A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.The authors wish to thank the core facilities (Biological Research Unit, Histopathology, Flow Cytometry and Microscopy) of the CRUK Cambridge Institute for advice and technical assistance. This work was funded by a CRUK Clinician Scientist Fellowship award (C37839/A12177).This is the final version. It was first published by NPG at http://www.nature.com/srep/2015/150605/srep11061/full/srep11061.html

Generation and Characterisation of a Pax8-CreERT2 Transgenic Line and a Slc22a6-CreERT2 Knock-In Line for Inducible and Specific Genetic Manipulation of Renal Tubular Epithelial Cells.

Genetically relevant mouse models need to recapitulate the hallmarks of human disease by permitting spatiotemporal gene targeting. This is especially important for replicating the biology of complex diseases like cancer, where genetic events occur in a sporadic fashion within developed somatic tissues. Though a number of renal tubule targeting mouse lines have been developed their utility for the study of renal disease is limited by lack of inducibility and specificity. In this study we describe the generation and characterisation of two novel mouse lines directing CreERT2 expression to renal tubular epithelia. The Pax8-CreERT2 transgenic line uses the mouse Pax8 promoter to direct expression of CreERT2 to all renal tubular compartments (proximal and distal tubules as well as collecting ducts) whilst the Slc22a6-CreERT2 knock-in line utilises the endogenous mouse Slc22a6 locus to specifically target the epithelium of proximal renal tubules. Both lines show high organ and tissue specificity with no extrarenal activity detected. To establish the utility of these lines for the study of renal cancer biology, Pax8-CreERT2 and Slc22a6-CreERT2 mice were crossed to conditional Vhl knockout mice to induce long-term renal tubule specific Vhl deletion. These models exhibited renal specific activation of the hypoxia inducible factor pathway (a VHL target). Our results establish Pax8-CreERT2 and Slc22a6-CreERT2 mice as valuable tools for the investigation and modelling of complex renal biology and disease.This work was supported by a Cancer Research UK Clinician Scientist Fellowship award (C37839/A12177) to AM. DA, BF, FY are funded by the Wellcome Trust Sanger Institute (grant number WT098051).This is the final version of the article. It first appeared from the Public Library of Science (PLOS) via https://doi.org/10.1371/journal.pone.014805

The WIRE study a phase II, multi-arm, multi-centre, non-randomised window-of-opportunity clinical trial platform using a Bayesian adaptive design for proof-of-mechanism of novel treatment strategies in operable renal cell cancer - a study protocol.

BACKGROUND: Window-of-opportunity trials, evaluating the engagement of drugs with their biological target in the time period between diagnosis and standard-of-care treatment, can help prioritise promising new systemic treatments for later-phase clinical trials. Renal cell carcinoma (RCC), the 7th commonest solid cancer in the UK, exhibits targets for multiple new systemic anti-cancer agents including DNA damage response inhibitors, agents targeting vascular pathways and immune checkpoint inhibitors. Here we present the trial protocol for the WIndow-of-opportunity clinical trial platform for evaluation of novel treatment strategies in REnal cell cancer (WIRE). METHODS: WIRE is a Phase II, multi-arm, multi-centre, non-randomised, proof-of-mechanism (single and combination investigational medicinal product [IMP]), platform trial using a Bayesian adaptive design. The Bayesian adaptive design leverages outcome information from initial participants during pre-specified interim analyses to determine and minimise the number of participants required to demonstrate efficacy or futility. Patients with biopsy-proven, surgically resectable, cT1b+, cN0-1, cM0-1 clear cell RCC and no contraindications to the IMPs are eligible to participate. Participants undergo diagnostic staging CT and renal mass biopsy followed by treatment in one of the treatment arms for at least 14 days. Initially, the trial includes five treatment arms with cediranib, cediranib + olaparib, olaparib, durvalumab and durvalumab + olaparib. Participants undergo a multiparametric MRI before and after treatment. Vascularised and de-vascularised tissue is collected at surgery. A ≥ 30% increase in CD8+ T-cells on immunohistochemistry between the screening and nephrectomy is the primary endpoint for durvalumab-containing arms. Meanwhile, a reduction in tumour vascular permeability measured by Ktrans on dynamic contrast-enhanced MRI by ≥30% is the primary endpoint for other arms. Secondary outcomes include adverse events and tumour size change. Exploratory outcomes include biomarkers of drug mechanism and treatment effects in blood, urine, tissue and imaging. DISCUSSION: WIRE is the first trial using a window-of-opportunity design to demonstrate pharmacological activity of novel single and combination treatments in RCC in the pre-surgical space. It will provide rationale for prioritising promising treatments for later phase trials and support the development of new biomarkers of treatment effect with its extensive translational agenda. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03741426 / EudraCT: 2018-003056-21

Chromosome 15q25 (CHRNA3-CHRNA5) Variation Impacts Indirectly on Lung Cancer Risk

Genetic variants at the 15q25 CHRNA5-CHRNA3 locus have been shown to influence lung cancer risk however there is controversy as to whether variants have a direct carcinogenic effect on lung cancer risk or impact indirectly through smoking behavior. We have performed a detailed analysis of the 15q25 risk variants rs12914385 and rs8042374 with smoking behavior and lung cancer risk in 4,343 lung cancer cases and 1,479 controls from the Genetic Lung Cancer Predisposition Study (GELCAPS). A strong association between rs12914385 and rs8042374, and lung cancer risk was shown, odds ratios (OR) were 1.44, (95% confidence interval (CI): 1.29–1.62, P = 3.69×10−10) and 1.35 (95% CI: 1.18–1.55, P = 9.99×10−6) respectively. Each copy of risk alleles at rs12914385 and rs8042374 was associated with increased cigarette consumption of 1.0 and 0.9 cigarettes per day (CPD) (P = 5.18×10−5 and P = 5.65×10−3). These genetically determined modest differences in smoking behavior can be shown to be sufficient to account for the 15q25 association with lung cancer risk. To further verify the indirect effect of 15q25 on the risk, we restricted our analysis of lung cancer risk to never-smokers and conducted a meta-analysis of previously published studies of lung cancer risk in never-smokers. Never-smoker studies published in English were ascertained from PubMed stipulating - lung cancer, risk, genome-wide association, candidate genes. Our study and five previously published studies provided data on 2,405 never-smoker lung cancer cases and 7,622 controls. In the pooled analysis no association has been found between the 15q25 variation and lung cancer risk (OR = 1.09, 95% CI: 0.94–1.28). This study affirms the 15q25 association with smoking and is consistent with an indirect link between genotype and lung cancer risk

Dynamic biomarker and imaging changes from a phase II study of pre- and post-surgical sunitinib.

Funder: Pfizer; Id: http://dx.doi.org/10.13039/100004319OBJECTIVE: To explore translational biological and imaging biomarkers for sunitinib treatment before and after debulking nephrectomy in the NeoSun (European Union Drug Regulating Authorities Clinical Trials Database [EudraCT] number: 2005-004502-82) single-centre, single-arm, single-agent, Phase II trial. PATIENTS AND METHODS: Treatment-naïve patients with metastatic renal cell carcinoma (mRCC) received 50 mg once daily sunitinib for 12 days pre-surgically, then post-surgery on 4 week-on, 2 week-off, repeating 6-week cycles until disease progression in a single arm phase II trial. Structural and dynamic contrast-enhanced magnet resonance imaging (DCE-MRI) and research blood sampling were performed at baseline and after 12 days. Computed tomography imaging was performed at baseline and post-surgery then every two cycles. The primary endpoint was objective response rate (Response Evaluation Criteria In Solid Tumors [RECIST]) excluding the resected kidney. Secondary endpoints included changes in DCE-MRI of the tumour following pre-surgery sunitinib, overall survival (OS), progression-free survival (PFS), response duration, surgical morbidity/mortality, and toxicity. Translational and imaging endpoints were exploratory. RESULTS: A total of 14 patients received pre-surgery sunitinib, 71% (10/14) took the planned 12 doses. All underwent nephrectomy, and 13 recommenced sunitinib postoperatively. In all, 58.3% (seven of 12) of patients achieved partial or complete response (PR or CR) (95% confidence interval 27.7-84.8%). The median OS was 33.7 months and median PFS was 15.7 months. Amongst those achieving a PR or CR, the median response duration was 8.7 months. No unexpected surgical complications, sunitinib-related toxicities, or surgical delays occurred. Within the translational endpoints, pre-surgical sunitinib significantly increased necrosis, and reduced cluster of differentiation-31 (CD31), Ki67, circulating vascular endothelial growth factor-C (VEGF-C), and transfer constant (KTrans , measured using DCE-MRI; all P < 0.05). There was a trend for improved OS in patients with high baseline plasma VEGF-C expression (P = 0.02). Reduction in radiological tumour volume after pre-surgical sunitinib correlated with high percentage of solid tumour components at baseline (Spearman's coefficient ρ = 0.69, P = 0.02). Conversely, the percentage tumour volume reduction correlated with lower baseline percentage necrosis (coefficient = -0.51, P = 0.03). CONCLUSION: Neoadjuvant studies such as the NeoSun can safely and effectively explore translational biological and imaging endpoints

<i>Slc22a6</i>-CreER^T2 mediated β-galactosidase expression.

<p>(A) Enzymatic X-gal staining of cryosections of kidneys (top panels) derived from 10 week old <i>Slc22a6</i>-CreER^T2/Rosa26R mice either induced with tamoxifen (a, renal cortex; b, medulla) or uninduced (c, renal cortex). Tamoxifen administration did not induce β-galactosidase expression in any of the other major organs examined (representative tissues presented in bottom panels). (B) β-galactosidase positivity was exclusively found in the tubular epithelium of the kidney of tamoxifen treated mice (a); kidneys from vehicle treated mice were negative (b). Co-localisation studies revealed specific X-gal expression within proximal tubular epithelium (AQP1) with no expression detected in distal tubules (THP) or collecting ducts (AQP2). Scale bars, 100μm.</p

Generation of <i>Slc22a6</i>-CreER^T2 knock-in mice.

<p>(A) Schematic diagram of the generation of Slc22a6-CreER^T2 knock-in mice. The exon-intron structure of the mouse <i>Slc22a6</i> locus is shown at the top. The targeting vector has a 4.3 kb 5′ arm that locates immediately upstream of the <i>Slc22a6</i> gene ATG, the CreER^T2-pA coding sequence, a PGK-hygromycin selection cassette flanked by FRT sites (green triangles) and a 5.3 kb 3′ arm that extends to intron 6. (B) Southern blots show a control (wild-type) and 4 correctly targeted ES cell clones (77, 79, 92, 185) that give expected hybridisation patterns. Clone 142 is incorrectly targeted. The location of 5’, 3’ and internal probes as well as expected band lengths are indicated in the diagrams on the right.</p

<i>Pax8</i>-CreER^T2 mediated β-galactosidase expression.

<p>(A) Enzymatic X-gal staining of cryosections of kidneys (top panels) derived from 10 week old <i>Pax8</i>-CreER^T2/Rosa26R mice either induced with tamoxifen (a, renal parenchyma; b, collecting ducts) or uninduced (c). Tamoxifen administration did not induce β-galactosidase expression in any of the other major organs examined (representative tissues presented in bottom panels). (B) β-galactosidase positivity was exclusively found in the tubular epithelium of the kidney of tamoxifen treated mice, with no staining observed in glomeruli or bloods vessels (a, framed area is shown enlarged in b). Kidneys from vehicle treated mice were negative (c). Co-localisation studies revealed X-gal expression within tubules of all renal tubular compartment (proximal tubules—AQP1, distal tubules–THP and collecting ducts–AQP2). Scale bars, 100μm.</p

Renal tubule specific models of <i>Vhl</i> deletion.

<p>(A) PCR analysis of recombination at the <i>Vhl</i> locus in the kidneys of mice with combinations of <i>Pax8</i>-CreER^T2, <i>Slc22a6</i>-CreER^T2 and the <i>Vhl</i> floxed (fl) and wild-type (+) alleles. The positions of the bands representing the <i>Vhl</i> floxed, wild type (Wt) and recombined (Δ) alleles are indicated. (B) Histological images of representative renal sections from 12 month old control, <i>Pax8</i>-CreER^T2/<i>Vhl</i>^Δ/Δ and <i>Slc22a6</i>-CreER^T2/<i>Vhl</i>^Δ/Δ mice (stains and antibodies as indicated, arrowheads indicate abnormal vascularisation). Scale bars, 100μm.</p