Comparative analysis of FV vectors with human α- or ß-globin gene regulatory elements for the correction of ß-thalassemia.

ß-Globin locus control region (LCR) sequences have been widely used for the regulated expression of the human ß-globin gene in therapeutic viral vectors. In this study, we compare the expression of the human ß-globin gene from either the HS2/HS3 ß-globin LCR or the HS40 regulatory element from the α-globin locus in the context of foamy virus (FV) vectors for the genetic correction of ß-thalassemia. Both regulatory elements expressed comparable levels of human ß-globin in a murine erythroleukemic line, whereas in murine hematopoietic stem cells the HS40.ß vector proved more efficient in ß-globin expression and correction of the ß-thalassemia phenotype. Following transplantation in the Hbb(th3/+) mouse model, the expression efficiency by the two vectors was similar, whereas the HS40.ß vector achieved relatively more stable transgene expression. In addition, in an ex vivo assay using CD34+ cells from thalassemic patients, both vectors achieved significant human ß-globin expression and restoration of the thalassemic phenotype as evidenced by enhanced erythropoiesis and decreased apoptosis. Our data suggest that FV vectors with the α-globin HS40 element can be used as alternative but equally efficient vehicles for human ß-globin gene expression for the genetic correction of ß-thalassemia.

Dual transgene expression by foamy virus vectors carrying an endogenous bidirectional promoter.

Several gene therapy applications require the transfer and simultaneous expression of multiple genes in the same cell. In this study, we analyzed the potential for coordinated expression of an endogenous bidirectional promoter located on chromosome X, which controls the expression of the heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2) and alpha-galactosidase (GLA) genes. The promoter was cloned in both transcriptional orientations in a foamy virus (FV) vector backbone, whereas the enhanced green fluorescent protein (EGFP) and low-affinity nerve growth factor receptor (DeltaLNGFR) reporter genes were cloned in the 5'-3' and 3'-5' transcriptional orientations, respectively. In all the cell lines tested, both vectors showed high levels of transgene coexpression that reached 76% of total positive cells (range from 76 to 18%). Comparison of EGFP and DeltaNGFR levels revealed that the side of the promoter that drives the expression of the HNRNPH2 gene in the genome was stronger and in accordance to its in situ activity. When tested with CD34(+) cells, transgene coexpression reached 35.3% of all positive cells in progenitor assays and 16.8% of all positive cells after transplantation in NOD/severe combined immunodeficient mice. In summary, we show that the endogenous promoter used in this study holds bidirectional activity in the context of FV vectors and can be used in gene therapy applications requiring synchronized expression of two genes.

Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12.

Cytokine-modified tumour cells have been used in clinical trials for immunotherapy of neuroblastoma, but primary tumour cells from surgical biopsies are difficult to culture. Autologous fibroblasts, however, are straightforward to manipulate in culture and easy to transfect using nonviral or viral vectors. Here we have compared the antitumour effect of fibroblasts and tumour cells transfected ex vivo to coexpress interleukin-2 (IL-2) and IL-12 in a syngeneic mouse model of neuroblastoma. Coinjection of cytokine-modified fibroblasts with Neuro-2A tumour cells abolished their in vivo tumorigenicity. Treatment of established tumours with three intratumoral doses of transfected fibroblasts showed a significant therapeutic effect with reduced growth or complete eradication of tumours in 90% of mice, associated with extensive leukocyte infiltration. Splenocytes recovered from vaccinated mice showed enhanced IL-2 production following Neuro-2A coculture, and increased cytotoxicity against Neuro-2A targets compared with controls. Furthermore, 100% of the tumour-free mice exhibited immune memory against tumour cells when rechallenged three months later. The potency of transfected fibroblasts was equivalent to that of tumour cells in all experiments. We conclude that syngeneic fibroblasts cotransfected with IL-2 and IL-12 mediate therapeutic effects against established disease, and are capable of generating immunological memory. Furthermore, as they are easier to recover and manipulate than autologous tumour cells, fibroblasts provide an attractive alternative immunotherapeutic strategy for the treatment of neuroblastoma.

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX.

Immune responses against an introduced transgenic protein are a potential risk in many gene replacement strategies to treat genetic disease. We have developed a gene delivery approach for hemophilia B based on lentiviral expression of human factor IX in purified hematopoietic stem cells. In both normal C57Bl/6J and hemophilic 129/Sv recipient mice, we observed the production of therapeutic levels of human factor IX, persisting for at least a year with tolerance to human factor IX antigen. Secondary and tertiary recipients also demonstrate long-term production of therapeutic levels of human factor IX and tolerance, even at very low levels of donor chimerism. Furthermore, in hemophilic mice, partial functional correction of treated mice and phenotypic rescue is achieved. These data show the potential of a stem cell approach to gene delivery to tolerize recipients to a secreted foreign transgenic protein and, with appropriate modification, may be of use in developing treatments for other genetic disorders.