No abstract

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.

Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair1 and restoration of vision through transplantation of photoreceptor precursors obtained from post-natal retinas into visually impaired adult mice2,3. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages4-6. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs6. We show that Rhop.GFP-selected rod precursors derived by this protocol integrate within degenerate retinae of adult mice and mature into outer segment–bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently than cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2

Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of functional photoreceptors within host retinae that express an array of donorspecific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor-host nuclear or cell-cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders.

SummaryTransplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.

These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Müller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 ± 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1 rd8/rd8 mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 ± 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 ± 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho −/− mouse; donor cell integration was significantly increased from 313 ± 58 cells without treatment to 919 ± 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies.