Green fluorescent protein (GFP) is a widely used intracellu-EGFP did lead to rapid development of disease in immunolar reporter molecule to assess gene transfer and deficient Nu/Nu mice. Mice surviving BM185/EGFP leukeexpression. A potential use for GFP is as a co-expressed mia challenge developed high cytotoxic T lymphocyte marker, to select and enrich gene-modified cells by flow (CTL) responses against EGFP-expressing cells. Furthercytometry. Processed peptides derived from GFP and more, immune stimulation against BM185/EGFP cells presented by the major histocompatibility complex on the could also be induced by immunization with EGFP+ transcell surface could potentially induce T cell immune duced dendritic cells. The effects of the co-expression of responses against GFP+ cells. Thus, clinical application of EGFP and immunomodulators (CD80 plus GM-CSF) were GFP is premature, since in vivo studies on its immunoalso investigated as an irradiated leukemia vaccine. EGFP genicity are lacking. Therefore, we investigated immune co-expression by the vaccine did not interfere with the responses against EGFP (enhanced-GFP) in two transdevelopment of CTLs against the parental leukemia or with plantable murine models: the BALB/c (H-2 that the immune response against EGFP may interfere with BM185 and EL-4 cell lines modified to express high levels its applicability in gene insertion/replacement strategies but of EGFP showed drastic reduction of disease development could potentially be employed for leukemia cell vaccines. when transplanted into immunocompetent mice. BM185/

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2°CFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2°CFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2°CFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2°CFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2°CFU-S from 8 recipients and expression in >50% of the vector-containing 2°CFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.

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Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5 end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.), and the G1Na plasmid was provided by P. Tolstechev (Genetic Therapy, Inc., Gaithersburg, Md.). The LN vector was constructed and packaged in the laboratory of A. Dusty Miller (Fred Hutchinson Cancer Center, Seattle, Wash.).The MPSV LTR was used to replace the 3Ј MoMuLV LTR of G1Na to make MPneo. The NCR was removed from the MPSV LTR as an NheI (at nucleotide 33 in the LTR)-to-Sau3a (at nucleotide 97 in the LTR) fragment. The cut ends of the LTR were ligated together after fill-in by Klenow DNA polymerase to make the MPncr 3Ј LTR; this was then used to replace the 3Ј LTR of G1Na, yielding MPncrneo. The Thy-1 fragment in the plasmid Bluescript was opened at the SmaI site immediately 3Ј of the insert, and a synthetic oligonucleotide encoding an XbaI site (New England Biolabs, Beverly, Mass.) was ligated in place. The Thy-1 piece was isolated as an XbaI-XbaI fragment and cloned into the NheI site of the MPSV LTR in Bluescript. The Thy-1-substituted MPSV LTR was inserted in place of the 3Ј LTR in G1Na to make MPthyneo.The 5Ј LTR and psi region from plasmid LN was subcloned as an EcoRI-EcoRI fragment. The KpnI-SpeI fragment encompassing the PBS was removed and replaced by the KpnI-SpeI fragment from dl587rev. The 5Ј LTR/leader region fragment containing the dl587rev PBS was then returned to the MPneo, MPthyneo, and MPncrneo plasmids to produce MPdlneo, MPthydlneo, and MPncrdlneo.To make LNncrneo, the NCR (NheI at position 33 to Sau3a at position 97; Fig.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months. Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied. In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2-6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

We examined the potential of generating an immune response against Philadelphia chromosome-positive acute lymphoblastic leukemia. The immunostimulatory molecules chosen for this study were the cytokines IL-2 and GM-CSF and the costimulatory ligand CD80 (B7.1). We used a murine model based on a BALB/c pre-B cell line, BM185wt, in which leukemia is induced by the p185 BCR-ABL oncogenic product, which reproduces Philadelphia chromosome-positive ALL. BM185wt cells were transduced with retroviral vectors and the transduced clones expressing mIL-2, mGM-CSF, or mCD80 were used for challenge. Expression of the immunomodulators by BM185 cells was correlated with delay in leukemia development in immunocompetent mice, but not in immunodeficient mice, indicating an immune response against the modified leukemia cells. Expression of CD80 caused leukemia rejection in 50% of the cohort, which was associated with the CD4+ and CD8+ T cell-dependent development of anti-leukemia cytotoxic T lymphocytes. Furthermore, mice surviving the BM185/CD80 challenge or preimmunized with irradiated BM185/CD80 cells developed an immune response against subsequent challenge with the parental leukemia. These studies provide evidence that immunotherapeutic approaches can be developed for the treatment of ALL.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a highly aggressive malignancy caused by the bcr-abl translocation oncogene. To explore alternative treatments for Ph+ ALL we tested gene-modified cell vaccines in the BALB/c-derived BM185 leukemia model. We compared the efficacy of BM185 cell vaccine expressing CD80 alone or in combination with IL-2 or GM-CSF. Mice injected with viable BM185 leukemia cells modified to express CD80 and GM-CSF (BM185/CD80+GM-CSF) showed the highest leukemia rejection rates. Cell vaccines consisting of irradiated BM185/CD80+GM-CSF cells administered subcutaneously stimulated a potent cytotoxic T lymphocyte (CTL) response against parental BM185. Histological examination of the vaccination site showed a large concentration of immune cells. Administration of the BM185/CD80+GM-CSF cell vaccine before intravenous challenge with parental cells caused strong inhibition of leukemia development. Vaccination after subcutaneous challenge with BM185 cells caused efficient elimination of leukemia promoting 40-60% long-term survival rates. The immunization efficacy of the BM185/CD80+ GM-CSF cell vaccine was directly correlated with the percentage of cells expressing the transgenes. In all, this preclinical study shows that leukemia cell vaccines coexpressing CD80 and GM-CSF can potentially be explored for immunotherapy in Ph+ ALL patients.

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