Replication-defective Retrovirus Research Articles

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.

A Novel Function of RNAs Arising From the Long Terminal Repeat of Human Endogenous Retrovirus 9 in Cell Cycle Arrest

The human genome contains approximately 50 copies of the replication-defective human endogenous retrovirus 9 (ERV-9) and thousands of copies of its solitary long term repeat (sLTR) element. While some sLTRs are located upstream of critical genes and have enhancer activity, other sLTRs are located within introns and may be transcribed as RNAs. We found that intronic RNAs arising from U3 sLTRs of ERV-9 were expressed as both sense (S) and antisense (AS) transcripts in all human cells tested but that expression levels differed in malignant versus nonmalignant cells. In nonmalignant cells, AS was expressed at higher levels than S and at higher levels than in malignant cells; in malignant cells, AS was expressed at amounts equivalent to those of S RNA. Critically, U3 AS RNA was found to physically bind to key transcription factors for cellular proliferation, including NF-Y, p53, and sp1, indicating that such RNA transcripts may function as decoy targets or traps for NF-Y and thus inhibit the growth of human cancer cells. Indeed, short U3 oligodeoxynucleotides (ODNs) based on these RNA sequences ably inhibited proliferation of cancer cell lines driven by cyclins B1/B2, the gene targets of NF-Y.

46IN - Immunotherapy for Advanced Prostate Cancer

ABSTRACT While the concept of immunotherapy for cancer has been proposed for many years, proof of principle did not exist. In the context of prostate cancer, the allogeneic cell based agent, GVAX has been tested. GVAX consists of a platform of irradiated hormone sensitive (LNCaP) and hormone resistant (PC-3) prostate cancer cell lines transduced with a replication-defective retrovirus bearing GM-CSF. Unfortunately, two phase III studies in men with metastatic castration resistant prostate cancer (CRPC) failed to demonstrate a survival advantage. More recently, the improved survival observed with sipuleucel-T, an autologous antigen presenting cell (APC)-based agent, for the treatment of patients with metastatic CRPC supports immunotherapy as a valid approach for this disease. Sipuleucel-T uses a fusion antigen of prostatic acid phosphatase and GM-CSF. Evidence exists to support activation of APCs and T cells in an antigen specific fashion and that the degree of activation correlates with overall survival. PROSTVAC-VF TRICOM, a poxvirus vector based PSA antigen targeted vaccine showed promising results in a randomized Phase II study and is now being tested in a international Phase III study. The CTLA-4 inhibiting human monoclonal antibody, ipilimumab, has extended survival in advanced melanoma. Early phase I clinical trials of ipilimumab have yielded clinical and PSA responses in advanced CRPC. A Phase I/II trial was conducted using ipilimumab 10 mg/kg every 3 weeks x 4 with or without prior priming by single fraction of radiation to a metastatic bony site. Results were promising enough to launch 2 phase III clinical trials in men with metastatic CRPC who were either chemo-naive or following prior chemotherapy. The outcomes of these 2 studies are pending. Disclosure P.W. Kantoff: Amgen USA consultant (con), Bayer con, Bellicum con, BIND Biosciences SAB, BN Immunotherapeutics con, Celgene DSMB, Dendreon con, Genetech con, Progenics Pharmaceuticals con, Janssen con, Takeda/Millenium DSMB, Oncogenex DSMB, Tokai con

Construction of retrovirus vector taking MDR1/ACBC1 and its transfection into human placenta derived mesenchymal stem cells

In the study, we used both the methods of perfusion and density gradient centrifugation to isolate and purify mesenchymal stem cells (MSCS) from placenta tissue, and constructed a retroviral vector with multiple drug resistant genes, and the green fluorescent protein (GFP) has been used as an indicative mark. The 293T cell was transfected by the retroviral vector PMX-flag-MDR1-GFP together with its peripheral membrane protein gene. After the infective and replication–defective retrovirus were acquired, we transfected them into human placenta-derived mesenchymal stem cells (HPMSCs). We successfully observed the expression of the reporter gene-GFP by using the green light fluorescence microscope and the p-glycoprotein (P-gp) expressed by exogenous gene MDR1 by Western Blotting. All these facts indicated that the retroviral vector PMX-flag-MDR1-GFP had successfully been transfected into HPMSCs and the exogenous gene multidrug resistance (MDR)1 was detected as normally expressed. The daunorubicin (DNR) pump experiment proved that P-gp of HPMSCs transfected with PMX-flag-MDR1-GFP was of biological activity. The result indicates that MDR1 retroviral vector can transfect the HPMSCs. Not only can the exogenous gene be expressed, but also the expression protein had the biological activity. The conclusion lays a solid foundation of the clinical application of MDR1 genetic therapy. Keywords: Transfect, human placenta-derived mesenchymal stem cells, multidrug resistance (MDR)1 gene.

Negative selection by an endogenous retrovirus promotes a higher-avidity CD4+ T cell response to retroviral infection.

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4+ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and diminished the response to FV infection. Surprisingly, however, CD4+ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4+ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4+ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4+ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.

BCL2A1 Is a Survival and Immortalization Factor for Primitive Hematopoietic Cells.

Abstract 2957Poster Board II-933We recently reported the development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2003). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene belonging to the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on hematopoiesis. To further investigate the impact of the BCL2A1 gene product on normal and malignant hematopoiesis, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentiviral vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins in vitro. We confirmed the role of BCL2A1 as an anti-apoptotic protein. The proliferation and survival of the BaF3 cells is dependant on IL-3. Upon removal of IL-3 from the media, BaF3 cells underwent an arrest in the G1 phase of the cycle. Untransduced cells or cells transduced with the empty lentiviral vector were 45% apoptotic, but this fraction decreased to 30 and 15% respectively with the cells transduced with murine and human BCL2A1. Similar results were obtained in murine 32Dcl3 cells and human UT7/Epo-S1 cells, two cells line that are respectively dependant on IL-3 and erythropoietin. In order to study the in vivo impact of BCL2A1 on hematopoiesis, C57/bl6 (Ly5.2) mice have been transplanted with primary bone marrow cells (from C57/bl6 (Ly5.1)) transduced with the BCL2A1 and control vectors, and have been followed for in vivo expansion of transduced clones and development of leukemia. The mice cohort consisted of 5 MOCK controls, 15 control vector expressing GFP only, and 15 murine BCL2A1. We frequently checked the blood counts and analyzed the lineage of blood by FACS. Mice transplanted with marrow cells transduced with the BCL2A1 vector had higher overall marking levels in the blood compared to the vector control (80% vs. 10% cells GFP+ respectively). Interestingly, preliminary histopathology revealed that 7 mice from the BCL2A1 group, but none of the control groups developed a further to be classified very poorly differentiated hematologic malignancy consisting of circulating blasts, splenomegaly, and lymphadenopathy. Cells responsible for this fatal disease were not stained by markers used for FACS analysis or by immunohistochemistry. The median overall survival was significantly different for mice treated with the vector (420 days) and mice treated with BCL2A1 (328 days). The median disease free survival was more striking as the median could not be defined for the vector while it was of 397 days for BCL2A1. Two hundred and fifty three days after the transplantation we selected 3 primary mice from BCL2A1 and vector control groups to perform secondary transplant. Less than a month after reinfusion all BCL2A1 mice developed a disease characterized by high white blood cell counts with a majority of undifferenciated cells, as well as splenomegaly, and hepatomegaly. These results were repeated in a second set of secondary transplant carried out 289 days after transplantation confirming that the disease is transplantable. The preliminary histopathology findings showed a similar phenotype to the lymphatic malignancy seen in the primary mice. In conclusion, we have confirmed the anti-apoptotic role of BCL2A1, and we are now investigating its role in hematopoiesis and leukemogenesis. We are investigating the exact phenotype of the disease seen in primary and secondary mice in order to clarify the role of BCL2A1 as a survival factor. Disclosures: Dunbar:ASH: Honoraria.

Osteogenic and adipogenic differentiation potential of an immortalized fibroblast-like cell line derived from porcine peripheral blood

Primary fibroblast-like cells isolated from the peripheral blood of a healthy pig were immortalized by transduction of cells with a replication-defective retrovirus vector expressing the E6/E7 proteins of human papillomavirus type 16 (pLXSN-16E6E7). The immortalized cells grow rapidly in cell culture and exhibit a distinct cell surface phenotype that was positive for CD90, CD44, collagen I, and vimentin and negative for CD14 and MHC II. Additionally, these immortalized blood derived-fibroblast-like cells had the potential to differentiate into osteoblasts and adipocytes in vitro as evidenced by the deposition of calcium, increased alkaline phosphatase activity, upregulated osteogenic and adipogenic marker gene expression, and accumulation of fat droplets in cells when osteogenic (dexamethasone, ascorbic acid, and β-glycerophosphate) or adipogenic supplements (dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, and insulin) were added to the culture. Overall, the results suggest that the immortalized blood-derived fibroblast-like cells exhibit some of the features of mesenchymal precursor cells, which may have implications in tissue repair and remodeling process.

A Short-term Rat Mammary Carcinogenesis Model for the Prevention of Hormonally Responsive and NonresponsiveIn situCarcinomas

Preclinical models that accurately reproduce specific aspects of human disease etiology are invaluable for the initial development and evaluation of chemopreventive agents. We developed a novel, short-term prevention model, which is particularly useful for assessing the efficacy of a compound to prevent hormonally responsive and nonresponsive in situ carcinomas. In this model, carcinogenesis is induced by a high titer of neu-containing, replication-defective retrovirus. The multiplicity and size of the resulting in situ carcinomas are scored in whole-mounted, aluminum carmine-stained mammary glands at 15 days postinfusion. These in situ carcinomas represent a distinct biological time point in the development of neu-induced mammary cancer in the rat. They are characterized by high rates of proliferation (40.0%; P < 0.0001) and apoptosis (2.8%; P < 0.005) compared with mammary carcinomas. The majority of in situ carcinomas regress spontaneously after 20 days postinfusion. The in situ carcinomas at 15 days postinfusion exhibit hormonal responsiveness. The effects of the chemoprevention agents tamoxifen, celecoxib, and targretin on hormonally responsive and nonresponsive in situ carcinomas recapitulate those observed on mammary carcinomas at 12 and 18 weeks postinfusion for intact and ovariectomized rats, respectively. Neu-induced in situ carcinomas in the rat represent etiologically relevant intermediate time points of mammary carcinogenesis. Our prevention model represents a cost-efficient in vivo system to determine whether the preventive effects of a compound extend to hormonally nonresponsive mammary lesions, for which new chemoprevention approaches are needed.

Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo ProductionIn Vitro

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNbeta-Z or LNbeta-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro. Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNbeta-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 +/- 6.4%; 12.0 +/- 5.7%) or EGFP (57.5 +/- 6.3%; 10.1 +/- 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 +/- 8.2%; 12.3 +/- 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.

Abstract 1582: Identification of BCAR genes relevant for breast cancer progression and endocrine therapy resistance

Abstract Endocrine therapy of breast cancer is effective and widely applied. In advanced disease, about half of the patients will experience a clinical benefit from tamoxifen treatment, but ultimately the disease will progress into a resistant phenotype. The molecular basis of this general failure of endocrine therapy is not understood yet. Although gene expression profiling experiments provide gene lists to identify patients who may benefit from specific treatments, these genes mostly do not define the underlying molecular mechanisms. We have executed a functional screen for the identification of BCAR genes causing anti-estrogen resistance in a human breast cancer model and evaluated their role in a large breast cancer cohort. Estrogen-dependent ZR-75-1 human breast cancer cells were subjected to insertion mutagenesis with replication-defective retroviruses and selected for proliferation in the presence of 4-hydroxy tamoxifen. In our panel of 79 tamoxifen-resistant cell lines, the target genes of the retroviruses have been identified and were functionally tested following transfection into ZR-75-1 cells. Seven BCAR genes (AKT1, AKT2, BCAR1, BCAR3, EGFR, GRB7 and TRERF1) causing the tamoxifen-resistant phenotype were identified. Furthermore, additional putative BCAR genes and loci have been pinpointed. To determine the relevance of the identified genes in clinical breast cancer, we have studied 561 estrogen receptor-positive primary breast cancers for tumor aggressiveness and tamoxifen therapy resistance. Quantitative RT-PCR of ten genes was performed to measure the tumor mRNA levels. The endpoint for tumor aggressiveness was metastasis-free survival (MFS) of a cohort of lymph node-negative patients. Therapy resistance was evaluated in a group of patients treated with tamoxifen as first-line therapy for recurrent disease and the endpoint of this analysis was progression-free survival (PFS). Statistically significant associations with MFS were found for AKT2, EGFR, TLE3 and TRERF1 mRNA levels. In multivariate analyses including the traditional prognostic factors, AKT2, EGFR and TRERF1 were independent prognostic factors for MFS and thus are associated with tumor aggressiveness. In the analyses of PFS, mRNA levels of BCAR3, ERBB2, GRB7, TLE3 and TRERF1 were associated with the duration of clinical benefit of tamoxifen therapy, independently of the classical predictive factors. Our functional screen identified a set of clinically relevant BCAR genes offering insight in the molecular and cellular pathways of breast tumor aggressiveness and tamoxifen resistance, and thereby provides novel targets for personalized treatment and prevention strategies. Supported by the Dutch Cancer Society, the Susan G Komen Breast Cancer Foundation and the Association for International Cancer Research.

Functional identification of genes causing estrogen independence of human breast cancer cells

Endocrine treatment of breast cancer is widely applied and effective. However, in advanced disease cases, the tumors will eventually progress into an estrogen-independent and therapy-resistant phenotype. To elucidate the molecular mechanisms underlying this endocrine therapy failure, we applied retroviral insertion mutagenesis to identify the main genes conferring estrogen independence to human breast cancer cells. Estrogen-dependent ZR-75-1 cells were infected with replication-defective retroviruses followed by selection with the anti-estrogen 4-hydroxy-tamoxifen. In the resulting panel of 79 tamoxifen-resistant cell lines, the viral integrations were mapped within the human genome. Genes located in the immediate proximity of the retroviral integration sites were characterized for altered expression and their capacity to confer anti-estrogen resistance when transfected into breast cancer cells. Out of 15 candidate BCAR (breast cancer anti-estrogen resistance) genes, seven (AKT1, AKT2, BCAR1, BCAR3, EGFR, GRB7, and TRERF1/BCAR2) were shown to directly underlie estrogen independence. Our results show that insertion mutagenesis is a powerful tool to identify BCAR loci, which may provide insights into the molecular and cellular mechanisms of breast tumor progression and therapy resistance thereby offering novel targets for the development of tailor-made therapeutical and prevention strategies.

A novel immortalization vector for the establishment of penaeid shrimp cell lines

Cell immortalization technology based on gene transfer has been successfully used to generate cell lines from a wide variety of cell types. The inability to stably introduce and express foreign genes has hampered application of this strategy in shrimp cells. We report here the use of replication-defective pantropic retrovirus to achieve a novel immortalization vector in which simian virus 40 large T antigen (SV40T) gene is expressed from Moloney murine leukemia virus (MoMLV) promoter. Data confirmed the presence of transferred SV40T gene and its stable mRNA expression in transduced lymphoid cells of Penaeus chinensis. The transduced cells showed a higher growth rate and a longer replication life-span compared with their untransduced counterparts. These results indicate the pantropic retrovirus-based immortalization-inducing gene delivery system is a potential tool for establishing cell lines from shrimp.

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells.

We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.

Development of Transgenic Chickens Expressing Human Parathormone Under the Control of a Ubiquitous Promoter by Using a Retrovirus Vector System

Transgenic chickens, ubiquitously expressing a human protein, could be a very useful model system for studying the role of human proteins in embryonic development as well as for efficiently producing pharmaceutical drugs as bioreactors. Human parathormone (hPTH) secreted from parathyroid glands plays a significant role in calcium homeostasis and is an important therapeutic agent for the treatment of osteoporosis in humans. Here, by using a robust replication-defective Moloney murine leukemia virus-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein, we generated transgenic chickens expressing hPTH under the control of a ubiquitous Rous sarcoma virus promoter. The recombinant retrovirus was injected into the subgerminal cavity of freshly laid eggs at the blastodermal stage. After 21 d of incubation, 42 chicks hatched from 473 retrovirus-injected eggs. All 42 living chicks were found to express the vector-encoded hPTH gene in diverse organs, as revealed by PCR and reverse transcription-PCR analysis by using primer pairs specific for hPTH. Four days after hatching, 6 chicks died and 14 chicks showed phenotypic deformities. At 18 wk of age, only 3 G0 chickens survived. They also released the hPTH hormone in their blood and transmitted the hPTH gene to G1 embryos. However, although the embryos were alive at d 18 of incubation, none hatched. An electrochemiluminescence immunoassay further showed that the hPTH expression level was markedly elevated in mammalian cells infected by the retrovirus vector. Thus, we demonstrated that transgenic chickens, expressing a human protein under the control of a ubiquitous promoter, not only could be an efficient bioreactor for the production of pharmaceutical drugs, but also could be useful for studies on the role of human proteins in embryonic development. To our knowledge, this is the first report on the production of a human protein (hPTH) in transgenic chickens under the control of a ubiquitous promoter by using a replication-defective Moloney murine leukemia virus-based retrovirus vector system.

Optimising stable retroviral transduction of primary human synovial fibroblasts

Fibroblast like synoviocytes are the main resident cells in normal joints and are known to play a major role in the pathogenesis of rheumatoid arthritis. Efficient gene targeting of fibroblast like synoviocytes (FLS) is a major goal of current ex vivo gene therapy approaches for the treatment of rheumatoid arthritis. However, there is a need to improve viral systems capable of delivering genes to human rheumatoid fibroblasts and attempts have been made to develop a protocol for high efficiency, reproducible gene transfer using a replication-defective retrovirus vector. The effects of different experimental conditions were examined as well as those related to cellular and viral features on the efficiency of transducing the retroviral-driven expression of enhanced green fluorescent protein (EGFP) to FLS harvested from patients with rheumatoid arthritis. The optimal method established involved a double round of infection by centrifugation with a resting period of 4 h between rounds. This approach led to the transduction of 30–70% of FLS obtained from nine patients with rheumatoid arthritis. Consistent transduction efficiencies were achieved in repeat assays such that it could be inferred that the variations observed were attributable to the specific characteristics of each cell line. This simple protocol renders a consistent and reproducible efficiency of rheumatoid fibroblast transduction and makes stable gene targeting using non-replicating retrovirus derived vectors an affordable option for the treatment of rheumatoid arthritis.

Cerebral ischemia induces neuronal expression of novel VL30 mouse retrotransposons bound to polyribosomes

Mammalian genomes are burdened with a large heterogeneous group of endogenous replication defective retroviruses (retrotransposons). Previously, we identified a transcript resembling a virus-like 30S ( VL30) retrotransposon increasing in mouse brain following transient cerebral ischemia. Paradoxically, this non-coding RNA was found bound to polyribosomes. Further analysis revealed that multiple retrotransposon species ( BVL-1-like and mVL30-1-like) were bound to polyribosomes and induced by ischemia. These VL30 transcripts remained associated with polyribosomes in the presence of 0.5 M KCl, indicating that VL30 mRNA was tightly associated with ribosomal subunits. Furthermore, the profile of BVL-1 distribution on polyribosomal profiles was distinct from those of translated and translationally repressed mRNA. Consistent with expectations, 5.0 kb VL30 transcripts were detected in ischemic brain with a temporal pattern of expression that was distinct from c- fos. Expression of VL30 was localized in neurons using a combination of in situ hybridization and immunocytochemistry. 3′-RACE-PCR experiments yielded two unique sequences ( VL30x-1 and VL30x-2) that were homologous to known VL30 genes. Phylogenetic analysis of VL30 promoter sequence (U3 region) resulted in the identification of two large VL30 subgroups. VL30x-1 and VL30x-2 were closely related and classified in a group that was distinct from the well-characterized VL30 genes BVL-1 and mVL30-1. The promoter regions of VL30x-1 and VL30x-2 did not possess the consensus sequences for either hypoxia or anoxia response elements, suggesting an alternative mechanism for induction. This is the first report that demonstrates ischemia-induced, neuronal expression of unique VL30 retrotransposons in mouse brain.