Regulated expansion of human pancreatic beta-cells.

Although pancreatic beta-cell transplantation may serve as a potential cure for diabetes mellitus (DM), limited donor tissue availability poses a major challenge. Thus, there is a great demand to find new sources for pancreatic beta-cells. Here, we present a lentiviral vector-based approach to achieve beta-cell proliferation through the beta-cell-specific activation of the hepatocyte growth factor (HGF)/cmet signaling pathway. The methodology is based on the beta-cell-specific expression of a ligand-inducible, chimeric receptor (F36Vcmet), under transcriptional control of the promoter from the human insulin gene, and its ability to induce HGF/cmet signaling in the presence of a synthetic ligand (AP20187). High transduction efficiency of human pancreatic islets was achieved utilizing this approach with chimeric receptor expression confined to the beta-cell population. In addition, specific proliferation of human pancreatic beta-cells was induced utilizing this approach. Selective, regulated beta-cell expansion may help to provide greater availability of cells for transplantation in patients with DM.

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion.

Adenosine deaminase (ADA)-deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose-dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.

In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors.

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.

Effective suicide gene therapy for leukemia in a model of insertional oncogenesis in mice.

The safety of gene therapy using hematopoietic stem cells may be increased by including a suicide gene in the therapeutic vector to eliminate adverse events like insertional oncogenesis while retaining the clinical benefits. We have developed a model of experimental insertional oncogenesis by transducing the murine factor-dependent leukemia cell line Ba/F3 with a bicistronic Moloney murine leukemia virus retroviral vector encoding a murine oncogene (cKit(D814V)) in addition to one of three suicide genes: Herpes simplex virus thymidine kinase (HSV-TK); SR39, an HSV-TK mutant with an increased affinity for the drug substrate Ganciclovir (GCV); or sc39, a splice-corrected version of SR39. Following intravenous challenge with transduced Ba/F3 clones and treatment with GCV, leukemia developed in mice given cells expressing HSV-TK, but not SR39 or sc39. In vitro GCV resistance was observed in heterogeneously transduced Ba/F3 pools at 2.5-14%, and single-nucleotide changes or partial loss of the suicide gene were identified as mechanisms of drug escape. However, GCV treatment resulted in 80-100% survival of mice challenged even with pools of partially resistant Ba/F3 cells expressing SR39 or sc39. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity.

Recombinant murine interleukin-12 elicits potent antileukemic immune responses in a murine model of Philadelphia chromosome-positive acute lymphoblastic leukemia.

Despite the success of chemotherapy regimens in the treatment of acute lymphoblastic leukemia (ALL), certain subsets of patients have a high rate of induction failure and subsequent relapse. One of these subsets of patients carry a translocation between chromosomes 9 and 22, the so called Philadelphia chromosome (Ph+). The result of this translocation is the fusion oncogene, Bcr-Abl, which is uniquely expressed in the leukemia clone, and as such has the potential to initiate antileukemic immune responses against the leukemia blasts. We utilized a murine model of Ph+ ALL to look at the ability of systemic interleukin 12 (IL-12) treatments to initiate antileukemic immune responses, and studied the mechanisms by which it does so. We found that IL-12 was able to eliminate pre-established leukemia, and that this protection was mediated by CD4, CD8, and NK cells in combination. While IL-12 was able to eliminate pre-established leukemia, it did not elicit immunologic memory. Consistent with previous work, vaccination with irradiated leukemia cells transduced with immunomodulator genes was able to establish long-term memory, and, when used with IL-12, was able to eradicate pre-existing disease and induce resistance to subsequent leukemia challenge. These studies demonstrate the feasibility of an immunotherapeutic approach towards the treatment of Ph+ ALL.

Requirement for NK cells in CD40 ligand-mediated rejection of Philadelphia chromosome-positive acute lymphoblastic leukemia cells.

We have previously developed a murine model of Philadelphia chromosome-positive acute lymphoblastic leukemia by i.v. injection of a pre-B ALL cell line (BM185) derived from Bcr-Abl-transformed BALB/c bone marrow. We are studying the potential to elicit autologous antileukemic immune responses by introducing genes encoding immunomodulators (CD40 ligand (CD40L), CD80, and GM-CSF) into leukemia cells. BM185 cells expressing CD40L or CD80 alone, when injected into BALB/c mice, were rejected in approximately 25% of mice, whereas cohorts receiving BM185 cells expressing two or more immunomodulator genes rejected challenge 50-76% of the time. The greatest protection was conferred in mice receiving BM185 cells expressing all three immunomodulators. Addition of murine rIL-12 treatments in conjunction with BM185/CD80/CD40L/GM-CSF vaccination allowed rejection of preestablished leukemia. BM185 cell lines expressing CD40L were rejected in BALB/c nu/nu (nude) mice, in contrast to cell lines expressing CD80 and/or GM-CSF. Nude mice depleted of NK cells were no longer protected when challenged with BM185/CD40L, demonstrating a requirement for NK cells. Similarly, NK cell depletion in immunocompetent BALB/c mice resulted in a loss of protection when challenged with BM185/CD40L, confirming the data seen in nude mice. The ability of CD40L to act in a T cell-independent manner may be important for clinical applications in patients with depressed cellular immunity following chemotherapy.

Lentivirus vectors incorporating the immunoglobulin heavy chain enhancer and matrix attachment regions provide position-independent expression in B lymphocytes.

In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (E micro ) with and without associated matrix attachment regions (E micro MAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34(+) progenitors in vitro transduced with E micro - and E micro MAR-containing lentivectors. Lastly, we evaluated the expression from the E micro MAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the E micro and E micro MAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the E micro MAR-containing vector and not other cells types or vectors. Proviral genomes with the E micro MAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the E micro MAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies.

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells.

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.