A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency.

Manufacturing of adeno-associated viruses (AAV) for gene and cell therapy applications has increased significantly and spurred development of improved mammalian and insect cell-based production systems. We developed a baculovirus-based insect cell production system-the SGMO Helper-with a novel gene architecture and greater flexibility to modulate the expression level and content of individual Rep and Cap proteins. In addition, we incorporated modifications to the AAV6 capsid sequence that improves yield, capsid integrity, and potency. Production of recombinant AAV 6 (rAAV6) using the SGMO Helper had improved yields compared to the Bac-RepCap helper from the Kotin lab. SGMO Helper-derived rAAV6 is resistant to a previously described proteolytic cleavage unique to baculovirus-insect cell production systems and has improved capsid ratios and potency, in vitro and in vivo, compared with rAAV6 produced using Bac-RepCap. Next-generation sequencing sequence analysis demonstrated that the SGMO Helper is stable over six serial passages and rAAV6 capsids contain comparable amounts of non-vector genome DNA as rAAV6 produced using Bac-RepCap. AAV production using the SGMO Helper is scalable using bioreactors and has improved yield, capsid ratio, and in vitro potency. Our studies demonstrate that the SGMO Helper is an improved platform for AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.

The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2.

Chemotherapy often generates intratumoral senescent cancer cells that strongly modify the tumor microenvironment, favoring immunosuppression and tumor growth. We discovered, through an unbiased proteomics screen, that the immune checkpoint inhibitor programmed cell death 1 ligand 2 (PD-L2) is highly upregulated upon induction of senescence in different types of cancer cells. PD-L2 is not required for cells to undergo senescence, but it is critical for senescent cells to evade the immune system and persist intratumorally. Indeed, after chemotherapy, PD-L2-deficient senescent cancer cells are rapidly eliminated and tumors do not produce the senescence-associated chemokines CXCL1 and CXCL2. Accordingly, PD-L2-deficient pancreatic tumors fail to recruit myeloid-derived suppressor cells and undergo regression driven by CD8 T cells after chemotherapy. Finally, antibody-mediated blockade of PD-L2 strongly synergizes with chemotherapy causing remission of mammary tumors in mice. The combination of chemotherapy with anti-PD-L2 provides a therapeutic strategy that exploits vulnerabilities arising from therapy-induced senescence.

Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype.

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). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.

A sensitive AAV transduction inhibition assay assists evaluation of critical factors for detection and concordance of pre-existing antibodies.

Pre-existing antibodies to viral capsids may have a negative impact on the efficacy and safety of adeno-associated virus (AAV)-based gene therapies. Total antibody (TAb) and/or cell-based transduction inhibition (TI) assays have been used to exclude seropositive individuals in clinical studies. Published AAV seroprevalence and patient enrollment criteria regarding antibody status lack comparability between assay formats, hindering a direct cross-study comparison. To identify critical factors impacting TI assay detection of AAV neutralizing antibodies (NAbs), we created a reporter construct expressing NanoLuc® luciferase (Nluc) that enabled a more sensitive and robust detection of AAV6 NAbs than using firefly luciferase. Assessment of additional factors including multiplicity of infection, cell lines, viral production, and capsid purity revealed the reporter is the major determinant of assay sensitivity impacting NAb detection. The Nluc reporter was further used to assess seroprevalence to AAV5, 8, and 9. Last, we compared AAV6 Nluc TI with two TAb assay formats. A higher correlation of Nluc TI was observed with direct binding (90%) than with the more sensitive bridging TAb assay (65%), suggesting both assay sensitivity and TAb formats contribute to AAV seropositivity concordance. Our results support a need to standardize assay formats to ensure proper assessment of pre-existing AAV immunity.

Human senescent fibroblasts trigger progressive lung fibrosis in mice.

Cell senescence has recently emerged as a potentially relevant pathogenic mechanism in fibrosing interstitial lung diseases (f-ILDs), particularly in idiopathic pulmonary fibrosis. We hypothesized that senescent human fibroblasts may suffice to trigger a progressive fibrogenic reaction in the lung. To address this, senescent human lung fibroblasts, or their secretome (SASP), were instilled into the lungs of immunodeficient mice. We found that: (1) human senescent fibroblasts engraft in the lungs of immunodeficient mice and trigger progressive lung fibrosis associated to increasing levels of mouse senescent cells, whereas non-senescent fibroblasts do not trigger fibrosis; (2) the SASP of human senescent fibroblasts is pro-senescence and pro-fibrotic both in vitro when added to mouse recipient cells and in vivo when delivered into the lungs of mice, whereas the conditioned medium (CM) from non-senescent fibroblasts lacks these activities; and, (3) navitoclax, nintedanib and pirfenidone ameliorate lung fibrosis induced by senescent human fibroblasts in mice, albeit only navitoclax displayed senolytic activity. We conclude that human senescent fibroblasts, through their bioactive secretome, trigger a progressive fibrogenic reaction in the lungs of immunodeficient mice that includes the induction of paracrine senescence in the cells of the host, supporting the concept that senescent cells actively contribute to disease progression in patients with f-ILDs.

A NIR fluorescent probe for the detection of renal damage based on overrepresentation of alanine aminopeptidase enzyme.

Kidney damage generates changes at the phenotypic and genotypic levels that allow its monitoring using different biomarkers in blood, urine or serum. Among these biomarkers, kidney failure causes the urine overrepresentation of the alanine aminopeptidase (APN) enzyme. Here, we describe the design of a molecular probe (NB-ALA) based on the Nile Blue fluorophore (NB), which can detect the APN enzyme in urine by simple fluorometric measurements.

Clinical enrollment assay to detect preexisting neutralizing antibodies to AAV6 with demonstrated transgene expression in gene therapy trials.

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.

First-in-human in vivo genome editing via AAV-zinc-finger nucleases for mucopolysaccharidosis I/II and hemophilia B.

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.

Regulatory Consideration for the Nonclinical Safety Assessment of Gene Therapies.

The nonclinical safety assessments for gene therapies are evolving, leveraging over 20 years of experimental and clinical experience. Despite the growing experience with these therapeutics, there are no approved harmonized global regulatory documents for developing gene therapies with only the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) S12 guidance on nonclinical biodistribution currently under discussion. Several health authorities have issued guidance over the last 15 years on the nonclinical safety aspects for gene therapy products, but many of the recommendations are limited to high-level concepts on nonclinical safety aspects or altogether silent on key topics. Historically, this approach was appropriately vague given our relatively small dataset of nonclinical experience, where a comprehensive and detailed regulatory guidance approach was unlikely to be appropriate to address all scenarios. However, harmonization of key considerations and assumptions can provide a consistent basis for developing the appropriate nonclinical safety development plans for individual programs, reducing uncertainty across regulatory regions and unnecessary animal use. Several key areas of nonclinical safety testing are nearing maturation for a harmonized approach, including species selection, certain aspects of study design, study duration, and unintended genomic integration risks. Furthermore, several emerging topics are unaddressed in current regulatory guidance for gene therapy products, which will become key areas of differentiation for the next generation of therapeutics. These topics include redosing, juvenile/pediatric safety, and reproductive/developmental safety testing, where relevant experience from other modalities can be applied. The rationale and potential study design considerations for these topics will be proposed, acknowledging that certain aspects of gene therapy development are not considered appropriate for harmonization. This article provides an overview of the current nonclinical safety regulatory landscape, summarizes typical nonclinical safety study designs, highlights areas of uncertainty, and discusses emerging topics that warrant consideration. Specific recommendations and perspectives are provided to inform future regulatory discussions and harmonization efforts.

ZFN-mediated in vivo gene editing in hepatocytes leads to supraphysiologic α-Gal A activity and effective substrate reduction in Fabry mice.

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 with <10% 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.

Reconstruction of Enriched Uranium Released to Air from the Former Apollo Facility, Apollo, Pennsylvania.

ABSTRACT: The former Apollo facility converted enriched uranium hexafluoride into uranium oxide for shipment to nuclear fuel fabrication plants from 1957 to 1983. This paper describes quantification of the source term from the Apollo facility in terms of quantities of uranium released, particle size, and solubility characteristics. Releases occurred through stacks, rooftop vents, and an incinerator that operated from 1964 to 1969. Incidental and accidental releases are addressed as part of this analysis. Atmospheric releases of uranium from plant operations were estimated from stack sampling and production records. Roof vents, both filtered and unfiltered, were the major emission points from the plant. The total estimated release of uranium activity (including 234U, 235U, and 238U) to the air was 28 GBq. Measurements by others found that the releases were primarily associated with large particles and that their solubility was variable but generally low (Class Y). The release estimates presented here and those findings were incorporated into a sophisticated atmospheric transport model to estimate atmospheric concentrations and soil contamination levels due to the releases and to reconstruct historical doses to individuals that lived in the vicinity of the former Apollo facility.

Genetic ablation of Cullin-RING E3 ubiquitin ligase 7 restrains pressure overload-induced myocardial fibrosis.

Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.

CERE-120 Prevents Irradiation-Induced Hypofunction and Restores Immune Homeostasis in Porcine Salivary Glands.

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.

AAV2/6 Gene Therapy in a Murine Model of Fabry Disease Results in Supraphysiological Enzyme Activity and Effective Substrate Reduction.

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.

Transmitted Home Oximetry and Duration of Home Oxygen in Premature Infants.

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.

Development of a Nursing Assignment Tool Using Workload Acuity Scores.

OBJECTIVE: To determine a just and consistent practice for creating nursing assignments. BACKGROUND: Traditional methods of assigning patients to nurses may lead to unbalanced nursing workload. This article describes the ongoing, hospital-wide effort to evaluate and implement a nursing assignment tool based on electronic health record (EHR) functionality and auto-calculated nursing workload scores. METHODS: EHR records of individual patient workload scores from all hospital units were collected from August 2017 to June 2018. A nurse-specific total workload score was summed for each staff. Then, each hospital unit's mean nurse workload score and standard deviation, along with the unit's nurse-to-patient ratio, were used to calculate levels of high, medium, and low nursing workload measurement (NWM). RESULTS: Mean patient-specific workload scores varied greatly across hospital units. Unit-specific nurse-to-patient ratios were factored into NWM scores to create ranges for assignments that were relatively consistent across the institution. CONCLUSION: The use of objective, electronically generated nursing workload scores, combined with traditional nurse-to-patient ratios, provides accurate real-time nurse staffing needs that can inform best practice in staffing. The confirmation of individual patient workload scores and an appreciation for the complexity of EHR vendor rules are necessary for successful implementation. Automation ensures patient safety, staff satisfaction, and optimal resource allocation.

Reconstruction of atmospheric concentrations of enriched uranium from the former Apollo facility, Apollo, Pennsylvania, USA.

The former Apollo facility in western Pennsylvania converted enriched uranium hexafluoride into uranium oxide for shipment to nuclear fuel fabrication plants from 1957 to 1983. Atmospheric releases of uranium from plant operations were estimated from stack sampling and production records. Releases occurred through stacks, rooftop vents, and an incinerator that operated from 1964 to 1969. Roof vents that exhausted workplace air was the major emission source from the plant. Total estimated release of uranium activity (including 234U, 235U, and 238U) to the air was 27.9 GBq. Atmospheric transport modeling was performed using a complex terrain model because the plant was located in an incised river valley. Almost two years of meteorological data were collected from a nearby 10-m tower, along with sounding from a collocated sodar. Light mean wind speed (1.56 m s-1) and predominately stable atmospheric conditions frequently resulted in poor dispersion conditions in the facility environs. Environmental sampling included continuous air monitoring data and depth profiles of uranium in soil that was deposited from airborne releases. Soil measurements exhibited a sharp drop-off in uranium concentrations with distance from the facility, indicating that large non-inhalable particles were emitted to the atmosphere. Large particles (~15-25 µm aerodynamic equivalent diameter) accounted for 17.5% of the total emissions. Soil measurements were used for model calibration and validation, while air measurements were used to evaluate model performance. Air concentrations were generally over-predicted for locations near the facility but showed only a slight positive bias for locations north of the facility. Predicted uranium activity air concentrations from Apollo sources averaged over 34 years were about three times greater than the background gross alpha activity value of 81 µBq m-3 in a ~0.5 km2 region surrounding the Apollo facility. The contribution of Apollo uranium to the gross alpha air concentration would have been negligible several kilometers from the facility.

ZFN-Mediated In Vivo Genome Editing Corrects Murine Hurler Syndrome.

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.

The emerging role of the corporate or system-level infection prevention director for integrated delivery networks.

BACKGROUND: One position in integrated delivery networks (IDNs) that provides centralized oversight to optimize patient safety is the corporate-level infection prevention (IP) director. After noting variability in their roles, responsibilities, and IP programs, a national network of IDN IP directors planned a member survey to better understand common and variable elements. Nine network members volunteered to design a survey to describe the current role, responsibilities, and resourcing of all members of the corporate IP director group. METHODS: A 17-question survey was designed using the Survey Monkey multiple-choice format with a comment option. The questions were reviewed by the entire network to ensure content validity. The survey was delivered to all 72 network members by e-mail, and a 44% response rate was achieved. RESULTS: Survey responses revealed variation and commonalities relative to role structure, responsibilities, resourcing, and level of physician support for corporate IP directors. In addition, advantages of the position were described. CONCLUSIONS: The results of the survey will serve as a foundation on which to build, supporting standardization and reliable design for the role, responsibilities, and resourcing of corporate IP directors, with the ultimate goal of improving patient safety.

Towards Personalized Medicine: An Improved De Novo Assembly Procedure for Early Detection of Drug Resistant HIV Minor Quasispecies in Patient Samples.

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.