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1.
Methods Mol Biol ; 1907: 103-114, 2019.
Article in English | MEDLINE | ID: mdl-30542994

ABSTRACT

Prostate cancer (PC) is the second leading cause of cancer-related deaths in US men, and progression to androgen-independent PC (AIPC) typically results in metastasis and is lethal. However, the mechanisms whereby PC progresses from androgen dependence to androgen independence are not completely understood. Mutagenesis screens to identify novel genes involved in the progression to AIPC have been performed using replication-incompetent lentiviral vectors (LVs). In this approach the LV acts both as a mutagen and as molecular tag to identify nearby genes that may have been dysregulated by the vector provirus, and are candidate AIPC genes. Here we describe protocols for generation of replication-incompetent LV preparations and performing a mutagenesis screen to identify AIPC genes in vitro.


Subject(s)
Androgens/metabolism , Genes, Neoplasm , Lentivirus/genetics , Mutagenesis , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/genetics , Virus Replication , Disease Progression , Genetic Vectors , High-Throughput Screening Assays , Humans , Male , Tumor Cells, Cultured
2.
J Gene Med ; 20(7-8): e3028, 2018 07.
Article in English | MEDLINE | ID: mdl-29935087

ABSTRACT

BACKGROUND: Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and also that insulators can improve FV vector safety. However, in a previous analysis of insulator effects on FV vector safety, strong viral promoters were used to elicit genotoxic events. In the present study, we developed and analyzed the efficacy and safety of a high-titer, clinically relevant FV vector driven by the housekeeping promoter elongation factor-1α and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). METHODS: Human CD34+ cord blood cells were exposed to an enhanced green fluorescent protein expressing vector, FV-EGW-A1, at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. RESULTS: FV-EGW-A1 resulted in high-marking, multilineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. CONCLUSIONS: An FV vector with an elongation factor-1α promoter and an A1 insulator is a promising vector design for use in the clinic.


Subject(s)
Gene Transfer Techniques , Genetic Vectors/genetics , Hematopoietic Stem Cells/metabolism , Spumavirus/genetics , Transduction, Genetic , Animals , Gene Expression , Gene Order , Genes, Reporter , Genetic Therapy , Genetic Vectors/administration & dosage , Genetic Vectors/adverse effects , Hematopoietic Stem Cell Transplantation , Heterografts , Humans , Mice , Transgenes
3.
Blood Adv ; 2(9): 987-999, 2018 05 08.
Article in English | MEDLINE | ID: mdl-29720491

ABSTRACT

Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.


Subject(s)
Dog Diseases , Genetic Therapy , Genetic Vectors/pharmacology , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization , Heterocyclic Compounds/pharmacology , Spumavirus , X-Linked Combined Immunodeficiency Diseases , Animals , Benzylamines , CD4-CD8 Ratio , Cyclams , Disease Models, Animal , Dog Diseases/blood , Dog Diseases/genetics , Dog Diseases/therapy , Dogs , Humans , Phosphoglycerate Kinase/genetics , X-Linked Combined Immunodeficiency Diseases/blood , X-Linked Combined Immunodeficiency Diseases/genetics , X-Linked Combined Immunodeficiency Diseases/therapy , X-Linked Combined Immunodeficiency Diseases/veterinary
4.
Oncotarget ; 9(21): 15451-15463, 2018 Mar 20.
Article in English | MEDLINE | ID: mdl-29643985

ABSTRACT

Replication-incompetent gammaretroviral (γRV) and lentiviral (LV) vectors have both been used in insertional mutagenesis screens to identify cancer drivers. In this approach the vectors stably integrate in the host cell genome and induce cancers by dysregulating nearby genes. The cells that contain a retroviral vector provirus in or near a proto-oncogene or tumor suppressor are preferentially enriched in a tumor. γRV and LV vectors have different integration profiles and genotoxic potential, making them potentially complementary tools for insertional mutagenesis screens. We performed screens using both γRV and LV vectors to identify driver genes that mediate progression of androgen-independent prostate cancer (AIPC) using a xenotransplant mouse model. Vector transduced LNCaP cells were injected orthotopically into the prostate gland of immunodeficient mice. Mice that developed tumors were castrated to create an androgen-deficient environment and metastatic tumors that developed were analyzed. A high-throughput modified genomic sequencing PCR (MGS-PCR) approach identified the positions of vector integrations in these metastatic tumors. OR2A14, FER1L6, TAOK3, MAN1A2, MBNL2, SERBP1, PLEKHA2, SPTAN1, ADAMTS1, SLC30A5, ABCC1, SLC7A1 and SLC25A24 were identified as candidate prostate cancer (PC) progression genes. TAOK3 and ABCC1 expression in PC patients predicted the risk of recurrence after androgen deprivation therapy. Our data shows that γRV and LV vectors are complementary approaches to identify cancer driver genes which may be promising potential biomarkers and therapeutic targets.

5.
Sci Rep ; 6: 36610, 2016 11 04.
Article in English | MEDLINE | ID: mdl-27812034

ABSTRACT

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.


Subject(s)
Genetic Vectors , Proto-Oncogenes , Spumavirus/genetics , Virus Integration , Cell Line , Humans , Male , Polymerase Chain Reaction
6.
Cancers (Basel) ; 8(11)2016 Oct 25.
Article in English | MEDLINE | ID: mdl-27792127

ABSTRACT

Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types.

7.
Nat Commun ; 7: 13173, 2016 10 20.
Article in English | MEDLINE | ID: mdl-27762266

ABSTRACT

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However, current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility, limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here, we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow, and reconstitute human haematopoiesis in immunocompromised mice. Importantly, nonhuman primate autologous gene-modified CD34+ cell products are capable of stable, polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy, there is enormous potential for this approach to treat patients on a global scale.


Subject(s)
Automation, Laboratory/instrumentation , Cell Separation/methods , Genetic Therapy/methods , Hematopoietic Stem Cells/cytology , Lentivirus/genetics , Transduction, Genetic/methods , Animals , Antigens, CD34/genetics , Antigens, CD34/metabolism , Automation, Laboratory/standards , Biomarkers/metabolism , Cell Separation/instrumentation , Cell Separation/standards , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Humans , Lentivirus/metabolism , Macaca nemestrina , Mice , Staining and Labeling/methods , Transduction, Genetic/standards
8.
Mol Ther Methods Clin Dev ; 3: 16048, 2016.
Article in English | MEDLINE | ID: mdl-27579335

ABSTRACT

Hematopoietic stem cell (HSC) gene therapy using retroviral vectors has immense potential, but vector-mediated genotoxicity limits use in the clinic. Lentiviral vectors are less genotoxic than gammaretroviral vectors and have become the vector of choice in clinical trials. Foamy retroviral vectors have a promising integration profile and are less prone to read-through transcription than gammaretroviral or lentiviral vectors. Here, we directly compared the safety and efficacy of foamy vectors to lentiviral vectors in human CD34(+) repopulating cells in immunodeficient mice. To increase their genotoxic potential, foamy and lentiviral vectors with identical transgene cassettes with a known genotoxic spleen focus forming virus promoter were used. Both vectors resulted in efficient marking in vivo and a total of 825 foamy and 460 lentiviral vector unique integration sites were recovered in repopulating cells 19 weeks after transplantation. Foamy vector proviruses were observed less often near RefSeq gene and proto-oncogene transcription start sites than lentiviral vectors. The foamy vector group were also more polyclonal with fewer dominant clones (two out of six mice) than the lentiviral vector group (eight out of eight mice), and only lentiviral vectors had integrants near known proto-oncogenes in dominant clones. Our data further support the relative safety of foamy vectors for HSC gene therapy.

9.
Curr Opin Virol ; 21: 41-46, 2016 12.
Article in English | MEDLINE | ID: mdl-27521874

ABSTRACT

Hematopoietic stem cell (HSC) gene therapy using retroviral vectors is a powerful and promising approach to permanently correct many hematopoietic disorders. Increasing the transduction of quiescent HSCs and reducing genotoxicity are major challenges in the field. Retroviral vectors, including lentiviral and foamy vectors, have been extensively modified resulting in improved safety and efficacy. This review will focus on recent advances to improve vector entry, transduction efficiency, control of transgene expression and approaches to improve safety by modifying the retroviral integration profile.


Subject(s)
Genetic Vectors , Hematopoietic Stem Cells/virology , Retroviridae/genetics , Animals , DNA Damage , Genetic Therapy/methods , Hematologic Diseases/therapy , Humans , Transduction, Genetic
10.
Mol Ther ; 24(7): 1237-46, 2016 08.
Article in English | MEDLINE | ID: mdl-27058824

ABSTRACT

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.


Subject(s)
Genetic Vectors/genetics , Hematopoietic Stem Cells/metabolism , Lentivirus/genetics , Membrane Glycoproteins/genetics , T-Lymphocytes/metabolism , Transduction, Genetic , Cell Culture Techniques , Gene Expression , Genes, Reporter , Genetic Engineering , HEK293 Cells , Humans , Lentivirus/metabolism , Membrane Glycoproteins/metabolism , Receptors, LDL/metabolism , Receptors, Virus/metabolism , Transgenes , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism , Viral Envelope Proteins/genetics
11.
Mol Carcinog ; 55(11): 1761-1771, 2016 Nov.
Article in English | MEDLINE | ID: mdl-26512949

ABSTRACT

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.


Subject(s)
Androgens/metabolism , Genes, Neoplasm , Lentivirus/genetics , Mutagenesis, Insertional/methods , Prostatic Neoplasms/genetics , Animals , Cell Line, Tumor , Disease Progression , Gene Expression Profiling/methods , Genetic Vectors/pharmacology , Humans , Male , Mice , Neoplasm Transplantation , Prostatic Neoplasms/metabolism
12.
Hum Gene Ther ; 27(3): 255-66, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26715244

ABSTRACT

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.


Subject(s)
Genetic Vectors/genetics , Insulator Elements , Spumavirus/genetics , Cell Line , Enhancer Elements, Genetic , Gene Order , Gene Transfer Techniques , Genes, Reporter , Genetic Vectors/adverse effects , Hematopoietic Stem Cells/metabolism , Humans , Plasmids/genetics , Transduction, Genetic , Virus Integration , Virus Replication
13.
Biomedicines ; 4(1)2016 Mar.
Article in English | MEDLINE | ID: mdl-28424756

ABSTRACT

Retroviral vector gene therapy is a promising approach to treating HIV-1. However, integrated vectors are mutagens with the potential to dysregulate nearby genes and cause severe adverse side effects. Leukemia has already been a documented severe adverse event in gene therapy clinical trials for the treatment of primary immunodeficiencies. These side effects will need to be reduced or avoided if retroviral vectors are to be used clinically for HIV-1 treatment. The addition of chromatin insulators to retroviral vectors is a potential strategy for reducing adverse side effects. Insulators have already been effectively used in retroviral vectors to reduce genotoxicity in pre-clinical studies. Here, we will review how insulators function, genotoxicity in gene therapy clinical trials, the design of insulated retroviral vectors, promising results from insulated retroviral vector studies, and considerations for the development of insulated retroviral treatment vectors for HIV-1 gene therapy.

14.
Biomedicines ; 4(2)2016 Mar 29.
Article in English | MEDLINE | ID: mdl-28536375

ABSTRACT

Stem cell gene therapy approaches for Human Immunodeficiency Virus (HIV) infection have been explored in clinical trials and several anti-HIV genes delivered by retroviral vectors were shown to block HIV replication. However, gammaretroviral and lentiviral based retroviral vectors have limitations for delivery of anti-HIV genes into hematopoietic stem cells (HSC). Foamy virus vectors have several advantages including efficient delivery of transgenes into HSC in large animal models, and a potentially safer integration profile. This review focuses on novel anti-HIV transgenes and the potential of foamy virus vectors for HSC gene therapy of HIV.

15.
Oncotarget ; 6(37): 39507-20, 2015 Nov 24.
Article in English | MEDLINE | ID: mdl-26506596

ABSTRACT

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.


Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Gammaretrovirus/genetics , Mutagenesis, Insertional , Ubiquitins/genetics , Animals , Base Sequence , Breast Neoplasms/pathology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Genetic Vectors/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kaplan-Meier Estimate , Molecular Sequence Data , Neoplasm Metastasis , Prognosis , RNA Interference , RNAi Therapeutics/methods , Trans-Activators , Transcription Factors , Transcriptional Coactivator with PDZ-Binding Motif Proteins , Xenograft Model Antitumor Assays/methods
16.
Hum Gene Ther Methods ; 26(6): 221-7, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26415022

ABSTRACT

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.


Subject(s)
Chromosome Breakpoints , High-Throughput Nucleotide Sequencing , Polymerase Chain Reaction , Retroviridae/physiology , Virus Integration , Animals , Computational Biology/methods , Genetic Vectors/genetics , Humans , Retroviridae/classification , Sequence Analysis, DNA/methods
17.
Oncotarget ; 6(31): 30664-74, 2015 Oct 13.
Article in English | MEDLINE | ID: mdl-26384344

ABSTRACT

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.


Subject(s)
Carcinogenesis/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Genetic Vectors/genetics , Leukemia, Myeloid, Acute/genetics , Myelodysplastic Syndromes/genetics , Animals , Blood Proteins/genetics , Cell Line, Tumor , HEK293 Cells , Humans , Leukemia, Myeloid, Acute/mortality , Mice , Myelodysplastic Syndromes/mortality , Myelodysplastic Syndromes/pathology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Proteins/genetics , Retroviridae/genetics , Virus Integration/genetics
18.
BMC Bioinformatics ; 16: 212, 2015 Jul 07.
Article in English | MEDLINE | ID: mdl-26150117

ABSTRACT

BACKGROUND: Analyzing the integration profile of retroviral vectors is a vital step in determining their potential genotoxic effects and developing safer vectors for therapeutic use. Identifying retroviral vector integration sites is also important for retroviral mutagenesis screens. RESULTS: We developed VISA, a vector integration site analysis server, to analyze next-generation sequencing data for retroviral vector integration sites. Sequence reads that contain a provirus are mapped to the human genome, sequence reads that cannot be localized to a unique location in the genome are filtered out, and then unique retroviral vector integration sites are determined based on the alignment scores of the remaining sequence reads. CONCLUSIONS: VISA offers a simple web interface to upload sequence files and results are returned in a concise tabular format to allow rapid analysis of retroviral vector integration sites.


Subject(s)
Genetic Vectors , Genome, Human , High-Throughput Nucleotide Sequencing/methods , Internet , Retroviridae/genetics , Software , Virus Integration/genetics , Humans
19.
Methods Mol Biol ; 1185: 321-44, 2014.
Article in English | MEDLINE | ID: mdl-25062639

ABSTRACT

Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods. Here, we describe an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. We also discuss methods to assess dominant/overrepresented gene-modified clones in preclinical and clinical models.


Subject(s)
Chromosome Mapping/methods , Genetic Therapy , Genetic Vectors/genetics , Genomics/methods , Retroviridae/genetics , Virus Integration/genetics , DNA/genetics , High-Throughput Nucleotide Sequencing , Humans , Polymerase Chain Reaction , Retroviridae/physiology , Sequence Analysis, DNA
20.
Mol Cancer ; 13: 120, 2014 May 23.
Article in English | MEDLINE | ID: mdl-24885513

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Neoplastic , Lentivirus/genetics , Mutagenesis, Insertional/methods , Prostate/metabolism , Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Clone Cells , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Loci , Genetic Vectors , High-Throughput Screening Assays , Humans , Lentivirus/metabolism , Male , Mice , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Molecular Chaperones/antagonists & inhibitors , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Androgen/deficiency , Signal Transduction , Terminal Repeat Sequences , Virus Replication , Xenograft Model Antitumor Assays
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