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1.
Gene Ther ; 29(10-11): 624-635, 2022 11.
Article in English | MEDLINE | ID: mdl-34853444

ABSTRACT

Sheep carrying a mutated CNGA3 gene exhibit diminished cone function and provide a naturally occurring large animal model of achromatopsia. Subretinal injection of a vector carrying the CNGA3 transgene resulted in long-term recovery of cone function and photopic vision in these sheep. Research is underway to develop efficacious vectors that would enable safer transgene delivery, while avoiding potential drawbacks of subretinal injections. The current study evaluated two modified vectors, adeno-associated virus 2-7m8 (AAV2-7m8) and AAV9-7m8. Intravitreal injection of AAV2-7m8 carrying enhanced green fluorescent protein under a cone-specific promoter resulted in moderate photoreceptor transduction in wild-type sheep, whereas peripheral subretinal delivery of AAV9-7m8 resulted in the radial spread of the vector beyond the point of deposition. Intravitreal injection of AAV2-7m8 carrying human CNGA3 in mutant sheep resulted in mild photoreceptor transduction, but did not lead to the clinical rescue of photopic vision, while day-blind sheep treated with a subretinal injection exhibited functional recovery of photopic vision. Transgene messenger RNA levels in retinas of intravitreally treated eyes amounted to 4-23% of the endogenous CNGA3 levels, indicating that expression levels >23% are needed to achieve clinical rescue. Overall, our results indicate intravitreal injections of AAV2.7m8 transduce ovine photoreceptors, but not with sufficient efficacy to achieve clinical rescue in CNGA3 mutant sheep.


Subject(s)
Color Vision Defects , Sheep/genetics , Animals , Humans , Color Vision Defects/genetics , Color Vision Defects/therapy , Intravitreal Injections , Genetic Vectors/genetics , Genetic Therapy/methods , Dependovirus/metabolism , Retina/metabolism , Transduction, Genetic , Cyclic Nucleotide-Gated Cation Channels/genetics , Cyclic Nucleotide-Gated Cation Channels/metabolism
2.
Sci Rep ; 11(1): 12603, 2021 06 15.
Article in English | MEDLINE | ID: mdl-34131223

ABSTRACT

Optogenetics has revolutionized neurosciences by allowing fine control of neuronal activity. An important aspect for this control is assessing the activation and/or adjusting the stimulation, which requires imaging the entire volume of optogenetically-induced neuronal activity. An ideal technique for this aim is fUS imaging, which allows one to generate brain-wide activation maps with submesoscopic spatial resolution. However, optical stimulation of the brain with blue light might lead to non-specific activations at high irradiances. fUS imaging of optogenetic activations can be obtained at these wavelengths using lower light power (< 2mW) but it limits the depth of directly activatable neurons from the cortical surface. Our main goal was to report that we can detect specific optogenetic activations in V1 even in deep layers following stimulation at the cortical surface. Here, we show the possibility to detect deep optogenetic activations in anesthetized rats expressing the red-shifted opsin ChrimsonR in V1 using fUS imaging. We demonstrate the optogenetic specificity of these activations and their neuronal origin with electrophysiological recordings. Finally, we show that the optogenetic response initiated in V1 spreads to downstream (LGN) and upstream (V2) visual areas.


Subject(s)
Brain/diagnostic imaging , Optogenetics , Ultrasonography , Visual Cortex/diagnostic imaging , Animals , Brain/physiology , Light , Neurons/physiology , Photic Stimulation , Rats , Visual Cortex/physiology
3.
Cell Death Differ ; 22(2): 323-35, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25257170

ABSTRACT

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4(flox/flox) animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.


Subject(s)
Axons/physiology , Myelin Proteins/genetics , Optic Nerve Injuries/therapy , Regeneration , Retinal Ganglion Cells/physiology , Signal Transduction , Animals , Dependovirus/genetics , Female , Genetic Vectors , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Sheath/physiology , Nerve Crush , Neuroglia/cytology , Neurons/cytology , Nogo Proteins
4.
Gene Ther ; 21(6): 585-92, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24694538

ABSTRACT

X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.


Subject(s)
Eye Proteins/genetics , Genetic Therapy/methods , Retina/cytology , Aging , Animals , Cell Adhesion Molecules/genetics , Disease Models, Animal , Electroretinography , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice, Inbred C57BL , Mice, Mutant Strains , Organ Culture Techniques , Photoreceptor Cells, Vertebrate/physiology , Retina/physiology , Retinoschisis/genetics , Retinoschisis/therapy
5.
Cell Death Dis ; 4: e734, 2013 Jul 18.
Article in English | MEDLINE | ID: mdl-23868067

ABSTRACT

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.


Subject(s)
Axons/physiology , Nerve Regeneration , Optic Nerve/physiology , STAT3 Transcription Factor/genetics , Signal Transduction , Amides/pharmacology , Animals , Aporphines , Cell Survival , Ciliary Neurotrophic Factor/physiology , Dependovirus/genetics , Male , Mice , Mice, Inbred C57BL , Optic Nerve/cytology , Pyridines/pharmacology , Retinal Ganglion Cells/physiology , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/physiology , Transcription, Genetic , Transduction, Genetic , rho GTP-Binding Proteins/antagonists & inhibitors , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolism
6.
Gene Ther ; 19(2): 176-81, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22011645

ABSTRACT

Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.


Subject(s)
Central Nervous System , Dependovirus/genetics , Genetic Therapy , Retina/pathology , Retinal Degeneration/therapy , Animals , Disease Models, Animal , Gene Expression Regulation, Developmental , Genetic Vectors/administration & dosage , Green Fluorescent Proteins , Humans , Mice , Mice, Inbred C57BL , Mutation , Promoter Regions, Genetic , Retina/cytology , Retina/growth & development , Retinal Degeneration/genetics , Retinal Rod Photoreceptor Cells/metabolism , Rhodopsin/genetics
7.
Cell Death Differ ; 19(7): 1096-108, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22193546

ABSTRACT

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.


Subject(s)
Apoptosis/drug effects , Endoplasmic Reticulum Stress , Myelin Proteins/metabolism , Neurons/metabolism , Retina/cytology , Up-Regulation , Animals , Annexin A5/metabolism , Axotomy , Cells, Cultured , Dependovirus/genetics , Lipoproteins/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Proteins/antagonists & inhibitors , Myelin Proteins/genetics , Neurites/physiology , Nogo Proteins , RNA Interference , RNA, Small Interfering/metabolism , Regeneration/drug effects , Retina/metabolism , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/metabolism , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolism
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