Appendix N. 
397 
recipients in order to suppress this organ rejection. Such 
immunosuppressive treatment has serious side effects including 
increased risks of infections and malignancies. In principle, ES cells 
can be created from a patient's nuclei using nuclear transfer. 
Because ntES cells will be genetically identical to the patient’s cells, 
the risks of immune rejection and the requirement for 
immunosuppression are eliminated. Moreover, ES cells provide a 
renewable source of replacement tissue allowing for repeated 
therapy whenever needed. Finally, if ES cells are derived from a 
patient carrying a known genetic defect, the mutation in question 
can be corrected in the ntES cells using standard gene targeting 
methods before introducing these ES cells (or derived tissue-specific 
stem cells) back into the patient's body. 
fii) Combining nuclear cloning with aene and cell therapy 
In a “proof of principle" experiment, nuclear cloning in combination 
with gene and cell therapy has been used to treat a mouse genetic 
disorder that has a human counterpart (Figure 4). To do so, the well- 
characterized Rag2 mutant mouse was used as "patient” (Rideout et 
al., 2002). This mutation causes severe combined immune deficiency 
(SCID), because the enzyme that catalyzes immune receptor 
rearrangements in lymphocytes is non-functional. Consequently, 
these mice are devoid of mature B and T cells, a disease resembling 
human Omeim syndrome (Rideout et al., 2002). 
In a first step, somatic (fibroblast) donor cells were isolated from 
the tails of Rag2-deficient mice and their nuclei were injected into 
enucleated eggs. The resultant embryos were cultured to the 
blastocyst stage and isogenic ES cells were isolated. Subsequently, 
one of the mutant Rag2 alleles was targeted by homologous 
recombination in ES cells to restore normal Rag2 gene structure and 
function. In order to obtain somatic cells for treatment, these 
genetically repaired ES cells were differentiated into embryoid 
bodies and further into hematopoietic precursors by expressing 
HoxB4, a transcription factor that is responsible for programming the 
behavior of the hematopoietic stem cells, i.e., those cells that are able 
to generate the full range of red and white cells in the blood. 
Resulting hematopoietic precursors were transplanted into irradiated 
Rag2-deficient animals in order to treat the disease caused by their 
Rag2 mutation. Initial attempts to engraft these cells were, however, 
unsuccessful because of an increased level of natural killer (NK) cells 
in the Rag mutant host. ES cell derived hematopoietic cells express 
low levels of the MHC antigens and thus are a preferred target for NK 
mediated destruction. Elimination of NK cells by antibody depletion 
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