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Retina Gene therapy delivery tools poised for success in ocular gene therapy Gearoid Tuohy PhD in Monte Carlo A NUMBER of years ago at an international gene therapy conference a leading academic researcher commented that the three great challenges for making gene therapy a realistic treatment option were “delivery, delivery, and delivery”. This unfortunately was not an over-statement. However, in the past 10 years significant strides have been made in developing technologies that are now capable of highly specific reproducible delivery of exogenous genes into a broad range of tissues and cells. At this year's EURETINA conference, two leading gene therapy researchers in ocular disease described the current stateof-the-art regarding gene delivery to the retina. These technologies recently culminated in the initiation of the first human gene therapy trial for the treatment of Leber congenital amaurosis (LCA), at London's Moorfields Eye Hospital. The medical and scientific community are now identifying therapeutic opportunities to deliver specific gene transcripts in an effort to correct the course of the underlying pathology. One of the keys to achieving such ambitious goals is “vector technology” – the technology used for getting foreign genes into cells and by far one of the most popular tools for gene delivery are virus vectors. Different vectors are now available for transfecting a wide range of tissues and cells. Each specific vector has advantages and disadvantages that chiefly depend on the gene that needs to be delivered and the target cell or tissue for delivery. Adenovirus has been the most used gene therapy vector in clinical trials to date. However, a large volume of research into applying gene therapy in ocular disease has focused on the use of adeno-associated vectors (AAV). Alberto Auricchio MD, based at the Telethon Institute of Genetics and Medicine (TIGEM) and Department of Paediatrics, “Federico II” University, Napoli, Italy, told delegates of recent progress made on adeno-associated virus (AAV) – a gene therapy vector which possesses several features which make it attractive for gene delivery to a range of organs in vivo, including the retina. AAV contains single stranded DNA and is non pathogenic to humans. The virus is able to transduce both dividing and nondividing cells such as those in the retina over the long term following a single subretinal administration. One of the most attractive features of AAV is that it exists in over 100 different “flavours”, called serotypes, which have been isolated in the last few years. Such a portfolio of serotypes is extremely valuable as it permits 48 researchers to take each serotype and convert it into a gene transfer vector capable of infecting specific cell types in a way that is both efficient and simple. The AAV particle is made of a genome encased in a protein from which biologists can derive gene therapy vectors by exchanging parts of the capsid protein. This is useful because each of the surface proteins on the capsid is the main determinant of a vector's ability to infect certain cell types and drive expression of the particular gene of interest. The ability of AAV vectors with different external surface proteins to transduce different cell types has been demonstrated by Dr Aurrichio's group and others using the retina as the target tissue.The broad range of different serotypes, some of which have been converted into gene therapy vectors, are currently being tested for their ability to transduce the retina. Turning genes on only where they are required Choosing a suitable capsid type represents just one way to direct or target gene expression in specific cell types. Another way to direct gene expression following subretinal administration or vitreal injection is to engineer the gene of interest with cell type specific promoters – sequences of DNA upstream of the gene that regulate gene expression. For example, expression of a given transgene may be restricted to a specific cell type such as the retinal pigment epithelia (RPE) or ganglion cells by using gene promoters specific to transcripts expressed in those cell types. Alternatively, using the rhodopsin or rhodopsin kinase promoter limits expression of a transgene to rod photoreceptors.This capability provides additional comfort in terms of regulatory safety concerns to insure that a given transgene is expressed only in the target cell. Dr Aurrichio showed examples of how AAV can be used to correct defects in some animal models including severe retinal diseases where most of the genes are expressed in photoreceptors and require gene replacement as a therapeutic strategy. For example in recessive RP, brought about by a homozygous mutation of the ≤ subunits of the phosphodiesterase (≤PDE) gene, delivery of a normal ≤PDE gene has brought about functional rescue from the disease state. In other ocular applications, for example in dominant disease, RNAi can be used to specifically knock down the mutated allele leaving the normal allele untouched.This approach has been tested in an ADRP (autosomal dominant retinitis pigmentosa) model carrying a Pro23His rhodopsin mutation whereby in vitro and in vivo Table of vectors used expression of RNAi can knock down the mutant rhodopsin by 70 per cent leaving the wild type rhodopsin gene untouched. Gene therapy may also be applied to animal models of ocular neovascularisation such as retinopathy of prematurity (ROP) and AMD. One model of ROP that induces neovascularisation exposes newborn mice to high levels of oxygen and then returns animals to normal oxygen levels at which point an AAV carrying an angiogenic gene can be introduced to block neovascularisation. Lentiviral vectors provide an alternative delivery tool A very different viral vector used for gene therapy applications was presented by Dr Kam Balaggan MRCOphth, specialist registrar at Moorfields Eye Hospital and research fellow in the Division of Molecular Therapy, Institute of Ophthalmology, London headed by Prof Robin Ali. Lentiviral vectors, unlike adenovirus and AAV, are RNA viruses with a capacity to efficiently transduce both dividing and non-dividing cells and are associated with minimal intraocular immune responses. The principal advantages of lentiviral vectors are that they accommodate a large carrying capacity and mediate high levels of transgene expression. For certain applications they can deliver genes too large for AAV to carry while also having predictable kinetics of expression, typically expressing at a very early stage following administration. Nearly all the different types of lentiviral vectors described to date can efficiently target ocular RPE cells with some possessing the capacity to also transduce photoreceptors and other neurosensory retinal cells with varying efficiency after conventional subretinal delivery. Lentiviral vectors may be derived from primate sources such as HIV and the simian equivalent (simian immunodeficiency virus) or from non-primate sources such as horse (equine infectious anaemia virus-EIAV), cat (feline immunodeficiency virus-FIV) and cow (bovine immunodeficiency virus-BIV). The principal advantage of such derivatives is that their use further minimises any potentially deleterious consequences given that their wild type forms are known to be non-pathogenic in humans. Lentiviral vectors integrate efficiently into the host cell genome and so the transgene may propagate to all daughter cells following cell division. The wild type HIV genome, typically around 9,700 base pairs in size, can be engineered into a gene therapy vector because most of the pathogenic components of these viruses can be safely removed without causing any diminution in either viral titre or activity.To create vector tools the viral genome may be split across three separate plasmids, coding for all the essential sequences necessary for transcription and integration of the viral genome, insertion of the gene of interest and a back bone with as little as 550 base pairs (down from the original 9,700 base pairs) so very little of the original pathogenic vector remains. As two of the plasmids do not have any encapsidation sequence they do not get incorporated into the viral particle but function purely to provide the structural proteins and enzymes necessary for correct functioning of the lentiviral particle.The natural tropism for wild type HIV-1 is the CD4+ lymphocyte. However, it is possible to expand this affinity beyond CD4+ cells by removing the original protein encoding the wild type viral envelope and replacing with a gene encoding the envelope protein from a different virus. Dr Balaggan and colleagues have tested a number of re-engineered vectors for delivery of genes to the retina in animal Retina models, using green fluorescent protein as a marker gene to track transduction and expression efficiencies. Using the EIAV vector Dr Balaggan's research group have stably and efficiently transduced retinal pigment epithelial cells in animal models resulting in high levels of GFP expression within just three days of subretinal administration and persisting for at least 16 months. The implications of maintaining transgene expression for 16 months following a single injection in itself illustrates the attractiveness of gene therapy because such an approach could be used for the longterm delivery of therapeutic proteins. For example in exudative AMD, genes encoding anti-VEGF proteins could be delivered resulting in sustained VEGF targeting after just a single injection, in contrast to the repeated delivery that is required of current pharmacological anti-VEGF agents including Lucentis and Avastin. As it is likely that life-long administration of pharmacological agents will be required to sustain therapeutic efficacy, the advantages of a gene-based approach for patients with exudative AMD could be immense. Clinical evaluation of lentiviral vector tools Use of viral vectors in clinical gene therapy has generally yielded few notable successes aside from two trials based on providing gene based therapies for SCID (severe combined immune deficiency), a disease of both cellular and humoral immunity, a form of which may be caused by a mutation in the IL2RG gene which encodes the 'alpha' chain of the interleukin-2 receptor. Research teams in France, and later in the UK, successfully used an integrating retroviral vector to deliver a correct functional copy of the IL2RG gene to autologous bone marrow cells which were then transplanted back into patients resulting in a dramatic recovery. Following treatment, patients produced normal numbers of T cells, did not need to live in specialised environments and responded well to childhood immunisation.This was a significant milestone in the history of gene therapy. However, even in advance of the SCID trials, there were concerns that apparent random integration of exogenous DNA into host cells could, in theory, activate a proto-oncogene or deactivate a tumour suppressor gene.This concern became a reality when four patients from one of the trials developed T cell leukaemia thought in at least two patients to arise from the vector genome inserting near to a protooncogene (LMO2) which is known to induce T cell leukaemia formation. To address the issue of insertional mutagenesis, a new generation of non- integrating lentiviral vectors has been developed and are currently undergoing testing.These “integration-deficient” lentiviral vectors are expected to substantially reduce the risk of insertional mutagenesis in addition to reducing the risk of inadvertent germ line transmission, for applications involving post-mitotic or essentially post-mitotic cells, such as those in the eye. Non-integrating lentivirus show equivalent efficiencies Dr Balaggan's group in collaboration with colleagues from Prof Adrian Thrasher's group at the Institute of Child Health, London have developed and tested selfinactivating advanced vectors carrying a single substitution mutation in one position of the gene encoding the integrase enzyme of HIV which catalyses the integration of the double-stranded DNA into the host cell chromosome.The result of this alteration reduces the integration frequency of HIV vector by 10,000 fold in vitro. “The implications of maintaining transgene expression for 16 months following a single injection in itself illustrates the attractiveness of gene therapy because such an approach could be used for the long-term delivery of therapeutic proteins” To test the feasibility of a gene therapy approach to choroidal neovascularisation Dr Balaggan and colleagues have used EIAV vectors to deliver angiostatic genes (endostatin and angiostatin) originally characterised for their tumour suppression characteristics now known to have multiple mechanisms of action including the inhibition of VEGF, and also imparting a positive effect on endothelial cell apoptosis. Animal studies showed that administration of the therapeutic genes inhibited angiogenesis in the order of 50-60 per cent. An additional efficacy measure of permeability was recorded by calculating the proportional degree of increased leakage from early phases to latter phases resulting in a reduction of approximately 25 per cent. In the therapeutic groups the size of the lesion is substantially smaller than that in the control groups as is the degree of leakage between early and late phases. Dr Balaggan's group also evaluated the efficacy of the newer non-integrating HIV1 vectors, in models of retinitis pigmentosa secondary to RPE defects. Analysis of two models of early onset retinal dystrophy, the RPE65 mutation which accounts for about 10 per cent of LCA cases and the MERTK deficiency modelled in the RCS rat, substantial rescue was observed in function in the RPE65 deficient mouse in the order of 300-500 per cent increase in 'b' wave amplitudes over fellow un-injected eyes. Similarly, in the MERTK deficient rat, preservation of function at six to eight weeks was recorded compared with uninjected eyes where no rescue was seen with control vectors.These technologies and pre-clinical results provide added confidence for the use of lentiviral vectors in gene therapy applications for a range of ocular diseases. [email protected] [email protected] Studies have demonstrated efficient and sustained transgene expression in vivo in both rodent ocular and brain tissue. In addition, Dr Balaggan and colleagues have shown substantial rescue of clinically relevant rodent models of retinal degeneration with such vectors. Dr Balaggan believes that “this shows that the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration”. A major advantage of gene therapy is its potential for localised sustained delivery of a therapeutic molecule following a single injection.This contrasts significantly with anti-VEGF treatments, which may require repeated intraocular injections, presenting a cumulative risk of ocular complications. 49