Salima Hacein-Bey

Gene therapy of severe combined immunodeficiencies

The concept that the outcome of a devastating disease can be modified by inserting a transgene into abnormal cells is appealing. However, the gene-transfer technologies that are available at present have limited the success of gene therapy so far. Nevertheless, severe combined immunodeficiencies are a useful model, because gene transfer can confer a selective advantage to transduced cells. In this way, a proof of concept for gene therapy has been provided.

Adenovirally Transduced Dendritic Cells Induce Bispecific Cytotoxic T Lymphocyte Responses Against Adenovirus and Cytomegalovirus pp65 or Against Adenovirus and Epstein-Barr Virus EBNA3C Protein: A Novel Approach for Immunotherapy

Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (Ad) cause significant morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. We have established a procedure to generate polyclonal cytotoxic T lymphocyte (CTL) populations with specificity against Ad and CMV or against Ad and EBV. Healthy donor-derived dendritic cells (DCs) were transduced with recombinant adenovirus encoding either CMV pp65 or EBV EBNA3C and used to stimulate autologous T cells. Stimulated T lymphocytes displayed specific simultaneous cytotoxicity against CMV and adenovirus and to a lesser extent against adenovirus and EBV. Recombinant vaccinia virus encoding individual adenovirus proteins showed that the T cell response to the adenovirus was directed mainly against the capsid protein hexon. The frequency of IFN-gamma-secreting T cells was 0.02% for adenovirus alone, and 0.05 and 0.14% for adenoviruses encoding EBNA3C and pp65, respectively. pp65-specific CTLs killed autologous fibroblasts infected with the laboratory strain CMV AD169. The culture conditions were specific as alloreactive T cells were not expanded. Therefore, this approach could be considered in order to generate efficient virus cytolytic T cells to be used as adoptive immunotherapy in transplanted patients.

Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the βA(T87Q)-Globin Gene

β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (βAT87Q-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. βAT87Q-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease.

Gene Therapy for Human Severe Combined Immunodeficiencies

Besides long-term analysis of treated cases, there is an obvious need to reproduce these results in a larger set of patients, including more cases in whom an endogenous abnormal γc protein is present in lymphocyte precursors as well as patients with partially preserved T cell development (Notarangelo et al., 2000xNotarangelo, L.D., Giliani, S., Mazza, C., Mella, P., Savoldi, G., Rodriguez-Perez, C., Mazzolari, E., Fiorini, M., Duse, M., Plebani, A. et al. Immunol. Rev. 2000; 178: 39–48Crossref | PubMedSee all References(Notarangelo et al., 2000). Also, and this is not a straightforward issue, methods for the large-scale production of clinical batches of vectors have to be developed. What about extension to the treatment of other immunodeficiencies? If one assumes that the most suitable disease was chosen, then treatment of other conditions raises additional difficulties (for a detailed discussion of the latter, see Candotti 2000xCandotti, F. Pediatr. Clin. North Am. 2000; 47: 1389–1407Abstract | Full Text | Full Text PDF | PubMedSee all References, Fischer et al. 2000xFischer, A., Hacein-Bey, S., Le Deist, F., Soudais, C., and Di Santo, J. Immunol. Rev. 2000; 178: 13–20Crossref | PubMedSee all References). This means that further advances in gene transfer technology in hematopoietic precursor cells will be necessary in this setting. Note, however, that for the treatment of conditions for which a selective advantage of transduced cells is expected, a high rate of CD34 (+) cell transduction might not be appropriate since: (1) a high rate of transduction is not necessary to achieve T lymphocyte development and (2) it will lead to the multiplication of the number of transgene copy integrations. The latter could potentially increase the risk of deleterious consequences of insertional mutagenesis. SCID conditions with an identical or closely related disease mechanism to SCID-X1, i.e., JAK-3 and IL-7Rα deficiency, are the most obvious next candidate diseases for gene therapy. Experimental gene therapy of JAK-3(-) mice has provided convincing results including proven efficacy in unirradiated mice (Bunting et al., 2000xBunting, K.D., Lu, T., Kelly, P.F., and Sorrentino, B.P. Hum. Gene Ther. 2000; 11: 2353–2364Crossref | PubMed | Scopus (33)See all References(Bunting et al., 2000). SCID conditions caused by Rag-1, Rag-2, or Artemis gene mutations are also worthwhile to consider, since allogeneic stem cell transplantation provides poorer results than for other SCID conditions. Genetic deficiencies in the immune system that impair further downstream T cell development will be more difficult to tackle since (1) a less potent selective advantage is to be expected and (2) considerations on transgene expression regulation (both spatial and temporal) will have to be addressed. In this respect however, the spectacular achievement of tissue-specific β-globin expression in erythroid cells in β thalassemic mice represents a significant advance. This result is based on the insertion in the lentiviral vector of a minimal version of the locus control region (LTR) of the β-globin gene (May et al., 2000xMay, C., Rivella, S., Callegari, J., Heller, G., Gaensler, K.M., Luzzatto, L., and Sadelain, M. Nature. 2000; 406: 82–86Crossref | PubMed | Scopus (372)See all References(May et al., 2000).One can, from the above, consider that proof of principle has been achieved in the genetic treatment of inherited disorders of the immune system. Much remains to be done by combining both an in depth appraisal of every single disease mechanism (i.e., in other words, understanding the function of the corresponding gene product) and development of new aspects in gene transfer technologies. It is obvious that a potentially safe use of lentiviral vectors able to efficiently target HSC would provide a major advance to the field (reviewed in Sadelain et al., 2000xSadelain, M., Frassoni, F., and Riviere, I. Curr. Opin. Hematol. 2000; 7: 364–377Crossref | PubMed | Scopus (20)See all ReferencesSadelain et al., 2000). Technologies of gene repair might also become a consideration in the future.

Optimization of Retroviral Gene Transfer Protocol to Maintain the Lymphoid Potential of Progenitor Cells

We have attempted to improve retrovirus-mediated gene transfer efficacy into hematopoietic progenitor cells (HPCs) without causing them to lose their lymphoid potential. Highly purified CD34(+) cells on CH-296 fibronectin fragments have been transduced with three different cytokine combinations. Murine CD2 was used as a marker gene. Transgene expression was assayed by FACS analysis shortly after transduction of CD34(+) cells and after long-term culture (LTC) extended by differentiation of various lymphoid lineages: NK cells, B cells, and dendritic cells. Compared with the historical cytokine mix, i.e., SCF (stem cell factor) + IL-3 (interleukin 3) + IL-6, the combination SCF + FL (Flt-3 ligand) + M-GDF (megakaryocyte growth and differentiation factor) + IL-3 significantly improved the total number of viable cells and CD34(+) cells after transduction and the long term-cultured progenitors after 6 weeks. In addition, the combination of SCF + FL + M-GDF + IL-3 maintained more efficiently the lymphoid potential of the progeny of transduced long term-cultured CD34(+) cells, as attested by the significantly higher number of CD56(+), CD19(+), and CD1a(+) cells recovered when FL and M-GDF were added to SCF + IL-3. Thus, even though additional improvements may still be needed in transduction of HPCs, these conditions were adopted for a clinical trial of gene therapy for X-linked severe combined immunodeficiency.

Thérapie génique du déficit immunitaire combiné sévère lié à l'X (DICS-X1)

X-linked severe combined immunodeficiency (SCID-X1) is a recessive hereditary disorder in which early T and Natural Killer (NK) lymphocyte development is blocked. The genetic disorder results from mutations in the common gamma c chain that participates in several cytokine receptors including the interleukin-2 (Il-2), Il-4, Il-7, Il-9, Il-15 receptors. SCID-X1 offers a reliable model for gene therapy as it is a lethal condition that is, in many cases, curable by allogeneic bone marrow transplantation. We have shown that retrovirus-mediated transfer of the gamma c cDNA induced gamma c chain expression and restored the function of the high-affinity IL-2 receptor on SCI-X1 EBV-transformed B-cell lines. We have the designed culture conditions to study NK-cell and T-cell development of CD34+ hematopoietic progenitor cells. In the culture systems, gamma c transduced CD34+ marrow cells from two SCID-X1 patients were able to mature into CD56+ and/or CD16+ NK cells and into CD4+ TCR alpha beta+ T cells. These preclinical results set the basis for a clinical study of ex-vivo gamma c gene transfer into CD34+ cells from SCID-X1 patients.