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Here, we describe the design of a molecular probe (NB-ALA) based on the Nile Blue fluorophore, which can detect alanine aminopeptidase enzyme (a kidney damage biomarker) in urine by simple fluorometric measurements.

Recombinant adeno-associated virus (AAV) vectors are the leading platform for gene delivery for a variety of clinical applications. Patients with preexisting antibodies to AAV are currently excluded from most AAV gene therapy trials to avoid vector neutralization and ensure response to therapy. Anti-AAV neutralizing antibodies (NAbs) are typically assessed by in vitro cell-based transduction inhibition (TI) assays. However, clinical relevance of the determined enrollment cutoff and the inherent variability of a cell-based assay present challenges for use as an enrollment screening test. Here, we describe an enrollment cutoff that was clinically validated and strategies to overcome assay challenges to enable long-term stable performance. A validated anti-AAV6 cell-based TI assay was used to support clinical enrollment across multiple investigational gene therapies and to evaluate AAV6 seroprevalence in healthy and disease populations. The clinical enrollment cutoff was determined statistically using samples collected from healthy donors, applying a 0.1% false error rate with the inclusion of a minimum significant ratio (MSR) metric and in consideration of results from in vivo mouse passive transfer studies. Our strategy for long-term monitoring and control of assay performance employed plate quality control samples flanking the predefined cutoff. An approach using donor samples was implemented to bridge different lots of critical reagents without the need to redefine the cutoff.

Anti-cancer therapies often result in a subset of surviving cancer cells that undergo therapy-induced senescence (TIS). Senescent cancer cells strongly modify the intratumoural microenvironment favoring immunosuppression and, thereby, tumour growth. An emerging strategy to optimise current therapies is to combine them with treatments that eliminate senescent cells. To this end, we undertook an unbiased proteomics approach to identify surface markers contributing to senescent cells immune evasion. Through this approach, we discovered that the immune checkpoint inhibitor PD-L2, but not PD-L1, is upregulated across multiple senescent human and murine cells. Importantly, blockade of PD-L2 strongly synergises with genotoxic chemotherapy, causing remission of solid tumours in mice. We show that PD-L2 inhibition prevents the persistence of chemotherapy-induced senescent cells, which exert cell-extrinsic immunomodulatory actions. In particular, upon chemotherapy, tumours deficient in PD-L2 fail to produce cytokines of the CXCL family, do not recruit myeloid-derived suppressor cells (MDSCs) and are eliminated in a CD8 T cell-dependent manner. We conclude that blockade of PD-L2 improves chemotherapy efficacy by reducing the intratumoural burden of senescent cells and their associated recruitment of immunosuppressive cells. These findings provide a novel strategy to exploit vulnerabilities arising in tumour cells as a result of therapy-induced damage and cellular senescence.

Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). However, the mechanisms involved in the conversion of damaged cells into senescent cells remain incompletely understood. Here, we report that multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries, through the release of iron, are efficient in triggering senescence and fibrosis. Interestingly, the accumulation of iron is an intrinsic property of senescent cells that does not require an abnormal surge in extracellular iron. Upon damage, cells initiate an iron accumulation response with abundant ferritin-bound iron within lysosomes and high levels of labile iron, the latter being a main driver of senescence-associated ROS and SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging (MRI) is a powerful non-invasive method to assess fibrotic burden in the kidneys of mice and patients with renal fibrosis. Our findings establish a central role for iron accumulation in senescence and fibrogenesis.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the alpha-galactosidase A (GLA) gene, which encodes the exogalactosyl hydrolase, alpha-galactosidase A (α-Gal A). Deficient α-Gal A activity results in the progressive, systemic accumulation of its substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), leading to renal, cardiac, and/or cerebrovascular disease and early demise. The current standard treatment for Fabry disease is enzyme replacement therapy, which necessitates lifelong biweekly infusions of recombinant enzyme. A more long-lasting treatment would benefit Fabry patients. Here, a gene therapy approach using an episomal adeno-associated viral 2/6 (AAV2/6) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette was evaluated in a Fabry mouse model that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. A detailed 3-month pharmacology and toxicology study showed that administration of a clinical-scale-manufactured AAV2/6 vector resulted in markedly increased plasma and tissue α-Gal A activities, and essentially normalized Gb3 and Lyso-Gb3 at key sites of pathology. Further optimization of vector design identified the clinical lead vector, ST-920, which produced several-fold higher plasma and tissue α-Gal A activity levels with a good safety profile. Together, these studies provide the basis for the clinical development of ST-920., Graphical Abstract

Zinc-finger nuclease (ZFN)-based in vivo genome editing is a novel treatment that can potentially provide lifelong protein replacement with single intravenous administration. Three first-in-human open-label ascending single-dose phase 1/2 studies were performed in parallel (starting November 2017) primarily to assess safety and tolerability of ZFN in vivo editing therapy in mucopolysaccharidosis I (MPS I) (n = 3), MPS II (n = 9), and hemophilia B (n = 1). Treatment was well tolerated with no serious treatment-related adverse events. At the 1e13 vg/kg dose, evidence of genome editing was detected through albumin-transgene fusion transcripts in liver for MPS II (n = 2) and MPS I (n = 1) subjects. The MPS I subject also had a transient increase in leukocyte iduronidase activity to the lower normal range. At the 5e13 vg/kg dose, one MPS II subject had a transient increase in plasma iduronate-2-sulfatase approaching normal levels and one MPS I subject approached mid-normal levels of leukocyte iduronidase activity with no evidence of genome editing. The hemophilia B subject was not able to decrease use of factor IX concentrate; genome editing could not be assessed. Overall, ZFN in vivo editing therapy had a favorable safety profile with evidence of targeted genome editing in liver, but no long-term enzyme expression in blood.

Fabry disease, a lysosomal storage disorder resulting from the deficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted in vivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated in vivo genome editing has the potential to be an effective one-time therapy for Fabry disease.

Rationale: Cellular senescence is a key mechanism implicated in the pathogenesis of pulmonary fibrosis. Objective: To test if the inoculation of senescent human fibroblasts in mice generates lung fibrosis, and whether this model is useful to assess antifibrotic or senolytic drugs in vivo. Methods: Immunodeficient mice were inoculated intratracheally with proliferating human lung fibroblasts (IMR90), senescent human lung fibroblasts induced by γ-irradiation (gIR-IMR90), or bleomycin as control. After 21 days, lung fibrosis was determined by immunohistochemistry (IHC), and quantified by hydroxyproline assay and Ashcroft score. Gene expression analysis was performed using senescence markers. Furthermore, a group of mice with established fibrosis were treated with pirfenidone, nintedanib or placebo administrated orally for 14 days. Another group was treated with navitoclax (ABT-263) or vehicle. Collagen deposition was measured by hydroxyproline assay and IHC. Results: Inoculation of gIR-IMR90 elicited a progressive lung remodeling process characterized by increased score of fibrosis (Ashcroft score, p Conclusion: Intratracheal inoculation of gIR-IMR90 generates progressive lung fibrosis in mice that is reduced by antifibrotic drugs. This model can be used to test novel drugs for pulmonary fibrosis in vivo.

OBJECTIVES: To determine if a home oxygen therapy (HOT) management strategy that includes analysis of recorded home oximetry (RHO) data, compared with standard monthly clinic visit assessments, reduces duration of HOT without harm in premature infants. METHODS: The RHO trial was an unmasked randomized clinical trial conducted in 9 US medical centers from November 2013 to December 2017, with follow-up to February 2019. Preterm infants with birth gestation ≤37 + 0/7 weeks, discharged on HOT, and attending their first pulmonary visit were enrolled. The intervention was an analysis of transmitted RHO between clinic visits (n = 97); the standard-care group received monthly clinic visits with in-clinic weaning attempts (n = 99). The primary outcomes were the duration of HOT and parent-reported quality of life. There were 2 prespecified secondary safety outcomes: change in weight and adverse events within 6 months of HOT discontinuation. RESULTS: Among 196 randomly assigned infants (mean birth gestational age: 26.9 weeks; SD: 2.6 weeks; 37.8% female), 166 (84.7%) completed the trial. In the RHO group, the mean time to discontinue HOT was 78.1 days (SE: 6.4), compared with 100.1 days (SE: 8.0) in the standard-care group (P = .03). The quality-of-life scores improved from baseline to 3 months after discontinuation of HOT in both groups (P = .002), but the degree of improvement did not differ significantly between groups (P = .75). CONCLUSIONS: RHO was effective in reducing the duration of HOT in premature infants. Parent quality of life improved after discontinuation. RHO allows physicians to determine which infants can be weaned and which need prolonged oxygen therapy between monthly visits.

Salivary gland hypofunction causes significant morbidity and loss of quality of life for head and neck cancer patients treated with radiotherapy. Preventing hypofunction is an unmet therapeutic need. We used an adeno-associated virus serotype 2 (AAV2) vector expressing the human neurotrophic factor neurturin (CERE-120) to treat murine submandibular glands either pre- or post-irradiation (IR). Treatment with CERE-120 pre-IR, not post-IR, prevented hypofunction. RNA sequencing (RNA-seq) analysis showed reduced gene expression associated with fibrosis and the innate and humoral immune responses. We then used a minipig model with CERE-120 treatment pre-IR and also compared outcomes of the contralateral non-IR gland. Analysis of gene expression, morphology, and immunostaining showed reduced IR-related immune responses and improved secretory mechanisms. CERE-120 prevented IR-induced hypofunction and restored immune homeostasis, and there was a coordinated contralateral gland response to either damage or treatment. CERE-120 gene therapy is a potential treatment for head and neck cancer patients to influence communication among neuronal, immune, and epithelial cells to prevent IR-induced salivary hypofunction and restore immune homeostasis., Graphical Abstract, Permanent salivary hypofunction is a major complication due to irradiation therapy in head and neck cancer patients. Lombaert et al. present a key translational advance where retro-ductal infusion of an adeno-associated virus serotype 2 (AAV2) vector expressing neurotrophic factor neurturin (CERE-120) into mouse and porcine salivary glands before irradiation reduces fibrosis, restores immune homeostasis, and prevents loss of saliva.

Mucopolysaccharidosis type I (MPS I) is a severe disease due to deficiency of the lysosomal hydrolase α-L-iduronidase (IDUA) and the subsequent accumulation of the glycosaminoglycans (GAG), leading to progressive, systemic disease and a shortened lifespan. Current treatment options consist of hematopoietic stem cell transplantation, which carries significant mortality and morbidity risk, and enzyme replacement therapy, which requires lifelong infusions of replacement enzyme; neither provides adequate therapy, even in combination. A novel in vivo genome-editing approach is described in the murine model of Hurler syndrome. A corrective copy of the IDUA gene is inserted at the albumin locus in hepatocytes, leading to sustained enzyme expression, secretion from the liver into circulation, and subsequent uptake systemically at levels sufficient for correction of metabolic disease (GAG substrate accumulation) and prevention of neurobehavioral deficits in MPS I mice. This study serves as a proof-of-concept for this platform-based approach that should be broadly applicable to the treatment of a wide array of monogenic diseases., In vivo genome editing following a single injection of ZFN and IDUA donor encoding AAV8 resulted in metabolic correction and neurological benefit in a murine model of MPS I. These results enable a currently open clinical trial to evaluate targeted genome engineering for MPS I in humans.

The third-generation sequencing technology, PacBio, has shown an ability to sequence the HIV virus amplicons in their full length. The long read of PaBio offers a distinct advantage to comprehensively understand the virus evolution complexity at quasispecies level (i.e. maintaining linkage information of variants) comparing to the short reads from Illumina shotgun sequencing. However, due to the highnoise nature of the PacBio reads, it is still a challenge to build accurate contigs at high sensitivity. Most of previously developed NGS assembly tools work with the assumption that the input reads are fairly accurate, which is largely true for the data derived from Sanger or Illumina technologies. When applying these tools on PacBio high-noise reads, they are largely driven by noise rather than true signal eventually leading to poor results in most cases. In this study, we propose the de novo assembly procedure, which comprises a positivefocused strategy, and linkage-frequency noise reduction so that it is more suitable for PacBio high-noise reads. We further tested the unique de novo assembly procedure on HIV PacBio benchmark data and clinical samples, which accurately assembled dominant and minor populations of HIV quasispecies as expected. The improved de novo assembly procedure shows potential ability to promote PacBio technology in the field of HIV drug-resistance clinical detection, as well as in broad HIV phylogenetic studies.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by deficiency of iduronate 2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs) in tissues of affected individuals, progressive disease, and shortened lifespan. Currently available enzyme replacement therapy (ERT) requires lifelong infusions and does not provide neurologic benefit. We utilized a zinc finger nuclease (ZFN)-targeting system to mediate genome editing for insertion of the human IDS (hIDS) coding sequence into a “safe harbor” site, intron 1 of the albumin locus in hepatocytes of an MPS II mouse model. Three dose levels of recombinant AAV2/8 vectors encoding a pair of ZFNs and a hIDS cDNA donor were administered systemically in MPS II mice. Supraphysiological, vector dose-dependent levels of IDS enzyme were observed in the circulation and peripheral organs of ZFN+donor-treated mice. GAG contents were markedly reduced in tissues from all ZFN+donor-treated groups. Surprisingly, we also demonstrate that ZFN-mediated genome editing prevented the development of neurocognitive deficit in young MPS II mice (6–9 weeks old) treated at high vector dose levels. We conclude that this ZFN-based platform for expression of therapeutic proteins from the albumin locus is a promising approach for treatment of MPS II and other lysosomal diseases., AAV-mediated in vivo delivery of ZFN and IDS donor resulted in site-specific gene insertion and dose-dependent IDS expression in a mouse model of MPS II. These results support a currently open clinical trial, the first ever in vivo human genome editing study to be conducted.

The clinical application and regulatory strategy of genome editing for ex vivo cell therapy is derived from the intersection of two fields of study: viral vector gene therapy trials; and clinical trials with ex vivo purification and engraftment of CD34+ hematopoietic stem cells, T cells, and tumor cell vaccines. This article covers the regulatory and translational preclinical activities needed for a genome editing clinical trial modifying hematopoietic stem cells and the genesis of this current strategy based on previous clinical trials using genome-edited T cells. The SB-728 zinc finger nuclease platform is discussed because this is the most clinically advanced genome editing technology.

A simulation program for staff at a long-term care facility helped increase their knowledge and awareness of the difficulties of performing the activities of daily living for patients who have dementia and other physical impairments.

Background: Fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. Recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. However, its relevance in the heart remains to be established. Objective: To investigate the role of premature senescence in myocardial fibrosis. Methods: Murine models of cardiac diseases and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. Loss-of-function and gain-of-function models of premature senescence were used to determine its pathophysiological role in myocardial fibrosis. Results: Senescence markers p21 CIP1/WAF1, senescence-associated s-galactosidase (SA-s-gal) and p16 INK4a were increased 2-, 8- and 20-fold (n=5-7; P < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction (TAC) when compared to sham-treated controls. Similar results were observed with cardiomyocyte-specific β1-adrenoceptor transgenic mice and human heart biopsies. Senescent cells were positive for platelet-derived growth factor receptor α (92.5 ± 1.8%), vimentin (92.1 ± 1.5%), and α-smooth muscle actin (65.2 ± 10.5%), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. Inactivation of the premature senescence program by genetic ablation of p53 and p16 INK4a (Trp53 -/- Cdkn2a -/- mice) resulted in aggravated fibrosis after TAC when compared to wild-type controls (49 ± 4.9% vs. 33 ± 2.7%, P < 0.01), and was associated with impaired cardiac function. Conversely, cardiac-specific expression of CCN1 (Cyr61), a potent inducer of premature senescence, by adeno-associated virus serotype 9 gene transfer, resulted in ~ 50% reduction of perivascular fibrosis after TAC when compared to mock- or dominant-negative CCN1-infected controls (11.9 ± 1.4% vs. 22.4±4.0% and 22.1±1.8%, respectively; P < 0.01), and improved cardiac function. Conclusions: Our data establish premature senescence of myofibroblasts as essential anti-fibrotic mechanism and potential therapeutic target in myocardial fibrosis.

The third-generation sequencing technology, PacBio, has shown an ability to sequence the HIV virus amplicons in their full length. The long read of PaBio offers a distinct advantage to comprehensively understand the virus evolution complexity at quasispecies level (i.e. maintaining linkage information of variants) comparing to the short reads from Illumina shotgun sequencing. However, due to the highnoise nature of the PacBio reads, it is still a challenge to build accurate contigs at high sensitivity. Most of previously developed NGS assembly tools work with the assumption that the input reads are fairly accurate, which is largely true for the data derived from Sanger or Illumina technologies. When applying these tools on PacBio high-noise reads, they are largely driven by noise rather than true signal eventually leading to poor results in most cases. In this study, we propose the de novo assembly procedure, which comprises a positivefocused strategy, and linkage-frequency noise reduction so that it is more suitable for PacBio high-noise reads. We further tested the unique de novo assembly procedure on HIV PacBio benchmark data and clinical samples, which accurately assembled dominant and minor populations of HIV quasispecies as expected. The improved de novo assembly procedure shows potential ability to promote PacBio technology in the field of HIV drug-resistance clinical detection, as well as in broad HIV phylogenetic studies.

Improved survival among preterm infants has led to an increase in diagnosis of chronic lung disease and infants discharged home from the NICU on supplemental oxygen. Despite this increased prevalence, no clearly defined guidelines for the management of home oxygen therapy (HOT) exist. This lack of consensus leads to significant variability in the duration of home oxygen therapy and a general paucity of evidence-based practice. Our team has identified recorded home oxygen therapy (RHO) as a potential new resource to guide clinical decision making in the outpatient pulmonology clinic. By recording extended O2 saturation data during the weaning process, RHO has the potential to save cost and improve the processes of HOT management. Our team is currently supporting a prospective, multi-center, randomized, controlled trial of RHO guided HOT weaning with the aims of determining effect upon duration of HOT, perceived parent quality of life and effect upon growth and respiratory outcomes. We plan to randomize 196 infants into one of two study arms evaluating standard HOT management versus RHO guided oxygen weaning. Our primary outcomes are total HOT duration and parental quality of life. This trial represents an unprecedented opportunity to test a novel home monitoring intervention for weaning within a vulnerable yet quickly growing population. If effective, the use of RHO may provide clinicians a tool for safe weaning.

The clinical application and regulatory strategy of genome editing for ex vivo cell therapy is derived from the intersection of two fields of study: viral vector gene therapy trials; and clinical trials with ex vivo purification and engraftment of CD34

Fabry disease (FD), an X-linked lysosomal storage disease, is caused by mutations in the GLA gene encoding α-galactosidase A (α-GalA). FD is characterized by progressive systemic accumulation of the enzyme’s substrates, globotriaosylceramide (Gb3) and lyso-Gb3, leading to renal, cardiac and/or cerebrovascular disease and culminating in premature demise. FD is treated by enzyme replacement therapy (ERT), however the short enzyme half-life necessitates life-long biweekly infusions. A more effective and long-lasting treatment would benefit FD patients. An AAV-mediated liver-targeted gene therapy was evaluated in a mouse model (GLAKO) that lacks α-GalA activity and accumulates high levels of Gb3/lyso-Gb3 in plasma and tissues. This strategy employs an episomal AAV (serotype 2/6) vector encoding human GLA cDNA (hGLA) driven by a liver-specific promoter. One-time administration of increasing amounts of AAV-hGLA cDNA generated using a clinical scale manufacturing process resulted in supraphysiological expression of plasma α-GalA (over 300-fold of WT) by study day 15, was well tolerated, and was stable for 3 months post-injection. Dose-dependent increases in α-GalA activities were achieved in liver, heart and kidney with a corresponding reduction of Gb3/lyso-Gb3. This initial vector was compared to an improved cDNA vector in a 1-month study using two different AAV doses in wild type C57BL/6 mice. The improved cDNA vector produced on average 7-fold higher levels of plasma α-GalA activity at study day 28 than mice administered the same dose of the initial cDNA. The high levels of α-GalA activity seen in these studies, along with the concomitant marked reduction in the accumulated Gb3/lyso-Gb3 in key tissues of the GLAKO mouse model, provide “proof-of-concept” for AAV-mediated targeting of hepatocytes to express therapeutic levels of human α-GalA. The clinical scale manufacturing process developed for these studies will enable rapid development and production in 2019 of clinical-grade material featuring the improved cDNA construct.

Procalcitonin (PCT)-a diagnostic serum parameter for bacterial infection and sepsis-is of great interest in the field of biosensors for point-of-care testing. Its detection needs specific biological recognition elements, such as antibodies. Herein, we describe the development and characterization of rat monoclonal antibodies (mAbs) for PCT, and their application in enzyme-linked immunosorbent assays (ELISAs) for the determination of PCT in patient serum samples. From about 50 mAbs, two mAbs, CALCA 2F3 and CALCA 4A6, were selected as a pair with high affinity for PCT in sandwich immunoassays. Both mAbs could be used either as capture or as detection mAb. They were Protein G-purified and biotinylated when used as detection mAb. The setup of two sandwich ELISAs with standards of human recombinant (hr) PCT, using either CALCA 2F3 (assay A) or CALCA 4A6 (assay B) as capture mAbs and the biotinylated mAbs CALCA 4A6 or CALCA 2F3, respectively, as detection mAbs, led to highly specific determinations of PCT without cross-reactivity to calcitonin and katacalcin. Test midpoints (IC(50)) of both assays were determined for hrPCT standards in 4% (w/v) human serum albumin and found with 2.5 (assay A) and 2.7 μg L(-1) (assay B). With both sandwich ELISAs a collection of eight patient serum samples have been determined in comparison to the determination by the Elecsys BRAHMS PCT assay. Good correlations between our prototype ELISAs and the BRAHMS assay could be demonstrated (R (2): assay A, 0.996 and assay B, 0.990). The use of these newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT(Δ69-83)), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.

The aim of this prospective study was to assess the value of speckle tracking echocardiographic (2D-STE) parameters to predict response to heart failure therapy in patients with dilated cardiomyopathy (DCM). Eighty-seven patients (mean age 51 ± 13 years) with DCM, defined as ejection fraction (EF)45 %, left ventricular (LV) end-diastolic diameter112 % of normal range derived from age and body surface area. Based on 2D-STE following parameters were extracted from three apical views of the LV: global longitudinal strain, systolic and diastolic strain rate (SRE). Mechanical dispersion was calculated as standard deviation of time-to-peak strain values including all LV segments. After receiving heart failure therapy (mean 39 ± 11 months, range 3-60 months) 50 patients reached combined endpoint defined as following: death, heart transplantation, rehospitalization due to heart failure, and absence of improvement in EF. On stepwise multivariate regression analysis, SRE was independently of EF and LV volumes predictive for combined endpoint (OR 0.44, 95 %CI 0.27-0.70, p = 0.001) with an area under the ROC-curve (AUC) of 0.91. In patients with cQRS duration ≤120 ms mechanical dispersion was predictive for combined endpoint with the highest AUC (OR 1.53, 95 %CI 1.08-2.16, p = 0.002; AUC = 0.94). In this study, SRE, a surrogate parameter of myocardial relaxation, was able to predict a response to heart failure therapy in patients with DCM. In patients with narrow QRS complex, mechanical dispersion yielded the highest predictive value. Parameters of 2D-STE may contribute to risk stratification in this patient population.

Hemophilia A, which is caused by a mutation in the Factor 8 (F8) gene resulting in a deficiency or lack of the Factor VIII (FVIII) protein, is the most common inherited bleeding disorder in humans with an estimated worldwide incidence of half a million people. The disorder is X-linked and occurs in approximately 1 in 5,000 males; however there is also a growing appreciation of the impact on carrier females having a single mutant allele, with at least 10% of hemophilia A female carriers having less than normal clotting activity. Even modest increases in Factor V III activity (>1% of normal) can have a positive impact on patient lives, thus making the disease an ideal candidate for liver-directed gene therapy. Recombinant AAV (rAAV) has been used extensively for nearly 20 years as a gene therapy vector in preclinical and clinical studies where rAAV delivery to non-dividing tissues such as liver, brain and muscle affords stable, long-term transgene expression. However, there has been a lag in the clinical translation of a rAAV gene therapy approach for Hemophilia A/human F8 (hF8) compared to Hemophilia B/human Factor 9 due to poor yields of rAAV encoding a F8 transgene at clinical scale, and a requirement for large doses of rAAV F8 vector to achieve therapeutically relevant levels of circulating human FVIII (hFVIII), with the attendant risk of inducing an AAV-directed immune response requiring transient immunosuppression. To address these issues we optimized a rAAV F8 cDNA vector cassette to improve both virus yields and liver-specific hFVIII expression. The rAAV F8 cDNA vector cassette optimization required multi-factorial modifications to the liver-specific promoter module, hF8 transgene, synthetic polyadenylation signal and vector backbone sequence. This iterative process resulted in improved vector yields at research scale and greater than five-fold improvement in vector yields at clinical scale using our proven manufacturing process. Administration of the optimized rAAV hF8 cDNA packaged in serotype AAV2/6 at a dose of ~7.2E+12 vg/kg to both wild type and Hemophilia A mice resulted in robust circulating hFVIII levels and activity (levels in wild type mice were 241.6% of normal, and activity in Hemophilia A mice were 330.9% of normal). An analysis of hF8 mRNA levels in different tissues following dosage with our optimized vector demonstrated that hF8 expression from the modified promoter module was restricted to the liver. Notably there was a striking impact on hemostasis in the Hemophilia A mice treated with the optimized rAAV hF8 cDNA, with a reduction in bleeding time from 38.3 minutes to 2.5 minutes in treated mice (n = 12, p-value < 0.0001), which is in line with bleeding times in wild type mice. Initial studies in non-human primates (NHPs) resulted in supraphysiological levels of circulating hFVIII with mean peak values of 400-600% of normal levels. A follow up dose-ranging study was performed in NHPs with a rAAV2/6 F8 cDNA vector manufactured using our GMP clinical manufacturing process. Administration of vector doses ranging from 6E+11 vg/kg to 6E+12 vg/kg resulted in therapeutic circulating levels of hFVIII that were 5% - 229% of normal levels. The peak circulating hFVIII levels achieved in this dose-ranging study using GMP clinical-scale vector exceeds the levels previously reported in NHPs by several fold on an AAV vector dose basis. The high potency of this enhanced rAAV F8 cDNA cassette could significantly reduce the dose required to achieve therapeutically relevant levels in human subjects and reduce the potential of developing immune responses to AAV capsid requiring immunosuppression. Disclosures Riley: Sangamo BioSciences Inc: Employment. Boonsripisal:Sangamo BioSciences Inc: Employment. Goodwin:Sangamo BioSciences Inc: Employment. Garces:Sangamo BioSciences Inc: Employment. Ballaron:Sangamo BioSciences Inc: Employment. Tran:Sangamo BioSciences Inc: Employment. Kang:Sangamo BioSciences Inc: Employment. Zhang:Sangamo BioSciences Inc: Employment. Meyer:Sangamo BioSciences Inc: Employment. Greengard:Sangamo BioSciences Inc: Employment. Surosky:Sangamo BioSciences Inc: Employment. Ando:Sangamo BioSciences Inc: Employment. Lillicrap:bayer: Research Funding; biogen: Research Funding; CSL: Research Funding; Octapharma: Research Funding; Sangamo Biosciences Inc: Research Funding. Nichol:Sangamo BioSciences Inc: Employment. Holmes:Sangamo BioSciences Inc: Employment.

Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).

Hunter syndrome (Mucopolysaccharidosis Type II, MPS II) is a rare X-linked lysosomal disorder caused by lack of functional iduronate-2 sulfatase (IDS) enzyme and subsequent accumulation of glycosaminoglycans (GAG) in affected individuals. Manifestations include skeleton dysplasia, splenohepatomegaly, cardiopulmonary obstruction, and shortened life expectancy. In severe cases there is also neurologic impairment. Enzyme replacement therapy (ERT) is currently the only FDA-approved treatment to manage disease progression; however, ERT does not affect neurological aspects of the disease and requires that patients receive long and costly infusions of replacement factor on a frequent basis. We have developed a zinc-finger nuclease (ZFN) approach to insert the human IDS (hIDS) coding sequence into the albumin locus using AAV2/8 vectors. In this study IDS-deficient MPS II mice (n= 8-13 per group, age 7-8 weeks) were treated by intravenous infusion of a mixture of ZFN-encoding AAV vectors along with an AAV vector encoding the hIDS partial cDNA flanked by albumin sequence homology arms at three different vector doses. Wild-type littermates, untreated MPS II mice, and MPS II animals infused only with the hIDS donor vector (without ZFN-encoding vectors) were included as controls. Successful insertion of the hIDS coding sequence will result in hIDS expression regulated by the endogenous albumin promoter. Plasma and tissue IDS activities as well as urine and tissue GAG contents were monitored throughout the study to evaluate the effectiveness of the treatment. Sufficient animals were maintained for neurobehavioral testing at four-months post-injection to determine whether the treatment is neurologically beneficial. We found that IDS activities in the plasma of the treated groups were 10- to 100-fold higher than wild-type and stably expressed through the entire study duration in a dose-dependent fashion, while only very low levels of IDS activity were found in the animals infused with hIDS donor vector alone. At 4 weeks post-treatment IDS activities in peripheral tissues ranged from 1% to 200% wild-type in a dose-dependent fashion, while in the hIDS donor-only group enzyme activity was not detected in any tissue except liver (10% that of wild-type). We observed up to 2% of the wild-type IDS activity in the brains of animals administered the complete set of AAV vectors, while no IDS activity was observed in the brains of animals infused with the IDS donor vector alone. Urine GAGs were reduced in all of the ZFN + Donor treatment groups regardless of the vector dose. Tissue GAGs in the treatment groups were also decreased, but GAG content in the brain was not different from untreated MPS II litter mates at the initial analysis conducted four weeks post-treatment. No tissue GAG reduction was observed in animals infused with hIDS donor vector alone. Before conclusion of this study, animals from all six groups will be tested in the Barnes Maze as neurobehavioral assessment to determine the neurological effect of targeted hIDS expression in the liver. These results together with hIDS expression and GAG level data from final necropsy tissues will be presented. These results demonstrate that ZFN can be effectively used to mediate in vivo insertion of the hIDS coding sequence into the albumin locus with resultant stable and high-level hIDS enzyme expression and metabolic correction in MPS II.

Hemophilia A is the most common inherited bleeding disorder in humans and is caused by a deficiency of blood coagulation factor VIII (FVIII). This disease is an ideal candidate for liver-directed gene therapy, as even modest increases in FVIII activity (>1% of normal) can ameliorate the severe bleeding phenotype. Adeno-associated viral (AAV) vectors have shown great promise in both preclinical and clinical trials to efficiently deliver therapeutic transgenes to the liver. Suboptimal virus yields of AAV comprising human FVIII (hFVIII) for use in human therapy have hampered clinical scale manufacturing, both from mammalian (HEK293) and insect cells (Baculovirus system). We optimized an hFVIII AAV vector cassette to improve both virus yields and liver-specific hFVIII expression. Compared to historical hFVIII AAV vector cassettes that routinely produce 20% of standard, non-hFVIII containing AAV2/6 virus preparations, the improved hFVIII AAV vector cassette produced yields 100% of standard AAV2/6, both from HEK293 cells at the cell factory (CF) scale, but also in the Baculovirus system at large scale. Improved yields were also observed using other AAV serotypes including hFVIII AAV2/8 and AAV2/9. Using virus produced from the improved hFVIII AAV cassette we transduced mice at 6E+10 vg/mouse (~2E+12 vg/kg). Peak levels of hFVIII protein were achieved in mouse plasma at day 14 and represented supraphysiological levels of normal hFVIII in humans with serotypes AAV2/8 and AAV2/9; up to 337% and 516% of normal hFVIII plasma levels respectively (1U = 200 ng/mL = 100%). Transducing mice with serotype AAV2/6, known to be inefficient at transducing mouse liver, at 6E+10 vg/mouse (~2E+12 vg/kg) we achieved a mean peak value of 91.9 % +/- 15.5 SEM (n=6) of normal hFVIII plasma levels in humans. At a higher dose representing ~6E+12 vg/kg we achieved a mean peak value of hFVIII plasma over six independent in vivo mouse studies of 169.2 % +/- 10.1 SEM (n = 36). Supraphysiological hFVIII plasma levels were also achieved in non-human primates at a dose of 2E+12 vg/kg using serotype AAV2/6, representing up to 840% of normal hFVIII levels (8.4U). The robust production of hFVIII from the enhanced hFVIII AAV vector could significantly reduce the dose required to achieve therapeutic levels in human subjects. Additionally the improved hFVIII AAV vector cassette will enable clinical scale manufacturing.

Negative feedback regulation of insulin signaling involves ubiquitin-dependent degradation of insulin receptor substrate 1 (IRS1).Cullin-RING E3 ubiquitin ligase 7 (CRL7) mediates the ubiquitination of IRS1 in hyperphosphorylated form.Multisite IRS1 phosphorylation triggers interactions with CRL7 for ubiquitin modification.Insulin signaling is self-restrained when its downstream effector kinases are hyperactivated to trigger the negative feedback inhibition. Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase S6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain PI3K activity and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser-307/Ser-312 and Ser-527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyperphosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1-260 led to substantial reduction of ubiquitination efficiency, suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and the N terminus of IRS1 for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.

Indications for prophylactic implantable cardioverter-defibrillator implantation in patients with nonischemic dilated cardiomyopathy (DCM) are based on left ventricular (LV) ejection fraction (LVEF), although LVEF has limited ability to predict arrhythmias. It has recently been shown that strain echocardiography can predict ventricular arrhythmias in patients after myocardial infarction. The aim of this study was to evaluate whether strain echocardiography may help in the risk stratification of ventricular arrhythmias in patients with DCM.Ninety-four patients with nonischemic DCM were prospectively included. By speckle-tracking strain echocardiography, global longitudinal strain was calculated as the average of peak longitudinal strain from a 16-segment LV model. The time interval from electrocardiographic peak R to peak negative strain was assessed in each LV segment. Mechanical dispersion was defined as the standard deviation of time to peak negative strain from 16 LV segments.After a median of 22 months of follow-up (range, 1-46 months), 12 patients (13%) had experienced arrhythmic events, defined as sustained ventricular tachycardia or cardiac arrest. LVEF and global longitudinal strain were reduced in patients with DCM with arrhythmic events compared with those without (28 ± 10% vs 38 ± 13%, P = .01, and -6.4 ± 3.3% vs -12.3 ± 5.2%, P.001, respectively). Global longitudinal strain showed greater area under the curve than LVEF to identify arrhythmic events in receiver operating characteristic curve analyses (P = .05). Patients with arrhythmic events had increased mechanical dispersion (98 ± 43 vs 56 ± 18 ms, P.001). Mechanical dispersion predicted arrhythmias independently of LVEF (hazard ratio, 1.28; 95% confidence interval, 1.11-1.49; P = .001).Global longitudinal strain is a promising marker of arrhythmias. Mechanical dispersion predicted arrhythmic events in patients with DCM independently of LVEF. Strain echocardiography may help in the risk stratification of patients with DCM not fulfilling current implantable cardioverter-defibrillator indications.

To determine association of anemia and red blood cell (RBC) transfusions with necrotizing enterocolitis (NEC) in preterm infants. A total of 111 preterm infants with NEC ⩾stage 2a were compared with 222 matched controls. In all, 28 clinical variables, including hematocrit (Hct) and RBC transfusions were recorded. Propensity scores and multivariate logistic regression models were created to examine effects on the risk of NEC. Controlling for other factors, lower Hct was associated with increased odds of NEC (odds ratio (OR)=1.10, P=0.01). RBC transfusion has a temporal relationship with NEC onset. Transfusion within 24 h (OR=7.60, P=0.001) and 48 h (OR=5.55, P=0.001) has a higher odds of developing NEC but this association is not significant by 96 h (OR=2.13, P=0.07), post-transfusion. Anemia may increase the risk of developing NEC in preterm infants. RBC transfusions are temporally related to NEC. Prospective studies are needed to better evaluate the potential influence of transfusions on the development of NEC.

Hemophilia is an attractive target for gene therapy, since activity levels as low as 1% to 2% of normal are beneficial and levels of ~5% prevent spontaneous bleeding. Our goal was to provide a single treatment that permanently enables hepatic production of therapeutic levels of hFIX activity to decrease or potentially eliminate the need for prophylactic treatment in hemophilia B patients. We performed targeted in vivo genome editing using 1) two zinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus, and 2) a human F9 donor template construct. The ZFNs and donor template are encoded on separate hepatotropic adeno-associated virus serotype 2/6 (AAV2/6) vectors injected intravenously, resulting in targeted insertion of a corrected copy of the hF9 gene into the albumin locus in a proportion of liver hepatocytes. The albumin locus was selected as a "safe harbor" as production of this most abundant plasma protein exceeds 10 g/day, and moderate reductions in those levels are well-tolerated. These genome edited hepatocytes produce normal hFIX in therapeutic quantities, rather than albumin, driven by the highly active albumin enhancer/promoter, to treat hemophilia B; the genetic modification is expected to be sustained even in the face of hepatocyte turnover, making this approach attractive for treating young children with hemophilia before the appearance of significant organ damage. Transformed and primary human hepatocytes transduced in vitro with AAV2/6 encoding human albumin ZFNs and a promoterless hF9 transgene were shown to secrete hFIX. Extensive molecular analyses demonstrated that this was due to targeted integration of the hF9 transgene at the albumin locus and splicing of this gene into the albumin transcript. By employing AAV2/6 delivery of murine-specific ZFNs in vivo, stable levels of hFIX were observed in blood of mice injected with the albumin ZFNs and hF9 transgene donor. C57BL/6 mice were administered vehicle (n=20) or AAV2/6 vectors (n=25) encoding mouse surrogate reagents at 1.0 x1013 vector genome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX in the treated mice showed peak levels of 50-1053 ng/mL that were sustained for the duration of the 6-month study. Analysis of FIX activity from mouse plasma confirmed bioactivity commensurate with expression levels. Next, we report the feasibility of this approach in non-human primates (NHPs), showing that a single intravenous co-infusion of AAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and a human F9 donor at 1.2x1013 vg/kg (n=5/group) resulted in >50 ng/mL (>1% of normal) in this large animal model. The use of higher AAV2/6 doses (up to 1.5x1014 vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or 20% of normal) in several animals and up to 2000 ng/ml (or 50% of normal) in a single animal, for the duration of the study (3 months). The treatment was well tolerated in mice and NHPs, with no significant toxicological findings related to AAV2/6 ZFN + donor treatment in either species at therapeutic doses. Together, these data support a clinical trial to determine if a single co-administration of ZFN and donor AAV vectors is sufficient to enable therapeutic and potentially lifelong production of the clotting factor for the treatment of Hemophilia B. Disclosures Wechsler: Sangamo BioSciences: Employment. Meyer:Sangamo Biosciences Inc: Employment. Spratt:Sangamo Biosciences Inc: Employment. Greengard:Sangamo Biosciences Inc: Employment. Santiago:Sangamo Biosciences Inc: Employment. Sproul:Sangamo Biosciences Inc: Employment. Surosky:Sangamo Biosciences Inc: Employment. Paschon:Sangamo Biosciences Inc: Employment. Dubois-Stringfellow:Sangamo Biosciences Inc: Employment. Ando:Sangamo Biosciences Inc: Employment. Nichol:Sangamo Biosciences Inc: Employment. Rebar:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment.

Pelvic floor outlet obstruction is a rare cause of severe constipation. Anal myectomy, subtotal colectomy, and medical therapy have limited success. The purpose of this study was to develop a short outpatient treatment using biofeedback techniques. Nine patients with severe constipation and straining resulting from pelvic floor outlet obstruction underwent complete investigation of the pelvic floor musculature and anal sphincter mechanism. Patients were unable to expel a 60-cc rectal balloon and had nonrelaxing puborectalis on defecography. The treatment protocol utilized anal surface electromyography to document improper straining and retrain pelvic floor muscles to relax during defecation. Sensory retraining with a rectal balloon, behavioral relaxation techniques, and defecation of simulated stool using a 120-cc Metamucil® (Procter & Gamble, Cincinnati, OH) slurry in the rectum allowed re-establishment of normal defecation in all nine patients. Repeat training was required in three patients during follow-up. Treatment of pelvic floor outlet obstruction with outpatient retraining techniques is possible.