The expansion and transduction of hematopoietic stem cells for gene therapy

Abstract

Gene therapy is a field in medical research that is being investigated for the treatment of genetic, non-communicable and infectious diseases. The results obtained from clinical trials that make use of gene therapy show promise; however, it has been determined that several procedural limitations hinder convincing outcomes. The genetic modification of host cells can be achieved using transfection (transfer of DNA fragments into the chromosomes of cells using chemical methods) or transduction (transfer of DNA fragments into the chromosomes of cells using viruses) procedures. Considerable work has been done to improve the safety of the viral vectors used to transfer genes, that are capable of providing therapeutic effects, into target cells and to improve the efficiency of the therapeutic genes. Differentiated cells can be used for a transient therapeutic effect or alternatively stem cells can be used which would provide a more long-term effect. The use of stem cells requires the isolation, gene modification and reintroduction of the cells into the patient without the stem cells losing their engraftment potential which is their ability to return to the original location and self-renew as well as differentiate. The majority of gene therapy clinical trials make use of hematopoietic stem cells (HSCs) that have been modified using molecular techniques; however, poor engraftment and low transduction efficiencies of long-term repopulating HSCs leads to minimal therapeutic effects. The majority of clinical trials make use of HSCs that were released from the bone marrow of patients or donors using mobilization techniques. Poor engraftment of genetically modified HSCs can be attributed to insufficient numbers of mobilized bone marrow CD34+ cells being obtained from some patients and to CD34+ cells that lose their repopulating potential during the transduction process. The transduction procedures used in gene therapy clinical trials do not promote the transduction of long-term repolulating HSCs. This study aimed to develop techniques that facilitate the improved transduction and expansion of the primitive HSC populations. The use of hypoxia and Stemregenin 1 (SR1) in the culturing of HSCs has been investigated to determine their benefit in the expansion of HSC cellular subpopulations. Also, the use of SR1 in the transduction protocol was studied to determine if it can benefit the engraftment potential of transduced HSCs. It was found that SR1 can be used to improve the expansion of the various HSC subpopulations, determined in vitro and in vivo. The use of SR1 with 1% O2 led to an increase in the number and ability of cells to repopulate NOD SCID gamma mice (NSG, NOD.Cg-Prkdc<scid> Il2rg<tm1Wjl>/SzJ). Also, SR1 promotes the transduction and expansion of the primitive HSC population during the transduction process and significantly increases the engraftment of HSCs transduced with therapeutic genes. SR1 may be used to benefit the field of gene therapy where HSC transduction is used. Gene therapy can be used in the treatment of monogenic disorders such as hemaglobinopathy or infectious diseases such as HIV-AIDS. South Africa is highly burdened with HIV-AIDS which can potentially be treated with gene therapy. However, the complexity of gene therapy requires the need for highly specialized expertise, infrastructure and supportive legislation that should be put into place in South Africa before gene therapy can be implemented. The barriers and opportunities in the implementing of gene therapy in South Africa have been investigated in this study and recommendations are made.