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.)

Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

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.)

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinaseexpressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.The liver is the principal site for the metabolism of xenobiotic compounds, including medical drugs. Because many hepatic enzymes are species specific, it is necessary to evaluate the metabolism of candidate pharmaceuticals using cultured primary human hepatocytes 1,2 . Today, hepatocytes are isolated primarily from cadaveric organs and then shipped to the location where testing will be performed. The condition (viability and state of differentiation) of hepatocytes from cadaveric sources is highly variable, and many cell preparations are of marginal quality. The availability of high-quality human hepatocytes is further hampered by the fact that they cannot be substantially expanded in tissue culture 3,4 . Hepatocytes from readily available mammalian species, such as the mouse, are not suitable for drug testing because they have a different complement of metabolic enzymes and

Competition between mammalian RNAi-related gene silencing pathways is well documented. It is therefore important to identify all classes of small RNAs to determine their relationship with RNAi and how they affect each other functionally. Here, we identify two types of 59-phosphate, 39-hydroxylated human tRNA-derived small RNAs (tsRNAs). tsRNAs differ from microRNAs in being essentially restricted to the cytoplasm and in associating with Argonaute proteins, but not MOV10. The first type belongs to a previously predicted Dicer-dependent class of small RNAs that we find can modestly down-regulate target genes in trans. The 59 end of type II tsRNA was generated by RNaseZ cleavage downstream from a tRNA gene, while the 39 end resulted from transcription termination by RNA polymerase III. Consistent with their preferential association with the nonslicing Argonautes 3 and 4, canonical gene silencing activity was not observed for type II tsRNAs. The addition, however, of an oligonucleotide that was sense to the reporter gene, but antisense to an overexpressed version of the type II tsRNA, triggered robust, >80% gene silencing. This correlated with the redirection of the thus reconstituted fully duplexed double-stranded RNA into Argonaute 2, whereas Argonautes 3 and 4 were skewed toward less structured small RNAs, particularly single-strand RNAs. We observed that the modulation of tsRNA levels had minor effects on the abundance of microRNAs, but more pronounced changes in the silencing activities of both microRNAs and siRNAs. These findings support that tsRNAs are involved in the global control of small RNA silencing through differential Argonaute association, suggesting that small RNA-mediated gene regulation may be even more finely regulated than previously realized.

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (F.IX). Previously, we established an experimental basis for gene transfer as a method of treating the disease in mice and hemophilic dogs through intramuscular injection of a recombinant adeno-associated viral (rAAV) vector expressing F.IX. In this study we investigated the safety of this approach in patients with hemophilia B. In an open-label dose-escalation study, adult men with severe hemophilia B (F.IX < 1%) due to a missense mutation were injected at multiple intramuscular sites with an rAAV vector. At doses ranging from 2 ؋ 10 11 vector genomes (vg)/kg to 1.8 ؋ 10 12 vg/ kg, there was no evidence of local or systemic toxicity up to 40 months after injection. Muscle biopsies of injection sites performed 2 to 10 months after vector administration confirmed gene transfer as evidenced by Southern blot and transgene expression as evidenced by immunohistochemical staining. Preexisting high-titer antibodies to AAV did not prevent gene transfer or expression. Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1%. Although more extensive transduction of muscle fibers will be required to develop a therapy that reliably raises circulating levels to more than 1% in all subjects, these results of the first parenteral administration of rAAV demonstrate that administration of AAV vector by the intramuscular route is safe at the doses tested and effects gene transfer and expression in humans in a manner similar to that seen in animals. (Blood.