P. Elizabeth Rakoczy

Adenovirus and Adeno-Associated Virus Vectors

Recombinant adenovirus (rAd) and recombinant adeno-associated virus (rAAV) are among the most extensively used vectors in gene therapy studies to date. These two vectors share some similar features such as a broad host range and ability to infect both proliferating and quiescent cells. However, they also possess their own unique set of properties that render them particularly attractive for gene therapy applications. rAd vectors can accommodate larger inserts, mediate transient but high levels of protein expression, and can be easily produced at high titers. Development of gutted rAd vectors has further increased the cloning capacity of these vectors. The gaining popularity of rAAV use in gene therapy can be attributed to its lack of pathogenicity and added safety due to its replication defectiveness, and its ability to mediate long-term expression in a variety of tissues. Site-specific integration, as occurs with wild-type AAV, will be a unique and valuable feature if incorporated into rAAV vectors, further improving their safety. This paper describes these properties of rAd and rAAV vectors, and discusses further development and vector improvements that continue to extend the utility of these vectors, such as cell retargeting by capsid modification, differential transduction by use of serotypes, and extension of the cloning capacity of rAAV vectors by dual vector heterodimerization.

Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue.

BackgroundLebers congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.MethodsrAAV.RPE65 was injected into the subretinal space of Rpe65-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection.ResultsrAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health.ConclusionThis work demonstrated the potential benefits of using the Rpe65-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina.

Preclinical Evaluation of a Phosphorothioate Oligonucleotide in the Retina of Rhesus Monkey

Overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization (CNV) in patients with age-related macular degeneration. In this study, a phosphorothioate oligonucleotide (PS-oligo) targeting both human and rat VEGF165 genes upstream of the translation initiation code, named DS135 in this study, was evaluated for its uptake dynamics and retinal tolerance after intravitreal (IV) and subretinal (SR) injections in the rhesus monkey. Intravitreal and SR injections of a fluorescent-labeled DS135 (FL-DS135) resulted in both dose- and time-dependent uptake and persistence, and FL-DS135 remained detectable in the retina for at least 3 weeks after injection. Ophthalmic examination showed transient vitreous haze after IV delivery of a high dose but not with a low dose of FL-DS135. Histologic examination showed no evidence of retinal degeneration with respect to IV delivery. After SR delivery, however, dose-related cellular infiltration, transient residual fluid, and slight distortion of the neuroretina were observed. The biologic efficacy of DS135 was further assessed in a laser-induced CNV model, and development of CNV was determined by fluorescein angiography and histologic examination. Incomplete inhibition of CNV formation was observed after IV and SR injection of DS135, but no statistically significant difference was achieved when compared with dose-matched control of PS-oligo. Analysis of fluorescein angiogram and histologic examination showed less than 30% incidence of CNV development in this monkey model. Our study demonstrated that PS-oligos can be successfully introduced into the retina, although with potential limitations, after SR delivery. DS135, a PS-oligo targeting the VEGF gene upstream of the translation initiation code, partially inhibited CNV formation. An improved CNV model is necessary for further confirmation of the full therapeutic potency of DS135 before clinical application.

Gene transfer in the RPE65 null mutation dog: relationship between construct volume, visual behavior and electroretinographic (ERG) results

In vivo gene transfer in a large group of RPE65 null mutation dogs have been recently performed. The present study was aimed at determining, through visual behavioral and electroretinographic (ERG) testing, if there is a volume effect of the gene construct administered. Eleven Beagle-Briard dogs homozygous for the RPE65 null mutation and two unaffected control dogs were included. Affected animals were unilaterally treated with either a high (70–100 μl; N = 6) or a low volume (30–60 μl; N = 5) of subretinally injected rAAV.RPE65 construct, at the age of 4 months to 2.5 years. Fellow eyes were treated with a subretinal injection of rAAV.GFP or sham operated and used as internal controls. Retinal function was measured pre- and 10–12 weeks post-surgically, using simultaneous bilateral full-field flash ERGs. A significant improvement in all ERG responses studied was identified for the high volume treated group compared to pre-surgical parameters. A significant improvement for the high intensity scotopic a-wave response for the low volume rAAV.RPE65 treated group was also found. Objective and subjective dim and day light visual maze testing, in eight of the affected treated animals, and the two control dogs, revealed better vision in daylight than in dim light for all animals. Vision in dogs treated with the high volume of gene construct was significantly better in day light than in dim light. No significant difference was noted between day and dim light testing for the control group or those animals treated with a low volume of the gene construct. Significantly better vision was noted in the control group when compared with the low volume group under dim light conditions, and the high volume group under day light conditions. No significant difference in functional vision could be identified between the high volume treated animals and control animals in day light conditions. These findings support the hypothesis that functional vision is improved by subretinal rAAV.RPE65 injection in a volume-dependent manner.

Long-term effect of therapeutic laser photocoagulation on gene expression in the eye

Microarray‐based gene expression analysis demonstrated that laser photocoagulation (LPC) of mouse eyes had a long‐term effect on the expression of genes functionally related to tissue repair, cell migration, proliferation, ion, protein and nucleic acid metabolism, cell signaling, and angiogenesis. Six structural genes, including five crystallins (Cryaa, Cryba1, Crybb2, Crygc, Crygs) and keratin 1–12 (Krt1–12), the anti‐angiogenic factor thrombospondin 1 (Tsp1), the retina‐ and brain‐specific putative transcription factor tubby‐like protein 1 (Tulp1), and transketolase (Tkt), a key enzyme in the pentose‐phosphate pathway, were all shown to be up‐regulated by real‐time PCR and/or Western blotting. Immunohistochemistry localized five of these proteins to the laser lesions and surrounding tissue within the retina and pigmented epithelium. This is the first study demonstrating long‐term changes in the expression of these genes associated with LPC. Therefore, it suggests that modulated gene expression might contribute to the long‐term inhibitory effect of LPC. In addition, these genes present novel targets for gene‐based therapies aimed at treating microangiopathies, especially diabetic retinopathy, a disease currently only treatable with LPC.

Practical considerations of recombinant adeno-associated virus-mediated gene transfer for treatment of retinal degenerations

Photoreceptor (PR) and retinal pigment epithelium (RPE) are the principal cell targets in retinal gene therapy. Recombinant adeno‐associated virus (rAAV) has emerged as a very promising vector for gene therapy in hereditary retinal diseases. Gene transfer at different stages of the disease is a practical consideration for future clinical application.

A model for a blinding eye disease of the aged

This work aims to investigate the effect of compromised lysosomal enzyme activity on the accumulation of photoreceptor-derived debris in the retinal pigment epithelial (RPE) cells and to examine if this accelerated debris accumulation can induce retinal abnormalities similar to those observed in aged individuals. A mutated, enzymatically inactive form of cathepsin D (CatD), generated by site-directed mutagenesis was used to produce stable cell lines and transgenic mice. There was a strong increase in enzymatically inactive CatD protein production in the mutated CatD DNA transfected D407 cells (D407MCD). The presence of the inactive CatD has been linked to an impairment in bovine rod outer segment(BROS) digestion and was confirmed by astatistically significant increase of undigested residual BROS in the medium ofD407MCD when compared to the control vector-transfected D407 cells (t-test,P ≤ 0.016, P ≤ 0.003) or untransfectedD407 cells (t-test, P ≤ 0.008,P ≤ 0.003). The impairment was also confirmed in vivo by demonstration of BROS-derived debris accumulation in the RPE cell layer of transgenic mice. These results demonstrated that the mutated and inactive CatD form could lead to impairment of photo receptorouter segments (POS) proteolysis. It is proposed that this initial impairment of POS proteolysis may result in the accumulation of CatD-opsin-like complexes in the pigment epithelium, which further compromises RPE cell functions and thus causes the changes observed in aging humans.

Treatments for choroidal and retinal neovascularization: a focus on oligonucleotide therapy and delivery for the regulation of gene function

Blinding eye diseases caused by neovascularization of the retinal tissue are the leading cause of blindness in Western societies. Current treatments, such as laser photocoagulation, are limited in their effectiveness at halting the progression of angiogenesis and are unable to reduce the number of vessels once they have developed. In addition, although complete blindness is often avoided, vision is often permanently impaired by the treatment itself. Several less invasive treatments are being developed and one of these is oligonucleotide gene therapy in which short stretches of nucleotides are being used as inhibitors of key, metabolic processes involved in angiogenesis. Combined with this is the development of new and improved nucleotide chemistries aimed at overcoming many of the problems associated with oligonucleotide gene therapy, such as poor longevity because of endonuclease activity. In addition, advancements in delivery systems have further enhanced the efficacy of oligonucleotide gene therapy by increasing cellular penetration and localizing delivery to specific cell types and organs.

Quantitative model demonstrating that recombinant adeno-associated virus and green fluorescent protein are non-toxic to the rat retina

Background: Recombinant adeno‐associated virus (rAAV) is one of the most promising recombinant viral vectors for delivering therapeutic agents to the retina. The present study aims to quantify any effect that an rAAV construct may have on the retina. To be able to use rAAV for therapeutic purposes, the potentially toxic effect of the vector and an associated green fluorescent protein (gfp) marker has to be investigated.

Effect of vascular endothelial growth factor upregulation on retinal gene expression in the Kimba mouse

Background:  The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice.