- Email: [email protected]
progenitor cells
62
Abstracts/Experimental Hematology 28 (2000) 31–131
constituting Fancc ⫺Ⲑ⫺ HSC expressed functional Fancc in vivo, 1⫻106 transduced cells were transplanted into lethally irradiated hosts and evaluated at 6 months. Fancc protein was detected in BM cells by Western analysis. Further Fancc ⫺Ⲑ⫺ progenitors transduced with MSCV-Fancc exhibited a MMC sensitivity similar to WT cells. These data support expression of functional Fancc in vivo. Competitive repopulation studies are in progress to compare repopulating ability of cells expressing recombinant Fancc to WT cells and mock transduced Fancc ⫺Ⲑ⫺ cells. Preliminary data 2 months after transplant suggest that short term repopulating ability was restored to WT levels with introduction of Fancc. These data have important implications regarding the application of gene transfer methodologies using Fancc ⫺Ⲑ⫺ mice as a preclinical model. 98
Monday, July 10, 2000 (9:45–11:15) Session III-4: Molecular Regulation of Hematopoiesis I
EXPRESSION OF CD41 ON CD34⫹ PRIMITIVE YOLK SAC HEMATOPOIETIC PROGENITORS Mervin C. Yoder, Mark Starr*, Xiaodong Xie* Departments of Pediatrics, Indiana University School of Medicine, Indianapolis, IN The phenotype of primitive hematopoietic cells varies during murine ontogeny. CD41 is an integrin alpha chain involved in platelet aggregation and generally considered a platelet lineage marker. Recently, CD41 has been identified as a cell surface marker of primitive hematopoietic cells in the chick. The purpose of the present experiments was to characterize CD41 expression on primitive hematopoietic cells (CD34⫹ cells including repopulating stem cells) in the murine yolk sac. CD41 was expressed on 65% of CD34⫹ cells and CD41⫹CD34⫹ represent 4.5% of fresh yolk sac cells. Cells were isolated by collagenaseⲐdispase digestion of yolk sacs from timed-pregnant females. Plating of 500 CD41⫹CD34⫹ and 5000 CD41-CD34⫹ cells in progenitor or high proliferative potential colony forming cell (HPP) assays revealed that essentially all HPP and committed progenitor activity resides in the CD41⫹CD34⫹ fraction of E9 yolk sac cells (HPP: 10⫾1 vs. 0 for CD41⫺CD34⫹). No HPP or progenitors were observed in the CD41⫹CD34⫺ or CD41⫺CD34⫺ populations. All megakaryocyte progenitors are present in the CD41⫹CD34⫹ population (BFU-Meg: 2⫾1, CFU-Meg 8⫾1). In ongoing studies, 500–1000 CD41⫹CD34⫹ or 10,000 CD41⫺CD34⫹ cells were transplanted into busulfan conditioned newborn congenic pups for evidence of repopulating ability (pending). Of interest, CD41 is expressed on 86% of cKit⫹CD34⫹ cells which contains long-term repopulating cells. In summary, CD41 is expressed on primitive hematopoietic cells and not restricted to the megakaryocyte lineage. 99
Sunday, July 9, 2000 (10:30–12:30) Session I-3: Cord Blood Transplantation
HIGHLY EFFICIENT RETROVIRAL TRANSDUCTION OF CORD BLOOD HEMATOPOIETIC STEM CELLS K. Theunissen* and C.M. Verfaillie Dept of Medicine, Cancer Center, Division of Hematology and Stem Cell Biology Program, Univesrity of Minnesota, Minneapolis, MN Retroviral transduction of hematopoietic stem cells(HSC) requires that these cells divide in vitro while retaining their primitive phenotype. We have recently developed the Myeloid-Lymphoid Initiating Cell (ML-IC) assay (Punzel et al, Blood 93(11)), an in
vitro culture system that allows for enumeration of single cells capable of generating secondary progenitors that can re-initiate long term myeloid and lymphoid cultures. The ML-IC is therefore believed to be closely related to HSC. CD34⫹CD38⫺CD33⫺ cord blood cells were plated in a stroma noncontact culture in CH296 coated transwells above AFT024 stromal feeders in a Flt 3, SCF, IL7 and Tepo containing medium. After 48 hours, and again after 72 hours, they were exposed for 6 hours to GALV pseudotyped MFG-eGFP containing viral supernatant. After a total of 5 days, CD34⫹CD38⫺CD33⫺ cells were single cell sorted to assess ML-IC frequency and transduction efficiency. On average there was a 2-fold expansion of total cell number. 14.6⫾11.3% retained the CD34⫹CD38⫺CD33⫺ phenotype, and of these 80.0⫾14.3% were GFP⫹, whereas only 39.7⫾16.1% of the total cell population was GFP⫹ (p⫽0.016). Within the CD34⫹ CD38⫺CD33⫺ fraction, the ML-IC frequency was 4.3⫾1.8%, and 73.9⫾15.7% of these were transduced. In 16% of the transduced ML-IC, GFP expression was silenced in the myeloid arm but present in the NK-IC. 5Ⲑ69 ML-IC arising from cells in the GFP⫺ population on day 5 proved transduced, i.e. GFP⫹, upon read-out. The generative potential of the ML-IC, defined as the fraction of ML-IC that generates 2 LTC-IC and 2 NK-IC, rather than just 1 of each, was similar for GFP⫹ and GFP⫺ ML-IC (39.6⫾20.5% vs. 51.3⫾11.8%, p⫽0.24). In conclusion, we describe a transduction protocol that efficiently targets primitive progenitors, while retaining their generative potential. Selection of GFP⫹ cells shortly after transduction may lead to underestimation of transduction efficiency. Finally, the ML-IC assay proves to be useful in studying differential silencing in different lineages arising from a single cell. 100
Sunday, July 9, 2000 (14:15–16:00) Session II-2: Gene Targeting and Gene Therapy
SPECIFIC TRANSGENE EXPRESSION IN ANTIGEN PRESENTING CELLS DERIVED FROM LENTIVIRALLY TRANSDUCED HEMATOPOIETIC STEMⲐPROGENITOR CELLS Yan Cui, Jonathan Golob, Xi Huang, Drew Pardoll and Linzhao Cheng Johns Hopkins Oncology Center, Baltimore, MD We previously demonstrated that we could achieve sustained transgene expression in dendritic cells (DCs) differentiated in vitro from retrovirally transduced human CD34⫹ hematopoietic stemⲐ progenitor cells (HSPC). We report here our attempt to specifically express a transgene in DCs and other antigen presenting cells (APC) derived from transduced HSPC. We used self-inactivating lentiviral vectors, in which the GFP reporter expression in transduced cells is solely controlled by the promoter of either human HLA-DR␣ (MHC II) gene or a housekeeping gene (PGK or EF1␣). The three vectors (DR.GFP, PGK.GFP and EF.GFP) were pseudotyped with the VSV-G envelope using 293T cells, and they can transduce a variety of human and murine cells efficiently. The levels of transgene expression by EF.GFP or PGK.GFP (3–8 fold lower) were equivalent in both MHC II⫹ and MHC II⫺ cells. In contrast, transgene expression mediated by DR.GFP is 10–20 fold lower in human MHC II⫺ cells (K562 and U937) than in MHC II⫹ cells (TF1). Similarly, the expression by DR.GFP vector was strong inthe murine MHC II⫹ cells (A20) but reduced drastically in murine MHC II⫺ cells (RENCA and CT26). We also monitored transgene expression in differentiated DC derived from mouse bone marrow (BM) cells in vitro. Fresh BM cells from normal
Abstracts/Experimental Hematology 28 (2000) 31–131
mice were transduced by either DR.GFP or EF.GFP, and then cultured for 8 days to allow DC differentiation. GFP expression was observed exclusively in the MHC II⫹ cell population derived from DR.GFP transduced BM cells, whereas transgene expression was observed equally in both MHC II⫹ and MHC II⫺ cell populations derived from EF.GFP transduced BM cells. Thus, we have developed a gene transduction system to allow stable and specific transgene expression in APC, which has many applications for immunobiology and immunotherapy. 101
Sunday, July 9, 2000 (10:30–12:30) Session I-5: Stem Cell Biology I
A CANDIDATE CYTOKINE GENE SELECTIVELY EXPRESSED IN HUMAN CD34⫹ CELLS Xuan Liu, Nick Rapp, Robert Deans, and Linzhao Cheng. Osiris Therapeutics, Inc. and Johns Hopkins University School of Medicine, Baltimore, MD Residing in bone marrow (BM) and neonatal cord blood (CB), a rare population of human cells bearing the CD34 cell surface marker (CD34⫹) functions as hematopoietic stemⲐprogenitor cells. In contrast, cells lacking CD34 expression (CD34⫺) are largely mature hematopoietic cells of various lineages. To elucidate molecular mechanisms governing functional differences between CD34⫹ and CD34⫺ cells, we set up a molecular screen to identify genes that are preferentially expressed in CD34⫹ cells. After 3 rounds of PCR-based subtraction (CB CD34⫹ vs. CD34⫺) using the RDA method, genes expressed preferentially in CD34⫹ cells were highly enriched (10% and 20% of cloned fragments were from the CD34 and c-kit gene, respectively). Among the 73 initially sequenced RDA fragments, 27 (37%) were novel, or homologous only to entries in EST databases. One of these NovelⲐEST clones (C17) was found 4 times (5.5%). By RT-PCR and Northern blot analyses, C17 gene expression was detected in CD34⫹ cells from CB, BM and G-CSF mobilized peripheral blood, but not in bulk CD34⫺ cells, peripheral blood leukocytes (PBL) or marrow stromal cells. The C17 cDNA is approximately 1.0 kb, and encodes a polypeptide of 136 amino acids. It has a putative signal peptide at its N-terminus. Transient expression in transfected human 293 cells demonstrated that the C17 peptide is indeed a secreted molecule. To date we have not found any existing molecule that shows significant homology to the amino acid sequence of the C17 gene. A secondary structure analysis predicts that the C17 peptide contains 4 alpha-helices, a characteristic of hematopoietic cytokines and interleukins. We are now investigating whether the C17 protein functions as a cytokine. 102
Monday, July 10, 2000 (12:45–14:15) Session IV-4: Lymphocyte Biology and Immunomodulation
LYMPHOID PROGENITORS EXHIBIT ACCELERATED THYMIMIC AND MARROW ENGRAFTMENT RELATIVE TO STEM CELLS S. Scott Perry*, L. Jeanne Pierce*, A. Elena Searles*, Mariluz P. Mojica*, William B. Slayton*, Gerald J. Spangrude Departments of Pathology, Human Genetics, Medicine, Pediatrics, and Oncological Sciences, University of Utah, Salt Lake City, UT We have recently described a mouse bone marrow population defined by the phenotype Thy1.1Neg, Sca1Pos, LinNeg (ThyNeg) which contains lymphoid progenitor cells. To evaluate the engraftment kinetics of these progenitors in comparison to Thy1.1Low, Sca1Pos,
63
LinNeg hematopoietic stemⲐprogenitor cells (HSPC), each population was transplanted intravenously into sublethally irradiated (6.5 Gy) Ly5 congenic mice at 103 cells per mouse. Over a course of 14 to 28 days post-transplant, both thymic lobes and one femur from each mouse were collected and assayed for the presence of donor cells by immunofluorescent staining and flow cytometry. Engraftment of CD19Pos B cells was observed in bone marrow of ThyNeg recipients by day 14, three days earlier than recipients of HSPC. Myeloid engraftment (Gr-1Pos) was also observed in HSPC transplant recipients by day 17, but was not observed in ThyNeg recipients. Thymic reconstitution was observed at significant levels (⬎106 cells per lobe) in 25% of ThyNeg recipients by day 14. In contrast, donor-derived thymocytes were not observed in recipients of HSPC until day 21, when 1 of 20 thymic lobes was positive for significant engraftment compared to 9 of 24 lobes assayed from ThyNeg recipients. HSPC engraftment proceeded rapidly after day 21, and by day 28 all of the lobes assayed were positive for ⬎106 donorderived thymocytes. ThyNeg recipients achieved this degree of engraftment in only 33% of lobes assayed. We conclude from these observations that the ThyNeg population contains lymphoid progenitors capable of more rapid thymic engraftment relative to HSPCs. This suggests that specific commitment steps have predisposed these cells to thymic homing and engraftment. The difference in engraftment kinetics will allow us to distinguish the activity of lymphoid progenitors from HSPCs. This assay will facilitate enrichment and characterization of bone marrow-derived T cell progenitors. 104
Sunday, July 9, 2000 (14:15–16:00) Session II-2: Gene Targeting and Gene Therapy
LONG-TERM, DRUG-DEPENDENT REGULATION OF ERYTHROPOIETIN AND HEMATOCRIT IN MICE FOLLOWING DELIVERY OF PLASMIDS TO SKELETAL MUSCLE V Mehta, RV Abruzzese, D Godin*, FC MacLaughlin*, LC Smith*, JL Nordstrom Valentis, Inc., The Woodlands, TX As gene therapy advances, the need for regulated gene therapy will become paramount for safety and efficacy. The GeneSwitchTM is a gene regulation system that utilizes hormonal levels of antiprogestins, like mifepristone (MFP), to specifically induce transgene expression. A 1:1 mixture of two plasmids (one for the GeneSwitch regulator protein, one for the inducible murine Epo transgene) was delivered to the hind-limb muscles of mice by direct injection followed by electroporation. We observed 9 rounds of induction of Epo expression by administering MFP (0.3 mgⲐkg) at different times during a 9-month period. Each time, serum Epo was induced from an undetectable level to 15–30 mUⲐmL. Peak expression occurred 24 hours following MFP administration, and returned to near basal levels after 72 hrs. We also observed five cycles of induction of hematocrit over a 9-month period. Each cycle, in which the hematocrit increased for 4–5 weeks, occurred in response to 5 successive days of MFP administration. Our data demonstrate that gene therapy with formulated plasmids delivered by electroporation can be used to provide a long-term, drug-controllable therapeutic benefit in a manner that had previously been achieved only with viral systems. The increased safety margin associated with nonviral gene therapy makes our plasmid-based GeneSwitch system extremely attractive for the controlled expression of systemic proteins of therapeutic interest.
Abstracts/Experimental Hematology 28 (2000) 31–131
constituting Fancc ⫺Ⲑ⫺ HSC expressed functional Fancc in vivo, 1⫻106 transduced cells were transplanted into lethally irradiated hosts and evaluated at 6 months. Fancc protein was detected in BM cells by Western analysis. Further Fancc ⫺Ⲑ⫺ progenitors transduced with MSCV-Fancc exhibited a MMC sensitivity similar to WT cells. These data support expression of functional Fancc in vivo. Competitive repopulation studies are in progress to compare repopulating ability of cells expressing recombinant Fancc to WT cells and mock transduced Fancc ⫺Ⲑ⫺ cells. Preliminary data 2 months after transplant suggest that short term repopulating ability was restored to WT levels with introduction of Fancc. These data have important implications regarding the application of gene transfer methodologies using Fancc ⫺Ⲑ⫺ mice as a preclinical model. 98
Monday, July 10, 2000 (9:45–11:15) Session III-4: Molecular Regulation of Hematopoiesis I
EXPRESSION OF CD41 ON CD34⫹ PRIMITIVE YOLK SAC HEMATOPOIETIC PROGENITORS Mervin C. Yoder, Mark Starr*, Xiaodong Xie* Departments of Pediatrics, Indiana University School of Medicine, Indianapolis, IN The phenotype of primitive hematopoietic cells varies during murine ontogeny. CD41 is an integrin alpha chain involved in platelet aggregation and generally considered a platelet lineage marker. Recently, CD41 has been identified as a cell surface marker of primitive hematopoietic cells in the chick. The purpose of the present experiments was to characterize CD41 expression on primitive hematopoietic cells (CD34⫹ cells including repopulating stem cells) in the murine yolk sac. CD41 was expressed on 65% of CD34⫹ cells and CD41⫹CD34⫹ represent 4.5% of fresh yolk sac cells. Cells were isolated by collagenaseⲐdispase digestion of yolk sacs from timed-pregnant females. Plating of 500 CD41⫹CD34⫹ and 5000 CD41-CD34⫹ cells in progenitor or high proliferative potential colony forming cell (HPP) assays revealed that essentially all HPP and committed progenitor activity resides in the CD41⫹CD34⫹ fraction of E9 yolk sac cells (HPP: 10⫾1 vs. 0 for CD41⫺CD34⫹). No HPP or progenitors were observed in the CD41⫹CD34⫺ or CD41⫺CD34⫺ populations. All megakaryocyte progenitors are present in the CD41⫹CD34⫹ population (BFU-Meg: 2⫾1, CFU-Meg 8⫾1). In ongoing studies, 500–1000 CD41⫹CD34⫹ or 10,000 CD41⫺CD34⫹ cells were transplanted into busulfan conditioned newborn congenic pups for evidence of repopulating ability (pending). Of interest, CD41 is expressed on 86% of cKit⫹CD34⫹ cells which contains long-term repopulating cells. In summary, CD41 is expressed on primitive hematopoietic cells and not restricted to the megakaryocyte lineage. 99
Sunday, July 9, 2000 (10:30–12:30) Session I-3: Cord Blood Transplantation
HIGHLY EFFICIENT RETROVIRAL TRANSDUCTION OF CORD BLOOD HEMATOPOIETIC STEM CELLS K. Theunissen* and C.M. Verfaillie Dept of Medicine, Cancer Center, Division of Hematology and Stem Cell Biology Program, Univesrity of Minnesota, Minneapolis, MN Retroviral transduction of hematopoietic stem cells(HSC) requires that these cells divide in vitro while retaining their primitive phenotype. We have recently developed the Myeloid-Lymphoid Initiating Cell (ML-IC) assay (Punzel et al, Blood 93(11)), an in
vitro culture system that allows for enumeration of single cells capable of generating secondary progenitors that can re-initiate long term myeloid and lymphoid cultures. The ML-IC is therefore believed to be closely related to HSC. CD34⫹CD38⫺CD33⫺ cord blood cells were plated in a stroma noncontact culture in CH296 coated transwells above AFT024 stromal feeders in a Flt 3, SCF, IL7 and Tepo containing medium. After 48 hours, and again after 72 hours, they were exposed for 6 hours to GALV pseudotyped MFG-eGFP containing viral supernatant. After a total of 5 days, CD34⫹CD38⫺CD33⫺ cells were single cell sorted to assess ML-IC frequency and transduction efficiency. On average there was a 2-fold expansion of total cell number. 14.6⫾11.3% retained the CD34⫹CD38⫺CD33⫺ phenotype, and of these 80.0⫾14.3% were GFP⫹, whereas only 39.7⫾16.1% of the total cell population was GFP⫹ (p⫽0.016). Within the CD34⫹ CD38⫺CD33⫺ fraction, the ML-IC frequency was 4.3⫾1.8%, and 73.9⫾15.7% of these were transduced. In 16% of the transduced ML-IC, GFP expression was silenced in the myeloid arm but present in the NK-IC. 5Ⲑ69 ML-IC arising from cells in the GFP⫺ population on day 5 proved transduced, i.e. GFP⫹, upon read-out. The generative potential of the ML-IC, defined as the fraction of ML-IC that generates 2 LTC-IC and 2 NK-IC, rather than just 1 of each, was similar for GFP⫹ and GFP⫺ ML-IC (39.6⫾20.5% vs. 51.3⫾11.8%, p⫽0.24). In conclusion, we describe a transduction protocol that efficiently targets primitive progenitors, while retaining their generative potential. Selection of GFP⫹ cells shortly after transduction may lead to underestimation of transduction efficiency. Finally, the ML-IC assay proves to be useful in studying differential silencing in different lineages arising from a single cell. 100
Sunday, July 9, 2000 (14:15–16:00) Session II-2: Gene Targeting and Gene Therapy
SPECIFIC TRANSGENE EXPRESSION IN ANTIGEN PRESENTING CELLS DERIVED FROM LENTIVIRALLY TRANSDUCED HEMATOPOIETIC STEMⲐPROGENITOR CELLS Yan Cui, Jonathan Golob, Xi Huang, Drew Pardoll and Linzhao Cheng Johns Hopkins Oncology Center, Baltimore, MD We previously demonstrated that we could achieve sustained transgene expression in dendritic cells (DCs) differentiated in vitro from retrovirally transduced human CD34⫹ hematopoietic stemⲐ progenitor cells (HSPC). We report here our attempt to specifically express a transgene in DCs and other antigen presenting cells (APC) derived from transduced HSPC. We used self-inactivating lentiviral vectors, in which the GFP reporter expression in transduced cells is solely controlled by the promoter of either human HLA-DR␣ (MHC II) gene or a housekeeping gene (PGK or EF1␣). The three vectors (DR.GFP, PGK.GFP and EF.GFP) were pseudotyped with the VSV-G envelope using 293T cells, and they can transduce a variety of human and murine cells efficiently. The levels of transgene expression by EF.GFP or PGK.GFP (3–8 fold lower) were equivalent in both MHC II⫹ and MHC II⫺ cells. In contrast, transgene expression mediated by DR.GFP is 10–20 fold lower in human MHC II⫺ cells (K562 and U937) than in MHC II⫹ cells (TF1). Similarly, the expression by DR.GFP vector was strong inthe murine MHC II⫹ cells (A20) but reduced drastically in murine MHC II⫺ cells (RENCA and CT26). We also monitored transgene expression in differentiated DC derived from mouse bone marrow (BM) cells in vitro. Fresh BM cells from normal
Abstracts/Experimental Hematology 28 (2000) 31–131
mice were transduced by either DR.GFP or EF.GFP, and then cultured for 8 days to allow DC differentiation. GFP expression was observed exclusively in the MHC II⫹ cell population derived from DR.GFP transduced BM cells, whereas transgene expression was observed equally in both MHC II⫹ and MHC II⫺ cell populations derived from EF.GFP transduced BM cells. Thus, we have developed a gene transduction system to allow stable and specific transgene expression in APC, which has many applications for immunobiology and immunotherapy. 101
Sunday, July 9, 2000 (10:30–12:30) Session I-5: Stem Cell Biology I
A CANDIDATE CYTOKINE GENE SELECTIVELY EXPRESSED IN HUMAN CD34⫹ CELLS Xuan Liu, Nick Rapp, Robert Deans, and Linzhao Cheng. Osiris Therapeutics, Inc. and Johns Hopkins University School of Medicine, Baltimore, MD Residing in bone marrow (BM) and neonatal cord blood (CB), a rare population of human cells bearing the CD34 cell surface marker (CD34⫹) functions as hematopoietic stemⲐprogenitor cells. In contrast, cells lacking CD34 expression (CD34⫺) are largely mature hematopoietic cells of various lineages. To elucidate molecular mechanisms governing functional differences between CD34⫹ and CD34⫺ cells, we set up a molecular screen to identify genes that are preferentially expressed in CD34⫹ cells. After 3 rounds of PCR-based subtraction (CB CD34⫹ vs. CD34⫺) using the RDA method, genes expressed preferentially in CD34⫹ cells were highly enriched (10% and 20% of cloned fragments were from the CD34 and c-kit gene, respectively). Among the 73 initially sequenced RDA fragments, 27 (37%) were novel, or homologous only to entries in EST databases. One of these NovelⲐEST clones (C17) was found 4 times (5.5%). By RT-PCR and Northern blot analyses, C17 gene expression was detected in CD34⫹ cells from CB, BM and G-CSF mobilized peripheral blood, but not in bulk CD34⫺ cells, peripheral blood leukocytes (PBL) or marrow stromal cells. The C17 cDNA is approximately 1.0 kb, and encodes a polypeptide of 136 amino acids. It has a putative signal peptide at its N-terminus. Transient expression in transfected human 293 cells demonstrated that the C17 peptide is indeed a secreted molecule. To date we have not found any existing molecule that shows significant homology to the amino acid sequence of the C17 gene. A secondary structure analysis predicts that the C17 peptide contains 4 alpha-helices, a characteristic of hematopoietic cytokines and interleukins. We are now investigating whether the C17 protein functions as a cytokine. 102
Monday, July 10, 2000 (12:45–14:15) Session IV-4: Lymphocyte Biology and Immunomodulation
LYMPHOID PROGENITORS EXHIBIT ACCELERATED THYMIMIC AND MARROW ENGRAFTMENT RELATIVE TO STEM CELLS S. Scott Perry*, L. Jeanne Pierce*, A. Elena Searles*, Mariluz P. Mojica*, William B. Slayton*, Gerald J. Spangrude Departments of Pathology, Human Genetics, Medicine, Pediatrics, and Oncological Sciences, University of Utah, Salt Lake City, UT We have recently described a mouse bone marrow population defined by the phenotype Thy1.1Neg, Sca1Pos, LinNeg (ThyNeg) which contains lymphoid progenitor cells. To evaluate the engraftment kinetics of these progenitors in comparison to Thy1.1Low, Sca1Pos,
63
LinNeg hematopoietic stemⲐprogenitor cells (HSPC), each population was transplanted intravenously into sublethally irradiated (6.5 Gy) Ly5 congenic mice at 103 cells per mouse. Over a course of 14 to 28 days post-transplant, both thymic lobes and one femur from each mouse were collected and assayed for the presence of donor cells by immunofluorescent staining and flow cytometry. Engraftment of CD19Pos B cells was observed in bone marrow of ThyNeg recipients by day 14, three days earlier than recipients of HSPC. Myeloid engraftment (Gr-1Pos) was also observed in HSPC transplant recipients by day 17, but was not observed in ThyNeg recipients. Thymic reconstitution was observed at significant levels (⬎106 cells per lobe) in 25% of ThyNeg recipients by day 14. In contrast, donor-derived thymocytes were not observed in recipients of HSPC until day 21, when 1 of 20 thymic lobes was positive for significant engraftment compared to 9 of 24 lobes assayed from ThyNeg recipients. HSPC engraftment proceeded rapidly after day 21, and by day 28 all of the lobes assayed were positive for ⬎106 donorderived thymocytes. ThyNeg recipients achieved this degree of engraftment in only 33% of lobes assayed. We conclude from these observations that the ThyNeg population contains lymphoid progenitors capable of more rapid thymic engraftment relative to HSPCs. This suggests that specific commitment steps have predisposed these cells to thymic homing and engraftment. The difference in engraftment kinetics will allow us to distinguish the activity of lymphoid progenitors from HSPCs. This assay will facilitate enrichment and characterization of bone marrow-derived T cell progenitors. 104
Sunday, July 9, 2000 (14:15–16:00) Session II-2: Gene Targeting and Gene Therapy
LONG-TERM, DRUG-DEPENDENT REGULATION OF ERYTHROPOIETIN AND HEMATOCRIT IN MICE FOLLOWING DELIVERY OF PLASMIDS TO SKELETAL MUSCLE V Mehta, RV Abruzzese, D Godin*, FC MacLaughlin*, LC Smith*, JL Nordstrom Valentis, Inc., The Woodlands, TX As gene therapy advances, the need for regulated gene therapy will become paramount for safety and efficacy. The GeneSwitchTM is a gene regulation system that utilizes hormonal levels of antiprogestins, like mifepristone (MFP), to specifically induce transgene expression. A 1:1 mixture of two plasmids (one for the GeneSwitch regulator protein, one for the inducible murine Epo transgene) was delivered to the hind-limb muscles of mice by direct injection followed by electroporation. We observed 9 rounds of induction of Epo expression by administering MFP (0.3 mgⲐkg) at different times during a 9-month period. Each time, serum Epo was induced from an undetectable level to 15–30 mUⲐmL. Peak expression occurred 24 hours following MFP administration, and returned to near basal levels after 72 hrs. We also observed five cycles of induction of hematocrit over a 9-month period. Each cycle, in which the hematocrit increased for 4–5 weeks, occurred in response to 5 successive days of MFP administration. Our data demonstrate that gene therapy with formulated plasmids delivered by electroporation can be used to provide a long-term, drug-controllable therapeutic benefit in a manner that had previously been achieved only with viral systems. The increased safety margin associated with nonviral gene therapy makes our plasmid-based GeneSwitch system extremely attractive for the controlled expression of systemic proteins of therapeutic interest.