Cell-Free DNA and Active Rejection in Kidney Allografts.

Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.

Biological Variation of Donor-Derived Cell-Free DNA in Renal Transplant Recipients: Clinical Implications.

BACKGROUND: Previous studies have demonstrated that donor-derived cell-free DNA (dd-cfDNA) found in circulating blood of transplant recipients may serve as a noninvasive biomarker of allograft rejection. To better interpret the clinical meaning of dd-cfDNA, it is essential to understand the biological variation of this biomarker in stable healthy recipients. This report establishes the biological variation and clinical reference intervals of dd-cfDNA in renal transplant recipients by using an analytically validated assay that has a CV of 6.8%. METHODS: We sampled venous blood at patient surveillance visits (typically at posttransplant months 1-4, 6, 9, and 12) in a 14-center observational study. Patients with stable renal allograft function spanning ≥3 serial visits were selected. We used AlloSure®, a targeted next-generation sequencing-based approach, to measure dd-cfDNA in the plasma and computed the intraindividual CV (CVI) and interindividual CV (CVG), the index of individuality (II), and reference change value (RCV). RESULTS: Of 93 patients, 61% were men, 56% were Caucasian, mean age was 49 years, and 63% were deceased donor kidney recipients. Of 380 blood samples, the dd-cfDNA median value was 0.21% (interquartile range 0.12%-0.39%) and the 97.5th percentile was 1.20%. In 18 patients with an average of 4.1 tests, the CVI was 21%, CVG was 37%, II was 0.57, and RCV was 61%. CONCLUSIONS: In a renal transplant recipient, a dd-cfDNA level above 1.2% is out of range and potentially abnormal. A serial increase of up to 61% in level of dd-cfDNA in a patient may be attributable to biological variation.Clinicaltrials.gov Identifier: NCT02424227.

Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients.

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells.

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening.

Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice.

A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment, and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies.

Induced pluripotent stem cells--opportunities for disease modelling and drug discovery.

The ability to generate induced pluripotent stem cells (iPSCs) from patients, and an increasingly refined capacity to differentiate these iPSCs into disease-relevant cell types, promises a new paradigm in drug development - one that positions human disease pathophysiology at the core of preclinical drug discovery. Disease models derived from iPSCs that manifest cellular disease phenotypes have been established for several monogenic diseases, but iPSCs can likewise be used for phenotype-based drug screens in complex diseases for which the underlying genetic mechanism is unknown. Here, we highlight recent advances as well as limitations in the use of iPSC technology for modelling a 'disease in a dish' and for testing compounds against human disease phenotypes in vitro. We discuss how iPSCs are being exploited to illuminate disease pathophysiology, identify novel drug targets and enhance the probability of clinical success of new drugs.

Induced pluripotent stem cells in the study of neurological diseases.

Five years after their initial derivation from mouse somatic cells, induced pluripotent stem (iPS) cells are an important tool for the study of neurological diseases. By offering an unlimited source of patient-specific disease-relevant neuronal and glial cells, iPS cell-based disease models hold enormous promise for identification of disease mechanisms, discovery of molecular targets and development of phenotypic screens for drug discovery. The present review focuses on the recent advancements in modeling neurological disorders, including the demonstration of disease-specific phenotypes in iPS cell-derived neurons generated from patients with spinal muscular atrophy, familial dysautonomia, Rett syndrome, schizophrenia and Parkinson disease. The ability of this approach to detect treatment effects from known therapeutic compounds has also been demonstrated, providing proof of principle for the use of iPS cell-derived cells in drug discovery.

Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.

Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES) cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.

Sex-lethal imparts a sex-specific function to UNR by recruiting it to the msl-2 mRNA 3' UTR: translational repression for dosage compensation.

MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein sex-lethal (SXL), which binds to the msl-2 mRNA 5' and 3' untranslated regions (UTRs) and blocks translation through distinct UTR-specific mechanisms. Here, we purify translationally silenced msl-2 mRNPs and identify UNR (upstream of N-ras) as a protein recruited to the 3' UTR by SXL. We demonstrate that SXL requires UNR as a corepressor for 3'-UTR-mediated regulation, imparting a female-specific function to the ubiquitously expressed UNR protein. Our results reveal a novel functional role for UNR as a translational repressor and indicate that UNR is a key component of a "fail-safe" dosage compensation regulatory system that prevents toxic MSL-2 synthesis in female cells.

A dual inhibitory mechanism restricts msl-2 mRNA translation for dosage compensation in Drosophila.

Drosophila MSL-2 is the limiting component of the dosage compensation complex. Female flies must inhibit msl-2 mRNA translation for survival, and this inhibition is mediated by Sex-lethal (SXL) binding to sites in both the 5' and the 3' untranslated regions (UTRs). Here, we uncover the mechanism by which SXL achieves tight control of translation initiation. SXL binding to the 3'UTR regulatory region inhibits the recruitment of 43S ribosomal preinitiation complexes to the mRNA. Ribosomal complexes escaping this block and binding to the 5' end of the mRNA are challenged by SXL bound to the 5'UTR, which interferes with scanning to the downstream initiation codon of the mRNA. This failsafe mechanism thus forms the molecular basis of a critical step in dosage compensation. The results also elucidate a two step principle of translational control via multiple regulatory sites within an mRNA.

A co-repressor assembly nucleated by Sex-lethal in the 3'UTR mediates translational control of Drosophila msl-2 mRNA.

Drosophila Sex-lethal (dSXL)-mediated translational repression of male-specific lethal 2 (msl-2) mRNA is essential for X-chromosome dosage compensation. Binding of dSXL to specific sites in both untranslated regions of msl-2 mRNA is necessary for inhibition of translation initiation. We describe the organization of dSXL as a translational regulator and show that the RNA binding and translational repressor functions are contained within the two RRM domains and a C-terminal heptapeptide extension. The repressor function is dormant unless dSXL binds to msl-2 mRNA with its own RRMs, because dSXL tethering via a heterologous RNA-binding peptide does not elicit translational inhibition. We reveal proteins that crosslink to the msl-2 3' untranslated region (3'UTR) and co-immunoprecipitate with dSXL in a fashion that requires its intact repressor domain and correlates with translational regulation. Translation competition and UV-crosslink experiments show that the 3'UTR msl-2 sequences adjacent to dSXL-binding sites are necessary to recruit titratable co-repressors. Our data support a model where dSXL binding to the 3'UTR of msl-2 mRNA activates the translational repressor domain, thereby enabling it to recruit co-repressors in a specific fashion.

A simple method for RNA isolation from formalin-fixed and paraffin-embedded lymphatic tissues.

Gene activation that lies beneath lymphoid cell differentiation has been one of the most explored issues in immunology in the recent years. However, the analysis of this molecular event in lymphoproliferative diseases is often hampered by the lack of fresh material. Most tissues available for routine histological investigation are formalin fixed and paraffin embedded. Gene expression in such specimens could be analyzed using reverse transcription of mRNA and the polymerase chain reaction (RT-PCR). Therefore we adjusted and established a method for mRNA isolation from such specimens by a combination of previously reported protocols and a modification of the phenol/chloroform extraction method. Given the significance of transcription factors in the human hemopoietic system, we investigated whether mRNA could be successfully isolated from archival tissue for a study on expression of Ikaros family transcription factors in lymphatic tissue. Although quantitative analysis of RNA isolated from archival tissue is probably not feasible due to the unpredictable degree of RNA isolation varying from sample to sample, we show here that screening analysis is possible and simple.

Drosophila sex-lethal inhibits the stable association of the 40S ribosomal subunit with msl-2 mRNA.

The inhibition of male-specific lethal-2 (msl-2) mRNA translation in female flies is essential for X chromosome dosage compensation in Drosophila melanogaster. Translational repression of msl-2 requires sex-lethal (SXL) binding to uridine-rich sequences in both the 5' and 3' untranslated regions (UTRs) of the message. We delineate the msl-2 mRNA sequence elements that are important for regulation by SXL and identify functionally critical sequences adjacent to regulatory SXL binding sites. We demonstrate that SXL inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA. These results elucidate a critical regulatory step for dosage compensation in Drosophila melanogaster.