FoxC1 activates limbal epithelial stem cells following corneal epithelial debridement.

Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6+/- mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6 +/- mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6 +/- mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.

In vitro modeling and rescue of ciliopathy associated with IQCB1/NPHP5 mutations using patient-derived cells.

Mutations in the IQ calmodulin-binding motif containing B1 (IQCB1)/NPHP5 gene encoding the ciliary protein nephrocystin 5 cause early-onset blinding disease Leber congenital amaurosis (LCA), together with kidney dysfunction in Senior-Løken syndrome. For in vitro disease modeling, we obtained dermal fibroblasts from patients with NPHP5-LCA that were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into retinal pigment epithelium (RPE) and retinal organoids. Patient fibroblasts and RPE demonstrated aberrantly elongated ciliary axonemes. Organoids revealed impaired development of outer segment structures, which are modified primary cilia, and mislocalization of visual pigments to photoreceptor cell soma. All patient-derived cells showed reduced levels of CEP290 protein, a critical cilia transition zone component interacting with NPHP5, providing a plausible mechanism for aberrant ciliary gating and cargo transport. Disease phenotype in NPHP5-LCA retinal organoids could be rescued by adeno-associated virus (AAV)-mediated IQCB1/NPHP5 gene augmentation therapy. Our studies thus establish a human disease model and a path for treatment of NPHP5-LCA.

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

A Common Outer Retinal Change in Retinal Degeneration by Optical Coherence Tomography Can Be Used to Assess Outcomes of Gene Therapy.

Identifying early disease hallmarks in animal models with slow disease progression may expedite disease detection and assessment of treatment outcomes. Using optical coherence tomography, a widely applied noninvasive method for monitoring retinal structure changes, we analyzed retinal optical sections from six mouse lines with retinal degeneration caused by mutations in different disease-causing genes. While images from wild-type mice revealed four well-separated hyper-reflective bands in the outer retina (designated as outer retina reflective bands, ORRBs) at all ages, the second band (ORRB2) and the third band (ORRB3) were merged in retinas of five mutant mouse lines at early ages, suggesting the pathological nature of this alteration. This ORRB change appeared to be degenerating photoreceptor related, and occurred before obvious morphological changes that can be identified on both hematoxylin and eosin-stained sections and electron microscopic sections. Importantly, the merging of ORRB2 and ORRB3 was reversed by treatment with adeno-associated viral vector-mediated gene replacement therapies, and this restoration occurred much earlier than measurable functional or structural improvement. Our data suggest that the ORRB change could be a common hallmark of early retinal degeneration and its restoration could be used for rapid and noninvasive assessment of therapeutic effects following gene therapy or other treatment interventions.

A CEP290 C-Terminal Domain Complements the Mutant CEP290 of Rd16 Mice In Trans and Rescues Retinal Degeneration.

Mutations in CEP290 cause ciliogenesis defects, leading to diverse clinical phenotypes, including Leber congenital amaurosis (LCA). Gene therapy for CEP290-associated diseases is hindered by the 7.4 kb CEP290 coding sequence, which is difficult to deliver in vivo. The multi-domain structure of the CEP290 protein suggests that a specific CEP290 domain may complement disease phenotypes. Thus, we constructed AAV vectors with overlapping CEP290 regions and evaluated their impact on photoreceptor degeneration in Cep290rd16/rd16 and Cep290rd16/rd16;Nrl-/- mice, two models of CEP290-LCA. One CEP290 fragment (the C-terminal 989 residues, including the domain deleted in mutant mice) reconstituted CEP290 function and resulted in cone preservation and delayed rod death. The CEP290 C-terminal domain also improved cilia phenotypes in mouse embryonic fibroblasts and iPSC-derived retinal organoids carrying the Cep290rd16 mutation. Our study strongly argues for in trans complementation of CEP290 mutations by a cognate fragment and suggests therapeutic avenues.

Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery.

This study evaluated the safety and tolerability of ocular RS1 adeno-associated virus (AAV8-RS1) gene augmentation therapy to the retina of participants with X-linked retinoschisis (XLRS). XLRS is a monogenic trait affecting only males, caused by mutations in the RS1 gene. Retinoschisin protein is secreted principally in the outer retina, and its absence results in retinal cavities, synaptic dysfunction, reduced visual acuity, and susceptibility to retinal detachment. This phase I/IIa single-center, prospective, open-label, three-dose-escalation clinical trial administered vector to nine participants with pathogenic RS1 mutations. The eye of each participant with worse acuity (≤63 letters; Snellen 20/63) received the AAV8-RS1 gene vector by intravitreal injection. Three participants were assigned to each of three dosage groups: 1e9 vector genomes (vg)/eye, 1e10 vg/eye, and 1e11 vg/eye. The investigational product was generally well tolerated in all but one individual. Ocular events included dose-related inflammation that resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in a dose-related fashion, but no antibodies against RS1 were observed. Retinal cavities closed transiently in one participant. Additional doses and immunosuppressive regimens are being explored to pursue evidence of safety and efficacy (ClinicalTrials.gov: NCT02317887).

Molecular codes for cell type specification in Brn3 retinal ganglion cells.

Visual information is conveyed from the eye to the brain by distinct types of retinal ganglion cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in a combinatorial code for RGC type specification, but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a- and/or Brn3b-positive RGCs, (ii) Brn3a- and/or Brn3b-dependent RGC transcripts, and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation, and synaptogenesis. We reveal a combinatorial code of TFs, cell surface molecules, and determinants of neuronal morphology that is differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.

Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice.

In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa.

A visual circuit uses complementary mechanisms to support transient and sustained pupil constriction.

Rapid and stable control of pupil size in response to light is critical for vision, but the neural coding mechanisms remain unclear. Here, we investigated the neural basis of pupil control by monitoring pupil size across time while manipulating each photoreceptor input or neurotransmitter output of intrinsically photosensitive retinal ganglion cells (ipRGCs), a critical relay in the control of pupil size. We show that transient and sustained pupil responses are mediated by distinct photoreceptors and neurotransmitters. Transient responses utilize input from rod photoreceptors and output by the classical neurotransmitter glutamate, but adapt within minutes. In contrast, sustained responses are dominated by non-conventional signaling mechanisms: melanopsin phototransduction in ipRGCs and output by the neuropeptide PACAP, which provide stable pupil maintenance across the day. These results highlight a temporal switch in the coding mechanisms of a neural circuit to support proper behavioral dynamics.

Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice.

X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

Retinal Structure and Gene Therapy Outcome in Retinoschisin-Deficient Mice Assessed by Spectral-Domain Optical Coherence Tomography.

PURPOSE: Spectral-domain optical coherence tomography (SD-OCT) was used to characterize the retinal phenotype, natural history, and treatment responses in a mouse model of X-linked retinoschisis (Rs1-KO) and to identify new structural markers of AAV8-mediated gene therapy outcome. METHODS: Optical coherence tomography scans were performed on wild-type and Rs1-KO mouse retinas between 1 and 12 months of age and on Rs1-KO mice after intravitreal injection of AAV8-scRS/IRBPhRS (AAV8-RS1). Cavities and photoreceptor outer nuclear layer (ONL) thickness were measured, and outer retina reflective band (ORRB) morphology was examined with age and after AAV8-RS1 treatment. Outer retina reflective band morphology was compared to immunohistochemical staining of the outer limiting membrane (OLM) and photoreceptor inner segment (IS) mitochondria and to electron microscopy (EM) images of IS. RESULTS: Retinal cavity size in Rs1-KO mice increased between 1 and 4 months and decreased thereafter, while ONL thickness declined steadily, comparable to previous histologic studies. Wild-type retina had four ORRBs. In Rs1-KO, ORRB1was fragmented from 1 month, but was normal after 8 months; ORRB2 and ORRB3 were merged at all ages. Outer retina reflective band morphology returned to normal after AAV-RS1 therapy, paralleling the recovery of the OLM and IS mitochondria as indicated by anti-ß-catenin and anti-COX4 labeling, respectively, and EM. CONCLUSIONS: Spectral-domain OCT is a sensitive, noninvasive tool to monitor subtle changes in retinal morphology, disease progression, and effects of therapies in mouse models. The ORRBs may be useful to assess the outcome of gene therapy in the treatment of X-linked retinoschisis patients.

Preclinical Dose-Escalation Study of Intravitreal AAV-RS1 Gene Therapy in a Mouse Model of X-linked Retinoschisis: Dose-Dependent Expression and Improved Retinal Structure and Function.

Gene therapy for inherited retinal diseases has been shown to ameliorate functional and structural defects in both animal models and in human clinical trials. X-linked retinoschisis (XLRS) is an early-age onset macular dystrophy resulting from loss of an extracellular matrix protein (RS1). In preparation for a human clinical gene therapy trial, we conducted a dose-range efficacy study of the clinical vector, a self-complementary AAV delivering a human retinoschisin (RS1) gene under control of the RS1 promoter and an interphotoreceptor binding protein enhancer (AAV8-scRS/IRBPhRS), in the retinoschisin knockout (Rs1-KO) mouse. The therapeutic vector at 1 × 10(6) to 2.5 × 10(9) (1E6-2.5E9) vector genomes (vg)/eye or vehicle was administered to one eye of 229 male Rs1-KO mice by intravitreal injection at 22 ± 3 days postnatal age (PN). Analysis of retinal function (dark-adapted electroretinogram, ERG), structure (cavities and outer nuclear layer thickness) by in vivo retinal imaging using optical coherence tomography, and retinal immunohistochemistry (IHC) for RS1 was done 3-4 months and/or 6-9 months postinjection (PI). RS1 IHC staining was dose dependent across doses ≥1E7 vg/eye, and the threshold for significant improvement in all measures of retinal structure and function was 1E8 vg/eye. Higher doses, however, did not produce additional improvement. At all doses showing efficacy, RS1 staining in Rs1-KO mouse was less than that in wild-type mice. Improvement in the ERG and RS1 staining was unchanged or greater at 6-9 months than at 3-4 months PI. This study demonstrates that vitreal administration of AAV8 scRS/IRBPhRS produces significant improvement in retinal structure and function in the mouse model of XLRS over a vector dose range that can be extended to a human trial. It indicates that a fully normal level of RS1 expression is not necessary for a therapeutic effect.

Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy.

Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development.

A long-term efficacy study of gene replacement therapy for RPGR-associated retinal degeneration.

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for >70% of X-linked retinitis pigmentosa (XLRP) and 15-20% of all inherited retinal degeneration. Gene replacement therapy for RPGR-XLRP was hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the full-length RPGR-ORF15 cDNA that includes a purine-rich 3'-coding region; however, its effectiveness has recently been demonstrated in four dogs with RPGR mutations. To advance the therapy to clinical stage, we generated new stable vectors in AAV8 or AAV9 carrying mouse and human full-length RPGR-ORF15-coding sequence and conducted a comprehensive long-term dose-efficacy study in Rpgr-knockout mice. After validating their ability to produce full-length proteins that localize to photoreceptor connecting cilia, we evaluated various vector doses in mice during a 2-year study. We demonstrate that eyes treated with a single injection of mouse or human RPGR-ORF15 vector at an optimal dose maintained the expression of RPGR-ORF15 throughout the study duration and exhibited higher electroretinogram amplitude, thicker photoreceptor layer and better targeting of opsins to outer segments compared with sham-treated eyes. Furthermore, mice that received treatment at an advanced age also showed remarkable preservation of retinal structure and function. Retinal toxicity was observed at high vector doses, highlighting the importance of careful dose optimization in future clinical experiments. Our long-term dose-efficacy study should facilitate the design of human trials with human RPGR-ORF15 vector as a clinical candidate.

Preclinical safety evaluation of a recombinant AAV8 vector for X-linked retinoschisis after intravitreal administration in rabbits.

X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and one of the most common causes of macular degeneration in young men. Currently, no FDA-approved treatments are available for XLRS and a replacement gene therapy could provide a promising strategy. We have developed a novel gene therapy approach for XLRS, based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal route. On the basis of our prior study in an Rs1-KO mouse, this construct transduces efficiently all the retinal layers, resulting in an RS1 expression similar to that observed in the wild-type and improving retinal structure and function. In support of a clinical trial, we carried out a study to evaluate the ocular safety of intravitreal administration of AAV8-scRS/IRBPhRS into 39 New Zealand White rabbits. Two dose levels of vector, 2e(10) and 2e(11) vector genomes per eye (vg/eye), were tested and ocular inflammation was monitored over a 12-week period by serial ophthalmological and histopathological analysis. A mild ocular inflammatory reaction, consisting mainly of vitreous infiltrates, was observed within 4 weeks from injection, in both 2e(10) and 2e(11) vg/eye groups and was likely driven by the AAV8 capsid. At 12-week follow-up, ophthalmological examination revealed no clinical signs of vitreitis in either of the dose groups. However, while vitreous inflammatory infiltrate was significantly reduced in the 2e(10) vg/eye group at 12 weeks, some rabbits in the higher dose group still showed persistence of inflammatory cells, histologically. In conclusion, intravitreal administration of AAV8-scRS/IRBPhRS into the rabbit eye produces a mild and transient intraocular inflammation that resolves, at a 2e(10) vg/eye dose, within 3 months, and does not cause irreversible tissue damages. These data support the initiation of a clinical trial of intravitreal administration of AAV8-scRS/IRBPhRS in XLRS patients.

Rd9 is a naturally occurring mouse model of a common form of retinitis pigmentosa caused by mutations in RPGR-ORF15.

Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy.

Potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28.

Adenovirus vaccine vectors derived from rare human serotypes have been shown to be less potent than serotype 5 (Ad5) at inducing immune responses to encoded antigens. To identify highly immunogenic adenovirus vectors, we assessed pro-inflammatory cytokine expression, binding to the CD46 receptor, and immunogenicity. Species D adenoviruses uniquely suppressed pro-inflammatory cytokines and induced high levels of type I interferon. Thus, it was unexpected that a vector derived from a representative serotype, Ad28, induced significantly higher transgene-specific T cell responses than an Ad35 vector. Prime-boost regimens with Ad28, Ad35, Ad14, or Ad5 significantly boosted T cell and antibody responses. The seroprevalence of Ad28 was confirmed to be <10% in the United States. Together, this shows that a rare human serotype-based vector can elicit strong immune responses, which was not predicted by in vitro results.

Clinical studies of X-linked retinitis pigmentosa in three Swedish families with newly identified mutations in the RP2 and RPGR-ORF15 genes.

PURPOSE: To describe new disease-causing RP2 and RPGR-ORF15 mutations and their corresponding clinical phenotypes in Swedish families with X-linked retinitis pigmentosa (XLRP) and to establish genotype-phenotype correlations by studying the clinical spectrum of disease in families with a known molecular defect. METHODS: Seventeen unrelated families with RP and an apparent X-linked pattern of disease inheritance were identified from the Swedish RP registry and screened for mutations in the RP2 and RPGR (for the RP3 disease) genes. These families had been previously screened for the RPGR exons 1-19, and disease-causing mutations were identified in four of them. In the remaining 13 families, we sequenced the RP2 gene and the newly discovered RPGR-ORF exon. Detailed clinical evaluations were then obtained from individuals in the three families with identified mutations. RESULTS: Mutations in RP2 and RPGR-ORF15 were identified in three of the 13 families. Clinical evaluations of affected males and carrier females demonstrated varying degrees of retinal dysfunction and visual handicap, with early onset and severe disease in the families with mutations in the ORF15 exon of the RPGR gene. CONCLUSIONS: A total of seven mutations in the RP2 and RPGR genes have been discovered so far in Swedish XLRP families. All affected individuals express a severe form of retinal degeneration with visual handicap early in life, although the degree of retinal dysfunction varies both in hemizygous male patients and in heterozygous carrier females. Retinal disease phenotypes in patients with mutations in the RPGR-ORF15 were more severe than in patients with mutations in RP2 or other regions of the RPGR.

Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing.

Conventional gene therapy of hemophilia A relies on the transfer of factor VIII (FVIII; encoded by the F8 gene) cDNA. We carried out spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutant FVIII mRNA. A pre-trans-splicing molecule (PTM) corrected endogenous FVIII mRNA in F8 knockout mice with the hemophilia A phenotype, producing sufficient functional FVIII to correct the hemophilia A phenotype. This is the first description of phenotypic correction of a genetic defect by RNA repair in a knockout animal model. Our results indicate the feasibility of using SMaRT to repair RNA for the treatment of genetic diseases.

Microarray analysis of gene expression in the aging human retina.

PURPOSE: To develop gene expression profiles of young and elderly human retinas and identify candidate genes for aging-associated retinal diseases. METHODS: Gene microarray slides containing 2400 human genes (primarily neuronal) were hybridized to biotin or dinitrophenyl (DNP)-labeled target cDNAs that were synthesized using total RNAs from young (13-14 years) and elderly (62-74 years) human retinas. Hybridization signals were visualized with cyanine (Cy)-5 or Cy-3 fluorescent reporter molecules, and the fluorescence intensities of the images were analyzed by computer. Northern blot analysis and real-time quantitative reverse transcription PCR (qRT-PCR) were performed to validate the microarray results. RESULTS: Of the 2400 genes represented on the microarray slides, more than 50% hybridized to the retinal cDNA targets. Expression of a majority of these genes was not altered during aging; nonetheless, changes in the expression of 24 genes were detected between young and elderly retinas. These genes could be clustered into four categories: energy metabolism, stress response, cell growth, and neuronal transmission/signaling. Northern blot analysis and qRT-PCR results confirmed the changes in expression of 8 of 10 genes examined. CONCLUSIONS: Using commercially available slide microarrays, the authors show that aging of the human retina is associated with changes in patterns of gene expression. This analysis suggests that pathways involved in stress response and energy metabolism play key roles in retinal aging. These studies demonstrate the utility of gene microarrays in identifying global patterns of retinal gene expression and lay the foundation for future studies defining the genetic basis of aging-associated retinal diseases, such as age-related macular degeneration.