Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system repair; photoreceptor degeneration initially leaves the inner retinal circuitry intact and new photoreceptors need only make single, short synaptic connections to contribute to the retinotopic map. So far, brain- and retina-derived stem cells transplanted into adult retina have shown little evidence of being able to integrate into the outer nuclear layer and differentiate into new photoreceptors. Furthermore, there has been no demonstration that transplanted cells form functional synaptic connections with other neurons in the recipient retina or restore visual function. This might be because the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation. We hypothesized that committed progenitor or precursor cells at later ontogenetic stages might have a higher probability of success upon transplantation. Here we show that donor cells can integrate into the adult or degenerating retina if they are taken from the developing retina at a time coincident with the peak of rod genesis. These transplanted cells integrate, differentiate into rod photoreceptors, form synaptic connections and improve visual function. Furthermore, we use genetically tagged post-mitotic rod precursors expressing the transcription factor Nrl (ref. 6) (neural retina leucine zipper) to show that successfully integrated rod photoreceptors are derived only from immature post-mitotic rod precursors and not from proliferating progenitor or stem cells. These findings define the ontogenetic stage of donor cells for successful rod photoreceptor transplantation.

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.

ABSTRACT

Intraocular delivery of a variety of neurotrophic factors has been widely investigated as a potential treatment for retinal dystrophy (RD). The most commonly studied factor, ciliary neurotrophic factor (CNTF), has been shown to preserve retinal morphology and to promote cell survival in a variety of models of RD. In order to evaluate CNTF as a potential treatment for RD, we used the Prph2 Rd2/Rd2 mouse. CNTF was expressed intraocularly using AAV-mediated gene delivery either by itself or, in a second treatment group, combined with AAV-mediated gene replacement therapy of peripherin2, which we have previously shown to improve photoreceptor structure and function. We confirmed in both groups of animals that CNTF reduces the loss of photoreceptor cells. Visual function, however, as assessed over a time course by electroretinography (ERG), was significantly reduced compared with untreated controls. Furthermore, CNTF gene expression negated the effects on function of gene replacement therapy. In order to test whether this deleterious effect is only seen when degenerating retina is treated, we recorded ERGs from wild-type mice following intraocular injection of AAV expressing CNTF. Here a marked deleterious effect was noted, in which the b-wave amplitude was reduced by at least 50%. Our results demonstrate that intraocular CNTF gene delivery may have a deleterious effect on the retina and caution against its application in clinical trials.