Quality-of-life scores improve after 96 weeks of PEG-IFNa-2a treatment of hepatitis D: An analysis of the HIDIT-II trial.

BACKGROUND & AIMS: Infection with the hepatitis D virus (HDV) causes the most severe form of viral hepatitis with a high risk to develop clinical complications of liver disease. In addition, hepatitis delta has been shown to be associated with worse patient-reported outcomes. Until recently, only pegylated interferon alfa could be used to treat hepatitis delta. METHODS: Here, we investigated quality of life (QOL) as assessed by the Short Form 36 Health Survey (SF-36) in patients undergoing antiviral therapy with pegylated interferon alfa (PEG-IFNa-2a)-based treatment in the HIDIT-II trial. HIDIT-II was a randomized prospective trial exploring PEG-IFNa-2a with tenofovir disoproxil (TDF) or placebo for 96 weeks in patients with compensated hepatitis delta. Surveys completed by 83 study participants before, during, and after treatments were available. RESULTS: Overall, we observed a reduced QOL of HDV patients compared with a reference population, both in physical as well as mental scores. Interestingly, PEG-IFNa-2a treatment showed only minor impairment of the QOL during therapy. Moreover, HDV-RNA clearance was not associated with relevant changes in physical or social SF-36 scores, whereas an improvement of fibrosis during treatment was associated with increased QOL. Overall, slight improvements of the QOL scores were observed 24 weeks after the end of treatment as compared with baseline. TDF co-treatment had no influence on QOL. CONCLUSIONS: Overall, our findings suggest that PEG-IFNa-2a was reasonably tolerated even over a period of 96 weeks by hepatitis D patients reporting SF-36 questionnaires. Of note, several patients may benefit from PEG-IFNa-2a-based therapies with off-treatment improvements in quality of life.

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin-binding IFT-B2 complex.

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αß-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.

Digoxin-mortality: randomized vs. observational comparison in the DIG trial.

AIMS: The Digitalis Investigation Group (DIG) trial, the only large randomized trial of digoxin in heart failure, reported a neutral effect on mortality and a significant reduction in heart failure hospitalizations. Recent observational studies reported increased mortality with digoxin treatment. We present further analyses of the DIG trial displaying the inability to control bias in observational treatment comparisons despite extensive statistical adjustments. METHODS AND RESULTS: Forty-four percent of the 6800 patients in the DIG trial had been treated with digoxin before randomization, and half of them were randomly withdrawn from digoxin treatment. We contrast the main randomization-based result of the DIG trial with the observational non-randomized comparison of patients pre-treated or not pre-treated with digoxin. Mortality [hazard ratio (HR) 1.22, 95% confidence interval (CI) 1.12-1.34; P < 0.001] and heart failure hospitalizations (HR 1.47, 95% CI 1.33-1.61; P < 0.001) were significantly higher in patients pre-treated with digoxin even after adjustment for baseline population differences. The higher risks for both outcomes in those who had previously received digoxin persisted even if they received placebo during the trial (HR 1.24, 95% CI 1.10-1.40; P < 0.001). This sharply contradicts the neutral effect on mortality and the significant reduction in heart failure hospitalizations observed in the randomized comparison. CONCLUSION: Prescription of digoxin is an indicator of disease severity and worse prognosis, which cannot be fully accounted for by covariate adjustments in the DIG trial where patients were well-characterized. It is unlikely that weaker research approaches (observational studies of administrative data or registries) can provide more reliable estimates of the effects of cardiac glycosides.

Comparison of relative and absolute rectal dose-volume parameters and clinical correlation with acute and late radiation proctitis in prostate cancer patients.

PURPOSE: To compare relative and absolute dose-volume parameters (DV) of the rectum and their clinical correlation with acute and late radiation proctitis (RP) after radiotherapy (RT) for prostate cancer (PCa). PATIENTS AND METHODS: 366 patients received RT for PCa. In total, 49.2% received definitive RT, 20.2% received postoperative RT and 30.6% received salvage RT for biochemical recurrence. In 77.9% of patients, RT was delivered to the prostate or prostate bed, and additional whole pelvic RT was performed in 22.1%. 33.9% received 3D-RT, and 66.1% received IMRT. The median follow-up was 59.5 months (18.0-84.0 months). The relative (in %) and absolute (in ccm) rectal doses from 20-75 Gy including the receiver operating characteristics curves (rAUC) from 30-65 Gy (in % and ccm) and several other clinical parameters were analyzed in univariate and multivariate analyses. We performed the statistical analyses separately for the entire cohort (n = 366), patients with (n = 81) and without (n = 285) pelvic RT, comparing RP vs. RP ≥ grade I. RESULTS: With the exception of the V50Gyccm (p = 0.02) in the univariate analyses for acute RP in the entire patient cohort, no absolute DV parameter (in ccm) was statistically significant associated with either acute or late RP. In the multivariate analyses, 3D-RT (p < 0.008) and rAUCV30-50 Gy% (p = 0.006) were significant parameters for acute RP for the entire cohort, and the V50Gy% (p = 0.01) was the significant parameter for patients with pelvic RT. The rAUCV40-50 Gy% (p = 0.004) was significant for RT to the prostate/prostate bed. Regarding the statistical analysis for late RP, the rAUCV30-65 Gy% (p = 0.001) was significant for the entire cohort, and rAUCV30-50 Gy% (p = 0.001) was significant for RT of the prostate/prostate bed. No parameter was significant in patients with pelvic RT. CONCLUSION: Absolute DV parameters in ccm are not required for RT in PCa patients.

Identification and quantification of domestic feline cytochrome P450 transcriptome across multiple tissues.

Understanding of cytochrome P450 (CYP) isoform distribution and function in the domestic feline is limited. Only a few studies have defined individual CYP isoforms across metabolically relevant tissues, hampering the ability to predict drug metabolism and potential drug-drug interactions. Using RNA sequencing (RNA-seq), transcriptomes from the 99 Lives Cat Genome Sequencing Initiative databank combined with experimentally acquired whole transcriptome sequencing of healthy, adult male (n = 2) and female (n = 2) domestic felines, expression of 42 CYP isoforms were identified in 20 different tissues. Thirty-seven of these isoforms had not been previously reported in cats. Depending on the tissue, three to twenty-nine CYP isoform transcripts were expressed. The feline genome annotations did not differentiate CYP2E1 and 2E2 genes, demonstrating poor annotation for this gene using the reference genome. As the majority of the sequences are based on automated pipelines, complete cDNA sequences for translation into CYP protein sequences could not be determined. This study is the first to identify and characterize 37 additional CYP isoforms in feline tissues, increasing the number of identified CYP from the previously reported seven isoforms to 42 across 20 tissues.

How to use prior knowledge and still give new data a chance?

A common challenge for the development of drugs in rare diseases and special populations, eg, paediatrics, is the small numbers of patients that can be recruited into clinical trials. Extrapolation can be used to support development and licensing in paediatrics through the structured integration of available data in adults and prospectively generated data in paediatrics to derive conclusions that support licensing decisions in the target paediatric population. In this context, Bayesian analyses have been proposed to obtain formal proof of efficacy of a new drug or therapeutic principle by using additional information (data, opinion, or expectation), expressed through a prior distribution. However, little is said about the impact of the prior assumptions on the evaluation of outcome and prespecified strategies for decision-making as required in the regulatory context. On the basis of examples, we explore the use of data-based Bayesian meta-analytic-predictive methods and compare these approaches with common frequentist and Bayesian meta-analysis models. Noninformative efficacy prior distributions usually do not change the conclusions irrespective of the chosen analysis method. However, if heterogeneity is considered, conclusions are highly dependent on the heterogeneity prior. When using informative efficacy priors based on previous study data in combination with heterogeneity priors, these may completely determine conclusions irrespective of the data generated in the target population. Thus, it is important to understand the impact of the prior assumptions and ensure that prospective trial data in the target population have an appropriate chance, to change prior belief to avoid trivial and potentially erroneous conclusions.

Diffusion-Weighted imaging and diffusion tensor imaging detect delayed graft function and correlate with allograft fibrosis in patients early after kidney transplantation.

PURPOSE: To combine diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI) for detection of allograft dysfunction in patients early after kidney transplantation and to correlate diffusion parameters with renal function and renal histology of allograft biopsies. MATERIALS AND METHODS: Between day 4 and 11 after kidney transplantation 33 patients with initial graft function and 31 patients with delayed graft function (DGF) were examined with a 1.5T magnetic resonance imaging (MRI) scanner. DTI and DWI sequences were acquired and fractional anisotropy (FA), apparent diffusion coefficient (ADCmono), pure diffusion (ADCdiff ), and the perfusion fraction (Fp) were calculated. Kidney biopsies in 26 patients were analyzed for allograft pathology, ie, acute tubular injury, inflammation, edema, renal fibrosis, and rejection. Histological results were correlated with MRI parameters. RESULTS: In the renal medulla FA (0.25 ± 0.06 vs. 0.29 ± 0.06, P < 0.01) and ADCmono (1.73 ± 0.13*10(-3) vs. 1.93 ± 0.16*10(-3) mm(2) /s, P < 0.001) were significantly reduced in DGF patients compared with patients with initial function. For ADCdiff and Fp similar reductions were observed. FA and ADCmono significantly correlated with renal function (r = 0.53 and r = 0.57, P < 0.001) and were inversely correlated with the amount of renal fibrosis (r = -0.63 and r = -0.65, P < 0.05). CONCLUSION: Combined DTI and DWI detected allograft dysfunction early after kidney transplantation and correlated with allograft fibrosis. J. Magn. Reson. Imaging 2016;44:112-121.

Getting tubulin to the tip of the cilium: one IFT train, many different tubulin cargo-binding sites?

Cilia are microtubule-based hair-like structures that project from the surfaces of eukaryotic cells. Cilium formation relies on intraflagellar transport (IFT) to move ciliary proteins such as tubulin from the site of synthesis in the cell body to the site of function in the cilium. A large protein complex (the IFT complex) is believed to mediate interactions between cargoes and the molecular motors that walk along axonemal microtubules between the ciliary base and tip. A recent study using purified IFT complexes has identified a tubulin-binding module in the two core IFT proteins IFT74 and IFT81 that likely serves to bind and transport tubulin within cilia. Here, we calculate the amount of tubulin required to support the observed cilium assembly kinetics and explore the possibility of multiple tubulin binding sites within the IFT complex.

Perioperative Bromelain Therapy after Wisdom Teeth Extraction - A Randomized, Placebo-Controlled, Double-Blinded, Three-Armed, Cross-Over Dose-Finding Study.

Reduction in postoperative edema and inflammatory reactions is the key to the posttraumatic regeneration process. Use of bromelain is well established in this indication, but there is some controversy with regard to the optimal dosing of this drug. The aim of our study was therefore to investigate the efficacy of dosage-dependent therapy with bromelain in patients after wisdom teeth extraction by comparing the registered dosage 1000 FIP (Fédération Internationale Pharmaceutique) against higher dosages of 3000 FIP and 4500 FIP. A total of 75 patients were randomized to one of the three dosage arms, and 68 of these patients were finally analyzed in the modified intention-to-treat population. Patients involved underwent two surgery sessions: one study period being conducted under treatment with bromelain and the other with placebo. Postoperative swelling determined by a 3D face scanning system was defined as the primary endpoint; further efficacy parameters were maximum swelling, pain, difficulty in swallowing, and use of analgesics. A superiority of treatment with 3000 FIP and 4500 FIP versus 1000 FIP could not be demonstrated. The analysis of pooled bromelain treatments versus placebo did, however, show a clear trend in favor of bromelain for all assessments. Adverse events did not occur more frequently under bromelain therapy compared with placebo. This study thus clearly supports the clinical relevance of treatment of postoperative conditions with bromelain, and the recommended daily dose was sufficiently effective in this trial and indication. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of an intensified prevention program for 4th graders with increased caries risk using ICDAS.

BACKGROUND: In order to investigate correlations between specific parameters and dental caries, it is useful to record incipient and dentine lesions. AIM: The aim of this study was to evaluate the effect of a selective intensified preventive program (SIP) on oral health of fourth graders using ICDAS. DESIGN: A cohort study was performed. ICDAS and DMFT index were recorded. Prevention and social factors were collected with psychometric questionnaires. The test group and the control group were parallelized using sociodemographic variables. RESULTS: The participants in the fluoride varnish program in the test region showed a significantly lower caries experience than pupils in the control region. The bivariate analysis revealed that a migrant background had a negative impact on oral health whereas fissure sealants, use of fluoridated table salt, use of fluoride tablets, early start of toothbrushing and high social status exerted a positive influence on dental health. Stepwise backward logistic regression analysis confirmed that fissure sealants, a high social status and an early start of toothbrushing had a significant positive impact on dental health. CONCLUSION: The results of our study suggest that a SIP impedes the primary occurrence of incipient lesions and the transition from incipient to advanced lesions.

Dissociating pathomechanisms of depression with fMRI: bottom-up or top-down dysfunctions of the reward system.

Depression is a debilitating psychiatric disorder characterized among other aspects by the inability to properly experience or respond to reward. However, it remains unclear whether patients with depression present impaired reward system due to abnormal modulatory mechanisms. We investigated the activation of the nucleus accumbens (NAcc), a crucial region involved in reward processing, with functional magnetic resonance imaging using the desire-reason-dilemma paradigm. This task allows tracking the activity of the NAcc during the acceptance or the rejection of previously conditioned reward stimuli. Patients were assigned into subgroups of lower (LA) or higher (HA) NAcc activation according to beta weights. LA patients presented significant hypoactivation in the ventral tegmental area in addition to bilateral ventral striatum, confirming impairments in the bottom-up input to the NAcc. Conversely, HA patients presented significant hyperactivation in prefrontal areas such as the rostral anterior cingulate cortex and the anterior ventral prefrontal cortex in addition to bilateral ventral striatum, suggesting disturbances in the top-down regulation of the NAcc. Demographic and clinical differences explaining the abnormal co-activations of midbrain and prefrontal regions were not identified. Therefore, we provide evidence for dysfunctional bottom-up processing in one potential neurobiological subtype of depression (LA) and dysfunctional top-down modulation in another subtype (HA). We suggest that the midbrain and prefrontal regions are more specific pathophysiological substrates for each depression subtype. Above all, our results encourage the segregation of patients by similar dysfunctional mechanisms of the dopaminergic system, which would finally contribute to disentangle more specific pathogeneses and guide the development of more personalized targets for future therapies.

Oxytocin facilitates the sensation of social stress.

Essentially all social species experience social stress which can be a catalyst for detriments in mental and physical health. The neuropeptide oxytocin (OXT) has been shown to produce anxiolytic and antistress effects, thereby qualifying the OXT system as a promising drug target in the treatment of stress-related disorders. However, recently it has been shown that OXT can have anxiogenic effects as well. In the present study, we used functional magnetic resonance imaging to scan the brains of 60 healthy men while they were exposed to social stress after they received either intranasal OXT (24 IU) or placebo treatment. Although OXT administration did not alter salivary cortisol levels as a surrogate marker of stress axis activity, our participants initially reported an increment in perceived social stress. This behavioral effect was paralleled on the neural level by increased activity in the precuneus and cingulate cortex. Taken together, our results support the hypothesis that OXT can induce a self-referential processing bias which facilitates the sensation of social stress in the absence of altered endocrine responses.

First appearance and location of catecholaminergic cells during experimental arthritis and elimination by chemical sympathectomy.

OBJECTIVE: The sympathetic nervous system is proinflammatory in early collagen-induced arthritis (CIA) and antiinflammatory in late disease. In late arthritis, sympathetic innervation of synovial and lymphoid tissue is markedly reduced. Thus, its suggested antiinflammatory role is difficult to explain. We hypothesized that newly discovered catecholamine-producing (catecholaminergic) cells are targets of chemical sympathectomy. However, in CIA, the time point of appearance, the location, and the possible chemical elimination of catecholaminergic cells have not been studied. The purpose of this study was to investigate the emergence and location of catecholamine-producing and -storing cells in different organs and joints of mice after induction of CIA and to determine whether catecholamine-producing cells can be depleted by 6-hydroxydopamine (6-OHDA) during the early and late phases of CIA in vivo. METHODS: The presence of cells positive for tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT-2) was evaluated immunohistologically in the lymph nodes, thymus, bone marrow, spleen, and joints of control and arthritic mice. Density was evaluated at different time points after early and late chemical sympathectomy. (131)I-metaiodobenzylguanidine ((131)I-MIBG) scintigraphy demonstrated functional activity of these cells in joint inflammation. RESULTS: The density of TH+ and VMAT-2+ cells was highest after arthritis onset (from day 28 onward) and was observed to occur in the following sequence: lymph nodes, thymus, joints, bone marrow, and spleen. Even before arthritis onset (days 5-21), these cells were already more numerous, particularly in the draining lymph nodes, thymus, and joints. (131)I-MIBG scintigraphy demonstrated catecholamine-storing cells in inflammatory hot spots in the paw. Chemical sympathectomy strongly reduced the density of catecholaminergic cells in vitro and in vivo. CONCLUSION: After disease onset, catecholaminergic cells are particularly present in primary and secondary lymphoid organs and joints. Since catecholaminergic cells have been reported to have antiinflammatory properties in arthritis, the proinflammatory role played by chemical sympathectomy in late arthritis, as we previously determined, is probably dependent on catecholaminergic cell elimination.

Delta-associated molluscan life and death assemblages in the northern Adriatic Sea: Implications for paleoecology, regional diversity and conservation.

Life-death (LD) studies of shelly macrofauna are important to evaluate how well a fossil assemblage can reflect the original living community, but can also serve as a proxy for recent ecological shifts in marine habitats and in practice this has to be distinguished using taphonomic preservation pattern and estimates of time-averaging. It remains to be rigorously evaluated, however, how to distinguish between sources of LD disagreement. In addition, death assemblages (DAs) also preserve important information on regional diversity which is not available from single censuses of the life assemblages (LAs). The northern Adriatic Sea is an ecosystem under anthropogenic pressure, and we studied the distribution and abundance of living and dead bivalve and gastropod species in the physically stressful environments (tidal flat and shallow sublittoral soft bottoms) associated with the delta of the Isonzo River (Gulf of Trieste). Specifically we evaluated the fidelity of richness, evenness, abundance, habitat discrimination and beta diversity. A total of 10,740 molluscs from fifteen tidal flat and fourteen sublittoral sites were analyzed for species composition and distribution of living and dead molluscs. Of 78 recorded species, only eleven were numerically abundant. There were many more dead than living individuals and rarefied species richness in the DA was higher at all spatial scales, but the differences are lower in habitats and in the region than at individual stations. Evenness was always higher in death assemblages, and probably due to temporally more variable LAs the differences are stronger in the sublittoral habitats. Distinct assemblages characterized intertidal and sublittoral habitats, and the distribution and abundance of empty shells generally corresponded to that of the living species. Death assemblages have lower beta diversity than life assemblages, but empty shells capture compositional differences between habitats to a higher degree than living shells. More samples would be necessary to account for the diversity of living molluscs in the study area, which is, however, well recorded in the death assemblages. There is no indication of a major environmental change over the last decades in this area, but due to the long history of anthropogenic pressure here, such a potential impact might be preserved in historical layers of the deeper sedimentary record.

Evaluation of a preventive program aiming at children with increased caries risk using ICDAS II criteria.

OBJECTIVES: A selective intensified prevention (SIP) was introduced at individual schools in deprived areas in Marburg County (Germany) in 1995. The outcome of the program was evaluated in sixth graders (mean age: 12.06 years) in comparison to a control region. MATERIALS AND METHODS: Caries experience was recorded by applying International Caries Detection and Assessment System (ICDAS) II criteria. Tooth brushing habits and other independent variables were examined psychometrically. To compare the mean caries scores, non parametric tests were applied. The influence of various independent variables on caries experience was assessed by stepwise backward logistic regression analysis. The matching criteria age, gender, ethnicity and maternal education were used to parallelize the samples. RESULTS: ICDAS scores of 2-6 were detected uniformly more often in the control region than in the test group. Combining ICDAS scores 3-6, children from the control region (mean D(3-6)MFT: 1.73) showed roughly double the caries experience compared to the test group (mean D(3-6)MFT: 0.88, p < 0.005). The D(5,6)MFT score of the test group amounted to 0.50, and the corresponding value of the reference group was 0.77 (p = 0.043). Multivariate analysis disclosed fissure sealants, early start of tooth brushing and topical fluoride application to be associated with the prevention of dental caries. High frequency of sugar intake was associated with the presence of dentine lesions. CONCLUSIONS: The results of our study confirm the positive effect of SIP on the dental health of 12-year-old pupils living in deprived areas. CLINICAL RELEVANCE: On the basis of ICDAS II, targeted preventive measures can be applied in children with increased caries risk. Frequent applications of fluoride varnish inhibit the progression of initial lesions in this group.

A chromosome-level reference genome and pangenome for barn swallow population genomics.

Insights into the evolution of non-model organisms are limited by the lack of reference genomes of high accuracy, completeness, and contiguity. Here, we present a chromosome-level, karyotype-validated reference genome and pangenome for the barn swallow (Hirundo rustica). We complement these resources with a reference-free multialignment of the reference genome with other bird genomes and with the most comprehensive catalog of genetic markers for the barn swallow. We identify potentially conserved and accelerated genes using the multialignment and estimate genome-wide linkage disequilibrium using the catalog. We use the pangenome to infer core and accessory genes and to detect variants using it as a reference. Overall, these resources will foster population genomics studies in the barn swallow, enable detection of candidate genes in comparative genomics studies, and help reduce bias toward a single reference genome.

Human and Insect Cell-Produced Recombinant Adeno-Associated Viruses Show Differences in Genome Heterogeneity.

In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy. In particular, the presence of empty AAV capsids and particles containing "inaccurate" vector genomes in preparations has been a subject of concern. Several methods exist to separate empty capsids from full particles; but thus far, no single technique can produce vectors that are free of empty or partial (non-unit length) capsids. Unfortunately, the exact genome compositions of full, intermediate, and empty capsids remain largely unknown. In this work, we used AAV-genome population sequencing to explore the compositions of DNase-resistant, encapsidated vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells (pTx/HEK293) and baculovirus expression vectors in Spodoptera frugiperda insect cells (rBV/Sf9). Intriguingly, our results show that vectors originating from the same construct design that were manufactured by the rBV/Sf9 system produced a higher degree of truncated and unresolved species than those generated by pTx/HEK293 production. We also demonstrate that empty particles purified by cesium chloride gradient ultracentrifugation are not truly empty but are instead packaged with genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). Our data suggest that the frequency of these "mutated" ITRs correlates with the abundance of inaccurate genomes in all fractions. These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated.

Long-term safety of satralizumab in neuromyelitis optica spectrum disorder (NMOSD) from SAkuraSky and SAkuraStar.

BACKGROUND: This analysis evaluated long-term safety findings from the SAkuraSky and SAkuraStar studies with satralizumab in patients with neuromyelitis optica spectrum disorder (NMOSD). METHODS: SAkuraSky (satralizumab in combination with baseline immunosuppressive therapy; IST) and SAkuraStar (satralizumab monotherapy) are international, multicenter, randomized, placebo-controlled, phase 3 studies consisting of a double-blind (DB) period followed by an open-label extension (OLE). The overall satralizumab treatment (OST) period safety population comprised patients receiving ≥1 dose of satralizumab in the DB and/or OLE periods (cut-off date: 22 February 2021). Safety was evaluated in the DB and OST periods. RESULTS: In the SAkuraSky DB period, patients received satralizumab (n = 41) or placebo (n = 42) in addition to stable baseline IST; 75 patients were included in the OST population. In the SAkuraStar DB period, 63 patients received satralizumab monotherapy and 32 received placebo; 91 patients were included in the OST population. Median treatment exposure in the OST period was 4.4 years (range 0.1-7.0) in SAkuraSky and 4.0 years (range 0.1-6.1) in SAkuraStar. Rates of adverse events (AEs per 100 patient-years) and serious AEs in the OST period were comparable with satralizumab and placebo in the DB periods of both studies. Similarly, overall rates of infections and serious infections were consistent between the OST and DB periods with satralizumab, with no increase in rates of infections or serious infections over time. In the OST periods, longer exposure to satralizumab was not associated with a higher risk of severe (grade ≥3) laboratory changes versus the DB periods. No deaths or anaphylactic reactions to treatment with satralizumab were reported during the OST periods of both studies. CONCLUSION: The safety profile of satralizumab as a monotherapy or in combination with IST was maintained in the OLE, and no new safety concerns versus the DB period were observed. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT02028884 (SAkuraSky) and NCT02073279 (SAkuraStar).

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity.

The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.