RESUMEN
Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.
Asunto(s)
Vectores Genéticos/genética , Lentivirus/genética , Mitosis/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , ADN Satélite/genética , Femenino , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Ratas , Secuencias Repetidas Terminales/genética , Factores de Transcripción/genética , Integración Viral/genéticaRESUMEN
At present, there are severe limitations to the successful migration and integration of stem cells transplanted into the degenerated retina to restore visual function. This study investigated the potential role of chondroitin sulfate proteoglycans (CSPGs) and microglia in the migration of human Müller glia with neural stem cell characteristics following subretinal injection into the Lister hooded (LH) and Royal College of Surgeons (RCS) rat retinae. Neonate LH rat retina showed minimal baseline microglial accumulation (CD68-positive cells) that increased significantly 2 weeks after transplantation (p < .001), particularly in the ganglion cell layer (GCL) and inner plexiform layer. In contrast, nontransplanted 5-week-old RCS rat retina showed considerable baseline microglial accumulation in the outer nuclear layer (ONL) and photoreceptor outer segment debris zone (DZ) that further increased (p < .05) throughout the retina 2 weeks after transplantation. Marked deposition of the N-terminal fragment of CSPGs, as well as neurocan and versican, was observed in the DZ of 5-week-old RCS rat retinae, which contrasted with the limited expression of these proteins in the GCL of the adult and neonate LH rat retinae. Staining for CSPGs and CD68 revealed colocalization of these two molecules in cells infiltrating the ONL and DZ of the degenerating RCS rat retina. Enhanced immune suppression with oral prednisolone and intraperitoneal injections of indomethacin caused a reduction in the number of microglia but did not facilitate Müller stem cell migration. However, injection of cells with chondroitinase ABC combined with enhanced immune suppression caused a dramatic increase in the migration of Müller stem cells into all the retinal cell layers. These observations suggest that both microglia and CSPGs constitute a barrier for stem cell migration following transplantation into experimental models of retinal degeneration and that control of matrix deposition and the innate microglial response to neural retina degeneration may need to be addressed when translating cell-based therapies to treat human retinal disease.
Asunto(s)
Inhibición de Migración Celular/fisiología , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Microglía/fisiología , Degeneración Retiniana/cirugía , Trasplante de Células Madre/métodos , Animales , Animales Recién Nacidos , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Proteoglicanos Tipo Condroitín Sulfato/genética , Femenino , Humanos , Microglía/citología , Embarazo , Ratas , Retina/citología , Retina/fisiología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Photoreceptor loss causes irreversible blindness in many retinal diseases. The identification of suitable donor cell populations is of considerable interest because of their potential use to replace the photoreceptors lost in disease. Stem or progenitor cells that give rise to neurons and glia have been identified in several regions of the brain, including the embryonic retina and the ciliary epithelium of the adult eye, raising the possibility of autologous transplantation. However, there has been little systematic investigation into precisely which regions of the large mammalian adult eye give rise to such cells. Here, we show for the first time using the porcine eye the presence of progenitor cells in additional regions of the adult eye, including the pars plana and iris, regions that, in the human, are readily accessible during routine eye surgery. When cultured in the presence of growth factors, these cells proliferate to form neurospheres comprised of cells expressing retinal progenitor markers. Using an adherent monolayer culture system, these cells could be readily expanded to increase their number more than 1 million-fold and maintain a progenitor phenotype. When grown on the substrate laminin in the presence of serum, cells derived from both spheres and monolayer cultures differentiated into neurons and glia. These results suggest that a population of cells derived from the adult iris, pars plana, and ciliary body of a large mammalian species, the pig, has progenitor properties and neurogenic potential, thereby providing novel sources of donor cells for transplantation studies. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Células Madre Adultas/citología , Cuerpo Ciliar/citología , Iris/citología , Sus scrofa/anatomía & histología , Animales , Biomarcadores , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Separación Celular , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Medios de Cultivo/farmacología , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Laminina , Masculino , Células Madre Multipotentes/citología , Neuroglía/citología , Neuronas/citología , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacosRESUMEN
Ciliary neurotrophic factor (CNTF) has been shown to protect ganglion cells in a variety of acute ischaemia models. Here we assess the efficacy of local CNTF gene transfer in protecting retinal ganglion cells when there is focal ischaemia combined with interruption of axoplasmic flow. This dual injury may be more representative of the pathological mechanisms operating in acute retinal diseases, such as vascular events acting at the optic nerve head. Fourteen rats received an intravitreal injection of an adeno-associated viral (AAV) vector expressing a secretable form of CNTF into the right eye and a control vector into the left eye. Three weeks later, each rat underwent a symmetrical small vertical 2mm standardised retinal crush injury approximately 2mm temporal to the optic disc. The injury also occluded the temporal retinal arteriole so that the axon crush was combined with an acute retinal infarction visible on fundoscopy. Changes in the damaged sector were compared histologically four weeks after injury and ganglion cell survival was estimated by comparing cell counts on retinal flat-mounts immunostained with RT-97 antibody. This mode of injury led to a profound loss of both the inner nuclear and ganglion cell layers, but was limited to the lesioned sector. In AAV.CNTF-treated eyes approximately 12% of ganglion cells survived compared with approximately 2% in control eyes (p=0.01). The scotopic electroretinogram (ERG), however, was reduced to about 50% in AAV.CNTF-treated eyes, both before and after injury. We therefore show that CNTF gene transfer confers neuroprotection to ganglion cells undergoing an acute ischaemic injury combined with interruption of axoplasmic flow. This approach may be relevant to optic nerve trauma and a variety of retinal vascular diseases that lead to swelling of the optic nerve head, provided CNTF can be delivered in a way that does not significantly suppress retinal function.
Asunto(s)
Factor Neurotrófico Ciliar/genética , Neuropatía Óptica Isquémica/patología , Retina/lesiones , Vasos Retinianos/patología , Animales , Apoptosis/fisiología , Arteriolas , Transporte Axonal/fisiología , Recuento de Células , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Electrorretinografía/métodos , Técnicas de Transferencia de Gen , Inmunohistoquímica/métodos , Infarto , Masculino , Neuropatía Óptica Isquémica/fisiopatología , Ratas , Ratas Long-Evans , Retina/patología , Células Ganglionares de la Retina/patologíaRESUMEN
While AAV- and lentivirus-mediated gene replacement therapy can produce structural and functional improvements in various animal models of inherited retinal degeneration, this approach often has very limited effects on the rate of photoreceptor cell loss. Neurotrophic factors such as ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) have been shown to prolong photoreceptor survival in rodent models of retinal degeneration, but AAV-mediated Cntf expression also results in suppression of electrophysiological responses from the retina. In this study using mice, we show that while the deleterious effects mediated by CNTF are dose-dependent, administering a dose of CNTF that does not adversely affect retinal function precludes its ability to delay photoreceptor cell death. In evaluating GDNF as a neuroprotective agent, we show that AAV-mediated Gdnf expression does not produce adverse effects similar to those of CNTF. In addition, we demonstrate the ability of AAV-mediated delivery of Gdnf to slow cell death in two rodent models of retinitis pigmentosa and to enhance retinal function in combination with the relevant gene replacement therapy. These data show for the first time that a combination of these approaches can provide enhanced rescue over gene replacement or growth factor therapy alone.
Asunto(s)
Factor Neurotrófico Ciliar/metabolismo , Dependovirus/genética , Expresión Génica , Terapia Genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Animales , Animales Modificados Genéticamente , Línea Celular , Supervivencia Celular , Factor Neurotrófico Ciliar/genética , Modelos Animales de Enfermedad , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Humanos , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Periferinas , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Ratas , Degeneración Retiniana/metabolismoRESUMEN
Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/genética , Lentivirus/genética , Animales , Encéfalo/citología , Proteínas Portadoras , Electrorretinografía , Proteínas del Ojo/metabolismo , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Epitelio Pigmentado Ocular/citología , Ratas , Retina/citología , Células Tumorales Cultivadas , Integración Viral/genética , cis-trans-IsomerasasRESUMEN
Memory lymphocytes are important mediators of the immune response. These cells are long-lived and undergo clonal expansion upon reexposure to specific antigen, differentiating into effector cells that secrete Ig or cytokines while maintaining a residual pool of memory T and B lymphocytes. Here, the ability of antigen-specific lymphocytes to undergo repeated cycles of antigen-driven clonal expansion and contraction is exploited in a therapeutic protocol aimed at regulating protein delivery. The principle of this strategy is to introduce genes encoding proteins of therapeutic interest into a small number of antigen-specific B lymphocytes. Output of therapeutic protein can then be regulated in vivo by manipulating the size of the responder population by antigen challenge. To evaluate whether such an approach is feasible, we developed a mouse model system in which Emu- and Iglambda-based vectors were used to express human erythropoietin (hEPO) gene in B lymphocytes. These mice were then immunized with the model antigen phycoerythrin (PE), and immune splenocytes (or purified PE-specific B lymphocytes) were adoptively transferred to normal or mutant (EPO-deficient) hosts. High levels of hEPO were detected in the serum of adoptively transferred normal mice after PE administration, and this responsiveness was maintained for several months. Similarly, in EPO-deficient anemic recipients, antigen-driven hEPO expression was shown to restore hematocrit levels to normal. These results show that antigen-mediated regulation of memory lymphocytes can be used as a strategy for delivering therapeutic proteins in vivo.