Human Adenosine Deaminase Gene Research Articles

Dissecting the Mechanism of Histone Deacetylase Inhibitors to Enhance the Activity of Zinc Finger Nucleases Delivered by Integrase-Defective Lentiviral Vectors

Integrase-defective lentiviral vectors (IDLVs) have been of limited success in the delivery of zinc finger nucleases (ZFNs) to human cells, due to low expression. A reason for reduced gene expression has been proposed to involve the epigenetic silencing of vector genomes, carried out primarily by histone deacetylases (HDACs). In this study, we tested valproic acid (VPA), a known HDAC inhibitor (HDACi), for its ability to increase transgene expression from IDLVs, especially in the context of ZFN delivery. Using ZFNs targeting the human adenosine deaminase (ADA) gene in K562 cells, we demonstrated that treatment with VPA enhanced ZFN expression by up to 3-fold, resulting in improved allelic disruption at the ADA locus. Furthermore, three other U.S. Food and Drug Administration-approved HDACis (vorinostat, givinostat, and trichostatin-A) exhibited a similar effect on the activity of ZFN-IDLVs in K562 cells. In primary human CD34(+) cells, VPA- and vorinostat-treated cells showed higher levels of expression of both green fluorescent protein (GFP) as well as ZFNs from IDLVs. A major mechanism for the effects of HDAC inhibitors on improving expression was from their modulation of the cell cycle, and the influence of heterochromatinization was determined to be a lesser contributing factor.

Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins

A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both β-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins.

Integrase-defective Lentiviral Vectors as a Delivery Platform for Targeted Modification of Adenosine Deaminase Locus

We investigated the use of integrase-defective lentiviral vectors (IDLVs) for transient delivery of zinc finger nucleases (ZFNs) and donor templates for site-specific modification of the human adenosine deaminase (hADA) gene. Initially, we constructed IDLVs carrying ZFN monomers (Single-IDLVs) and found them to be able to deliver their gene-editing payload to K562 cells successfully upon cotransduction, with minimal cytotoxicity. To simplify delivery, we designed an IDLV construct to deliver both ZFN monomers from the same vector (Double-IDLV). However, this construct in its original state was prone to rearrangements of the vector genome, resulting in greatly reduced functionality; this was due to recombination between highly similar ZFN monomers arranged in tandem. We modified the Double-IDLV constructs to reduce recombination and restored simultaneous delivery of both ZFNs. We also tested an IDLV construct for delivery of donor templates and demonstrated its efficacy for gene modification. In summary, we highlighted the importance of modifying vector design for co-delivery of highly similar sequences inherent to genome-editing nucleases, and demonstrated significant improvement in the use of IDLVs for delivery of ZFNs and donor templates for genome modification.

The β-Globin Locus Control Region in Combination With the EF1α Short Promoter Allows Enhanced Lentiviral Vector-mediated Erythroid Gene Expression With Conserved Multilineage Activity

Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the β-globin gene (β-LCR). We show that the β-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3-7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the β-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the β-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010 ). Currently, rAAV vectors are being utilized in phase I/II clinical trials for cystic fibrosis, α-1 antitrypsin deficiency, Canavan's disease, Parkinson's disease, hemophilia, limb-girdle muscular dystrophy, arthritis, Batten's disease, and Leber's congenital amaurosis (Flotte et al., 1996 , 2004 ; Kay et al., 2000 ; Aitken et al., 2001 ; Wagner et al., 2002 ; Manno et al., 2003 ; Snyder and Francis, 2005 ; Maguire et al., 2008 ; Cideciyan et al., 2009 ). In this study, we present preclinical data to support the viability of an rAAV-based gene transfer strategy for cure of ADA-SCID. We report efficient transduction of a variety of postmitotic target tissues in vivo, subsequent human ADA (hADA) expression, and enhanced hADA secretion in tissues and blood, with increasing peripheral lymphocyte populations over time.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

BackgroundMurine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.MethodsA MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.ResultsVectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.ConclusionThese studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Temporal Regulation of Enhancer Function in Intestinal Epithelium: A ROLE FOR ONECUT FACTORS

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2-3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.

Cdx Binding Determines the Timing of Enhancer Activation in Postnatal Duodenum

In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.

Adenosine deaminase, a key enzyme in DNA precursors control, is a new p73 target.

The discovery of the p73 and p63 genes, homologous to p53 tumor suppressor has uncovered a family of transcription factors and widened the scenario of cell cycle control and apoptosis. We have identified a putative p53-responsive element in the human adenosine deaminase (ADA) gene, an important enzyme involved in nucleotide metabolism, the deficit of which causes the inhibition of DNA synthesis and repair. Here, we demonstrate that the ectopic expression of p73 isoforms leads to the ADA gene upregulation, showing for the first time a correlation between p73 and ADA. We found that p73 promotes ADA gene expression following a dNTP unbalance generated by ADA enzyme deficiency and 2'deoxyadenosine accumulation. The abrogation of p73 transcriptional activity by the specific dominant-negative p73DD abolishes ADA induction. By contrast, the ADA gene does not appear to be a functional p53 target in the physiological conditions we tested. On the whole, our results contribute to the emerging picture that p73 could play a different role from p53 in normal growth and development by inducing alternative target genes, which are not shared by p53.

Linkage disequilibrium analysis of the human adenosine deaminase ( ada) gene provides evidence for a lack of correlation between hot spots of equal and unequal homologous recombination

The linkage disequilibrium (LD) pattern within the adenosine deaminase ( ADA) gene was analyzed by studying 13 polymorphic loci in 137 families from two European and three African populations. Evidence for the presence of a 12-kb meiotic crossover hot spot, spanning part of the first and the second intron and flanked by regions of reduced recombination activity, was obtained. Moreover, segregation analysis of 113 informative meioses revealed two recombination events that are internal or overlap the 12-kb region, thus suggesting a recombination rate for the hot-spot region about 50-fold higher than the mean rate across the human genome. Within the hot spot, a 144-bp palindromic sequence was also identified and its possible involvement in the recombination process is discussed. The 12-kb region characterized by the low degree of LD does not include the 3.2-kb region that is deleted, as a result of recurrent unequal homologous recombination between two Alu elements, in patients affected by autosomal severe combined immunodeficiency. This observation provides the first evidence for an absence of correlation between hot spots of equal and unequal homologous recombination.

High-level activation by a duodenum-specific enhancer requires functional GATA binding sites.

The purine metabolic gene adenosine deaminase (ADA) is expressed at high levels in a well-defined spatiotemporal pattern in the villous epithelium of proximal small intestine. A duodenum-specific enhancer module responsible for this expression pattern has been identified in the second intron of the human ADA gene. It has previously been shown that binding of the factor PDX-1 is essential for function of this enhancer. The studies presented here examine the proposed roles of GATA factors in the enhancer. Site-directed mutagenesis of the enhancer's GATA binding sites crippled enhancer function in 10 lines of transgenic mice, with 9 of the lines demonstrating <1% of normal activity. Detailed studies along the longitudinal axis of mouse small intestine indicate that GATA-4 and GATA-5 mRNA levels display a reciprocal pattern, with low levels of GATA-6 throughout. Interestingly, gel shift studies with duodenal nuclear extracts showed binding only by GATA-4.

PDX-1 Is Required for Activation in Vivo from a Duodenum-specific Enhancer

The purine metabolic gene adenosine deaminase (ADA) is expressed along a defined spatiotemporal pattern in the developing mammalian small intestine, where high-level expression is limited to the villous epithelium of the duodenum. This activation is observed in rodents as the intestine completes the final maturation resulting in adult crypt-villus structures at 2-3 weeks postpartum. A regulatory module responsible for this pattern of expression has been identified in the second intron of the human ADA gene. Of the multiple duodenal proteins that can interact with this small duodenal enhancer region, the studies contained in this work describe the identification of five of these proteins as the dispersed homeobox protein PDX-1. This transcription factor exhibits a profile of expression in the small intestine similar to that observed for ADA, making it an ideal candidate factor for the duodenum-specific ADA enhancer. Loss of PDX-1 binding, via a PDX-1 mutated enhancer transgenic construction, resulted in complete loss of high-level activation in the duodenum, demonstrating the absolute requirement for this factor in vivo. However, co-transfection experiments suggest that other proteins that bind the enhancer are also required for enhancer function because PDX-1 alone was incapable of significant transactivation.

Human RNA-specific adenosine deaminase ( ADAR1) gene specifies transcripts that initiate from a constitutively active alternative promoter

The human ADAR1 gene specifies two size forms of RNA-specific adenosine deaminase, an interferon (IFN) inducible ∼150 kDa protein and a constitutively expressed N-terminally truncated ∼110 kDa protein, encoded by transcripts with alternative exon 1 structures that initiate from different promoters. We have now identified a new class of ADAR1 transcripts, with alternative 5′-structures and a deduced coding capacity for the ∼110 kDa protein. Nuclease protection and 5′-rapid amplification of cDNA ends (5′-RACE) revealed five major ADAR1 transcriptional start sites that mapped within the previously identified and unusually large (∼1.6 kb) exon 2. These transcripts were observed with RNA from human amnion U cells and placenta tissue. Their abundance was not affected by IFN-α treatment of U cells in culture. Transfection analysis identified a functional promoter within human genomic DNA that mapped to the proximal exon 2 region of the ADAR1 gene. Promoter activity was not affected by IFN. These results suggest that transcripts encoding the constitutively expressed ∼110 kDa form of the ADAR1 editing enzyme are initiated from multiple promoters, including one within exon 2, that collectively contribute to the high basal level of deaminase activity observed in nuclei of mammalian cells.

A duodenum-specific enhancer regulates expression along three axes in the small intestine.

Adenosine deaminase (ADA) is expressed at high levels in the epithelium of proximal small intestine. Transgenic mice were used to characterize the regulatory region governing this activation. A duodenum-specific enhancer is located in intron 2 of the human ADA gene at the central site among a cluster of seven DNase I-hypersensitive sites present in duodenal DNA. Flanking DNA, including the remaining hypersensitive sites, is required for consistent high-level enhancer function. The enhancer activates expression in a pattern identical to endogenous ADA along both the anterior-posterior axis of the small intestine and the crypt-villus differentiation axis of the intestinal epithelium. Timing of activation by the central enhancer mimics endogenous mouse ADA activation, occurring at 2-3 wk of age. However, two upstream DNA segments, one proximal and one distal, collaborate to change enhancer activation to a perinatal time point. Studies with duodenal nuclear extracts identified five distinct DNase I footprints within the enhancer. Protected regions encompass six putative binding sites for the transcription factor PDX-1, as well as proposed CDX, hepatocyte nuclear factor-4, and GATA-type sites.

Clone-forming activity of embryonal stem hemopoietic cells after transplantation to newborn or adult sublethally irradiated mice.

Hemopoietic activity of stem hemopoietic cells from the liver of embryos was studied at different terms of intrauterine development. The fate of individual clones of hemopoietic cells marked by human adenosine deaminase gene was followed up in sublethally irradiated or newborn recipients. The efficiency of marker gene incorporation in primitive stem hemopoietic cells from the liver of 12-, 13-, and 17-day embryos was not high. Gene transfer was performed without cell prestimulation to division, and hence, these data show that primitive stem cells proliferate even in 17-day embryos. Cells from embryonal liver in all terms maintain hemopoiesis both in newborn and adult microenvironment, hemopoiesis being realized according to the clonal succession model, i. e. in the some way after transplantation of the bone marrow from adult mice.

Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides.

A sequence-specific genomic delivery system for the correction of chromosomal mutations was designed by incorporating two different binding domains into a single-stranded oligonucleotide. A repair domain (RD) contained the native sequence of the target region. A third strand-forming domain (TFD) was designed to form a triplex by Hoogsteen interactions. The design was based upon the premise that the RD will rapidly form a heteroduplex that is anchored synergistically by the TFD. Deoxyoligonucleotides were designed to form triplexes in the human adenosine deaminase (ADA) and p53 genes adjacent to known point mutations. Transfection of ADA-deficient human lymphocytes corrected the mutant sequence in 1-2% of cells. Neither the RD or TFD individually corrected the mutation. Transfection of p53 mutant human glioblastoma cells corrected the mutation and induced apoptosis in 7.5% of cells.

Successful transfer of ADA gene in vitro into human peripheral blood CD34 + cells by transfecting EBV-based episomal vectors

We report a novel non-viral system for transfecting human immature hematopoietic cells in vitro. Epstein-Barr virus (EBV)-based episomal vectors carrying human adenosine deaminase (ADA) gene cDNA were transfected by electroporation into human peripheral blood (PB) CD34 + cells. The transgene-specific mRNA were detected from 37 to 100% of CFU-c (colony forming unit in culture) colonies derived from the transfected cells. A two-fold increase in enzyme activity was also found. These results indicate the successful transfer and expression of genes in human immature hematopoietic cells using the EBV-based episomal vector system.

Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo.

We introduced the locus control region (LCR) of the human CD2 gene in eight different ways into retroviral constructs containing the human adenosine deaminase (hADA) gene. Two of these constructs were tested in transgenic mice. Neither expressed hADA in any examined tissue, nor did control vectors without the LCR insert. Amphotropic retrovirus vector producer cell clones were isolated and analyzed for provirus integrity and vector titer. Three constructs yielded recombined proviruses in 20-100% of transduced clones, whereas the other five constructs always rendered viruses that remained stable upon replication and gave vector titers comparable to the control lacking an LCR. Human ADA-deficient T cells and murine fibroblasts transduced with these recombinant viruses showed considerable hADA expression levels that were, however, not significantly different from those of cells transduced with the control vector lacking an LCR insert. Furthermore, no difference in hADA expression levels could be detected in spleen, thymus and bone marrow of long-term repopulated mice that had received bone marrow cells transduced with either the control vector or one of two different CD2-LCR containing vectors. In conclusion, the CD2-LCR does not alleviate the expression block for recombinant retroviruses in the germ line and does not enhance the LTR-driven expression in T cells.

Regulation of Duodenal Specific Expression of the Human Adenosine Deaminase Gene

Formation of the mammalian gastrointestinal tract is an ordered process of development and differentiation. Yet, the adult small intestine also retains the plasticity to respond to cues both internal and environmental to modulate intestinal function. The components that regulate this development, differentiation, and modulation at the molecular level are only now being elucidated. We have used the human adenosine deaminase (ADA) gene as a model to identify potential cis-regulatory components involved in these processes within the small intestine. In mammals, high levels of ADA in the small intestine are limited specifically to the differentiated enterocytes within the duodenal region. These studies describe the identification of a region of the human ADA gene, completely distinct from the previously identified T-cell enhancer, which is capable of directing the human intestinal expression pattern in the intestine of transgenic mice. The reporter gene expression pattern observed in these transgenic mice is identical to the endogenous gene along both the cephalocaudal and crypt/villus axis of development. Timing of this transgene activation, however, varies from that of the endogenous mouse gene in that the transgene is activated approximately 2 weeks earlier in development. Even so, this precocious activation is also limited to the epithelium of the developing villi strictly within the duodenal region of the small intestine.

The number of functioning hemopoietic clones in transplantation-restored bone marrow of mice depends on the dose of transplanted cells

The clone composition of mouse bone marrow restored by transplantations of different doses of hemopoietic cells containing the unique genetic markers at the expense of integration of human adenosine deaminase gene is analyzed. Low dose of donor hemopoietic cells accelerates reversion of hemopoiesis to the recipient type and decreases the number of functioning clones. This finding confirms that a stem hemopoietic cell cannot maintain itself and its proliferative potential is limited. For long and stable repopulation of the recipient subjected to gene therapy, the maximum possible number of transduced stem hemopoietic cells should be transplanted.