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Mosaicism of GFP expression in lentivirus transduced hematopoietic colonies. (Top) GFP ϩ methylcellulose colonies 6 days after transduction of Lin Ϫ c-kit ϩ Sca1 ϩ cells by a lentivirus vector with EF-1 ␣ as an internal promoter. Two colonies are shown with phase-contrast microscopy (lower panels) and fluores- cent microscopy (upper panels). In each colony it can be seen that only a fraction of the cells are expressing the GFP transgene. (Bottom) FACS analysis of GFP expression within individual GFP ϩ methylcellulose colonies. Each circle repre- sents a single colony.
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Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin(-) c-kit(+) Sca1(+) primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for G...
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Context 1
... into clinical use will require preclinical trials in animal models. This will be invaluable for in vivo testing of new lentivirus vectors, e.g., to achieve optimal expression levels in differentiated progeny cells or to provide lineage-specific or regulatable expression of the transgene. The animal disease models are also crucial for testing the effects of lentivirus gene transfer in vivo , as well as assessing the safety of the vector system in immunocompetent hosts. The aim of this work was to study the efficiency of lentivirus gene transfer into murine hematopoietic stem cells under qui- escent and proliferating conditions. In this study we have dem- onstrated that purified Lin Ϫ c-kit ϩ Sca1 ϩ primitive murine hematopoietic progenitor cells can be transduced by lentivirus vectors under both quiescent as and cytokine-stimulated con- ditions and that high transgene expression levels can be achieved using the elongation factor 1 ␣ (EF-1 ␣ ) promoter. However, the transduction efficiency is consistently higher if the target cells are transduced with cytokine support. Further- more, the final gene transfer efficiency in the daughter cells is compromised by a latency of lentivirus vector integration, and optimal gene transfer of primitive murine hematopoietic pro- genitor cells depends on adjustment of the cytokine stimula- tion and proliferation kinetics of the target cell during and after transduction. hematopoietic cells. To select an optimal expression cassette for high expression of lentivirus-vectors in murine hematopoi- etic cells, 10 different expression vectors were tested in murine hematopoietic cells (Fig. 1). The vectors EF-1 ␣ , EF-1 ␣ – WPRE, PGK, PGK-SIN, PGK-WPRE-SIN, CMV, CMV- WPRE, CMV-SIN, CAG, and CAG-WPRE were named ac- cording to the internal promoter and whether the WPRE or the SIN deletion was present. The purified hematopoietic cells were transduced, grown in liquid culture in the presence of IL-3, IL-6, and SCF for 3 days, and then analyzed by FACS. The vector containing the EF-1 ␣ promoter generated the high- est mean fluoresence intensity (MFI) among the GFP ϩ cells by FACS (EF-1 ␣ , 179 Ϯ 8; PGK, 58 Ϯ 3; CMV, 39 Ϯ 2; CAG, 40 Ϯ 0.5; n ϭ 3 experiments) and was comparable to the MFI (162 Ϯ 2) generated by the oncoretrovirus, vector MGIN (Fig. 3). Addition of the WPRE did not provide an improvement in murine hematopoietic cells, in contrast to the human cell lines HeLa and 293T, where two- to threefold improvement was seen (data not shown). Inclusion of the SIN deletion did not have a clear effect on the expression levels in murine hemato- poietic cells (data not shown). Therefore, we chose the EF-1 ␣ vector without addition of the WPRE element and without the SIN deletion for all further experiments presented below. Gene transfer efficiencies of lentivirus vectors in Lin ؊ c-kit ؉ Sca1 ؉ murine hematopoietic progenitor cells. Lentivirus gene transfer efficiency in Lin Ϫ c-kit ϩ Sca1 ϩ clonogenic progenitors was analyzed in methylcellulose colony assays. The lentivirus vector transduction efficiency, as scored by the percentage of GFP ϩ colonies in methylcellulose, was high under all 20-h transduction conditions tested. The scoring was initially per- formed by microscopy and confirmed by FACS analysis of the individual GFP ϩ colonies. If the transduction was performed without cytokines or serum, the transduction efficiency with the lentivirus vector was 27.3 Ϯ 6.7%, whereas there was no sig- nificant transduction with the oncoretrovirus control (1.26 Ϯ 0.8% with the concentrated supernatant under serum-free con- ditions, or 1.7 Ϯ 0.3% with the unconcentrated supernatant with a final serum concentration of 3%) (Fig. 4). The lentivirus transduction efficiency within the clonogenic progenitors was higher if cytokine support was used. When SCF was added, lentivirus transduction generated 42.0 Ϯ 5.5% GFP ϩ colonies, in contrast to the concentrated and unconcentrated oncoret- rovirus MGIN controls, which resulted in 3.3 Ϯ 1.8 and 9.7 Ϯ 1.8% GFP ϩ colonies, respectively. When transduction was per- formed with SCF, IL-6, and IL-3, 53.3 Ϯ 1.8% of the lentivirus- transduced colonies were positive for GFP, in comparison to 9.3 Ϯ 1.2% (serum free) and 39.3 Ϯ 9.4% (with serum) of the oncoretrovirus controls. Heterogeneity of GFP expression within GFP ؉ colonies. Al- though lentivirus transduction resulted in a high percentage of GFP ϩ colonies, microscopy and FACS analysis of individual GFP ϩ colonies revealed that only a portion of the cells within each colony expressed the GFP gene (Fig. 5). The ratio of GFP ϩ cells within each colony was lowest when no cytokines were used during transduction (19.2 Ϯ 5.0%), in comparison with 34.2 Ϯ 6.0% when SCF was used alone and 45 Ϯ 11% when SCF, IL-6, and IL-3 were used during transduction. In contrast, 87.8 Ϯ 5.2% of the cells in GFP ϩ colonies transduced in the presence of IL-3, IL-6, and SCF with the MGIN on- coretrovirus, control vector were GFP ϩ (Fig. 5, bottom). Mo- saicism of GFP expression was observed in transduced colonies from all lentivirus-vectors tested, irrespective of the nature of the internal promoter (data not shown). Analysis of secondary hematopoietic colonies for GFP ex- pression and presence of the vector genome. To study whether the heterogeneity of GFP expression in the methylcellulose colonies was due to transcriptional silencing or true genetic mosaicism with respect to the presence of the proviral vector genome, individual GFP ϩ colonies were plated further for secondary colony assays. Purified hematopoietic progenitors were transduced in the presence of IL-3, IL-6, and SCF, then plated into methylcellulose cultures, and GFP ϩ hematopoietic colonies of various types or morphologies (colony-forming units mix [CFU-mix], colony-forming units granulocyte-mac- rophage [CFU-GM], etc.) were picked at days 5 to 6 and plated into secondary methylcellulose cultures. Individual secondary colonies were then scored for GFP expression by FACS and for the presence of vector genome in the daughter cells by PCR. Within the 30 secondary colonies that derived from 7 different primary GFP ϩ colonies, only 42% were GFP positive by both FACS and PCR (Table 1). None of the colonies were positive by FACS without being PCR positive, demonstrating the high sensitivity of the PCR assay. Approximately half of the colonies (46%) were negative both by FACS and by PCR. Some of the colonies (13%) were positive by PCR without demonstrating any expression of GFP by FACS, representing either integrated copies where the expression from the EF-1 ␣ promoter was silenced or cases where the GFP gene was am- plified by PCR from a nonintegrated vector. The presence of GFP Ϫ PCR Ϫ daughter colonies in the progeny of primary GFP ϩ colonies indicates that the lentivirus gene transfer is not fully completed between the time of transduction and the time when the transduced progenitor divides. Therefore, the vector genome seems to integrate after proliferation in the methyl- cellulose culture starts, and as a consequence, vector integra- tion is seen in only a portion of each progenitor’s progeny cells. In contrast, all secondary colonies derived from the oncoret- rovirus-transduced colonies expressed GFP and contained the proviral DNA, as detected by PCR. The difference between the number of secondary colonies containing the lentiviral and oncoretroviral DNA, respectively, is highly significant by the chi-square test (P Ͻ 0.01). efficiency. To study the time course needed for completion of lentivirus gene transfer in mouse hematopoietic progenitor cells, the transduced cells were cultured for an extended period (an additional 24 to 72 h) before plating into methylcellulose clonal assays. By delaying the start of a clonal assay after transduction in SCF, the degree of mosaicism was reduced. The percentage of GFP ϩ cells within the GFP ϩ colonies in- creased from 34 Ϯ 8% at day 0 to 55 Ϯ 6% when the cells were plated at day 1 posttransduction and to 61 Ϯ 9 and 68 Ϯ 11% at days 2 and 3 posttransduction, respectively (Fig. 6A). The difference between day 0 and day 3 was significant ( P Ͻ 0.02), as was the difference between day 0 and day 2 ( P Ͻ 0.05), by Student’s t test. The difference between day 0 and day 1 was not statistically significant ( P ϭ 0.11). In an effort to analyze the effect of cytokine stimulation and proliferation kinetics after transduction on the final gene trans- fer efficiency as judged by the total percentage of GFP ϩ cells in the progeny, half of the transduced cells were grown for addi- tional 48 h in liquid culture before plating into methylcellulose with SCF, IL-6, and IL-3, whereas half of the sample was plated directly after a 20-h transduction. Extended culture in SCF for an additional 48 h increased the percentage of GFP ϩ cells in methylcellulose culture twofold, from 12 Ϯ 3% to 23 Ϯ 2% (Fig. 6B). This difference is statistically significant ( P Ͻ 0.03). In contrast, when the transduction and extended culture were performed with more-efficient cytokine stimulation (SCF, IL-6, and IL-3), the initial gene transfer was higher but the extended culture did not provide any further significant in- crease in the ratio of GFP ϩ cells in methylcellulose (19 Ϯ 5% when cells were plated directly in comparison to 22 Ϯ 2% with extended culture) (Fig. 6B). Likewise, when the cells were transduced in the presence of SCF alone and split following 20 h of transduction in two parts, liquid culture with SCF alone or with the three cytokines SCF, IL-6, and IL-3 for an addi- tional 5 days, the cells cultured under low proliferating condi- tions (SCF alone) showed a much higher percentage of GFP ϩ cells by FACS than the subgroup cultured under high prolif- erating conditions (21 Ϯ 4% versus 7 Ϯ 1%, respectively). These results demonstrate that the initial transduction effi- ciency is higher when the cells are stimulated with the three cytokines. However, ...
Context 2
... in immunodeficient NOD/SCID mice which act as hosts for the transplantation of human hematopoietic cells (30). Although the NOD/SCID mouse assay is the most commonly used assay so far for the study of human candidate stem cells, it is limited by the short life span of the recipients as well as by the inability to support differentiation to all hematopoietic lineages. The development of lentiviral gene therapy into clinical use will require preclinical trials in animal models. This will be invaluable for in vivo testing of new lentivirus vectors, e.g., to achieve optimal expression levels in differentiated progeny cells or to provide lineage-specific or regulatable expression of the transgene. The animal disease models are also crucial for testing the effects of lentivirus gene transfer in vivo , as well as assessing the safety of the vector system in immunocompetent hosts. The aim of this work was to study the efficiency of lentivirus gene transfer into murine hematopoietic stem cells under qui- escent and proliferating conditions. In this study we have dem- onstrated that purified Lin Ϫ c-kit ϩ Sca1 ϩ primitive murine hematopoietic progenitor cells can be transduced by lentivirus vectors under both quiescent as and cytokine-stimulated con- ditions and that high transgene expression levels can be achieved using the elongation factor 1 ␣ (EF-1 ␣ ) promoter. However, the transduction efficiency is consistently higher if the target cells are transduced with cytokine support. Further- more, the final gene transfer efficiency in the daughter cells is compromised by a latency of lentivirus vector integration, and optimal gene transfer of primitive murine hematopoietic pro- genitor cells depends on adjustment of the cytokine stimula- tion and proliferation kinetics of the target cell during and after transduction. hematopoietic cells. To select an optimal expression cassette for high expression of lentivirus-vectors in murine hematopoi- etic cells, 10 different expression vectors were tested in murine hematopoietic cells (Fig. 1). The vectors EF-1 ␣ , EF-1 ␣ – WPRE, PGK, PGK-SIN, PGK-WPRE-SIN, CMV, CMV- WPRE, CMV-SIN, CAG, and CAG-WPRE were named ac- cording to the internal promoter and whether the WPRE or the SIN deletion was present. The purified hematopoietic cells were transduced, grown in liquid culture in the presence of IL-3, IL-6, and SCF for 3 days, and then analyzed by FACS. The vector containing the EF-1 ␣ promoter generated the high- est mean fluoresence intensity (MFI) among the GFP ϩ cells by FACS (EF-1 ␣ , 179 Ϯ 8; PGK, 58 Ϯ 3; CMV, 39 Ϯ 2; CAG, 40 Ϯ 0.5; n ϭ 3 experiments) and was comparable to the MFI (162 Ϯ 2) generated by the oncoretrovirus, vector MGIN (Fig. 3). Addition of the WPRE did not provide an improvement in murine hematopoietic cells, in contrast to the human cell lines HeLa and 293T, where two- to threefold improvement was seen (data not shown). Inclusion of the SIN deletion did not have a clear effect on the expression levels in murine hemato- poietic cells (data not shown). Therefore, we chose the EF-1 ␣ vector without addition of the WPRE element and without the SIN deletion for all further experiments presented below. Gene transfer efficiencies of lentivirus vectors in Lin ؊ c-kit ؉ Sca1 ؉ murine hematopoietic progenitor cells. Lentivirus gene transfer efficiency in Lin Ϫ c-kit ϩ Sca1 ϩ clonogenic progenitors was analyzed in methylcellulose colony assays. The lentivirus vector transduction efficiency, as scored by the percentage of GFP ϩ colonies in methylcellulose, was high under all 20-h transduction conditions tested. The scoring was initially per- formed by microscopy and confirmed by FACS analysis of the individual GFP ϩ colonies. If the transduction was performed without cytokines or serum, the transduction efficiency with the lentivirus vector was 27.3 Ϯ 6.7%, whereas there was no sig- nificant transduction with the oncoretrovirus control (1.26 Ϯ 0.8% with the concentrated supernatant under serum-free con- ditions, or 1.7 Ϯ 0.3% with the unconcentrated supernatant with a final serum concentration of 3%) (Fig. 4). The lentivirus transduction efficiency within the clonogenic progenitors was higher if cytokine support was used. When SCF was added, lentivirus transduction generated 42.0 Ϯ 5.5% GFP ϩ colonies, in contrast to the concentrated and unconcentrated oncoret- rovirus MGIN controls, which resulted in 3.3 Ϯ 1.8 and 9.7 Ϯ 1.8% GFP ϩ colonies, respectively. When transduction was per- formed with SCF, IL-6, and IL-3, 53.3 Ϯ 1.8% of the lentivirus- transduced colonies were positive for GFP, in comparison to 9.3 Ϯ 1.2% (serum free) and 39.3 Ϯ 9.4% (with serum) of the oncoretrovirus controls. Heterogeneity of GFP expression within GFP ؉ colonies. Al- though lentivirus transduction resulted in a high percentage of GFP ϩ colonies, microscopy and FACS analysis of individual GFP ϩ colonies revealed that only a portion of the cells within each colony expressed the GFP gene (Fig. 5). The ratio of GFP ϩ cells within each colony was lowest when no cytokines were used during transduction (19.2 Ϯ 5.0%), in comparison with 34.2 Ϯ 6.0% when SCF was used alone and 45 Ϯ 11% when SCF, IL-6, and IL-3 were used during transduction. In contrast, 87.8 Ϯ 5.2% of the cells in GFP ϩ colonies transduced in the presence of IL-3, IL-6, and SCF with the MGIN on- coretrovirus, control vector were GFP ϩ (Fig. 5, bottom). Mo- saicism of GFP expression was observed in transduced colonies from all lentivirus-vectors tested, irrespective of the nature of the internal promoter (data not shown). Analysis of secondary hematopoietic colonies for GFP ex- pression and presence of the vector genome. To study whether the heterogeneity of GFP expression in the methylcellulose colonies was due to transcriptional silencing or true genetic mosaicism with respect to the presence of the proviral vector genome, individual GFP ϩ colonies were plated further for secondary colony assays. Purified hematopoietic progenitors were transduced in the presence of IL-3, IL-6, and SCF, then plated into methylcellulose cultures, and GFP ϩ hematopoietic colonies of various types or morphologies (colony-forming units mix [CFU-mix], colony-forming units granulocyte-mac- rophage [CFU-GM], etc.) were picked at days 5 to 6 and plated into secondary methylcellulose cultures. Individual secondary colonies were then scored for GFP expression by FACS and for the presence of vector genome in the daughter cells by PCR. Within the 30 secondary colonies that derived from 7 different primary GFP ϩ colonies, only 42% were GFP positive by both FACS and PCR (Table 1). None of the colonies were positive by FACS without being PCR positive, demonstrating the high sensitivity of the PCR assay. Approximately half of the colonies (46%) were negative both by FACS and by PCR. Some of the colonies (13%) were positive by PCR without demonstrating any expression of GFP by FACS, representing either integrated copies where the expression from the EF-1 ␣ promoter was silenced or cases where the GFP gene was am- plified by PCR from a nonintegrated vector. The presence of GFP Ϫ PCR Ϫ daughter colonies in the progeny of primary GFP ϩ colonies indicates that the lentivirus gene transfer is not fully completed between the time of transduction and the time when the transduced progenitor divides. Therefore, the vector genome seems to integrate after proliferation in the methyl- cellulose culture starts, and as a consequence, vector integra- tion is seen in only a portion of each progenitor’s progeny cells. In contrast, all secondary colonies derived from the oncoret- rovirus-transduced colonies expressed GFP and contained the proviral DNA, as detected by PCR. The difference between the number of secondary colonies containing the lentiviral and oncoretroviral DNA, respectively, is highly significant by the chi-square test (P Ͻ 0.01). efficiency. To study the time course needed for completion of lentivirus gene transfer in mouse hematopoietic progenitor cells, the transduced cells were cultured for an extended period (an additional 24 to 72 h) before plating into methylcellulose clonal assays. By delaying the start of a clonal assay after transduction in SCF, the degree of mosaicism was reduced. The percentage of GFP ϩ cells within the GFP ϩ colonies in- creased from 34 Ϯ 8% at day 0 to 55 Ϯ 6% when the cells were plated at day 1 posttransduction and to 61 Ϯ 9 and 68 Ϯ 11% at days 2 and 3 posttransduction, respectively (Fig. 6A). The difference between day 0 and day 3 was significant ( P Ͻ 0.02), as was the difference between day 0 and day 2 ( P Ͻ 0.05), by Student’s t test. The difference between day 0 and day 1 was not statistically significant ( P ϭ 0.11). In an effort to analyze the effect of cytokine stimulation and proliferation kinetics after transduction on the final gene trans- fer efficiency as judged by the total percentage of GFP ϩ cells in the progeny, half of the transduced cells were grown for addi- tional 48 h in liquid culture before plating into methylcellulose with SCF, IL-6, and IL-3, whereas half of the sample was plated directly after a 20-h transduction. Extended culture in SCF for an additional 48 h increased the percentage of GFP ϩ cells in methylcellulose culture twofold, from 12 Ϯ 3% to 23 Ϯ 2% (Fig. 6B). This difference is statistically significant ( P Ͻ 0.03). In contrast, when the transduction and extended culture were performed with more-efficient cytokine stimulation (SCF, IL-6, and IL-3), the initial gene transfer was higher but the extended culture did not provide any further significant in- crease in the ratio of GFP ϩ cells in methylcellulose (19 Ϯ 5% when cells were plated directly in comparison to 22 Ϯ 2% with extended culture) (Fig. 6B). Likewise, when the cells were transduced in the presence of SCF alone and split following 20 h of transduction in two parts, liquid culture with SCF alone or with the three cytokines SCF, ...
Citations
... These processes are inherently stochastic and contribute to the high heterogeneity of responses within the targeted cell population Schwake et al., 2010;Ligon et al., 2014). Similarly, viral delivery and natural viral infection involve substantial cell-to-cell variation (Snijder et al., 2009;Zhu et al., 2020;Mikkola et al., 2000;Brandt et al., 2020;Russell et al., 2018). Regardless of these limitations, even ideal, fully controlled gene transfer would allow for overexpression of only a few transgenes with limited command over the exact levels and kinetics of the expressed proteins. ...
Therapeutic genome modification requires precise control over the introduced therapeutic functions. Current approaches of gene and cell therapy fail to deliver such command and rely on semi-quantitative methods with limited influence on timing, contextuality and levels of transgene expression, and hence on therapeutic function. Synthetic biology offers new opportunities for quantitative functionality in designing therapeutic systems and their components. Here, we discuss synthetic biology tools in their therapeutic context, with examples of proof-of-principle and clinical applications of engineered synthetic biomolecules and higher-order functional systems, i.e. gene circuits. We also present the prospects of future development towards advanced gene-circuit therapy.
... Prkdc gene editing in scid HSPC and transplantation. To attempt Prkdc gene editing in HSPC we used lin − scid bone marrow cells 29 . Using IPLV-delivered ZFN we detected up to 18% target gene cutting according to a Cel-I assay (Fig. 3a). ...
Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.
... is accompanied with genome-wide integration of vectors. This was described to result in heterogeneous transgene expression in individual cell clones of cell populations [9][10][11][12]19 . It was shown that transduction with UCOE-containing vectors resulted in significantly less heterogeneity than populations of cells with UCOE-free vectors 9, 10, 15 . ...
Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element.
... Prkdc gene editing in scid HSPC and transplantation. To attempt Prkdc gene editing in HSPC we used lin − scid bone marrow cells 29 . Using IPLV-delivered ZFN we detected up to 18% target gene cutting according to a Cel-I assay (Fig. 3a). ...
... Both LVs and AAVs have high transduction effi ciencies targeting many human and experimental animal cell types, including stem cells, neurons and glia, muscle, skin, liver, pancreas, lung, primary T lymphocytes, and fetal tissues ( 68,(71)(72)(73)(74)(75)(76)(77)(78)(79)(80) . Because LVs integrate into the host genome, transgene expression persists through cell division of nonquiescent cells and is observed in daughter cells ( 81 ) . This becomes an important consideration when targeting cells that undergo frequent mitosis such as epithelial cells of the lungs and gastrointestinal tract, splenocytes, epidermal cells, monocytes, and stem cells. ...
Viral-vector-based gene therapy is a powerful tool that allows experimental studies of species that previously were not amenable to genetic manipulation. Nonhuman primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the diffi culty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this diffi culty by infecting and transducing dividing or nondividing cells, without eliciting an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells and are able to maintain long-term expression. In this chapter, we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. In addition, we describe in detail the lentiviral vector production protocol we use and provide specifi c examples of how this vector can be employed to target peripheral tissues and the brain. Finally, we conclude by comparing and contrasting the use of lentiviral vectors with adeno-associated viral vectors with a particular emphasis on the use of these vectors in preclinical and clinical studies.
... This result is consistent with those of Mikkola et al. concerning murine HSC transduction by a VSVg-pseudotyped lentiviral vector, in which a mismatch was reported between the transduction rate of cells (almost 25%) and the transduction rate of myeloid colonies (almost 60%). These authors highlighted the occurrence of mosaicism in GFP gene expression in colonies obtained following the myeloid differentiation of CD34 + cells [63], possibly due to a delay in the integration of the transgene during differentiation, resulting in the formation of clusters of GFP-positive cells within a single myeloid colony. ...
Background Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques. Results The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% ± 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colonyforming cells (37% ± 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery. Conclusion The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T-and B-lymphocyte, thrombocyte and red blood cell compartments.
... This result is consistent with those of Mikkola et al. concerning murine HSC transduction by a VSVg-pseudotyped lentiviral vector, in which a mismatch was reported between the transduction rate of cells (almost 25%) and the transduction rate of myeloid colonies (almost 60%). These authors highlighted the occurrence of mosaicism in GFP gene expression in colonies obtained following the myeloid differentiation of CD34+ cells[63], possibly due to a delay in the integration of the transgene during differentiation, resulting in the formation of clusters of GFP-positive cells within a single myeloid colony. ...
Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques.
The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% +/- 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% +/- 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery.
The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.
... 131 It remains to be established whether internal LTRs pose a greater oncogenic risk than internal mammalian promoters, for the reasons stated above. The elongation factor-1a (EF-1a) promoter is among the strongest promoters in vitro, 129,130,132 showing three-to four-fold stronger activity than the PGK promoter in a human CD34 þ cell line 130 and in cultured cord blood cells. 129,130 Continued in vivo expression of the EF-1a promoter over 15 weeks was observed in the progeny of CD34 þ cells engrafted into NOD/SCID mice. ...
Recent studies on the integration patterns of different categories of retroviral vectors, the genotoxicity of long-terminal repeats (LTRs) and other genetic elements, the rise of lentiviral technology and the emergence of regulated vector systems providing tissue-restricted transgene expression and RNA interference, are profoundly changing the landscape of stem cell-based therapies. New developments in vector design and an increasing understanding of the mechanisms underlying insertional oncogenesis are ushering in a new phase in hematopoietic stem cell (HSC) engineering, thus bringing the hitherto exclusive reliance on LTR-driven, gamma-retroviral vectors to an end. Based on their ability to transduce non-dividing cells and their genomic stability, lentiviral vectors offer new prospects for the manipulation of HSCs. Tissue-specific vectors, as exemplified by globin vectors, not only provide therapeutic efficacy, but may also enhance safety, insofar that they restrict transgene expression in stem cells, progenitor cells and blood cells in all but the transcriptionally targeted lineage. This review provides a survey of these advances as well as several remaining challenges, focusing in particular on the importance of achieving adequate levels of protein expression from a limited number of vector copies per cell-ideally one to two.
... While we could not categorically exclude rare events of donor-host cell fusion, this clearly did not account for the great majority of GFP-marked flow cytometric events. We noted relatively low levels of GFP fluorescence intensity in cells from these animals, likely related to known EF1␣ promoter performance characteristics (22) or perhaps as a result of selective immune elimination of cells with higher expression levels (32). Analysis of fluorescence intensities over time in leukocyte subsets of a large cohort of recipients indicated that fluorescence levels remained low but clearly persisted. ...
... However, while attachment to the primary target (i.e., carrier) cell is not pseudotype restricted, the susceptibility of secondary tissue targets remains subject to the specific interaction of vector envelope protein with its cognate cell surface receptor. Thus, for in vivo applications of vectorexposed cells, the combination of extended VSV-G tissue tropism and the ability of lentivirus vectors to transduce nondividing cells may lead to off-target transduction (3,22,37). Recent promising Env-based targeting approaches conferring tissue-restricted entry may be suitable in reducing off-target transduction in secondary tissues (41). Our experimental transduction culture and transplantation design is representative of other investigators' strategies for stem cell gene therapy, and the implications of our study go beyond issues of biosafety or the potential for vector transmission to the germ line. ...
Human immunodeficiency virus type 1-derived lentivirus vectors bearing the vesicular stomatitis virus G (VSV-G) envelope glycoprotein
demonstrate a wide host range and can stably transduce quiescent hematopoietic stem cells. In light of concerns about biosafety
and potential germ line transmission, they have been used predominantly for ex vivo strategies, thought to ensure the removal
of excess surface-bound particles and prevent in vivo dissemination. Studies presented here instead reveal prolonged particle
adherence after ex vivo exposure, despite serial wash procedures, with subsequent transduction of secondary target cells in
direct and transwell cocultures. We explored the critical parameters affecting particle retention and transfer and show that
attachment to the cell surface selectively protects virus particles from serum complement-mediated inactivation. Moreover,
studies with nonmyeloablated murine recipients show that transplantation of vector-exposed, washed hematopoietic cells results
in systemic dissemination of functional VSV-G/lentivector particles. We demonstrate genetic marking by inadvertent transfer
of vector particles and prolonged expression of transgene product in recipient tissues. Our findings have implications for
biosafety, vector design, and cell biology research.
... On murine HSC, CD117 (c-kit or stem cell factor receptor (SCF-R)) is the main surface marker. 4 Recent studies have shown that the lentivirus protein has nuclear localization signals that facilitate its entry into the nuclei of cells that are not dividing. 5 This enables the lentivirus to infect HSC efficiently. ...
... 10 Mikkola et al. have reported successful gene transfer to murine haematopoietic progenitor cells by second-generation lentivirus. 4 So, the purpose of the present study was to investigate the gene transfer efficiency of lentivirus (prepared by tat-defective packaging construct pCMVD8.93) into murine HSC and to investigate haemophilia B gene therapy by lentivirus-infected HSC. ...
... In 2000, Mikkola et al. reported gene transfer in murine HSC using second-generation lentivirus. 4 After that, some investigators made an effort to improve the biosafety of the lentivirus. In 2005, Siapati et al. used third-generation lentivirus in the gene transfer of murine and human HSC. 2 The FUGW and FUXW lentivirus used in the present study, whose packaging construct pCMVD8.93 was tat-defective, also offer significant advantages because of their predicted biosafety. ...
Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX ( hFIX ) gene, respectively, to infect HSC.
High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate‐mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro . Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence‐activated cell sorting, whereas the expression of hFIX was detected by ELISA.
The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)‐3, IL‐6 and stem cell factor.
Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [ ⁶⁰ Co]‐irradiated non‐obese diabetic/severe combined immunodeficiency (NOD‐SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 ± 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice.
The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.
