Long-term safety and efficacy outcomes of valoctocogene roxaparvovec gene transfer up to 6 years post-treatment.

INTRODUCTION: Valoctocogene roxaparvovec uses an adeno-associated virus serotype 5 (AAV5) vector to transfer a factor VIII (FVIII) coding sequence to individuals with severe haemophilia A, providing bleeding protection. AIM: To assess safety and efficacy of valoctocogene roxaparvovec 5-6 years post-treatment. METHODS: In a phase 1/2 trial, adult male participants with severe haemophilia A (FVIII ≤1 IU/dL) without FVIII inhibitors or anti-AAV5 antibodies received valoctocogene roxaparvovec and were followed for 6 (6 × 1013 vg/kg; n = 7) and 5 (4 × 1013 vg/kg; n = 6) years. Safety, including investigation of potential associations between a malignancy and gene therapy, and efficacy are reported. RESULTS: No new treatment-related safety signals emerged. During year 6, a participant in the 6 × 1013  vg/kg cohort was diagnosed with grade 2 parotid gland acinar cell carcinoma; definitive treatment was uncomplicated parotidectomy with lymph node dissection. Target enrichment sequencing of tumour and adjacent healthy tissue revealed low vector integration (8.25 × 10-5 per diploid cell). Integrations were not elevated in tumour samples, no insertions appeared to drive tumorigenesis, and no clonal expansion of integration-containing cells occurred. During all follow-ups, >90% decreases from baseline in annualised treated bleeds and FVIII infusion rates were maintained. At the end of years 6 and 5, mean FVIII activity (chromogenic assay) was 9.8 IU/dL (median, 5.6 IU/dL) and 7.6 IU/dL (median, 7.1 IU/dL) for the 6 × 1013 and 4 × 1013 vg/kg cohorts, respectively, representing proportionally smaller year-over-year declines than earlier timepoints. CONCLUSIONS: Valoctocogene roxaparvovec safety and efficacy profiles remain largely unchanged; genomic investigations showed no association with a parotid tumour.

Reaching people and managing membership in community-based dementia support groups: the Get Real with Meeting Centres realist evaluation part 1.

OBJECTIVES: There is a need to improve the provision and reach of community services for people living with dementia, a goal in which community-based support groups can play a key role. The Get Real with Meeting Centres project aimed to explore factors involved in the success and sustainability of Meeting Centres (MCs) a form of community-based support proliferating in the UK. This is the first of two linked articles outlining learning from this realist evaluation of MCs, which focusses on findings around reach and membership. METHOD: Semi-structured interviews and focus group discussions were conducted with 77 participants across three case study MC sites in England and Wales, including people living with dementia, informal carers, staff, volunteers, trustees, and supporting professionals/practitioners. Data were themed, then analysed using both soft systems methodology and realist logic of analysis. RESULTS: Fifty-two 'context-mechanism-outcome' statements were generated, explaining how background circumstances might trigger responses/processes to produce wanted or unwanted outcomes regarding four key areas for MC sustainability: Referrals and the dementia care pathway; Reaching people and membership; Carer engagement and benefit; and Venue and location. CONCLUSION: Strong links with formal services and a well-functioning dementia care pathway are essential to sustaining community-based group support such as MCs; group support is also well-placed to assist work to improve pathway issues. Clarity of offer (including benefit to carers), and a wide range of activities, are key to appeal and reach; transport to, and use of, venue are challenges, as are pressures to support people with more advanced dementia.

Strategic and operational issues in sustaining community-based dementia support groups: the Get Real with Meeting Centres realist evaluation part 2.

OBJECTIVES: Support for people with dementia in their communities is neither robust nor consistent in the UK, often bolstered by third sector/grass-roots initiatives facing formidable challenges in sustaining long-term. The Get Real with Meeting Centres project explored factors involved in sustaining one such form of community-based support. This is the second of two linked articles outlining learning from this realist evaluation of Meeting Centres (MCs) for people with dementia and carers, which focusses on findings regarding their operational and strategic running. METHOD: Semi-structured interviews and focus group discussions were conducted with 77 participants across three MC sites in England and Wales, including people living with dementia, informal carers, staff, volunteers, trustees, and supporting professionals/practitioners. Data were themed, then analysed using soft systems methodology and realist logic of analysis. RESULTS: Forty-two 'context-mechanism-outcome' statements were generated, explaining how background circumstances might trigger responses/processes to produce wanted or unwanted outcomes regarding three key areas for MC sustainability: External relationships and collaboration; Internal relationships and practices; and Finances and funding. CONCLUSION: Collaboration is essential to sustaining community-based initiatives such as MCs, particularly between local community and regional level. MCs need to be vigilant in mitigating pressures that create 'mission drift', as targeting a gap in the care pathway and maintaining a person-centred ethos are central to MCs' appeal. Stable, ongoing funding is needed for stable, ongoing community dementia support. More formal recognition of the value of social model community-based initiatives, helped by improved data collection, would encourage more robust and consistent community dementia support.

Understanding 'value' in the context of community-based interventions for people affected by dementia: A concept analysis.

AIM: This study aimed to conduct a concept analysis of value in the context of community-based interventions for people affected by dementia. BACKGROUND: Concepts of value play a critical role in shaping the delivery and distribution of community-based health interventions through related concepts. However, the use and meaning of 'value' is rarely clarified limiting the term's utility in practice and research. Increasing need for community healthcare and scarce public resources means developing understanding of value in community-based interventions for people affected by dementia is timely, and may support more informed approaches to exploring, explaining and delivering value. DESIGN: Evolutionary Concept Analysis was used to systematically determine the characteristics of value. DATA SOURCES: Peer-reviewed and grey literature databases were searched between April and July 2021, with 32 pieces of literature from different disciplines included in the final sample. No limits were set for the years of literature retrieved. METHODS: Literature was thematically analysed for information on the antecedents, attributes and consequences of value. RESULTS AND DISCUSSION: The analysis uncovered a need and/or desire to understand the experience of people affected by or that affect interventions; and to demonstrate, prove/disprove the (best) quality and nature of results of interventions as antecedents of value. Attributes of value were stakeholder/person centred, measurable, time and context dependent and multidimensional. Consequences of the concept included shared decision-making, valuation of interventions and internal/external investment and development of interventions. CONCLUSION: Through concept analysis value can now be better understood and applied. The development of a conceptual model to illustrate the constituent elements and relationships of the concept adds transparency to where, why and how concepts of value are enabled that supports future concept development. PATIENT AND PUBLIC CONTRIBUTION: No patient or public contribution.

Factors associated with satisfaction of the australian rural resident medical officer cadetship program: results from a cross-sectional study.

BACKGROUND: Australian Rural Resident Medical Officer Cadetships are awarded to medical students interested in a rural medical career. The Rural Residential Medical Officer Cadetship Program (Cadetship Program) is administered by the Rural Doctors Network on behalf of the NSW Ministry of Health. This study aimed to assess the overall experience of medical students and key factors that contributed to their satisfaction with the Cadetship Program. METHODS: A quantitative cross-sectional study was conducted among 107 former cadets who had completed the Cadetship Program. Data on medical students' experience with the Cadetship Program (outcome variable) and potential explanatory variables were collected using a structured self-administered questionnaire. Explanatory variables included gender, geographical location, rural health club membership, rural clinical school attendance, financial support, mentorship benefits, networking opportunities, influence on career decisions, opportunity for preferential placements, and relocation. Both bivariate (Pearson's chi-squared test) and multiple logistic regression analysis were employed to identify the factors associated with medical students' overall experience with the Cadetship Program. The non-linear analysis was weighted to represent the rural/remote health workforce, in Stata/SE 14.1. RESULTS: Our results indicate that 91% of medical students were satisfied with the Cadetship Program. The logistic regression model identified two significant predictors of a positive experience with the Cadetship Program. Medical students who perceived financial support as beneficial were significantly more likely to report a satisfactory program experience (aOR = 6.22, 95% CI: 1.36-28.44, p = 0.019) than those who perceived financial support as not beneficial. Similarly, those who valued networking opportunities were more likely to have a positive view of their cadetship experience (aOR = 10.06, 95% CI: 1.11-91.06, p = 0.040) than their counterparts. CONCLUSION: Our study found that students who valued financial support and networking opportunities had the most positive views of the Cadetship Program. These findings demonstrate that the Cadetship Program may be most helpful for those who need financial support and for students who seek networking opportunities. These findings increase our knowledge about the characteristics of medical students who have the most positive experiences with the Cadetship Program. They help us to understand the mechanisms of influence of such programs on individuals' decisions to be part of the future rural health workforce.

Multiyear Follow-up of AAV5-hFVIII-SQ Gene Therapy for Hemophilia A.

BACKGROUND: Adeno-associated virus (AAV)-mediated gene therapy is under investigation as a therapeutic option for persons with hemophilia A. Efficacy and safety data include 3 years of follow-up after a single administration of AAV5-hFVIII-SQ. METHODS: We report durable efficacy, long-term safety, and clinical and biologic results in 15 adults with severe hemophilia A (factor VIII level, ≤1 IU per deciliter) who had received a single infusion of AAV5-hFVIII-SQ at various dose levels. We evaluated the factor VIII level, annualized rate of bleeding events, use of factor VIII, safety, expression kinetics, and biologic markers of AAV transduction for up to 3 years. RESULTS: Three years after infusion, two participants (one who had received 6×1012 vector genomes [vg] per kilogram of body weight and one who had received 2×1013 vg per kilogram) had factor VIII expression of less than 1 IU per deciliter, as assessed on chromogenic assay. Seven participants (who had received 6×1013 vg per kilogram) had a median factor VIII expression of 20 IU per deciliter; the median number of annualized treated bleeding events was 0, and the median use of exogenous factor VIII was reduced from 138.5 infusions to 0 infusions per year. Bleeding in all target joints (major joints with ≥3 bleeding events within 6 months) in this cohort resolved (≤2 bleeding events within 12 months). Two years after infusion, six participants (who had received 4×1013 vg per kilogram) had a median factor VIII expression of 13 IU per deciliter; the median annualized rate of bleeding events was 0, and the median use of factor VIII was reduced from 155.5 infusions to 0.5 infusions per year. Bleeding in target joints resolved in five of six participants. The factor VIII pharmacodynamic profiles reflected cellular turnover in the blood and molecular events leading to episomal DNA stabilization for persistent expression, findings that are consistent with previous observations in two model systems. Transgene-derived human factor VIII (hFVIII) protein activity mirrored native hFVIII in hemostatic ability. No inhibitor development, thromboses, deaths, or persistent changes in liver-function tests were observed. CONCLUSIONS: Gene therapy with AAV5-hFVIII-SQ vector in participants with hemophilia A resulted in sustained, clinically relevant benefit, as measured by a substantial reduction in annualized rates of bleeding events and complete cessation of prophylactic factor VIII use in all participants who had received 4×1013 vg per kilogram or 6×1013 vg per kilogram of the gene therapy. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov number, NCT02576795; EudraCT number, 2014-003880-38.).

Cerebrospinal fluid and serum glycosphingolipid biomarkers in canine globoid cell leukodystrophy (Krabbe Disease).

Globoid cell leukodystrophy (GLD, Krabbe disease, Krabbe's disease) is caused by genetic mutations in the gene encoding, galactosylceramidase (GALC). Deficiency of this enzyme results in central and peripheral nervous system pathology, and is characterized by loss of myelin and an infiltration of globoid cells. The canine model of GLD provides a translational model which faithfully recapitulates much of the human disease pathology. Targeted lipidomic analysis was conducted in serum and cerebrospinal fluid (CSF) over the lifetime of GLD affected and normal canines, and in brain tissue at humane endpoint to better understand disease progression and identify potential biomarkers of disease. Psychosine, a substrate of GALC and primary contributor to the pathology in GLD, was observed to be significantly elevated in the serum and CSF by 2 or 4 weeks of age, respectively, and steadily increased over the lifetime of affected animals. Importantly, psychosine concentration strongly correlated with disease severity. Galactosylceramide, glucosylceramide, and lactosylceramide were also found to be elevated in the CSF of affected animals and increased with age. Psychosine and galactosylceramide were found to be significantly increased in brain tissue at humane endpoint. This study identified several biomarkers which may be useful in the development of therapeutics for GLD.

Short-term transcriptional response to IL-17 receptor-A antagonism in the treatment of psoriasis.

BACKGROUND: IL-17 antagonists induce impressive clinical benefits in psoriasis, but it is unknown to what extent cellular and molecular psoriasis characteristics are suppressed by a clinically relevant dose/schedule of any IL-17-receptor antagonist. OBJECTIVE: We sought to examine the effects of the IL-17 receptor-A antagonist brodalumab, on clinical and molecular psoriasis features over a 12-week period. METHODS: A subset of patients (n = 116) enrolled in 3 phase-3 randomized clinical trials (AMAGINE -1 [Efficacy, Safety, and Withdrawal and Retreatment With Brodalumab in Moderate to Severe Plaque Psoriasis Subjects], -2 [P3 Study Brodalumab in Treatment of Moderate to Severe Plaque Psoriasis], and -3 [Efficacy and Safety of Brodalumab Compared With Placebo and Ustekinumab in Moderate to Severe Plaque Psoriasis in Subjects]) participated in a mechanistic substudy where punch biopsies were collected (lesional and nonlesional skin) between baseline and 12 weeks. This cohort included moderate-to-severe psoriasis patients treated with 140 mg (n = 46), 210 mg (n = 41) brodalumab, or placebo (n = 29). Key epidermal psoriatic features, including T-cell and dendritic cell subsets, were examined using immunohistochemistry. Treatment-induced changes in lesional skin gene expression profiles were evaluated using Affymetrix arrays. RESULTS: IL-17 receptor-A antagonism caused extensive improvements in clinical, histologic, and transcriptomic features of psoriasis. Cellular infiltrates (CD3+, CD8+, CD11c+, CD163+), markers of keratinocyte proliferation (Ki67+, KRT16), and inflammatory cytokines (IL-17A/C/F, IL-23A, IL-12B) decreased progressively, reaching close to nonlesional levels, paralleled by decreases in epidermal thickness. Psoriasis transcriptome gene expression improved ∼85% to 95% in responders whose psoriasis area severity index improved by 75% from baseline by week 12 (n = 63), compared with ∼30% to 65% in nonresponders (n = 12), while the residual disease genomic profile was 10% of the psoriasis transcriptome, which is less than for earlier generation drugs. IL-17-dependent gene expression, including keratinocyte genes, improved earlier and more extensively following brodalumab treatment compared with ustekinumab treatment (anti-IL-23/-IL-12). CONCLUSIONS: The clinically approved dose and schedule for brodalumab leads to nearly complete resolution of clinical, histologic, and transcriptomic features of psoriasis. Evidently, IL-17-induced release of keratinocyte-derived inflammatory mediators is a key driver of psoriasis pathogenesis.

The effect of melatonin on ultrasound markers of follicular development: A double-blind placebo-controlled randomised trial.

BACKGROUND: Melatonin is a potent oxygen scavenger and is capable of altering blood flow in various vascular beds. AIMS: We aimed to determine the effect of melatonin on ovarian vascular indices during ovarian stimulation for in vitro fertilisation (IVF). MATERIALS AND METHODS: This is a pilot double-blind placebo-controlled randomised trial. Sixty-nine women (mean age 35.8 ± 4.3 years) undergoing their first cycle of IVF were randomised to receive either placebo, 2, 4 or 8 mg of melatonin, twice a day. Each participant underwent a transvaginal ultrasound at days 6-10 assessing follicular number and size. The vascularisation index (VI), flow index (FI) and vascularisation-flow index (VFI) were measured. These indices were then correlated with embryological outcomes. Informed consent was obtained from participants. This trial was registered with the Australia New Zealand Clinical Trials Registry (ACTRN12613001317785). RESULTS: The number of follicles did not differ between groups (P = 0.4). There were no differences in the VI (P = 0.4), FI (P = 0.1) or VFI (P = 0.3) in the right ovary or the FI (P = 0.3) or VFI (P = 0.3) in the left ovary between groups. When comparing placebo to any dose of melatonin, there were no differences in any measured parameter. While there was correlation between the number of follicles on ultrasound and all measured embryological outcomes, there was no correlation between ovarian vascular indices and these important clinical outcomes. CONCLUSIONS: Melatonin does not appear to change ovarian vascular indices during ovarian stimulation. In addition, such vascular indices cannot predict the number or quality of oocytes or embryos obtained in an IVF cycle. These findings require confirmation in future larger studies.

Capability ... what's in a word? Rural Doctors Network of New South Wales Australia is shifting to focus on the capability of rural health professionals.

Rural health services, and the workforces that provide those services, are under unprecedented pressure due to insufficient health workforce numbers and distribution of health workforce weighted to urban areas. This creates health service access issues in rural areas, compounding existing health inequalities between rural and urban people. Many approaches to date have aimed to rectify these issues, with moderate success. In this article we present a call to action to pursue a complementary approach: supporting the capability of the rural health workforce. We hypothesise that further exploring what it means to be a 'capable' rural health professional and what processes or conditions support or erode capability may additionally bolster efforts toward strong rural and remote health systems. The Capability Approach is a theory proposed by Amartya Sen, who was awarded the Nobel Memorial Prize in Economic Sciences in 1998 for this work. Although the Capability Approach inspired, for instance, the UN's Human Development Index, it has not been deeply explored in the context of rural health workforce. While still untested, a focus on capability may assist us in taking a broader view, which encompasses functioning and the freedom to pursue different functioning combinations. The feasible freedom and opportunities are paramount to the concept of capability. We posit that competence is static and the responsibility of the practitioner (and their education), but that capability is fluid and multi-dimensional and the responsibility of the practitioner, community and system. Therefore, we hypothesise that a focus on a Capability Approach, which modulates the relation between the contextual factors and outcomes, may provide us with greater understanding and avenues for action when we aim to improve outcomes such as rural health service sustainability. Developing a list of appropriate capabilities and setting strategies to support capability and its more nuanced domains may present unique opportunities for influence, and these may have positive effects on the rural health workforce. Of course it will need to be determined if improving rural primary health professionals' capability has positive impacts upon quality and access to care, and whether supporting capability is sustainable and worthy of investment.

A central nervous system-restricted isoform of the interleukin-1 receptor accessory protein modulates neuronal responses to interleukin-1.

Interleukin-1 (IL-1) has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified an isoform of the IL-1 receptor (IL-1R) accessory protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1R but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild-type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild-type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest that it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival.

A Randomized, Double-Blind, Placebo-Controlled Phase 2 Study of Brodalumab in Patients With Moderate-to-Severe Crohn's Disease.

OBJECTIVES: To assess the safety and efficacy of brodalumab, a human anti-interleukin-17 receptor monoclonal antibody, in patients with moderate-to-severe Crohn's disease (CD). METHODS: Phase 2, randomized, double-blind, placebo-controlled, dose-ranging study in patients with moderate-to-severe CD and evidence of active inflammation. Patients were randomized 1:1:1:1 to receive brodalumab (210, 350, or 700 mg at baseline and week 4) or placebo. The primary end point was proportion of patients achieving Crohn's disease activity index (CDAI) remission (≤150) at week 6. Secondary end points included proportion of patients with CDAI response (reduction from baseline of ≥100) at week 6 and change from baseline in CDAI at week 6. RESULTS: The study was terminated early based on an imbalance in worsening CD in active treatment groups. At the time of termination, 130 patients had been randomized. At week 6, remission rates were 3% (210 mg), 15% (350 mg), 9% (700 mg), and 3% (placebo) and CDAI response occurred in 16% (210 mg), 27% (350 mg), 15% (700 mg), and 13% (placebo) of patients. Mean change in CDAI at week 6 was -8.7 (95.3) (210 mg), -35.4 (105.6) (350 mg), -0.6 (105.9) (700 mg), and -28.2 (86.0) (placebo). Besides worsening of CD, overall incidences of adverse events were similar across treatment groups. CONCLUSIONS: Treatment with brodalumab resulted in a disproportionate number of cases of worsening CD in patients with active CD and no evidence of meaningful efficacy. These analyses did not suggest additional safety risks of brodalumab beyond worsening of CD symptoms in patients with active CD.

Effect of IL-17 receptor A blockade with brodalumab in inflammatory diseases.

IL-17 cytokines are expressed by a variety of cells and mediate host defence against extracellular pathogens. IL-17 is upregulated at sites of inflammation and can synergize with other cytokines, such as TNF-α, to amplify the inflammatory response. Activation of these signalling pathways has been hypothesized to contribute to the underlying pathogenesis of several inflammatory diseases, including psoriasis, RA, PsA and asthma. Thus the IL-17 signalling pathway is an attractive target for the development of therapeutic agents to modulate aberrant inflammatory responses. This review of the clinical development of therapeutic agents that target IL-17 signalling pathways in inflammatory diseases focuses on brodalumab, a human anti-IL-17 receptor A mAb. The cumulative findings of early clinical studies with anti-IL-17 agents, including brodalumab, secukinumab and ixekizumab, provide strong evidence for the role of IL-17 signalling in the pathophysiology of certain inflammatory diseases and support the potential use of these agents in treating these diseases.

The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses.

Psoriasis is a complex inflammatory process resulting from activation of the well-defined interleukin (IL)-23/T17 cytokine axis. We review the role of key cytokines IL-17 and IL-23 in psoriasis, as well as tumor necrosis factor (TNF)α, focusing on therapeutic cytokine interventions and what they reveal about psoriatic inflammation. The potential role of recently described epidermal IL-36RN and CARD14 genetic mutations in psoriasis pathogenesis is also explored, because they augment keratinocyte responses to proinflammatory cytokines. The discovery of these genetic mutations in familial and pustular psoriasis suggests new links between cytokine-induced gene products and IL-1 family members from keratinocytes, which may regulate features of the disease, including epidermal hyperplasia and neutrophil infiltrating responses.

Gene expression profiles normalized in psoriatic skin by treatment with brodalumab, a human anti-IL-17 receptor monoclonal antibody.

The IL-17 pathway is an established driver of psoriasis pathogenesis. We examined the detailed molecular and cellular effects of blockade of IL-17 signaling in human psoriatic skin before and following treatment with brodalumab, a competitive inhibitor of the IL-17 Receptor A subunit. Thousands of aberrantly expressed genes in lesional skin normalized within 2 weeks following brodalumab treatment, with conversion of the lesional psoriasis transcriptome to resemble that seen in nonlesional skin. Keratinocyte-expressed genes appeared to normalize rapidly, whereas T cell-specific normalization occurred over six weeks. The three IL-17 ligand genes that are upregulated in lesional skin, IL17A, IL17C, and IL17F, were all downregulated in a dose-dependent manner following brodalumab treatment. Cellular measures also showed a similar pattern with dramatic decreases in keratinocyte hyperplasia within one week, and decreases in infiltrating leukocytes occurred over a longer timescale. Individuals with the highest brodalumab exposure showed normalization of both IL-17-responsive genes and the psoriasis transcriptome, whereas subjects with lower exposures showed transient or incomplete molecular responses. Clinical and molecular response appeared dependent on the extent of brodalumab exposure relative to the expression of IL-17 ligand genes, and reduction of IL-17 signaling into the nonlesional range was strongly correlated with normalization of the psoriasis transcriptome. These data indicate that blockade of IL-17 signaling in psoriatic skin leads to rapid transcriptomal changes initially in keratinocyte-expressed genes, followed by normalization in the leukocyte abnormalities, and demonstrates the essential role of the IL-17R on keratinocytes in driving disease pathogenesis.

Brodalumab, an anti-interleukin-17-receptor antibody for psoriasis.

BACKGROUND: In this phase 2, randomized, double-blind, placebo-controlled, dose-ranging study, we assessed the efficacy and safety of brodalumab (AMG 827), a human anti-interleukin-17-receptor monoclonal antibody, for the treatment of moderate-to-severe plaque psoriasis. METHODS: We randomly assigned patients with a score of 12 or higher on the psoriasis area-and-severity index (PASI, on which scores range from 0 to 72, with higher scores indicating more severe disease) and with 10% or more of their body-surface area affected by psoriasis to receive brodalumab (70 mg, 140 mg, or 210 mg at day 1 and weeks 1, 2, 4, 6, 8, and 10 or 280 mg monthly) or placebo. The primary end point was the percentage improvement from baseline in the PASI score at week 12. Secondary end points included improvement of at least 75% and at least 90% in the PASI score and the score on the static physician's global assessment at week 12. RESULTS: A total of 198 patients underwent randomization. At week 12, the mean percentage improvements in the PASI score were 45.0% among patients receiving 70 mg of brodalumab, 85.9% among those receiving 140 mg, 86.3% among those receiving 210 mg, 76.0% among those receiving 280 mg, and 16.0% among those receiving placebo (P<0.001 for all comparisons with placebo). An improvement of at least 75% and at least 90% in the PASI score at week 12 was seen in 77% and 72%, respectively, of the patients in the 140-mg brodalumab group and in 82% and 75%, respectively, of the patients in the 210-mg group, as compared with 0% in the placebo group (P<0.001 for all comparisons). The percentage of patients with a static physician's global assessment of clear or minimal disease was 26%, 85%, 80%, and 69% with the 70-mg, 140-mg, 210-mg, and 280-mg doses, respectively, of brodalumab, as compared with 3% with placebo (P<0.01 for all comparisons with placebo). Two cases of grade 3 neutropenia were reported in the 210-mg brodalumab group. The most commonly reported adverse events in the combined brodalumab groups were nasopharyngitis (8%), upper respiratory tract infection (8%), and injection-site erythema (6%). CONCLUSIONS: Brodalumab significantly improved plaque psoriasis in this 12-week, phase 2 study. (Funded by Amgen; ClinicalTrials.gov number, NCT00975637.).

Cellular responses to individual amino-acid depletion in antibody-expressing and parental CHO cell lines.

Depletion of two nonessential amino acids, asparagine (Asn) and glutamine (Gln), occurred during a fed-batch production process with a CHO cell line expressing a recombinant antibody. This depletion coincided with growth suppression and the onset of the stationary phase. Experimental withdrawal of Asn led to cell cycle arrest of cell line A in G0/G1 phase. On a mechanistic level, withdrawal of either Asn or Gln stimulated the amino-acid response (AAR) pathway, indicating that depletion of nonessential amino acids can induce AAR in this cell line. Compared to withdrawal of an essential amino acid, leucine (Leu), withdrawal of either Asn or Gln induced fewer changes in downstream effectors of mammalian target of rapamycin (mTOR) signaling involved in regulation of global protein synthesis. Global transcriptional analysis followed by pathway analysis revealed that the cultures experienced a down-regulation of cell-cycle progression, DNA replication and nucleotide biosynthesis in an E2F-dependent manner, as well as a down-regulation of lipid metabolism in a SREBP1/2-dependent manner as a result of individual amino-acid withdrawal. Timing and magnitude of observed phenotypic and transcriptional responses to amino-acid withdrawal differed between essential (Leu) and nonessential (Asn and Gln) amino acids examined. Observed responses were similar in parental (CS9 and CHOK-1) and two other antibody-producing CHO cell lines, but the magnitude of the transcriptional response was both cell-line and amino-acid dependent. Overall, these results suggest that depletion of nonessential amino acids in cell culture plays a role in the onset of the stationary phase of production process and offer mechanistic insights into the observed growth attenuation phenotype.

Blood biodistribution and vector shedding of valoctocogene roxaparvovec in people with severe hemophilia A.

Following systemically-administered adeno-associated virus (AAV) gene therapy, vector particles are widely distributed, which has raised concerns about horizontal or germline transmission of vector. Characterization of biodistribution and kinetics of vector DNA in body fluids can address these concerns and provide insights into vector behavior in accessible samples. We investigated the biodistribution and vector shedding profile of valoctocogene roxaparvovec in men with severe hemophilia A enrolled in the phase 3 GENEr8-1 trial. Participants (n=134) received a single 6E13 vg/kg infusion and were assessed over 3 years. Vector DNA was measured with 4 different assays. Total vector DNA was evaluated in blood, saliva, stool, semen, and urine by quantitative (q)PCR. Encapsidated vector DNA was measured in plasma and semen with immunocapture-based qPCR. Contiguity of vector genomes and assembly of inverted terminal repeat fusions were measured in whole blood and peripheral blood mononuclear cells (PBMCs) using multi-color digital PCR. Median peak vector DNA levels observed 1 to 8 days after dosing were highest in blood, followed by saliva, semen, stool, and urine. Concentrations then declined steadily. Encapsidated vector DNA cleared faster than total vector DNA, achieving clearance by <=12 weeks in plasma and semen. Predominant vector genome forms transitioned from non-contiguous to full-length over time in whole blood and PBMCs, indicating formulation of stable circularized episomes within nucleated cells. The replication-incompetent nature of valoctocogene roxaparvovec, coupled with the steady clearance of total and encapsidated vector DNA from shedding matrices, indicates risk of transmission is low. This trial is registered at www.clinicaltrials.gov as NCT03370913.

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A.

Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial ( NCT02576795 ), liver biopsy samples were collected 2.6-4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability.