[PMC free article] [PubMed] [Google Scholar]Deyle DR

[PMC free article] [PubMed] [Google Scholar]Deyle DR., and, Russell DW. initial 5.6?Kb construct (Construct A-Original) that used the insulin-like growth factor binding protein (IGFBP) promoter (184?bp), two 1-microglobulin/bikunin enhancers (E)(100?bp) and -globin intron (I)(175bp) with an SV40 polyadenylation signal (Poly A1)(263?bp). The 5.471?Kb Construct A-New contains the same elements as the Construct A-Original, however, it was modified during the cloning to eliminate unnecessary nucleotides and to include new restriction sites to facilitate the generation of the new smaller constructs. The hepatic control region-human alpha-1 anti-trypsin (HCR-hAAT) promoter constructs (Construct E and J) utilize a Zaldaride maleate shortened version of the hepatic control region of the human apolprotein ExxC-1 gene locus (E2). A 65?bp SV40 intron (I2) and a 134?bp SV40 polyadenylation (Poly A2) sequence is also used. These enhancer/promoter elements and the polyadenylation sequences were systematically altered by altering one regulatory element at a time (Construct B-E) and then multiple elements at a time Zaldaride maleate (Construct F-J) to determine if removal of any individual element may have a detrimental effect on FVIII expression. mt2010240x1.doc (92K) GUID:?89A26199-19A6-47C0-9E81-553DB69074F6 Physique S2: Hydrodynamic delivery of pAAV-cFVIII gene constructs. The pAAV plasmid constructs (n=10) were compared in vitro by transient transfection in HepG2 cells (data not shown) and in vivo by hydrodynamic infusion in hemophilia A mice. (a) cFVIII plasmid constructs that have a single regulatory element altered (Construct B-E, Supplementary Physique S1) were compared to the initial 5.6?Kb (Construct A-Original) or the new 5.5?Kb version (Construct A) after hydrodynamic infusion in HA mice (n = 3-5 per group). cFVIII activity was determined by Coatest assay. (b) cFVIII plasmid constructs that have multiple regulatory elements altered (Construct F-J, Supplementary Physique S1) were compared to the initial 5.6?kb (Construct A-Original) after hydrodynamic infusion in HA mice (n = 3-5 per group). Coatest assay was used to determine cFVIII activity. Error bars represent mean SD. Overall, none of the modifications resulted in significant decreases in FVIII expression. Three constructs that were the best performers in terms of expression in these studies were selected for AAV production (Construct G, H and J)(Physique 1). mt2010240x2.doc (74K) GUID:?9E443AB4-F106-48D3-A8A4-AC9CEE64E7C9 Figure S3: Thromboelastography of HA dogs after single chain delivery of cFVIII. Thromboelastographs were performed after administration of AAV8-cFVIII. The y-axis is the clot amplitude (mm) and the x-axis is usually time (min). R (min) is the time to initial fibrin formation Zaldaride maleate and the maximum amplitude (MA)(mm) is the amplitude of the clot. Prior to vector administration TEG analysis demonstrated that this R was not reached after 60 minutes and there was no clot present (MA and angle were not measurable). In the normal doggie the R value is usually 6-9 minutes, angle is usually 62-72 degrees and the MA is usually 66-72?mm. The % cFVIII activity is usually shown for that time point on each graph. TEG analysis exhibited a distinct dose response among dogs expressing different levels of FVIII. Thus, M06 had 8% cFVIII activity at the time of the TEG and had parameters that were closer to normal values than L51 that was expressing 1% cFVIII activity at the time of TEG analysis. The TEG tracings did not RAF1 fully normalize in the AAV treated dogs, demonstrating that this TEG can distinguish a wide Zaldaride maleate range of FVIII activity. mt2010240x3.doc (67K) GUID:?715AF3AF-8BEA-413C-BE04-2F7F41883B4E Table S1: Clinical blood chemistries after vector administration. mt2010240x4.doc (143K) GUID:?0EE012B4-5110-4B45-A532-732FD736D135 Materials and Methods. mt2010240x5.doc (40K) GUID:?F3B5C84A-DC36-4B3C-96F2-8CBD3D3E3D4B Abstract Developing adeno-associated viral (AAV)Cmediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the ((cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (= 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 the portal vein expressed long-term, vector doseCdependent levels of.