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1.  Mid-stage intervention achieves similar efficacy as conventional early-stage treatment using gene therapy in a pre-clinical model of retinitis pigmentosa 
Human Molecular Genetics  2013;23(2):514-523.
Deficiencies in rod-specific cyclic guanosine monophosphate (cGMP) phosphodiesterase-6 (PDE6) are the third most common cause of autosomal recessive retinitis pigmentosa (RP). Previously, viral gene therapy approaches on pre-clinical models with mutations in PDE6 have demonstrated that the photoreceptor cell survival and visual function can be rescued when the gene therapy virus is delivered into the subretinal space before the onset of disease. However, no studies have currently been published that analyze rescue effects after disease onset, a time when human RP patients are diagnosed by a clinician and would receive the treatment. We utilized the AAV2/8(Y733F)-Rho-Pde6α gene therapy virus and injected it into a pre-clinical model of RP with a mutation within the alpha subunit of PDE6: Pde6αD670G. These mice were previously shown to have long-term photoreceptor cell rescue when this gene therapy virus was delivered before the onset of disease. Now, we have determined that subretinal transduction of this rod-specific transgene at post-natal day (P) 21, when approximately half of the photoreceptor cells have undergone degeneration, is more efficient in rescuing cone than rod photoreceptor function long term. Therefore, AAV2/8(Y733F)-Rho-Pde6α is an effective gene therapy treatment that can be utilized in the clinical setting, in human patients who have lost portions of their peripheral visual field and are in the mid-stage of disease when they first present to an eye-care professional.
PMCID: PMC3869365  PMID: 24101599
2.  Therapeutic Margins in a Novel Preclinical Model of Retinitis Pigmentosa 
The Journal of Neuroscience  2013;33(33):13475-13483.
The third-most common cause of autosomal recessive retinitis pigmentosa (RP) is due to defective cGMP phosphodiesterase-6 (PDE6). Previous work using viral gene therapy on PDE6-mutant mouse models demonstrated photoreceptors can be rescued if administered before degeneration. However, whether visual function can be rescued after degeneration onset has not been addressed. This is a clinically important question, as newly diagnosed patients exhibit considerable loss of rods and cones in their peripheral retinas. We have generated and characterized a tamoxifen inducible Cre-loxP rescue allele, Pde6bStop, which allows us to temporally correct PDE6-deficiency. Whereas untreated mutants exhibit degeneration, activation of Cre-loxP recombination in early embryogenesis produced stable long-term rescue. Reversal at later time-points showed partial long-term or short-lived rescue. Our results suggest stable restoration of retinal function by gene therapy can be achieved if a sufficient number of rods are treated. Because patients are generally diagnosed after extensive loss of rods, the success of clinical trials may depend on identifying patients as early as possible to maximize the number of treatable rods.
PMCID: PMC3742933  PMID: 23946405
3.  Gene therapy provides long-term visual function in a pre-clinical model of retinitis pigmentosa 
Human Molecular Genetics  2012;22(3):558-567.
Approximately 36 000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are caused by a loss in phosphodiesterase (PDE6) function. In the preclinical Pde6αnmf363 mouse model of this disease, defects in the α-subunit of PDE6 result in a progressive loss of photoreceptors and neuronal function. We hypothesized that increasing PDE6α levels using an AAV2/8 gene therapy vector could improve photoreceptor survival and retinal function. We utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)-Rho-Pde6α, to transduce Pde6αnmf363 retinas and monitored its effects over a 6-month period (a quarter of the mouse lifespan). We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. These results suggest that RP due to PDE6α deficiency in humans, in addition to PDE6β deficiency, is also likely to be treatable by gene therapy.
PMCID: PMC3542865  PMID: 23108158
4.  Mice with a D190N Mutation in the Gene Encoding Rhodopsin: A Model for Human Autosomal-Dominant Retinitis Pigmentosa 
Molecular Medicine  2012;18(1):549-555.
Rhodopsin is the G protein–coupled receptor in charge of initiating signal transduction in rod photoreceptor cells upon the arrival of the photon. D190N (RhoD190n), a missense mutation in rhodopsin, causes autosomal-dominant retinitis pigmentosa (adRP) in humans. Affected patients present hyperfluorescent retinal rings and progressive rod photoreceptor degeneration. Studies in humans cannot reveal the molecular processes causing the earliest stages of the condition, thus necessitating the creation of an appropriate animal model. A knock-in mouse model with the D190N mutation was engineered to study the pathogenesis of the disease. Electrophysiological and histological findings in the mouse were similar to those observed in human patients, and the hyperfluorescence pattern was analogous to that seen in humans, confirming that the D190N mouse is an accurate model for the study of adRP.
PMCID: PMC3388123  PMID: 22252712
5.  PARP1 Gene Knock-Out Increases Resistance to Retinal Degeneration without Affecting Retinal Function 
PLoS ONE  2010;5(11):e15495.
Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.
PMCID: PMC2990765  PMID: 21124852

Results 1-5 (5)