AAV-mediated and pharmacological induction of Hsp70 expression stimulates survival of retinal ganglion cells following axonal injury.

We evaluated the effect of AAV2- and 17-AAG (17-N-allylamino-17-demethoxygeldanamycin)-mediated upregulation of Hsp70 expression on the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC). AAV2-Hsp70 expression in the retina was primarily observed in the ganglion cell layer. Approximately 75% of all transfected cells were RGCs. RGC survival in AAV2-Hsp70-injected animals was increased by an average of 110% 2 weeks after the axonal injury compared with the control. The increase in cell numbers was not even across the retinas with a maximum effect of approximately 306% observed in the inferior quadrant. 17-AAG-mediated induction of Hsp70 expression has been associated with cell protection in various models of neurodegenerative diseases. We show here that a single intravitreal injection of 17-AAG (0.2 ug ul(-1)) results in an increased survival of ONC-injured RGCs by approximately 49% compared with the vehicle-treated animals. Expression of Hsp70 in retinas of 17-AAG-treated animals was upregulated approximately by twofold compared with control animals. Our data support the idea that the upregulation of Hsp70 has a beneficial effect on the survival of injured RGCs, and the induction of this protein could be viewed as a potential neuroprotective strategy for optic neuropathies.

AAV-mediated expression of human PRELP inhibits complement activation, choroidal neovascularization and deposition of membrane attack complex in mice.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Approximately 50% of AMD patients have a polymorphism in the negative regulator of complement known as Factor H. Individuals homozygous for a Y402H polymorphism in Factor H have elevated levels of membrane attack complex (MAC) in their choroid and retinal pigment epithelium relative to individuals homozygous for the wild-type allele. An inability to form MAC due to a polymorphism in C9 is protective against the formation of choroidal neovascularization (CNV) in AMD patients. Hence, blocking MAC in AMD patients may be protective against CNV. Here we investigate the potential of human proline/arginine-rich end leucine-rich repeat protein (PRELP) as an inhibitor of complement-mediated damage when delivered via the subretinal route using an AAV2/8 vector. In a fluorescence-activated cell sorting (FACS) lysis assay, PRELP inhibited normal human serum-mediated lysis of Hepa-1c1c7 cells by 18.7%. Unexpectedly, PRELP enhanced the formation of tubes by human umbilical vein endothelial cells (HUVECs) by approximately 240%, but, when delivered via an AAV vector to the retina of mice, PRELP inhibited laser-induced CNV by 60%. PRELP reduced deposition of MAC in vivo by 25.5%. Our results have implications for the development of complement inhibitors as a therapy for AMD.

Mutation-independent rescue of a novel mouse model of Retinitis Pigmentosa.

Retinitis Pigmentosa (RP) is the leading cause of inherited blindness in the developed world, affecting approximately 1 in 3000 individuals. Although there is currently no cure for RP, the genetic pathology has been well established. In this study, we developed a novel mouse model of RP (huRhoP347S) expressing a pathogenic human rhodopsin gene with a Pro347Ser (P347S) mutation on a rhodopsin knockout background. These mice undergo severe retinal degeneration at 1 month of age. In contrast to prior studies, this model was administered a gene therapy treatment at 19 days postnata. We evaluated several self-complementary adeno-associated virus (AAV) serotypes for photoreceptor tropism, including scAAV2/2, scAAV2/5, scAAV2/6.2 and scAAV2/9, and found that scAAV2/9 transduced photoreceptors with greater efficiency and expression than other vectors. We engineered an scAAV2/9 vector to contain a microRNA sequence specifically targeting the human rhodopsin gene and demonstrated its ability to silence rhodopsin by 60.2±8.2% in vitro. In addition, we constructed an scAAV2/9 vector to contain a replacement 'codon-modified' rhodopsin transgene (RhoR2) that was resistant to degradation by the microRNA. We found that delivery of the RhoR2 by scAAV2/9 is capable of restoring vision to rhodopsin knockout mice, and rescuing our novel transgenic huRhoP347S mouse model of dominant RP. Average a-wave responses of RhoR2-injected eyes were 1.8-fold higher than those of control-injected eyes. We found that delivery of the microRNA and replacement rhodopsin in a 1:2 ratio produced an average electroretinography (ERG) a-wave response of 17.4±2.9 compared to 6.5±2.8 µV for eyes injected with negative control virus.

Adenovirus-mediated delivery of CD46 attenuates the alternative complement pathway on RPE: implications for age-related macular degeneration.

Activation of the alternative pathway of the complement system has been implicated in the pathogenesis of age-related macular degeneration. Membrane attack complex (MAC) has been identified mainly on the Bruch's membrane and drusen underlying the retinal pigment epithelium (RPE). Membrane cofactor protein (CD46) preferentially regulates the alternative pathway of complement. The aim of this study was to evaluate the potential of increasing CD46 expression on RPE cells using an adenovirus as a gene therapy approach to reduce alternative pathway-mediated damage to RPE cells. We generated a recombinant adenovirus vector expressing human CD46 (hCD46) and delivered the vector to murine hepatocytes and RPE cells in vitro. After incubation in human serum in conditions in which the classical pathway of complement was blocked, we measured alternative pathway-mediated damage of these cells by quantifying lysis and MAC formation. Adenovirus expressing hCD46 was delivered to the subretinal space of adult mice, and 1 week later, ocular flat mounts were challenged with human serum and the levels of complement-mediated damage was quantified. Adenovirus-mediated delivery of hCD46 localizes to the basal and lateral surfaces of RPE cells where it offers protection from alternative pathway-mediated damage, but not classical, allowing the classical pathway to function unhindered.

Towards mutation-independent silencing of genes involved in retinal degeneration by RNA interference.

More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation-independent therapies for these diseases. We describe use of RNA interference (RNAi) and specifically short hairpin RNAs (shRNAs) expressed from DNA templates to silence both normal and mutant (P23H) human rhodopsin alleles by 94.34+/-2.17 and 94.9+/-1.9%, respectively, in human embryonic retinoblasts. Degeneracy of the genetic code was used to engineer a codon-exchanged mRNA (cmRNA) that demonstrated complete resistance to silencing by the shRNA. Simulation of autosomal dominant retinitis pigmentosa in cell culture through triple transfection of DNAs expressing a cmRNA, a P23H mRNA and an shRNA revealed shRNA-mediated silencing, specifically of P23H rhodopsin by 90.64+/-5.19% and no loss of rhodopsin translation from the cmRNA in those cells. In addition, we present data on two alternative shRNA sequences targeting human rhodopsin. Our results have implications for the treatment of a very large variety of retinal degenerations in a mutation-independent manner.

Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration.

First (DeltaE1/E3) and second (DeltaE1+DeltaE2/E3/E4) generation adenovirus (Ad) vectors have been shown previously to be of limited use in the treatment of human genetic diseases due to the induction of a host cytotoxic T-cell mediated immune response against virally expressed genes. In addition, a limited cloning capacity of approximately 8 kb does not cater for the incorporation of large upstream sequences essential for regulated tissue-specific expression or inclusion of multiple gene-expression cassettes. In this study we have exploited our recently developed Ad-based vector, the encapsidated adenovirus mini-chromosome (EAM) from which all of the viral genes have been deleted. EAMs contain only the inverted terminal repeats required for replication and five cis -acting Ad encapsidation signals necessary for packaging. We have shown previously that EAMs can efficiently transduce a variety of cell types in vitro. In this study we demonstrate that EAMs can transduce and rescue cells from the neurosensory retina in vivo. EAM-mediated delivery of the beta subunit of cyclic GMP phosphodiesterase (PDE) cDNA to mice affected with retinal degeneration (rd) allows prolonged transgene expression and rescue of rod photoreceptor cells. RT-PCR analysis from the injected retina indicates that transgene products are present for at least 18 weeks post-injection. Both the alpha and beta subunits of PDE could be detected up to 90 days postnatal in EAM-injected rd retina by western analysis. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal. Examination of outer nuclear thickness showed significant differences up to 12 weeks post-injection. These results demonstrate an improved level of rescue over first-generation adenoviral vectors and suggest the possibility of successful EAM-mediated treatment of some retinal diseases in humans.

Improved adenoviral vectors for gene therapy of Duchenne muscular dystrophy.

We have been exploring the feasibility of gene therapy for Duchenne muscular dystrophy by characterizing parameters important for the design of therapeutic protocols. These studies have used transgenic mice to analyze expression patterns of multiple dystrophin vectors, and have been accompanied by the development of viral vectors for gene transfer to dystrophic mdx mouse muscle. Analysis of transgenic mdx mice indicates that greater than 50% of the fibers in a muscle group must express dystrophin to prevent development of a significant dystrophy, and that low-level expression of truncated dystrophins can function very well. These results suggest that gene therapy of DMD will require methods to transduce the majority of fibers in critical muscle groups with vectors that express moderate levels of dystrophin proteins. Strategies for the development of viral vectors able to deliver dystrophin genes to muscle include the use of muscle specific regulatory sequences coupled with deletion of viral gene sequences to limit virus-induced immune rejection of transduced tissues. These strategies should enable production of adenoviral vectors expressing full-length dystrophin proteins in muscle.

Clinical and molecular genetic characterisation of a family segregating autosomal dominant retinitis pigmentosa and sensorineural deafness.

AIMS/BACKGROUND: To characterise clinically a large kindred segregating retinitis pigmentosa and sensorineural hearing impairment in an autosomal dominant pattern and perform genetic linkage studies in this family. Extensive linkage analysis in this family had previously excluded the majority of loci shown to be involved in the aetiologies of RP, some other forms of inherited retinal degeneration, and inherited deafness. METHODS: Members of the family were subjected to detailed ophthalmic and audiological assessment. In addition, some family members underwent skeletal muscle biopsy, electromyography, and electrocardiography. Linkage analysis using anonymous microsatellite markers was performed on DNA samples from all living members of the pedigree. RESULTS: Patients in this kindred have a retinopathy typical of retinitis pigmentosa in addition to a hearing impairment. Those members of the pedigree examined demonstrated a subclinical myopathy, as evidence by abnormal skeletal muscle histology, electromyography, and electrocardiography. LOD scores of Zmax = 3.75 (theta = 0.10), Zmax = 3.41 (theta = 0.10), and Zmax = 3.25 (theta = 0.15) respectively were obtained with the markers D9S118, D9S121, and ASS, located on chromosome 9q34-qter, suggesting that the causative gene in this family may lie on the long arm (q) of chromosome 9. CONCLUSIONS: These data indicate that the gene responsible for the phenotype in this kindred is located on chromosome 9 q. These data, together with evidence that a murine deafness gene is located in a syntenic area of the mouse genome, should direct the research community to consider this area as a candidate region for retinopathy and/or deafness genes.

Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells.

Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of approximately 8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (approximately 14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a beta-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1 + E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (> 10(8)/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.

Evidence for genetic heterogeneity in Best's vitelliform macular dystrophy.

Best's vitelliform macular dystrophy is an early onset, autosomal dominant macular degeneration. Linkage analysis has previously mapped a disease locus in this disorder to the pericentromeric region of chromosome 11. We examined two families, one of German and one of Irish origin, both affected with this disorder. The Irish family (BTMD1) showed strong evidence for linkage to the previously reported locus on chromosome 11. Linkage of the disease locus to the same region of chromosome 11 has been significantly excluded in the German family (Fam E), thereby providing evidence of locus heterogeneity in this clinically unique condition.

Isolation and genetic mapping of four microsatellite repeats from chromosome 3p21 using 40 CEPH pedigrees.

We have isolated and characterized the microsatellite markers D3S1447, D3S1448, D3S1449, and D3S1450, with PIC values of 0.771, 0.644, 0.633, and 0.552, respectively, from a hamster/human chromosome 3 library. By typing 40 CEPH pedigrees with each of these novel markers, we have sublocalized each microsatellite to 3p21 and with multipoint analyses have shown that the likelihood of the order centromere-D3S13-24.24 cM-D3S1450-9.41 cM-D3S1447-7.05 cM-D3F15S2-3 cM-[D3S1448-2.2 cM-D3S1449]-6.13 cM-D3S1100- telomere significantly exceeds any other order (8.0 x 10(3):1). These finely mapped markers will be extremely useful in aiding in the physical characterization of 3p21, which has been implicated in a range of tumor tissues.

Exclusion of the involvement of all known retinitis pigmentosa loci in the disease present in a family of Irish origin provides evidence for a sixth autosomal dominant locus (RP8).

Retinitis Pigmentosa (RP) is the most prevalent degenerative retinal disease of mendelian origin, currently affecting approximately 1.5 million people worldwide. To date it has been established that a minimum of five different genes maybe involved in the pathogenesis of autosomal dominant forms of RP (adRP). The genes encoding two retinal specific proteins, rhodopsin and peripherin/RDS, have been implicated in causing adRP due to the observation of many different mutations in these genes in patients suffering from RP. The three remaining adRP genes have been mapped to specific regions of human chromosomes but as yet are uncharacterized. We have investigated if there is evidence for the presence of another locus in the genome which when mutated causes adRP. We have utilised polymorphic genetic markers which have previously been mapped to each of the regions known to harbour adRP genes, to test for the exclusion or linkage of the disease gene segregating in a pedigree of Irish origin and find no evidence for linkage. Hence we provide definitive evidence for the involvement of yet another locus. The implications of high levels of genetic heterogeneity inherent in adRP are discussed in relation to diagnosis, prognosis and future therapies.

Localization of an autosomal dominant retinitis pigmentosa gene to chromosome 7q.

Retinitis pigmentosa is a group of clinically and genetically heterogeneous retinopathies and a significant cause of worldwide visual handicap. We have typed DNA from members of a Spanish family segregating an autosomal dominant form of retinitis pigmentosa (adRP) using a large series of simple sequence polymorphic markers. Positive two-point lod scores have been obtained with fifteen markers including D7S480 (theta max = 0.00, Zmax = 7.22). Multipoint analyses using a subset of these markers gave a lod score of 7.51 maximizing at D7S480. These data provide definitive evidence for the localisation of an adRP gene on chromosome 7q, and highlight the extensive genetic heterogeneity that exists in the autosomal dominant form of this disease.