Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics.

Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Clinical and Analytical Validation of a Novel Urine-Based Test for the Detection of Allograft Rejection in Renal Transplant Patients.

In this clinical validation study, we developed and validated a urinary Q-Score generated from the quantitative test QSant, formerly known as QiSant, for the detection of biopsy-confirmed acute rejection in kidney transplants. Using a cohort of 223 distinct urine samples collected from three independent sites and from both adult and pediatric renal transplant patients, we examined the diagnostic utility of the urinary Q-Score for detection of acute rejection in renal allografts. Statistical models based upon the measurements of the six QSant biomarkers (cell-free DNA, methylated-cell-free DNA, clusterin, CXCL10, creatinine, and total protein) generated a renal transplant Q-Score that reliably differentiated stable allografts from acute rejections in both adult and pediatric renal transplant patients. The composite Q-Score was able to detect both T cell-mediated rejection and antibody-mediated rejection patients and differentiate them from stable non-rejecting patients with a receiver-operator characteristic curve area under the curve of 99.8% and an accuracy of 98.2%. Q-Scores < 32 indicated the absence of active rejection and Q-Scores ≥ 32 indicated an increased risk of active rejection. At the Q-Score cutoff of 32, the overall sensitivity was 95.8% and specificity was 99.3%. At a prevalence of 25%, positive and negative predictive values for active rejection were 98.0% and 98.6%, respectively. The Q-Score also detected subclinical rejection in patients without an elevated serum creatinine level but identified by a protocol biopsy. This study confirms that QSant is an accurate and quantitative measurement suitable for routine monitoring of renal allograft status.

Targeted Urine Metabolomics for Monitoring Renal Allograft Injury and Immunosuppression in Pediatric Patients.

Despite new advancements in surgical tools and therapies, exposure to immunosuppressive drugs related to non-immune and immune injuries can cause slow deterioration and premature failure of organ transplants. Diagnosis of these injuries by non-invasive urine monitoring would be a significant clinical advancement for patient management, especially in pediatric cohorts. We investigated the metabolomic profiles of biopsy matched urine samples from 310 unique kidney transplant recipients using gas chromatography-mass spectrometry (GC-MS). Focused metabolite panels were identified that could detect biopsy confirmed acute rejection with 92.9% sensitivity and 96.3% specificity (11 metabolites) and could differentiate BK viral nephritis (BKVN) from acute rejection with 88.9% sensitivity and 94.8% specificity (4 metabolites). Overall, targeted metabolomic analyses of biopsy-matched urine samples enabled the generation of refined metabolite panels that non-invasively detect graft injury phenotypes with high confidence. These urine biomarkers can be rapidly assessed for non-invasive diagnosis of specific transplant injuries, opening the window for precision transplant medicine.

A urine score for noninvasive accurate diagnosis and prediction of kidney transplant rejection.

Accurate and noninvasive monitoring of renal allograft posttransplant is essential for early detection of acute rejection (AR) and to affect the long-term survival of the transplant. We present the development and validation of a noninvasive, spot urine-based diagnostic assay based on measurements of six urinary DNA, protein, and metabolic biomarkers. The performance of this assay for detecting kidney injury in both native kidneys and renal allografts is presented on a cohort of 601 distinct urine samples. The urinary composite score enables diagnosis of AR, with a receiver-operator characteristic curve area under the curve of 0.99 and an accuracy of 96%. In addition, we demonstrate the clinical utility of this assay for predicting AR before a rise in the serum creatinine, enabling earlier detection of rejection than currently possible by standard of care tests. This noninvasive, sensitive, and quantitative approach is a robust and informative method for the rapid and routine monitoring of renal allografts.

Non-radiological assessment of kidney stones using the kidney injury test (KIT), a spot urine assay.

OBJECTIVES: To evaluate the utility of kidney injury test (KIT) assay urinary biomarkers to detect kidney stones and quantify stone burden. PATIENTS AND METHODS: A total of 136 spot urine samples from 98 individuals, with and without kidney stone disease, were processed in a predefined assay to measure six DNA and protein markers in order to generate a risk score for the non-invasive detection of nephrolithiasis. From this cohort, 56 individuals had spot, non-timed urine samples collected at the time of radiographically confirmed kidney stones, and 54 demographically matched, healthy controls without kidney stone disease also provided spot, non-timed urine samples. Sixteen individuals with persistent stone disease had more than one urine sample. Using a proprietary microwell-based KIT assay, we measured cell-free DNA (cfDNA), methylated cfDNA, clusterin, creatinine, protein and CXCL10. A KIT stone score was computed across all markers using the prior locked KIT algorithm. The KIT stone score, with a scale of 0 to 100, was then correlated with demographic variables, kidney stone burden, obstructive kidney stone disease, and urine solutes in 24-h urine collections. RESULTS: The scaled KIT stone score, a composite of all six biomarkers, readily discriminated individuals with current or prior radiographically confirmed kidney stones from healthy controls without kidney stone disease (P < 0.001). In individuals with nephrolithiasis, KIT stone score also correlated with radiologically measured stone size (P = 0.017) and differentiated patients with a clinical radiological diagnosis of obstructive nephrolithiasis associated with upper renal tract dilatation (P = 0.001). Stone burden as assessed by KIT stone score, however, did not correlate with the any of the traditional measures of 24-h urine solutes or the 24-h urine supersaturation levels. In patients with persistent stone disease, where multiple urine samples were collected over time and after different interventions, the use of KIT stone score could non-invasively track stone burden over time through a spot urine, non-timed urine sample. CONCLUSIONS: A random, spot urine-based assay, KIT stone score, can non-invasively detect, quantify and monitor current stone burden, and may thus minimize radiographic exposure for kidney stone detection. The KIT stone score assay may also help monitor stone recurrence risk for patients with nephrolithiasis, without the requirement for 24-h urine collections.

Noninvasive Urinary Monitoring of Progression in IgA Nephropathy.

Standard methods for detecting and monitoring of IgA nephropathy (IgAN) have conventionally required kidney biopsies or suffer from poor sensitivity and specificity. The Kidney Injury Test (KIT) Assay of urinary biomarkers has previously been shown to distinguish between various kidney pathologies, including chronic kidney disease, nephrolithiasis, and transplant rejection. This validation study uses the KIT Assay to investigate the clinical utility of the non-invasive detection of IgAN and predicting the progression of renal damage over time. The study design benefits from longitudinally collected urine samples from an investigator-initiated, multicenter, prospective study, evaluating the efficacy of corticosteroids versus Rituximab for preventing progressive IgAN. A total of 131 urine samples were processed for this study; 64 urine samples were collected from 34 IgAN patients, and urine samples from 64 demographically matched healthy controls were also collected; multiple urinary biomarkers consisting of cell-free DNA, methylated cell-free DNA, DMAIMO, MAMIMO, total protein, clusterin, creatinine, and CXCL10 were measured by the microwell-based KIT Assay. An IgA risk score (KIT-IgA) was significantly higher in IgAN patients as compared to healthy control (87.76 vs. 14.03, p < 0.0001) and performed better than proteinuria in discriminating between the two groups. The KIT Assay biomarkers, measured on a spot random urine sample at study entry could distinguish patients likely to have progressive renal dysfunction a year later. These data support the pursuit of larger prospective studies to evaluate the predictive performance of the KIT-IgA score in both screening for non-invasive diagnosis of IgAN, and for predicting risk of progressive renal disease from IgA and utilizing the KIT score for potentially evaluating the efficacy of IgAN-targeted therapies.

A urinary Common Rejection Module (uCRM) score for non-invasive kidney transplant monitoring.

A Common Rejection Module (CRM) consisting of 11 genes expressed in allograft biopsies was previously reported to serve as a biomarker for acute rejection (AR), correlate with the extent of graft injury, and predict future allograft damage. We investigated the use of this gene panel on the urine cell pellet of kidney transplant patients. Urinary cell sediments collected from patients with biopsy-confirmed acute rejection, borderline AR (bAR), BK virus nephropathy (BKVN), and stable kidney grafts with normal protocol biopsies (STA) were analyzed for expression of these 11 genes using quantitative polymerase chain reaction (qPCR). We assessed these 11 CRM genes for their abundance, autocorrelation, and individual expression levels. Expression of 10/11 genes were elevated in AR when compared to STA. Psmb9 and Cxcl10could classify AR versus STA as accurately as the 11-gene model (sensitivity = 93.6%, specificity = 97.6%). A uCRM score, based on the geometric mean of the expression levels, could distinguish AR from STA with high accuracy (AUC = 0.9886) and correlated specifically with histologic measures of tubulitis and interstitial inflammation rather than tubular atrophy, glomerulosclerosis, intimal proliferation, tubular vacuolization or acute glomerulitis. This urine gene expression-based score may enable the non-invasive and quantitative monitoring of AR.

A Novel Multi-Biomarker Assay for Non-Invasive Quantitative Monitoring of Kidney Injury.

The current standard of care measures for kidney function, proteinuria, and serum creatinine (SCr) are poor predictors of early-stage kidney disease. Measures that can detect chronic kidney disease in its earlier stages are needed to enable therapeutic intervention and reduce adverse outcomes of chronic kidney disease. We have developed the Kidney Injury Test (KIT) and a novel KIT Score based on the composite measurement and validation of multiple biomarkers across a unique set of 397 urine samples. The test is performed on urine samples that require no processing at the site of collection and without target sequencing or amplification. We sought to verify that the pre-defined KIT test, KIT Score, and clinical thresholds correlate with established chronic kidney disease (CKD) and may provide predictive information on early kidney injury status above and beyond proteinuria and renal function measurements alone. Statistical analyses across six DNA, protein, and metabolite markers were performed on a subset of residual spot urine samples with CKD that met assay performance quality controls from patients attending the clinical labs at the University of California, San Francisco (UCSF) as part of an ongoing IRB-approved prospective study. Inclusion criteria included selection of patients with confirmed CKD and normal healthy controls; exclusion criteria included incomplete or missing information for sample classification, logistical delays in transport/processing of urine samples or low sample volume, and acute kidney injury. Multivariate logistic regression of kidney injury status and likelihood ratio statistics were used to assess the contribution of the KIT Score for prediction of kidney injury status and stage of CKD as well as assess the potential contribution of the KIT Score for detection of early-stage CKD above and beyond traditional measures of renal function. Urine samples were processed by a proprietary immunoprobe for measuring cell-free DNA (cfDNA), methylated cfDNA, clusterin, CXCL10, total protein, and creatinine. The KIT Score and stratified KIT Score Risk Group (high versus low) had a sensitivity and specificity for detection of kidney injury status (healthy or CKD) of 97.3% (95% CI: 94.6-99.3%) and 94.1% (95% CI: 82.3-100%). In addition, in patients with normal renal function (estimated glomerular filtration rate (eGFR) ≥ 90), the KIT Score clearly identifies those with predisposing risk factors for CKD, which could not be detected by eGFR or proteinuria (p < 0.001). The KIT Score uncovers a burden of kidney injury that may yet be incompletely recognized, opening the door for earlier detection, intervention and preservation of renal function.

Cell-Free DNA and CXCL10 Derived from Bronchoalveolar Lavage Predict Lung Transplant Survival.

Standard methods for detecting chronic lung allograft dysfunction (CLAD) and rejection have poor sensitivity and specificity and have conventionally required bronchoscopies and biopsies. Plasma cell-free DNA (cfDNA) has been shown to be increased in various types of allograft injury in transplant recipients and CXCL10 has been reported to be increased in the lung tissue of patients undergoing CLAD. This study used a novel cfDNA and CXCL10 assay to evaluate the noninvasive assessment of CLAD phenotype and prediction of survival from bronchoalveolar lavage (BAL) fluid. A total of 60 BAL samples (20 with bronchiolitis obliterans (BOS), 20 with restrictive allograft syndrome (RAS), and 20 with stable allografts (STA)) were collected from 60 unique lung transplant patients; cfDNA and CXCL10 were measured by the ELISA-based KIT assay. Median cfDNA was significantly higher in BOS patients (6739 genomic equivalents (GE)/mL) versus STA (2920 GE/mL) and RAS (4174 GE/mL) (p < 0.01 all comparisons). Likelihood ratio tests revealed a significant association of overall survival with cfDNA (p = 0.0083), CXCL10 (p = 0.0146), and the interaction of cfDNA and CXCL10 (p = 0.023) based on multivariate Cox proportional hazards regression. Dichotomizing patients based on the median cfDNA level controlled for the mean level of CXCL10 revealed an over two-fold longer median overall survival time in patients with low levels of cfDNA. The KIT assay could predict allograft survival with superior performance compared with traditional biomarkers. These data support the pursuit of larger prospective studies to evaluate the predictive performance of cfDNA and CXCL10 prior to lung allograft failure.

The common rejection module in chronic rejection post lung transplantation.

RATIONALE: Recent studies suggest that similar injury mechanisms are in place across different solid organ transplants, resulting in the identification of a common rejection module (CRM), consisting of 11 genes that are overexpressed during acute and, to a lesser extent, chronic allograft rejection. OBJECTIVES: We wanted to evaluate the usefulness of the CRM module in identifying acute rejection (AR) and different phenotypes of chronic lung transplant rejection (CLAD), i.e., bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), using transbronchial brushings, broncho-alveolar lavage (BAL) samples, and explant tissue. METHODS: Gene expression measurements for the 11 CRM genes (CD6, TAP1, CXCL10, CXCL9, INPP5D, ISG20, LCK, NKG7, PSMB9, RUNX3, and BASP1) were performed via qRT-PCR in 14 transbronchial brushings (AR, n = 4; no AR, n = 10), 32 BAL samples (stable, n = 13; AR, n = 8; BOS, n = 9; RAS, n = 10), and 44 tissue specimens (unused donor lungs, n = 15; BOS, n = 13; RAS, n = 16). A geometric mean score was calculated to quantitate overall burden of immune injury and a new computational model was built for the most significant genes in lung transplant injury. RESULTS: Acute rejection showed a significant difference in almost every gene analysed, validating previous observations from microarray analysis. RAS tissue demonstrated a higher geometric mean score (6.35) compared to donor tissue (4.09, p = 0.018). Analysis of individual CRM genes showed an increased expression of ISG20, CXCL10 and CXCL9 in RAS. In BAL samples, no differences were detected in gene expression or geometric mean scores between the various groups (stable, 5.15; AR, 5.81; BOS, 5.62; RAS, 7.31). A newly modelled 2-gene tissue CRM score did not demonstrate any difference between BOS and RAS (p>0.05). However, the model was able to discriminate RAS from BOS tissue (AUC = 0.75, 95% CI = 0.55-0.94, p = 0.025). CONCLUSION: Transcriptional tissue analysis for CRM genes in CLAD can identify acute rejection and distinguish RAS from BOS. The immune activation in RAS seems similar to acute rejection after kidney/liver/heart transplantation.

Retrospective evaluation of the efficacy and safety of belatacept with thymoglobulin induction and maintenance everolimus: A single-center clinical experience.

Belatacept use has been constrained by higher rates of acute rejection. We hypothesized that belatacept with low-dose rATG and initial mycophenolate maintenance with conversion to everolimus at 1 month post-transplant ± corticosteroids would improve efficacy and maintain safety. Retrospective single-center analysis of the first 44 low immunologic risk kidney transplant recipients treated with this regimen. The cohort was 59% male, mean age at transplant of 57 years. Diabetes was the most common cause of ESRD (39%). The mean 1-year eGFR was 61.4 (SD 18.4) mL/min/1.73 m2 . There were five acute cellular rejections (11.4%) that occurred in patients who had changed from everolimus to mycophenolate mofetil due to side effects. Thirty-two percent developed BK viremia and 12% developed CMV viremia. There were no cases of PTLD. A novel belatacept regimen with rATG induction and maintenance everolimus demonstrated a low acute rejection rate and maintained an excellent 1-year eGFR.

Transplant genetics and genomics.

Ever since the discovery of the major histocompatibility complex, scientific and clinical understanding in the field of transplantation has been advanced through genetic and genomic studies. Candidate-gene approaches and recent genome-wide association studies (GWAS) have enabled a deeper understanding of the complex interplay of the donor-recipient interactions that lead to transplant tolerance or rejection. Genetic analysis in transplantation, when linked to demographic and clinical outcomes, has the potential to drive personalized medicine by enabling individualized risk stratification and immunosuppression through the identification of variants associated with immune-mediated complications, post-transplant disease or alterations in drug-metabolizing genes.