GENE TRANSFER INTO HAEMTOPOIETIC STEM CELL (HSC): Haematopoietic stem cells are an attractive target for gene therapy because these cells are easily accessible and readily delivered back to patients by established autologous transplantation methods. They are long-lived and have an enormous potential for self-renewal. Their pluripotency offers the possibility of correcting defects in all haemopoietic lineages following gene transfer into a small number of HSC.
The potential plasticity of HSC may also broaden their therapeutic potential beyond haematological disorders (Theise, 2004). Apart from mono-genetic disorders, the targets for gene therapy have extended to providing HSC with new or enhanced properties such as those that confer resistance to chemotherapy or lead to expression of tumour-specific antigen or cytokine.
Preclinical studies in Murine models showed that oncoretroviral vectors were highly efficient at transducing Murine HSC, resulting in the generation of gene-marked myeloid and lymphoid progenitors in the circulation of mice reconstituted with transduced bone marrow (Sorrentino and Nienhuis, 1999), this studies laid a foundation for clinical trails of retroviral transduction of human HSC in more than 200 subjects. However, because of the fact that retroviruses cannot penetrate quiescent HSC, therapeutic inefficacy, gene transfer inefficiency and low expression of the genes the early clinical trials were disappointing. This limitation had been overcome by;
- Administration of 5-fluorouracil prior to harvest of bone marrow which causes apoptosis of the committed dividing progenitors, thereby forcing the quiescent HSC to replicate.
- using specific domains of fibronetin, in a recombinant molecule (CH-296 or Retronectin) to assist in the interaction of the viral envelope protein and target cell receptors (Moritz et al, 1996). An unexpected benefit of the CH-296 molecule is its ability to protect HSC from under going apoptosis.
- studies carried out by some scientists have shown that switching the viral envelope protein to those obtained from the endogenous RD114 feline virus or murine leukaemia virus 10A1, gibbon ape leukaemia virus mediated significantly higher gene transfer into human HSC (Kelly et al, 2000).
These technological advances have substantially improved the current prospects of HSC gene therapy.
Recent success of HSC gene transfer and GENE THERAPY
Cure of X-linked severe combined immunodeficiency (SCID-XI)
SCID-XI is an immunodeficiency disorders in which the body produces very little T-cell and NK cells. In the absence of T-helper cells, β-cells become defective (Fisher et al., 2002). It is an X-linked recessive trait stemming from a mutated (abnormal) version of the 1L2-RG gene located at Xq 13.1 on the X-Chromosome which is shared between receptors for 1L-2, 1L-4, 7, 9, 15 and 1L-21. (1L2RG) (Buckley, 2000; Puck, et al., 1996). It affects mostly male child whose mother is a carrier (heterozygous) for the disorder. From its inception, SCID-XI has been regarded as an attractive target for gene therapy for several reasons which include;
- It is the result of a defect in a single gene involving the common gamma chain (YC), subunit of the interleukin 2 cytokine receptor family.
- In addition, gene-modified cells were predicted to have a selective growth and survival advantage therefore; highly efficient transduction of HSC would not be a pre-requisite for success.
Without treatment; the condition is invariably fatal in first year of life. Treatment by transplantation from a human leucoyte antigen-matched sibling bone-marrow donor results in good immunological reconstitution and is associated with a 90% long-term survival rate. In a situation whereby there is no donor, gene therapy becomes the option.
Cavazzana-calvo and his group from the Necker Hospital in Paris, reported successful treatment of ten SCID-XI infants with gene therapy nine of the ten were cured of X-SCID (Cavazzana-calvo, 2000) while two developed leukaemia.
In their gene transfer protocol, cytokine stimulated, bone-marrow-derived, autologous CD34+ cells were repeated transduced with an oncoretroviral vector encoding the normal yc gene. Approximately, 15-20 x 106 CD34+cells/kg, with a mean bulk transduction efficiency of approximately 40%, were infused back to the patient without any conditioning.
Significant levels of immune reconstitution occurred in all except one infant, who was ill from a pre-existing Bacille calmette gurine infection and did not engraft. T-cell levels increased from near undetectable levels to within normal limits within 3 months of infusion of gene-modified cells. Importantly, these gene-modified T-cells were able to mount an appropriate response to a variety of antigens. The retroviral transgenes was detectable in almost 100% of circulating T-cell, but in only a minority of neutrophils(6.1-1%). This dearly demonstrates that genetic modification of a small number of haematopoietic progenitors would be sufficient to reconstitute the T-lymphocyte compartment because of the selective advantage conferred to these cells as a result of normal yc expression. There was also evidence of partial restoration of β-cells to levels where the patients were not dependent on intravenous infusions of immunoglobulin. Similarly, some recovery of natural killer cells (NK) activity was also observed. (Amit et al., 2004).