Current Gene Therapy - Volume 10, Issue 5, 2010

This special issue was meant to provide a comprehensive review on AAV-mediated retinal gene therapy from vector design to current clinical applications. There has been a great progress over the past decade in generating and characterizing animal models for retinal disorders, developing and testing strategies for ameliorating the disease process and moving the promising proof-of-concept data into clinical trials. The recent success of three separate Phase I clinical trials investigating an AAV2/2 -mediated gene therapy for the treatment of Leber's Congenital Amaurosis (LCA) due to RPE65 mutations [1-4] provides a great incentive to extend these accomplishments to other retinal diseases. The strategy to treat autosomal recessive diseases, in which the mutated gene product is not existent or does not cause toxic effects, is to provide the cell with a correct allele of the mutated gene (ex : rpe65). The choice of vector and promoter for the expression cassette will depend on the targeted cells that is in most of the cases either the retinal pigmented epithelium (RPE) cells or the photoreceptors. The strategy to treat autosomal dominant diseases, which are caused by mutations which result in a toxic “gain of function” effect of the encoded protein (ex: Rhodopsin), is to suppress the production of the toxic protein using either ribozymes or siRNA in conjunction with efficient replacement. A third strategy consists in gene supply, which is the delivery of a gene that can prevent or arrest disease progression without being directly implicated in the disease pathogenesis. This approach involves antiapoptotic factors to prevent retina neurons degeneration (Retinitis Pigmentosa, LCA, glaucoma), and anti-angiogenic molecules to inhibit retinal neovascularization (age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity). These retinal gene transfer strategies as well as other considerations such as AAV vector design, AAV vector production, immune response and issues relating to clinical trials are discussed in this special issue. On behalf of the Current Gene Therapy journal, I would like to thank the authors for their outstanding articles that truly illustrate the impressive breakthroughs achieved in AAV-mediated gene therapies for the retina, including the first evidence of a therapeutic effect in humans.

Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.

Recombinant adeno-associated virus (AAV) -based vectors expressing therapeutic gene products have shown great promise for human gene therapy. A recent milestone has been the safety and efficacy observed using recombinant AAV2 expressing retinal pigment epithelial associated 65KDa protein for Leber Congenital Amaurosis. This review summarizes manufacturing and characterization of ‘AAV2-hRPE65v2’, the vector used in one completed Phase I/II clinical trial. Regulatory challenges and strategies that were successfully used for this groundbreaking trial are described.

Retinal blinding disorders together have a prevalence of 1 in 2000 humans world wide and represent a significant impact on the quality of life as well as the possibility to attain personal achievements. Mutations in genes that are expressed either in RPE cells, photoreceptors or bipolar cells can cause varying forms of degenerative or stationary retinal disorders, as the presence of the encoded proteins is crucial for normal function, maintenance and synaptic interaction. The degree of damage caused by different mutations depends upon the type of mutation within the gene, resulting in either total absence or the presence of a non-functional or potentially toxic protein. Potential treatment strategies require the identification of the cell type, in which the mutated gene is expressed for later targeting by viral vector mediated gene transfer. In the first part of this review, the authors present different cellular pathways that take place either in the RPE, photoreceptors, or bipolar cells. Furthermore, the authors demonstrate why genetic and molecular testing methods, which clearly identify the disease causing mutations, are crucial for attaining the correct diagnosis in order to indentify patients suitable to be treated by upcoming new therapeutic methods. In the second part, a short clinical classification of the most important forms of retinal blinding disorders is given, together with clinical aspects concerning the problems that arise when facing low residual visual perception and the enormous heterogeneity of symptoms within these disorders.

Common blinding diseases that are currently untreatable include conditions characterized by progressive neuronal degeneration, such as retinitis pigmentosa, Leber congenital amaurosis or glaucoma, and characterized by ocular neovascularization, like wet age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. The pathogenic mechanisms underlying either neuronal degeneration or new vessel formation may be similar and independent of the mutation underlying the disease, thus allowing to test therapeutic strategies acting downstream of the primary causative event. Gene supply is the delivery of a gene that can prevent or arrest disease progression without being directly implicated in the disease pathogenesis. To this end, one of the most efficient and safe retinal gene delivery vehicles derives from the small adeno-associated virus (AAV). We review studies on AAV-mediated gene supply of: neurotrophic/ antiapoptotic factors to prevent retinal neurons degeneration, and anti-angiogenic molecules to inhibit retinal neovascularization. Successful gene supply may represent a one-fit-all treatment for inherited and acquired blinding diseases.

Over the past two decades, significant progress has been made in defining the molecular pathogenesis of hereditary retinal degenerations. Many of these are characterised by immense genetic heterogeneity. For example, in retinitis pigmentosa (RP), the most common form of this group of disorders, approximately 50 disease causing genes have been implicated, 20 of which are inherited in an autosomal dominant manner. Knowledge of the underlying genetic pathogenesis together with the availability of animal models and vectors for delivery has enabled exploration of gene-based therapies for inherited retinopathies. Notably, many studies have focused on treatment of recessive forms of these disorders and significant progress including ongoing clinical trials has been achieved. Progress in developing gene therapies for dominant retinopathies has been slower. One reason for this is that gene therapies for many dominant diseases, which are targeted to correcting the primary genetic defect, are likely to require suppression of the mutant gene. Alternative therapeutic approaches, which involve modulating secondary features associated with the disease pathology (such as ER stress or apoptosis) are also being explored. This review is focused on the development of gene-based therapies for dominantly inherited retinopathies. The main topics discussed are suppression technologies, preclinical animal models, retinal gene delivery and therapeutic strategies.

Inherited retinal diseases are non-lethal and have a wide level of genetic heterogeneity. Many of the genes involved have now been identified and their function elucidated, providing a major step towards the development of genebased treatments. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because they mediate long-term transgene expression in a variety of retinal cell types and elicit minimal immune responses. Extensive preclinical evaluation of gene transfer strategies in small and large animal models is key to the development of successful gene-based therapies for the retina. These preclinical studies have already allowed the field to reach the point where gene therapy to treat inherited blindness has been brought to clinical trial. In this manuscript, we focus on recombinant AAV-mediated specific gene therapy for recessive retinal degenerative diseases we describe the preclinical studies for the treatment of retinal degeneration caused by retinal pigmented epithelium cells or photoreceptor defects and the immune response induced by retinal rAAV gene transfer.

Gene therapy utilizing retroviral vectors is being postulated as a real therapeutic alternative for many hemopoietic inherited diseases, such as β-thalassemia or sickle cell disease. A major limitation of current vectors is their inability to achieve efficient gene transfer into quiescent cells, such as human CD34+ cells that reside in the Go phase of the cell cycle and are highly enriched in hemopoietic stem cells. For that reason, lentiviral vectors (LVs) were proven to be more efficient than oncoretroviral vectors. Additional problems of these vectors are a) the low titers observed due to regulatory elements of the β-globin locus, used for the improvement of the transgene's expression, b) the eventual silencing of the transgene and c) the toxicity posed on CD34+ cells due to the usage of VSV-G as an envelope protein. These facts hamper their application for gene therapy of hematopoietic cells. Thus, the major current drawbacks of the field affecting therapeutic efficacy, include 1) insufficient transduction efficiency of the target hemopoietic stem cells, 2) inconsistent expression of the transgene, 3) putative aberrant expression near integration sites raising safety issues and 4) lack of long term expression of the transgene exhibiting eventual silencing. This review presents the current status of globin gene therapy for the hemoglobin disorders, reviews the recent results and discusses how the knowledge gained from these trials can be used to develop a safe and effective gene therapy approach for the treatment of β-thalassemia and SCD.