Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin () gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of -adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RNA, while the human replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with -adRP.

Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adenoassociated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.

Mutations in the gene for rhodopsin, RHO, cause autosomal dominant retinitis pigmentosa, a disease characterized by death of rod photoreceptor cells. At the end stage, when most rods are gone, cones die too, taking central vision with them. One goal of gene therapy, therefore, is to preserve central vision by promoting rod survival in the vicinity of the macula. Dominance in RHO mutations is associated with two phenomena: interference with the function of normal rhodopsin and intrinsic toxicity of the mutant protein. In the case of interference, increased production of the wild-type protein may be therapeutic, but in the case of toxicity, suppression of the mutant protein may also be needed. RHO augmentation has made use of advances in gene delivery to the retina using adeno-associated virus (AAV). Several strategies have been developed for suppression of rhodopsin expression, but because of the heterogeneity of RHO mutations they are not specific for the mutant allele: They suppress both mutant and wild-type RHO. Experiments in autosomal dominant retinitis pigmentosa (adRP) mouse models suggest that both RHO augmentation and supplementation plus suppression preserve the survival of rod cells.

ABSTRACT.Purpose: Four-port bimanual vitrectomy is a surgical technique that facilitates removal of epiretinal membranes in severe proliferative diabetic retinopathy (PDR). As the illumination is held by the assistant through the fourth scleral incision, fibrovascular membranes are removed by bimanual manipulation techniques. The objective of this study was to evaluate the safety and efficacy of four-port bimanual 23-gauge vitrectomy for patients with tractional retinal detachment (TRD) in severe PDR. Methods: Retrospective, comparative, consecutive, interventional case series. Sixty-six eyes of 58 consecutive patients who underwent primary vitrectomy for severe diabetic TRD. Thirty-six eyes of 31 cases that were treated with four-port 23-gauge vitrectomy were compared with 30 eyes of 27 cases that were treated with 23-gauge pars plana vitrectomy (PPV). Main outcome measures were bestcorrected visual acuity (BCVA), retinal status, intraocular pressure, and incidence of intraoperative and postoperative complications with at least 6 months of follow-up. Results: The primary and ultimate anatomic success rates (94.4% versus 93.3%, and 100% in both groups, respectively) and the mean BCVA changes did not differ significantly between groups. The whole surgical time and the membrane removal time were significantly (p < 0.001, respectively) shorter in the four-port 23-gauge group than in the 23-gauge group. There was no difference in the incidence of intraoperative and postoperative complications in both groups. Conclusions: Four-port bimanual 23-gauge vitrectomy offers comparable anatomic success and shortens the surgical time compared with conventional 23-gauge PPV in patients with TRD resulting from severe PDR.