ABSTRACT
OBJECTIVES: Pulmonary hypertension (PH) is a leading cause of death in patients with systemic sclerosis (SSc). The purpose of this study was to determine the prognostic significance of pericardial effusion in patients with SSc-PH. METHODS: Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma (PHAROS) is a prospective multicentre registry which enrolled patients with newly diagnosed SSc-PH from 2005 to 2016. The prognostic impact of pericardial effusion status, including those who ever or never had pericardial effusion, and those who had persistent or intermittent pericardial effusion, was analyzed. Kaplan-Meier survival analyses, log-rank test, and multivariable Cox proportional hazards regression were performed. RESULTS: Of the 335 patients with SSc-PH diagnosed by right heart catheterization and documentation of pericardial effusion presence or absence on echocardiogram, 166 (50%) ever had pericardial effusion. Ever having pericardial effusion was not predictive of survival (Log-rank test p= 0.49). Of the 245 SSc-PH patients who had at least two echocardiograms, 44% had a change in pericardial effusion status over an average of 4.3 years of follow up. Having a persistent pericardial effusion was an independent predictor of survival (adjusted hazard ratio [aHR] = 2.34, 95% CI 1.20-4.64, p= 0.002), while intermittent pericardial effusion was not a predictor of survival (aHR = 0.89, 95% CI 0.52-1.56, p= 0.68), in a multivariable-adjusted analysis. CONCLUSION: Persistent pericardial effusion, but not ever having had pericardial effusion or intermittent pericardial effusion, was independently associated with poorer survival. Incorporating information from serial echocardiograms may help clinicians better prognosticate survival in their SSc-PH patients.
ABSTRACT
Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examined HLA associations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk, and the HLA-DRB1*08:04 allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and HLA-DRB1*07:01 alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13:01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 allele frequency in multiple populations was observed (r = 0.98, P = 3 × 10-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/ß allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.
Subject(s)
Autoantibodies/immunology , Autoantigens/genetics , HLA Antigens/genetics , Molecular Mimicry/immunology , Scleroderma, Systemic/genetics , Black or African American/genetics , Alleles , Amino Acid Sequence/genetics , Antigens, Viral/genetics , Antigens, Viral/immunology , Autoantigens/immunology , Computational Biology , Datasets as Topic , Female , Genetic Predisposition to Disease , HLA Antigens/immunology , Humans , Male , Mimiviridae/immunology , Phycodnaviridae/immunology , Protein Structure, Secondary/genetics , Risk Assessment , Scleroderma, Systemic/epidemiology , Scleroderma, Systemic/immunology , Sequence Homology, Amino Acid , White People/geneticsABSTRACT
OBJECTIVES: We sought to examine temporal trends in adverse pregnancy outcomes among SSc pregnancies in a large nationwide sample. METHODS: We used the National Inpatient Sample (NIS) database from 2000 - 2017 to derive national estimates of delivery-associated hospitalizations in the United States among patients with SSc. Each SSc delivery was matched to 100 non-SSc deliveries by age, delivery year, and race. We evaluated adverse pregnancy outcomes (APOs) including maternal and fetal death, cesarean delivery, hospital length of stay, preterm delivery, intrauterine growth restriction, and hypertensive disorders of pregnancy. We used multivariable regression models with an interaction term between SSc and year and adjusting for race, advanced maternal age, diabetes mellitus, and pre-existing hypertension to evaluate temporal trends in APOs among SSc and non-SSc deliveries. RESULTS: From 2000 to 2017, there were 3740 delivery-associated hospitalizations for women with SSc. SSc was associated with an increased risk of all APOs compared to non-SSc deliveries. Fetal death declined in SSc deliveries from 49.0 per 1000 delivery-related admissions in 2000 - 2005 to 16.2 per 1000 in 2012 - 2017. There was a significant difference in trends for fetal death between SSc and non-SSc deliveries (p = 0.043), but the trends for other APOs did not differ between the two groups. CONCLUSIONS: In this large nationwide sample, the risk of fetal death among women with SSc markedly improved over the past 18 years. The risk for other APOs remained high in SSc deliveries compared to non-SSc deliveries, and further studies are needed to determine what strategies can improve these outcomes.
Subject(s)
Hypertension , Scleroderma, Systemic , Pregnancy , Infant, Newborn , Humans , Female , United States/epidemiology , Pregnancy Outcome/epidemiology , Cesarean Section , Fetal Death , Hospitalization , Scleroderma, Systemic/complications , Scleroderma, Systemic/epidemiologyABSTRACT
OBJECTIVE: Despite efforts at early detection, patients with systemic sclerosis (SSc) pulmonary hypertension (PH) present with advanced disease. We sought to determine whether endothelial biomarkers (asymmetric dimethylarginine [ADMA], soluble endoglin [sEng], and pentraxin-3 [PTX-3]) can determine SSc-PH risk or differentiate between SSc-PH subgroups. METHODS: ADMA, sEng, and PTX-3 were measured by enzyme-linked immunosorbent assay in four groups: 1) 18 healthy controls, 2) 74 patients with SSc-PH, 3) 44 patients at high risk for PH features, and 4) 10 patients with low risk for PH features. High-risk features included a diffusion capacity (DLco) less than 55% with a forced vital capacity (FVC) greater than 70%, an FVC/DLco ratio of >1.6, or a right ventricular systolic pressure on an echocardiogram greater than or equal to 40 mm Hg. ADMA, sEng, and PTX-3 were compared between these four groups as well as stratified based on the three SSc-PH clinical classification groups (pulmonary arterial hypertension [PAH], left-heart disease, and interstitial lung disease [ILD]). RESULTS: PTX-3 was significantly lower in subjects with SSc at low risk for PH (median 27.0 pg/ml [interquartile range (IQR) 19.0-47.3]; P < 0.003) than the other groups. The area under the receiver operating characteristic curve was 0.87 (95% confidence interval 0.76-0.98, P = 0.0002) to differentiate low risk from high risk for patients with PH. PTX-3 was significantly lower in SSc-PH from disease of the left side of the heart (57.5 pg/ml [IQR 39.8-79.0]; P < 0.01) compared to SSc-PH from either PAH (85.5 pg/ml [IQR 56.3-104.5]) or ILD (90.3 pg/ml [IQR 74.9-111.0]). Neither ADMA nor sEng differed between the four groups. CONCLUSION: PTX-3 is a promising biomarker of PH risk status in patients with SSc as well as a possible marker of precapillary PH, which should be validated in an external cohort.
ABSTRACT
Mutations in over 80 identified genes can induce apoptosis in photoreceptors, resulting in blindness with a prevalence of 1 in 3,000 individuals. This broad genetic heterogeneity of disease impacting a wide range of photoreceptor functions renders the design of gene-specific therapies for photoreceptor degeneration impractical and necessitates the development of mutation-independent treatments to slow photoreceptor cell death. One promising strategy for photoreceptor neuroprotection is neurotrophin secretion from Müller cells, the primary retinal glia. Müller glia are excellent targets for secreting neurotrophins as they span the entire tissue, ensheath all neuronal populations, are numerous, and persist through retinal degeneration. We previously engineered an adeno-associated virus (AAV) variant (ShH10) capable of efficient and selective glial cell transduction through intravitreal injection. ShH10-mediated glial-derived neurotrophic factor (GDNF) secretion from glia, generates high GDNF levels in treated retinas, leading to sustained functional rescue for over 5 months. This GDNF secretion from glia following intravitreal vector administration is a safe and effective means to slow the progression of retinal degeneration in a rat model of retinitis pigmentosa (RP) and shows significant promise as a gene therapy to treat human retinal degenerations. These findings also demonstrate for the first time that glia-mediated secretion of neurotrophins is a promising treatment that may be applicable to other neurodegenerative conditions.
Subject(s)
Dependovirus/genetics , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Retinitis Pigmentosa/therapy , Animals , Apoptosis , Disease Models, Animal , Genetic Engineering , Genetic Therapy/methods , Genetic Vectors , Glial Cell Line-Derived Neurotrophic Factor/analysis , Glial Cell Line-Derived Neurotrophic Factor/genetics , Mutation , Neuroglia/metabolism , Photoreceptor Cells, Vertebrate/pathology , Rats , Retina/metabolism , Retinitis Pigmentosa/physiopathologyABSTRACT
Inherited retinal degeneration results from many different mutations in either photoreceptor-specific or nonphotoreceptor-specific genes. However, nearly all mutations lead to a common blinding phenotype that initiates with rod cell death, followed by loss of cones. In most retinal degenerations, other retinal neuron cell types survive for long periods after blindness from photoreceptor loss. One strategy to restore light responsiveness to a retina rendered blind by photoreceptor degeneration is to express light-regulated ion channels or transporters in surviving retinal neurons. Recent experiments in rodents have restored light-sensitivity by expressing melanopsin or microbial opsins either broadly throughout the retina or selectively in the inner segments of surviving cones or in bipolar cells. Here, we present an approach whereby a genetically and chemically engineered light-gated ionotropic glutamate receptor (LiGluR) is expressed selectively in retinal ganglion cells (RGCs), the longest-surviving cells in retinal blinding diseases. When expressed in the RGCs of a well-established model of retinal degeneration, the rd1 mouse, LiGluR restores light sensitivity to the RGCs, reinstates light responsiveness to the primary visual cortex, and restores both the pupillary reflex and a natural light-avoidance behavior.
Subject(s)
Blindness/therapy , Receptors, Glutamate/metabolism , Animals , Blindness/genetics , Dependovirus/genetics , Electroretinography , Light , Mice , Mice, Inbred C57BL , Receptors, Glutamate/genetics , Retinal Ganglion Cells/metabolism , Visual Cortex/metabolism , Visual Cortex/radiation effectsABSTRACT
OBJECTIVE: To investigate whether obstetric complications prior to systemic sclerosis (SSc) diagnosis are more common in SSc patients compared to the general obstetric population. METHODS: A case-control study was performed at Kaiser Permanente Northern California to compare prior obstetric complications in adult women who later developed SSc (cases) with women from the general obstetric population who did not develop SSc (controls; matched 10:1 by age and year of delivery) from 2007 to 2016. Exposures included past hypertensive disorders of pregnancy (preeclampsia, eclampsia, gestational hypertension), premature rupture of membranes (PROM), intrauterine growth restriction (IUGR), maternal infections, neonatal intensive care unit (NICU) admission, and preterm birth. Fischer's exact tests were used to compare categorical variables. Conditional logistic regression models estimated the odds ratio (OR), and corresponding 95% confidence intervals (95% CIs) for the outcome SSc. RESULTS: Seventeen SSc cases and 170 non-SSc controls were identified, with median maternal age at delivery 34 years (range 23-46 years) and median time from delivery to SSc diagnosis 2 years (range 0.2-7.3 years). Women with SSc were more likely to be Hispanic and Black. Prior obstetric complications appeared higher in women with an eventual SSc diagnosis compared to controls (70.6% versus 50%), including hypertensive disorders (17.7% versus 9.4%), PROM (11.8% versus 4.1%), IUGR (5.9% versus 1.8%), maternal infection (29.4% versus 14.1%), NICU admissions (23.5% versus 7.7%), and preterm delivery (29.4% versus 21.8%). Women with SSc had a higher odds of delivering infants requiring NICU admission (OR 4.7 [95% CI 1.2-18.8]). CONCLUSION: Women who eventually develop SSc had trends toward more complicated pregnancy histories before overt diagnosis.
Subject(s)
Pregnancy Complications , Premature Birth , Scleroderma, Systemic , Adult , Case-Control Studies , Female , Fetal Growth Retardation/diagnosis , Fetal Growth Retardation/epidemiology , Fetal Growth Retardation/etiology , Humans , Infant , Infant, Newborn , Middle Aged , Pregnancy , Pregnancy Complications/diagnosis , Pregnancy Complications/epidemiology , Pregnancy Complications/etiology , Pregnancy Outcome/epidemiology , Premature Birth/epidemiology , Premature Birth/etiology , Retrospective Studies , Scleroderma, Systemic/complications , Scleroderma, Systemic/diagnosis , Scleroderma, Systemic/epidemiology , Young AdultABSTRACT
BACKGROUND: Pulmonary arterial hypertension (PAH) affects approximately 10% of patients with systemic sclerosis (SSc) and is a leading cause of death. We sought to identify serum cytokine signatures that risk stratify SSc patients for this potentially fatal complication. METHODS: Subjects at high risk for PAH and with incident PAH based on right heart catheterization (RHC) were enrolled in the multi-center prospective registry, Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma (PHAROS). Low-risk SSc patients were enrolled at Stanford and had normal pulmonary function test and echocardiogram parameters. Serum was available from 71 high-risk patients, 81 incident PAH patients, 10 low-risk patients, and 20 healthy controls (HC). Custom 14- and 65-plex arrays were used for cytokine analysis. Cytokine expression was compared between patient groups by principal component analysis and Tukey's test result. A multiple hypotheses corrected p value <0.05 was considered significant. RESULTS: Exploratory analysis using principal components showed unique clustering for each patient group. There was a significant difference in cytokine expression in at least one group comparison for every cytokine. Overall, there was very little difference in cytokine expression comparing high-risk and PAH patient groups; however, these groups had substantially different cytokine profiles compared to low-risk patients and HC. CONCLUSION: These data suggest that cytokine profiles can distinguish SSc patients who are at high-risk for or have PAH from SSc patients who may be at lower risk for PAH and HC. However, high-risk and PAH patients had very similar cytokine profiles, suggesting that these patients are on a disease continuum.
Subject(s)
Hypertension, Pulmonary , Pulmonary Arterial Hypertension , Scleroderma, Systemic , Cytokines , Familial Primary Pulmonary Hypertension/complications , Humans , Hypertension, Pulmonary/etiologyABSTRACT
The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.
Subject(s)
Behavior, Animal/physiology , Lighting/methods , Neurons/physiology , Receptors, Kainic Acid/physiology , Action Potentials/physiology , Action Potentials/radiation effects , Animals , Animals, Genetically Modified , Animals, Newborn , Behavior, Animal/radiation effects , Cells, Cultured , Cysteine/genetics , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Excitatory Postsynaptic Potentials , Hippocampus/cytology , Larva , Leucine/genetics , Mutation , Neurons/drug effects , Neurons/radiation effects , Patch-Clamp Techniques/methods , Physical Stimulation/methods , Rats , Receptors, Kainic Acid/genetics , Transfection/methods , Zebrafish , GluK2 Kainate ReceptorABSTRACT
Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy.
Subject(s)
Cell Membrane/metabolism , Dependovirus/genetics , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Retina/metabolism , Vitreous Body/metabolism , Animals , Cell Membrane Permeability , Electroretinography , Fluorescein Angiography , Green Fluorescent Proteins/metabolism , Pronase/metabolism , Rats , Rats, Sprague-Dawley , Retina/cytology , Retina/virology , Transduction, Genetic , Vitreous Body/virologyABSTRACT
BACKGROUND: Pulmonary arterial hypertension (PAH) is a leading cause of death in patients with systemic sclerosis (SSc). The purpose of this study was to assess long-term outcomes in patients with SSc-PAH. METHODS: Pulmonary Hypertension Assessment and Recognition of Outcomes in Scleroderma is a prospective registry of patients with SSc at high risk for or with incident pulmonary hypertension from right heart catheterization. Incident World Health Organization group I PAH patients were analyzed. Kaplan-Meier survival curves were generated for the overall cohort and those who died of PAH. Multivariate Cox regression models identified predictors of mortality. RESULTS: Survival in 160 patients with incident SSc-PAH at 1, 3, 5, and 8 years was 95%, 75%, 63%, and 49%, respectively. PAH accounted for 52% of all deaths. When restricted to deaths from PAH, respective survival rates were 97%, 83%, 76%, and 76%, with 93% of PAH-related deaths occurring within 4 years of diagnosis. Men (hazard ratio [HR], 3.11; 95% CI, 1.38-6.98), diffuse disease (HR, 2.12; 95% CI, 1.13-3.93), systolic pulmonary artery pressure (PAP) on ECG (HR, 1.06 95% CI, 1.01-1.11), mean PAP on right heart catheterization (HR, 1.03; 95% CI, 1.001-1.07), 6-min walk distance (HR, 0.92; 95% CI, 0.86-0.99), and diffusing capacity for carbon monoxide (HR, 0.65; 95% CI, 0.46-0.92) significantly affected survival on multivariate analysis. CONCLUSIONS: Overall survival in PHAROS was higher than other SSc-PAH cohorts. PAH accounted for more than one-half of deaths and primarily within the first few years after PAH diagnosis. Optimization of treatment for those at greatest risk of early PAH-related death is crucial.
Subject(s)
Hypertension, Pulmonary/etiology , Scleroderma, Systemic/complications , Epidemiologic Methods , Female , Forced Expiratory Volume/physiology , Humans , Hypertension, Pulmonary/mortality , Male , Middle Aged , Prognosis , Scleroderma, Systemic/mortality , United States/epidemiology , Vital Capacity/physiologyABSTRACT
OBJECTIVE: The idiopathic inflammatory myopathies dermatomyositis (DM) and polymyositis (PM) are autoimmune diseases that can affect females of childbearing potential. We assessed pregnancy outcomes in DM and PM patients compared with the general obstetric population. METHODS: The Nationwide Inpatient Sample (NIS) (1993-2007) was used to identify delivery-associated hospitalizations in women with DM or PM (DM/PM, n = 853). Controls were from the general obstetric population delivery-associated hospitalizations matched to each case by year of delivery. Pregnancy outcomes included hospital length of stay (LOS), hypertensive disorders (HTN), premature rupture of membranes (PROM), intrauterine growth restriction (IUGR), and cesarean delivery. Multivariate regression analyses were performed using maternal age, race/ethnicity, and diabetes mellitus as covariates. RESULTS: On multivariate analysis, patients with DM/PM had longer LOS compared to controls (p < 0.001). DM/PM was associated with an increased risk of hypertensive disorders compared to controls (OR = 2.90, 95% CI: 2.00-4.22). There were no differences in rates of PROM, IUGR, or cesarean section in patients with DM/PM compared with controls. Independent of a DM/PM diagnosis, African-American race, older age, and diagnosis of diabetes increased the hospital LOS (p < 0.001). African-American race and diabetes increased the risk of hypertensive disorders (OR = 1.38, 95% CI: 1.19-1.60; OR = 2.94, 95% CI: 2.04-4.23, respectively) compared to controls. CONCLUSION: These data suggest that patients with inflammatory myopathies are at increased risk of hypertensive disorders of pregnancy and longer length of hospitalization. Vigilant monitoring of blood pressure is advisable in pregnant patients with DM or PM.
Subject(s)
Dermatomyositis/complications , Hypertension, Pregnancy-Induced , Pregnancy Complications , Adult , Delivery, Obstetric , Female , Hospitalization , Humans , Maternal Age , Pregnancy , Pregnancy Outcome , Risk FactorsABSTRACT
OBJECTIVE: Whole-exome sequencing (WES) studies in systemic sclerosis (SSc) patients of European American (EA) ancestry have identified variants in the ATP8B4 gene and enrichment of variants in genes in the extracellular matrix (ECM)-related pathway that increase SSc susceptibility. This study was undertaken to evaluate the association of the ATP8B4 gene and the ECM-related pathway with SSc in a cohort of African American (AA) patients. METHODS: SSc patients of AA ancestry were enrolled from 23 academic centers across the US under the Genome Research in African American Scleroderma Patients consortium. Unrelated AA individuals without serologic evidence of autoimmunity who were enrolled in the Howard University Family Study were used as unaffected controls. Functional variants in genes reported in the 2 WES studies in EA patients with SSc were selected for gene association testing using the optimized sequence kernel association test (SKAT-O) and pathway analysis by Ingenuity Pathway Analysis in 379 patients and 411 controls. RESULTS: Principal components analysis demonstrated that the patients and controls had similar ancestral backgrounds, with roughly equal proportions of mean European admixture. Using SKAT-O, we examined the association of individual genes that were previously reported in EA patients and none remained significant, including ATP8B4 (P = 0.98). However, we confirmed the previously reported association of the ECM-related pathway with enrichment of variants within the COL13A1, COL18A1, COL22A1, COL4A3, COL4A4, COL5A2, PROK1, and SERPINE1 genes (corrected P = 1.95 × 10-4 ). CONCLUSION: In the largest genetic study in AA patients with SSc to date, our findings corroborate the role of functional variants that aggregate in a fibrotic pathway and increase SSc susceptibility.
Subject(s)
Black or African American/genetics , Gene Regulatory Networks/genetics , Genetic Predisposition to Disease/ethnology , Scleroderma, Systemic/ethnology , Scleroderma, Systemic/genetics , Adenosine Triphosphatases/genetics , Adult , Extracellular Matrix Proteins/genetics , Female , Genetic Variation , Humans , Male , Middle Aged , Principal Component Analysis , White People/genetics , Exome SequencingABSTRACT
PURPOSE: Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans. METHODS: In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters. RESULTS: AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-ß-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters--human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)--AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis. CONCLUSIONS: The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.
Subject(s)
Dependovirus/genetics , Genetic Vectors , Green Fluorescent Proteins/genetics , Microglia/metabolism , Retinal Ganglion Cells/metabolism , Transduction, Genetic , Actins/genetics , Animals , Axons/metabolism , Connexins/genetics , Gene Expression , Genes, Reporter , Intravitreal Injections , Macaca , Microscopy, Confocal , Synapsins/genetics , Gap Junction delta-2 ProteinABSTRACT
Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state.