CRISPR-Mediated VHL Knockout Generates an Improved Model for Metastatic Renal Cell Carcinoma.

Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC

Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism

Mucosal gene therapy using a pseudotyped lentivirus vector encoding murine interleukin-10 (mIL-10) suppresses the development and relapse of experimental murine colitis

BACKGROUND: Therapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. This study investigates the safety and therapeutic benefit of a locally administered lentiviral vector encoding murine interleukin-10 in altering the onset and relapse of dextran sodium sulfate induced murine colitis. METHODS: Lentiviral vectors encoding the reporter genes firefly-luciferase and murine interleukin-10 were administered by intrarectal instillation, either once or twice following an ethanol enema to facilitate mucosal uptake, on Days 3 and 20 in Balb/c mice with acute and relapsing colitis induced with dextran sulfate sodium (DSS). DSS colitis was characterized using clinical disease activity, macroscopic, and microscopic scores. Bioluminescence optical imaging analysis was employed to examine mucosal lentiviral vector uptake and transgene expression. Levels of tumor necrosis factor-α and interleukin-6 in homogenates of rectal tissue were measured by ELISA. Biodistribution of the lentiviral vector to other organs was evaluated by real time quantitative PCR. RESULTS: Mucosal delivery of lentiviral vector resulted in significant transduction of colorectal mucosa, as shown by bioluminescence imaging analysis. Lentiviral vector-mediated local expression of interleukin-10 resulted in significantly increased levels of this cytokine, as well as reduced levels of tumor necrosis factor-α and interleukin-6, and significantly reduced the clinical disease activity, macroscopic, and microscopic scores of DSS colitis. Systemic biodistribution of locally instilled lentiviral vector to other organs was not detected. CONCLUSIONS: Topically-delivered lentiviral vectors encoding interleukin-10 safely penetrated local mucosal tissue and had therapeutic benefit in this DSS model of murine colitis

Improved Therapeutic Efficacy of Cancer Gene Therapy using Retroviral Replicating Vectors Designed for Multiple Transgene Transduction

Retroviral Replicating Vectors (RRVs) carrying the human codon optimized yeast cytosine deaminase (yCD2) suicide gene are currently in Phase I/II clinical trials for treatment of high-grade glioma. These vectors are highly tumor specific due to their intrinsic inability to infect non-dividing cells, and their restriction by the innate immune system, which is often suppressed in the tumor microenvironment. Currently, this system relies on delivery of a single transgene, yCD2, to the tumor mass. However, the current ‘gold standard’ in medical care is to treat cancer with a multi-drug regimen to combat the heterogeneous nature of tumors. As RRV therapy progresses, it must also address the issue of heterogeneity and seek to deliver more therapeutics to the cancer. These studies explore the possibility of using RRVs armed with multiple transgenes to improve the therapeutic efficacy of RRV-mediated cancer gene therapy.We examined the use of independent intrinsic promoter to drive RRV transgene expression in multiple cell lines. As transgene space in limited in RRVs, a smaller promoter was studied. Super Core Promoter (SCP1) was found to have varying degrees of protein expression across different cell lines. As certain immune factors are confined to a therapeutic window, low expression of transgene by SCP1 was used in U87 cells to express cytokine mGM-CSF along with yCD2. Protein levels of yCD2 dropped, but the transgene still remained functional, while levels of mGM-CSF remained within the therapeutic range.The MLV 4070A envelope, suffers from “superinfection resistance”, a phenomenon in which a virus is unable to infect cells that have already been infected by a virus expressing the same envelope protein. By switching out the envelope of RRV for different viral envelopes, the efficiency of cotransduction was studied across multiple cell lines. Results showed that cotransduction efficiencies varied between cell lines, potentially as a function of receptor availability or intrinsic viral immunity. Based on results from these studies, prime candidates for cotransduction of U87 cells were established. Simultaneous cotransduction using two RRVs, expressing the noninterfering 4070A (AC3) and gibbon ape leukemia virus (GALV) envelopes, resulted in increased transgene expression from AC3-RRV, whereas transgene expression from GALV-RRV decreased slightly when compared to transduction with a single RRV.In order to increase transgene expression of larger transgenes without compromising transgene stability, P2A was attached to the end of 4070A and GALV envelope proteins. P2A proved to increase protein expression in most cell lines compared to SCP1, however, the vector copy number per cell decreased. GALV pseudotyped RRV with P2A was then used to cotransduce cells in combination with AC3-yCD2. Expression of the yCD2 transgene increased, while expression of the GALV-RRV transgene decreased slightly. Functional assays revealed that cotransduction with two RRVs resulted in increased cell death, except at the highest concentrations of ganciclovir. Thus use of pseudotyped RRVs to deliver multiple transgenes has the potential to increase the therapeutic efficacy of RRV-mediated gene therapy

Potent dual block to HIV-1 infection using lentiviral vectors expressing fusion inhibitor peptide mC46- and Vif-resistant APOBEC3G

Gene therapy strategies that effectively inhibit HIV-1 replication are needed to reduce the requirement for lifelong antiviral therapy and potentially achieve a functional cure. We previously designed self-activating lentiviral vectors that efficiently delivered and expressed a Vif-resistant mutant of APOBEC3G (A3G-D128K) to T cells, which potently inhibited HIV-1 replication and spread with no detectable virus. Here, we developed vectors that express A3G-D128K, membrane-associated fusion inhibitor peptide mC46, and O6-methylguanine-DNA-methyltransferase (MGMT) selectable marker for in vivo selection of transduced CD34+ hematopoietic stem and progenitor cells. MGMT-selected T cell lines MT4, CEM, and PM1 expressing A3G-D128K (with or without mC46) potently inhibited NL4-3 infection up to 45 days post infection with no detectable viral replication. Expression of mC46 was sufficient to block infection >80% in a single-cycle assay. Importantly, expression of mC46 provided a selective advantage to the A3G-D128K-modified T cells in the presence of replication competent virus. This combinational approach to first block HIV-1 entry with mC46, and then block any breakthrough infection with A3G-D128K, could provide an effective gene therapy treatment and a potential functional cure for HIV-1 infection

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy

Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches