Elio F. Vanin

Jak2 Is Essential for Signaling through a Variety of Cytokine Receptors

A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis. Fetal liver myeloid progenitors, although present based on the expression of lineage specific markers, fail to respond to erythropoietin, thrombopoietin, interleukin-3 (IL-3), or granulocyte/macrophage colony-stimulating factor. In contrast, the response to granulocyte specific colony-stimulating factor is unaffected. Jak2-deficient fibroblasts failed to respond to interferon gamma (IFNgamma), although the responses to IFNalpha/beta and IL-6 were unaffected. Lastly, reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation. Therefore, Jak2 plays a critical, nonredundant role in the function of a specific group of cytokines receptors.

High-Efficiency Transduction and Long-Term Gene Expression with a Murine Stem Cell Retroviral Vector Encoding the Green Fluorescent Protein in Human Marrow Stromal Cells

Bone marrow stromal cells (MSCs) are unique mesenchymal cells that have been utilized as vehicles for the delivery of therapeutic proteins in gene therapy protocols. However, there are several unresolved issues regarding their potential therapeutic applications. These include low transduction efficiency, attenuation of transgene expression, and the technical problems associated with drug-based selection markers. To address these issues, we have developed a transduction protocol that yields high-level gene transfer into human MSCs, employing a murine stem cell virus-based bicistronic vector containing the green fluorescent protein (GFP) gene as a selectable marker. Transduction of MSCs plated at low density for 6 hr per day for 3 days with high-titer viral supernatant resulted in a gene transfer efficiency of 80+/-6% (n = 10) as measured by GFP fluorescence. Neither centrifugation nor phosphate depletion increased transduction efficiency. Assessment of amphotropic receptor (Pit-2) expression by RT-PCR demonstrated that all MSCs expressing the receptor were successfully transduced. Cell cycle distribution profiles measured by propidium iodide staining showed no correlation with the susceptibility of MSCs to transduction by the retroviral vector. Human MSCs sequentially transduced with an adenoviral vector encoding the ecotropic receptor and ecotropic retroviral vector encoding GFP demonstrated that all MSCs are susceptible to retroviral transduction. We further showed that both genes of bicistronic vector are expressed for at least 6 months in vitro and that transgene expression did not affect the growth or osteogenic differentiation potential of MSCs. Future studies will be directed toward the development of gene therapy protocols employing this strategy.

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/107particles) and adenovirus. Transduction of CD34+ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34+ cells were resistant to transduction (less than 4%), but transduction increased to 23 ± 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor α (51 ± 4%) at a multiplicity of infection of 106. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34+ and CD34+, CD38− subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 ± 7% of CD34+ progenitors and 21 ± 6% of CD34+, CD38− progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient.

Jak3 Selectively Regulates Bax and Bcl-2 Expression To Promote T-Cell Development

ABSTRACT Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.

RD114-Pseudotyped Oncoretroviral Vectors

Abstract: Limited functional expression of the viral envelope receptor is a recognized barrier to efficient oncoretroviral mediated gene transfer. To circumvent this barrier we evaluated a number of envelope proteins with respect to gene transfer efficiency into primitive human hematopoietic stem cell populations. We observed that oncoretroviral vectors pseudotyped with the envelope protein of feline endogenous virus (RD114) could efficiently transduce human repopulating cells capable of establishing multilineage hematopoiesis in immunodeficient mice after a single exposure to RD114‐pseudotyped vector. Comparable rates of gene transfer with amphotropic and GALV‐pseudotyped vectors have been reported, but only after multiple exposures to the viral supernatant. Oncoretroviral vectors pseudotyped with the RD114 or the amphotropic envelopes had similar stability in vitro, indicating that the increased efficiency in gene transfer is at the receptor level likely due to increased receptor expression or an increased receptor affinity for the RD114 envelope. We also found that RD114‐pseudotype vectors can be efficiently concentrated, thereby removing any adverse effects of the conditioned media to the long‐term repopulating potential of the target human hematopoietic stem cell. These studies demonstrate the potential of RD114‐pseudotyped vectors for clinical use.

Creation of Polarized Cells Coexpressing CYP3A4, NADPH Cytochrome P450 Reductase and MDR1/P-glycoprotein

AbstractPurpose. To develop model polarized cell systems expressingcytochrome P4503A4, NADPH P450 reductase, and P-glycoprotein (Pgp). Methods. LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp,the product of the multidrug resistance gene (MDR1), were transfectedstably using either a mammalian neomycin selectable expression vector(CYP3A4-Neo) or an episomal vector based on Epstein—Barr virus(CYP3A4-Hygro). These CYP3A4 expressing cells were compared withLLC-PK1, L-MDR1, or Caco-2 cells transduced with Adenovirus-3A4vector (Ad3A4) with or without simultaneous Adenovirus-P450 Reductase(AdRed) transduction. Cells were characterized for expression of CYP3A4protein and CYP3A4 mediated metabolism towards midazolam and testosterone. Analysis of membrane integrity and drug transport assays wereperformed to determine whether infection with recombinant Ad3A4 ±AdRed affected Pgp function. Results. The rank order of optimal CYP3A4 expression and activitiesin LLC-PK1 and L-MDR1 cells from highest to lowest was cellsco-transduced with Ad3A4 plus AdRed >> Ad3A4 >>>CYP3A4-Hygro > CYP3A4-Neo. Similarly, coexpression of Ad3A4 plus AdRedled to enhanced CYP3A4 mediated metabolism in Caco-2 cells overcells with Ad3A4 alone. Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detectable metabolitein the medium. In microsomes from Caco-2 and LLC-PK1 cells, eachco-transduced with Ad3A4/AdRed, Vmax values for testosterone6β-hydroxylase activity ranged from 414 to 1350 pmoles/min/mg,respectively. For either Caco-2 or LLC-MDR1 cells, TEER values and therate of apical to basal and basal to apical transport of vinblastine ordigoxin were similar in cells with and without Ad3A4/Red transduction. Conclusions. Polarized cellular systems coexpressing Ad3A4, AdRed,and the MDR1/Pgp transporter were developed and characterized. Theresults document the utility of these polarized model systems forsimultaneous drug transport/drug metabolism studies. Since the experimentalapproach can be adapted to study the interplay of multipleenzyme/transporting systems, it may find significant application as a screeningtool for the pharmaceutical industry and as a more basic research toolto study the kinetics of intestinal drug bioavailability.

Neuroblastoma regression and immunity induced by transgenic expression of interleukin-12

PURPOSE Interleukin-12 (IL-12) is a cytokine with potent antitumor effects. The authors sought to assess its capacity to increase tumor immunogenicity when expressed by tumor cells in a murine model of neuroblastoma. METHODS Syngeneic A/J mice were inoculated subcutaneously with 2 x 10(6) cells from a murine neuroblastoma-derived cell line (neuro-2a). In situ transduction of the neuroblastoma cells was achieved by intratumoral injection of an adenoviral vector encoding both subunits of the murine IL-12 heterodimer. Growth of the IL-12 gene-modified tumor cells was compared with untreated neuro-2a cells. Tumor immunity was assessed by rechallenging mice that had rejected their tumor with unmodified neuroblastoma cells. The contribution of cytotoxic T lymphocytes (CTLs) was evaluated through cytotoxicity assays. RESULTS Eighteen (72%) of 25 tumor-bearing mice treated with the mlL-12 adenoviral vector exhibited tumor regression, with 12 mice (48%) completely rejecting their tumors over 2 to 3 weeks. None of the mice that had rejected their tumor and were rechallenged with unmodified neuro-2a cells subsequently developed new tumors. Pooled splenocytes from mice rejecting their tumors showed significant tumor killing (>20% cytolysis) in vitro in 51Cr release assays. CONCLUSIONS Adenoviral-mediated IL-12 expression by tumor cells in a murine neuroblastoma model produced a significant antitumor response. Most treated tumors demonstrated at least transient regression, whereas many completely regressed. Cured mice exhibited protective immunity and CTL activity against the tumor. These data confirm the immunomodulatory efficacy of IL-12 as part of a vaccine-based antineuroblastoma strategy.

Vector development: A major obstacle in human gene therapy

Gene therapy has been proposed for a wide variety of human conditions including monogenic disorders, such as the haemoglobinopathies and immunodeficiency syndromes, cancer and many other diseases. Prerequisites for the success of this approach include the ability to deliver the therapeutic gene intact to the target cell, persistent levels of transgene expression sufficient to correct the disease phenotype, lack of unwanted side-effects associated with vector exposure or gene transfer and relative simplicity allowing the widespread use of this methodology. Although substantial progress has been made in animal models since the inception of genetic therapy in the early 1980s, significant obstacles remain for human therapy, most notably in the area of vector development. The first generation of gene therapy vectors has failed to overcome many of the biological hurdles cited above necessitating the development of alternate means of gene delivery and expression.

Retroviral vector-producer cell-mediated in vivo gene transfer of TIMP-3 restricts angiogenesis and neuroblastoma growth in mice.

Destruction and remodeling of the extracellular matrix occurs during the formation of new blood vessels that are required for tumor growth. We sought to determine whether gene-therapy mediated in vivo delivery of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), using retroviral vector-producer cells, could suppress angiogenesis and subsequent tumor growth in a murine neuroblastoma model. Tumor volume 28 days after coinjection of tumor cells with producer cells generating TIMP-3-encoding retroviral vectors was 21% that of controls, as was the mean tumor vascular index, a measure of blood vessel maturity. When tumors were allowed to reach a mean volume of 0.05 cm3 before treatment, their size 2 weeks later was 47% relative to controls; larger tumors were not significantly affected. When producer cells were injected at surgical sites following excision of subcutaneous tumors, local recurrence 14 days later was only 22% in TIMP-3 producer cell treated mice as compared to 71% in controls. Unsuccessful transduction of melanoma cells in situ, another tumor of neural crest origin, resulted in unimpaired tumor growth, despite the fact that these tumors are susceptible to TIMP-3 overexpression, demonstrating the importance of tumor cell transduction in this approach. Thus, retroviral vector-producer cell-mediated in vivo gene transfer of TIMP-3 to tumor cells can significantly restrict tumor-induced angiogenesis and tumor growth. This approach may be an effective adjuvant in the treatment of neuroblastoma and other solid tumors refractory to traditional therapy, although it appears to be most effective in smaller tumors or in the setting of minimal residual disease, and the tumor cells must be susceptible to retroviral vector-mediated transduction.

Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector

The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice. Gene transfer efficiency as reflected by the number of genetically modified cells in hematopoietic tissues varied among the five monkeys studied from low levels (<1%) in three animals to much higher levels in two (20-60%). An animal that exhibited extremely high levels for several weeks was found by vector genome insertion site analysis to have reconstitution predominantly with a single clone of cells. This variability among animals is in keeping with computer simulations of reconstitution with limiting numbers of stem cells genetically modified at about 10% efficiency. Our studies provide insights into the biology of hematopoietic reconstitution and suggest approaches for increasing stem cell targeted gene transfer efficiency.