes had TUNEL-positive cells in the fetal vasculature. CONCLUSIONS: These data demonstrate that Bax and Bak serve overlapping functions in fetal vasculature regression, emphasizing the importance of apoptosis in developmental remodeling. Clinical Relevance Disruption of Bax and Bak results in persistent fetal vasculature in knockout mice, providing a model of the human disease persistent fetal vasculature to investigate its etiology and potential therapies.

Animals↗
PubMed2005

Imaging oxygen pressure in the retina of the mouse eye.

The phosphorescence lifetime imaging system previously used to image oxygen in the retina of the cat eye was modified to allow imaging of phosphorescence lifetimes in the much smaller mouse eye. Following the lead of Shonat and coworkers, a frequency domain approach was used in which the excitation light source was modulated in a 50% on: 50% off square wave while the gate of the intensified CCD camera was similarly modulated but delayed with respect to the excitation. These were analyzed by fitting the intensity at each pixel to a sinusoid. The phase of the phosphorescence relative to the excitation was determined and from the phase shift and frequency, the phosphorescence lifetime was calculated. The Stern-Volmer relationship was then used to calculate the oxygen pressure at each pixel of the image array. High resolution maps of phosphorescence lifetime and oxygen pressure in the retina of the mouse eye have been attained. The retinal veins draining into the optic head appear as large, highly phosphorescent vessels against a lower phosphorescence background with a network of smaller vessels. The oxygen pressure in the retinal veins is typically from 20 to 30 mm Hg while the background has somewhat higher oxygen pressures. Experiments are underway to resolve the oxygen in the choroid from that in the retina. The arteries on the retinal surface can be observed, but their small diameter, relatively high oxygen pressures (> 90 mm Hg), and surrounding tissue with much lower oxygen pressures, makes accurate determination of the oxygen pressure a challenge.

Animals↗
PubMed2005

Long-term inducible gene expression in the eye via adeno-associated virus gene transfer in nonhuman primates.

Adeno-associated viral gene therapy has shown promise for the treatment of inherited and degenerative diseases in a variety of animal models. Some of the most dramatic results have been obtained in the field of ocular gene therapy, where efficacy has been tremendous in inherited and acquired retinal disorders. For the promise of this approach to be realized it will be necessary to create vectors capable of pharmacologic or physiologic regulation of the transgene. We describe in this paper a dimerizer-inducible viral expression system that is able to reproducibly drive expression of the reporter gene erythropoietin in the eyes of nonhuman primates over a period of 2.5 years. The expression profiles were characterized by minimal basal expression in the absence of inducer and dose-responsive maximal expression in the presence of inducer drug.

Animals↗
PubMed2005

Herpes simplex virus type 1 glycoprotein e is required for axonal localization of capsid, tegument, and membrane glycoproteins.

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promotes cell-to-cell spread at basolateral surfaces of epithelial cells, but its activity in neurons is less clear. We used the mouse retina infection model and neuronal cell cultures to define the spread phenotype of gE mutant viruses. Wild-type (WT) and gE-null (NS-gEnull) viruses both infected retina ganglion cell neurons; however, NS-gEnull viral antigens failed to reach the optic nerve, which indicates a defect in axonal localization. We evaluated two Fc receptor-negative gE mutant viruses containing four amino acid inserts in the gE ectodomain. One mutant virus failed to spread from the retina into the optic nerve, while the other spread normally. Therefore, the gE ectodomain is involved in axonal localization, and the Fc receptor and neuronal spread are mediated by overlapping but distinct gE domains. In the retina infection model, virus can travel to the brain via the optic nerve from presynaptic to postsynaptic neurons (anterograde direction) or via nerves that innervate the iris and ciliary body from postsynaptic to presynaptic neurons (retrograde direction). WT virus infected the brain by anterograde and retrograde routes, whereas NS-gEnull virus failed to travel by either pathway. The site of the defect in retrograde spread remains to be determined; however, infection of rat superior cervical ganglia neurons in vitro indicates that gE is required to target virion components to the axon initial segment. The requirement for gE in axonal targeting and retrograde spread highlights intriguing similarities and differences between HSV-1 and pseudorabies virus gE.

Animals↗