Publications by authors named "Prateek Buch"

11 Publications

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Long-term preservation of cones and improvement in visual function following gene therapy in a mouse model of leber congenital amaurosis caused by guanylate cyclase-1 deficiency.

Hum Gene Ther 2011 Oct 10;22(10):1179-90. Epub 2011 Aug 10.

Department of Genetics, University College London, Institute of Ophthalmology, London, United Kingdom.

Leber congenital amaurosis (LCA) is a severe retinal dystrophy manifesting from early infancy as poor vision or blindness. Loss-of-function mutations in GUCY2D cause LCA1 and are one of the most common causes of LCA, accounting for 20% of all cases. Human GUCY2D and mouse Gucy2e genes encode guanylate cyclase-1 (GC1), which is responsible for restoring the dark state in photoreceptors after light exposure. The Gucy2e(-/-) mouse shows partially diminished rod function, but an absence of cone function before degeneration. Although the cones appear morphologically normal, they exhibit mislocalization of proteins involved in phototransduction. In this study we tested the efficacy of an rAAV2/8 vector containing the human rhodopsin kinase promoter and the human GUCY2D gene. Following subretinal delivery of the vector in Gucy2e(-/-) mice, GC1 protein was detected in the rod and cone outer segments, and in transduced areas of retina cone transducin was appropriately localized to cone outer segments. Moreover, we observed a dose-dependent restoration of rod and cone function and an improvement in visual behavior of the treated mice. Most importantly, cone preservation was observed in transduced areas up to 6 months post injection. To date, this is the most effective rescue of the Gucy2e(-/-) mouse model of LCA and we propose that a vector, similar to the one used in this study, could be suitable for use in a clinical trial of gene therapy for LCA1.
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http://dx.doi.org/10.1089/hum.2011.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3205803PMC
October 2011

Dominant cone-rod dystrophy: a mouse model generated by gene targeting of the GCAP1/Guca1a gene.

PLoS One 2011 Mar 28;6(3):e18089. Epub 2011 Mar 28.

University College London Institute of Ophthalmology, London, United Kingdom.

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018089PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065489PMC
March 2011

Guanylate cyclases and associated activator proteins in retinal disease.

Mol Cell Biochem 2010 Jan 26;334(1-2):157-68. Epub 2009 Nov 26.

UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.

Two isoforms of guanylate cyclase, GC1 and GC2 encoded by GUCY2D and GUCY2F, are responsible for the replenishment of cGMP in photoreceptors after exposure to light. Both are required for the normal kinetics of photoreceptor sensitivity and recovery, although disease mutations are restricted to GUCY2D. Recessive mutations in this gene cause the severe early-onset blinding disorder Leber congenital amaurosis whereas dominant mutations result in a later onset less severe cone-rod dystrophy. Cyclase activity is regulated by Ca(2+) which binds to the GC-associated proteins, GCAP1 and GCAP2 encoded by GUCA1A and GUCA1B, respectively. No recessive mutations in either of these genes have been reported. Dominant missense mutations are largely confined to the Ca(2+)-binding EF hands of the proteins. In a similar fashion to the disease mechanism for the dominant GUCY2D mutations, these mutations generally alter the sensitivity of the cyclase to inhibition as Ca(2+) levels rise following a light flash.
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http://dx.doi.org/10.1007/s11010-009-0331-yDOI Listing
January 2010

Subretinal delivery of adeno-associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice.

J Gene Med 2009 Jun;11(6):486-97

Division of Molecular Therapy, UCL Institute of Ophthalmology, London, UK.

Background: Adeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product.

Methods: We evaluated cellular and humoral responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.

Results: Following subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low-dose AAV.hRPE65.hRPE65 to both eyes of RPE65(-/-) mice resulted in transgene expression and functional rescue, but re-administration of high-dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.

Conclusions: These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose-dependent. Low-dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration.
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http://dx.doi.org/10.1002/jgm.1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841821PMC
June 2009

Neuroprotective gene therapy for the treatment of inherited retinal degeneration.

Curr Gene Ther 2007 Dec;7(6):434-45

Division of Molecular Therapy, UCL Institute of Ophthalmology, Bath St, London EC1V 9EL, UK.

Inherited retinal degeneration (IRD) affects around 1/3000 of the population in Europe and the United States. It is a diverse group of conditions that results from mutations in any one of over 100 different genes. Many of the genes have now been identified and their functions elucidated, providing a major impetus to develop gene-based treatments. Whilst gene replacement and gene silencing strategies offer prospects for the treatment of specific inherited retinal disorders, other disorders may be less amenable to these corrective approaches. These conditions include, in particular, those associated with abnormal retinal development and those in which retinal degeneration is advanced at birth. Furthermore, the development of individualized corrective gene therapy strategies for patients with disorders due to very rare mutations may be unfeasible. However, generic gene therapy strategies that aim not to correct the gene defect but to ameliorate its consequences offer the possibility of therapies that are widely applicable across a range of conditions. One potential strategy in these cases is to halt or delay the process of cell death, so that useful visual function can be maintained throughout the lifetime of an affected individual. It has been shown in variety of experimental models over the last three decades, that neurotrophic factors have the potential to delay neuronal apoptosis. Neurotrophic factors are small proteins which have relatively short half lives and a requirement for repeated administration has limited their clinical application. Since these proteins do not ordinarily cross the blood-brain barrier, previous approaches have relied upon intrathecal infusion pumps or similar complex devices to sustain elevated neurotrophin levels within the central nervous system (CNS). However, sustained delivery through viral vector mediated expression of genes encoding neurotrophic factors may circumvent the potential side effects of repeated administration. In this review we shall explore some of the concepts of neurotrophic gene therapy and how this might be applicable to preserving vision in inherited retinal degenerations.
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http://dx.doi.org/10.2174/156652307782793531DOI Listing
December 2007

In contrast to AAV-mediated Cntf expression, AAV-mediated Gdnf expression enhances gene replacement therapy in rodent models of retinal degeneration.

Mol Ther 2006 Nov 26;14(5):700-9. Epub 2006 Jul 26.

Division of Molecular Therapy, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.

While AAV- and lentivirus-mediated gene replacement therapy can produce structural and functional improvements in various animal models of inherited retinal degeneration, this approach often has very limited effects on the rate of photoreceptor cell loss. Neurotrophic factors such as ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) have been shown to prolong photoreceptor survival in rodent models of retinal degeneration, but AAV-mediated Cntf expression also results in suppression of electrophysiological responses from the retina. In this study using mice, we show that while the deleterious effects mediated by CNTF are dose-dependent, administering a dose of CNTF that does not adversely affect retinal function precludes its ability to delay photoreceptor cell death. In evaluating GDNF as a neuroprotective agent, we show that AAV-mediated Gdnf expression does not produce adverse effects similar to those of CNTF. In addition, we demonstrate the ability of AAV-mediated delivery of Gdnf to slow cell death in two rodent models of retinitis pigmentosa and to enhance retinal function in combination with the relevant gene replacement therapy. These data show for the first time that a combination of these approaches can provide enhanced rescue over gene replacement or growth factor therapy alone.
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http://dx.doi.org/10.1016/j.ymthe.2006.05.019DOI Listing
November 2006

CNTF gene transfer protects ganglion cells in rat retinae undergoing focal injury and branch vessel occlusion.

Exp Eye Res 2006 Nov 10;83(5):1118-27. Epub 2006 Jul 10.

Division of Molecular Therapy, Institute of Ophthalmology, University College London, London EC1V 9EL, UK.

Ciliary neurotrophic factor (CNTF) has been shown to protect ganglion cells in a variety of acute ischaemia models. Here we assess the efficacy of local CNTF gene transfer in protecting retinal ganglion cells when there is focal ischaemia combined with interruption of axoplasmic flow. This dual injury may be more representative of the pathological mechanisms operating in acute retinal diseases, such as vascular events acting at the optic nerve head. Fourteen rats received an intravitreal injection of an adeno-associated viral (AAV) vector expressing a secretable form of CNTF into the right eye and a control vector into the left eye. Three weeks later, each rat underwent a symmetrical small vertical 2mm standardised retinal crush injury approximately 2mm temporal to the optic disc. The injury also occluded the temporal retinal arteriole so that the axon crush was combined with an acute retinal infarction visible on fundoscopy. Changes in the damaged sector were compared histologically four weeks after injury and ganglion cell survival was estimated by comparing cell counts on retinal flat-mounts immunostained with RT-97 antibody. This mode of injury led to a profound loss of both the inner nuclear and ganglion cell layers, but was limited to the lesioned sector. In AAV.CNTF-treated eyes approximately 12% of ganglion cells survived compared with approximately 2% in control eyes (p=0.01). The scotopic electroretinogram (ERG), however, was reduced to about 50% in AAV.CNTF-treated eyes, both before and after injury. We therefore show that CNTF gene transfer confers neuroprotection to ganglion cells undergoing an acute ischaemic injury combined with interruption of axoplasmic flow. This approach may be relevant to optic nerve trauma and a variety of retinal vascular diseases that lead to swelling of the optic nerve head, provided CNTF can be delivered in a way that does not significantly suppress retinal function.
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http://dx.doi.org/10.1016/j.exer.2006.05.019DOI Listing
November 2006

Corrigendum to "Local Administration of an Adeno-Associated Viral Vector Expressing IL-10 Reduces Monocyte Infiltration and Subsequent Photoreceptor Damage During Experimental Autoimmune Uveitis".

Mol Ther 2006 Apr 3;13(4):829. Epub 2005 Nov 3.

Division of Molecular Therapy, Institute of Ophthalmology, University College, London, 11-43 Bath Street, London EC1V 9EL, UK; Molecular Immunology Unit, Institute of Child Health, London, UK. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2005.09.013DOI Listing
April 2006

Effective gene therapy with nonintegrating lentiviral vectors.

Nat Med 2006 Mar 19;12(3):348-53. Epub 2006 Feb 19.

Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.
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http://dx.doi.org/10.1038/nm1365DOI Listing
March 2006

Gene replacement therapy rescues photoreceptor degeneration in a murine model of Leber congenital amaurosis lacking RPGRIP.

Invest Ophthalmol Vis Sci 2005 Sep;46(9):3039-45

Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA.

Purpose: Retinitis pigmentosa GTPase regulator (RPGR) is a photoreceptor protein anchored in the connecting cilia by an RPGR-interacting protein (RPGRIP). Loss of RPGRIP causes Leber congenital amaurosis (LCA), a severe form of photoreceptor degeneration. The current study was an investigation of whether somatic gene replacement could rescue degenerating photoreceptors in a murine model of LCA due to a defect in RPGRIP.

Methods: An RPGRIP expression cassette, driven by a mouse opsin promoter, was packaged into recombinant adeno-associated virus (AAV). The AAV vector was delivered into the right eyes of RPGRIP(-/-) mice by a single subretinal injection into the superior hemisphere. The left eyes received a saline injection as a control. Full-field electroretinograms (ERGs) were recorded from both eyes at 2, 3, 4, and 5 months after injection. After the final follow-up, retinas were analyzed by immunostaining or by light and electron microscopy.

Results: Delivery of the AAV vector led to RPGRIP expression and restoration of normal RPGR localization at the connecting cilia. Photoreceptor preservation was evident by a thicker cell layer and well-developed outer segments in the treated eyes. Rescue was more pronounced in the superior hemisphere coincident with the site of delivery. Functional preservation was demonstrated by ERG.

Conclusions: AAV-mediated RPGRIP gene replacement preserves photoreceptor structure and function in a mouse model of LCA, despite ongoing cell loss at the time of intervention. These results indicate that gene replacement therapy may be effective in patients with LCA due to a defect in RPGRIP and suggest that further preclinical development of gene therapy for this disorder is warranted.
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http://dx.doi.org/10.1167/iovs.05-0371DOI Listing
September 2005

Local administration of an adeno-associated viral vector expressing IL-10 reduces monocyte infiltration and subsequent photoreceptor damage during experimental autoimmune uveitis.

Mol Ther 2005 Aug;12(2):369-73

Division of Molecular Therapy, Institute of Ophthalmology, University College, London, 11-43 Bath Street, London EC1V 9EL, UK.

Autoimmune posterior uveitis is a chronic, potentially blinding inflammatory disease of the eye. It is commonly treated with immunosuppressive drugs that have adverse long-term effects. Advances in gene transfer techniques have enabled long-term, stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. Here we report for the first time that subretinal injection of rAAV-2 encoding murine IL-10 into the retina of C57BL/6 mice significantly decreases the median experimental autoimmune uveitis (EAU) disease severity. This protection is shown to be due to a decrease in the number and activation status of infiltrating monocytes during EAU, as determined by costimulatory molecule expression and nitrotyrosine detection. No differences within splenocyte proliferative responses or serum antibody levels were detected, emphasizing the potential of gene therapy strategies in ameliorating autoimmune responses in local microenvironments without unwanted systemic effects.
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http://dx.doi.org/10.1016/j.ymthe.2005.03.018DOI Listing
August 2005