ABSTRACT

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Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

Objective The aim of this study was to examine worldwide chronic venous disease (CVD) burden and evaluate effectiveness, adherence and satisfaction with conservative treatment. Materials and methods The VEIN Act Program (VAP) was an international, prospective, observational study that enrolled adult outpatients with lower-limb pain consulting for any CVD-related clinical presentation. Patients' CVD symptoms, Clinical Etiological Anatomic Pathophysiologic (CEAP) classification, and symptom intensity were recorded. After prescribing conservative treatments, patients returned for a follow-up visit. Result A total of 567 physicians enrolled 7987 patients, 7397 of whom were included in the analysis. Patients were mainly female (79.3%), mean age was 54.0 ± 15.1 years and mean body mass index was 26.8 ± 4.8 kg/m 2. CEAP clinical classifications were C 0s (3.6%), C 1 (19.4%), C 2 (21.8%), C 3 (32.0%) and C 4-C 6 (23.2%). Symptoms were reported by 89.6% of patients and were present across all CEAP classes. Mean intensity (10 cm visual analog scale) was > 5 cm for all symptoms. Only 30.7% of patients had previously consulted for leg problems, and 24.3% had previously received treatment. Conservative treatment alone was prescribed to 78.3% of patients, and in association with operative treatment in 21.6% of patients, and consisted mostly of venoactive drugs (VADs) [99.6%, mostly micronized purified flavonoid fraction] and compression therapy (63.4%) in combination with lifestyle changes such as weight loss. Adherence to prescribed duration was 65.2% for VADs, but only 29.1% wore compression therapy as prescribed. Symptom relief was high with VADs and compression therapy (> 96%), and the majority of patients were satisfied (94.1%). Conclusion Conservative treatments were beneficial across the spectrum of CVD, highlighting the importance of identifying patients early in the disease course.