BACKGROUND Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS We infused a single dose of a serotype-8–pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid–specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.)

BACKGROUND In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose–response relationship, and the level of persistent or late toxicity. METHODS We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×1012 vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.)

We have established 51 solid tumor and 10 ALL in vivo models. The models identify vincristine and cyclophosphamide as having broad-spectrum activity. The PPTP tumor panels appear to generally recapitulate the activity of these agents against specific childhood cancers and to have the potential for identifying novel agents having significant clinical activity.

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We present the first crystal structures of a human protein bound to analogs of cocaine and heroin. Human carboxylesterase 1 (hCE1) is a broad-spectrum bioscavenger that catalyzes the hydrolysis of heroin and cocaine, and the detoxification of organophosphate chemical weapons, such as sarin, soman and tabun. Crystal structures of the hCE1 glycoprotein in complex with the cocaine analog homatropine and the heroin analog naloxone provide explicit details about narcotic metabolism in humans. The hCE1 active site contains both specific and promiscuous compartments, which enable the enzyme to act on structurally distinct chemicals. A selective surface ligand-binding site regulates the trimer-hexamer equilibrium of hCE1 and allows each hCE1 monomer to bind two narcotic molecules simultaneously. The bioscavenger properties of hCE1 can likely be used to treat both narcotic overdose and chemical weapon exposure.

• 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...

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

Background-MLN8237 is a small molecule inhibitor of Aurora Kinase A (AURKA) that is currently in early phase clinical testing. AURKA plays a pivotal role in centrosome maturation and spindle formation during mitosis.