Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. SUMMARY: Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

Correction of human hemophilia A whole blood abnormalities with a novel bypass agent: zymogen-like FXa(I16L).

BACKGROUND: Approximately 30% of hemophilia A (HA) and 5% of hemophilia B patients develop inhibitors to protein replacement therapy, and this is the major cause of disease-related morbidity in the developed world. We previously developed zymogen-like factor Xa (FXa) molecules with impaired active site maturation, enabling a greater half-life than wild-type FXa while maintaining full procoagulant function in the prothrombinase complex. Here we evaluated the ability of zymogen-like FXa(I16L) to correct whole blood thromboelastometry abnormalities of severe HA subjects with and without inhibitors. METHODS: Fourteen severe HA subjects without and five with inhibitors were enrolled at baseline ( FVIII: C < 1%) > 5 half-lives from factor or bypass therapy. The subjects' whole blood was evaluated by thromboelastography (ROTEM(®) ) using INTEM analysis with two concentrations of FXa(I16L) or recombinant factor VIIa (rFVIIa). RESULTS: With 0.1 nm FXa(I16L) , clot time (CT, in minutes [min]) among HA subjects without and with inhibitors (mean = 2.87 min, 95% CI = 2.58-3.15 min, and mean = 2.9 min, 95% CI = 2.07-3.73 min, respectively) did not significantly differ from control CT (mean = 2.73 min, 95% CI = 2.62-2.85 min). Addition of 20 nm rFVIIa, simulating a 90-µg/kg dose, resulted in significantly prolonged CTs for HA subjects without and with inhibitors (mean = 5.43 min, 95% CI = 4.53-6.35 min, and mean = 4.25 min, 95% CI = 3.32-5.17 min, respectively) relative to controls. CONCLUSIONS: FXa(I16L) restored thromboelastometry CT to control values in severe HA subjects with and without inhibitors. The findings corroborate previous animal data and demonstrate the first evidence of zymogen-like FXa(I16L) correcting human HA subjects' whole-blood abnormalities and support the use of FXa(I16L) as a novel hemostatic agent.

Maternal obesity and increased nutrient intake before and during gestation in the ewe results in altered growth, adiposity, and glucose tolerance in adult offspring.

We evaluated the effects of preconception and gestational obesity in the ewe on offspring growth, metabolism, and glucose homeostasis. From 60 d before conception through parturition, multiparous ewes were fed 100% (control; n = 8) or 150% (obese, OB; n = 10) of NRC (1985) recommendations. Ewes on the OB diet increased BW by 30% from diet initiation to mating (P = 0.03) and by 52% by d 135 of gestation (P = 0.04), whereas control ewes increased BW by 7% (P = 0.65) from diet initiation to d 135 of gestation. Lambs were weaned at 120 d of age and were maintained as a group. At 19.5 ± 0.5 mo of age, offspring from control and OB ewes were individually penned and subjected to a 12-wk ad libitum feeding challenge. At the beginning and end of the feeding challenge, dual x-ray absorptiometry was used to determine percentage of body fat, and a frequently sampled intravenous glucose tolerance test (FSIGT) with minimal model analysis was used to assess insulin and glucose homeostasis. At the beginning of the feeding challenge, BW and percentage of body fat were similar for control and OB offspring, averaging 69.0 ± 1.5 kg and 5.3 ± 0.5%, respectively. At the initial FSIGT, glucose effectiveness and insulin sensitivity were reduced (P < 0.05) in offspring from OB compared with control ewes. During the feeding challenge, plasma concentrations of leptin were increased (P < 0.05) in offspring from OB compared with control ewes. Fasted plasma glucose before the feeding challenge tended to be greater (P = 0.06) in the OB offspring compared with the control offspring (83.3 ± 1.4 vs. 79.0 ± 1.6 mg/dL, respectively). At the end of the feeding challenge, fasted plasma glucose and insulin were increased (P < 0.05) in the OB offspring compared with the control offspring (84.0 ± 1.4 vs. 79.5 ± 1.5 mg/dL and 30.1 ± 2.1 vs. 23.4 ± 2.2 µIU/mL, respectively). During the feeding challenge, offspring from OB ewes consumed approximately 10% more feed (P < 0.05) and tended to have increased BW gain (approximately 14%; P = 0.08) compared with offspring from control ewes. At the final dual x-ray absorptiometry scan, percentage of body fat was greater (P < 0.05) for offspring from OB ewes than for offspring from control ewes (16.5 ± 1.2 vs. 10.8 ± 1.1%). At the final FSIGT, offspring from OB ewes had a decreased (P ≤ 0.05) acute insulin response to glucose, disposition index, and glucose effectiveness, and tended (P = 0.10) to have a decreased insulin sensitivity compared with offspring from control ewes. Maternal obesity induced before and during gestation leads to alterations in appetite, glucose and insulin regulation, and adiposity of mature offspring.

Insulin sensitivity and glucose dynamics during pre-weaning foal development and in response to maternal diet composition.

Nutritional management of animals during pregnancy can affect glucose and insulin dynamics in the resulting offspring through influences on fetal development. Additionally, high starch feeding in mature horses is associated with reduced insulin sensitivity and an increased risk for diseases such as obesity and laminitis. However, no study has yet evaluated the effect of feeding a high starch diet to pregnant mares on glucose and insulin dynamics in their offspring. Twenty late-gestation mares maintained on pasture were provided two-thirds of digestible energy requirements from isocaloric, isonitrogenous low starch (LS, n=10) or high starch (HS, n=10) feed. Their foals were assessed with an insulin-modified frequently sampled intravenous glucose tolerance test at 5, 40, 80, and 160 d of age. Baseline glucose concentrations, insulin sensitivity, and insulin-independent glucose clearance in 5-d foals were all greater than values observed in mature horses and declined towards mature values as foals reached 160 d of age. Baseline glucose concentrations were all within normal range, but higher in foals born from HS mares through 80 d of age. Insulin sensitivity was not different between dietary groups until a trend for lower insulin sensitivity in HS foals emerged at 160 d of age. These data are the first to characterize decreasing insulin sensitivity and glucose tolerance in Thoroughbred foals from 5 to 160 d of age. This study also presents the first data examining glucose and insulin dynamics in developing foals in response to maternal high starch diet.

cDNA cloning and characterisation of an alpha-glucosidase gene from potato (Solanum tuberosum L.).

Using an Arabidopsis thaliana expressed sequence tag with sequence similarity to human lysosomal alpha-glucosidase as a probe, a potato cDNA was isolated. The cDNA encodes a polypeptide with an Mr value of 105,400 and the most significant matches of the deduced amino acid sequence are with members of family 31 of glucosyl transferase. The potato cDNA was expressed in a strain of Saccharomyces cerevisiae that is deficient in maltase activity and unable to grow using maltose as a carbon source (ABYSMAL81). Expression of the potato cDNA in the mutant yeast strain restores its ability to use maltose as a carbon source for growth. Additionally, alpha-glucosidase activity could be measured in extracts of the yeast cells following complementation. A range of maltodextrins were substrates for this activity. The steady-state expression level of the potato alpha-glucosidase gene was low in most tissues examined, the highest levels occurring in sprouting tubers and source leaves.

Surface roughness of glass-ceramic insert-composite restorations: assessing several polishing techniques.

The authors compared the effectiveness of seven polishing methods on glass-ceramic insert-composite restorations placed in plastic resin squares. The polishing methods used carbide dental finishing burs and diamond polishing paste, diamond abrasive finishing burs and diamond polishing paste, diamond abrasive finishing burs and composite resin finishing disks, diamond abrasive finishing burs and composite resin polishing points, diamond abrasive finishing burs only, diamond abrasive finishing burs followed by resin impregnated disks and an aluminum oxide polishing abrasive paste, and diamond abrasive finishing burs followed by diamond polishing paste. All systems achieved comparable smoothness except the carbide finishing burs, which damaged the insert-composite margin.

Reduction of marginal gaps in composite restorations by use of glass-ceramic inserts.

The objective of this study was to evaluate the effects of glass-ceramic inserts on reducing the marginal gaps caused by polymerization shrinkage in composite restorations. A light microscope was used to measure the largest gap at margins around restorations made in glass cylinders and tooth cavities with and without adhesion promoters. Where the cylinder was not silanated, the average gap was less in samples containing an insert than in those without. Two preparations were made in the dentin of 20 human molars. In each molar one cavity was restored with a dentin bonding agent and composite and the other with a dentin bonding agent and an insert seated in the composite. The average maximum gap width of restorations containing inserts was statistically less than for those with only composite (paired t-test, P<0.0001). When considering the volume of composite displaced by the insert, these results that the use of a glass-ceramic insert decreased the marginal gaps resulting form polymerization shrinkage.

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato.

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.

Mechanical properties of direct and post-cured composites.

Surface mechanical properties (diametral tensile strength, Knoop hardness, and Rockwell Superficial indentation and recovery) and bulk mechanical properties (compressive strength and modulus of elasticity in compression) of seven composites were measured in vitro under two curing conditions (light-curing only and light-curing plus manufacturer's recommended post-curing). Post-curing improved the Knoop hardeness by 7 to 46% and diametral tensile strength by 15 to 39% of some of the composites. None of the composites had improved compressive strength and only two had an improved modulus of elasticity.

An esthetic glass-ceramic for use in composite restoration inserts.

The objective of the preliminary work reported here was to prepare an improved formulation of intrinsically colored microcrystalline glass-ceramic. Applications could include "megafillers" for direct composite restorations, precision castings, and CAD-CAM prostheses. The experimental glass-ceramic reported here contained SiO2 56.9, AI2O3 19, LiO2 7, ZnO 6, MgO 5, TiO22, ZrO22, P2O52, and CeO20.1 mole%. The batch materials were melted and stirred at 1,610 degrees C for 2 h, quenched in water and also formed into a block of a clear, slightly yellow glass. To identify the crystalline phases that developed during transformation of the glass to the ceramic, x-ray diffraction was used on ten aliquots taken during 15 h of stepwise heating from 750 to 1050 degrees C. With heating, the yellow color deepened to a very translucent "dark yellow" dental shade, then lightened with gradually increasing opacity during formation of secondary crystalline phases. X-ray opacity was approximately equivalent to that of dental enamel. The refractive index of the glass, nD1.554, increased during nucleation and growth of the crystalline phases to a maximum of 1.586. Intrinsic coloration of these glass-ceramic materials can be controlled by varying the heat treatment and/or composition to match typical dental shades.

Differential Expression and Sequence Analysis of Ribosomal Protein Genes Induced in Stolon Tips of Potato (Solanum tuberosum L.) during the Early Stages of Tuberization.

cDNA clones of two genes, TUBS19 and TUBL7, which show a 15- to 20-fold increase in transcript level in the stolon tip during the early stages of tuberization, have been isolated by differential screening. These genes are also expressed in leaves, stems, and roots, and the expression pattern in these organs changes on tuberization. Southern analysis shows that there are similar sequences in the genome of nontuberizing wild-type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUBS19 cDNA and the eukaryotic S19 ribosomal protein gene. TUBL7 cDNA shows similarity to another eukaryotic ribosomal protein gene, L7.

Isolation and molecular characterisation of a tuberisation-related cDNA clone from potato (Solanum tuberosum L.).

A cDNA clone of a gene which shows a large increase in transcript level in the stolon tip during the early stages of tuberisation in potato (Solanum tuberosum) has been isolated by differential screening. This gene is also expressed at low levels in other parts of the plant including leaves and stems. Sequence analysis and comparison did not reveal any significant similarity with other gene sequences in the EMBL database. DNA-blot analysis indicates that the gene is present as a single copy in the potato genome and a restriction fragment length polymorphism exists between wild type and cultivated potatoes.