Severe delayed hypersensitivity reactions to IL-1 and IL-6 inhibitors link to common HLA-DRB1*15 alleles.

OBJECTIVES: Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, delayed hypersensitivity reaction (DHR). We observed DRESS to inhibitors of interleukin 1 (IL-1) or IL-6 in a small group of patients with Still's disease with atypical lung disease. We sought to characterise features of patients with Still's disease with DRESS compared with drug-tolerant Still's controls. We analysed human leucocyte antigen (HLA) alleles for association to inhibitor-related DHR, including in a small Kawasaki disease (KD) cohort. METHODS: In a case/control study, we collected a multicentre series of patients with Still's disease with features of inhibitor-related DRESS (n=66) and drug-tolerant Still's controls (n=65). We retrospectively analysed clinical data from all Still's subjects and typed 94/131 for HLA. European Still's-DRESS cases were ancestry matched to International Childhood Arthritis Genetics Consortium paediatric Still's cases (n=550) and compared for HLA allele frequencies. HLA association also was analysed using Still's-DRESS cases (n=64) compared with drug-tolerant Still's controls (n=30). KD subjects (n=19) were similarly studied. RESULTS: Still's-DRESS features included eosinophilia (89%), AST-ALT elevation (75%) and non-evanescent rash (95%; 88% involving face). Macrophage activation syndrome during treatment was frequent in Still's-DRESS (64%) versus drug-tolerant Still's (3%; p=1.2×10-14). We found striking enrichment for HLA-DRB1*15 haplotypes in Still's-DRESS cases versus INCHARGE Still's controls (p=7.5×10-13) and versus self-identified, ancestry-matched Still's controls (p=6.3×10-10). In the KD cohort, DRB1*15:01 was present only in those with suspected anakinra reactions. CONCLUSIONS: DRESS-type reactions occur among patients treated with IL-1/IL-6 inhibitors and strongly associate with common HLA-DRB1*15 haplotypes. Consideration of preprescription HLA typing and vigilance for serious reactions to these drugs are warranted.

The 3'tsRNAs are aminoacylated: Implications for their biogenesis.

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG3´tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3´tsRNAs remains elusive and it is unclear if the 3´-ends of 3´tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3´charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG3´tsRNA, and two other 3´tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNALeu concentration but a reduction in both the charged tRNALeu and LeuCAG3´tsRNA, suggesting the 3´tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNALeu mutants were also shown to affect the generation of the LeuCAG3´tsRNA further suggesting they are created in a highly regulated process. The fact that the 3´tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery.

Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing.

Despite early successes using recombinant adeno-associated virus (rAAV) vectors in clinical gene therapy trials, limitations remain making additional advancements a necessity. Some of the challenges include variable levels of pre-existing neutralizing antibodies and poor transduction in specific target tissues and/or diseases. In addition, readministration of an rAAV vector is in general not possible due to the immune response against the capsid. Recombinant adeno-associated virus (AAV) vectors with novel capsids can be isolated in nature or developed through different directed evolution strategies. However, in most cases, the process of AAV selection is not well understood and new strategies are required to define the best parameters to develop more efficient and functional rAAV capsids. Therefore, the use of barcoding for AAV capsid libraries, which can be screened by high-throughput sequencing, provides a powerful tool to track AAV capsid evolution and potentially improve AAV capsid library screens. In this study, we examined how different parameters affect the screen of two different AAV libraries in two human cell types. We uncovered new and unexpected insights in how to maximize the likelihood of obtaining AAV variants with the desired properties. The major findings of the study are the following. (1) Inclusion of helper-virus for AAV replication can selectively propagate variants that can replicate to higher titers, but are not necessarily better at transduction. (2) Competition between AAVs with specific capsids can take place in cells that have been infected with different AAVs. (3) The use of low multiplicity of infections for infection results in more variation between screens and is not optimal at selecting the most desired capsids. (4) Using multiple rounds of selection can be counterproductive. We conclude that each of these parameters should be taken into consideration when screening AAV libraries for enhanced properties of interest.

Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors.

While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional ß cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell-derived ß cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.

Emergent high fatality lung disease in systemic juvenile arthritis.

OBJECTIVE: To investigate the characteristics and risk factors of a novel parenchymal lung disease (LD), increasingly detected in systemic juvenile idiopathic arthritis (sJIA). METHODS: In a multicentre retrospective study, 61 cases were investigated using physician-reported clinical information and centralised analyses of radiological, pathological and genetic data. RESULTS: LD was associated with distinctive features, including acute erythematous clubbing and a high frequency of anaphylactic reactions to the interleukin (IL)-6 inhibitor, tocilizumab. Serum ferritin elevation and/or significant lymphopaenia preceded LD detection. The most prevalent chest CT pattern was septal thickening, involving the periphery of multiple lobes ± ground-glass opacities. The predominant pathology (23 of 36) was pulmonary alveolar proteinosis and/or endogenous lipoid pneumonia (PAP/ELP), with atypical features including regional involvement and concomitant vascular changes. Apparent severe delayed drug hypersensitivity occurred in some cases. The 5-year survival was 42%. Whole exome sequencing (20 of 61) did not identify a novel monogenic defect or likely causal PAP-related or macrophage activation syndrome (MAS)-related mutations. Trisomy 21 and young sJIA onset increased LD risk. Exposure to IL-1 and IL-6 inhibitors (46 of 61) was associated with multiple LD features. By several indicators, severity of sJIA was comparable in drug-exposed subjects and published sJIA cohorts. MAS at sJIA onset was increased in the drug-exposed, but was not associated with LD features. CONCLUSIONS: A rare, life-threatening lung disease in sJIA is defined by a constellation of unusual clinical characteristics. The pathology, a PAP/ELP variant, suggests macrophage dysfunction. Inhibitor exposure may promote LD, independent of sJIA severity, in a small subset of treated patients. Treatment/prevention strategies are needed.

miR-122 removal in the liver activates imprinted microRNAs and enables more effective microRNA-mediated gene repression.

miR-122 is a highly expressed liver microRNA that is activated perinatally and aids in regulating cholesterol metabolism and promoting terminal differentiation of hepatocytes. Disrupting expression of miR-122 can re-activate embryo-expressed adult-silenced genes, ultimately leading to the development of hepatocellular carcinoma (HCC). Here we interrogate the liver transcriptome at various time points after genomic excision of miR-122 to determine the cellular consequences leading to oncogenesis. Loss of miR-122 leads to specific and progressive increases in expression of imprinted clusters of microRNAs and mRNA transcripts at the Igf2 and Dlk1-Dio3 loci that could be curbed by re-introduction of exogenous miR-122. mRNA targets of other abundant hepatic microRNAs are functionally repressed leading to widespread hepatic transcriptional de-regulation. Together, this reveals a transcriptomic framework for the hepatic response to loss of miR-122 and the outcome on other microRNAs and their cognate gene targets.

Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle.

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

A transfer-RNA-derived small RNA regulates ribosome biogenesis.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

Global transcriptome analysis of formalin-fixed prostate cancer specimens identifies biomarkers of disease recurrence.

Prostate cancer remains the second leading cause of cancer death in American men and there is an unmet need for biomarkers to identify patients with aggressive disease. In an effort to identify biomarkers of recurrence, we performed global RNA sequencing on 106 formalin-fixed, paraffin-embedded prostatectomy samples from 100 patients at three independent sites, defining a 24-gene signature panel. The 24 genes in this panel function in cell-cycle progression, angiogenesis, hypoxia, apoptosis, PI3K signaling, steroid metabolism, translation, chromatin modification, and transcription. Sixteen genes have been associated with cancer, with five specifically associated with prostate cancer (BTG2, IGFBP3, SIRT1, MXI1, and FDPS). Validation was performed on an independent publicly available dataset of 140 patients, where the new signature panel outperformed markers published previously in terms of predicting biochemical recurrence. Our work also identified differences in gene expression between Gleason pattern 4 + 3 and 3 + 4 tumors, including several genes involved in the epithelial-to-mesenchymal transition and developmental pathways. Overall, this study defines a novel biomarker panel that has the potential to improve the clinical management of prostate cancer.