Dose Range Finding Studies with Two Transgenes in a Canine Model of X-Linked Retinitis Pigmentosa Treated with Subretinal Gene Therapy.

Recombinant adeno-associated viral (rAAV) vector-mediated gene therapy is being developed to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator () gene. In preparation for a clinical gene therapy trial, we conducted dose range finding (DRF) studies with an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF) vector administered by subretinal injection in a naturally occurring -mutant canine model (XLPRA2) to compare two different human () transgenes and to establish a reasonable starting dose for a clinical trial. Different dose levels of two candidate vectors (0.

Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid.

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses.

Characterization of intraocular immunopathology following intracameral inoculation with alloantigen.

Purpose: Anterior chamber-associated immune deviation (ACAID) is a form of peripheral tolerance achieved via intracameral antigen inoculation. It is well known that ACAID effectively down-regulates potentially destructive immunities such as delayed-type hypersensitivity (DTH) at extraorbital sites. However, what has not been specifically addressed is whether local intraocular tissues are negatively affected from intracamerally placed antigen.

Cone-specific expression using a human red opsin promoter in recombinant AAV.

Purpose: To determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector.

Methods: An rAAV vector was constructed that contains a 2.1kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression.

Restoration of cone vision in a mouse model of achromatopsia.

Loss of cone function in the central retina is a pivotal event in the development of severe vision impairment for many prevalent blinding diseases. Complete achromatopsia is a genetic defect resulting in cone vision loss in 1 in 30,000 individuals. Using adeno-associated virus (AAV) gene therapy, we show that it is possible to target cones and rescue both the cone-mediated electroretinogram response and visual acuity in the Gnat2 ( cpfl3 ) mouse model of achromatopsia.

Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors.

Purpose: To develop a gene therapy system that specifically targets transgene expression to S-cones of the mammalian retina, the authors coupled recombinant AAV-mediated delivery with the use of a human blue-opsin (HB) promoter to drive expression.

Methods: Two regions of the HB promoter sequence, HB569 and HB996, were amplified from human DNA, cloned into an AAV vector cassette upstream of the green fluorescent protein (GFP) gene, and packaged into AAV2 and AAV5 capsids. Eyes of postnatal day (P) 40 to P48 Sprague-Dawley rats were subretinally injected with 2 muL vector.

Does recombinant adeno-associated virus-vectored proximal region of mouse rhodopsin promoter support only rod-type specific expression in vivo?

Purpose: We have previously found that the -385 to +86 portion of the mouse rod opsin promoter (mOP500) can limit recombinant adeno-associated virus (rAAV)-mediated transgene expression to photoreceptor cells when delivered subretinally. However, the photoreceptor (PR) subtype-specificity of expression remains unclear. Here, we evaluated whether the presence of certain cis-elements in this proximal promoter, such as the rod-specific, neural retina leucine zipper protein (NRL) response element (NRE), can render it a driver of rod-specific expression.

Ribozyme knockdown of the gamma-subunit of rod cGMP phosphodiesterase alters the ERG and retinal morphology in wild-type mice.

Purpose: To generate an animal model of retinal degeneration by using AAV-mediated ribozyme knockdown of the gamma-subunit of the rod cGMP phosphodiesterase (PDEgamma) mRNA in the retina of wild-type mice.

Methods: Two hammerhead ribozymes, HRz35 and HRz42, were designed to target the PDEgamma gene in wild-type C57BL/6 mice. The efficiency and specificity of the ribozyme cleavage was tested in vitro against three different types of target: short synthetic RNA oligomers, longer targets transcribed from clones, and full-length mRNA from total retinal RNA extracts.

Prolonged recovery of retinal structure/function after gene therapy in an Rs1h-deficient mouse model of x-linked juvenile retinoschisis.

X-linked juvenile retinoschisis (RS) is a common cause of juvenile macular degeneration in males. RS is characterized by cystic spoke-wheel-like maculopathy, peripheral schisis, and a negative (b-wave more reduced than a-wave) electroretinogram (ERG). These symptoms are due to mutations in the RS1 gene in Xp22.

Preventing stem cell incorporation into choroidal neovascularization by targeting homing and attachment factors.

Purpose: The primary cause of vision loss in people more than 50 years of age in developed nations is age-related macular degeneration (ARMD). The wet form of ARMD is characterized by choroidal neovascularization (CNV). A prior study has shown that adult hematopoietic stem cells (HSCs) contribute to approximately 50% of newly formed vasculature in CNV.

AAV-mediated intravitreal gene therapy reduces lysosomal storage in the retinal pigmented epithelium and improves retinal function in adult MPS VII mice.

The beta-glucuronidase-deficient mucopolysaccharidosis type VII (MPS VII) mouse accumulates partially degraded glycosaminoglycans in many cell types, including retinal pigmented epithelial (RPE) cells in the eye. This lysosomal storage in RPE cells leads to progressive retinal degeneration and reduced function as measured by flash electroretinography (ERG). The impact of AAV-mediated intraocular gene therapy on pathology and retinal function was examined in normal and MPS VII mice treated at 4 weeks of age, when lysosomal storage is evident but functional impairment is minimal in affected animals.

Range of retinal diseases potentially treatable by AAV-vectored gene therapy.

Viable strategies for retinal gene therapy must be designed to cope with the genetic nature of the disease and/or the primary pathologic process responsible for retinal malfunction. For dominant gene defects the aim must be to destroy the presumably toxic gene product, for recessive gene defects the direct approach aims to provide a wild-type copy of the gene to the affected retinal cell type, and for diseases of either complex or unknown genetic origin, more general cell survival strategies that deal with preserving affected retinal cells are often the best and only option. Hence examples of each type of therapy will be briefly discussed in several animal models, including ribozyme therapy for autosomal dominant retinitis pigmentosa in the transgenic P23H opsin rat, beta-PDE gene augmentation therapy for autosomal recessive retinitis pigmentosa in the rd mouse, glial cell-derived neurotrophic factor (GDNF) gene therapy for autosomal dominant RP in the transgenic S334ter opsin rat and pigment epithelial cell-derived neurotrophic factor (PEDF) gene therapy for neovascular retinal disease in rodents.

A muscleblind knockout model for myotonic dystrophy.

The neuromuscular disease myotonic dystrophy (DM) is caused by microsatellite repeat expansions at two different genomic loci. Mutant DM transcripts are retained in the nucleus together with the muscleblind (Mbnl) proteins, and these abnormal RNAs somehow interfere with pre-mRNA splicing regulation. Here, we show that disruption of the mouse Mbnl1 gene leads to muscle, eye, and RNA splicing abnormalities that are characteristic of DM disease.

Decreased expression of the insulin-like growth factor 1 receptor by ribozyme cleavage.

Purpose: Insulin-like growth factor (IGF)-1 and its receptor (IGF-1R) are associated with abnormal retinal neovascularization. Ribozymes were designed that selectively decreased the expression of the IGF-1R and these ribozymes were tested in angiogenesis models in vitro and in vivo.

Methods: Two hammerhead ribozymes were designed that cleave the human IGF-1R mRNA.

Anti-apoptotic effects of CNTF gene transfer on photoreceptor degeneration in experimental antibody-induced retinopathy.

Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy syndrome, a paraneoplastic disease associated with retinal degeneration. We have previously shown that anti-recoverin autoantibodies induced photoreceptor apoptotic cell death after injection into the vitreous of Lewis rats. Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of a number of neuronal cell types, including photoreceptors.

Inactivation of the Basigin gene impairs normal retinal development and maturation.

5A11/Basigin is an immunoglobulin-like glycoprotein expressed on the surface of Müller cells, the apical and basal surfaces of the retinal pigmented epithelium, and photoreceptor cell bodies and their inner segments. Disruption of the 5A11/Basigin gene in the mouse results in photoreceptor degeneration and a corresponding decrease in electroretinogram amplitudes in mature mice. The purpose of this study was to examine the electrophysiology of the 5A11/Basigin null mouse retina at earlier ages than previously examined.