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Chapter 19 : Gene Therapy in CD4+ T Lymphocytes in SCID-hu Mice

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Abstract:

Development of gene therapy strategies for the treatment of human T-lymphocyte disorders, including AIDS, requires an in vivo system in which transduced human hematopoietic stem cells can be used to reconstitute the T-lymphoid compartment. Combination of HIV-1 pathology and CD34 progenitor cell transduction taking place within a relatively short time in the SCID-hu mouse provides an experimental system in which to address many of the issues that are critical to successful gene therapy approaches for AIDS but are not easily tested in clinical trials. This chapter reviews the recent progress in the use of the SCID-hu system to further one's understanding of vector transduction and expression in modeling potential therapeutic approaches in stem cells. Most anti-AIDS gene therapy strategies are based on the assumption that direct cell killing is the primary pathogenic mechanism for HIV-1 CD4 cell destruction. One of the key issues in human gene therapy has been the development of retroviral vectors with optimal gene expression in differentiated hematopoietic cells. Gene therapy strategies aimed at inhibiting HIV infection or replication require vectors that are expressed in cells infected by HIV, the human CD4 T lymphocytes. Absence of an immune response in SCID-hu mice has two advantages. First, viral pathogenesis can be studied in the absence of an immune response, thereby shortening the time frame for onset of disease. Second, while it is likely that introduction of a foreign protein elicits a host immune response in patients, the effects of therapeutic reagents is assessable independent of the immune response.

Citation: Withers-Ward E, Chen I. 1995. Gene Therapy in CD4+ T Lymphocytes in SCID-hu Mice, p 287-299. In Cooper G, Temin R, Sugden B (ed), The DNA Provirus. ASM Press, Washington, DC. doi: 10.1128/9781555818302.ch19

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Figures

Image of Figure 1
Figure 1

Two-color flow cytometric analysis of Thy-Liv implants. At 6 weeks postinfection, implants were biopsied and stained with monoclonal antibodies to human CD8 and CD4 directly conjugated with fluorescein isothiocyanate or phycoerythrin. (Left) Cells from a mock-infected implant; (right) cells from an HIV-1-infected implant.

Citation: Withers-Ward E, Chen I. 1995. Gene Therapy in CD4+ T Lymphocytes in SCID-hu Mice, p 287-299. In Cooper G, Temin R, Sugden B (ed), The DNA Provirus. ASM Press, Washington, DC. doi: 10.1128/9781555818302.ch19
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Image of Figure 2a
Figure 2a

Reconstitution of SCID-hu Thy-Liv implants with LNL6-transduced CD34 cells. (A) Quantitative PCR analysis of transduced sequences in input CD34+ cells and in reconstituted irradiated implants. CD34 cells were transduced in vitro by cocultivation with the amphotropic PA317 clone 8 producer line ( ). For engraftment and reconstitution, the Thy-Liv implants of irradiated (200 rads) SCID mice were directly injected with 5 × 10 LNL6-transduced CD34 cells within 24 h of irradiation. Sequential biopsies of injected implants were taken at 4 and 6 weeks posttransplantation and analyzed by quantitative PCR and flow cytometry (see panel B). Analysis of the input CD34 cells and two representative reconstituted implants are shown. In each case, PCR analysis for sequences (upper panels) was performed in parallel with PCR analysis for -globin sequences (lower panels). The copy number was determined by comparison with standard reactions containing known amounts of cell or plasmid DNA, which are shown to the right of panel A. Mock control implants were irradiated but did not receive transduced CD34 cells. (B) Representive flow cytometric analysis of control and reconstituted irradiated implants. Panels on the left show forward versus side scatter plots and gating of the live thymocyte population, and panels on the right show the flow cytometric analysis for human CD4 (horizontal axis) and human CD8 (vertical axis) antigens. (C) RT-PCR analysis of control and LNL6-transduced implants. A longer exposure of the gel shown in this panel revealed mRNA expression in both of the reconstituted Thy-Liv implants (shown in panel A) at 6 weeks postreconstitution. RT-PCR analysis of serial dilutions of mRNA isolated from the PA317 producer line is shown at the right. This figure was reprinted from Akkina et al. ( ) with permission from the publisher.

Citation: Withers-Ward E, Chen I. 1995. Gene Therapy in CD4+ T Lymphocytes in SCID-hu Mice, p 287-299. In Cooper G, Temin R, Sugden B (ed), The DNA Provirus. ASM Press, Washington, DC. doi: 10.1128/9781555818302.ch19
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Image of Figure 2b
Figure 2b

Reconstitution of SCID-hu Thy-Liv implants with LNL6-transduced CD34 cells. (A) Quantitative PCR analysis of transduced sequences in input CD34+ cells and in reconstituted irradiated implants. CD34 cells were transduced in vitro by cocultivation with the amphotropic PA317 clone 8 producer line ( ). For engraftment and reconstitution, the Thy-Liv implants of irradiated (200 rads) SCID mice were directly injected with 5 × 10 LNL6-transduced CD34 cells within 24 h of irradiation. Sequential biopsies of injected implants were taken at 4 and 6 weeks posttransplantation and analyzed by quantitative PCR and flow cytometry (see panel B). Analysis of the input CD34 cells and two representative reconstituted implants are shown. In each case, PCR analysis for sequences (upper panels) was performed in parallel with PCR analysis for -globin sequences (lower panels). The copy number was determined by comparison with standard reactions containing known amounts of cell or plasmid DNA, which are shown to the right of panel A. Mock control implants were irradiated but did not receive transduced CD34 cells. (B) Representive flow cytometric analysis of control and reconstituted irradiated implants. Panels on the left show forward versus side scatter plots and gating of the live thymocyte population, and panels on the right show the flow cytometric analysis for human CD4 (horizontal axis) and human CD8 (vertical axis) antigens. (C) RT-PCR analysis of control and LNL6-transduced implants. A longer exposure of the gel shown in this panel revealed mRNA expression in both of the reconstituted Thy-Liv implants (shown in panel A) at 6 weeks postreconstitution. RT-PCR analysis of serial dilutions of mRNA isolated from the PA317 producer line is shown at the right. This figure was reprinted from Akkina et al. ( ) with permission from the publisher.

Citation: Withers-Ward E, Chen I. 1995. Gene Therapy in CD4+ T Lymphocytes in SCID-hu Mice, p 287-299. In Cooper G, Temin R, Sugden B (ed), The DNA Provirus. ASM Press, Washington, DC. doi: 10.1128/9781555818302.ch19
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Image of Figure 3
Figure 3

Determination of the number of LNL6-transduced stem cells required for reconstitution of irradiated Thy-Liv implants. Irradiated implants were injected with various amounts of LNL6-transduced CD34 cells as indicated. Reconstituted implants were biopsied at 6 weeks posttransplantation and analyzed for the presence of sequences by quantitative PCR. In each case, PCR analysis for sequences (upper panel) was performed in parallel with PCR analysis for -globin sequences (lower panels). The copy number was determined by comparison with standard reactions containing known amounts of cell or plasmid DNA, which are shown to the right. Mock control implants were irradiated but did not receive transduced CD34 cells. Each lane represents a separate implant. This figure was reprinted from Akkina et al. ( ) with permission from the publisher.

Citation: Withers-Ward E, Chen I. 1995. Gene Therapy in CD4+ T Lymphocytes in SCID-hu Mice, p 287-299. In Cooper G, Temin R, Sugden B (ed), The DNA Provirus. ASM Press, Washington, DC. doi: 10.1128/9781555818302.ch19
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Image of Figure 4
Figure 4

Detection of the transduced neo gene in the various thymocyte populations of reconstituted implants as they mature into end-stage cells. Cells were obtained from an implant reconstituted with 50,000 LNL6-transduced CD34+ cells at 4 weeks postreconstitution (see Fig. 3 ) and were sorted into CD4+/CD8– CD4–/CD8+ , and CD4+/CD8+ subsets after staining with fluoresceinated monoclonal antibodies specific for the human CD4 and CD8 surface markers. Each population was analyzed by quantitative PCR to determine the percentage of cells in each subgroup that contained the neo gene. Upper panels show PCR analysis for neo sequences, which was performed in parallel with PCR analysis for β-globin sequences (lower panels). The copy number was determined by comparison with standard reactions, which are shown to the right in each case. This figure was reprinted from Akkina et al. (1) with permission from the publisher.

Citation: Withers-Ward E, Chen I. 1995. Gene Therapy in CD4+ T Lymphocytes in SCID-hu Mice, p 287-299. In Cooper G, Temin R, Sugden B (ed), The DNA Provirus. ASM Press, Washington, DC. doi: 10.1128/9781555818302.ch19
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