Manuel Caruso

Enhanced Ganciclovir Killing and Bystander Effect of Human Tumor Cells Transduced with a Retroviral Vector Carrying a Herpes Simplex Virus Thymidine Kinase Gene Mutant

Gene transfer of the herpes simplex virus thymidine kinase (TK) gene associated with ganciclovir (GCV) treatment can lead to death of TK-expressing cells, and of neighboring TK- cells because of the bystander effect. Thus, a small proportion of TK+ cells in a tumor can lead to its complete regression after GCV treatment. However, a lack of efficacy of gene transfer into tumors associated with low GCV sensitivity and poor bystander effect of human cancer cells currently limit the clinical use of this suicide gene therapy approach. To increase the potency of suicide gene therapy, we have tested the GCV sensitivity and the bystander effect of TK mutants that have been previously described. After retroviral transduction of the TK mutants into human tumor cells of various origins, we have found a strong killing effect of GCV with cells expressing the mutants TK30 or TKF161C. The GCV sensitivity of several human tumor cell types expressing TK30 was 9- to 500-fold higher than cells containing wild-type TK. Furthermore, TK30-expressing cells were able to kill bystander cells much more efficiently than TK-expressing cells. Thus, TK30 mutant is a promising candidate for suicide gene therapy clinical trials.

High translation efficiency is mediated by the encephalomyocarditis virus internal ribosomal entry sites if the natural sequence surrounding the eleventh AUG is retained.

Internal ribosomal entry sites (IRES) allow cap-independent translation and are sequences that are often used in gene therapy for strategies in which several genes need to be expressed. In this study, two different sequences of encephalomyocarditis virus (EMCV) IRES were compared for their translation efficiency in the context of retroviral vectors. When the sequence surrounding the 11th AUG of the IRES was conserved (IRESg), the translation efficiency was significantly higher than if the AUG of the downstream gene started with the 11th AUG of the IRES (IRESb). The translation efficiency with IRESg was influenced by the cell type and also by the nature of the transgene.

Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media

Several patients with severe combined immunodeficiency-X1 disease and adenosine deaminase deficiency have been cured by retroviral-mediated gene therapy. Despite the earlier success, the production of retroviral vectors for clinical gene therapy is cumbersome, costly and lacks safety features because of the adherent nature of packaging cells and the necessity to supplement the culture media with bovine serum. The aim of this study was to generate a retrovirus packaging cell line that could be used for the production of large clinical batch vectors. Bicistronic vectors containing an internal ribosomal entry site followed by a selection gene were used to express Moloney murine leukemia gag-pol and amphotropic envelope viral proteins in HEK293 cells. The candidate clone (293GP-A2) that was selected as the packaging cell line could release recombinant green fluorescent protein retroviruses at 4 × 107 infectious viral particles per ml. Similar titers were achieved after these cells were adapted to grow in suspension and serum-free media. Furthermore, using the same culture conditions viral titers proved to be stable for a 3-month culture period. The 293GP-A2 packaging cell line has the potential to be cultured in bioreactors, opening the possibility for large-scale use of retroviral vectors in late stage clinical trials.

Efficient human hematopoietic cell transduction using RD114- and GALV-pseudotyped retroviral vectors produced in suspension and serum-free media.

Retroviral vectors derived from the Moloney murine leukemia virus have been used in successful and promising gene therapy clinical trials. However, platforms for their large-scale production must be further developed. As a proof of principle, we reported the generation of a packaging cell line that produces amphotropic retroviral vectors in suspension and serum-free medium (SFM). In the present study, we have constructed and characterized two retroviral packaging cell lines designed for gene transfer in hematopoietic cells. These cell lines grow in suspension and SFM, and produce high-titer RD114- and gibbon ape leukemia virus (GALV)-pseudotyped vectors for a 3-month culture period. Viral particles released are as robust during repeated freeze-thaw cycles and on thermal inactivation at 37 degrees C as their counterparts produced in cells cultured adherently with serum. We also show that RD114- and GALV-pseudotyped vectors produced in suspension and SFM efficiently transduce human lymphocytes and hematopoietic stem cells. As these retroviral packaging cell lines distinctively maintain high vector titers while growing in suspension and SFM, we conclude that these cell lines are uniquely suitable for large-scale clinical-grade vector production for late-phase clinical trials involving gene transfer into hematopoietic cells.

Retrovirus-mediated gene transfer of the human multidrug resistance-associated protein into hematopoietic cells protects mice from chemotherapy-induced leukopenia.

Utilization of chemotherapy for the treatment of tumors is mainly limited by its hematological toxicity. Because of the low-level expression of drug resistance genes, transduction of hematopoietic progenitors with multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes should provide protection from chemotherapeutic agent toxicity. Successful transfer of drug resistance genes into hematopoietic cells may allow the administration of higher doses of chemotherapy and, thus, increase regression of chemosensitive tumors. The interest in the use of MRP as an alternative to MDR1 for bone marrow protection lies in its different modulation. This would allow, in the same patient, the use of MDR1 reversal agents to decrease MDR1 tumor resistance without reversing bone marrow (BM) protection of the MRP-transduced hematopoietic cells, since MRP expression is not reversed by these agents. We have constructed MRP-containing retroviral vectors using the phosphoglycerate kinase promoter and generated ecotropic producer cells. Lethally irradiated mice were engrafted with BM cells transduced by coculture with MRP producer cells. Evidence of long-term (9 months) gene transfer was provided by PCR of peripheral blood from MRP-transduced mice. Southern blot analysis confirmed the integrity of the provirus in the MRP-transduced mice. Long-term MRP expression (>5 months) was detected by RT-PCR and fluorescence-activated cell sorting of blood from living mice. High-level expression of MRP in murine hematopoietic cells reduces doxorubicin-induced leukopenia and mortality. Furthermore, we show in vivo selection of MRP-transduced cells following doxorubicin administration, with better and more significant chemoprotection after the second chemotherapy cycle. These data indicate that MRP retroviral gene transfer may be useful for chemoprotection and selection.

Immunosuppressive Effect of Isopropanol: Down-Regulation of Cytokine Production Results from the Alteration of Discrete Transcriptional Pathways in Activated Lymphocytes

Isopropanol (IPA) is widely used in household applications and constitutes a leading cause of acute alcohol intoxication second only to ethanol. Although the effects of ethanol on the immune system have been extensively studied, far fewer data are available on IPA. Given the structural similarity between the two molecules, we hypothesized that IPA could as well have immunomodulatory properties. We report here that acute IPA exposure is detrimental to human T lymphocyte and NK cell activity in vitro in concentrations as low as 0.08–0.16% (13–26 mM). IPA treatment did not affect receptor-mediated early signaling but had a reproducible and dose-dependent effect on the nuclear translocation of NFAT and AP-1. Furthermore, we show in a model of acute IPA intoxication that animals became immunosuppressed as judged by their reduced ability to release IL-2 and IFN-γ in the serum in response to staphylococcal enterotoxin B. This effect was also associated to the down-regulation of TNF-α production and was sufficiently strong to rescue susceptible animals from enterotoxin-induced toxic shock. Our results suggest that IPA is potentially immunosuppressive to the adaptive and innate immune system and have broad significance given the exposure of the general population to this ubiquitous chemical.

A Dominant-Negative Approach That Prevents Diphthamide Formation Confers Resistance to Pseudomonas Exotoxin A and Diphtheria Toxin

Diphtheria toxin (DT), Pseudomonas aeruginosa Exotoxin A (ETA) and cholix toxin from Vibrio cholerae share the same mechanism of toxicity; these enzymes ADP-rybosylate elongation factor-2 (EF-2) on a modified histidine residue called diphthamide, leading to a block in protein synthesis. Mutant Chinese hamster ovary cells that are defective in the formation of diphthamide have no distinct phenotype except their resistance to DT and ETA. These observations led us to predict that a strategy that prevents the formation of diphthamide to confer DT and ETA resistance is likely to be safe. It is well documented that Dph1 and Dph2 are involved in the first biochemical step of diphthamide formation and that these two proteins interact with each other. We hypothesized that we could block diphthamide formation with a dominant negative mutant of either Dph1 or Dph2. We report in this study the first cellular-targeted strategy that protects against DT and ETA toxicity. We have generated Dph2(C-), a dominant-negative mutant of Dph2, that could block very efficiently the formation of diphthamide. Cells expressing Dph2(C-) were 1000-fold more resistant to DT than parental cells, and a similar protection against Pseudomonas exotoxin A was also obtained. The targeting of a cellular component with this approach should have a reduced risk of generating resistance as it is commonly seen with antibiotic treatments.

Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase

The treatment of solid tumors by retroviral delivery of the herpes simplex virus thymidine kinase (TK) followed by ganciclovir (GCV) treatment has so far shown only limited success in patients. One major drawback in this approach is the lack of efficient in vivo gene delivery to cancer cells. Although, the transduction of every single tumor cell is not a requirement since the bystander effect (BE) mediated by gap junctions allows the diffusion of the toxic GCV metabolites from TK-expressing cells toward untransduced cells. To render the TK/GCV approach more potent, and independent of the level of gap junctions, we have tested the efficiency of a TK mutant (TK30) fused to VP22, a herpes simplex protein that seems to be capable of intercellular trafficking. We failed to detect an increase in the BE with cells expressing VP22 fused to TK30 versus cells containing TK30 alone, and this result forced us to reinvestigate the trafficking properties of VP22. Using very sensitive Western blot and fluorescence assays, we were not able to detect the spread of VP22 fused either to TK30 or GFP. These results indicate that VP22 cannot be used as a cargo to translocate TK30 or GFP.

RD114‐pseudotyped retroviral vectors kill cancer cells by syncytium formation and enhance the cytotoxic effect of the TK/GCV gene therapy strategy

Wild‐type RD114 virus is capable of generating syncytia during its replication, and it is believed that cell‐free viruses direct the fusion of neighboring cells. The RD114 envelope (Env) that mediates this fusion event is now widely used to pseudotype retroviral and lentiviral vectors in gene therapy. Indeed, vectors pseudotyped with RD114 Env are very efficient to transfer genes into human hematopoietic cells, and they are resistant to human complement inactivation. In this study, we have tested the potential of RD114‐pseudotyped vectors produced from the FLYRD18 packaging cell line to induce syncytia.

Nilotinib and imatinib inhibit cytarabine cellular uptake: implications for combination therapy.

The tyrosine kinase inhibitor (TKI) imatinib has been used for a decade to treat chronic myeloid leukemia (CML). A very efficient response is obtained with patients in chronic phase, but its efficacy in late phase patients is often transient. The combination of imatinib or of the new TKI nilotinib with cytarabine is a new treatment approach proposed for CML. We have investigated the effect of imatinib and nilotinib on cytarabine uptake, and have found that both molecules inhibit cytarabine transport. These results should impact on the design of clinical trials that investigate the combination of TKIs and nucleoside analogs.