Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats

Background: Vectors based on adeno-associated virus-8 (AAV8) have shown efficiency and efficacy for liver-directed gene therapy protocols following intravascular injection, particularly in relation to haemophilia gene therapy. AAV8 has also been proposed for gene therapy targeted at skeletal and cardiac muscle, again via intravascular injection. It is important to assess vector targeting at the level of virion accumulation and transgene expression in multiple species to ascertain potential issues relating to species variation in infectivity profiles. Methods: We used AAV8 vectors expressing human factor IX (FIX) from the liver-specific LP-1 promoter and administered this virus via the intravascular route of injection into 12 week old Wistar Kyoto rats. We assessed FIX levels in serum by ELISA and transgene expression at sacrifice by immunohistochemistry using anti-FIX antibodies. Vector DNA levels in organs we determined by real time PCR. Results: Administration of 1 × 1011 or 5 × 1011 scAAV8-LP1-hFIX vector particles/rat resulted in efficient production of physiological hFIX levels, respectively in blood assessed 4 weeks post-injection. This was maintained for the 4 month duration of the study. At 4 months we observed liver persistence of vector with minimal non-hepatic distribution. Conclusion: Our results demonstrate that AAV8 is a robust vector for delivering therapeutic genes into rat liver following intravascular injection

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway.

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5alpha dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver

Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques

The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 × 1012 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B. © 2002 by The American Society of Hematology

Influence of real-world characteristics on outcomes for patients with methicillin-resistant Staphylococcal skin and soft tissue infections:a multi-country medical chart review in Europe

BACKGROUND: Patient-related (demographic/disease) and treatment-related (drug/clinician/hospital) characteristics were evaluated as potential predictors of healthcare resource use and opportunities for early switch (ES) from intravenous (IV)-to-oral methicillin-resistant Staphylococcus aureus (MRSA)-active antibiotic therapy and early hospital discharge (ED). METHODS: This retrospective observational medical chart study analyzed patients (across 12 European countries) with microbiologically confirmed MRSA complicated skin and soft tissue infections (cSSTI), ≥3 days of IV anti-MRSA antibiotics during hospitalization (July 1, 2010-June 30, 2011), and discharged alive by July 31, 2011. Logistic/linear regression models evaluated characteristics potentially associated with actual resource use (length of IV therapy, length of hospital stay [LOS], IV-to-oral antibiotic switch), and ES and ED (using literature-based and expert-verified criteria) outcomes. RESULTS: 1542 patients (mean ± SD age 60.8 ± 16.5 years; 61.5% males) were assessed with 81.0% hospitalized for MRSA cSSTI as the primary reason. Several patient demographic, infection, complication, treatment, and hospital characteristics were predictive of length of IV therapy, LOS, IV-to-oral antibiotic switch, or ES and ED opportunities. Outcomes and ES and ED opportunities varied across countries. Length of IV therapy and LOS (r = 0.66, p < 0.0001) and eligibilities for ES and ED (r = 0.44, p < 0.0001) showed relatively strong correlations. IV-to-oral antibiotic switch patients had significantly shorter length of IV therapy (−5.19 days, p < 0.001) and non-significantly shorter LOS (−1.86 days, p > 0.05). Certain patient and treatment characteristics were associated with increased odds of ES (healthcare-associated/ hospital-acquired infection) and ED (patient living arrangements, healthcare-associated/ hospital-acquired infection, initiating MRSA-active treatment 1–2 days post cSSTI index date, existing ED protocol), while other factors decreased the odds of ES (no documented MRSA culture, ≥4 days from admission to cSSTI index date, IV-to-oral switch, IV line infection) and ED (dementia, no documented MRSA culture, initiating MRSA-active treatment ≥3 days post cSSTI index date, existing ES protocol). CONCLUSIONS: Practice patterns and opportunity for further ES and ED were affected by several infection, treatment, hospital, and geographical characteristics, which should be considered in identifying ES and ED opportunities and designing interventions for MRSA cSSTI to reduce IV days and LOS while maintaining the quality of care. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2334-14-476) contains supplementary material, which is available to authorized users

Polonium-210 poisoning: a first-hand account

Background: Polonium-210 (210Po) gained widespread notoriety after the poisoning and subsequent death of Mr Alexander Litvinenko in London, UK, in 2006. Exposure to 210Po resulted initially in a clinical course that was indistinguishable from infection or exposure to chemical toxins, such as thallium. Methods: A 43-year-old man presented to his local hospital with acute abdominal pain, diarrhoea, and vomiting, and was admitted to the hospital because of dehydration and persistent gastrointestinal symptoms. He was initially diagnosed with gastroenteritis and treated with antibiotics. Clostridium difficile toxin was subsequently detected in his stools, which is when he first raised the possibility of being poisoned and revealed his background and former identity, having been admitted under a new identity with which he had been provided on being granted asylum in the UK. Within 6 days, the patient had developed thrombocytopenia and neutropenia, which was initially thought to be drug induced. By 2 weeks, in addition to bone marrow failure, he had evidence of alopecia and mucositis. Thallium poisoning was suspected and investigated but ultimately dismissed because blood levels of thallium, although raised, were lower than toxic concentrations. The patient continued to deteriorate and within 3 weeks had developed multiple organ failure requiring ventilation, haemofiltration, and cardiac support, associated with a drop in consciousness. On the 23rd day after he first became ill, he suffered a pulseless electrical activity cardiorespiratory arrest from which he could not be resuscitated and was pronounced dead. Findings: Urine analysis using gamma-ray spectroscopy on day 22 showed a characteristic 803 keV photon emission, raising the possibility of 210Po poisoning. Results of confirmatory analysis that became available after the patient's death established the presence of 210Po at concentrations about 109-times higher than normal background levels. Post-mortem tissue analyses showed autolysis and retention of 210Po at lethal doses in several organs. On the basis of the measured amounts and tissue distribution of 210Po, it was estimated that the patient had ingested several 1000 million becquerels (a few GBq), probably as a soluble salt (eg, chloride), which delivered very high and fatal radiation doses over a period of a few days. Interpretation: Early symptoms of 210Po poisoning are indistinguishable from those of a wide range of chemical toxins. Hence, the diagnosis can be delayed and even missed without a high degree of suspicion. Although body surface scanning with a standard Geiger counter was unable to detect the radiation emitted by 210Po, an atypical clinical course prompted active consideration of poisoning with radioactive material, with the diagnosis ultimately being made with gamma-ray spectroscopy of a urine sample

Potential limits of AAV-based gene therapy with the use of new transgenes expressing factor IX fusion proteins

Introduction: The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV‐based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half‐life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). / Aim: Adeno‐associated viral vectors (AAV) mediating expression of hFIX‐Alb and hFIX‐Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half‐life translates to higher plasma levels of FIX. / Methods: Single‐stranded cross‐packaged AAV2/8 vectors expressing hFIX‐Alb, hFIX‐Fc and hFIX were evaluated in vitro, and in mice. / Results: Both hFIX‐Alb and hFIX‐Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV‐mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX‐fusion proteins were comparable to wild‐type hFIX. However, their expression levels were threefold lower than wild‐type hFIX in vivo most likely due to inefficient secretion. / Conclusion: This, the first, evaluation of hFIX‐fusion proteins in the context of AAV gene transfer suggests that the hFIX‐fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches

Weight-based antibiotic dosing in a real-world European study of complicated skin and soft-tissue infections due to methicillin-resistant <i>Staphylococcus aureus</i>

AbstractWe aimed to characterize real-world dosing of weight-based intravenous (IV) antibiotic therapy in patients hospitalized for methicillin-resistant Staphylococcus aureus (MRSA) complicated skin and soft-tissue infections (cSSTIs). This was a subgroup analysis of a retrospective chart review that captured data from 12 European countries. The study included patients ≥18 years old, hospitalized with an MRSA cSSTI between 1 July 2010 and 30 June 2011 and discharged alive by 31 July 2011. Patients treated with IV vancomycin, teicoplanin or daptomycin at any stage during hospitalization were included in this analysis. Analyses were conducted at the regimen level (dosing in mg/kg or in mg, frequency, and total daily dose (TDD)), with potentially multiple regimens per patient, and the patient level, categorizing patients into low, standard (labelled) and high dosing groups according to their initial MRSA-targeted regimen. Among the 1502 patients in the parent study, 998 patients contributed a total of 1050 daptomycin, teicoplanin or vancomycin regimens. Across all regimens, the mean initial TDDs were 6.3 ± 1.9 mg/kg for daptomycin, 10.5 ± 4.9 mg/kg for teicoplanin and 28.5 ± 11.5 mg/kg for vancomycin. A total of 789 patients received first-line therapy with one of the above antibiotics. The majority of patients receiving first-line teicoplanin and daptomycin (96% and 80%, respectively) received higher than labelled cSSTI doses, whereas vancomycin doses were lower than labelled doses in >40% of patients. These real-world data reveal significant deviation from labelled antibiotic dosing in 12 European countries and the potential for suboptimal outcomes in patients with MRSA cSSTIs

Comparison of vancomycin and linezolid in patients with peripheral vascular disease and/or diabetes in an observational European study of complicated skin and soft-tissue infections due to methicillin-resistant <i>Staphylococcus aureus</i>

AbstractSuboptimal antibiotic penetration into soft tissues can occur in patients with poor circulation due to peripheral vascular disease (PVD) or diabetes. We conducted a real-world analysis of antibiotic treatment, hospital resource use and clinical outcomes in patients with PVD and/or diabetes receiving linezolid or vancomycin for the treatment of methicillin-resistant Staphylococcus aureus complicated skin and soft-tissue infections (MRSA cSSTIs) across Europe. This subgroup analysis evaluated data obtained from a retrospective, observational medical chart review study that captured patient data from 12 European countries. Data were obtained from the medical records of patients ≥ 18 years of age, hospitalized with an MRSA cSSTI between 1 July 2010 and 30 June 2011 and discharged alive by 31 July 2011. Hospital length of stay and length of treatment were compared between the treatment groups using inverse probability of treatment weights to adjust for clinical and demographic differences. A total of 485 patients had PVD or diabetes and received treatment with either vancomycin (n = 258) or linezolid (n = 227). After adjustment, patients treated with linezolid compared with vancomycin respectively had significantly shorter hospital stays (17.9 ± 13.6 vs. 22.6 ± 13.6 days; p < 0.001) and treatment durations (12.9 ± 7.9 vs. 16.4 ± 8.3 days; p < 0.001). The proportions of patients prescribed oral, MRSA-active antibiotics at discharge were 43.2% and 12.4% of patients in the linezolid and vancomycin groups, respectively (p < 0.001). The reduction in resource use may result in lower hospital costs for patients with PVD and/or diabetes and MRSA cSSTIs if treated with linezolid compared with vancomycin

Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), the detection of minimal residual disease (MRD) correlates with outcome in the trial setting. However, MRD assessment does not guide routine clinical management and its assessment remains complex. We incorporated detection of the B cell, tumor-specific antigen CD160 to develop a single-tube, flow cytometry assay (CD160FCA) for CLL MRD to a threshold of 10(−4) to 10(−5). One hundred and eighty-seven patients treated for CLL were enrolled. Utilizing the CD160FCA methodology, there was a high level of comparison between blood and bone marrow (R=0.87, P<0.001). In a validation cohort, CD160FCA and the international standardised approach of the European Research Initiative on CLL group demonstrated high concordance (R=0.91, P<0.01). Patients in complete remission (CR) and CD160FCA negative had longer event-free survival (EFS) (63 vs 16 months, P<0.01) and prolonged time to next treatment (60 vs 15 months, P<0.001) vs MRD positive patients; with a median time to MRD positivity of 36 months. In multivariate analysis, CD160FCA MRD detection was independently predictive of EFS in patients in CR and even predicted EFS in the good-risk cytogenetic subgroup. CD160FCA offers a simple assay for MRD detection in CLL and gives prognostic information across different CLL risk groups