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1.  Modulation of the rate of retinal degeneration in T17M RHO mice by reprogramming the Unfolded Protein Response 
The goal of this study is to validate whether reprogramming of the UPR via modulation of pro-apoptotic caspase-7 and CHOP proteins could be an effective approach to slow down the rate of retinal degeneration in ADRP mice. In order to pursue our goal we created the T17M RHO CASP7 and T17M RHO CHOP mice to study the impact of the CASP7 or CHOP ablations in T17M RHO retina by ERG, SD-OCT, histology and western blot analysis. The scotopic ERG demonstrated that the ablation of the CASP7 in T17M RHO retina leads to significant preservation of the function of photoreceptors compared to control. Surprisingly, the ablation of pro-apoptotic CHOP protein in T17M RHO mice led to a more severe form of retinal degeneration. Results of the SD-OCT and histology were in agreement with the ERG data. The further analysis demonstrated that the preservation of the structure and function or the acceleration of the onset of the T17M RHO photoreceptor degeneration occurred via reprogramming of the UPR. In addition, the CASP7 ablation leads to the inhibition of cJUN mediated apoptosis, while the ablation of CHOP induces an increase in the HDAC. Thus, manipulation with the UPR requires careful examination in order to achieve a therapeutic effect.
PMCID: PMC4083741  PMID: 24664731
ADRP; UPR; Caspase-7; CHOP; apoptosis
2.  Triple Combination of siRNAs Targeting TGFβ1, TGFβR2, and CTGF Enhances Reduction of Collagen I and Smooth Muscle Actin in Corneal Fibroblasts 
Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA).
Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict “effective” and “ineffective” triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry.
Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%–80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability.
Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.
An optimized triple combination of siRNAs targeting TGFβ1, TGFβR2, and CTGF genes produced very high knockdown of target and downstream scarring genes (collagen and alpha smooth muscle actin) without cell toxicity, suggesting a new approach for reducing fibrosis in the cornea and other tissues.
PMCID: PMC3867185  PMID: 24282226
siRNA; RNA interference; combination therapy; CTGF; TGFβ1
3.  Delivery of Antioxidant Enzyme Genes to Protect against Ischemia/Reperfusion-Induced Injury to Retinal Microvasculature 
Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice.
I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated.
Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration.
Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.
PMCID: PMC3756491  PMID: 19628743
4.  Pathological Consequences of Long-Term Mitochondrial Oxidative Stress in the Mouse Retinal Pigment Epithelium 
Experimental eye research  2012;101:60-71.
Oxidative stress in the retinal pigment epithelium (RPE) is hypothesized to be a major contributor to the development of age-related macular degeneration (AMD). Mitochondrial manganese superoxide dismutase (MnSOD) is a critical antioxidant protein that scavenges the highly reactive superoxide radical. We speculated that specific reduction of MnSOD in the RPE will increase the level of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis similar to geographic atrophy. To test this hypothesis, an Sod2-specific hammerhead ribozyme (Rz), delivered by AAV2/1 and driven by the human VMD2 promoter was injected subretinally into C57BL/6J mice. Dark-adapted full field electroretinogram (ERG) detected a decrease in the response to light. We investigated the age-dependent phenotypic and morphological changes of the outer retina digital fundus imaging and SD-OCT measurement of ONL thickness. Fundus microscopy revealed pigmentary abnormalities in the retina and these corresponded to sub-retinal and sub-RPE deposits seen in SD-OCT B-scans. Light and electron microscopy documented the localization of apical deposits and thickening of the RPE. In RPE flat-mounts we observed abnormally displaced nuclei and regions of apparent fibrosis in the central retina of the oldest mice. This region was surrounded by enlarged and irregular RPE cells that have been observed in eyes donated by AMD patients and in other mouse models of AMD.
PMCID: PMC3419481  PMID: 22687918
retinal pigment epithelium; oxidative stress; superoxide dismutase; mitochondria; mouse model; age related macular degeneration
5.  Proteolytic Processing of Connective Tissue Growth Factor in Normal Ocular Tissues and during Corneal Wound Healing 
Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas.
Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5′ Rapid Amplification of cDNA Ends (RACE).
HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle “hinge” region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11.
Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.
Proteolytic processing of connective tissue growth factor (CTGF, 38 kDa) alters its biological effects. C-terminal fragments of CTGF (21 kDa and 25 kDa) are abundant in normal ocular tissues, and peak 11 days after excimer laser ablation in rat corneas. Processing of CTGF during corneal wound healing may impact scar formation.
PMCID: PMC3522436  PMID: 23139278
6.  Hammerhead Ribozyme-Mediated Knockdown of mRNA for Fibrotic Growth Factors: Transforming Growth Factor-Beta 1 and Connective Tissue Growth Factor 
Excessive scarring (fibrosis) is a major cause of pathologies in multiple tissues, including lung, liver, kidney, heart, cornea, and skin. The transforming growth factor- β (TGF- β) system has been shown to play a key role in regulating the formation of scar tissue throughout the body. Furthermore, connective tissue growth factor (CTGF) has been shown to mediate most of the fibrotic actions of TGF- β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. Currently, no approved drugs selectively and specifically regulate scar formation. Thus, there is a need for a drug that selectively targets the TGF- β cascade at the molecular level and has minimal off-target side effects. This chapter focuses on the design of hammerhead ribozymes, measurement of kinetic activity, and assessment of knockdown mRNAs of TGF- β and CTGF in cell cultures.
PMCID: PMC3660091  PMID: 22131029
Ribozymes; TGF- β; CTGF; Scar formation; Transduction; Oligonucleotides
7.  Long-Term Rescue of Retinal Structure and Function by Rhodopsin RNA Replacement with a Single Adeno-Associated Viral Vector in P23H RHO Transgenic Mice 
Human Gene Therapy  2012;23(4):356-366.
Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adeno-associated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.
Mao and colleagues show that a single subretinal injection of AAV serotype 5 (AAV5) vector encoding both a small interfering RNA targeted against a mutant form of rhodopsin (P23H RHO) and a replacement cDNA for the nonmutant form of RHO preserves retinal structure and leads to full recovery of retinal function in a mouse model of autosomal dominant retinitis pigmentosa.
PMCID: PMC3327607  PMID: 22289036
8.  Suppression of rds Expression by siRNA and Gene Replacement Strategies for Gene Therapy Using rAAV Vector 
Small interfering RNA (siRNA) is a promising tool for the treatment of dominant diseases. Autosomal dominant eye disease like retinitis pigmentosa, are a leading cause of blindness. Mutations in rds/peripherin lead to the degeneration of photoreceptors and are associated with several autosomal retinal diseases. Our goal is to develop a gene therapy for rds mutations. We describe a siRNA based mutation-independent approach, targeting rds in which levels of endogenous mutant and wild-type mRNA were reduced, and a siRNA-resistant version of rds gene was supplied simultaneously. siRNAs and resistant rds were delivered to the photoreceptors by recombinant adeno-associated virus (rAAV) vector through subretinal injections. The retinal phenotype was examined, both structurally and functionally at different time points after rAAV delivery. We demonstrate suppression of rds transcript by up to 50% with concomitant expression of replacement transcript in the retina of mice in vivo. These results validate the concept of suppression of rds and replacement strategies of gene therapy with rAAV vectors containing siRNA.
PMCID: PMC3556273  PMID: 22183336
9.  Gene Delivery of Wild-Type Rhodopsin Rescues Retinal Function in an Autosomal Dominant Retinitis Pigmentosa Mouse Model 
Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations within the gene for the opsin of rod photoreceptor cells. Studies on transgenic mice, carrying mutated rhodopsin (RHO) transgene on different genetic backgrounds suggested that that an increased amount of wild-type RHO in ADRP photoreceptors attenuated the impact of the mutant transgene. Therefore, we employed a gene therapy approach with a help of Adeno-associated virus (AAV) to treat mice expressing a P23H mutant human RHO transgene. Knowing that AAV5 primarily transduces photoreceptor cells, we designed “hardened” form of the rhodopsin gene (RHO301) that expressed normal rhodopsin and was specifically resistant to degradation by the previously tested siRNA301. AAV5 RHO301 was subretinaly injected into the right eyes of P23H RHO mice at post-natal day 15. Animals were analyzed monthly by electroretinography (ERG) for 6 months. Analysis of the full field scotopic electroretinogram (ERG) demonstrated that increased expression of opsin slowed the rate of retinal degeneration in P23H mice with increased amplitudes in both a-wave and b-wave amplitudes compared to control eyes. An increase in the ERG amplitudes was correlated with improvement of retinal structure. The thickness of the outer nuclear layer in AAV-RHO301 injected eyes was increased by 80% compared to control eyes. This finding indicates that wild -type RHO could rescue the retinal degeneration in transgenic mice carrying a dominant RHO mutation and that increased production of normal rhodopsin could suppress the effect of the mutant protein. These findings suggest that wild-type RHO can used as a therapeutic agent to retard retinal degeneration in ADRP caused by different mutations of RHO via increased production of normal rhodopsin protein.
PMCID: PMC3392039  PMID: 22183334
10.  Characterization of a Transgenic Short Hairpin RNA-Induced Murine Model of Tafazzin Deficiency 
Human Gene Therapy  2010;22(7):865-871.
Barth's syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS.
Barth's syndrome (BTHS) is an X-linked mitochondrial disease that has been associated with loss-of-function mutations in the Tafazzin (TAZ) gene. There is no mammalian animal model of BTHS. In this study, Soustek and colleagues describe an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model. They report that this model exhibits some of the molecular and clinical characteristics of patients with BTHS and may ultimately serve as an important tool in developing therapeutic strategies for BTHS.
PMCID: PMC3166794  PMID: 21091282
11.  Functional Rescue of P23H Rhodopsin Photoreceptors by Gene Delivery 
The mechanism of autosomal dominant retinitis pigmentosa (ADRP) caused by the P23H mutation in rhodopsin is tightly associated with misfolded rhodopsin (RHO) which causes endoplasmic reticulum overload (ER stress), activates the unfolded protein response (UPR) and triggers apoptosis. In efforts to create a therapy for ADRP caused by the P23H mutation, we have explored different approaches leading to survival of photoreceptor (PR) cells. The direct approach involves the modulation of the level of wild-type RHO, while the indirect approach involves reprogramming the UPR and increasing the expression of heat shock proteins (HSPs). Taking the direct approach, we found that over-expression of wild-type RHO rescues scotopic ERG responses partially. However, greater therapeutic effects were obtained by manipulation of the UPR in P23H RHO rat PRs treated with the endoplasmic reticulum protein BiP/Grp78. In vitro study revealed that the pro-survival effect of Bip gene was not associated with its function as a molecular chaperone, but rather with its regulation of the UPR. Another indirect approach was the over-expression of the Hsf-1 gene, a transcriptional regulator of the heat shock response. AAV-delivery of Hsf-1 resulted in an increase of scotopic ERG amplitudes by over 35%. Taken together these data suggest viable therapeutic treatments for ADRP.
PMCID: PMC3365920  PMID: 22183333
12.  AAV Delivery of Wild-Type Rhodopsin Preserves Retinal Function in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa 
Human Gene Therapy  2010;22(5):567-575.
Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / +  or RHO + /– backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.
In this study, Mao and colleagues examine the efficacy of AAV5-mediated gene transfer of the wild-type rhodopsin (RHO) gene in a mouse model of autosomal dominant retinitis pigmentosa. The authors find that this approach leads to the production of normal RHO protein and to the rescue of retinal degeneration.
PMCID: PMC3131806  PMID: 21126223
13.  The 5HT1a Receptor Agonist 8-Oh DPAT Induces Protection from Lipofuscin Accumulation and Oxidative Stress in the Retinal Pigment Epithelium 
PLoS ONE  2012;7(4):e34468.
Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT1A receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT1A receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H2O2 was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H2O2-treated cells compared to controls and protected against H2O2-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT1A agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT1A receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.
PMCID: PMC3317995  PMID: 22509307
14.  Mutant NADH dehydrogenase subunit 4 gene delivery to mitochondria by targeting sequence-modified adeno-associated virus induces visual loss and optic atrophy in mice 
Molecular Vision  2012;18:1668-1683.
Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed.
The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription–polymerase chain reaction (RT–PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy.
The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT–PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT–PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident.
Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON.
PMCID: PMC3388991  PMID: 22773905
15.  Gene therapy in animal models of autosomal dominant retinitis pigmentosa 
Molecular Vision  2012;18:2479-2496.
Gene therapy for dominantly inherited genetic disease is more difficult than gene-based therapy for recessive disorders, which can be treated with gene supplementation. Treatment of dominant disease may require gene supplementation partnered with suppression of the expression of the mutant gene either at the DNA level, by gene repair, or at the RNA level by RNA interference or transcriptional repression. In this review, we examine some of the gene delivery approaches used to treat animal models of autosomal dominant retinitis pigmentosa, focusing on those models associated with mutations in the gene for rhodopsin. We conclude that combinatorial approaches have the greatest promise for success.
PMCID: PMC3472929  PMID: 23077406
16.  The Importance of Mitochondria in Age-Related and Inherited Eye Disorders 
Ophthalmic Research  2010;44(3):179-190.
Mitochondria are critical for ocular function as they represent the major source of a cell's supply of energy and play an important role in cell differentiation and survival. Mitochondrial dysfunction can occur as a result of inherited mitochondrial mutations (e.g. Leber's hereditary optic neuropathy and chronic progressive external ophthalmoplegia) or stochastic oxidative damage which leads to cumulative mitochondrial damage and is an important factor in age-related disorders (e.g. age-related macular degeneration, cataract and diabetic retinopathy). Mitochondrial DNA (mtDNA) instability is an important factor in mitochondrial impairment culminating in age-related changes and pathology, and in all regions of the eye mtDNA damage is increased as a consequence of aging and age-related disease. It is now apparent that the mitochondrial genome is a weak link in the defenses of ocular cells since it is susceptible to oxidative damage and it lacks some of the systems that protect the nuclear genome, such as nucleotide excision repair. Accumulation of mitochondrial mutations leads to cellular dysfunction and increased susceptibility to adverse events which contribute to the pathogenesis of numerous sporadic and chronic disorders in the eye.
PMCID: PMC2952187  PMID: 20829642
Mitochondria; Ocular function; Reactive oxygen species
17.  Efficiency and Safety of AAV-Mediated Gene Delivery of the Human ND4 Complex I Subunit in the Mouse Visual System 
To evaluate the efficiency and safety of AAV-mediated gene delivery of a normal human ND4 complex I subunit in the mouse visual system.
A nuclear encoded human ND4 subunit fused to the ATPc mitochondrial targeting sequence and FLAG epitope were packaged in AAV2 capsids that were injected into the right eyes of mice. AAV-GFP was injected into the left eyes. One month later, pattern electroretinography (PERG), rate of ATP synthesis, gene expression, and incorporation of the human ND4 subunit into the murine complex I were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed compared with green fluorescent protein (GFP)-injected eyes.
Rates of ATP synthesis and PERG amplitudes were similar in ND4FLAG- and GFP-inoculated eyes. PERG latency was shorter in eyes that received ND4FLAG. Immunoprecipitated murine complex I gave the expected 52-kDa band of processed human ND4FLAG. Confocal microscopy revealed perinuclear expression of FLAG colocalized with mitochondria-specific fluorescent dye. Transmission electron microscopy revealed FLAG immunogold within mitochondria. Compared with Thy1.2-positive retinal ganglion cells (RGCs), quantification was 38% for FLAG-positive RGCs and 65% for GFP-positive RGCs. Thy1.2 positive-RGC counts in AAV-ND4FLAG were similar to counts in control eyes injected with AAV-GFP.
Human ND4 was properly processed and imported into the mitochondria of RGCs and axons of mouse optic nerve after intravitreal injection. Although it had approximately two-thirds the efficiency of GFP, the expression of normal human ND4 in murine mitochondria did not induce the loss of RGCs, ATP synthesis, or PERG amplitude, suggesting that allotopic ND4 may be safe for the treatment of patients with Leber hereditary optic neuropathy.
PMCID: PMC3085487  PMID: 19387075
18.  rAAV2/5 gene-targeting to rods: dose-dependent efficiency and complications associated with different promoters 
Gene therapy  2010;17(9):1162-1174.
A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that affect primarily rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken beta actin proximal promoter CBA) were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, while the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 1011 vg/ml were used.
PMCID: PMC2914811  PMID: 20428215
rAAV vectors; promoters; rod; retina; gene transfer; dog
20.  Unexpected off-targeting effects of anti-huntingtin ribozymes and siRNA in vivo 
Neurobiology of disease  2007;29(3):446-455.
Gene transfer strategies to reduce levels of mutant huntingtin (mHtt) mRNA and protein by targeting human Htt have shown therapeutic promise in vivo. Previously, we have reported that a specific, adeno-associated viral vector (rAAV)-delivered short-hairpin RNA (siHUNT-2) targeting human Htt mRNA unexpectedly decreased levels of striatal-specific transcripts in both wild-type and R6/1 transgenic HD mice. The goal of this study was to determine whether the siHUNT-2-mediated effect was due to adverse effects of RNA interference (RNAi) expression in the brain. To this end, we designed two catalytically active hammerhead ribozymes directed against the same region of human Htt mRNA targeted by siHUNT-2 and delivered them to wild-type and R6/1 transgenic HD mice. After 10 weeks of continuous expression, these ribozymes, like siHUNT-2, negatively impacted the expression of a subset of genes in the striatum. This effect was independent of rAAV transduction and specific to the targeting of a unique sequence in human Htt mRNA. After consideration of the known potential RNAi-specific toxic mechanisms, only cleavage of an unintended RNA target can account for the data reported herein. Thus, long-term rAAV-mediated RNAi in the brain does not, in and of itself, negatively affect striatal gene expression. These findings have important implications in the development of therapeutic RNAi for the treatment of neurological disease.
PMCID: PMC2695881  PMID: 18166484
21.  Intrastriatal rAAV-Mediated Delivery of Anti-huntingtin shRNAs Induces Partial Reversal of Disease Progression in R6/1 Huntington’s Disease Transgenic Mice 
Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by the presence of an abnormally expanded polyglutamine domain in the N-terminus of huntingtin. We developed a recombinant adeno-associated viral serotype 5 (rAAV5) gene transfer strategy to posttranscriptionally suppress the levels of striatal mutant huntingtin (mHtt) in the R6/1 HD transgenic mouse via RNA interference. Transient cotransfection of HEK293 cells with plasmids expressing a portion of human mHtt derived from R6/1 transgenic HD mice and a short-hairpin RNA directed against the 5′ UTR of the mHtt mRNA (siHUNT-1) resulted in reduction in the levels of mHtt mRNA (−75%) and protein (−60%). Long-term in vivo rAAV5-mediated expression of siHUNT-1 in the striatum of R6/1 mice reduced the levels of mHtt mRNA (−78%) and protein (−28%) as determined by quantitative RT-PCR and Western blot analysis, respectively. The reduction in mHtt was concomitant with a reduction in the size and number of neuronal intranuclear inclusions and a small but significant normalization of the steady-state levels of preproenkephalin and dopamine- and cAMP-responsive phosphoprotein 32 kDa mRNA. Finally, bilateral expression of rAAV5-siHUNT-1 resulted in delayed onset of the rear paw clasping phenotype exhibited by the R6/1 mice. These results suggest that a reduction in the levels of striatal mHtt can ameliorate the HD phenotype of R6/1 mice.
PMCID: PMC2656966  PMID: 16019264
brain; adeno-associated virus; gene therapy; viral gene transfer; RNA interference; short hairpin RNA; small interfering RNA; transcriptional dysregulation; polyglutamine; neuronal intranuclear inclusions
22.  Reduction in Severity of a Herpes Simplex Virus Type 1 Murine Infection by Treatment with a Ribozyme Targeting the UL20 Gene RNA▿  
Journal of Virology  2008;82(15):7467-7474.
Hammerhead ribozymes were designed to target mRNA of several essential herpes simplex virus type 1 (HSV-1) genes. A ribozyme specific for the late gene UL20 was packaged in an adenovirus vector (Ad-UL20 Rz) and evaluated for its capacity to inhibit the viral replication of several HSV-1 strains, including that of the wild-type HSV-1 (17syn+ and KOS) and several acycloguanosine-resistant strains (PAAr5, tkLTRZ1, and ACGr4) in tissue culture. The Ad-UL20 Rz was also tested for its ability to block an HSV-1 infection, using the mouse footpad model. Mouse footpads were treated with either the Ad-UL20 Rz or an adenoviral vector expressing green fluorescent protein (Ad-GFP) and then infected immediately thereafter with 104 PFU of HSV-1 strain 17syn+. Ad-UL20 ribozyme treatment consistently led to a 90% rate of protection for mice from lethal HSV-1 infection, while the survival rate in the control groups was less than 45%. Consistent with this protective effect, treatment with the Ad-UL20 Rz reduced the viral DNA load in the feet, the dorsal root ganglia, and the spinal cord relative to that of the Ad-GFP-treated animals. This study suggests that ribozymes targeting essential genes of the late kinetic class may represent a new therapeutic strategy for inhibiting HSV infection.
PMCID: PMC2493317  PMID: 18508896
23.  Down-regulation of expression of rat pyruvate dehydrogenase E1α gene by self-complementary adeno-associated virus-mediated small interfering RNA delivery 
Mitochondrion  2007;7(4):253-259.
Mutations in the E1 α subunit gene (PDHA1) of the pyruvate dehydrogenase complex (PDC) are common causes of congenital lactic acidosis. An animal model of E1α deficiency could provide insight into the pathological consequences of mutations and serve to test potential therapies. Small interfering RNAs (siRNAs) were designed to cleave the messenger RNA (mRNA) of the E1 α subunit and were tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co-transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1 α mRNA. Quantitative PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24 hours and up to 65% by 56 hours, compared to negative and positive controls. Since oligonucleotide-mediated siRNA delivery provided only transient suppression, we next selected two siRNA candidates and generated self-complementary, double-stranded adeno-associated virus (scAAV) vectors (serotypes 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53–80% 72 hours after infection and 54–70% 10 days after infection in RLF cultures. The expression of E1 α and the specific activity of pyruvate dehydrogenase were also decreased at 10 days after infection in RLF cultures. Thus, scAAV siRNA-mediated knockdown of PDHA1 gene expression provides a strategy that may be applied to create a useful animal model of PDC deficiency.
PMCID: PMC1973157  PMID: 17392036
AAV; Gene therapy; RNAi; Pyruvate dehydrogenase deficiency
24.  Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus 
Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs.
PMCID: PMC2222599  PMID: 18070352
25.  Inhibition of RNA Synthesis as a Therapeutic Strategy against Aspergillus and Fusarium: Demonstration of In Vitro Synergy between Rifabutin and Amphotericin B 
We investigated the in vitro antifungal activity of amphotericin B, alone and in combination with rifabutin, an inhibitor of bacterial RNA polymerase, against 26 clinical isolates of Aspergillus and 25 clinical isolates of Fusarium. Synergy or additivism between these drugs was demonstrated against all isolates tested. Amphotericin B MICs were reduced upon combination with rifabutin from a mean of 0.65 μg/ml to a mean of 0.16 μg/ml against Aspergillus, and from a mean of 0.97 μg/ml to a mean of 0.39 μg/ml against Fusarium (P < 0.000001 for both). Similarly, the MICs of rifabutin were reduced upon combination with amphotericin B from a mean of >32 μg/ml to a mean of 1.1 μg/ml against both fungi (P < 0.000001 for both). These positive interactions were corroborated by a colony count study with two Fusarium isolates, for which treatment with the combination of subinhibitory concentrations of amphotericin B (at concentrations 2- and 4-fold less than the MIC) and rifabutin (at concentrations ranging from 4- to 64-fold less than the MIC) resulted in 3.2-log reductions in colony counts compared to those after treatment with either drug alone. Inhibition of RNA synthesis was shown to be the mechanism of antifungal activity. These results suggest that inhibition of fungal RNA synthesis might be a potential target for antifungal therapy.
PMCID: PMC105490  PMID: 9517924

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