Corinne Kostic

Isolation of Multipotent Neural Precursors Residing in the Cortex of the Adult Human Brain

Multipotent precursors able to generate neurons, astrocytes, and oligodendrocytes have previously been isolated from human brain embryos and recently from neurogenic regions of the adult human brains. The isolation of multipotent neural precursors from adult human should open new perspectives to study adult neurogenesis and for brain repair. The present study describes the in vitro isolation from adult human brains of a progenitor responsive to both epidermal and basic fibroblast growth factors that forms spheres as it proliferates. Single spheres derived from various regions of the brain generate in vitro neurons, astrocytes, and oligodendrocytes. The clonal origin of the spheres was revealed by genomic viral insertion using lentiviral vector. Interestingly, this vector appears to be a potent tool for gene transfer into human neural progeny. Ninety-six percent of the spheres investigated were multipotent. Multipotent precursors were isolated from all brain regions studied, including the temporal and the frontal cortex, the amygdala, the hippocampus, and the ventricular zone. This study is the first evidence that primitive precursors such as multipotent precursors exist in the adult human cortex and can reside far from the ventricles. Neurogenesis derived from adult human progenitors differ to murine neurogenesis by the requirement of laminin for oligodendrocyte generation and by the action of basic-fibroblast growth factor and platelet derived growth factor that prevented the formation of oligodendrocytes and neurons. Moreover, the differentiation of human adult precursors seems to differ from fetal ones: adult precursors do not necessitate the removal of mitogen for differentiation. These results indicate that the study of adult multipotent precursors is a new platform to study adult human neurogenesis, potentially generate neural cells for transplantation, and design protocols for in vivo stimulation.

Bmi1 Loss Produces an Increase in Astroglial Cells and a Decrease in Neural Stem Cell Population and Proliferation

The polycomb transcriptional repressor Bmi1 promotes cell cycle progression, controls cell senescence, and is implicated in brain development. Loss of Bmi1 leads to a decreased brain size and causes progressive ataxia and epilepsy. Recently, Bmi1 was shown to control neural stem cell (NSC) renewal. However, the effect of Bmi1 loss on neural cell fate in vivo and the question whether the action of Bmi1 was intrinsic to the NSCs remained to be investigated. Here, we show that Bmi1 is expressed in the germinal zone in vivo and in NSCs as well as in progenitors proliferating in vitro, but not in differentiated cells. Loss of Bmi1 led to a decrease in proliferation in zones known to contain progenitors: the newborn cortex and the newborn and adult subventricular zone. This decrease was accentuated in vitro, where we observed a drastic reduction in NSC proliferation and renewal because of NSC-intrinsic effects of Bmi1 as shown by the means of RNA interference. Bmi1-/- mice also presented more astrocytes at birth, and a generalized gliosis at postnatal day 30. At both stages, colocalization of bromodeoxyuridine and GFAP demonstrated that Bmi1 loss did not prevent astrocyte precursor proliferation. Supporting these observations, Bmi1-/- neurospheres generate preferentially astrocytes probably attributable to a different responsiveness to environmental factors. Bmi1 is therefore necessary for NSC renewal in a cell-intrinsic mode, whereas the altered cell pattern of the Bmi1-/- brain shows that in vivo astrocyte precursors can proliferate in the absence of Bmi1.

Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis.

Background RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. Methods and Findings Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. Conclusions By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65 −/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.

Delivery of ciliary neurotrophic factor via lentiviral-mediated transfer protects axotomized retinal ganglion cells for an extended period of time.

Ciliary neurotrophic factor (CNTF) has recently been demonstrated to be one of the most promising neurotrophic factors to improve both the survival and regeneration of injured retinal ganglion cells (RGCs). In the present study, we used optic nerve transection as an in vivo model to evaluate the effectiveness of a self-inactivating, replication-deficient lentiviral-mediated transfer of human ciliary neurotrophic factor (SIN-PGK-CNTF) on the survival of axotomized adult rat RGCs. Counts of dextran-fluorescein isothiocyanate conjugated (D-FITC)-retrogradely labeled RGCs revealed that the percentage of RGCs was drastically reduced (<90% cell death) 21 days after optic nerve transection. Retinal sections stained with X-gal revealed that intravitreal injection of the control LacZ-expressing lentiviral vector (LV-LacZ) resulted in the transduction of RGCs and retinal pigment epithelium (RPE) cells. A single intravitreal injection of LV-CNTF at the time of axotomy significantly enhanced RGC survival at 14 and 21 days postaxotomy compared to controls. These results demonstrate for the first time that rapid and prolonged delivery of CNTF using lentiviral-mediated gene transfer to the retina is an effective treatment for rescuing axotomized RGCs for an extended period of time. These results suggest that early and continuous administration of CNTF could serve as a potential treatment for retinal disorders involving optic neuropathy and RGC injury such as in glaucoma.

Activity analysis of housekeeping promoters using self-inactivating lentiviral vector delivery into the mouse retina

For most retinal degeneration disorders, no efficient treatment exists to preserve photoreceptors (PRs) and, consequently, to maintain vision. Gene transfer appears to be a promising approach to prevent PR loss. In order to design adequate vectors to target specific retinal cell types, we have analyzed the expression pattern of three different promoters (mouse phosphoglycerate kinase 1 (PGK), elongation factor-1 (EFS), rhodopsin (Rho)) in newborn and adult DBA/2 mice retinas using self-inactivating lentiviral vectors. At 7 days after intraocular injection and in optimal conditions, cell transduction was observed up to 1.5 mm from the injection site. PGK promoter expression was predominant in the retinal pigment epithelium (RPE), especially in adult mice, whereas the EFS promoter allowed a broad expression in the retina. Finally, as expected, the Rho promoter was specifically expressed in PRs. Differences in the cell types transduced and in transduction efficiency were observed between newborn and adult injected eyes emphasizing the importance of such basic studies for further gene therapy approaches as well as for understanding the transcriptional changes during retinal maturation. Thus, for future attempts to slow or rescue retinal degeneration by lentiviral delivery, PGK and EFS are more suitable to control the expression of a supporting secreted factor, PGK being mainly expressed in RPE and EFS in different cell types throughout the entire retina, whereas Rho should allow to specifically deliver the therapeutic gene to PRs.

High Yield of Cells Committed to the Photoreceptor Fate from Expanded Mouse Retinal Stem Cells

The purpose of the present work was to generate, from retinal stem cells (RSCs), a large number of cells committed toward the photoreceptor fate in order to provide an unlimited cell source for neurogenesis and transplantation studies. We expanded RSCs (at least 34 passages) sharing characteristics of radial glial cells and primed the cells in vitro with fibroblast growth factor (FGF)‐2 for 5 days, after which cells were treated with the B27 supplement to induce cell differentiation and maturation. Upon differentiation, cells expressed cell type‐specific markers corresponding to neurons and glia. We show by immunocytochemistry analysis that a subpopulation of differentiated cells was committed to the photoreceptor lineage given that these cells expressed the photoreceptor proteins recoverin, peripherin, and rhodopsin in a same ratio. Furthermore, cells infected during the differentiation procedure with a lentiviral vector expressing green fluorescent protein (GFP) under the control of either the rhodopsin promoter or the interphotoreceptor retinoid‐binding protein (IRBP) promoter, expressed GFP. FGF‐2 priming increased neuronal differentiation while decreasing glia generation. Reverse transcription‐polymerase chain reaction analyses revealed that the differentiated cells expressed photoreceptor‐specific genes such as Crx, rhodopsin, peripherin, IRBP, and phosphodiesterase‐α. Quantification of the differentiated cells showed a robust differentiation into the photoreceptor lineage: Approximately 25%–35% of the total cells harbored photoreceptor markers. The generation of a significant number of nondifferentiated RSCs as well as differentiated photoreceptors will enable researchers to determine via transplantation studies which cells are the most adequate to integrate a degenerating retina.

Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA

Strategies leading to the long‐term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age‐related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD.

FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.

Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors.

Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

Retinal Degeneration Progression Changes Lentiviral Vector Cell Targeting in the Retina

In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb) with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.