We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.

Hepatic adeno-associated virus (AAV)-serotype 2 mediated gene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid-specific memory CD8(+) T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid-specific CD8(+) T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.

BACKGROUND The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX–R338L) transgene at a dose of 5×1011 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P = 0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P = 0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092.)

Intron retention (IR) is widely recognized as a consequence of mis-splicing that leads to failed excision of intronic sequences from pre-messenger RNAs. Our bioinformatic analyses of transcriptomic and proteomic data of normal white blood cell differentiation reveal IR as a physiological mechanism of gene expression control. IR regulates the expression of 86 functionally related genes, including those that determine the nuclear shape that is unique to granulocytes. Retention of introns in specific genes is associated with downregulation of splicing factors and higher GC content. IR, conserved between human and mouse, led to reduced mRNA and protein levels by triggering the nonsense-mediated decay (NMD) pathway. In contrast to the prevalent view that NMD is limited to mRNAs encoding aberrant proteins, our data establish that IR coupled with NMD is a conserved mechanism in normal granulopoiesis. Physiological IR may provide an energetically favorable level of dynamic gene expression control prior to sustained gene translation.

Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe β-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zincfinger domain as mammalian CTCF genes and thus the same DNAbinding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1184-4) contains supplementary material, which is available to authorized users.