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To determine safety and efficacy of subretinal gene therapy in the RPE65 form of Leber congenital amaurosis using recombinant adeno-associated virus 2 (rAAV2) carrying human RPE65 gene.
Open-label, dose-escalation Phase I study of 15 patients (11-30 years) evaluated after subretinal injection of rAAV2-hRPE65 to the worse-functioning eye. Five cohorts represented four dose levels and two different injection strategies.
Primary outcomes were systemic and ocular safety. Secondary outcomes assayed visual function with dark-adapted full-field sensitivity testing and ETDRS visual acuity. Further assays included immune responses to the vector, static visual fields, pupillometry, mobility performance and OCT.
No systemic toxicity was detected; ocular adverse events were related to surgery. Visual function improved in all patients to different degrees; improvements were localized to treated areas. Cone and rod sensitivities increased significantly in study eyes but not control eyes. Minor acuity improvements were recorded in many study and control eyes. Major acuity improvements occurred in study eyes with the lowest entry acuities and parafoveal fixation loci treated with subretinal injections. Other patients with better foveal structure lost retinal thickness and acuity after subfoveal injections.
RPE65-LCA gene therapy is sufficiently safe and substantially efficacious to the extrafoveal retina. There is no benefit and some risk in treating the fovea. No evidence of age-dependent effects was found. Our results point to specific treatment strategies for subsequent phases.
Gene therapy for inherited retinal disease has the potential to become a future part of clinical practice.
Treatments for previously incurable hereditary retinal degenerations are emerging (1). A gene-based therapy is currently being evaluated in clinical trials for the autosomal recessive retinal disease Leber congenital amaurosis (LCA) resulting from RPE65 (retinal pigment epithelium-specific-65-kDa) deficiency. This disorder interrupts the visual-retinoid cycle (2,3); visual pigment is not available to photoreceptors through this key pathway; and vision is severely compromised. In a canine model of RPE65 deficiency, subretinal delivery of a gene-viral vector agent led to a remarkable activation of retinal and postretinal function (4,5). Mice with RPE65 deficiency showed the same dramatic treatment effect (6-11). Following further proof-of-concept studies, dose-response data, and toxicity testing (12-14), human gene therapy in RPE65-LCA seemed to be the next worthy step and clinical trials began.
Early results of three contemporaneous human clinical trials were reported in 2008 and these preliminary results showed safety and modest efficacy after subretinal injections of AAV2-RPE65 (15-18); more recently, a fourth trial was initiated and early results published (19). Questions about the longevity of the safety and efficacy in gene therapy for RPE65-LCA have started to be addressed (20-23). Beyond the initial increases in visual sensitivity post-treatment, we detected a slow and progressive movement of fixation over many months from the anatomical fovea to the treated retinal region. The region of therapy had become a preferred locus for use in this eye under certain conditions, suggesting cortical adaptations to the restored vision (24).
We have now performed gene vector administration in five cohorts of patients, representing children and adults, in a dose-escalation study using a single subretinal injection in the first three cohorts and two injections in the same eye at the time of surgery in the last two cohorts. Safety and efficacy results from our initially-reported cohort (17,18) now represent a 3-year interval since treatment. These results of relatively long-term follow-up taken together with those from patients with shorter-term follow-up lead to a perspective on how best to advance this trial and other retinal gene therapy clinical trials.
The clinical trial was performed at Scheie Eye Institute of the University of Pennsylvania (UP) and at University of Florida/Shands (UF). The subjects had a clinical diagnosis of LCA. RPE65 mutations were determined by the John and Marcia Carver Nonprofit Genetic Testing Laboratory (University of Iowa). Study eligibility and protocol; regulatory approvals and oversight; cGMP vector production, purification and titering; and surgical procedure for vector administration have been reported in earlier studies of Cohort 1 (17,18,20). Trial conduct was in a manner consistent with the ICH—E6 Good Clinical Practice guideline document, and was reviewed by the United States Food and Drug Administration (Investigational New Drug application BB-IND 12824) and the National Institutes of Health Recombinant DNA Advisory Committee (Protocol #0410-677). Approvals were obtained from the Institutional Review Boards (IRBs) and Institutional Biosafety Committees of UP and UF, the Vice Provost Research Review Committee of UP, the Western Institutional Review Board and the General Clinical Research Center of UF. A Data and Safety Monitoring Committee, appointed by the National Institutes of Health, monitored the trial. The tenets of the Declaration of Helsinki were followed. Informed consent or assent was obtained from all subjects. Brief summaries of the methods are given below and expanded methods are in the eSupplement.
The eye with worse visual function was chosen for vector administration in all subjects except P10 (Table 1). Both eyes of P10 were severely affected but the eye with worse function had keratoconus and the contralateral eye was chosen for the procedure. Two methods of anesthesia were used. For the first two cohorts (n=6, ages 20-30 years), the procedure was performed with retrobulbar anesthesia. For the subsequent three cohorts (n=9) that included 6 patients under 18 years of age, general anesthesia was used. A standard 3-port 23-gauge vitrectomy was performed in Cohorts 1-3 as previously described (17); in Cohorts 4 and 5, a 25-gauge vitrectomy was performed. The vector was introduced into the subretinal space with a 39 gauge injection cannula (Synergetics Inc., O'Fallon, MO). A single injection was used in the first three cohorts while two injection sites were used in the last two cohorts (Table 1).
Ocular safety was assessed with standard eye examinations. To quantify severity of inflammatory response, standard grading systems were used (25-27). To document fundus appearance, fundus photographs (using an infrared camera to avoid excess visible light exposure) were taken at baseline and at post-treatment visits. Systemic safety was evaluated with physical examinations at baseline and postoperative visits. Routine hematology, serum chemistry, prothrombin time (with INR), partial thromboplastin time, and urinalysis were performed at baseline and postoperatively. The schedule of study visits and list of measured parameters for each timepoint from baselines to 3 years post-operative have been published (17). In all safety and efficacy studies, the examiners were not masked to which eye was the study eye.
Anti-AAV2 antibody titers. Serum samples from the patients were assayed for circulating antibodies to AAV2 capsid proteins at baseline, at days 14 and 90, and at years 1, 2 and 3. Details of the assay were previously described (17). Antigen-specific response. Anti-AAV2 antigen-specific lymphocyte proliferation responses were assessed as previously described (17). AAV DNA in peripheral blood. Procedures for biodistribution of patient samples in this trial were described (17). Ex vivo and cultured Interferon-enzyme-linked immunospot assays (ELISpot). Details of blood collection and the IFN-γ ELISA were described for Cohort 1 patients (17). Further details are provided for all immunology parameters (eMethods).
Best-corrected visual acuity (VA) was measured using ETDRS (Early Treatment Diabetic Retinopathy Study) methodology (28) at all baseline and postoperative visits (17,20). BCVA was scored as the number of letters correctly read after adjusting for distance and expressed as logMAR. The fixation locus of each eye at each visit was determined by video imaging the retina under invisible near-infrared (NIR) light (MP1, Nidek Incorporated, Fremont, CA) while the subject was gazing at a target (18,20,29). Further details are provided (eMethods).
Full-field stimulus testing (FST) was performed using a LED-based ganzfeld stimulator (Colordome, Diagnosys LLC, Littleton, MA) as previously described (5,17,30,31). Briefly, blue and red stimuli were used for testing monocularly under two dark adapted conditions: standard dark adaptation of <2 hours and extended dark adaptation of >3 hours. The latter condition draws from our previous observations of prolonged kinetics of rod but not cone dark adaptation in RPE65-related LCA treated with gene therapy (18). Further details are provided (eMethods).
Dark-adapted static visual field testing with computerized perimetry was able to be performed in 12 of 15 patients; three patients could not distinguish where in the visual field a light stimulus originated. A modified automated perimeter (Humphrey Field Analyzer; Zeiss Meditec Inc, Dublin, CA) was used (18,20,32,33). To summarize the data, maps were constructed of loci showing significant sensitivity changes postoperatively. Further details are provided (eMethods).
The direct transient pupillary light reflex (TPLR) was elicited and recorded as previously described (5,18,34). Luminance-response functions were derived from TPLR amplitude to increasing intensities (from −6.6 to 2.3 log10 scot-cd.m−2) of green stimuli with short duration (0.1 s) presented monocularly in the dark-adapted state. TPLR luminance response functions were recorded during the short-term postoperative timepoints at 1 (P2, P3, P13, P14, and P15), 3 (P1, P4-P10, P12) or 6 (P11) months. Further details are provided (eMethods).
A mobility performance task was used to quantify the ability of the patients to move through an indoor obstacle course and determine if there was any difference in this behavior before and after treatment (n=5) or between treated and untreated eyes postoperatively (n=15). The task is a version of published methods (35-40) and further details are in eMethods.
Differences between baseline and postoperative values of efficacy parameters (FST blue, FST red, TPLR, and VA) were evaluated for control and study eyes with paired, two-sided t-tests. Results from multiple visits within baseline and postoperative timepoints were averaged before performing the t-tests. Repeated measures analyses of variance (ANOVA) were performed with a test of interaction comparing the magnitudes of differences between treated and control eyes. Repeated measures factors included were eyes (study versus control), and visits (baseline versus postoperative). Between subjects factors included in the ANOVA were age (younger, 11-20 years or older, 21-30 years), for the analyses of all parameters, and fixation (foveal versus extrafoveal) for the analysis of VA. All group statistics are specified as mean (SE).
Cross-sectional imaging using optical coherence tomography (OCT) was used to assess retinal structure before and after administration of the agent in the treated eye; comparable data were also acquired in the untreated eye. Ultra-high speed and high resolution OCT imaging with a spectral-domain (SD) OCT instrument (RTVue-100, Optovue Inc., Fremont, CA) was used, as described (17,20). Foveal thickness measurements were performed as described and statistical comparisons made between data from different visits (17,41).
There were 15 patients (8 female and 7 male subjects) in the trial (Table 1). Other than a sibling pair (P9, P12), the patients were unrelated. P10 and P13 had the same mutant alleles (homozygous for R91W) but were not known to be related. Ninety-day and 1-year post-injection data from the three patients in Cohort 1 have been published (17,18,20,24). The first three cohorts received single subretinal injections (Figure 1). Cohorts 1 and 2 were young adult patients (ages 20-30 years) and dose-escalation occurred by doubling the initial injection volume of 150 μl of vector (Cohort 1) to 300 μl (Cohort 2). Cohort 3 patients were <18 years of age and we were advised by a UP regulatory body to reduce dosage in this first cohort of children, hence the 225 μl volume for these 3 patients. Cohorts 4 and 5 had two injections of 225 μl each (total 450 μl; Figure 1), first in young adults (ages 24 and 27 years) and then in ≤18 year-old patients. P15 had two injections but less total volume was injected. Infrared views of all 15 study eyes are shown with superimposed locations of the injection site and estimated boundaries of retinal detachments caused by the subretinal injections. The treated eyes are all portrayed as left eyes for comparison (Figure 1); the actual eye treated is tabulated (Table 1). The postoperative course was similar in 13 of 15 patients with absorption of the subretinal fluid within 48 hours and no evidence of intraocular inflammation. P7 and P11 were exceptions with a second retinal detachment and choroidal effusions, respectively (see below). By 30-60 days postoperatively, all eyes were quiet and have remained so. Systemic safety parameters including physical examinations and blood (hematology, serum chemistry, coagulation) and urine testing showed no clinically significant abnormalities after gene transfer in all patients.
Among the postoperative adverse events were retinal detachment, choroidal effusions, ocular hypotension in the immediate postoperative period and ocular hypertension associated with the administration of topical steroids. P7, Cohort 3, had a retinal detachment in the region of the single subretinal injection, one day after it was deemed flat by ophthalmoscopy. This was surgically repaired and there has not been further complication. P11, Cohort 4, was detected to have choroidal effusions on the 3rd day after surgery. The choroidals were treated with topical cycloplegics and increased topical steroids. The lack of resolution of the choroidals at day 30 led to a 3-week course of systemic steroids. By 149 days postoperatively, the choroidals had resolved by clinical examination and ultrasound. There was no measured ocular hypotension from the first measurement (postoperative day 3) through day 238. This patient, however, developed increased intraocular pressure which was detected on day 149 and presumed to be secondary to the extended use of topical steroids. Cessation of steroids followed and topical beta blockers were added. After a period of quiescence, the choroidals re-appeared on day 190 and were treated with further cycloplegics and a short course of topical steroids. There was resolution by day 238. Ocular hypotension was documented in four patients (P4, P9, P12, P15) during the early postoperative period (days 2-5; intraocular pressures were ≤6 mm Hg with an interocular difference of 5-10 mm Hg). By postoperative day 7, pressure in the study eyes increased and interocular asymmetry decreased in all patients. Ocular hypertension was also documented after topical steroid use in the postoperative period in P8, P12 and P15. Cessation of steroids and treatment with topical beta blockers led to the eyes becoming normotensive.
Humoral immune responses were monitored by measuring levels of circulating antibody to AAV2 capsid at baseline and at postoperative days 14, 90, 270 and years 1, 2 and 3, depending on the treatment cohort (Table 2). All patients exhibited titers at baseline and at all post-treatment timepoints well below the normal population mean of 2,148,715 mU/ml (n=99 random samples) except P12 with a titer of 2,789,606 mU/ml at day 90, which is ~60% above this patient's baseline value. Ten of the 14 patients with at least day 14 data showed no increase in antibody titer greater than two-fold from baseline;, and most experienced a decline. Of the remaining four, P2 exhibited a four-fold increase at day 14, and returned to below baseline at day 90 and years 1 and 3. P5 experienced a 3-fold increase at day 14, returned to baseline at day 90 and year 1, but then spiked again at year 2. This pattern of episodic antibody spikes over multiple years with a return to baseline is not consistent with a humoral immune response to a one-time vector administration, and is more likely a result of periodic re-exposure to wild type AAV2 through natural viral infections. P6 experienced a 7-fold titer increase at day 14 that peaked at 11-fold at day 60, and has subsequently declined to just over 2-fold at year 2. P8 also showed an increase in titer at day 14 (3-fold) that peaked at day 90 (4-fold) and is diminishing at year 1 (2-fold over baseline). P6 and P8 are circumstantially the only patients with serum antibody titer behaviors potentially consistent with a response to the vector, but coincidental exposure to wild type AAV2 either directly or by reactivation of latent AAV2 through natural Adenoviral or Herpes viral infection cannot be ruled out. Overall, the patterns of patient serum antibody titers to AAV2 over time post-treatment suggest limited or no systemic immune response to subretinal AAV2 vector delivery.
AAV2 capsid antigen-specific reactivity of peripheral lymphocytes (ASR) was monitored at baseline and at post-treatment days 14 and 90 and years 1, 2 and 3 (eTable 1). Only P1 and P2 of the patients with ASR data exhibited a significant increase in stimulation index (SI) at any timepoint (minimal level of significance for SI ranges from 2 to 3). For both patients, the change in SI was only marginally significant and only a small increase from baseline: P1 at year 1 had a SI of 2.02, a small increase from the baseline value of 1.62, and P2 at day 90 showed a SI of 2.10, also a small change from 1.89 at baseline. We conclude that the AAV2 capsid antigen-specific lymphocyte proliferation response to a single subretinal AAV2 treatment elicits neither a consistent nor pronounced AAV2 antigen-specific immune response.
The T cell immune response to AAV2 capsid was monitored by IFN-γ ELISPOT assays. PBMCs from subjects at baseline and at days 14 and 90 and years 1, 2 and 3 after treatment, depending on the patient, were stimulated with AAV2 peptide library pools and assayed for IFN-γ secretion. With one exception, there were no positive responses to AAV2 peptide pools at any of the timepoints tested in the subjects thus far (eTable 2). P9 exhibited a response to peptide pool 2C at day 90, but neither before nor after. This is inconsistent with T cell response to AAV2 vector, and a clear reason for this modest and transient increase at day 90 is not apparent. The study of AAV2-specfic memory T cells measured by the cultured ELISpot showed that some patients had pre-existing AAV2-specific T cells at baseline (Patients 2, 6, 9, 11-14; eTable 3). Most of the patients that were positive at baseline remained positive by the cultured ELISpot and most of the patients that were negative remained negative. Only P5 and P7, who were negative at baseline, became positive post-treatment by cultured ELISpot but not by ex-vivo ELISpot. Although pre-existing AAV2-specific memory T cells were found in some patients, AAV2 administration to the eye did not expand these resting cells as demonstrated by the negative data obtained by the ex vivo ELISpot. The data suggest that even in the presence of peripheral AAV2-specfic memory T cells, AAV2 administration to the eye is not sufficient to activate them.
Biodistribution of vector in the peripheral blood was monitored at baseline and at days 1, 3 and 14 post-treatment by quantitative PCR with spike-in assays to control for PCR inhibition in specific samples. For all patients at all timepoints, there were no vector genome copies detectable, thus confirming the lack of escape of subretinally administered AAV2 vector into the circulation.
Visual function was assessed before and after treatment using FST, TPLR, dark-adapted static visual field testing, mobility performance and ETDRS visual acuity. We did not assume that the vector used in each patient was bioactive but tested it. At the end of each surgery, unused residual vector was injected subretinally into rd12 (Rpe65-deficient) mice and an ERG bioassay performed to quantify vector activity (18,42,43). In all patients, the residual vector was proven active by this method (eFigure 1).
Visual function was measured psychophysically using FST in dark-adapted eyes with blue and red flashes (Figure 2A, eFigure 2, Table 3). At baseline, the mean (SE) FST sensitivity with blue flashes in all RPE65-LCA eyes (study and control) was 1.17(0.087) log10, a value that was substantially reduced compared to 6.61 (0.10) log10 in normal eyes. At baseline, there were no significant differences (p=0.16) between control and study eyes of trial patients. Intervisit test-retest variability of the FST measure at baseline in trial patients was similar to that published previously (30,31). FST sensitivity to red flashes in all RPE65-LCA eyes at baseline was 0.72 log10 (0.11), which is greatly reduced compared to the normal value of 4.37 (0.08) log10 measured under dark-adapted conditions, or the normal value of 2.48 (0.09) log10 measured at the cone plateau (eFigure 2, Table 3). Chromatic differences defined the photoreceptor type mediating FST responses. In all patients, red FST flashes were detected by cones whereas blue FST flashes could be detected by rods (P1, P3, P5, P7, P8, P9, P10), or cones (P11, P12, P15), or rods and cones (P2, P4, P6, P13, P14) at baseline (eFigure 2).
In the postoperative period, FST sensitivities to blue flashes showed highly significant differences compared to baseline in study eyes but not in control eyes (Figure 2A, Table 3, p<0.001, repeated measures ANOVA test of interaction). The postoperative improvement in study eyes was 1.59 (0.25) log10. Chromatic differences supported mediation of blue FST flashes by rods postoperatively in all study eyes except for P6 (eFigure 2).
FST sensitivities to red flashes showed highly significant differences compared to baseline in study eyes but not in control eyes (p=0.008, repeated measures ANOVA test of interaction). The postoperative improvement in study eyes was 0.45 (0.10) log10 (Table 3). Chromatic differences supported mediation of red FST flashes by cones postoperatively in all study eyes (eFigure 2). The ages of the clinical trial participants did not have a significant effect on blue FST (P=.25, overall; P=.53, magnitude) or on red FST (P=.10, overall; P=.63, magnitude).
The transmission of information from retina to the brainstem was quantified objectively with the TPLR (Figure 2C,D). Measurements were made under fully dark-adapted conditions and luminance-response functions were available in 27 of 30 eyes. For P10 at baseline, the control eye showed a sub-criterion contraction whereas the study eye showed no contraction to maximal stimulation; his sensitivities were assigned to the reciprocal of the maximum stimulus luminance for statistical purposes. For the control eye of P13, TPLR was not recorded due to time constraints. At baseline, RPE65-LCA eyes required on average 5.6 log10 unit higher luminance of a full-field green flash in order to produce a criterion pupillary contraction compared to normal eyes (RPE65-LCA= −0.88 (0.15) log10 versus normal= 4.74 (0.064) log10). The magnitude of this defect was similar to the 5.5 log10 difference observed with blue FST between RPE65-LCA and normal eyes. At baseline, there were no significant differences (p=0.81) between the control and study eyes of trial patients (Figure 2C).
In the postoperative period (1-6 months), TPLR sensitivities showed highly significant differences compared to baseline in study eyes but not in control eyes (Figure 2C, Table 3, p<0.001, repeated measures ANOVA test of interaction). The magnitude of the postoperative TPLR sensitivity improvement in study eyes was 1.17 (0.20) log10 (Figure 2D, Table 3) which corresponded to an intermediate value between the blue and red FST improvements observed. The ages of the clinical trial participants did not have a significant effect on the TPLR (P=.67, overall; P=.56, magnitude of the treatment effect).
The significantly increased light sensitivity postoperatively in treated eyes using FST and TPLR prompted us to ask whether we could localize the increases in the visual field and how any localization of function was related to the sites of subretinal injection (Figure 3). Visual field maps of sensitivity change from baseline in the study eyes showed good correspondence between the loci where there were significantly increased responses to stimuli and the estimated region where the retinal detachment with subretinal injection of agent occurred. Patients in Cohorts 4 and 5, who had a second subretinal injection site in the nasal retina, showed loci with significant responding in the temporal visual field. In summary, 11 of the 12 patients with visual field maps showed correspondence between most detected loci and the area of injection; these include P1-P4, P7-P9, P11, P12, P14 and P15. It is of interest that some of the detected loci in the patients, however, were outside the estimated injection area. The basis for this effect remains uncertain. It is to be noted that most of these unexpectedly responsive loci were in the far periphery. The results of P6 indicated response at a single peripheral locus but no evidence of a response in the subretinal injection area. This temporal inferior peripheral field locus was consistently detected and it is likely to be the source of the FST and TPLR responses in this patient (Figure 2), who described this location of perception and its appearance postoperatively.
We also asked whether these localized changes from baseline had any impact on the ability of the subjects to negotiate an obstacle course. In 5 patients, representing Cohorts 4 and 5, we performed the study both before and after treatment (Figure 4A). A comparison of mobility performance for study eyes relative to postoperative values (first row) indicates overall a better performance after treatment for ambient illuminations between 0.2 to 4 lux. For the 100 lux illumination, however, patients were able to navigate the course practically without errors both at baseline and postoperatively and regardless of which eye was used. For the control eyes (second row) there were less pronounced differences in performance for the lower illumination levels, which suggests a learning effect. We also determined if the difference in performance between eyes (interocular difference, IOD) changed after treatment (third row). The results in P11 and P13 indicate greater IOD after treatment, with better performance of the treated eye relative to the control eye, at the lower illumination levels. P12 performed better with the treated eye only at the lowest illumination level; P10, P14 and P15 did not show notable effects.
All study participants were assessed by the difference in performance between eyes, averaged across all post-treatment visits (Figure 4B). Results indicate a consistently lower number of incidents while navigating with the treated eye, with varying degrees of performance gain across participants. The mobility performance IOD results for all participants are summarized (Figure 4C). Mean differences between study and control eyes were significantly different from zero for the four lower illumination levels indicating that patients as a group navigated more efficiently when using their treated eyes under such conditions.
At baseline, VA in control eyes was 0.96 (SE, 0.13) logMAR (corresponding to a mean Snellen acuity of 20/182; range 20/39 to worse than 20/2000) as compared to study eyes of 1.09 (0.11) logMAR (=20/246; range 20/43 to 20/1824) (Table 1, Table 3). Postoperatively, the mean VA increased to 0.91 (0.13) logMAR in control eyes and to 0.97 (0.11) logMAR in study eyes, and both changes were significant (Table 3). In contrast to other psychophysical and to pupillometric measurements, repeated measures ANOVA showed no indication (p=0.16) of a difference in the magnitude of the postoperative acuity change between study and control eyes of −0.12 (0.05) and −0.05 (0.02) logMAR, respectively. Furthermore, in the great majority (28 of 30) of the eyes, mean postoperative acuity change did not reach or surpass the 0.30 logMAR (3-line) halving of visual angle limit which is a commonly accepted criterion for clinical significance (44). It was noted that the second baseline VA was better than the first baseline measurement in 10 study eyes (range: −0.18 logMAR better to 0.08 logMAR worse) and in 9 control eyes (range: −0.10 logMAR better to 0.10 logMAR worse). Regression to the mean and learning curve effects could have potentially contributed to this tendency. Thus, an alternative analysis was performed using only the second baseline VA. This led to a decrease in the logMAR improvement postoperatively in both the study eyes (mean (SE) = −0.09 (0.05), p=0.099) and the control eyes (mean (SE) = −0.04 (0.02), p=0.077).
In order to understand better any possible VA changes resulting from gene therapy, fixation properties were analyzed in each eye at each visit. At baseline, in 12 of 15 study eyes mean fixation location corresponded to the anatomical foveal depression (Figure 5A) and not unexpectedly these eyes had the highest VA (Table 1). Instability of fixation (including high frequency nystagmus and lower frequency wandering eye movements) around the mean was 1.97 (0.21) degrees and correlated (r2=0.42) inversely with VA as previously published in this patient population (29). Control eyes showed less fixation instability with a mean of 1.54 (0.14) degrees (data not shown) consistent with the better acuities recorded; the difference was borderline significant (p=0.052). Postoperatively, fixation remained foveal in both study and control eyes in this subset of 12 patients (the study eye of P2 developed a secondary fixation locus which was detectable with dimmer stimuli, reference 24), and there were no significant changes to fixation instability (Figure 5B).
Three study eyes (P4, P5, and P10) with the worst baseline acuities (Table 1) fixated parafoveally at mean eccentricities of 2.1, 5.1 and 3.4 degrees, respectively, from the anatomical foveal depression (Figure 5D). Their fixation instability was 2.3, 3.7, and 2.2 degrees, respectively. Control eyes of these three patients also fixated eccentrically; mean locus of fixation was 2.0 and 2.3 degrees eccentric for P4 and P5, respectively, whereas it could not be quantified in P10. Postoperatively, fixation remained extrafoveal for all three patients (Figure 5D).
Next, VA changes were considered in the context of the type of fixation. There was a larger VA improvement in patients with extrafoveal fixation (−0.30 (0.14) logMAR, Figure 5E) compared to those with foveal fixation (−0.09 (0.04) logMAR, Figure 5C). A repeated measures ANOVA demonstrated a statistically significant difference (p=0.036, test of interaction) between the magnitude of postoperative VA improvement in the patients with the two types of fixation. In subgroup analyses, acuity change in foveal-fixating study eyes, or extrafoveal-fixating study and control eyes did not achieve statistical significance (p=0.07, 0.17, 0.56, respectively); counterintuitively, foveal-fixating control eyes showed a significant (p=0.02) improvement of VA. Of note were the large VA improvements (P4=−0.29 and P10=−0.55 logMAR) in two of the extrafoveal-fixating eyes where the central retina including the fixation was involved in the subretinal injection, as compared to the lack of any such large improvement in the extrafoveal-fixating eye without a central detachment (P5=−0.06 logMAR), or in two foveal-fixating eyes with a foveal detachment (P1=0.20, P13=−0.11 logMAR). Until there are physiologically-based hypotheses for improvements in untreated control eyes or in eyes without foveal detachment, parsimony would suggest that small VA improvements in foveal-fixating eyes may have been influenced by the high expectations and motivation in patients taking part in an open-label study as well as a possible learning effect (45). Clinically significant unilateral VA improvements in extrafoveal-fixating eyes, on the other hand, appear consistent with independent data (18,24) showing improvements in extrafoveal cone sensitivity following gene therapy.
Retinal laminar architecture across the fovea was quantified in all patients. Foveal thickness in both eyes of the 15 patients pre- and post-operatively are summarized (Figure 6A). For control eyes, changes from baseline are within published intervisit variability for a retinal degeneration population (17,41). For study eyes, there are two notable examples of foveal thinning in the short-term: P1 and P13. Long-term follow-up in P1 showed that foveal thinning was still present. In both P1 and P13, the fovea was detached in the subretinal injection (Figure 1). P4, another patient with foveal detachment showed less pronounced but still significant thinning long-term but not short-term. P4's control eye also showed long-term thinning but not to the degree as in the study eye. As a counter example, P5 who did not have foveal detachment showed similar results as P4. The other patients with a foveal detachment, P6 and P10, showed no such effects. All other study eyes without foveal detachment did not have thinning within the time period studied.
Representative horizontal OCT cross-sections and longitudinal reflectivity profiles through the fovea (highlighted and labeled for outer retinal laminae and with histograms of layer thickness) are shown for four study eyes at baseline and at early and later postoperative times (Figure 6B,C). P7, a patient without foveal detachment, shows no remarkable changes at 30 days and 18 months postoperatively. P1, P13 and P6 had foveal detachments and all showed disturbance of the IS/OS laminar architecture at 30 days postoperatively but recovery at later visits. Unlike P6, however, P1 and P13 also showed loss of ONL at 30 days and later timepoints. How do these structural findings relate to visual acuity? The only patient with clinically significant loss of visual acuity was P1 at last visit (Figure 5) and this patient had the most prominent foveal abnormalities by OCT.
This five-cohort 15-patient RPE65-LCA retinal gene therapy clinical trial can be summarized as follows: there were no detectable systemic safety concerns; certain ocular adverse events occurred and these were attributable to the surgical procedure (46); and improved visual function (by FST) was present in all patients to different degrees. Like the other concurrent early phase RPE65-LCA clinical trials (15,16,21,22), our trial of relatively small numbers of patients has had limitations. Study design did not involve randomization of eyes and there was an inherent imbalance between eyes in variables related to outcome since study eyes by definition had worse vision. The statistical approach was thus confounded by such issues. Examiners were not masked to the study eye versus control eye (study eyes showed residual conjunctival injection for weeks postoperatively). Later phase trials in this disease and in other rare genetic retinal degenerations may be able to confront these issues. Despite limitations of the ongoing RPE65-LCA clinical trials, the fact remains that the longstanding concept that genetic retinal degenerations are incurable and vision cannot be improved has been revised. The advantages of beginning the era of treating rare inherited retinal diseases with RPE65-LCA are discussed in the eComment.
Some key questions to consider in planning future phases of gene therapy clinical trials in RPE65-LCA are as follows: Where in the retina should we treat and where should we not treat? Is there sufficient evidence for "age-dependent effects" (21) of this gene therapy to recruit younger and younger RPE65-LCA patients in future cohorts? Is cone-based vision improved by the currently used vectors?
All 15 patients in this trial and a further 15 patients reported by two other trials (15,21) received subretinal injections to one eye. Where have the injections been delivered? Sixteen of 30 (53%) procedures detached the macula and fovea. All but two of these 16 patients were reported (15) to have some measure of efficacy from the treatment. The motivation to include the fovea in these macular injections would be two-fold: 1) to determine if there was any safety issue such as a deleterious effect to the foveal cones, which are known to be abnormally reduced at all ages in RPE65-LCA (29); and 2) to ask whether this treatment affects the visual cycle at the fovea and leads to efficacy such as improved visual acuity, which is reduced in nearly all of the patients (8,29 33).
Beginning with safety, two of our 5 patients with foveal inclusion in the macular detachment (P1,P13) showed foveal thinning at early postoperative timepoints; in P1, long-term data indicated that this process continued. A third patient (P4) had less dramatic long-term thinning of the fovea, and this degree of thinning also occurred in patients without foveal injections (P5) or in untreated eyes (P2). We postulate that the less dramatic long-term foveal thinning likely represents the natural history of foveal change in RPE65-LCA, albeit asymmetrical, whereas short-term effects are a complication of subfoveal injection. Also, in two of the patients with foveal detachments (P1,P6) and long-term follow-up, there was evidence that photoreceptor IS/OS structure was disrupted in the short-term postoperatively but did recover later. Experimental retinal detachment also shows that loss of OS is one of the earliest features to occur post-detachment but there is reversal with reattachment (47).
Of possible relevance, predictors of poor visual outcome in macula-off rhegmatogenous retinal detachment after reattachment surgery are persistent subretinal fluid and pre-operative increased thickness (48). There was no persistent subretinal fluid in this trial. P1 had the thickest fovea at baseline, compared to the four other patients with foveal detachments; P13 ranked third in thickness. We previously postulated that foveal thinning in P1 was due to proximity of the subretinal injection site (17) and there was similarity of this injection site to that in a patient who developed a macular hole in another RPE65-LCA trial (16). The site of injection in P13, however, was near the superior vessel arcade. Another explanation is that some patients with RPE65-LCA are vulnerable to surgical trauma of the fovea. Of the 5 patients with retinal detachments that included the fovea, the two patients with most foveal photoreceptors (foveal thickness) to lose were the ones that lost them – P1 and P13. The other 3 patients had more severe central disease with foveal atrophic lesions (P4, P10) or a thinned ONL layer at the fovea (P6). Other RPE65-LCA clinical trial reports have displayed but not measured OCTs in patients with foveal inclusion in the detachment (15,16,21).
We conclude that there is a risk to foveal cone cells in retinal detachments that include the fovea. Is there proven benefit, specifically increased visual acuity or slower natural history of foveal cone loss independent of functional gain? There is no consensus among the ongoing trials on this point. In the present study, group statistics for visual acuity indicated statistical improvement compared to baseline. The improvements in most eyes, however, were substantially less than the 0.3 logMAR (15 letter) limit generally accepted as clinically significant. Further, both study and control eyes showed improvement; and there was no statistically significant difference in the magnitude of improvement between treated study eyes and untreated control eyes (21,22). Two patients (P4, P10) showed an increase of >0.3 logMAR postoperatively. These two patients had the two lowest acuities at baseline in our study, had foveal inclusion in the detachments, and improved by maintaining extrafoveal fixation loci in the treated areas. Another trial reported no improvement in acuity (15) whereas group statistics in a third trial showed significant changes in study eyes (see Suppl. Table 5 in reference 21); individual results suggest improvements in many control eyes but group statistics were not provided (21,22). Using the 0.3 logMAR criterion as a clinically significant change in visual acuity, there was improvement in 4 of 8 patients with macular injections that included the fovea in this third trial (16,21,22); and 2 of 12 untreated control eyes also showed large improvements in acuity (16,21,22). Fixation was not reported. Complicating the risk-benefit issue for this trial is the report of a decrease in acuity in a 10-year-old subject's treated eye (21).
Taking a conservative, patient safety-first approach, the lack of consensus in visual acuity results within and between the contemporaneous trials and our evidence of foveal thickness loss lead to the recommendation that the fovea should not be included in macular subretinal injections except when there is foveal atrophy (by OCT) or very reduced visual acuity and fixation is parafoveal (readily detectable on OCT). This approach also opens the door to treatment of patients at later disease stages when there is foveal atrophy but preserved extrafoveal ONL, best exemplified by P4 and P10 in the current study.
Our two-site injection protocol in Cohorts 4 and 5 was safe in adults and children to date. This is a positive step in the direction of a subretinal injection protocol with the goal of increasing even further the visual area affected by vector. Most of our single injections in this study were directed at the superior retina in order to subserve inferior visual field function. When two injections were used, a nasal retinal injection was added to enhance temporal peripheral visual function that can be detected in untreated RPE65-LCA patients even at relatively late stages (33). We propose that a next step in previously-untreated patients would be to administer the agent to three sites of injection: 1) superior retina involving the macula but not the fovea unless there is foveal atrophy and extrafoveal fixation; 2) nasal-superior retina; and 3) temporal retina. Ideally, a preoperative map of remaining ONL would help guide the injections (8,49) or at least a pre-operative visual field (using sufficiently bright stimuli) would help to determine where residual vision is detectable (33).
To date, we have elected to use the eye with worse vision as the study eye. In some cases, this non-preferred eye has been strabismic and possibly amblyopic. This strategy was motivated by safety, but has also been convenient for patients who in the postoperative period can continue to be visually active despite a protective occluder worn on the operated eye. For the initial 5 cohorts in this safety trial, that decision was appropriate. Some patients, however, have continued to use their preferred (control) eye after treatment and are only aware of visual gain in the treated eye when asked to occlude the control eye for our studies. The three-injection protocol proposed above would ideally be used in the preferred eye. Factoring in the remarkable visual improvements in our Cohort 1 who received 150 ul at a single site and the lack of toxicity to date of two-site injections totaling 450 ul, we recommend three injections of 150 ul each. A single eye treated with such a protocol will test the safety and efficacy of this approach and leave the contralateral eye for further advances in the field (50).
The RPE65-LCA phenotype includes not only a severe visual dysfunction but also a progressive retinal degeneration (8) and the relationship between severity of retinal degeneration and age can be complex. On an individual patient basis, there is certainly age dependence of severity of disease; visual field extent decreases with age when followed longitudinally over more than a decade (33). But this longitudinal progression in individual patients does not simply translate to cross-sectional studies across different patients at different ages, and this is especially true in the first three decades of life (33). Our studies of visual function and photoreceptor topography have indicated that severity of dysfunction and degeneration can be as profound in some young patients in the first decade of life as in some patients in the third decade of life (33,49). Given comparable numbers of photoreceptors remaining in the retina (49) and placement of the vector injection(s) in the region(s) of these photoreceptors, there should be an equal chance of efficacy of therapy and this would be independent of age (assuming RPE health is similar). Did we find an age dependent effect of gene therapy in our study, as reported in another trial (21)? For FST, TPLR and VA, age did not have a statistically significant effect either overall or on the magnitude of the treatment effect in our trial. As previously discussed, the most dramatic visual acuity increases were in two of our older patients (ages 24 and 30 at time of procedure) and this was consistent with no significant relationship between age and the visual acuity outcome reported in another trial (21). So, what is the basis of the previously reported conclusion of age dependence of gene therapy outcomes (21)? It seems to be a matter of emphasis. For example, the improved light sensitivity of an 8-year-old patient (CH08; Fig 2C, reference 21) is emphasized but there is equal improvement in light sensitivity in a 35-year old patient in the same trial (CH13; Fig 2C, reference 21). Mobility performance of younger patients is also emphasized, but these patients are studied uniocularly and with lower room illuminations whereas most older patients are only studied binocularly at 250 lux light levels (see reference 21, Supplementary Table 6), a condition we found does not reveal any differences in performance before and after treatment or between eyes. Considerable heterogeneity of disease severity in RPE65-LCA is a fact and when determining candidacy for this therapy there should be evaluation on an individual basis, independent of age. The extent of retinal disease in human RPE65-LCA at different ages has not been as predictable as, for example, a murine model (51). The temptation should be resisted to assume there is a common natural history among humans without measuring it. With greater understanding of the human disease, a better evidence-based formula may emerge for candidacy, in which age is one of several parameters.
There is consensus from all current trials that some measures of visual function improve in response to gene therapy in RPE65-LCA patients (15-24). Evidence of rods and extrafoveal cones responding to the vector-gene has been presented in our previous work (18,20) and this is confirmed and extended in the present work to larger numbers of patients. Importantly, foveal cones have not been proven conclusively to show either increased vision from the current vector-gene treatments or protection from further cell loss. It is thus possible that there is a difference in targeting efficiency of foveal versus extrafoveal RPE in RPE65-LCA with the current vectors, or there is localized toxicity (directly or indirectly) to foveal cones. Foveal and extrafoveal cones, for example, do not have the same relationship with RPE apical processes (52). Alternatively or additionally, contributions of chromophore required from retinal and RPE visual cycle pathways may also differ between foveal and extrafoveal cones (2,3,53). There is also evidence of RPE65 localization in cones (54,55) suggesting a pathway that may need to be subserved by vectors capable of expressing RPE65 not only in the RPE but also in cones. In this context, using a promoter in the vector that does not limit expression to just the RPE may turn out to be beneficial.
We thank the members of the Data and Safety Monitoring Committee: Maryann Redford, DDS, MPH (Program Director, Collaborative Clinical Research, National Eye Institute, National Institutes of Health); Stephen Gange, PhD (Chair); Karl Csaky, MD, PhD; Herbert Goldstein, PhD, PA; Donald Hood, PhD; Ruth Macklin, PhD; Jerry Mendell, MD; Daniel Salomon, MD; and Donald Zack, MD, PhD. The DSMC approved this manuscript. We are grateful for the expertise and all the kindness shown to our patients by members of the UF CTSI (GCRC) during the early postoperative period.
Funding: The clinical trial was supported by the National Eye Institute (National Institutes of Health, Department of Health and Human Services) grant U10 EY017280. SGJ had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Author Disclosure Statement B.J.B., W.W.H. and the University of Florida have a financial interest in the use of AAV therapies, and own equity in a company (AGTC Inc.) that might, in the future, commercialize some aspects of this work. University of Pennsylvania, University of Florida and Cornell University hold a patent on the described gene therapy technology (United States Patent 20070077228, "Method for Treating or Retarding the Development of Blindness").
Trial Registration: clinicaltrials.gov Identifier: NCT00481546.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Well, now the Toronto Blue Jays have gone and done it. Mentioned the kid's name again, on Thursday night. Talked about how they'd like to see Brett Lawrie in the majors in August, before the rosters expand.
Then Jose Bautista gets hurt. Twists his right ankle on a slide at third on an inning-ending double play and, well, perhaps we should put an embargo on mentioning Lawrie until he's actually here, no?
Lawrie returned to active duty Thursday, playing third base in an injury rehabilitation assignment at Single-A Dunedin, for the first time since sustaining a non-displaced fracture on the back of his left hand on May 31. That injury occurred just hours after manager John Farrell and general manager Alex Anthopoulos sent out signals that the Langley, B.C., native was on the verge of being called up. Bautista, who made the All-Star Game as a right fielder, was forced to move to third to keep the spot warm for Lawrie. Now he's day to day, after much pregame conjecture about Lawrie's ETA. Detect a trend?
It is understandable, this fixation with Lawrie. A home-grown star? A player who can ensure Edwin Encarnacion's glove never again sees the light of a major league day? We're all about that! Yet while Anthopoulos shares Farrell's belief that there is a value to having a young player evaluated during the dog days of August instead of simply waiting for the September call-up period – "You definitely get a better evaluation in August than September," he said – he also has started the process of managing expectations.
"Any young player that we call up, I expect them to be optioned down some time," Anthopoulos said as the Blue Jays took batting practice before the first game of a four-game series against the New York Yankees. "It's not that I want them to. It's just that reality dictates it. People have talked about Brett Lawrie coming up here and staying, but look at the numbers. Look at this field right now. Almost everybody's been optioned: Romero, Snider, Morrow – he's been optioned. Eric Thames has been optioned. Position players, you're likely going to burn one option. Starters? Three, maybe four options."
On several levels, it will be a shame if Bautista's injury is serious or turns into one of those petulant, nagging aches. Because proximity to a healthy Bautista could not hurt Lawrie's development. According to the Elias Sports Bureau, Bautista is the first American League player in the expansion era to lead the first half of a season in home runs, walks, slugging and on-base percentage. Bautista is the embodiment of the vagaries of the game – from living on the waiver wire for much of his career to hobnobbing with Justin Timberlake at awards shows … to, yes, hobbling off the field 24 hours later.
There's a great deal waiting for Lawrie up here. Case in point? Saturday's Yankees starter, CC Sabathia, has held Bautista hitless in 15 career at-bats, with seven strikeouts. Sabathia carved up Bautista with sliders and change-ups in a 5-4 Yankees win on May 24. It took just 11 pitches to hang an 0-for-4 on Bautista, who also stranded five base runners.
Farrell thinks the slight hitch in Sabathia's delivery messes with hitters' timing. "That [the hitch]was something Dick Pole introduced to me when he was my pitching coach in Cleveland," Sabathia said. "It's not really for deception. It was just something to give me more time to gather myself, to help me with my balance."
Sabathia, Bautista said, doesn't have any "pronounced tendencies like some guys." If he did? Trust us: Bautista would find them.
"Look, I develop a game plan and try to put it to work," Bautista said. "If it works, it works. If it doesn't, I don't lose any sleep over it … because it works most of the time. I don't change my approach because somebody's had success against me or because I've had success against them."
Sop there you go, Brett. Can't wait for you to get here. Stay healthy, Godspeed – and bring a notebook.Report Typo/Error
Follow us on Twitter: @Globe_Sports

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Great hotel and service. The staff at this hotel will go out of their way to deliver service that exceeds your expectations. Would definitely stay again, again and again. The night before my departure, my husband called to ask if he could get something special (chocolate) sent to my room with a note. The staff member stated he would take care of and don't worry about it. My husband asked how can I pay you and he responded "no problem" for he will take care of it. He ended up buying a bag of M&M's (my favorite) that made my night so special.
Wow, what customer service. The conversation and action by the team at the hotel has made it to a life long impression that I will to tell everybody how wonderful the team at the Dallas Forth Worth Holiday Inn was.
- Official Description (provided by the hotel):
- Dallas - Fort Worth Airport Hotel Offers Ideal Location Welcome to the Holiday Inn Dallas - Fort Worth Airport. Lodging at our Fort Worth hotel puts you close to many area businesses and attractions. In addition, we are just a mile from the Dallas - Fort Worth International Airport (DFW). Vacationing families enjoy staying at our DFW hotel because of its close proximity to area attractions in Fort Worth. Our hotel is within just a few miles of famous Six Flags Over Texas, offering thrills for all ages. Other nearby attractions include the Dallas Cowboys' Stadium, Texas Motor Speedway, the Kennedy 6th Floor Museum, Grapevine Mills Mall, Lone Star Horse Racing Park and the C.R. Smith Aviation Museum. Business travelers enjoy staying with us because of our location near the Arlington Convention Center. Our Fort Worth Airport hotel is also close to corporations and businesses that include American Airlines, Regent Aero Space, GM, Chase Bank, Pfizer and State Farm. Whether for an overnight business stay or a week long family vacation, you can enjoy our many amenities to make you feel comfortable. For your convenience, our complimentary hotel shuttle travels to the Dallas - Fort Worth International Airport and within four miles of our accommodations. Other amenities include an outdoor pool, high-speed Internet access, an on-site restaurant, a convenience store and fitness center. Reserve your hotel room in Fort Worth, TX today. ... more less
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text:
I don't own pride and prejudice or anything to do with it.
Summery: Lady Catherine always wanted Darcy to Marry Lady Anne right keep the money in the family, What if instead he married another cousin. Lady Elizabeth Fitzwilliam the Colonel Fitzwilliams younger sister. Charles Bingley has just rented an estate in Hertfordshire and everyone is headed their for the year. What happens when they chance upon Miss Jane Bennet. Pairings. Jane/Bingley Darcy/Elizabeth Fitzwilliam/Georgiana and Charlotte/Mr. Collins. Yes there are two cousin's pairings if you don't approve please don't read. Please remember the time period this would be quite normal.
"Now Charles what could possess you to make us go all the way to Hertfordshire couldn't you find a place closer to Pemberley?" she pouted and Darcy had to chuckle at the look on his cousin's face. Lady Elizabeth Fitzwilliam was a playful women she loved to tease all of her friends and acquaintances.
"Well now Lady Elizabeth I could have always went to Kent, I heard there was a place for rent in close distance to Rosings. His blue eyes were alight with mirth.
Elizabeth shivered at the very thought. "Now Mr. Bingley don't tease my dear Cousin," Georgiana chided playfully.
"Yes Charles don't be ridiculous who'd ever want to be around that women," Caroline Bingley snapped her fan shut. Georgiana and Elizabeth locked eyes and tried to hide their smiles at the irony of that statement. Colonel Fitzwilliam wasn't as successful and let out a small chuckle trying to cover it with a cough.
"Now dear brother would you like some water," Elizabeth teased getting ready to grab the chilled container that contained the water for their journey.
"No sister dear I'm quite fine thank you," he sat back his lips still quirked slightly.
Darcy hide his own mirth behind the book he'd been pursuing on the long journey.
"Charles how much longer until we get to this new place you decided upon," Caroline practically wined much to the amusement of the other occupants of the carriage.
"Caroline we'll be there within the hour," Charles sighed and repressed the urge to roll his eyes at his sister, ever the impatient one.
"So Georgie what do you think we'll be able to do in this place?" Elizabeth asked her favorite cousin well besides William but no one could know of the fervor she carried for her elder cousin.
"Well I'm hoping they'll have some quaint little shops I do need some new sheet music and a new ribbon or two wouldn't be remiss. She fingered her green shot silk dress.
"Yes, I'm looking for a new shawl and maybe some fabric for a new dress maybe they have a dress maker," Elizabeth got excited.
"Oh I wouldn't let Madame Lovin hear you say that she'd be scandalized and she's the best seamstress in London." Georgiana giggled.
"Well you guys can have your country seamstress I'll stick with Madame Lovin thank you very much." Caroline stuck her noes up at the conversation.
"we didn't invite you anyway," Elizabeth muttered under her breath so low that Caroline couldn't hear but Darcy who had been sitting beside her heard and wasn't sure whether to laugh or scold her. But out loud she said, "That's fine Miss. Bingley we can do just fine on our own."
This stopped Caroline in her tracks she'd just been called her Miss Bingley in an informal setting this wasn't good Elizabeth was a lady the daughter of an Earl it wouldn't do to upset her.
"Well I'm sure you can, Lady Elizabeth," Caroline bowed her head slightly.
Colonel Fitzwilliam leaned over slightly and whispered into his sisters ear, "Elizabeth stop intimidating Miss. Bingley," Elizabeth looked over at her brother bashfully but she could also hear the laughter in his tone.
"I mean Charles why couldn't we go to Ramsgate like a normal person, "Caroline lamented. Everyone except for the women that spoke the question stiffened at the mention of the seaside town.
"Caroline its to crowded now be quite," Charles snapped looking over at Georgiana who had stiffened herself. Colonel Fitzwilliam had been sitting beside her and grabbed her hand gently. Caroline seemed oblivious to the turmoil her question had caused with in the small carriage. Just as she was about to answer back the carriage came to a stop and they all looked out at the place they'd call home for a while Netherfield.
A/N please review but don't flame.


text:
In addition to complimentary wireless Internet access, this hotel provides complimentary parking, a lobby with 24-hour reception and check-out service, and coffee in the lobby. Furthermore, a newspaper stand, breakfast room and room service are provided.Check-in time is 1 pm. Check-out time is 12 pm. Check-in age is 21 years or older.The hotel is renovated with all new rooms featuring contemporary furniture, new en suite bathrooms with granite countertops, a comfortable easy chair and more. All rooms offer free wireless Internet access, a shower, hairdryer, direct dial telephone, satellite/cable TV and a radio. The rooms are also equipped with tea and coffee making facilities, an ironing set and individually regulated air conditioning and heating. Selected rooms offer a microwave and mini fridge unit. Rooms with either double or king-size beds are available.
From Downtown Louisville: Travel east on I-64 to exit 15-C. Turn left at the Olive Garden. Turn left at the stop sign and then right onto Blairwood Road. The hotel is located at the end of the road on the left side.


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text: Anderson Cooper Posts Childhood Photo On The 29th Anniversary Of His Brother's Death July 25, 2017 • 5:05 PM ET The journalist will never forget his brother. Read Blog Rob Lowe's Sons Are Not Only Adorable Like Their Famous Dad, But Also Really Brainy by Tamara Palmer July 25, 2017 • 4:02 PM ET You may have noticed that Rob Lowe is currently promoting The Lowe Files, his forthcoming adventure reality series where he uncovers spooky legends and unsolved mysteries with his dashing sons Matthew and John Owen. Rob Lowe and... Read more Yolanda Hadid Is Baring Her Bottom and Not Afraid to Show It by Jocelyn Vena July 25, 2017 • 4:02 PM ET These days, Yolanda Hadid is looking fiercer than ever. And that just might explain why The Real Housewives of Beverly Hills alum is baring her bum on Instagram. Of course, she paired the cheeky moment with an update on her life and health. She... Read more Erika Jayne Clearly Knows How to Party and These Photos Prove It by Jocelyn Vena July 25, 2017 • 3:02 PM ET Whether she's out and about as Erika Girardi or Erika Jayne, it's clear from scrolling through The Real Housewives of Beverly Hills singer's Instagram feed that she really knows how to raise a glass and paint the town a sizzling shade of red. We... Read more Ciara's Three-Year-Old Son is a Model July 25, 2017 • 2:31 PM ET This little nugget is capitalizing on his cuteness. Read Blog Mercedes "MJ" Javid Is Going to Handle Her Future Pregnancy Differently Than Asa Soltan Rahmati by Laura Rosenfeld July 25, 2017 • 2:16 PM ET Mercedes "MJ" Javid and Asa Soltan Rahmati's relationship was strained at the beginning of this season of Shahs of Sunset , and some major personal news hasn't really done much to smooth things over between the two. Yes, it seems as though Asa's... Read more Mary-Kate and Ashley Olsen Always Seem to Pop Up in the Most Unexpected Places by Jocelyn Vena July 25, 2017 • 1:49 PM ET There they are! Just when you thought appearances by Mary-Kate and Ashley Olsen were only reserved for weird photo opps with Nicki Minaj at the Met Gala, the twin moguls go and show up in an unexpected place. And by unexpected, we mean totally... Read more Load More


text: Zipper Making Machines Showing 1 - 7 of Total 7 Manufacturers/ Suppliers All Pack Machinery Send Trade Enquiry BUSINESS PROFILE Nature of Business: Manufacturers And ExportersProducts Offered: Manufacturers and suppliers of packaging machine like multitrack form fill and seal machine, zipper and standy pouch making machine, flex tube machinery, hologram machinery, sealing and cutting machine, wrapper machine etc. CONTACT DETAILS Street Address: Plot No. 4, Malenrna Road, Near Bala Ji Public School, Ballabgarh City: Faridabad State: Haryana PIN: 121 004 Country: India Phone: +(91)-(129)-3242330 Send Trade Enquiry Now Atics Industrial Corporation Send Trade Enquiry BUSINESS PROFILE Nature of Business: Manufacturers And ExportersProducts Offered: Wholesale manufacturer and exporter of zipper making machines. CONTACT DETAILS Street Address: 12th Floor, 62 Fuhsing S. Road, Section 1 City: Taipei State: PIN: 106 Country: Taiwan Phone: +(886)-(2)-27817077/27518506 Fax: +(886)-(2)-27713447/27810765 Send Trade Enquiry Now Wenli Kinitting Machinery Trading Co. Send Trade Enquiry BUSINESS PROFILE Nature of Business: ExportersProducts Offered: Exporters of zipper making machine. CONTACT DETAILS Street Address: No. 4-6, 10 Zone Zipper Profess Street City: Yiwu State: PIN: 322 000 Country: China Phone: +(86)-(579)-5551525/13906895564 Fax: +(86)-(579)-5551010 Send Trade Enquiry Now Shanghai Guangsu Plastic Machinery Manufacture Co. Limited Send Trade Enquiry BUSINESS PROFILE Nature of Business: ManufacturersProducts Offered: Manufacturer of all kinds of injection moulding machines, including thermoplastic machines, thermosetting machines, zipper machines, glasses-making machines, and related accessories. CONTACT DETAILS Street Address: No.888, Kungang Road, Songjiang Sci. And Tech. Park, City: Shanghai State: Shanghai PIN: 201 614 Country: China Phone: +(86)-(21)-57855656 Fax: +(86)-(21)-57850725 Send Trade Enquiry Now Kao Shing Zipper Co. Limited. (foshan Nanhai Chao Shing Zipper Co. Limited.) Send Trade Enquiry BUSINESS PROFILE Nature of Business: ExportersProducts Offered: Wholesale sellers of invisible zipper, shoes zipper, slider, zipper raw material and zipper making machine and hook. CONTACT DETAILS Street Address: 7-F-12, No. 77, Shih-jen N. 1st Road. City: Taipei State: Taichung Hsien PIN: Country: Taiwan Phone: +(886)-(4)-22516881) Fax: +(886)-(4)-22516883) Send Trade Enquiry Now Lung Meng Machinery Co. Limited Send Trade Enquiry CONTACT DETAILS Street Address: 20, Huan Kung Road, Yungkang Industrail Area City: Tainan Hsien State: PIN: Country: Taiwan Phone: +(886)-(6)-2321126 Fax: +(886)-(6)-2320776 Send Trade Enquiry Now Pham Srl Send Trade Enquiry CONTACT DETAILS Street Address: 6, V. Lombardia City: Carpi State: PIN: 41012 Country: Italy Phone: +(39)-(59)-642511 Fax: +(39)-(59)-642374 Send Trade Enquiry Now


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text: Politics 18 July 2005 Cooking up a rural fantasy Food for thought: Mrs Beeton Sign up for our weekly email * The frontispiece of Mrs Beeton's Book of Household Management of 1861 looks uncannily like the photographs on the walls of branches of Fresh 'n' Wild, which show the store's various artisanal and ethical suppliers in their natural habitats. In Mrs Beeton's version, an extended family group clusters around the door of a cottage at harvest time. The men are plump John Bulls. The principal female figure is serving them beer, probably brewed from her own grain. Ducks dabble, hens peck and cows drowse under a tree. The caption says the scene represents "The Free Fair Homes of England"; a time before industrialisation scarred the land, cutting off a generation of urban Britons from the source of their nourishment. You just know that Mrs Beeton would love to step in to that picture. Her book is saturated with a longing for an agrarian world that had already slipped into extinction but just might, by some enormous effort, be brought back. So, in her instructions for making a syllabub, she suggests mixing up some sugar and nutmeg and then simply squirting the milk from a cow's udder straight into the bowl. Animals destined for the table are described in their natural habitat with such lulling, lyrical grace that you feel you are watching them from a hot, summer meadow. Mrs Beeton describes how a sheep "indolently and luxuriously chews his cud with closed eyes and blissful satisfaction, only rising when his delicious repast is ended to proceed silently and without emotion to repeat the pleasing process of laying in more provender". Pigs, meanwhile, snuffle in well-kept sties while deer bound through the heather. Such soft-focus rural fantasy was only possible because Mrs Beeton was a sharp-edged daughter of the industrial age. Her guidelines for domestic bliss have less to do with the farmhouse than the factory. Briskly, she divides the working day into segments and allots each household member a set of tasks that read like a time-and-motion study. The labour is specialised, repetitive, often mechanised. Kitchen equipment is described like industrial plant. Despite her dewy-eyed gestures to the days when households produced their own butter, eggs, bread and wine, she recommends short-cuts such as commercially bottled sauces. As for baking, she is ambivalent about whether you should even bother to do it. The illustration to "General Observations on Bread, Biscuits and Cakes" may show a pyramid of rustic-looking loaves, yet a few pages later Mrs Beeton writes enthusiastically about a new system for mass-producing bread. None of this makes her rusticism phony, despite her vision lacking any mention of intensive farming, seasonal unemployment and poverty among the rural working class. What she shares with our own age is a niggling feeling that something about Britain's relationship with its own food supplies had gone badly wrong. But what that "something" was exactly, and whether it was too high a price to pay for convenience, safety and comfort, was something she hardly considered. Whirling not so much like a dervish, more a cog in an intricate machine, she pressed on, determined to finish her 1,112 pages in record time. "The Fair Free Homes of England" remained a lovely, compensating dream. Kathryn Hughes's biography of Mrs Beeton will be published in October by 4th Estate Subscribe from just £1 per issue More


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Kidzone – Kids learning center is a multi-page web design which suit for any type of school such as: learning center, kindergarten, primary, secondary, high school and universities.
The theme comes with bright and cute design that easy to grab attention from visitors. Not only about it design, this theme also built in with lots of advanced features to support users easily manage their WordPress site such as: Slider revolution, visual composer, SEO friendly, fully responsive, various home page, etc.
1.0.1 |30th Jan 2016|
- [FIX] Fix problem from WooCommerce 2.5.1


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by nancy
(santee, California)
Please Jesus send your angels, to help my 17yr.old granddaughter and her 1yr.baby. She has no guidance as her mom left her sister, her, and her dad. She hardly feeds her, does'nt dress her accordingly for the weather and is starting to neglect her all together. They sleep on the floor and don't have money, not really even for diapers. They are all hungry in the house. My son goes to work but it is'nt enough. I ride a bycicle and have emphasema and scoliosis. If I was'nt in pain all the time, I could get there more often. Please God help all of them before something bad happens to the baby. She does'nt listen to anyone, and their so sad, no ones thinking logically. In Jesus name we pray.
Join in and write your own page! It's easy to do. How? Simply click here to return to Catholic Prayer Request.
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text: strawberry-milk Play via YouTube OPEN IN APP NSFW Safe Is this playlist safe for work? comatose September 08, 2012 | 9 tracks | 1,489 94 | Stats Balam Acab Bon Iver Giraffage electronicchillwaveambient Liked Collect Share Edit Liked! Share this mix with friends: comatose Wrong video Collect Share Liked! Share this mix with friends:


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"However, more often than not, people fall into one or more traps – and the whole KM process is compromised. Here's what to avoid to ensure your knowledge management initiatives are successful.1. Lack of a clear ROI2. Resistance to change3. The irritating tendency to transform KM into IT projects4. Ruining KM with a bad tool5. Lack of Accessibility
|Scooped by Karen du Toit


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Chandra Turner
Founder & President
Chandra Turner (CHAN-druh) has an accomplished career as an editor at the nation's top women's magazines. She's currently the Executive Editor at Parents magazine, the country's leading parenting magazine with 12 million readers. Before Parents, she was the Executive Editor of CosmoGirl. Chandra has also been an editor at Ladies' Home Journal, Cosmopolitan, YM, and Glamour. She started her career as an ASME intern at American Baby and then as an editorial assistant at Good Housekeeping.
Chandra is the founder & president of Ed2010.com. She founded Ed2010 in 1998 when she was an assistant editor at Glamour. Ed2010 began as a group of a dozen friends who met periodically at bars in New York City and exchanged emails about job openings and industry gossip. The group grew and stayed connected via an email newsletter, then a geocities chat board, and now the lifestyle and jobs website it is today. Via Ed2010, Chandra speaks frequently to college students, recent graduates, and junior editors about how to get started in the industry and how to move up the editorial ladder.
Chandra is also the founder of Talent Fairy, a talent recruitment agency that places editors and writers in creative jobs in all areas of publishing/media. The service is free for all registered Ed2010 users.
You can reach Chandra at email@example.com.


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Articles about NoSQL
Introducing FaunaDB Serverless Cloud by 1 Posted on Jun 14, 2017
Introducing Reladomo - Enterprise Open Source Java ORM, Batteries Included! (Part 2) by Posted on Jun 13, 2017
Pascal Desmarets on NoSQL Data Modeling Best Practices by 1 Posted on May 01, 2017
Introducing Reladomo - Enterprise Open Source Java ORM, Batteries Included! by 5 Posted on Mar 28, 2017
Big Data Processing Using Apache Spark - Part 6: Graph Data Analytics with Spark GraphX by 2 Posted on Mar 14, 2017
Analysis and Mitigation of NoSQL Injections by Posted on Jan 18, 2017
Cassandra: The Definitive Guide, 2nd Edition Book Review and Interview by Posted on Jan 05, 2017
From Raw Data to Data Science: Adding Structure to Unstructured Data to Support Product Development by Posted on Nov 25, 2016
Chris Fregly on the PANCAKE STACK Workshop and Data Pipelines by Posted on Aug 29, 2016
Virtual Panel: Current State of NoSQL Databases by 2 Posted on Aug 02, 2016
|All articles
Presentations about NoSQL
|All presentations
News about NoSQLAmazon DynamoDB Accelerator (DAX) Released in Preview: In-Memory Cache for Read-Intensive Workloads by Posted on May 16, 2017 MongoDB Atlas Expands AWS Footprint by Posted on May 02, 2017 Emerging Technologies for the Enterprise Conference 2017: Day One Recap by Posted on Apr 24, 2017 Causal Consistency for Large Neo4j Clusters by Posted on Apr 15, 2017 FaunaDB: A New Distributed Database from the Team That Scaled Twitter by Posted on Mar 24, 2017 Amazon Adds Time to Live (TTL) Support to AWS DynamoDB by Posted on Mar 18, 2017 Lightbend Speaks to InfoQ on Their Acquisition of OpsClarity by Posted on Feb 24, 2017 Apache HBase 1.3 Ships with Multiple Performance Improvements by Posted on Jan 30, 2017 Kuzzle – An On-Premises Document Back-End by Posted on Jan 12, 2017 Neo4j 3.1 Supports Causal Clustering and Security Enhancements by Posted on Dec 31, 2016 Julien Le Dem on the Future of Column-Oriented Data Processing with Apache Arrow by 1 Posted on Dec 08, 2016
|All news
Featured Blog Post
New — Live Migrations, More AWS Regions, Queryable Backups & More in MongoDB Atlas
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Littleton Vacation Packages
From Littleton, Portland is 85 miles (137 kilometers) to the southeast, while Manchester is 90 miles (145 kilometers) to the south.
It comes as no surprise that Littleton is so popular with travelers from all over the world; the surrounding region is an Aladdin's Cave of captivating cultural and natural attractions. Roughly 17 miles (27 kilometers) away is Bretton Woods. Head here to visit the Omni Mount Washington and Mount Washington Cog Railway.
About 35 miles (56 kilometers) to the southeast of Littleton is North Conway, a regional center where you can explore Attitash, Conway Scenic Railway and North Conway Depot Railroad Museum. If you're still looking for things to do in the city centre, be sure to visit Cranmore Mountain Resort and Settlers' Green Outlet Village before you head back to Littleton.
There are some great exciting adventures to be had. Around Northern New Hampshire. Head out and go mountain biking or rock climbing to really get your blood pumping. If you are in the area during winter, have a go at some of the local wintersports including snow shoeing and skiing. Both of these are on offer in the Northern New Hampshire.
When it's time to say farewell to Littleton, head to the nearest major airport situated in Lebanon, 55 miles (89 kilometers) away.


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Each month, Rated People connects over 50,000 homeowners with quality tradesmen - simply enter your job details in the form above to get started.
We'll match your job to the relevant tradesmen in your area - those interested will pay to see your contact details and call you to quote.
As each tradesman expresses interest in quoting on your job, we'll send you their profile and ratings to help you choose the right one for you.
After the work is complete, return to the site and rate your tradesman based on their quality of work, reliability and value for money.
Added 14/01/2017
My front door currently has a BG lock. However, the door knob has broken and i might need to...
Searching for tradespeople
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To fit a push button lock, I need a price for two adjustable door closers
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I have a D lock on my bike, which I've lost the key for, and it needs cutting off. The bike...
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Simple job. I want my front door lock replaced because builders who were doing some work have...
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Change key locks since we've taken up residence. Also want some extra security such as a digital...
Went the extra mile to make good my old gate and made sure I was able to unlock without any difficulty. He will be boarding my loft in a week's time as well.
Managing your job is easier with the Rated People Homes App, download it today.


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Characters part 8
EditDr. Gero
The head Scientist of the now dead Red Ribbon Army, Dr. Gero survived the assault made by Gokou years ago when he was a kid and ever since then, he's held a psychotic grudge on him, vowing to someday get revenge and kill him. After being warned by Trunks about him, since the real timeline had Dr. Gero's Plan working, Gokou and the Z Senshi prepare for the Androids and end up foiling his plans in the end, but not after a long and grueling battle that did indeed end with Gokou dying. Although he's strong, he's nothing compared to the Z Senshi and his other Androids and even in the game he's not the best fighter, but depending on how you use him, he could be quite deadly.
His Renzoku Energy Dan costs only 6% of Ki to do and it homes in, plus his Ki Blast costs 16% of Ki to do, which are semi strong, but they aren't the best and they don't home in. Due to not being able to gather Ki, since he's a machine, he has to steal it from the opponent or absorb their attacks. In his later Levels, he can slowly gather Ki, but it's VERY slow. An average fighter over all, if you can use him well, he can be quite deadly.
Level 1 Attacks
- Normal Moves
- Grapple - B Button.
- Knock Away - A Button. (Note: If you use this on a Renzoku Energy Dan or Ki Blast your opponent fires at you, if you time it right, you can knock it back at them.)
- Ki Shield (Block.) - Hold A/B Button. (B Button for weak attacks, A Button for strong.)
- Power Absorbtion - Hold R Button. (If used while a Renzoku Energy Dan or Ki Blast is fired at you, when it hits, you'll absorb it and gain the Ki.)
- Renzoku Energy Dan/Rapid Fire Ki - Hold R Button + Tap B Button.
- Ki Blast - Hold R Button + Tap OR Hold B Button.
- Power Absorbtion - A + B Buttons. (Gives you 50% of Ki.)
- Hikou/Flying Rush - R Button + Hold D-Pad in the direction you want to go in.
- Dodge/Rush - Tap D-Pad in the direction you want to go twice.
Desperation Moves
- Energy Blast - 50-99% Ki, Left or Right of the opponent, R Button + A And B Buttons.
- Chou/Super Energy Blast - 100% Ki, Left or Right of the opponent, R Button + A And B Buttons.
- Eye Beams - 50-99% Ki, Above the opponent, R Button + A And B Buttons.
- Shougekiha/Invisible Ki Wave - 50% Ki, Below the opponent, R Button + A And B Buttons.
Level 2 Attacks
- Normal Moves
- Same as Level 1.
Desperation Moves
- Same as Level 1.
Level 3 Attacks
- Normal Moves
- Same as Level 1.
Desperation Moves
- Same as Level 1.
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Pop open a cold one and relax in the man cave with this sophisticated wall-mounted beer bottle opener.
This cool beer bottle opener is made of quality wood and comes with cap catcher at the bottom for the convenience.
Mounting screws not included.
Not for outdoor use.
Out of Stock
Warning: Last items in stock!


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Student evaluation module - In Development
The Student evaluation module (SEM) is a flexible evaluation tool for teachers to make yearly (or shorter period) evaluation report of the student. The SEM works as a tool for communicating the status of a student to the parents and the student itself.
The teacher (or admin) can build a template for the report out of different building blocks/ components. These can be blocks providing data from assessments made in itslearning, data from national goals and simple empty text fields.
The teacher will then edit and publish the report that holds the automated content. Parents and student will be able to comment on the report.
In Denmark this report is build up around the national goals and used as basis for a yearly talk to the parents of the student.
The SEM can be used for other kinds of evaluation reports since it is flexible in the manner the template is build out of components.


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It used to be the hot state of affairs to clothes genitor and youngster like. This day and age, however, the minor man robed is four-legged and furry. The little contemporaries is having smaller amount family and gratifying their genitor desires with soft pooches. So, here are a bulky figure of nation looking to infect their dogs and themselves by dishing out the intermixture on interior decorator dog wearing apparel. Just suchlike human clothing, dog garb designs get their incentive on runways say the international and front masses conflicting styles and colors to be the in point this period of time.
The ingenious flag and designs on models in Milan, Paris and New York have found their way into dog get-up boutiques in circles the world. Designers have only a short and sweet juncture after the quality vogue shows to cultivate their new lines to go on with what the parents will be exhausting. "The dog journal of clothing essential not lone clash quality styles, it essential besides be structural for the dog" same Onika Carroll, a engineer for Elizabeth Austin. All of these challenges metallic element to a versatile listings of dog uniform travel from the attractive and crude to the haute dressmaking.
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Kevin Hale is a partner at Y Combinator, which provides seed funding to startups. Their companies have a combined valuation of over $30B. Among their successful investments are Dropbox, Stripe, 9GAG, Code Academy, AIRBNB, Reddit and Homejoy. He works with their portfolio of companies on user experience design, product development and customer support. In 2006, he co-founded Wufoo, which was one of the earliest Y Combinator funded companies. Wufoo is a software company in Palo Alto, California.
"You gotta say no a lot to get to a billion" - Kevin Hale
Kevin shared his insights on how to pitch your own company. Start by saying in a clear and precise way what you do and not why you do it! A good idea can always be explained in one single sentence and is understandable for everyone.
To continue, he states that a successful startup is based on a team which is enthusiastic and smart. Taking this into consideration, the business model does not matter on first glance because nobody can predict how a market will develop in the future.
In order to participate in the Y Combinator program you need to convince the investors that you are both, clear in your vision but also shapeable to become a better entrepreneur. Therefore, young founders tend to have higher chances to be pitched since they are rather open for new concepts. In a nutshell, pitching is an art that combines soft skills and full devotion for your project.