John E.J. Rasko

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

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 × 1012 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 ∼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*.

CD8 + T-cell responses to adeno-associated virus capsid in humans

Hepatic adeno-associated virus (AAV)-serotype 2 mediatedgene 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.

BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma

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-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding 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.

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells.

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.

Orchestrated intron retention regulates normal granulocyte differentiation.

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.

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B0. We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G → A and 0.001 for 718C → T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers

Inositol polyphosphate 4-phosphatase-II (INPP4B) is a regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B expression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas.

Predicting microRNA targets and functions: traps for the unwary.

To the Editor: microRNAs (miRNAs) are short RNAs that are important in gene regulation in many organisms. In mammals, they guide the RNA-induced silencing complex to target sites typically located in the 3′ untranslated regions (UTR) of mRNAs, causing translational repression and/or mRNA degradation1. Despite recent advances in large-scale target screening techniques, experimental validation of miRNA targets remains cumbersome, and computational approaches remain the most commonly used method to identify putative target genes. Most algorithms use sequences collected from either the Ensembl2 or University of California Santa Cruz (UCSC)3 databases and then preprocess these sequences to correct for genes that do not have a predicted 3′ UTR of sufficient length. However, these databases use different criteria to define 3′ UTR boundaries (Supplementary Fig. 1 online) and these boundaries will change as more sequences from varied tissue types are added to them4. To determine whether these differences can alter target prediction, we applied four popular algorithms, miRanda5, TargetScan6, RNA22 (ref. 7) and PITA8 (Supplementary Table 1 online), to two 3′ UTR databases obtained from the Ensembl and the UCSC servers (Table 1 and Supplementary Methods online). When we ran TargetScan and Miranda on sequences from the same database, the results overlapped by 39.5%, whereas when we ran the same two algorithms on two different databases, the overlap was 11.5% (Supplementary Fig. 2 online). This result demonstrates the importance of using 3′ UTR sequences from both sources when comparing results between algorithms as optimally the overlap should approach 100%. Notably, MiRBase9 uses a modified version of miRanda on Ensembl data and TargetScan uses UCSC data. Current databases of miRNA target genes provide a list of hundreds of predictions for each miRNA. To reduce the number of predictions, investigators often consider only those targets that are predicted by multiple algorithms and consider this overlap as a higher-quality subset of predictions. To test the efficiency of intersecting multiple predictions, we performed an enrichment analysis on predictions from five commonly used databases: picTar10, TargetScan, RNA22, miRBase and PITA. The rationale of our analysis was the following: if the intersection of two of these datasets is enriched in true targets, then predictions taken from this intersection are more likely to be present in the intersection of two other datasets than in their exclusion (Supplementary Fig. 3 online). We found that this relation was not significantly true in 29/30 of the permutations tested and was of borderline significance in one (P = 0.03). From this analysis we concluded that the enrichment in true targets of overlapping predictions was weak at best. Admittedly, our test would be more reliable if we compared each overlap with a dataset of experimentally validated targets. However, such a set of quality verified targets, in which target site mutagenesis is shown to reduce the efficiency of miRNA regulation, is still too small to be used as a benchmark dataset (only 48 such targets are reported in the miRecords database; http://mirecords.umn.edu/miRecords/). We suggest that the routine identification of an overlap between miRNA target prediction algorithms should be discouraged owing to a lack of utility and rationale. Two more-logical approaches to filtering miRNA target predictions would be to examine coexpression of miRNA-target pairs (Supplementary Table 2 online) or to identify instances of multitargeting: genes that are targeted multiple times by the same miRNA. Four commonly used algorithms (picTar, TargetScan, miRanda and RNA22) can detect a number of multitargeting occurrences that is much higher than the number expected by chance (Supplementary Fig. 4 online). This demonstrates that multitargeting is widespread and nonrandom. In practical terms, picTAR and RNA22 are likely to reliably predict target genes when a given miRNA target site appears three or more times in the same 3′ UTR even though this method may omit many true targets. Because miRNAs can repress many genes, it is of interest to identify groups of genes targeted by the same miRNA that share a biological function or localization. This functional profiling of miRNA targets can be performed through the Gene Ontology (GO) website (http://www.geneontology.org/) and its associated tools. In addition to the known biases inherent in GO enrichment analyses, the choice of algorithm used to predict miRNA targets can be decisive in the outcome of functional profiling (Supplementary Fig. 5 online). To test how often different target prediction algorithms will predict a different enriched GO function, we created a program called MirGO (Supplementary Data online). This program uses publicly available data to discover the GO enriched function of a set of predicted targets of a given miRNA predicted by miRanda, TargetScan or picTar. Running MirGO 1,000 times on randomly selected miRNAs (Supplementary Fig. 6 online) showed that these three popular algorithms predicted different and unrelated functions in 94% (942/1,000) of cases tested. Notably, when we ran the same experiment considering only enriched GO functions predicted with very low P values (<0.001), their predicted function was discordant in only 4% (42/1,000) of the runs, even though the predicted genes were different for each of the algorithms. The approach used in MirGO has been implemented in many online resources such as

Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans

We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences immediately downstream of transcription start sites. Here, using cytoplasmic and nuclear small RNA high-throughput sequencing datasets, we report the identification of a second class of nuclear-specific ∼17- to 18-nucleotide small RNAs whose 3′ ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed genes and show evidence of developmental stage– and region–specific expression. We also show that tiRNAs are localized to the nucleus, are enriched at chromatin marks associated with transcription initiation and possess a 3′-nucleotide bias. Additionally, we find that microRNA-offset RNAs (moRNAs), the miR-15/16 cluster previously linked to oncosuppression and most small nucleolar RNA (snoRNA)-derived small RNAs (sdRNAs) are enriched in the nucleus, whereas most miRNAs and two H/ACA sdRNAs are cytoplasmically enriched. We propose that nuclear-localized tiny RNAs are involved in the epigenetic regulation of gene expression.

ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer

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.