Human hematopoietic stem cells in gene therapy: pre-clinical and clinical issues.
ABSTRACT Hematopoietic stem and progenitor cells (HSC) have been widely used in allogeneic transplant procedures, therefore their intrinsic characteristics, the biology of their niche in the bone marrow, and the mobilization and homing processes have been extensively investigated. With the development of gene therapy strategies, new therapeutic options based on autologous HSC have become available which may reduce the morbidity and mortality associated to allogeneic transplantation, but require an ex vivo manipulation of the cells to be corrected before re-infusion. For the success of these approaches it is necessary to optimize culture conditions in order to achieve efficient cell transduction while preserving the biological properties of the stem cells. We review here the factors critical for achieving efficient HSC transduction and maintenance of HSC stemness and homing capacity upon ex vivo culture. When HSC gene therapy is used in genetic disorders, permanent integration of therapeutic genes into the chromosomes of affected cells is needed. Indeed, by use of integrating vectors, such as retroviruses, gene therapy has met significant success in immunodeficiency syndromes characterized by a selective advantage of the transduced cells. However, retroviral integration can take place in stem cells at a variety of chromosomal sites, and examples have been reported of integration of therapeutic vectors causing cancer in patients. The clinical benefit arising from the long-term correction of the genetic defect, due to vector integration into the HSC genome, and the adverse consequences of these events are also here discussed, together with the new and challenging perspectives of HSC gene therapy.
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ABSTRACT: Clinical trials in cystic fibrosis (CF) patients established proof-of-principle for transfer of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells. However, the limited efficacy of gene transfer vectors as well as extra- and intracellular barriers have prevented the development of a gene therapy-based treatment for CF. Here, we review the use of new viral and nonviral gene therapy vectors, as well as human artificial chromosomes, to overcome barriers to successful CFTR expression. Pre-clinical studies will surely benefit from novel animal models, such as CF pigs and ferrets. Prenatal gene therapy is a potential alternative to gene transfer to fully developed lungs. However, unresolved issues, including the possibility of adverse effects on pre- and postnatal development, the risk of initiating oncogenic or degenerative processes and germ line transmission require further investigation. Finally, we discuss the therapeutic potential of stem cells for CF lung disease.Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 06/2011; 10 Suppl 2:S114-28. · 3.19 Impact Factor
Article: Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.[show abstract] [hide abstract]
ABSTRACT: Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.PLoS ONE 01/2011; 6(12):e29110. · 4.09 Impact Factor