National Institute for Health Research, Guys and St Thomas' Charity, Chief Scientist Office Scotland, Tommy's Charity.
• Novel, more potent codonoptimized human FVIII variant (codop-hFVIII-V3).• Codop-hFVIII-V3 is safe and efficacious in mice and nonhuman primates, thus improving the prospects of gene therapy for hemophilia A.Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAVexpression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 6 162% of normal) in HA knockout mice following administration of 2 3 10 12 vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 6 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy. (Blood. 2013;121(17):3335-3344) Introduction Hemophilia A (HA), the most common inherited bleeding disorder, caused by a deficiency of factor VIII (FVIII) is well suited for a gene replacement approach, primarily because a modest increase in the level of FVIII (.1% of normal) can ameliorate the severe bleeding phenotype.1 Several gene transfer strategies for FVIII replacement have been evaluated. 2 However, adeno-associated viral (AAV) vectors currently show the greatest promise because of their excellent safety profile and ability to direct long-term transgene expression from postmitotic tissues such as the liver.3-5 Indeed, our ongoing gene therapy clinical trial for hemophilia B, a related bleeding diathesis, has demonstrated that a single peripheral vein administration of AAV vector leads to stable (.30 months) expression of human factor IX (FIX) at levels between 1% to 6% of normal. This is sufficient for conversion of the hemophilia phenotype from severe to moderate or mild.5 Two-thirds of the participants in this study have discontinued prophylaxis and remain free of spontaneous hemorrhage. The other participants have increased the interval between FIX prophylaxes. The use of AAV vectors for HA gene therapy, however, poses new challenges because of the distinct molecular and biochemical properties of human FVIII (hFVIII). Compared with other proteins of similar size, expression of hFVIII is highly inefficient.6 Bioengineeri...
PurposeOlder adults are more likely to be vitamin D deficient. The aim of the study was to determine whether these patients have worse outcomes with COVID-19.MethodsWe conducted a prospective cohort study between 1 March and 30 April 2020 to assess the importance of vitamin D deficiency in older patients with COVID-19. The cohort consisted of patients aged ≥65 years presenting with symptoms consistent with COVID-19 (n=105). All patients were tested for serum 25-hydroxyvitamin D (25(OH)D) levels during acute illness. Diagnosis of COVID-19 was confirmed via viral reverse transcriptase PCR swab or supporting radiological evidence. COVID-19-positive arm (n=70) was sub-divided into vitamin D-deficient (≤30 nmol/L) (n=39) and -replete groups (n=35). Subgroups were assessed for disease severity using biochemical, radiological and clinical markers. Primary outcome was in-hospital mortality. Secondary outcomes were laboratory features of cytokine storm, thoracic imaging changes and requirement of non-invasive ventilation (NIV).ResultsCOVID-19-positive arm demonstrated lower median serum 25(OH)D level of 27 nmol/L (IQR=20–47 nmol/L) compared with COVID-19-negative arm, with median level of 52 nmol/L (IQR=31.5–71.5 nmol/L) (p value=0.0008). Among patients with vitamin D deficiency, there was higher peak D-dimer level (1914.00 μgFEU/L vs 1268.00 μgFEU/L) (p=0.034) and higher incidence of NIV support and high dependency unit admission (30.77% vs 9.68%) (p=0.042). No increased mortality was observed between groups.ConclusionOlder adults with vitamin D deficiency and COVID-19 may demonstrate worse morbidity outcomes. Vitamin D status may be a useful prognosticator.
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