Retargeted Foamy Virus Vectors Integrate Less Frequently Near Proto-oncogenes.

Retroviral gene therapy offers immense potential to treat many genetic diseases and has already shown efficacy in clinical trials. However, retroviral vector mediated genotoxicity remains a major challenge and clinically relevant approaches to reduce integration near genes and proto-oncogenes are needed. Foamy retroviral vectors have several advantages over gammaretroviral and lentiviral vectors including a potentially safer integration profile and a lower propensity to activate nearby genes. Here we successfully retargeted foamy retroviral vectors away from genes and into satellite regions enriched for trimethylated histone H3 at lysine 9 by modifying the foamy virus Gag and Pol proteins. Retargeted foamy retroviral vectors integrated near genes and proto-oncogenes less often (p < 0.001) than controls. Importantly, retargeted foamy retroviral vectors can be produced at high, clinically relevant titers (>107 transducing units/ml), and unlike other reported retargeting approaches engineered target cells are not needed to achieve retargeting. As proof of principle for use in the clinic we show efficient transduction and retargeting in human cord blood CD34+ cells. The modified Gag and Pol helper constructs we describe will allow any investigator to simply use these helper plasmids during vector production to retarget therapeutic foamy retroviral vectors.

Lentiviral vector-mediated insertional mutagenesis screen identifies genes that influence androgen independent prostate cancer progression and predict clinical outcome.

Prostate cancer (PC) is the second leading cause of cancer related deaths in US men. Androgen deprivation therapy (ADT) improves clinical outcome, but tumors often recur and progress to androgen independent prostate cancer (AIPC) which no longer responds to ADT. The progression to AIPC is due to genetic alterations that allow PC cancer cells to grow in the absence of androgen. Here we performed an insertional mutagenesis screen using a replication-incompetent lentiviral vector (LV) to identify the genes that promote AIPC in an orthotopic mouse model. Androgen sensitive PC cells, LNCaP, were mutagenized with LV and injected into the prostate of male mice. After tumor development, mice were castrated to select for cells that proliferate in the absence of androgen. Proviral integration sites and nearby dysregulated genes were identified in tumors developed in an androgen deficient environment. Using publically available datasets, the expression of these candidate androgen independence genes in human PC tissues were analyzed. A total of 11 promising candidate AIPC genes were identified: GLYATL1, FLNA, OBSCN, STRA13, WHSC1, ARFGAP3, KDM2A, FAM83H, CLDN7, CNOT6, and B3GNT9. Seven out the 11 candidate genes; GLYATL1, OBSCN, STRA13, KDM2A, FAM83H, CNOT6, and B3GNT6, have not been previously implicated in PC. An in vitro clonogenic assay showed that knockdown of KDM2A, FAM83H, and GLYATL1 genes significantly inhibited the colony forming ability of LNCaP cells. Additionally, we showed that a combination of four genes, OBSCN, FAM83H, CLDN7, and ARFGAP3 could significantly predicted the recurrence risk in PC patients after prostatectomy (P = 5.3 × 10-5 ). © 2015 Wiley Periodicals, Inc.

Insulated Foamy Viral Vectors.

Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34(+) cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy.

A novel gammaretroviral shuttle vector insertional mutagenesis screen identifies SHARPIN as a breast cancer metastasis gene and prognostic biomarker.

Breast cancer (BC) is the second leading cause of malignancy among U.S. women. Metastasis results in a poor prognosis and increased mortality, but the molecular mechanisms by which metastatic tumors occur are not well understood. Identifying the genes that drive the metastatic process could provide targets for improved therapy and biomarkers to improve BC patient outcomes. Using a forward mutagenesis screen, BC cells mutagenized with a replication-incompetent gammaretroviral vector (γRV) were xenotransplanted into the mammary fat pad of immunodeficient mice. In this approach the vector provirus dysregulates nearby genes, providing a selective advantage to transduced cells to form metastases. Metastatic tumors were analyzed for proviral integration sites to identify nearby candidate metastasis genes. The γRV has a transgene cassette that allows for rescue in bacteria and rapid identification of vector integration sites. Using this approach, we identified the previously described metastasis gene WWTR1 (TAZ), and three other novel candidate metastasis genes including SHARPIN. SHARPIN was independently validated in vivo as a BC metastasis gene. Analysis of patient data showed that SHARPIN expression predicts metastasis-free survival after adjuvant therapy. Our approach has broad potential to identify genes involved in oncogenic processes for BC and other cancers. We show here it can identify both known (WWTR1) and novel (SHARPIN) BC metastasis genes.

Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites.

High-throughput mapping of retroviral vector integration sites (RIS) has become an invaluable tool to evaluate novel gene therapy vectors and to track clonal contribution in preclinical and clinical studies. Beard et al. (Methods Mol Biol 2014;1185:321-344) described an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. Here we describe adaptations to the previously published modified genomic sequencing PCR (MGS-PCR) protocol using the Illumina MiSeq paired-end sequencing platform. Lentiviral, gammaretroviral, and foamy virus vector integrations were analyzed. MGS-PCR using the MiSeq platform allows for the use of merged paired-end reads, which allows for efficient localization of RIS to published genomes.

A novel retroviral mutagenesis screen identifies prognostic genes in RUNX1 mediated myeloid leukemogenesis.

Using a novel retroviral shuttle vector approach we identified genes that collaborate with a patient derived RUNX1 (AML1) mutant. RUNX1 mutations occurs in 40% of myelodysplastic syndromes (MDS). MDS are a group of hematopoietic stem cell disorders that are characterized by dysplasia that often progress to acute myeloid leukemia (AML). Our goal was to identify genes dysregulated by vector-mediated genotoxicity that may collaborate with the RUNX1 mutant (D171N). D171N expressing cells have a survival and engraftment disadvantage and require additional genetic lesions to survive and persist. By dysregulating genes near the integrated vector provirus, the shuttle vector can promote transformation of D171N cells and tag the nearby genes that collaborate with D171N. In our approach, a gammaretroviral shuttle vector that expresses D171N is used to transduce CD105+, Sca-1+ mouse bone marrow. Mutagenized cells are expanded in liquid culture and vector integration sites from surviving cells are then identified using a retroviral shuttle vector approach. We repeatedly recovered integrated vector proviruses near genes (Itpkb, Ccdc12, and Nbeal2). To assess the prognostic significance of the genes identified we examined differential expression, overall survival, and relapse free survival of AML patients with alteration in the genes identified using The Cancer Genome Atlas (TCGA) AML data set. We found that ITPKB functions as an independent factor for poor prognoses and RUNX1 mutations in conjunction with ITPKB, CCDC12, and NBEAL2 have prognostic potential in AML.

A novel approach to identify driver genes involved in androgen-independent prostate cancer.

BACKGROUND: Insertional mutagenesis screens have been used with great success to identify oncogenes and tumor suppressor genes. Typically, these screens use gammaretroviruses (γRV) or transposons as insertional mutagens. However, insertional mutations from replication-competent γRVs or transposons that occur later during oncogenesis can produce passenger mutations that do not drive cancer progression. Here, we utilized a replication-incompetent lentiviral vector (LV) to perform an insertional mutagenesis screen to identify genes in the progression to androgen-independent prostate cancer (AIPC). METHODS: Prostate cancer cells were mutagenized with a LV to enrich for clones with a selective advantage in an androgen-deficient environment provided by a dysregulated gene(s) near the vector integration site. We performed our screen using an in vitro AIPC model and also an in vivo xenotransplant model for AIPC. Our approach identified proviral integration sites utilizing a shuttle vector that allows for rapid rescue of plasmids in E. coli that contain LV long terminal repeat (LTR)-chromosome junctions. This shuttle vector approach does not require PCR amplification and has several advantages over PCR-based techniques. RESULTS: Proviral integrations were enriched near prostate cancer susceptibility loci in cells grown in androgen-deficient medium (p < 0.001), and five candidate genes that influence AIPC were identified; ATPAF1, GCOM1, MEX3D, PTRF, and TRPM4. Additionally, we showed that RNAi knockdown of ATPAF1 significantly reduces growth (p < 0.05) in androgen-deficient conditions. CONCLUSIONS: Our approach has proven effective for use in PCa, identifying a known prostate cancer gene, PTRF, and also several genes not previously associated with prostate cancer. The replication-incompetent shuttle vector approach has broad potential applications for cancer gene discovery, and for interrogating diverse biological and disease processes.